EA041410B1 - APPLICATION OF MINERALOCORTICOID RECEPTOR ANTAGONIST FOR TREATMENT OF CHRONIC KIDNEY DISEASE AND METHOD FOR TREATMENT OF CHRONIC KIDNEY DISEASE - Google Patents

Description

The field of technology to which the present invention relates

The present invention relates to the field of pharmaceutical technology and, in particular, describes a pharmaceutical composition containing a mineralocorticoid receptor antagonist, the use of the composition in the preparation of a medicament for the treatment and/or prevention of chronic kidney disease, and a method for treating a patient with chronic kidney disease using the composition.

Background of the Invention

Chronic kidney disease (CKD) is a type of disease characterized by (1) 3 or more months of kidney damage with or without reduced glomerular filtration rate (GFR); or (2) a glomerular filtration rate of less than 60 ml/min/1.73 m ² for 3 months or longer with or without kidney damage; while kidney damage is defined as pathological abnormalities or markers of damage, including abnormalities in blood or urine tests or imaging studies [The National Kidney Foundation, NKF KDOQI Clinical Practice Guidelines for Chronic Kidney Disease: Evaluation, Classification, and Stratification]. The most common clinical symptoms include proteinuria (foamy urine), hematuria, edema, hypertension, increased night urine, anemia, and the like. Chronic kidney disease in the late stages leads to chronic kidney failure and a certain clinical syndrome that develops as a systemic disorder, manifested in the accumulation of metabolites, water / electrolyte imbalance, acidemia, etc.

Aldosterone is a steroid hormone with mineralocorticoid activity, which is produced mainly by the zona glomeruli of the adrenal glands [Kidney International (2012), 81, 955-968]. Aldosterone mainly functions to regulate Na ⁺ reabsorption and K ⁺ excretion in the distal nephron, maintaining electrolyte balance and volumetric homeostasis. In addition to sodium retention, aldosterone can also cause pathological progression leading to inflammation, remodeling, and fibrosis. Aldosterone activates the mineralocorticoid receptor (MR) by acting on the blood vessels, causing vasoconstriction. Aldosterone has also been shown to cause damage to renal tissue resulting in increased proteinuria/albuminuria. Excessive levels of aldosterone lead to hypertension, heart failure (HF) and chronic kidney disease (CKD) [Expert Opin. Investig. Drugs (2015), 24(8), 1-13].

A mineralocorticoid receptor antagonist (MRA) binds to the mineralocorticoid receptor to block the interaction of aldosterone with the mineralocorticoid receptor [INSPRA guidelines, Pfizer, 2002]. MRA has been approved clinically for patients with heart failure with reduced ejection fraction, hypertension and/or chronic kidney disease [Curr Opin Nephrol Hypertens, 2015, 24:417-424].

So far, only two steroidal mineralocorticoid receptor antagonists have been developed for clinical use. Spironolactone, a first generation MRA with relatively high potency, is characterized by side effects such as gynecomastia, impotence, and menstrual irregularities due to its progesterone-like structure. Eplerenone, a second generation MRA, has improved selectivity but reduced potency [Expert Opin. Investig. Drugs (2015), 24(8), 1-13]. Both spironolactone and eplerenone may reduce hospitalization and/or mortality in patients with heart failure with reduced ejection fraction; reduce urinary albumin or urinary albumin to creatinine ratio (UACR) and slow progression in patients with chronic kidney disease. However, the risk of hyperkalemia has limited the use of these two MRAs, especially in patients with kidney injury [Expert Opin. Investig. Drugs (2015), 24(8), 1-13; Kedney International, 2012, 81:955-968]. Spironolactone is contraindicated in patients with severe kidney injury [Aldactone® (spironolactone tablets, USP)], while eplerenone is contraindicated in patients with moderate to severe kidney disease with hypertension, and in patients with severe kidney injury [INSPRA® statement (eplerenone), oral tablets (USP)].

Finerenone is a non-steroidal MRA developed by Bayer and has better MR selectivity than spironolactone and higher MR affinity than eplerenone [Expert Opin. Investig. Drugs (2015), 24(8), 1-13]. Finerenone was investigated in clinical trials for the treatment of diabetic nephropathy (ARTS-DN), with UACR reduced by 21%, 24%, 33% and 38% in the groups administered finerenone at doses of 7.5, 10, 15 and 20 mg/day. day respectively. However, the onset of hyperkalemia results in treatment discontinuation in these groups at rates of 2.1%, 0%, 3.2%, and 1.7%, respectively [JAMA, 2015, 314(9):884-894]. The problem of hyperkalemia manifests itself in dosage groups in which the therapeutic effect is unsatisfactory.

Compound I, 2-chloro-4-[(3S,3aR)-3-cyclopentyl-7-(4-hydroxylpiperidine-1-carbonyl)-3,3a,4,5tetrahydro-2H-pyrazolo[3,4-f] quinolin-2-yl]benzonitrile having the following formula is disclosed in international patent publications WO 2012022121 A1 and WO 2014094664 A1. It's co

- 1 041410 Unity is also a non-steroidal mineralocorticoid receptor antagonist (MRA) that exhibits relatively high selectivity and affinity for MR and thus is useful in the treatment of chronic kidney disease. However, since MR-targeting drugs always induce hyperkalemia, the prior art has not disclosed any product containing Compound I or any method using Compound I that has proven to be safe and effective.

Compound I

In healthy subjects, approximately 90% of potassium is excreted by the kidneys, with the rest excreted in sweat and feces. Patients with chronic kidney disease with impaired potassium ion modulatory capacity are more likely to develop hyperkalemia, particularly in patients with moderate to severe kidney disease.

Elevated serum potassium causes damage in patients, especially damage to the heart. If the condition is severe, abnormal heart rhythms can occur, which can lead to cardiac arrest and even death. In another aspect, high serum potassium can also make the neuromuscular system less agitated, leading to flaccid paralysis, or can cause deterioration of the digestive system, leading to abdominal pain, nausea, vomiting, or the like.

Hyperkalemia leads to lesions in patients with chronic kidney disease. It negatively affects the depolarization and repolarization of cardiomyocytes and thus causes delayed conduction of electrical waves and cardiac arrhythmia. Untreated, severe hyperkalemia can cause ventricular fibrillation and cardiac arrest, leading to sudden cardiac death.

At the present stage, there is no clinically safe and effective drug for the treatment of chronic kidney disease, despite the constant need for it. Although the mineralocorticoid receptor antagonist (MRA) has shown effects in the treatment of kidney disease, its clinical use is limited due to high serum potassium due to its underlying mechanism of action.

Therefore, the technical challenge is to find a safe and effective drug or method for treating chronic kidney disease while avoiding elevated serum potassium levels.

Brief summary of the present invention

The present inventors found that Compound I cannot be used for the safe and effective treatment of chronic kidney disease because (1) Compound I is a mineralocorticoid receptor antagonist that is highly likely to cause elevated serum potassium due to its target action; and (2) the absorption of a pharmaceutical composition containing Compound I varies among patients when it is prepared by conventional techniques.

Thus, the therapeutic window of Compound I is quite narrow in the treatment of patients with chronic kidney disease. In other words, a drug manufactured using conventional technical means does not meet clinical requirements.

In view of the foregoing, the inventors of the present invention conducted extensive tests and finally solved the problem. In particular, the present inventors found a correlation between the occurrence of elevated serum potassium and the area under the plasma concentration-time curve (AUC), and also found a safety window that can produce a therapeutic effect without causing elevated serum potassium. More specifically, the safe and effective AUC of Compound I is in the range of 188 to 3173 nghh/ml when administered to patients with chronic kidney disease.

In order to achieve the safety window mentioned above, the present inventors have found a safe and effective dosage range and a pharmaceutical composition providing such a dosage range when administered to subjects. The pharmaceutical composition according to the present invention is characterized by a bioavailability of 50% or more in mammals. When the pharmaceutical composition of the present invention is administered to a patient with chronic kidney disease in the dosage range of the present invention, the safe and effective AUC of Compound I is in the range of 188 to 3173 nghh/mL.

The present invention relates to the use of compound I for the treatment of a chronic disease

- 2 041410 human kidneys.

When a compound I of the present invention is orally administered to a patient with chronic kidney disease, the effective and safe area under the plasma concentration-time curve (AUC) of compound I is in the range of 188 to 3173 nghh/mL. According to one embodiment of the present invention, an effective and safe area under the plasma concentration versus I curve is in the range of 188 to 2893 nghh/mL. According to one embodiment of the present invention, an effective and safe area under the plasma concentration-time curve (AUC) of Compound I is in the range of 188 to 2613 nghh/mL. According to one embodiment of the present invention, an effective and safe area under the plasma concentration-time curve (AUC) of Compound I is in the range of 188 to 1117 nghh/mL. According to one embodiment of the present invention, an effective and safe area under the plasma concentration-time curve (AUC) of Compound I is in the range of 188 to 885 nghh/mL.

Considering that the bioavailability of the pharmaceutical composition according to the present invention is 50% or more in mammals, in order to obtain a safe and effective AUC value in patients with chronic kidney disease, according to the present invention, the use of the compound in a daily dose of 0.1 to 1.0 mg is proposed.

In one embodiment, the present invention provides the use of Compound I at a daily dose of 0.1 to 1.0 mg per patient to obtain the safe and effective AUC value mentioned above. Optionally, the daily dose of Compound I is in the range of 0.1 to 0.9 mg. Optionally, the daily dose of Compound I is in the range of 0.1 to 0.8 mg. Optionally, the daily dose of Compound I is in the range of 0.1 to 0.7 mg. Optionally, the daily dose of Compound I is in the range of 0.1 to 0.6 mg. Optionally, the daily dose of Compound I is in the range of 0.1 to 0.5 mg. Optionally, the daily dose of compound I is in the range of 0.15 to 1 mg. Optionally, the daily dose of Compound I is in the range of 0.15 to 0.9 mg. Optionally, the daily dose of Compound I is in the range of 0.15 to 0.8 mg. Optionally, the daily dose of Compound I is in the range of 0.15 to 0.7 mg. Optionally, the daily dose of Compound I is in the range of 0.15 to 0.6 mg. Optionally, the daily dose of Compound I is in the range of 0.15 to 0.5 mg. Optionally, the daily dose of Compound I is in the range of 0.2 to 1 mg. Optionally, the daily dose of Compound I is in the range of 0.2 to 0.9 mg. Optionally, the daily dose of Compound I is in the range of 0.2 to 0.8 mg. Optionally, the daily dose of Compound I is in the range of 0.2 to 0.7 mg. Optionally, the daily dose of Compound I is in the range of 0.2 to 0.6 mg. Optionally, the daily dose of Compound I is in the range of 0.2 to 0.5 mg. Optionally, the daily dose of Compound I is in the range of 0.25 to 1 mg. Optionally, the daily dose of Compound I is in the range of 0.25 to 0.9 mg. Optionally, the daily dose of Compound I is in the range of 0.25 to 0.8 mg. Optionally, the daily dose of Compound I is in the range of 0.25 to 0.7 mg. Optionally, the daily dose of Compound I is in the range of 0.25 to 0.6 mg. Optionally, the daily dose of Compound I is in the range of 0.25 to 0.5 mg. Optionally, the daily dose of Compound I is 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1 mg.

Compound I is insoluble in water and has a medium penetration rate. The pharmaceutical composition of said compound, produced by conventional techniques, provides low bioavailability and there is great inter-subject variability upon administration. To obtain a clinically acceptable effect, ie. for effective treatment of all patients, it is generally accepted to increase the dose. However, due to great inter-individual variability, in some patients after the administration of compound I, the AUC value is too high, which leads to an increased risk of elevated serum potassium levels. In particular, in patients with chronic kidney disease, the ability to modulate potassium ions is impaired, and elevated serum potassium may lead to higher health risks.

The use of Compound I at the indicated daily dose satisfies clinical safety and efficacy requirements by increasing bioavailability to reduce individual variability in AUC.

The present invention provides the use of compound I for the manufacture of a pharmaceutical composition for the treatment of chronic kidney disease in humans at a dose of 0.1-1.0 mg.

According to one embodiment, the use of compound I for the manufacture of a pharmaceutical composition for the treatment of chronic kidney disease in humans at a dose of 0.1 to 0.9 mg is proposed. Optionally, the daily dose of Compound I is in the range of 0.1 to 0.8 mg. Optionally, the daily dose of Compound I is in the range of 0.1 to 0.7 mg. Optionally, the daily dose of Compound I is in the range of 0.1 to 0.6 mg. Optionally, the daily dose of Compound I is in the range of 0.1 to 0.5 mg. Optionally, the daily dose of compound I is in the range of 0.15 to 1 mg. Optionally, the daily dose of Compound I is in the range of 0.15 to 0.9 mg. Optional su

- 3 041410 the exact dose of compound I is in the range of 0.15 to 0.8 mg. Optionally, the daily dose of Compound I is in the range of 0.15 to 0.7 mg. Optionally, the daily dose of Compound I is in the range of 0.15 to 0.6 mg. Optionally, the daily dose of Compound I is in the range of 0.15 to 0.5 mg. Optionally, the dose of Compound I is in the range of 0.2 to 1 mg. Optionally, the dose of Compound I is in the range of 0.2 to 0.9 mg. Optionally, the dose of Compound I is in the range of 0.2 to 0.8 mg. Optionally, the dose of Compound I is in the range of 0.2 to 0.7 mg. Optionally, the dose of Compound I is in the range of 0.2 to 0.6 mg. Optionally, the dose of Compound I is in the range of 0.2 to 0.5 mg. Optionally, the daily dose of Compound I is in the range of 0.25 to 1 mg. Optionally, the daily dose of Compound I is in the range of 0.25 to 0.9 mg. Optionally, the daily dose of Compound I is in the range of 0.25 to 0.8 mg. Optionally, the daily dose of Compound I is in the range of 0.25 to 0.7 mg. Optionally, the daily dose of Compound I is in the range of 0.25 to 0.6 mg. Optionally, the daily dose of Compound I is in the range of 0.25 to 0.5 mg.

Optionally, the daily dose of Compound I is in the range of 0.3 to 1 mg. Optionally, the daily dose of Compound I is in the range of 0.3 to 0.9 mg. Optionally, the daily dose of Compound I is in the range of 0.3 to 0.8 mg. Optionally, the daily dose of Compound I is in the range of 0.3 to 0.7 mg. Optionally, the daily dose of Compound I is in the range of 0.3 to 0.6 mg. Optionally, the daily dose of Compound I is in the range of 0.3 to 0.5 mg. Optionally, the daily dose of Compound I is 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2 or 2.5 mg.

When the pharmaceutical composition of the present invention is administered to a patient at the dose mentioned above, the effective and safe area under the plasma concentration-time curve (AUC) of Compound I is in the range of 188 to 3173 nghh/mL. According to one embodiment of the present invention, an effective and safe area under the plasma concentration-time curve (AUC) of Compound I is in the range of 188 to 2893 nghh/mL. According to one embodiment of the present invention, an effective and safe area under the plasma concentration-time curve (AUC) of Compound I is in the range of 188 to 2613 nghh/mL. According to one embodiment of the present invention, an effective and safe area under the plasma concentration-time curve (AUC) of Compound I is in the range of 188 to 1117 nghh/mL. According to one embodiment of the present invention, an effective and safe area under the plasma concentration-time curve (AUC) of Compound I is in the range of 188 to 885 nghh/mL.

Compound I is insoluble in water and has a medium penetration rate. The pharmaceutical composition of said compound, produced by conventional techniques, provides low bioavailability and there is great inter-subject variability upon administration. To obtain a clinically acceptable effect, ie. for effective treatment of all patients, it is generally accepted to increase the dose. However, due to significant inter-individual variability, the AUC of Compound I is too high in some patients, leading to an increased risk of elevated serum potassium levels. In particular, in patients with chronic kidney disease, the ability to modulate potassium ions is impaired, and elevated serum potassium leads to higher health risks.

The present pharmaceutical composition satisfies clinical safety and efficacy requirements by improving bioavailability to effectively reduce individual variability in AUC.

In order to increase the bioavailability of the pharmaceutical composition to a level of 50% or more in mammals, according to one embodiment of the present invention, the particle size of compound I is reduced in the pharmaceutical composition of the present invention. In one embodiment of the present invention, the particle size distribution parameter D ₉₀ of compound I is 25 µm or less. According to one embodiment of the present invention, the particle size distribution parameter D ₉₀ of compound I is 21.7 µm or less. According to one embodiment of the present invention, the particle size distribution parameter D ₉₀ of compound I is 10 μm or less. According to one embodiment of the present invention, the particle size distribution parameter D ₉₀ of compound I is 5 μm or less.

Compound I in a pharmaceutical composition with various particle sizes can be obtained by grinding, extruding, impinging, cutting, mechanical fine grinding, vibratory fine grinding, fluidized bed grinding, ultrasonication, high pressure grinding, chemical precipitation, or the like.

To increase the bioavailability of the pharmaceutical composition to a level of 50% or more in mammals, according to one embodiment of the present invention, a surfactant is added to the pharmaceutical composition.

- 4 041410

The surfactant is one or more selected from the group consisting of the following: benzalkonium chloride, sodium lauryl sulfonate, sodium dodecyl sulfate, glycerin, cholic acid, poloxamer, polyvinyl alcohol, polysorbate 80, PVP K ₃₀ and polyethylene glycol. Preferably the surfactant is one or more selected from the group consisting of benzalkonium chloride, sodium lauryl sulfonate and sodium dodecyl sulfate. Preferably the surfactant is benzalkonium chloride, sodium lauryl sulfonate or sodium dodecyl sulfate.

In the pharmaceutical composition according to the present invention, the weight ratio of compound I to surfactant is 1:0.1-1:20, preferably 1:1-1:20 and more preferably 1:5-1:20.

The present invention relates to a pharmaceutical composition containing Compound I and a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention can be formulated as an oral formulation, more preferably tablets, extended release tablets, capsules, granules, soft capsules, micropills, microcapsules, microspheres, liposomes, self-emulsifying drug delivery systems, solid dispersions, orally soluble micelles. cavities of tablets, solutions, suspensions or emulsions.

A single use pharmaceutical composition according to the present invention contains 0.1 to 1 mg of Compound I, optionally 0.1 to 0.9 mg of Compound I, optionally 0.1 to 0.8 mg of Compound I, optionally 0.1 to 0 .7 mg of compound I, optionally 0.1 to 0.6 mg of compound I, optionally 0.15 to 1 mg of compound I, optionally 0.15 to 0.9 mg of compound I, optionally 0.15 to 0.8 mg of compound I, optionally 0.15 to 0.7 mg of compound I, optionally 0.15 to 0.6 mg of compound I, optionally 0.15 to 0.5 mg of compound I, optionally 0.2 to 1 mg of compound I, optionally 0 2 to 0.9 mg of Compound I, optionally 0.2 to 0.8 mg of Compound I, optionally 0.2 to 0.7 mg of Compound I, optionally 0.2 to 0.6 mg of Compound I, optionally 0.2 up to 0.5 mg of compound I, optionally 0.25 to 1 mg of compound I, optionally 0.25 to 0.9 mg of compound I, optionally 0.25 to 0.8 mg of compound I, optionally 0.25 to 0.7 mg compound I, optionally 0.25 to 0 .6 mg of compound I, optionally 0.25 to 0.5 mg of compound I, optionally 0.3 to 1 mg of compound I, optionally 0.3 to 0.9 mg of compound I, optionally 0.3 to 0.8 mg of compound I, optionally 0.3 to 0.7 mg of compound I, optionally 0.3 to 0.6 mg of compound I, optionally 0.3 to 0.5 mg of compound I, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35 mg, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1 mg of compound I.

The pharmaceutical composition of the present invention can easily block the binding of the mineralocorticoid receptor to aldosterone. According to the main pharmacological action, the pharmaceutical composition can be used specifically for the treatment and/or prevention of chronic kidney disease selected from the group consisting of the following: hypertensive nephropathy, diabetic nephropathy, glomerulonephritis, renal failure, albuminuria, acute kidney injury and kidney cyst; chronic kidney disease with hypertension, chronic kidney disease with heart failure, chronic kidney disease with hypertension and heart failure, chronic kidney disease with obesity, chronic kidney disease with hyperlipidemia, chronic kidney disease with diabetes mellitus and cardiorenal syndrome; cardiovascular diseases selected from the group consisting of the following: hypertension, heart failure (i.e. congestive heart failure selected from the group consisting of the following; heart failure with reduced ejection fraction, heart failure with normal ejection fraction and acute heart failure) , myocardial infarction, angina pectoris, cardiac hypertrophy, myocarditis, cardiac vascular fibrosis, baroreceptor dysfunction, hypervolemia and cardiac arrhythmia, hyperlipidemia and obesity; endocrine diseases selected from the group consisting of the following: primary and secondary hyperaldosteronism, Addison's disease, Itsenko-Cushing's syndrome and Bartter's syndrome. In particular, the pharmaceutical composition according to the present invention can be used for the treatment and/or prevention of chronic kidney disease, including the following: diabetic nephropathy and hypertensive nephropathy; chronic kidney disease with hypertension and/or heart failure; heart failure and/or hypertension.

In another embodiment, the present invention relates to a method of treating chronic kidney disease comprising administering to a subject in need a therapeutically effective amount of Compound I at a dose of 0.1 to 1 mg.

The subject of the present invention may be a mammal, preferably a human, in particular those characterized by the presence of a disease selected from the group consisting of the following: chronic kidney disease selected from the group consisting of the following: hypertensive nephropathy, diabetic nephropathy, glomerulonephritis, renal insufficiency, albuminuria, kidney cyst and glomerulosclerosis; chronic kidney disease with hypertension, chronic kidney disease with heart failure, chronic kidney disease with hypertension and heart failure, chronic kidney disease with obesity, chronic kidney disease with hyperlipidemia, chronic kidney disease with diabetes mellitus and cardiorenal syndrome; heart

- 5 041410 but-vascular diseases selected from the group consisting of the following: hypertension, heart failure (i.e. congestive heart failure, selected from the group consisting of the following: heart failure with reduced ejection fraction, heart failure with normal ejection fraction and acute heart failure), myocardial infarction, angina pectoris, cardiac hypertrophy, myocarditis, cardiac vascular fibrosis, myocardial ischemia, coronary heart disease, atherosclerotic heart disease, baroreceptor dysfunction, cardiac hypervolemia and arrhythmia, hyperlipidemia and obesity; endocrine diseases selected from the group consisting of the following: primary and secondary hyperaldosteronism, Addison's disease, Itsenko-Cushing's syndrome and Bartter's syndrome. In particular, the subject is a subject characterized by the presence of a disease selected from the following: chronic kidney disease selected from the group consisting of the following: hypertensive nephropathy, diabetic nephropathy, glomerulonephritis, renal failure, albuminuria, kidney cyst and glomerulosclerosis; chronic kidney disease with hypertension, chronic kidney disease with heart failure, chronic kidney disease with hypertension and heart failure, chronic kidney disease with obesity, chronic kidney disease with hyperlipidemia, chronic kidney disease with diabetes mellitus and cardiorenal syndrome.

According to an embodiment, the pharmaceutical composition of the present invention may be administered once or several times a day. Preferably the pharmaceutical composition is administered once a day at the dose mentioned above. The pharmaceutical composition according to the present invention can be administered at any time of the day.

The pharmaceutical composition of the present invention may be used to treat a subject in need thereof in combination with certain other agents, which may be one or two selected from the group consisting of an antihypertensive agent, an antilipemic agent, and an antidiabetic agent.

The antihypertensive agent may be an angiotensin-converting enzyme inhibitor, an angiotensin II receptor blocker, a renin inhibitor, a calcium channel blocker, a diuretic, a beta receptor blocker, or an alpha receptor blocker. In particular, the antihypertensive agent includes, without limitation, an angiotensin-converting enzyme inhibitor selected from the group consisting of the following: captopril, enalapril, benazepril, delapril, lisinopril, and perindopril; an angiotensin II receptor blocker selected from the group consisting of the following: losartan, valsartan, irbesartan, candesartan, telmisartan, eprosartan and irbesartan; a renin inhibitor selected from the group consisting of the following: aliskiren and aliskiren; a calcium channel blocker selected from the group consisting of the following: nifedipine, amlodipine, lercanidipine, nimodipine, nicardipine, nitrendipine, nisoldipine, felodipine, benidipine, lacidipine, diltiazem, verapamil, flunarizine, cinnarizine, and lidoflazin; a diuretic selected from the group consisting of the following: chlorthiazide, chlorthalidone and furosemide; a beta receptor blocker selected from the group consisting of the following: atenolol, metoprolol, sotalol hydrochloride, propranolol hydrochloride and carvedilol; and an alpha receptor blocker selected from the group consisting of the following: phentolamine, tolazoline, phenoxybenzamine and prazosin.

The antilipemic agent is selected from the group consisting of the following: atorvastatin, lovastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin, ciprofibrate, bezafibrate, fenofibrate, and gemfibrozil.

The antidiabetic agent is selected from the group consisting of the following: an insulinotropic agent, metformins, an alpha-glucosidase inhibitor, a thiazolidinedione sensitizer, a meglitinide insulinotropic agent, a GLP-1 receptor agonist, and a DPP-4 inhibitor. The antidiabetic agent includes, without limitation, a sulfonylurea insulinotropic agent selected from the group consisting of the following: glipizide, gliclazide, glibenclamide, glibornuride, glimepiride, and gliquidone; a non-sulphonylurea insulinotropic agent, a meglitinide derivative, selected from the group consisting of repaglinide and nateglinide; metformins selected from metformin; an alpha-glucosidase inhibitor selected from the group consisting of Bose-100, acarbose and voglibose; an insulin sensitizing agent selected from rosiglitazone and pioglitazone; a dipepityl peptidase 4 (DPP-4) inhibitor selected from the group consisting of sitagliptin, saxagliptin and vildagliptin; and a GLP-1 receptor agonist selected from exenatide and liraglutide.

Compound I is a mineralocorticoid receptor antagonist. Its clinical use poses a risk of elevated serum potassium due to the mechanism underlying its action. A patient with chronic kidney disease, due to the deterioration of the ability to modulate potassium ions, is highly likely to develop hyperkalemia when this compound is administered. Hyperkalemia leads to severe damage or even a fatal risk for a patient with chronic kidney disease. Thus, the use of MRA drugs is largely limited and no MRA drugs have been approved to date for the treatment of chronic kidney disease and its associated complications.

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For the safe and effective use of Compound I in the clinical treatment of chronic kidney disease, for which there has been no clinically approved drug to date, the present inventors conducted extensive experiments on Compound I and finally found a window of safety and efficacy. Later, based on the window of safety and efficacy, they found a safe and effective dosage range for Compound I and invented a pharmaceutical composition having a bioavailability of 50% or more in mammals.

I. The pharmaceutical composition of the present invention has a narrow window of safety and efficacy.

Compound I has been found in clinical trials to have a very narrow safety window in patients with chronic kidney disease, ie. it is safe and effective when the AUC is in the range of 188-3173 nghh/mL.

i. A wide safety window was found in SD rats.

In a toxicity test experiment and a pharmacokinetic study in which male SD rats were intragastrically treated with test compound for 13 weeks followed by a recovery period of 4 weeks, Compound I was administered at a high dose of 30 mg/kg/day, steady state AUC _o . ₂ 4 ₄ was approximately 49900 nghh/ml. No overt side effects were observed and serum potassium was not elevated.

ii. A wide safety window was observed in healthy volunteers.

In one clinical trial, healthy volunteers were given a single dose of Compound I in a dose range of 0.5-30 mg/day. The AUC _o _ _{24 h} was in the range of 162.5-5016 nghh/ml and no elevated serum potassium was observed.

In another clinical trial, 6 healthy volunteers were given multiple doses of Compound I at a dose of 5 mg/day. The mean steady state AUC _tau was found to be 6373±1026 nghh/mL and elevated serum potassium was observed in 3 subjects, but these were transient.

In an additional clinical trial, Compound I was given to healthy volunteers at a dose of 2.5 mg/day in a multiple dose regimen. The mean steady state AUC _tau was found to be 2863±822 nghh/mL and no elevated serum potassium was observed.

iii. A very narrow safety window was found in patients with chronic kidney disease.

Patients with chronic kidney disease responded differently. When exposed to multiple doses of compound I at a dose of 2.5 mg/day, the safe steady state AUCtau was 2613±280 nghh/ml. In one subject, serum potassium was slightly elevated.

Pharmaceutical effects of compound I were observed when patients with chronic kidney disease were given repeated doses of the compound at a dose of 0.5 mg/day. The mean steady state AUC _tau was 652.5±232.2 nghh/mL and no elevated serum potassium was observed.

In view of the above, Compound I has shown a wide safety window in animal and healthy subjects, but a very narrow safety window in patients with chronic kidney disease. The clinical safety window could not be predicted from animal or healthy human trials. In other words, the window of safety and efficacy of compound I in patients with chronic kidney disease is unpredictable.

In statistics, the 95% confidence interval in a normally distributed sample ranges from the mean minus two times the standard deviation to the mean plus twice the standard deviation. Based on the clinical trials in CKD patients mentioned above, the lower limit of the AUC value of Compound I that shows effects in CKD patients administered the pharmaceutical composition is defined as the mean AUC minus two-fold standard deviation, measured at a dose of 0.5 mg, those. 188 nghh/ml; and the upper limit of the AUC value of compound I that is safe in CKD patients with is the mean AUC value plus two times the standard deviation, measured at a dose of 2.5 mg, i.e. 3173 nghh/ml; accordingly, the safe and effective AUC of compound I is in the range of 188 to 3173 nghh/ml. Preferably, the lower limit of the AUC value of Compound I that exhibits post-absorption effects in patients is the mean AUC value minus two times the standard deviation, measured at a dose of 0.5 mg, i.e. 188 nghh/ml, and the upper limit of the AUC value of compound I that is safe in patients with CKD is the mean AUC value plus standard deviation measured at a dose of 2.5 mg, i.e. 2893 nghh/ml; accordingly, the safe and effective AUC of compound I is in the range of 188 to 2893 nghh/ml. Preferably, the lower limit of the AUC value of Compound I that exhibits post-absorption effects in patients is the mean AUC value minus two times the standard deviation, measured at a dose of 0.5 mg, i.e. 188 nghh/ml, and the upper limit of the AUC value of Compound I that is safe in patients with CKD is the mean AUC value measured at a dose of

- 7 041410

2.5 mg i.e. 2613 nghh/ml; accordingly, the safe and effective AUC of compound I is in the range of 188-2613 nghh/ml. Preferably, the lower limit of the AUC value of Compound I that exhibits post-absorption effects in patients is the mean AUC value minus two times the standard deviation, measured at a dose of 0.5 mg, i.e. 188 nghh/ml, and the upper limit of the AUC value of Compound I that is safe in patients with CKD is the mean AUC value plus two times the standard deviation, measured at a dose of 0.5 mg, i.e. 1117 nghh/ml; accordingly, the safe and effective AUC value of compound I is in the range of 188-1117 nghh/ml. Preferably, the lower limit of the AUC value of Compound I that exhibits post-absorption effects in patients is the mean AUC value minus two times the standard deviation, measured at a dose of 0.5 mg, i.e. 188 nghh/ml, and the upper limit of the AUC value of Compound I that is safe in patients with CKD is the mean AUC value plus standard deviation, measured at a dose of 0.5 mg, i.e. 885 nghh/ml; accordingly, the safe and effective AUC of compound I is in the range of 188-885 nghh/ml.

In a pharmacodynamic experiment using salt-sensitive rats with high salt-induced kidney injury, a curve showing the relationship between change in SBP or UACR from baseline and AUC ₀ _ ₂₄ (see Figs. 1 and 2) reveals the point undulating change at 100 hng/ml AUC, after which the curve tends to be flat. This indicates that the drug begins to act when the AUC reaches 100 hng/ml.

On the first day when CKD patients were dosed at 0.5 mg/day, a steady-state AUC _tau was found to be 105.6 hng/mL or more in these subjects, with UACR reduced by 30.5% or more compared with baseline, indicating that the administration of the drug had an effect.

A pharmacokinetic study in the group given multiple doses of compound I at a dose of 0.5 mg/day was used to predict the steady state AUC _tau in the other dosing groups. When a pharmaceutical composition with a bioavailability of 50% in dogs was given to CKD patients at a dose of 0.1 mg/day, the AUC _tau was 130.5 hng/ml, which was above the lower effective AUC _tau limit of 100 hng/ml. ml, indicating that the administration of the drug at a dose of 0.1 mg/day had an effect. In other words, a daily dose of 0.1 mg had an effect.

II. Enormous individual variability has been found in relation to the absorption of Compound I.

i. A large individual variability in the absorption of the compound was observed.

Individuals respond differently to a particular drug, which is called inter-drug variability in drug effect. For example, some people are not sensitive to the drug and the standard dose may not be effective in obtaining a therapeutic effect. On the other hand, some other people are particularly sensitive to the drug and a very low dose may produce an obvious effect, while a standard dose may produce an unusually intense effect or even toxicity.

In addition, the absorption of the drug is also affected by dietary conditions. For example, the absorption of a drug may be affected by the state of fasting or satiety, or the composition of the food. In the state of satiety, the absorption of the drug will be delayed and the rate of elimination will slow down. Consumption of water, tea, alcohol, and high-fat foods also affect drug absorption to some extent.

Compound I is insoluble in water and has a medium penetration rate. The patient's condition and diet will affect drug absorption. The pharmaceutical composition of this compound, produced using conventional technical means, provides low bioavailability, and patients with CKD experience a huge individual variability in y. Studies have shown that the lower the bioavailability, the greater the individual variability. In the meantime, Compound I has a narrow safety window in patients with chronic kidney disease, creating a high safety risk when using this compound in these patients. Alcohol and a high-fat diet will particularly enhance compound I absorption, further increasing individual variability and posing a greater safety risk to patients.

Thus, even if the range of clinical doses is narrow, bioavailability must be improved to reduce individual variability in order to safely and effectively clinical use of this compound.

Therefore, the safe and effective use of Compound I is closely related to the bioavailability and dosage of the pharmaceutical composition of the present invention.

ii. Effect of compound I with different particle size on absorption.

a) With the aim of clinically safe and effective drug administration, the authors have

- 8 041410 of the present invention found through extensive research that the smaller the particle size of the compound I, the higher will be the bioavailability when administered orally. Below is the absorption of compound I, characterized by different particle sizes in rats.

The bioavailability is 13.2% when the particle size distribution parameter D ₉₀ of compound I is 72.0 μm;

the bioavailability is 34.8% when the particle size distribution parameter D ₉₀ of compound I is 41.5 μm;

the bioavailability is 54.6% when the particle size distribution parameter D ₉₀ of compound I is 21.7 μm;

the bioavailability is 66.9% when the particle size distribution parameter D ₉₀ of compound I is 3.8 μm;

the bioavailability is 82.3% when the particle size distribution parameter D ₉₀ of compound I is 538 nm;

the bioavailability is 76.9% when Compound I in a solid dispersion is administered orally;

the bioavailability is 81.6% when Compound I is administered orally as a solution.

When compound I is produced by conventional methods, compound I has a D ₉₀ of 72.2 μm. Its bioavailability is only 13.2% when given orally to rats. Bioavailability reaches 54.6% when D ₉₀ is reduced to 21.7 microns. When Compound I is formulated as a solid dispersion or solution, it distributes as single molecules and will have a bioavailability of 76.9% and 81.6%, respectively, when given orally to rats.

When Compound I has a D90 of 25 μm or less, its availability is 50% or more, which satisfies clinical safety and efficacy requirements.

b) Absorption of the pharmaceutical composition according to the present invention in dogs.

Capsule 5 prepared in Example 2 was tested for its pharmacokinetic properties in beagle dogs. The bioavailability was 50% or more when the D ₉₀ of Compound I was 5 μm, which met the requirements for clinical safety and efficacy.

iii. Effect of surfactant on absorption.

In a pharmacokinetic study in rats, when no surfactants were used, the bioavailability was 34.8% with a D ₉₀ of compound I of 41.5 μm. After adding sodium dodecyl sulfate (SDS) to compound I as a surfactant in a ratio of 10:1 (SDS: compound I), the bioavailability was 64.7% with a D ₉₀ of compound I of 52.5 μm.

In a pharmacokinetic study in beagle dogs using capsule 5 prepared in Example 2 without any surfactant, the mean bioavailability was 56.5%. When tablet 3 was prepared in Example 2 with sodium dodecyl sulfate (SDS), the bioavailability was proven to be 77.4%.

All this indicates that the bioavailability can be greatly improved by adding a surfactant to the pharmaceutical formulation of the present invention, which can effectively reduce inter-individual variability.

vi. Absorption of a pharmaceutical composition in human clinical trials.

The capsule prepared in Example 2 as the preferred embodiment (ie, capsule 5 having a D90 of 5 μm and an average bioavailability of 56.5% in beagle dogs) was used in clinical trials.

In clinical trials, Compound I capsule was administered in multiple doses at a daily dose of 2.5 mg. With respect to individual variability in AUC in healthy volunteers, the AUC in the subject with the highest AUC level was 2.37 times that of the subject with the lowest level. In patients with CKD, the AUC in the subject with the highest AUC was 2.27 times that of the subject with the lowest.

In clinical trials, Compound I capsules were also given to CKD patients in multiple doses at a daily dose of 0.5 mg. However, the AUC of the subject with the highest AUC was still 2.14 times that of the subject with the lowest.

When formulated as described according to a preferred embodiment of the present invention, Compound I had a bioavailability of 56.5% in dogs. However, when this compound formulation was given to healthy subjects and patients with CKD, there was still inter-individual variability in AUC (the AUC value of the subject with the highest level was more than twice that of the subject with the lowest level).

According to the above embodiments relating to the bioavailability of Compound I in mammals, the capsule obtained in Example 2 as a preferred embodiment had a bioavailability of 50% or more in dogs. This capsule of choice was then administered to patients with CKD in multiple doses at a daily dose of 0.5 or 2.5 mg, individual variability was observed (AUC value in the subject with the most

- 9 041410 high level was more than twice that of the subject with the lowest level). In addition to all this, Compound I was found to induce high serum potassium levels in CKD patients. Thus, it was determined that a bioavailability of 50% or more in mammals was the main requirement, i.e. a bioavailability of 50% or more in beagle dogs was required for the pharmaceutical composition of the present invention. Said requirement for bioavailability and the dosage range of the present invention should be combined in consideration of safe and effective drug administration.

It has been found through experimentation that the problems of too high AUC levels in some patients due to individual variability can be avoided if the bioavailability of the pharmaceutical composition of the present invention can be increased to 50% or more in mammals. Thus, the risk of developing high serum potassium levels will be under control. For a safe and effective clinical use of a drug, the pharmaceutical composition of the present invention must be characterized by its increased bioavailability in patients with CKD, so as to reduce inter-individual variability and ensure the safety of the drug.

Improved bioavailability with reduced particle size.

To increase the bioavailability of the pharmaceutical composition of the present invention to a level of 50% or more in mammals, in one embodiment, the particle size distribution parameter D ₉₀ of Compound I is determined to be 25 µm or less. According to one embodiment of the present invention, the particle size distribution parameter D ₉₀ of compound I is 21.7 µm or less. According to one embodiment of the present invention, the particle size distribution parameter D ₉₀ of compound I is 10 μm or less. According to one embodiment of the present invention, the particle size distribution parameter D90 of compound I is 5 μm or less.

Compound I having different particle sizes can be obtained by grinding, extruding, impinging, cutting, mechanical fine grinding, vibratory fine grinding, fluid bed grinding, ultrasonic treatment, high pressure grinding, chemical precipitation, or the like.

Improved bioavailability by adding a surfactant.

To increase the bioavailability of the pharmaceutical composition of the present invention to a level of 50% or more in mammals, in one embodiment, the pharmaceutical composition of the present invention contains a surfactant.

The surfactant is one or more selected from the group consisting of the following: benzalkonium chloride, sodium lauryl sulfonate, sodium dodecyl sulfate, glycerin, cholic acid, poloxamer, polyvinyl alcohol, polysorbate 80, PVP K ₃₀ and polyethylene glycol. Preferably the surfactant is one or more selected from the group consisting of benzalkonium chloride, sodium lauryl sulfonate and sodium dodecyl sulfate. Preferably the surfactant is benzalkonium chloride, sodium lauryl sulfonate or sodium dodecyl sulfate.

In the pharmaceutical composition according to the present invention, the weight ratio of compound I to surfactant is 1:0.1-1:20, preferably 1:1-1:20 and more preferably 1:5-1:20.

Compound I according to the present invention may be in any form. For example, compound I may be in an amorphous, crystalline, or mixed crystal structure.

III. The pharmaceutical composition according to the present invention is characterized by a specific dosage range.

After administration of the pharmaceutical composition according to the present invention, the AUC level of Compound I in humans is sufficiently high, indicating a sufficiently good efficacy of the drug. In clinical use, a very low dose will produce a therapeutic effect.

Clinical doses of spironolactone tablets and eplerenone tablets, the two available mineralocorticoid receptor antagonists, are 40-400 and 25-50 mg/day, respectively. The doses are quite high.

In testing the pharmaceutical composition of the present invention, healthy volunteers were administered a single dose of 0.5-30 mg, or alternatively administered in multiple doses in a daily dose of 2.5 mg, and showed very good tolerance. When healthy volunteers were administered in multiple doses at a dose of 5 mg/day, relatively good tolerance was also observed despite single elevated serum potassium levels.

The pharmaceutical composition of the present invention, when given to patients with

- 10 041410

Multiple doses of CKD at a daily dose of 0.5 mg showed good therapeutic activity, with no elevated serum potassium levels observed. When the pharmaceutical composition was administered in multiple doses at a dose of 2.5 mg/day, fluctuations in serum potassium were found in one subject, indicating that there is no safety risk for patients with CKD at this dose.

In addition, from a pharmacodynamic experiment using salt-sensitive rats with salt-induced kidney injury, as well as from a clinical trial in patients with CKD, it appears that a daily dose of 0.1 mg showed therapeutic effects in patients with chronic kidney disease. .

Administration of the pharmaceutical composition of the present invention induces a very high AUC of Compound I in humans, indicating a relatively good therapeutic activity. On the other hand, a very low dose of 0.1 mg in clinical use has a therapeutic effect, which means that the effective dose is quite low. In addition, elevated serum potassium levels were not found at a daily dose of 0.1-0.5 mg. Thus, the dosage range for the safe and effective use of the pharmaceutical composition according to the present invention has finally been found. The dose for clinical use of the pharmaceutical composition according to the present invention cannot be predicted from drugs available on the market or from a clinical trial involving healthy volunteers.

Thus Compound I may induce elevated serum potassium levels due to the mechanism underlying the action of the mineralocorticoid receptor antagonist. MRA drugs available on the market take very high clinical doses, and Compound I has also shown a wide safety window in animal experiments and clinical trials in healthy volunteers. Thus, it was expected that the clinical dose of Compound I would be quite high in patients with chronic kidney disease. However, in clinical trials in patients with CKD, serum potassium levels increased at a daily dose of 2.5 mg, indicating that the dose range in CKD patients would be very narrow.

The present inventors have discovered a window of safety and efficacy for Compound I through a series of experiments. In addition, based on this window, a dosage range for safe and effective use of the pharmaceutical composition according to the present invention was found. Furthermore, the present inventors have invented a pharmaceutical composition having a bioavailability of 50% or more in mammals. When the pharmaceutical composition of the present invention is administered to a patient with chronic kidney disease in the dosage range disclosed herein, the AUC of Compound I is controlled to a safe and effective level, so that clinical use is safe and effective.

Bioavailability is a measure of the rate and extent to which a drug reaches the systemic circulation when administered via non-intravenous routes and is an important parameter for assessing drug absorption. Bioavailability includes absolute bioavailability and relative bioavailability, the former comparing the absorption of the active drug after non-intravenous administration with intravenous administration, and the latter measuring the absorption of a given formulation compared to another formulation. Bioavailability in this document refers to absolute bioavailability.

Particle size may also be referred to herein as particle size. In the pharmaceutical composition according to the present invention, compound I has a particle size of 100 µm or less, of the order of a micron or nanometer, or exists as molecules.

The term D ₉₀ in this document refers to the size that separates the particle size distribution of 10% above and 90% below this diameter.

The pharmaceutical composition according to the present invention can effectively block the binding of the mineralocorticoid receptor to aldosterone, and thus, according to the mechanism underlying the action, it can be used, in particular, in the treatment and/or prevention of chronic kidney disease. Chronic kidney disease is selected from the group consisting of the following: hypertensive nephropathy, diabetic nephropathy, glomerulonephritis, renal failure, albuminuria, kidney cyst and glomerulosclerosis; chronic kidney disease with hypertension, chronic kidney disease with heart failure, chronic kidney disease with hypertension and heart failure, chronic kidney disease with obesity, chronic kidney disease with hyperlipidemia, chronic kidney disease with diabetes mellitus and cardiorenal syndrome; cardiovascular diseases selected from the group consisting of the following: hypertension, heart failure (i.e., congestive heart failure selected from the group consisting of heart failure with reduced ejection fraction, heart failure with normal ejection fraction, and acute heart failure) , acute myocardial infarction, angina pectoris, cardiac hypertrophy, myocarditis, vascular fibrosis of the heart, myocardial ischemia, coronary atherosclerotic heart disease, atherosclerotic heart disease, baroreceptor dysfunction, hypervolemia and

- 11 041410 cardiac arrhythmia, hyperlipidemia and obesity; endocrine diseases selected from the group consisting of the following: primary and secondary hyperaldosteronism, Addison's disease, Itsenko-Cushing's syndrome and Bartter's syndrome. According to an embodiment, the present invention also relates to the use of the pharmaceutical composition in the preparation of medicaments for the treatment of chronic kidney disease, heart failure and/or hypertension.

According to another embodiment, the present invention relates to a method for treating chronic kidney disease, heart failure and/or hypertension, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition according to the present invention.

According to an embodiment of the present invention, the pharmaceutical composition according to the present invention may be administered one or more times a day, preferably may be administered once with the dose described above. The pharmaceutical composition according to the present invention can be administered at any time of the day.

The pharmaceutical composition of the present invention may be administered to patients in need thereof in combination with other drugs, which may be one or two selected from the group consisting of an antihypertensive agent, an antilipemic agent and an antidiabetic agent.

The antihypertensive agent is selected from the group consisting of an angiotensin converting enzyme inhibitor, an angiotensin II receptor blocker, a renin inhibitor, a calcium channel blocker, a diuretic, a beta receptor blocker, and an alpha receptor blocker. In particular, the antihypertensive agent includes, without limitation, an angiotensin converting enzyme inhibitor selected from the group consisting of the following: captopril, enalapril, benazepril, delapril, lisinopril, and perindopril; an angiotensin II receptor blocker selected from the group consisting of the following: losartan, valsartan, irbesartan, candesartan, telmisartan, eprosartan and irbesartan; a renin inhibitor selected from the group consisting of aliskiren and aliskiren; a calcium channel blocker selected from the group consisting of the following: nifedipine, amlodipine, lercanidipine, nimodipine, nicardipine, nitrendipine, nisoldipine, felodipine, benidipine, lacidipine, diltiazem, verapamil, flunarizine, cinnarizine, and lidoflazin; a diuretic selected from the group consisting of chlorothiazide, chlorthalidone and furosemide; a beta receptor blocker selected from the group consisting of the following: atenolol, metoprolol, sotalol hydrochloride, propranolol hydrochloride and carvedilol; and an alpha receptor blocker selected from the group consisting of phentolamine, tolazoline, phenoxybenzamine and prazosin; an antilipemic agent selected from the group consisting of the following: atorvastatin, lovastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin, ciprofibrate, bezafibrate, fenofibrate and gemfibrozil.

The antilipemic agent is selected from the group consisting of the following: atorvastatin, lovastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin, ciprofibrate, bezafibrate, fenofibrate and gemfibrozil.

The antidiabetic agent is selected from the group consisting of the following: an insulinotropic agent, metformins, an α-glucosidase inhibitor, a thiazolidinedione sensitizer, a meglitinide insulinotropic agent, a GLP-1 receptor agonist, and a DPP-4 inhibitor. The antidiabetic agent includes, without limitation, a sulfonylurea insulinotropic agent selected from the group consisting of the following: glipizide, gliclazide, glibenclamide, glibornuride, glimepiride, and gliquidone; a non-sulphonylurea insulinotropic agent, a meglitinide derivative, selected from the group consisting of repaglinide and nateglinide; metformins selected from metformin; an alpha-glucosidase inhibitor selected from the group consisting of Bose-100, acarbose and voglibose; an insulin sensitizing agent selected from rosiglitazone and pioglitazone; a dipepityl peptidase 4 (DPP-4) inhibitor selected from the group consisting of sitagliptin, saxagliptin and vildagliptin; and a GLP-1 receptor agonist selected from exenatide and liraglutide.

The pharmaceutical composition of the present invention comprises Compound I and a pharmaceutically acceptable carrier, and may be administered by appropriate routes including, but not limited to, oral, parenteral, intraperitoneal, intravenous, transdermal, sublingual, intramuscular, rectal, nasal, and subcutaneous routes.

The pharmaceutical composition according to the present invention is preferably an oral formulation and more preferably tablets, extended release tablets, capsules or granules. The pharmaceutical composition of the present invention may also be soft capsules, micropills, microcapsules, microspheres, liposomes, self-emulsifying drug delivery systems, solid dispersions, micelles, orodispersible tablets, solutions, suspensions or emulsions.

The pharmaceutically acceptable carrier in the pharmaceutical composition of the present invention is one or more non-toxic pharmaceutical carriers. Said carriers may be compatible with other ingredients in the pharmaceutical composition and do not cause any harm to subjects receiving the composition.

Media.

The pharmaceutical composition of the present invention is formulated as an oral formulation and contains one or more carriers including diluents, excipients, lubricants, glidants, binders, disintegrators, surfactants, and the like. The carriers used in the pharmaceutical composition of the present invention can be selected and combined to provide the best properties, safe, effective and controllable pharmacokinetics when the composition is administered to a subject, and meet the requirements of drug administration.

The diluent or excipient used to bulk up the single dose formulation includes, but is not limited to, calcium phosphate, calcium sulfate, microcrystalline cellulose, lactose, mannitol, sorbitol, starch, and the like.

A lubricant used to reduce friction between the granules and the mold wall during compression or discharge, to prevent the granules from attaching to the tablet punching machine, and/or to eject the granules from the tablet punching machine, includes, but is not limited to, talc, stearic acid, calcium stearate , zinc stearate, magnesium stearate, vegetable oils, etc.

The glidant added to improve the mobility of the granules includes, but is not limited to, talc, silica, and corn starch.

The binder includes, but is not limited to, pyrrolidone, polyvinylpyrrolidone, xanthan gum, cellulose gum (such as carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose), gelatin, and starch.

A disintegrating agent by which tablets can rapidly disintegrate into small particles in the gastro-intestinal juice so that the active ingredients are dissolved and absorbed to exert effects includes, but is not limited to, starch, clay, cellulose, alginates, pregelatinized starch, crospolyvinylpyrrolidone , croscarmellose sodium, sodium carboxymethyl starch, vegetable glue, etc.

A surfactant capable of changing interfacial tension to provide wetting and solubilizing effects includes, but is not limited to, poloxamer, sodium dodecyl sulfate, polysorbates, polyethylene glycol octanoate, glyceryl caprate, polyethylene glycol glyceryl laurate, polyethylene glycol glyceryl stearate, and the like.

Other carrier materials include, without limitation, preservatives, antioxidants, and other carrier materials commonly used in the pharmaceutical industry.

Extended release tablets may contain one or more carrier materials selected from the group consisting of the following: cellulose ether derivatives, including hydroxypropyl methyl cellulose, ethyl cellulose, sodium carboxymethyl cellulose, and hydropropyl cellulose; acrylic polymers including carbomer and acrylic resin; chitin and derivatives, lactic acid polymer, and the like. Other additives may be included in addition, commonly used additives include wetting agents (such as ethanol, water, etc.), coloring agents (such as iron III oxides), preservatives, anti-sagging agents (such as glycine), surfactants (such as sodium dodecyl sulfate), pH adjusters (such as sodium citrate or sodium hydroxide), bulking agents (microcrystalline cellulose, etc.), and disintegrators (croscarmellose sodium, etc.).

Commonly used additives for solution formulation, such as the oral formulation of the present invention, include additives to improve taste, improve clarity, and enhance stability. The flavor enhancer generally includes four classes, i.e. sweetener, flavors, slime and effervescent. The sweetener contains naturally occurring and artificial sweeteners. The naturally occurring sweetener may be sucrose, sugar syrup, and flavor syrup, while the artificial sweetener may be sodium saccharin, aspartame, and the like. The fragrances include lemon essence, peppermint oil, apple essence and rubber essence. Commonly used mucus may be sodium alginate, gum arabic, gelatin, methylcellulose, sodium carboxymethylcellulose, and the like. The effervescent agent may be citric acid, tartaric acid, and the like. The transparency enhancer is, for example, chitosan, juice brightener 101, ZTC1+1 naturally occurring brightener, gelatin, tannic acid, egg whites, and the like. The stability enhancing agent may be parahydroxybenzoate esters, organic acids and salts thereof, and other additives such as chlorhexidine di(acetate), domifene, and the like.

Commonly used softgel additives can be vegetable oils, aromatic esters, organic acids, glycerin, isopropanol, polyethylene glycol, propanediol, and surfactants.

The micropills may contain one or more base materials selected from the group

- 13 041410 consisting of a water-soluble base, a water-insoluble base and a mixed base. The water soluble base mainly includes polyethylene glycol 4000, polyethylene glycol 6000, polyvinylpyrrolidone (PVP), S-40 (polyoxyethylene monostearate), sodium stearate, glycerin, gelatin, urea, poloxamer, PEG together with surfactants and polyethers . The main water-insoluble bases are as follows: stearic acid, glyceryl monostearate, insect wax, hydrogenated vegetable oils, stearyl alcohol, cetyl alcohol and semi-synthetic fatty acids. The mixed base may be, for example, polyethylene glycol together with S-40 or a poloxamer. The mixed base is used to increase the amount of drug to be dispensed by adjusting the distribution time limit or the disintegration time limit, which can be useful for micropill formulation.

The suspensions may contain one or more carrier materials selected from the group consisting of the following: a suspending agent, a wetting agent, a flocculating agent, and a deflocculating agent.

Frequently used suspending agents include, but are not limited to, small molecule suspending agents such as glycerol and syrups; and polymers such as 1) a naturally occurring polymer which is mainly a vegetable gum such as gum arabic, gum tragacanth and peach gum and a vegetable polysaccharide such as sodium alginate, agar and starch paste; 2) synthetic or semi-synthetic polymers, including celluloses such as methylcellulose, sodium carboxymethylcellulose, and hydropropylcellulose, and others such as carbopol, povidone, and glucan; 3) diatomite; and 4) thixotropic.

The wetting agent includes, but is not limited to, polysorbates, polyoxyethylene castor oil, and poloxamer.

The flocculating agent includes, without limitation, inorganic flocculating agents such as aluminum sulfate, aluminum chloride, iron(III) sulfate, and iron(III) chloride; modified monocationic inorganic flocculating agents such as aluminum polysilicate (ferrite) and aluminum polyphosphate (ferrite); modified polycationic inorganic flocculating agents such as iron(III) polyaluminum chloride sulfate, iron(III) polysilicate, iron(III)-aluminum copolymer, and the like; organic polymers such as polyacrylamide; composite flocculating agents such as polyaluminum chloride and iron(III) polysulphate; and flocculating agents of microbiological origin such as Phodococcus erythropolis and NOC-1 containing them.

The deflocculating agent includes, but is not limited to, sodium citrate, tartrates, phosphates, carbonates, glycinates, magnesium succinate, and sodium dehydrocholate.

The microcapsules may contain one or more carrier materials selected from the group consisting of the following: gelatin, gum arabic, albumin, starch, chitosan, alginates, methylcellulose, ethylcellulose, carboxymethylcellulose, hydropropylmethylcellulose, lactic acid polymer, lactic acid-glycolic acid copolymer, polyalkyl cyanoacrylate , polyamide, poly(vinyl alcohol) and polyacrylic resin.

The microspheres may contain one or more carrier materials selected from the group consisting of the following: naturally occurring polymeric microspheres such as starch microspheres, albumin microspheres, gelatin microspheres, chitosans, and the like; and synthetic polymeric microspheres such as polylactic microspheres and the like.

Liposomes may contain one or more carrier materials selected from the group consisting of phospholipids and cholesterols. Phospholipids include naturally occurring and synthetic phospholipids. The main naturally occurring phospholipid is lecithin (phosphatidylcholine, PC), while the synthetic phospholipid is mainly DPPC (dipalmitoylphosphatidylcholine), DPPE (dipalmitoylphosphoethanolamine), and DSPC (distearoylphosphatidylcholine).

The self-emulsifying drug delivery system may contain one or more carrier materials selected from the group consisting of oils, surfactants, and co-surfactants.

Oils are divided into naturally occurring vegetable oils (such as soybean oil, peanut oil, etc.), medium chain triglycerides and semi-synthetic medium chain derivatives such as C8/C10 coconut oil triglycerides, C8/C10 coconut oil monoglycerides or diglycerides, sorbitan oleate, glyceryl oleate-propylene glycol, glyceryl oleate, propylene glycol monooctanoate, glyceryl tributyrate and purified acetylated monoglycerides.

Surfactants are mainly polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitanoleate, polyoxyethylene triglycerides, PEG-8 caprylic/capric acid glycerides, C8/C10 polyglycolized coconut oil glycerides, PEG lauric acid glyceride, Labrafil M 1944CS and Lavrafil M-22125CS.

- 14 041410

Co-surfactants include, but are not limited to, ethanol, propylene glycol, glycerin, isopropanol, polyethylene glycol, and propenylethylene glycol laurate.

There are three trips of carrier materials for solid dispersions. They are water-soluble carrier materials including polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), surfactants, organic acids, saccharides, alcohols, and the like; insoluble carrier materials including cellulose and polyacrylic resin; and enteric materials, including cellulose and polyacrylic resins.

The micelles contain surfactants, including, but not limited to, hexadecyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, dodecyltrimethylammonium bromide, dodecylpyridinium bromide, sodium octanesulfonate, sodium octyl sulfate, sodium dodecyl sulfate, sodium tetradecyl sulfate, sodium hexadecyl sulfate, sodium octadecyl sulfate, potassium stearate, potassium oleate, sodium dotacyl sulfonate, potassium dodecyl laurate polyoxyethylene(b) lauryl ether, polyoxyethylene(9) lauryl ether, polyoxyethylene(12) lauryl ether, polyoxyethylene(6) myristyl ether, dioctyl sodium sulfosuccinate, dodecylammonium chloride, sodium dodecylbenzenesulfonate, sucrose laurate, sucrose palmitate, sucrose stearate, Tween-20, Tween- 40, Tween-60, Tween-65, Tween 80 and Tween 85.

Orally dissolving tablets may contain one or more materials selected from the group consisting of the following: surfactants such as sodium dodecyl sulfate, lecithin, Tween and Span; long chain polymers such as polypeptides (gelatin or dehydrated gelatin); saccharides and derivatives such as dextran, glucans, sorbitol, mannitol and starch; gels such as gum arabic, xanthan gum and vegetable glue; cellulose; alginates; PVP; and polyvinyl alcohol. Several other additives may be included, including a wetting agent (such as ethanol), a coloring agent (such as ferric oxides), a preservative, an antioxidant, an anti-fall agent (such as glycine), a penetration enhancer (such as sodium dodecyl sulfate), a pH (such as sodium citrate or sodium hydroxide), flavors and sweetener.

Brief description of graphic materials

Fig. 1 is a curve showing the correlation between SBP variability based on baseline levels and AUC _0-24 in a DSS model with high salt-induced hypertension and kidney injury in rats.

Fig. 2 is a curve showing the correlation between UACR variability based on baseline levels and AUC0-24 in a DSS model of high salt-induced hypertension and kidney injury in rats.

Detailed disclosure of the present invention

The present invention includes, without limitation, the following examples. Other embodiments for implementing the technical solution according to the present invention fall within the scope of the present invention.

Example 1 Preparation of Compound I

2-chloro-4-[(3S,3aR)-3-cyclopentyl-7-(4-hydroxylpiperidine-1-carbonyl)-3,3a,4,5-tetrahydro-2Hpyrazolo[3,4-f]quinoline-2 -yl]benzonitrile (herein referred to as compound I) with the structure as shown below.

Compound I

Compound I can be obtained by the method described in international patent publication WO 2012022121 A1 or WO 2014094664 A1, or using other synthetic methods.

Example 2 Exemplary formulations of a pharmaceutical composition according to the present invention.

Examples according to the present invention are part of the compositions of the pharmaceutical composition, which are applicable for the introduction of the pharmaceutical composition according to the present invention. It should be noted that the composition in this document is not limited to the following, other compositions with a bioavailability of 50% or more fall within the declared scope of the present invention.

Exemplary tablet formulations are shown below.

- 15 041410

Composition 1. Each tablet contained 0.5 mg of Compound I.

Compound I with D90 constituting

0.5 g C28H30CIN5O2

538 nm

Microcrystalline cellulose

PH101

Mannitol70 g

Croscarmellose sodium 6 g

Polyvinylpyrrolidone KZO 2.15 g

Magnesium stearate2 g

Silicon dioxide2 g

Formulated in 1000 Tablets

Preparation method.

(1) Polyvinylpyrrolidone K30 was formulated into an aqueous solution at a concentration of 5% (w/w) to be used as a binder.

(2) Microcrystalline cellulose PH101, mannitol, and croscarmellose sodium were weighed in the amounts as described above and sufficiently mixed for 15 minutes to obtain mixture 1.

(3) A suspension containing 0.5 g of nano-sized compound I was weighed and then added to and mixed with mixture 1; an aqueous solution of polyvinylpyrrolidone K30, a binder, was added to the resulting mixture to obtain a soft material, which was processed with a 24 mesh sieve to obtain wet granules.

(4) Wet granules were dried at 55±5°C for 2-3 hours, controlled to keep the water content of the granules below 2.5%.

(5) The dried granules were again sifted through a 24 mesh sieve.

(6) Magnesium stearate and silica were added to the granules in the amounts described above, and then mixed for 15 minutes.

(7) Tablet pressing was carried out while controlling that the hardness of the tablet was 6-10 kg.

(8) The tablets were coated.

(9) Tablets were packaged and placed in a warehouse.

Composition 2. Each tablet contained 0.5 mg of Compound I.

Compound I with D90 constituting 4.0 µm 0.5 g C28H30CIN5O2 Sodium dodecyl sulfate 5 g Microcrystalline cellulose 40 g PH101 Lactose monohydrate 60 g Croscarmellose sodium 3.5 g Hydroxypropylcellulose 5 g magnesium stearate 2.0 g Silica 1.0 g Compiled in 1000 tablets

Composition 3. Each tablet contained 2.5 mg of Compound I.

Compound I with D90 of 4.0 µm 2.5 g C28H30CIN5O2 Sodium dodecyl sulfate 25 g Microcrystalline cellulose PH101 40 g Lactose monohydrate 60 g Croscarmellose sodium 3.5 g Hydroxypr opilcellu l o z a 5 g magnesium stearate 2.0 g Silica 1.0 g

Formulated in 1000 Tablets

Preparation method.

(1) The starting materials were sifted through a 30 mesh sieve and stored until the next use.

- 16 041410 (2) Compound I, sodium dodecyl sulfate, microcrystalline cellulose PH101, lactose monohydrate, croscarmellose sodium and hydroxypropyl cellulose were sufficiently mixed for a minute.

(3) Water was added to the mixture to obtain a soft material, which was processed with a 24 mesh sieve to obtain wet granules.

(4) Wet granules were dried at 55±5°C for 2-3 hours, controlled to keep the water content of the granules below 2.5%.

(5) The dried granules were again sifted through a 24 mesh sieve.

(6) Magnesium stearate and silica were added to the granules in the amounts described above, and then mixed for 15 minutes.

(7) Tablet pressing was carried out while controlling that the hardness of the tablet was 6-10 kg.

(8) The tablets were coated.

(9) Tablets were packaged and placed in a warehouse. Exemplary capsule formulations are shown below.

Composition 1. Each capsule contained 0.1 mg of Compound I.

Compound I with D90 constituting

0.1 g C28H30CIN5O2 µm

Microcrystalline cellulose

30.0 g

PH101

Mannitol 70.0 g

Croscarmellose sodium 6.0 g

Polyvinylpyrrolidone KZ 02.15g

Magnesium stearate2.0 g

Silicon dioxide2.0 g

Formulated in 1000 capsules

Composition 2 Each capsule contained 0.25 mg of Compound I.

Compound I with D90 constituting

0.25 g C28H30CIN5O2 µm

Microcrystalline cellulose

30.0 g

PH101

Mannitol 70.0 g

Croscarmellose sodium 6.0 g

Polyvinylpyrrolidone KZ 02.15g

Magnesium stearate2.0 g

Silicon dioxide2.0 g

Formulated in 1000 capsules

Preparation method.

(1) Polyvinylpyrrolidone K30 was formulated into an aqueous solution at a concentration of 5% (w/w) to be used as a binder.

(2) Compound I, microcrystalline cellulose PH101, mannitol and croscarmellose sodium were weighed in the amounts described above and sufficiently mixed for 15 minutes; the water content of the resulting mixture was measured.

(3) An aqueous solution of polyvinylpyrrolidone K30 was added to the resulting mixture as a binder to obtain a soft material, which was processed with a 24 mesh sieve to obtain wet granules.

(4) Wet granules were dried at 55±5°C for 2-3 hours, controlled to keep the water content of the granules below 2.5%.

(5) The dried granules were again sifted through a 24 mesh sieve.

(6) Magnesium stearate and silica were added to the granules in the amounts described above, and then mixed for 15 minutes.

(7) The amount of the obtained mixture to be introduced into the capsule was calculated, and the mixture was weighed in this amount and placed in the capsule.

(8) The capsules were placed in high-density polyethylene vials for solid oral drugs and the vials were sealed.

(9) The vials were placed in the warehouse.

- 17 041410

Formulation 3 Each capsule contained 0.5 mg of Compound I.

Compound I with D90 constituting

0.5 g C28H30CIN5O2 µm

Microcrystalline cellulose 30.0 g

PH101

Mannitol 70.0 g

Croscarmellose sodium 6.0 g

Polyvinylpyrrolidone KZ 02.15g

Magnesium stearate2.0 g

Silicon dioxide2.0 g

Formulated in 1000 capsules

Formulation 4 Each capsule contained 2.5 mg of Compound I.

Compound I with D90 constituting

2.5 g C2 ₈ H3CM02 µm

Microcrystalline cellulose 30.0 g

PH101

Mannitol 70.0 g

Croscarmellose sodium 6.0 g

Polyvinylpyrrolidone KZ 02.15g

Magnesium stearate2.0 g

Silicon dioxide2.0 g

Formulated in 1000 capsules

Composition 5 Each capsule contained 10 mg of Compound I.

Compound I with D90 constituting 10 g C28H30CIN5O2 5 µm Microcrystalline cellulose 30.0 g RN101 Mannitol 70.0 g Croscarmellose sodium 6.0 g Polyvinylpyrrolidone K30 2.15 g magnesium stearate 2.0 g Silica 2.0 g

Formulated in 1000 capsules

Preparation method.

(1) Preparation of binder solution: Polyvinylpyrrolidone K30 was formulated into an aqueous solution at a concentration of 3-5% (w/w) to be used as a binder.

(2) Mixing: Microcrystalline cellulose PH101, mannitol and croscarmellose sodium were passed through a 30 mesh sieve and then transferred with Compound I to a high shear granulator where they were mixed for 2-3 minutes at a stirring speed of 180-220 rpm min, and a cutting speed of 475-525 rpm.

(3) Granulation: The binder solution was added to the mixture obtained in step (2) within 5-7 minutes after completion of step (2), and the mixture was subjected to granulation at a stirring speed of 180-220 rpm and a cutting speed , component 475-525 rpm, followed by granulation for 0.5-1.5 min at a mixing speed of 180-220 rpm and a cutting speed of 1800-2200 rpm.

(4) Drying: The wet granules were dried at 55±5° C., the water content of the granules was controlled to be below 2.5%.

(5) Crushing: the dried granules were placed in a Fitz mill and passed through a No. 0033 sieve at a rotation speed of 840-960 rpm; the crushing step can be carried out using other methods or devices, provided that the same effect can be obtained.

(6) Blending: magnesium stearate and silica were sieved, respectively, using a 30 mesh sieve or some other device; silica was then placed in a V-shaped mixer and mixed for 12 min at 20 rpm; magnesium stearate was added and mixed with silica for 3 minutes at 20 rpm.

- 18 041410 (7) Capsule filling: capsules were filled with mixed powder using a capsule filling machine (MF-30).

(8) Polishing: The capsules were polished using a capsule polishing machine.

(9) Packaging and labeling: capsules were placed in HDPE vials whose caps were tightly screwed and sealed using an electromagnetic sealing machine; vials were labeled later.

Composition 6 Each capsule contained 2.5 mg of Compound I.

Compound I with D90 constituting

2.5 g C28H30CIN5O2

21.7 µm

Microcrystalline cellulose

40.0 g

PH101

Lactose monohydrate 60.0g

Croscarmellose sodium 3.0 g

Magnesium stearate2.0 g

Silicon dioxide2.0 g

Formulated in 1000 capsules

Preparation method.

(1) The starting materials were passed through a 30 mesh sieve and stored until future use.

(2) Mixing: Compound I, microcrystalline cellulose PH101, lactose monohydrate, croscarmellose sodium, magnesium stearate and silica were uniformly mixed for 15 minutes.

(3) The amount to be introduced into a capsule was calculated, and the mixture in this amount was put into capsules.

(4) The capsules were placed in high-density polyethylene vials for oral solid drugs, and the vials were sealed.

(5) The vials were placed in the warehouse.

An exemplary self-emulsifying drug delivery system (SEDDS) is shown below.

Composition of SEDDS.

Compound I: ethanol: Kolliphor EL: Miglyol 812N = 10 mg: 1: 5: 4 g.

Preparation method.

Compound I was dissolved in ethanol, the resulting was added and mixed with Kolliphor EL and Miglyol 812N to obtain an emulsified mixture.

Exemplary soft capsule formulations are shown below.

Composition 1. Each soft capsule contained 0.25 mg of Compound I.

Compound I 0.25 g C28H30CIN5O2

Polyethylene glycol 400240 g

Propyl englycol 30 g

Formulated in 1000 capsules

Formulation 2 Each softgel contained 0.5 mg of Compound I.

Compound I 0.5 g C28H30CIN5O2

Polyethylene glycol 400240 g

Propyl englycol 30 g

Formulated in 1000 capsules

Formulation 3 Each softgel contained 1.0 mg of Compound I.

Compound! 1.0 g C28H30CIN5O2

Polyethylene glycol 400240 g

Propyl englycol 30 g

Formulated in 1000 capsules

Preparation method.

(1) Compound I was mixed with polyethylene glycol 400 and propylene glycol and dissolved in them.

(2) The mixture was compressed into soft capsules.

(3) Soft capsules were packed and placed in a warehouse.

Exemplary slurry formulations are shown below.

Composition 1. The suspension contained 0.25 mg of Compound I per ml.

Compound I 0.25 g C28H30CIN5O2

Methylcellulose 5 g

Water added up to 1000 ml

Compounded in 1000 ml

- 19 041410

Preparation method.

(1) Methylcellulose was dissolved in water, whose amount was approximately 80% of the total used; the solution was stored for future use.

(2) Compound I was weighed and placed in solution; after sufficient mixing, compound I was suspended in solution.

(3) Water was added to a total volume of 1000 ml, and sufficient mixing was carried out.

Formulation 2 The suspension contained 0.5 mg of Compound I per ml.

Compound I 0.5 g C28H30CIN5O2

Methylcellulose 5 g

Water added up to 1000 ml

Compounded in 1000 ml

Formulation 3 The suspension contained 1.0 mg of Compound I per ml.

Compound! 1.0 g C28H30CIN5O2

Methylcellulose

Water added up to 1000 ml

Compiled in 1000 ml Exemplary emulsion formulations are shown below. Composition 1. The emulsion contained 0.25 mg of Compound I per ml. Compound I 0.25 g C28H30CIN5O2 Polyethylene glycol 400 50 g Span 80 (sorbitan oleate) 3 g Tween 80 6 g Soybean oil 50 g Glyceryl monostearate 10 g Water added to 1000 ml Compiled in 1000 ml

Preparation method.

(1) Preparation of an aqueous phase: compound I and polyethylene glycol 400 were heated to 60° C. so that compound I was dissolved; the solution was kept warm for future use.

(2) Obtaining the oil phase: glyceryl monostearate and soybean oil were heated to 60°C and dissolved; the solution was kept warm for future use.

(3) The aqueous phase was added and mixed at 60°C with the oil phase and the resulting mixture was added and mixed at 60°C with Span 80 and Tween 80; water was added to a total volume of 1000 ml; the resulting mixture was ground to a uniform size using a colloid mill.

Composition 2 The emulsion contained 0.5 mg of Compound I per ml.

Compound I 0.5 g C28H30CIN5O2 Polyethylene glycol 400 50 g

span 80 3 g Tween 80 6 g Soybean oil 50 g

Glyceryl monostearate 10 g Water added to 1000 ml

Compounded in 1000 ml

Composition 3 The emulsion contained 1.0 mg of Compound I per ml.

Compound I 1.0 g C28H30CIN5O2 Polyethylene glycol 400 50 g span 80 3 g Tween 80 6 g Soybean oil 50 g Glyceryl monostearate 10 g Water added to 1000 ml Compiled in 1000 ml

Exemplary micropill formulations are shown below.

- 20 041410

Composition 1. Each pill contained 0.1 mg of Compound I. Compound I with D90 of

5 µm 0.1 g C28H30CIN5O2 Polyethylene glycol 4000 60.0 g Polyethylene glycol 6000 30.0 g

Formulated in 1000 pills

Composition 2 Each pill contained 0.25 mg of Compound I.

Compound I with D90 constituting

5 µm 0.25 g C28H30CIN5O2

Polyethylene glycol 4000

60.0 g

Polyethylene glycol 6000

30.0 g

Compiled in

1000 pills

Composition 3. Each pill contained 0.5 mg of Compound I.

Compound I with D90 constituting

0.5 g C28H30CIN5O2 µm

Polyethylene glycol 4000 60.0 g

Polyethylene glycol 6000 30.0 g

Compiled in

1000 pills

Formulation 4 Each pill contained 1.0 mg of Compound I.

Compound I with D90 constituting

1.0 g C28H30CIN5O2 µm

Polyethylene glycol 4000

60.0 g

Polyethylene glycol 6000 30.0 g

Formulated in 1000 pills

Preparation method.

(1) Compound I, polyethylene glycol 4000 and polyethylene glycol 6000 were weighed in the amounts set forth above for future use.

(2) Polyethylene glycol 4000 and polyethylene glycol 6000 were melted in a water bath at 80°C, and then Compound I was added, which was dissolved with stirring; the mixture was stored at 80°C for future use.

(3) At a temperature of 80°C, the mixture was added dropwise to the condensed dimethicone, where the mixture was condensed into solid pills.

(4) The pills were removed and dried, and then placed in polyethylene vials, which were properly sealed.

Exemplary oral tablet formulations are shown below.

Formulation 1. Each orodispersible tablet contained 0.25 mg of Compound I.

Compound I 0.25 g C28H30CIN5O2

Microcrystalline cellulose 10 g

RN101

Mannitol 80 g Croscarmellose sodium Water magnesium stearate Silica Compiled in Preparation method. 10 g how much will it take 1.0 g 1.0 g 1000 tablets

(1) Compound I, microcrystalline cellulose PH101, mannitol and croscarmellose sodium were uniformly mixed.

(2) Water was added to the granulation mixture, and the obtained granules were dried.

(3) Magnesium stearate and silica were added to mix uniformly with the granules.

(4) Conducted panning.

(5) The resulting tablets were coated and then packaged.

-21 041410

Formulation 2 Each orodispersible tablet contained 0.5 mg of Compound I.

Compound I

0.5 g C28H30CIN5O2

Microcrystalline cellulose South

RN101

Mannitol 80 g Croscarmellose sodium 10 g Water how much will it take magnesium stearate 1.0 g Silica 1.0 g

Formulated in 1000 Tablets

Formulation 3. Each orodispersible tablet contained 1.0 mg of Compound I. Compound! 1.0 g C28H30CIN5O2

Microcrystalline cellulose 10 g

RN101

Mannitol 80 g Croscarmellose sodium Water magnesium stearate Silica 10 g how much will it take 1.0 g 1.0 g

Formulated in 1000 Tablets

Exemplary extended release tablet formulations are shown below.

Formulation 1 Each extended release tablet contained 0.25 mg of Compound I.

Compound I 0.25 g C28H30CIN5O2

Microcrystalline cellulose 20 g

PH101

Lactose 70 g Hydroxypropyl methylcellulose Polyvinylpyrrolidone K30 Magnesium stearate 40 g 6 g 1.0 g Silica 1.0 g

Formulated in 1000 Tablets

Preparation method.

(1) Compound I and additives were weighed.

(2) Sufficiently mixed for 15 minutes.

(3) Conducted panning.

(4) The resulting tablets were coated and then packaged. Formulation 2 Each extended release tablet contained 0.5 mg of Compound I. Compound I 0.5 g C28H30CIN5O2 Microcrystalline cellulose 20 g PH101 Lactose 70 g Hydroxypropyl methylcellulose 40 g Polyvinylpyrrolidone K30 6 g magnesium stearate 1.0 g Silica 1.0 g

Compiled in

1000 tablets

-22041410

Formulation 3 Each extended release tablet contained 1.0 mg of Compound I.

Compound I 1.0 g C28H30CIN5O2 Microcrystalline cellulose 20 g

PH101

Lactose 70 g Hydroxypropyl methylcellulose Polyvinylpyrrolidone K30 magnesium stearate Silica Compiled in 40 g 6 g 1.0 g 1.0 g 1000 tablets

Exemplary microcapsule formulations are shown below.

Composition 1. Each gram of microcapsules contained 0.25 mg of Compound I.

Compound I 0.25 g C28H30CIN5O2

Stearic acid 900 g

10% ethyl cellulose in ethanol 1000 ml

95% ethanol as needed

Compiled in 1000g

Preparation method. (1) Stearic acid was weighed and melted in a water bath.

(2) Compound I was weighed and then stirred with 10% ethylcellulose in ethanol and dissolved in it.

(3) Ethanol (as required) was added to the mixture obtained in step (2), and the resulting mixture was added to the molten stearic acid; the mixture was left to heat in a water bath until a homogeneous liquid was obtained.

(4) The liquid was sprayed and cooled, while the microcapsules settled.

(5) Microcapsules were collected.

Composition 2. Each gram of microcapsules contained 0.5 mg of Compound I. Compound I 0.5 g of C28H30CIN5O2

Stearic acid 900 g 10% ethyl cellulose in ethanol 1000 ml 95% ethanol how much will it take Compiled in 1000 g

Composition 3. Each gram of microcapsules contained 1.0 mg of Compound I.

Compound I 1.0 g C28H30CIN5O2 Stearic acid 900 g 10% ethylcellulose to ethanol 1000 ml

95% ethanol how much will it take Compiled in 1000 g

An exemplary liposome composition is shown below.

Composition 1. Each ml of liposomes contained 0.25 mg of compound I.

Compound I soy lecithin 0.25 g C28H30CIN5O2 20 g

Cholesterol 5 g

Polyethylene glycol 4000 100 ml

ethanol how much will it take Compiled in Manufacturing Method. 1000 ml

(1) Soy lecithin and cholesterol were weighed and dissolved in ethanol (as required).

(2) Compound I was weighed and dissolved by sonication in polyethylene glycol 4000; water was added to the solution to a total volume of 800 ml.

(3) The solution obtained in step (1) was slowly introduced into the solution obtained in step (2), and the resulting mixture was stirred at 55°C; ethanol was completely removed and water was added to a total volume of 1000 ml.

An exemplary micelle composition is shown below.

- 23 041410

Composition 1. Each ml of micelles contained 0.25 mg of compound I. Compound I 0.25 g C28H30CIN5O2 soy lecithin 10 g ethanol 100 g Polyethylene glycol 4000 50 g Water added to 1000 ml

Compounded in 1000 ml

Preparation method.

(1) Compound I, soy lecithin and polyethylene glycol 4000 were weighed and dissolved in ethanol.

(2) Water was added to a total volume of 1000 ml to dilute the resulting mixture.

Exemplary solution formulations are shown below.

Composition 1. Each 5 ml of solution contained 0.1 mg of compound I.

Compound I with D90 constituting

0.1 g C ₂₈ H ₃ oSM02 µm

Sodium dodecyl sulfate 1.0 g Edetate disodium 1.0 g Lemon acid 2.0 g Aspartame 5.0 g Water for injections Added up to 5000 ml Compiled in 1000 ampoules

Composition 2. Each 5 ml of solution contained 0.25 mg of compound I.

Compound I with D90 constituting

0.25 g C ₂ 8H ₃ oC1N ₅ 02 µm

Dodecyl sulfate sodium Edetate disodium Lemon acid 2.5 g 1.0 g 2.0 g aspartame 5.0 g Water for injections Added up to 5000 ml

Compiled in 1000 ampoules

Composition 3. Each 5 ml of solution contained 0.5 mg of compound I. Compound I with D90,

component 5 µm 0.5 g C28H30CIN5O2

Sodium dodecyl sulfate Edetate disodium Lemon acid 5.0 g 1.0 g 2.0 g Aspartame 5.0 g Water for injections Added up to 5000 ml Compiled in 1000 ampoules

Composition 4. Each 5 ml of solution contained 1.0 mg of compound I.

Compound I with D90 constituting

1.0 g C28H30CIN5O2 µm

Sodium dodecyl sulfate 10.0 g Edetate disodium 1.0 g Lemon acid 2.0 g Aspartame 5.0 g Water for injections Added up to 5000 ml Compiled in 1000 ampoules Preparation method.

(1) Compound I and additives were weighed.

(2) Sodium dodecyl sulfate was dissolved in water for injection (as needed) and then Compound I was added, stirred to dissolve; then disodium edetate, citric acid and aspartame were added to the resulting solution and dissolved in it.

(3) The resulting solution was filtered and sterilized.

(4) Ampoules were filled with a solution of 5 ml each.

(5) Ampoules were packed.

- 24 041410

Example 3 Effect of particle size on absorbance.

Example 3-1. Effect of particle size on absorption.

Compound I with different formulations and/or different D9 ₀ values were compared with respect to absorption rates in male SD rats.

Test samples: Compound I was prepared using the method described in Example 1 of International Patent Publication WO 2014094664 A1 and then finely ground to obtain Compound I samples with D90 of 538 nm, 3.8, 21.7, 41.5 and 71.6 µm, respectively; immediately before testing, these samples were suspended in 0.5% MS to obtain 1 mg/ml suspensions. Solutions: 5% DMSO+95% (solution containing 6% HP-e-CD). Solid dispersions: P-polyvinylpyrrolidone compound K30=1:8 (w/w).

SD rats were intragastrically injected with a single dose of test samples at a dose of 1.0 mg/kg, and blood was collected before administration and 30 minutes, 1, 2, 4, 6, 8, 10 and 24 hours after administration.

The intravenous injection formulation was a solution where Compound I was dissolved in 5% DMSO+95% (6% HP-e-CD solution) and administered to SD rats at a dose of 2 mg/kg. Blood samples from rats were collected before administration and 5, 15, 30 min, 1, 2, 4, 6, 8 and 24 hours after administration.

Blood sampling: animals were fixed and their tails were heated in a water bath 10 min before blood sampling; approximately 100 μl of blood was collected via the tail vein for each animal and placed in an anticoagulant tube containing heparin; blood samples were subjected to centrifugation at 8000 rpm for 6 min at 4°C to obtain plasma samples, which must be obtained no later than 30 min after blood sampling; plasma samples were stored in a freezer at -80°C for future testing.

Sample analysis: 50 μl of each plasma sample from the freezer was transferred to a centrifuge tube; 100 µl of water and 400 µl of MTBE standard solution (50 ng/ml) were added to the tube and mixed well with plasma; the resulting mixture was mixed for 10 minutes using a vortex mixer and then subjected to centrifugation for 10 minutes (4000 rpm); 300 μl of the supernatant was transferred to another centrifuge tube and dried with a stream of nitrogen gas; the resulting substance was dissolved in 200 μl of a solution consisting of methanol and water in a ratio of 1:1, of which 20 μl was used for testing LC-MS/MS.

Data processing: Compound concentration was derived using Analyst 1.6.1 (AB Sciex); means, standard deviations and coefficients of variation were calculated using Microsoft Excel (calculations were not required if these parameters were directly derived using Analyst 1.6.1) and pharmacokinetic parameters were determined using NCA analysis performed in Pharsight Phoenix 6.3.

Results and discussion.

Table 1

Absorption of Compound I with Different Particle Sizes in Male SD Rats

Test sample AUCinf (ng*h/mL) VA%

Solution 1276 81.6 Solid dispersion 1202 76.9 Compound I with D90, 1287 82.3 component 538 nm Compound I with D90, 1045 66.9 component 3.8 µm Compound I with D90, 854 54.6 component 21.7 µm Compound I with D90, 545 34.8 component 41.5 µm Compound I with D90, 207 13.2 component 72.0 µm Note. Solution for administration by using intravenous

injection at a dose of 2.0 mg/kg resulted in

AUCINF, which is 3126 nghh/ml.

Conclusions.

From the table above, it can be seen that the lower the particle size of Compound I, the higher the bioavailability will be. When Compound I had a particle size of 21.7 μm, the bioavailability was 54.6%, which satisfies clinical requirements.

Example 3-2. Effect of particle size on absorption.

Test sample: Compound I with a D90 of 5 μm was suspended in 0.5% MC immediately prior to testing to give a 0.25 mg/ml suspension.

Animals: three beagle dogs.

Way.

Drug administration and blood sampling.

(1) All animals were fasted for 12 hours or more before drug administration and food was provided 4 hours after drug administration with unlimited access to water at all times. Animals were intragastrically injected with test samples at a single dose of 0.5 mg/kg, and 200 μl of blood was collected for each animal, through the small saphenous vein, immediately before administration (t=0) and after 10, 30, 45 min. 1, 2, 4, 6, 8, 12, 24, 30, 48 and 72 hours after administration. The collected blood was stored in a dry tube with heparin (isotonic sodium chloride solution containing 0.1% sodium heparin).

(2) Plasma preparation: Whole blood samples collected in step (1) were centrifuged at low speed (8000 rpm, 6 min, 4°C) to separate plasma (whole blood was stored in a mobile freezer at approximately 0- 4°C, and the plasma had to be separated no later than 30 min after blood sampling), and the plasma was stored in the dark in a freezer at 70°C (or lower) for future analysis.

Sample analysis: 50 μl of each plasma sample from the freezer was transferred to a centrifuge tube; 100 µl of water and 400 µl of MTBE standard solution (50 ng/ml) were added to the tube and mixed well with plasma; the resulting mixture was mixed for 10 minutes using a vortex mixer and then subjected to centrifugation for 5 minutes (12,000 rpm); 300 μl of the supernatant was transferred to another centrifuge tube and dried with a stream of nitrogen gas; the resulting substance was dissolved in 200 μl of a solution consisting of methanol and water in a ratio of 1:1, of which 20 μl was used for LC-MS/MS measurement.

Data processing: Compound concentration was derived using Analyst 1.6.1 (AB Sciex); means, standard deviations and coefficients of variation were calculated using Microsoft Excel (calculations were not required if these parameters were directly derived using Analyst 1.6.1) and pharmacokinetic parameters were determined using NCA analysis performed in Pharsight Phoenix 6.3.

Results.

Table 2 ________________ Absorption in Beagle Dogs ________________ Test Sample Dose (mg/kg) AUCo-® (ng*h/ml) VA%

Compound I with D90,

0.5 506 57.4 5 µm

Note. The solution, when administered by intravenous injection at a dose of 1.0 mg/kg, resulted in an AUCINF of 1762 nghh/ml.

Example 3-3. The additives had no effect on absorption.

Using the methods described in Example 3-1, the capsule formulation of Capsule 6 obtained in Example 2 was added to water to obtain a suspension at a concentration of 0.1 mg/mL, which was administered intragastrically to SD rats in a single dose at a dose of 10 ml/kg (with an actual dose of 0.95 mg/kg).

The AUC _{of INF} was proven to be 821 nghh/ml (a solution administered by intravenous injection at a dose of 2.0 mg/kg resulted in an AUCINF of 3126 nghh/ml), with a bioavailability of 52.5% .

Example 3-4. Effect of particle size on absorption.

Test specimens: Capsule of Capsule Formulation 5 prepared in Example 2, 10 mg Compound I per capsule.

Solutions: 5% DMSO+95% (6% HP-e-CD solution).

Animals: four beagle dogs.

Way.

Drug administration and blood sampling.

(1) Introduction of capsules. All animals were fasted for 12 hours or more before drug administration and food was provided 4 hours after drug administration, always with ad libitum access to water. Beagle dogs were orally given the capsule formulation of Capsule 5 prepared in Example 2 (two capsules for each Beagle dog, 10 mg of Compound I per capsule) in a single dose, and 200 μl of blood was collected for each animal, via the small saphenous vein, just before administration (0 h) and 0.17, 0.50, 0.75, 1.0, 2.0, 4.0, 6.0, 8.0, 12 and 24 h after administration. The collected blood was stored in a dry tube with heparin (isotonic sodium chloride solution containing 0.1% sodium heparin).

(2) Obtaining plasma. Whole blood samples collected in step (1) were centrifuged

- 26 041410 low speed (8000 rpm, 6 min, 4°C) to separate plasma (whole blood was stored in a mobile freezer at approximately 0-4°C and plasma had to be separated no later than 30 min after blood sampling) and the plasma was stored in the dark in a freezer at 70°C (or lower) for future analysis.

Sample analysis: 50 μl of each plasma sample from the freezer was transferred to a centrifuge tube; 100 µl of water and 400 µl of MTBE standard solution (50 ng/ml) were added to the tube and mixed well with plasma; the resulting mixture was mixed for 10 minutes using a vortex mixer and then subjected to centrifugation for 5 minutes (12,000 rpm); 300 μl of the supernatant was transferred to another centrifuge tube and dried with a stream of nitrogen gas; the resulting substance was dissolved in 200 μl of a solution consisting of methanol and water in a ratio of 1:1, of which 20 μl was used for LC-MS/MS measurement.

Data processing: Compound concentration was derived using Analyst 1.6.1 (AB Sciex); means, standard deviations and coefficients of variation were calculated using Microsoft Excel (calculations were not required if these parameters were directly derived using Analyst 1.6.1) and pharmacokinetic parameters were determined using NCA analysis performed in Pharsight Phoenix 6.3.

Results.

Table 3

Capsule Absorption in Beagle Dogs

Test sample Dose (mg/kg) AUCo-® (ng*h/ml) VA%

5 Capsule Composition Capsule 2.67 2361 50.2 5 Capsule Composition Capsule 2.63 2611 56.3 5 Capsule Composition Capsule 2.63 2314 49.9 5 Capsule Composition Capsule 2.78 3349 68.4 Average 2.67 2659 56.5

Note. Solutions administered by intravenous injection at a dose of 1.0 mg/kg resulted in an AUCINF of 1762 nghh/ml.

Example 3-5. Effect of particle size on absorption.

Test sample: Capsule of Capsule Formulation 6 prepared in Example 2, 2.5 mg Compound I per capsule.

Animals: four beagle dogs.

Beagles were orally administered the capsule of Capsule Formulation 6 prepared in Example 2, one capsule for each Beagle dog, 2.5 mg Compound I per capsule. The experiments were carried out using the methods described in example 3-3.

Table 4

Capsule Absorption in Beagle Dogs Test sample Dose (mg/kg) AUCo-® (ng*h/ml) VA% Capsule Composition Capsule 6 0.23 239 59.0

Note. Solutions administered by intravenous injection at a dose of 1.0 mg/kg resulted in an AUCINF of 1762 nghh/ml.

Example 4 Effect of Surfactants on Absorption.

Example 4-1. Effect of surfactants on absorption.

Test samples: 1 mg/ml suspension was made up by mixing compound I and surfactant in ratios of 1:5, 1:10 and 1:20, respectively.

SD rats were intragastrically injected with test samples at a single dose of 1.0 mg/kg and blood was collected before administration and 30 minutes, 1, 2, 4, 6, 8, 10 and 24 hours after administration.

Blood sampling: animals were fixed and their tails were heated in a water bath 10 min before blood sampling; approximately 100 μl of blood was collected via the tail vein for each animal and placed in an anticoagulant tube containing heparin; blood samples were subjected to centrifugation at 8000 rpm for 6 min at 4°C to obtain plasma samples, which must be obtained no later than 30 min after blood sampling; plasma samples were stored in a freezer at -80°C for future testing.

Sample analysis: 50 μl of each plasma sample from the freezer was transferred to a centrifuge tube; 100 µl of water and 400 µl of MTBE standard solution (50 ng/ml) were added to the tube and mixed well with plasma; the resulting mixture was mixed for 10 minutes using a vortex mixer and then subjected to centrifugation for 5 minutes (12,000 rpm); 300 µl

- 27 041410 the supernatant was transferred to another centrifuge tube and dried with a stream of nitrogen gas; the resulting substance was dissolved in 200 μl of a solution consisting of methanol and water in a ratio of 1:1, of which 20 μl was used for LC-MS/MS measurement.

Data processing: Compound concentration was derived using Analyst 1.6.1 (AB Sciex); means, standard deviations and coefficients of variation were calculated using Microsoft Excel (calculations were not required if these parameters were directly derived using Analyst 1.6.1) and pharmacokinetic parameters were determined using NCA analysis performed in Pharsight Phoenix 6.3.

Results.

Table 5

Effect of surfactant on absorption (compound I:surfactant=1:10)

Surface-active substance Dose (mg/kg) AUCinf (ng*h/ml) VA% Glycerol 0.87 657 48.3 Pro propylene glycol 0.89 457 32.9 polyvinyl alcohol 0.87 603 44.3 Polyethylene glycol 400 0.97 613 40.0 Sodium dodecylsulfonate 0.99 831 53.7 Sodium dodecyl sulfate 1.01 964 61.1 Polyoxyl (40) stearate 0.88 427 31.0 PVP Kzo 0.94 608 41.4 Carbomer 0.91 289 20.1 Polysorbate 80 0.9 654 46.5 Poloxamer 188 0.97 712 47.0 Hydroxypropylcellulose 0.92 535 37.2 Hydroxypropyl methylcellulose 0.91 483 33.6 Kolliphor HS15 0.99 536 34.6 Cholic acid 0.96 701 46.7 Benzalkonium chloride 0.83 768 59.2 Hydroxypropyl-3-cyclodextrin 0.91 350 24.6

Note. The solution, when administered by intravenous injection at a dose of 2.0 mg/kg, resulted in an AUCINF of 3126 nghh/ml.

Table 6

Effect of Surfactant on Absorption (Compound Psurfactant=1:20)

Surface-active substance Dose (mg/kg) AUCinf (ng*h/ml) VA% Benzalkonium chloride 0.93 1402 96.5 Sodium dodecylsulfonate 0.98 953 62.2 Sodium dodecyl sulfate 0.93 1297 89.2

Note. The solution, when administered by intravenous injection at a dose of 2.0 mg/kg, resulted in an AUCINF of 3126 nghh/ml.

Table 7

Effect of surfactant on absorption (compound I:surfactant=1:5)

Ratio Surfactant substance Dose (mg/kg) AUCinf (ng*h/ml) VA% 1:5 Benzalkonium chloride 0.80 855 68.4 1:5 Sodium dodecylsulfonate 0.96 968 64.5

Note. The solution, when administered by intravenous injection at a dose of 2.0 mg/kg, resulted in an AUCINF of 3126 nghh/ml.

- 28 041410

Example 4-2. Effect of surfactants on absorption.

Test specimens: tablet of tablet formulation 3 prepared in Example 2, 2.5 mg of Compound I per tablet.

Solutions: 5% DMSO + 95% (6% HP-e-CD solution), intravenous (IV) administration.

Animals: Eight beagle dogs, four for each group.

Way.

Drug administration and blood sampling.

(1) All animals were fasted for 12 hours or more prior to drug administration and food was provided 4 hours after drug administration, always with ad libitum access to water. Beagle dogs were orally given the tablets prepared in Example 2, two tablets for each beagle dog, 2.5 mg Compound I per tablet, and 200 µl of blood was collected from each animal via the small saphenous vein at 30, 45 min, 1, 2, 4, 6, 8, 12 and 24 hours after administration. The collected blood samples were stored in a dry tube with K2EDTA.

For the intravenous (IV) administration group, blood was collected at 5, 15, 30 minutes, 1, 2, 4, 6, 8, 12 and 24 hours after administration.

(2) Plasma preparation: Whole blood samples collected in step (1) were centrifuged at low speed (8000 rpm, 6 min, 4°C) to separate plasma (whole blood was stored in a mobile freezer at approximately 0- 4°C, and the plasma had to be separated no later than 30 min after blood sampling), and the plasma was stored in the dark in a freezer at 70°C (or lower) for future analysis.

Sample analysis: 50 μl of each plasma sample from the freezer was transferred to a centrifuge tube; 100 µl of water and 400 µl of MTBE standard solution (50 ng/ml) were added to the tube and mixed well with plasma; the resulting mixture was mixed for 10 minutes using a vortex mixer and then subjected to centrifugation for 5 minutes (12,000 rpm); 300 μl of the supernatant was transferred to another centrifuge tube and dried with a stream of nitrogen gas; the resulting substance was dissolved in 200 μl of a solution consisting of methanol and water in a ratio of 1:1, of which 20 μl was used for LC-MS/MS measurement.

Data processing: Compound concentration was derived using Analyst 1.6.1 (AB Sciex); means, standard deviations and coefficients of variation were calculated using Microsoft Excel (calculations were not required if these parameters were directly derived using Analyst 1.6.1) and pharmacokinetic parameters were determined using NCA analysis performed in Pharsight Phoenix 6.3.

Results.

Table 8

Absorption in Beagle dogs

Compound Dose (mg/kg) AUCinf (ng*h/ml) F% Composition of tablet 3 0.59 1100 77.4 Group IV administration 0.6 1445 ΝΑ

Example 4-3. Effect of surfactants on absorption.

Test item: Self-Emulsifying Drug Delivery System (SEDDS): Compound I: Ethanol: Kolliphor EL: Miglyol 812N=10 mg:1:5:4 g.

Solutions: 5% DMSO+95% (6% HP-e-CD solution).

SD rats were intragastrically injected with test samples at a single dose of 1.0 mg/kg and blood was collected before administration and 30 minutes, 1, 2, 4, 6, 8, 10 and 24 hours after administration.

The intravenous injection formulation was a solution where Compound I was dissolved in 5% DMSO+95% (6% HP-e-CD solution) and administered to SD rats at a dose of 2 mg/kg. Blood samples from rats were collected before administration and 5, 15, 30 min, 1, 2, 4, 6, 8 and 24 hours after administration.

Blood sampling: animals were fixed and their tails were heated in a water bath 10 min before blood sampling; approximately 100 μl of blood was collected via the tail vein for each animal and placed in an anticoagulant tube containing heparin; blood samples were subjected to centrifugation at 8000 rpm for 6 min at 4°C to obtain plasma samples, which must be obtained no later than 30 min after blood sampling; plasma samples were stored in a freezer at -80°C for future testing.

Sample analysis: 50 μl of each plasma sample from the freezer was transferred to a centrifuge tube; 100 µl of water and 400 µl of MTBE standard solution (50 ng/ml) were added to the tube and mixed well with plasma; the resulting mixture was mixed for 10 minutes using a vortex mixer and then subjected to centrifugation for 5 minutes (12,000 rpm); 300 μl of the supernatant was transferred to another centrifuge tube and dried with a stream of nitrogen gas; the resulting substance was dissolved in 200 μl of a solution consisting of methanol and water in the ratio

- 29 041410 component 1:1, of which 20 µl was used for LC-MS/MS measurement.

Data processing: Compound concentration was derived using Analyst 1.6.1 (AB Sciex); means, standard deviations and coefficients of variation were calculated using Microsoft Excel (calculations were not required if these parameters were directly derived using Analyst 1.6.1) and pharmacokinetic parameters were determined using NCA analysis performed in Pharsight Phoenix 6.3.

Results and conclusions.

Rats treated orally with SEDDS had an AUCINF of 1297 nghh/mL (solution given intravenously at a dose of 2.0 mg/kg resulted in an AUCINF of 3126 nghh/mL), with a bioavailability of 83 .0%.

Example 5 Toxicity and pharmacokinetic studies in SD rats treated intragastrically with Compound I for 13 weeks with a recovery period of 4 weeks.

Test specimen: solid dispersion of compound I (compound I:PVPK30=1:8 (w/w)).

Animals: SD rats, SPF level.

Way.

220 SD rats were used in the studies. Three groups, i.e. the high dose group, the intermediate dose group and the low dose group, were administered Compound I at the following doses, 20 males and 20 females in each group. Specifically, male animals were administered the suspension at doses of 3, 9, and 30 mg/kg/day, respectively, while females were administered at doses of 1, 3, and 10 mg/kg/day, respectively. Animals in the control group, 20 males and 20 females, were given purified water. All animals were fasted overnight before blood sampling and anatomical experiments.

In addition, for the pharmacokinetic study, 9 males and 9 females were included in the administration group, with 3 males and 3 females for the control group.

Results and conclusions.

Table 9

Serum potassium (K ⁺ ) levels and AUC levels _{0-24 h} in SD rats treated intragastrically with Compound I for 13 weeks

Floor Dose (mg/kg) K ⁺ (mmol/l) (average±80) AUCo-244 (ng*h/ml) 0 5.1±0.3 / Male 3 5.0±0.3 5430 9 5.0±0.3 16500 thirty 5, GCD 49900 0 4.2±0.3 / Female 1 4.4±0.4 4080 3 4.4±0.3 15500 10 4.5±0.2 43600

Note. / means n.a. (not applicable).

Based on the above results, the NOAEL (maximum dose without observed adverse effects) was 30 mg/kg/day in male animals and 10 mg/kg/day in female animals with corresponding AUC _{0-24 hours} of 49,900 and 43,600 nghh/ ml, respectively, and below this AUC level, no obvious abnormal serum potassium was observed.

Example 6 Efficacy Test in Salt-Sensitive Rats with Salt-Induced Kidney Injury.

Animals: Dahl/ss male rats.

Investigated product and composition.

Solid dispersions of compound I (compound I:PVPK30=1:8 (w/w)): solid dispersions of compound I were formulated with appropriate amounts of sterile water for injection in suspension at concentrations of 0.03, 0.10, 0, 30 and 1.00 mg/ml, respectively. Suspensions were prepared immediately before use.

Dahl/ss rats were randomly divided into 6 groups based on pre-drug blood pressure values, ie. control group 1 with normal animals (n=10), control group 2 with animals with induced kidney injury (4% NaCl, n=12), treatment groups with doses of 0.3 mg/kg/day (n=11 ), 1 mg/kg/day (n=11), 3 mg/kg/day (n=11), and 10 mg/kg/day (n=11), respectively, where n is the number of animals.

Way.

Dahl/ss rats were fed research diet AIN-93G containing 4% NaCl to induce hypertensive nephropathy in order to test in vivo pharmacodynamic

- 30 041410 activity of compound I.

One week before the experiment, blood pressure values were measured twice in rats by measuring blood pressure with a tail cuff so that the rats could adjust to the blood pressure monitoring operation. Then, immediately before the test, blood pressure was measured and used as a baseline. Rats were randomly divided into groups according to baseline blood pressure values. The following day, animals in control group 2 and treatment groups were fed the research diet AIN-93G containing 4% NaCl and water, ad libitum, to induce a kidney injury model. Building the model took 42 days. Animals in control group 1 were given a normal diet of AIN-93G.

Animals in treatment groups were intragastrically administered Compound I twice daily at doses of 0.3, 1, 3 and 10 mg/kg/day, respectively, as suspensions of Compound I administered in a volume of 5 ml/kg per dose. The two control groups were given an equal amount of sterile water for injection.

Measurement of blood pressure (systolic blood pressure, SBP): blood pressure was measured for 6 weeks, once a week, and analyzed for changes in blood pressure in these groups.

Pathological examination of the kidneys and heart: at the end of the study, rats were painlessly sacrificed, bilateral kidney and heart sampling was performed for histopathological analysis. Kidneys were stained using hematoxylin-eosin (HE) for semi-quantitative assessment of kidney damage. Left ventricular wall thickness was measured for each heart to analyze heart damage.

In addition, blood samples at 0, 1, 2, 4, 6, 8, 10 and 24 hours after the last injection were collected from the tail vein of rats until the end of the study. The blood samples were placed in a low temperature high speed centrifuge and centrifuged for 6 min at 4°C, 8000 rpm. Plasma samples were collected and stored in a -80°C freezer until plasma drug concentration was measured.

Results and conclusions.

According to the test results, blood pressure (SBP) or urinary albumin to creatinine ratio (UACR) versus AUC _0-24 was plotted (see FIGS. 1 and 2).

The curve of SBP or UACR versus AUC _0-24 showed a ripple point at 100 hng/mL AUC, after which the curve tended to be flat and SBP and UACR decreased significantly. This indicated that Compound I had good efficacy and was effective when the AUC reached 100 hng/ml.

EXAMPLE 7 Single dose safety, tolerability and pharmacokinetic studies in healthy subjects.

Five groups of healthy subjects were given capsules at doses of 0.5 mg (1 capsule, 0.5 mg Compound I per capsule), 1.0 mg (2 capsules, 0.5 mg Compound I per capsule), 2.5 mg (1 capsule, 2.5 mg Compound I per capsule), 10 mg (1 capsule, 10 mg Compound I per capsule) and 30 mg (3 capsules, 10 mg Compound I per capsule), each group containing 8 subjects. The introduction of the drug was carried out only once.

The capsules mentioned above were the capsules prepared in Example 2, i.e. capsules of Capsule Formulation 3 (0.5 mg Compound I per capsule), Capsule Formulation 4 (2.5 mg Compound I per capsule), and Capsule Formulation 5 (10 mg Compound I per capsule).

Plasma samples were collected to measure plasma drug concentration and pharmacokinetic parameters were analyzed using a non-compartmental model. Serum samples were collected to measure serum potassium levels.

Results.

In healthy subjects administered at a dose of 0.5-30 mg/day, the AUC _0-24 of compound I was 162.5-5016 nghh/ml and T _1/2 was approximately 60 hours, with an increased content of potassium was not observed in serum.

Table 10

Pharmacodynamic results

Pharmacodynamic parameters Dose 0.5 mg 1.0 mg 2.5 mg 10 mg 30 mg AUCo-24 (h*ng/ml) 162.5 396.7 639.8 3077 5016 Т1/2 (H) 61.5 60.9 53.3 52.8 73.9

Example 8 Multiple Dose Safety, Tolerability and Pharmacokinetic Studies in Healthy Subjects.

Two groups of healthy subjects were given capsules once daily at daily doses of 2.5 mg (1 capsule for each administration, 2.5 mg Compound I per capsule) and 5.0 mg (2 capsules for each administration, 2. 5 mg of compound I per capsule), respectively, with each group containing 6 subjects. The introduction was carried out for 14 days in a row. The capsules used in this study were the capsule formulation 4 prepared in Example 2.

Plasma samples were collected to measure plasma drug concentration and pharmacokinetic parameters were analyzed using a non-compartmental model. Serum samples were collected to measure serum potassium levels.

Results.

In healthy subjects administered in a multiple dose regimen with a daily dose of 2.5 mg/day, the mean steady state AUC ₀ _ ₂₄ was 2865±821 nghh/ml, with no observed elevated serum potassium.

In healthy subjects administered on a multiple dose regimen with a daily dose of 5.0 mg/day, the mean steady-state AUC ₀ _ ₂₄ was 6376±1028 nghh/ml, with transient elevated serum potassium observed in three subjects.

Example 9 Safety, tolerability and pharmacokinetic studies in patients with chronic kidney disease.

The patients in the present example were chronic kidney disease patients with symptoms of renal failure, proteinuria, acute kidney injury, glomerular nephritis, renal cyst, urinary frequency, kidney stone, obstructive uropathy, etc. These patients are also characterized by the presence of other diseases or complications such as diabetes mellitus, hyperlipidemia, hypercholesterolemia, hypertension, peripheral vascular disease, atherosclerotic heart disease, etc.

These patients continued to take the drugs they were taking prior to this clinical trial, the drugs being, for example, angiotensin-converting enzyme inhibitors such as lisinopril, benazepril, enalapril and enalapril maleate; angiotensin II receptor blockers such as valsartan and losartan; calcium channel blockers such as amlodipine and nifedipine; diuretics such as furosemide; betareceptor blockers such as metoprolol succinate, metoprolol tartrate, carvedilol and atenolol; and antilipemic drugs such as simvastatin, atorvastatin, fenofibrate, pravastatin and rosuvastatin.

In the present example, the dose administered referred to the amount of Compound I although the pharmaceutical composition was administered. The two groups of subjects were administered once daily for 56 consecutive days at daily doses of 0.5 mg (1 capsule for each administration, 0.5 mg Compound I per capsule) and 2.5 mg (1 capsule for each administration , 2.5 mg Compound I per capsule), respectively, 6 subjects in the 0.5 mg/day dose group and 6 subjects in the 2.5 mg/day dose group. The capsules used in this test were those prepared in Example 2, i.e. capsules of capsule formulation 3 (capsule containing 0.5 mg of compound I) and capsule formulation 4 (capsule containing 2.5 mg of compound I).

Plasma samples were collected to measure plasma drug concentration and pharmacokinetic parameters were analyzed using a non-compartmental model. Serum samples were collected to measure serum potassium levels.

Results.

In patients with CKD administered in multiple doses with a daily dose of 0.5 mg, the mean steady-state AUC _tau was 652.5 ± 232.2 nghh/mL, with no observed elevated serum potassium.

In patients with CKD administered at multiple doses with a daily dose of 2.5 mg, a modest increase in serum potassium was observed in one subject. The mean safe steady-state AUC _tau in patients was 2613±280 nghh/ml.

In these CKD patients, after multiple doses of 0.5 mg/day or 2.5 mg/day, a significant reduction in blood pressure and urinary albumin to creatinine ratio (UACR) was observed. At week 8, UACR decreased by 58.9 and 50.7% in the two groups, respectively. The pharmaceutical composition according to the present invention showed a clear effect on the protection of the kidneys.

Patients with CKD had a steady state AUC _tau of 105.6 nghh/mL or greater when administered over one day at a daily dose of 0.5 mg and UACR decreased by 30.5% or greater from baseline, indicating on the effectiveness of the pharmaceutical composition according to the present invention.

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Claims (17)

Table 11 Pharmacodynamic results Pharmacodynamic parameter AUCtau (ng*h/ml) Dose (mg) 0.1 0.15 0.2 0.25 0.3 0.5 1.0 2.0 2.5 Actual AUC _tau / / / / / 652.5 / / 2613 Forecast AUCtau 130.5 195.8 261 326.3 391.5 / 1305 2610 3262.5 In CKD patients taking the pharmaceutical composition of the present invention, the AUCtau value of Compound I was in the range of 188-3173 nghh/mL, indicating excellent efficacy. In addition, elevated serum potassium levels were not observed. Thus, the pharmaceutical composition according to the present invention satisfies the requirements of clinical safety and efficacy. CLAIM 1. Application of compound I for the treatment of chronic kidney disease in humans at a daily dose of 0.1 to 1.0 mg. 2. Use according to claim 1, in which the daily dose is from 0.1 to 0.5 mg. 3. Use according to claim 2, in which the daily dose is from 0.2 to 0.5 mg. 4. A method of treating chronic kidney disease in a human, comprising administering compound I to a human in a daily dose of 0.1-1.0 mg. 5. The method according to claim 4, in which the daily dose is 0.1-0.5 mg. 6. The method according to claim 5, in which the daily dose is 0.2-0.5 mg. 7. Use of Compound I for the manufacture of a pharmaceutical composition for the treatment of chronic kidney disease in humans at a dose of 0.1-1.0 mg. 8. Use according to claim 7, in which the dose is 0.1-0.5 mg. 9. Use according to claim 8, in which the dose is 0.2-0.5 mg. 10. Use according to claim 7, wherein the pharmaceutical composition comprises Compound I and one or more pharmaceutically acceptable carriers, and wherein the pharmaceutical composition is a single use formulation containing 0.1 to 1.0 mg of Compound I. 11. Application according to compound I. 12. Application according to compound I. 13. Application according to 14. Application according to 10, and the composition for single use contains from 0.1 to 0.5 mg 11, and the composition for single use contains from 0.2 to 0.5 mg 10, wherein the _D90 of the compound I particles is 25 µm or less. 13, wherein the D9 ₀ of the compound I particles is 10 µm or less. 15. Use according to claim 10, wherein the pharmaceutical composition contains one or more surfactants selected from the group consisting of benzalkonium chloride, sodium lauryl sulfonate, sodium dodecyl sulfate, glycerin, cholic acid, poloxamer, polyvinyl alcohol, polysorbate 80, PVP K ₃₀ and polyethylene glycol. 16. Use according to claim 15, wherein the pharmaceutical composition contains one or more surfactants selected from the group consisting of benzalkonium chloride, sodium lauryl sulfonate and sodium dodecyl sulfate. 17. Use according to claim 16, wherein the pharmaceutical composition contains a surfactant -