WO2022048614A1 - 一种含氮的饱和杂环化合物的应用 - Google Patents

一种含氮的饱和杂环化合物的应用 Download PDF

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WO2022048614A1
WO2022048614A1 PCT/CN2021/116338 CN2021116338W WO2022048614A1 WO 2022048614 A1 WO2022048614 A1 WO 2022048614A1 CN 2021116338 W CN2021116338 W CN 2021116338W WO 2022048614 A1 WO2022048614 A1 WO 2022048614A1
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formula
hypertension
compound
malate
kidney disease
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PCT/CN2021/116338
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French (fr)
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夏广新
苏威
王雪松
柯樱
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上海医药集团股份有限公司
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Priority to KR1020237011168A priority Critical patent/KR20230058506A/ko
Priority to US18/024,807 priority patent/US20230330093A1/en
Priority to JP2023515103A priority patent/JP2023539779A/ja
Priority to EP21863678.5A priority patent/EP4209218A4/en
Publication of WO2022048614A1 publication Critical patent/WO2022048614A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C59/235—Saturated compounds containing more than one carboxyl group
    • C07C59/245—Saturated compounds containing more than one carboxyl group containing hydroxy or O-metal groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs

Definitions

  • the present invention relates to the application of a nitrogen-containing saturated heterocyclic compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and/or preventing chronic kidney disease.
  • Chronic kidney disease has the characteristics of high prevalence, low awareness, poor prognosis and high medical costs. It is another disease that seriously endangers human health after cardiovascular and cerebrovascular diseases, diabetes and malignant tumors. In recent years, the prevalence of CKD has increased year by year, and the prevalence of the global general population has reached 14.3%. A cross-sectional epidemiological study in my country shows that the prevalence of CKD in people over 18 years old is 10.8%.
  • Chronic kidney disease is a progressive disease. If there is no timely and effective treatment, the disease will deteriorate and progress. As the disease progresses, patients with chronic kidney disease will develop renal failure. If artificial dialysis or kidney transplantation is not performed, survival will be changed. difficult. At present, there is no particularly effective method for the treatment of chronic kidney disease. Therefore, it is of great clinical value to actively search for drugs that are effective in the treatment of chronic kidney disease and have less toxic and side effects.
  • CN103562191B discloses a nitrogen-containing saturated heterocyclic compound with the following structural formula or a pharmaceutically acceptable salt thereof, which exhibits an inhibitory effect on renin and can be used for the treatment of hypertension.
  • R 1 represents a cycloalkane group, etc.
  • R 22 represents an aryl group that may be substituted, etc.
  • R represents a lower alkane group, etc.
  • T represents a carbonyl group
  • Z represents -O-, etc.
  • R 3 , R 4 , R 5 and R 6 is the same or different, and represents a hydrogen atom or the like.
  • CN106928218A discloses novel pharmaceutically acceptable salts of morpholine derivatives (nitrogen-containing saturated heterocycles), including malate, tartrate, hydrochloride, acetate and naphthalene diphosphate, wherein the tartrate has There are three crystalline salt forms of A crystal form, B crystal form and dihydrate, one crystalline salt form each of malate, hydrochloride and acetate, and naphthalene diphosphate is amorphous.
  • the salts of morpholine derivatives have one or more improved properties, such as better crystallinity, greatly improved water solubility, photostability and thermal stability.
  • the above-mentioned morpholine derivative salt or its crystalline form can be used for the treatment and/or prevention of hypertension.
  • the technical problem to be solved by the present invention is to provide a nitrogen-containing saturated heterocyclic compound or a pharmaceutically acceptable salt thereof in the preparation for the treatment and / or the application of drugs for the prevention of chronic kidney disease.
  • a nitrogen-containing saturated heterocyclic compound represented by the following formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicine for the treatment and/or prevention of chronic kidney disease:
  • the pharmaceutically acceptable salts are hydrochloride, sulfate, phosphate, hydrobromide, acetate, fumarate, oxalate, citric acid salt, mesylate, benzenesulfonate, p-toluenesulfonate, maleate, malate, tartrate, acetate or naphthalene disulfonate.
  • the pharmaceutically acceptable salt is malate, tartrate, hydrochloride, acetate or naphthalene disulfonate.
  • the pharmaceutically acceptable salt is the malate salt of the compound of formula I, which is a compound formed by the compound of formula I and malic acid in a molar ratio of 1:1, and its structural formula is as follows:
  • the malate salt is a crystal
  • its X-ray powder diffraction pattern at 2 ⁇ is 7.767° ⁇ 0.2°, 13.897° ⁇ 0.2°, 14.775° ⁇ 0.2°, 17.098° ⁇ 0.2° , 18.999° ⁇ 0.2°, 20.153° ⁇ 0.2°, 20.960° ⁇ 0.2°, 21.423° ⁇ 0.2°, 26.348° ⁇ 0.2°, 27.892° ⁇ 0.2° have characteristic peaks.
  • the X-ray powder diffraction pattern of the malate crystal is 2 ⁇ of 5.598° ⁇ 0.2°, 7.357° ⁇ 0.2°, 10.395° ⁇ 0.2°, 11.108° ⁇ 0.2°, 16.037° ⁇ 0.2°, 16.523° ⁇ 0.2°, 19.410° ⁇ 0.2°, 22.645° ⁇ 0.2°, 26.630° ⁇ 0.2°, 26.891° ⁇ 0.2°, 27.380° ⁇ 0.2°, 31.056° ⁇ 0.2°, 33.306° ⁇ 0.2°, 33.775°
  • the malate salt crystals of the compound of formula I have an X-ray powder diffraction pattern as shown in FIG. 1 .
  • the chronic kidney disease includes hypertension combined with kidney disease, hypertension combined with kidney disease with abnormal glucose metabolism, chronic renal insufficiency combined with chronic heart failure or chronic kidney disease with abnormal glucose metabolism.
  • the chronic kidney disease refers to the G1, G2, G3a, G3b, G4 stage of chronic kidney disease, preferably the G2, G3a or G3b stage.
  • the hypertension in the hypertension complicated with renal disease is grade 1 hypertension, grade 2 hypertension or grade 3 hypertension, preferably grade 1 hypertension or grade 2 hypertension.
  • the dosage forms of the medicine include tablets, capsules, intravenous injections, inhalants, aerosols, freeze-dried preparations, patches, gels, sprays, or suppositories, preferably for tablets.
  • the medicament is a unit dose.
  • the unit dose of the medicine contains 25mg-200mg of the nitrogen-containing saturated heterocyclic compound represented by the above formula I or a pharmaceutically acceptable salt thereof, such as 25mg, 50mg, 100 mg, 150 mg, 200 mg of the nitrogen-containing saturated heterocyclic compound represented by the above formula I or a pharmaceutically acceptable salt thereof.
  • Kidney Disease Outcomes Quality Initiative KDOQI
  • Kidney Foundation NSF
  • Chronic kidney disease was defined as abnormal kidney structure or function for >3 months.
  • the diagnostic criteria for chronic kidney disease were any one of the indicators in Table 1 below for more than 3 months.
  • AER urinary albumin excretion rate
  • ACR urinary albumin to creatinine ratio
  • GFR glomerular filtration rate
  • Chronic kidney disease staging Chronic kidney disease is divided into 5 stages according to the glomerular filtration rate (GFR), as shown in Table 2 below.
  • GFR glomerular filtration rate
  • SBP systolic blood pressure
  • DBP diastolic blood pressure
  • SBP ⁇ 140mmHg and DBP ⁇ 90mmHg are isolated systolic hypertension.
  • the patient has a history of hypertension and is currently taking antihypertensive drugs. Although the blood pressure is lower than 140/90mmHg, hypertension should still be diagnosed.
  • Hypertension was further divided into grades 1, 2, and 3 based on the level of blood pressure elevation. Blood pressure levels are categorized and defined in Table 3 below.
  • Heart failure is a clinical syndrome defined as a complex group of clinical syndromes in which the ability of the ventricle to fill or eject blood is impaired due to any structural or functional abnormality of the heart. Its main clinical manifestations are dyspnea and fatigue (limited activity tolerance), and fluid retention (pulmonary congestion and peripheral edema).
  • heart failure is divided into heart failure with reduced ejection fraction (heart failure with reduced ejection fraction, HFrEF), heart failure with preserved ejection fraction (heart failure with preserved Ejection fraction, HFpEF) and heart failure with mid-range ejection fraction (HFmrEF), the three types of heart failure are defined in Table 4 below:
  • Heart failure with reduced ejection fraction in HFrEF heart failure with median ejection fraction in HFmrEF; heart failure with preserved ejection fraction in HFpEF; a B-type natriuretic peptide (BNP)>35ng/L and/or N-terminal B-type Pro-natriuretic peptide (NT-proBNP)>125ng/L; b E/e' ⁇ 13, average e' (interventricular septum and free wall) ⁇ 9cm/s; -none
  • disorders of glucose metabolism include prediabetes and diabetes.
  • Pre-diabetes is the impaired glucose regulation, which means that the blood sugar level has exceeded the normal range, but has not yet reached the diagnostic criteria of diabetes, that is, the intermediate state between normal people and diabetic patients.
  • Diabetes mellitus is a group of common metabolic diseases characterized by hyperglycemia, glucosuria, impaired glucose tolerance, and abnormal insulin release tests.
  • the positive and progressive effect of the present invention is that: the study on the efficacy and safety of nitrogen-containing saturated heterocyclic compound salts for 8 weeks on chronic renal insufficiency combined with chronic heart failure rhesus monkeys (middle-aged/elderly) shows that the present invention provides
  • the nitrogen-containing saturated heterocyclic compound or a pharmaceutically acceptable salt thereof can effectively prevent or treat chronic kidney disease, and the chronic kidney disease includes hypertension complicated with nephropathy, hypertension with nephropathy accompanied by abnormal glucose and lipid metabolism, and chronic renal insufficiency. Combined with chronic heart failure or chronic kidney disease with abnormal glucose and lipid metabolism.
  • the nitrogen-containing saturated heterocyclic compound or the pharmaceutically acceptable salt thereof provided by the present invention has good safety, and no adverse events related to administration are observed.
  • Fig. 1 is the XRPD pattern of the malate salt of the compound of formula I used in the examples of the present invention.
  • FIG. 2 is a concentration-time graph of the compound of formula I in monkeys following intravenous (iv) and oral (po) administration in Example 3.
  • Example 1 Study on the efficacy and safety of compound malate of formula I administered continuously for 8 weeks on rhesus monkeys (middle-aged/elderly) with chronic renal insufficiency and chronic heart failure
  • ENDPOINT Primary efficacy endpoint: 1. Improvement of renal function: glomerular filtration rate eGFR creat-cys , cystatin C (CysC), creatinine and UACR; 2. Improvement of cardiac function: echocardiographic index analysis of the heart Changes in structure and systolic and diastolic function before and after administration; 3. Safety indicators: blood pressure, serum potassium, glucose and lipid metabolism-related indicators, etc.
  • Macaca mulatta (Rhesus Macaque)
  • Grade Normal grade. Quarantine before the test is qualified, including physical examination, 2 times of Mycobacterium tuberculosis test, parasite, Salmonella, Herba and B virus inspection.
  • Animal identification A stainless steel number plate with Arabic numerals is worn on the neck ring, and the chest is tattooed.
  • Animal certificate number 0016710, 0016944, 0016966.
  • abnormal glucose metabolism is fasting blood glucose (FPG)>4.8mmol/l, vs. age-matched control group 4.1 ⁇ 0.3mmol/l;
  • A2-A3 urinary albumin/creatinine ratio
  • HFrEF LVEF 30%-49% (normal value: 50%-70% Simpson biplane method) or HFpEF (moderately impaired): Ea ⁇ 8 or E/Ea>10 (based on clinical diagnostic criteria).
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • Table 5 shows the underlying etiology and characteristics of rhesus monkeys with chronic DKD renal insufficiency tested in baseline-various dose groups.
  • Table 5 Baseline period - underlying etiology and characteristics of rhesus monkeys with chronic DKD renal insufficiency in each dose group
  • Table 5 Baseline period - underlying etiology and characteristics of rhesus monkeys with chronic DKD renal insufficiency tested in each dose group (continued)
  • the first stage consisted of two groups: 3 animals (#257, #4721, #287) in the compound malate of formula I group (#257, #4721, #287), and 2 animals in the placebo group (# 1029, #4713), Phase II compound of formula I malate 5 mg/kg group 3 animals (#144, #5073, #6651), Valsartan group 3 animals (#2091, #4861, #6645), placebo Dosage group 2 animals (#6653, #231), the third stage compound of formula I malate salt 2 mg/kg group 5 animals (#4829, #6501, #287, #4721, #2091), compound of formula I apple Salt 1mg/kg group 5 animals (#1567, #4861, #5453, #6653, #5073),
  • Valsartan group was administered once a day in the first to second week, and twice a day in the third to eighth week.
  • Dosing frequency the malate salt of the compound of formula I was once a day, and the Valsartan group was administered once a day in the first to second weeks, and twice a day in the third to eighth weeks.
  • Dosage calculation Calculate the dose for the next week based on the body weight of each weighing.
  • Administration time 08:00-09:00 administration.
  • Glomerular filtration rate eGFR creat-cys Detect creatinine (Cr-P), urea nitrogen (BUN), cystatin (CysC), and calculate the eGFR value. 1 time before administration and once every two weeks after administration.
  • eGFR male 135 ⁇ min(Cr/0.9,1) -0.207 ⁇ max(Cr/0.9,1) -0.601 ⁇ min(CysC/0.8,1) -0.375 ⁇ max(CysC/0.8,1) -0.711 ⁇ 0.995 Age ⁇ 3
  • eGFR female 135 ⁇ min(Cr/0.7,1) -0.284 ⁇ max(Cr/0.7,1) -0.601 ⁇ min(CysC/0.8,1) -0.375 ⁇ max(CysC/0.8,1) -0.711 ⁇ 0.995 Age ⁇ 3 ⁇ 0.969
  • Blood pressure was measured after anesthesia, including systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP) and heart rate (HR). 1 time before administration, 1 time at 4 weeks and 1 time at 8 weeks after administration.
  • SBP systolic blood pressure
  • DBP diastolic blood pressure
  • MBP mean blood pressure
  • HR heart rate
  • Serum potassium 2 times before administration, once every 2 weeks during administration period
  • Glycolipid metabolism and liver function FPG, FRA, LDL-c, HDL-c, TG, TC, NT-proBNP, ALT, AST, TBIL, etc., 2 times before administration, 1 each at 4 weeks and 8 weeks Second-rate;
  • Body weight once before dosing, once a week during the dosing period;
  • Hematology testing instrument Siemens ADVIA 2120i Hematolagy Systems.
  • Blood biochemical and urine index detection equipment Roche cobas6000 analyzer series C501 module detection, NT-proBNP is detected by ELISA kit.
  • Anesthesia method 15mg/kg ketamine hydrochloride was injected intramuscularly for anesthesia. According to the animal's anesthesia state, supplementary anesthesia was performed after the veterinarian's judgment. The dose of each supplementary anesthesia was 1/2 of the initial dose.
  • Detection method The animals were placed in the left lateral decubitus position, and the 6S-RS probe (frequency 2.7-8.0MHz) was used for image acquisition.
  • echoPAC Software was used to repeatedly detect cardiac systolic and diastolic function indicators in three consecutive cardiac cycles.
  • Testing equipment GE Vivid S5 color Doppler ultrasound diagnostic instrument.
  • Detection method The animals were anesthetized by intramuscular injection of 15 mg/kg ketamine hydrochloride. After anesthesia, the animals were placed in a supine position. The hair on the left upper arm was shaved, and an appropriate size cuff was tied according to the standard. The blood oxygen probe was clamped on the animal's finger or toe. On (except the left hand), the red photosensitive surface is on the side of the belly of the finger, and the blood pressure of the animal is detected 3 times continuously using the automatic mode, with an interval of 1 min each time. If the DBP, SBP and MBP of blood pressure for more than three times have little difference (the difference between the highest value and the lowest value is less than 15mmHg), the measurement is over.
  • Detection indicators include systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP) and heart rate (HR).
  • SBP systolic blood pressure
  • DBP diastolic blood pressure
  • MBP mean blood pressure
  • HR heart rate
  • Testing equipment GE B40i electrophysiological monitor.
  • Observation content injection site, skin, coat, eyes, ears, nose, mouth, chest, abdomen, genitourinary, limbs, etc., as well as changes in breathing, exercise, urination, defecation, and behavior.
  • Feeding method 8:00 am to 9:00 am, 10:00 am, 2:00 pm and 4:00 pm, each time, to ensure that there is food in the food box, free to eat, the next morning 07:40- At 08:00 the remaining feed was removed.
  • Feeding amount about 250-500g / animal / day, in the test, ensure that there is feed in the food box for 24 hours, and the animals can eat freely.
  • Measuring instrument METTLER TOLEDO electronic platform scale.
  • Compound of formula I malate (5mg/kg qd) group (n 6): compared with baseline, W8 glomerular filtration rate eGFR creat-cys increased by an average of 6 ⁇ 6ml/min/1.73m 2 after administration, with The change value ratio in the placebo group was significantly increased (p ⁇ 0.05), and the curative effect was time-sensitive.
  • Compound of formula I malate (1 mg/kg qd) group (n 5): compared with baseline, W8 glomerular filtration rate eGFR creat-cys increased by an average of 3 ⁇ 4 ml/min/1.73 m 2 after administration, and There was no statistically significant change in the value of the placebo group.
  • the change value ratio in the placebo group was significantly decreased (p ⁇ 0.01), and the curative effect was time-sensitive.
  • the change value ratio in the placebo group was significantly decreased (p ⁇ 0.01), and the curative effect was time-sensitive.
  • Table 17 Influence of formula I compound malate administration for 8 weeks on the main indicators of cardiac function in rhesus monkeys with spontaneous chronic kidney disease and evaluation of efficacy
  • the level of abnormal diastolic function is determined after comprehensive consideration of Ea, E/Ea, Ea/Aa and other functional indicators; when the level of abnormal diastolic function of the test animal is improved compared with the baseline, it is regarded as "benefit”. Otherwise, it was considered “invalid”; the #257 animal had a deformity of the thorax, and echocardiographic images of the heart could not be acquired.
  • Table 18 shows the effect of compound of formula I malate on NT-proBNP.
  • the NT-proBNP levels of animals in each administration group did not change significantly after 8 weeks of administration.
  • Table 18 Effects of 8-week administration of compound of formula I malate on NT-proBNP in rhesus monkeys with spontaneous chronic kidney disease
  • the compound of formula I malate at 2-5 mg/kg qd can significantly improve proteinuria (UACR) and glomerular filtration rate eGFR creat-cys after 8 weeks of administration, and the curative effect is comparable to that of the Valsartan group.
  • Formula I compound malate salt 1-5mg/kg qd administered for 8 weeks, showed the activity of lowering blood pressure. At the same time, there was no risk of hyperkalemia under the conditions of this test.
  • the compound of formula I was prepared and the structure confirmed according to the method in CN103562191B.
  • the solvent is 0.5% sodium carboxymethyl cellulose (Sodium carboxymethyl cellulose, CMC-Na).
  • Compounds of formula I are stored at room temperature, protected from light and humidity.
  • test substances were suspended in 0.5% CMC-Na at relevant concentrations. Prepared suspensions were analyzed for homogeneity, concentration, and one-week stability to ensure correct administration of reported doses.
  • the active pharmaceutical ingredient (API) was tested for six-month accelerated stability.
  • Healthy male and female cynomolgus monkeys aged 3-4 were provided by Hainan Xinyuan Biotechnology Co., Ltd. (Hainan, China). At the start of treatment, males ranged from 3.2–5.8 kg and females ranged from 2.5–3.7 kg. Before the study, they were housed for a 36-day acclimation period. Once a day in the morning, animals were given free access to food and water.
  • Cynomolgus monkeys (5/sex/group) were selected using a weight-based computer randomization program and fed a compound of formula I (dose levels 0 (control), 20, 100, 450 mg/kg/day in a volume of 5 ml/day by nasal gavage) kg). Individual doses were adjusted weekly according to the body weight of the animals. For all groups, 2/3 animals were randomly selected and euthanized on day 28. The remaining animals were euthanized 28 days after drug withdrawal.
  • Mortality and clinical signs were assessed daily from the isolation period. Each animal was examined at least twice a day for behavior, post-dose response, or the development of disease.
  • Observations include but are not limited to the following: skin and fur changes; eyes and mucous membranes; breathing, circulation, autonomic, central nervous system and behavioral patterns. Rectal temperature was measured before dosing (twice), 1 and 24 hours after the first dose, and before and 1 and 24 hours after the first dose on Day 26, respectively. Measure body weight weekly. The monkeys' food consumption was estimated daily. Ophthalmoscopy was performed on monkeys using a portable slit lamp (YZ2) and direct ophthalmoscope (GFJY-01B).
  • YZ2 portable slit lamp
  • GFJY-01B direct ophthalmoscope
  • Table 20 Parameters evaluated in hematology, serum chemistry and urinalysis
  • ECG analysis was performed on day 56, hour 1, hour 24).
  • the ECG system used derived DII (ECG-6951E, Shanghai, China) to record P waves, R waves, T waves, PR interval, Q-T interval, QRS duration and heart rate.
  • necropsies were performed on all animals by visual inspection. Trim, weigh and assess absolute weight and percentage of final body weight or brain weight for the following selected organs: brain, heart, kidneys, kidneys, liver, spleen, thymus, testes, epididymis, uterus, ovaries, adrenal glands and thyroid (including thyroid) parathyroid glands).
  • Lung tissue was infected with fixative at necropsy. All preserved tissues were embedded, sectioned, stained with hematoxylin and eosin (HE), and examined microscopically. Morphological examination of bone marrow cells was performed on both smear (monkey sternum) and paired-embedded sternum sections.
  • Blood samples were collected at 0 hours, 0.5 hours, 1.5 hours, 3 hours, 5 hours, 7 hours, and 24 hours on day 1 and day 26 post-dose. Approximately 1.0 mL of blood was drawn from the vein at a time and collected into EDTA-K 2 anticoagulant tubes. Blood samples were placed on ice and centrifuged (3500 rpm, 5 minutes) at 4°C to obtain plasma samples, which were frozen at approximately -80°C until analysis. The toxicokinetic assay of the compound of formula I was carried out at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.
  • Elevated NEUT% and decreased LYMPH% were observed in all treated females and high-dose males. Elevated Fbg was also observed in mid- and high-dose animals, and APTT was elevated in high-dose females (Table 21).
  • Table 21 Hematological data of monkeys treated with compounds of formula I for 4 weeks.
  • Serum chemical parameters CPK, BUN and CREA were significantly increased in male and female high-dose cynomolgus monkeys, especially in monkeys that died early on day 23 (day 14 data, BUN: 34.99; CREA: 526). CHOL was significantly reduced in high-dose males. Serum Na levels in high-dose males decreased slightly, but were statistically significant at day 26. Other statistically significant findings were isolated events that were not considered toxicologically significant (Table 22).
  • Table 24 Organ weights of cynomolgus monkeys treated with compounds of formula I for 4 weeks.
  • Systemic exposure (represented by plasma AUC 0-24h and Cmax of the parent compound and major metabolites) increased proportionally more than the dose over the 20-450 mg/kg dose range. After 28 consecutive days of dosing, no accumulation was seen.
  • the compound of formula I was prepared and the structure confirmed according to the method in CN103562191B.
  • Cynomolgus monkeys (3/sex/group: body weight 3-5 kg, age 5-6 years) were from Suzhou Xishan Zhongke Experimental Animal (Shanghai, China). Animals were acclimated for 7 days before the study. Monkeys were individually caged in animal rooms with controlled temperature (18–26 °C) and humidity (50 ⁇ 20%) with an air flow of 10 and fresh air changes every hour. The light cycle was 12 hours on and 12 hours off, and the monkeys were fed chow and water once every morning. All animal experiments were performed in accordance with the Guidelines for the Care and Use of Animals developed by the AMMS Animal Care and Use Committee.
  • Compounds of formula I were administered intravenously or orally to cynomolgus monkeys that were fasted overnight. The doses were 1 mg/kg (intravenous), 1 mg/kg, 3 mg/kg, and 9 mg/kg (oral). Blood samples (0.2 mL per time point) were collected from the vein at 0, 5, and 15 minutes post-dose at 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours, respectively. Immediately after collection, all blood samples were centrifuged at 10,000 x g for 5 min, and plasma was stored at -75 ⁇ 10 °C until analysis. Plasma samples were prepared by protein precipitation. The concentration of the compound of formula I in plasma was determined by a validated liquid chromatography-tandem mass spectrometry method.
  • Pharmacokinetic parameters such as area under the plasma concentration-time curve (AUC), maximum plasma concentration ( Cmax ), time to Cmax ( Tmax ), half-life (T1 /2 ), systemic plasma clearance (CL ), mean residence time (MRT) and volume of distribution at steady state (V ss ) were calculated by non-compartmental analysis with WinNonlin v1.3 (Pharsight, Mountain View, CA, USA).
  • AUC area under the plasma concentration-time curve
  • Cmax maximum plasma concentration
  • Tmax time to Cmax
  • T1 /2 half-life
  • CL systemic plasma clearance
  • MRT mean residence time
  • V ss volume of distribution at steady state
  • Table 25 and Figure 2 show the pharmacokinetic data following intravenous and oral administration in cynomolgus monkeys.
  • the drug concentration decreased bi-exponentially, the clearance rate was 1.96 L/h/kg, and the volume of distribution in monkeys was 1.86 L/kg.
  • the compound of formula I reached Cmax in 1.0-1.30h, and the T1 /2 in plasma disappeared in 2.73-5.99h.
  • the oral bioavailability of the compound of formula I in monkeys was calculated to be 3.3-11.3% from AUC 0-24h after intravenous and oral administration, suggesting that efflux transporters are involved.

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Abstract

本发明提供了一种含氮的饱和杂环化合物的应用。具体地，本发明提供了由下式（I）所示的含氮的饱和杂环化合物或其药学上可接受的盐在制备治疗和/或预防慢性肾病的药物中的应用

Description

一种含氮的饱和杂环化合物的应用 技术领域
本发明涉及一种含氮的饱和杂环化合物或其药学上可接受的盐在制备用于治疗和/或预防慢性肾病的药物中的应用。
背景技术
慢性肾病(chronic kidney disease，CKD)具有患病率高、知晓率低、预后差和医疗费用高等特点，是继心脑血管疾病、糖尿病和恶性肿瘤之后，又一严重危害人类健康的疾病。近年来CKD患病率逐年上升，全球一般人群患病率已高达14.3％，我国横断面流行病学研究显示，18岁以上人群CKD患病率为10.8％。慢性肾病(CKD)是一种进展性疾病，如未能及时有效救治，导致病情恶化进展，则随病程进展，慢性肾病患者将发展为肾衰竭，如果不进行人工透析或肾脏移植，生存将变得困难。目前对于治疗慢性肾病尚无特别有效的方法，因此，积极寻找治疗慢性肾病有效并且毒副作用小的药物，具有较大的临床价值。
CN103562191B公开了一种结构式如下所示的含氮的饱和杂环化合物或其药学上可接受的盐，显示出了对肾素的抑制作用，可用于治疗高血压。
Figure PCTCN2021116338-appb-000001
式中，R 1表示环烷烃基等，R 22表示可以被取代的芳基等，R表示低级烷烃基等，T表示羰基，Z表示－O－等，R 3、R 4、R 5和R 6相同或不同，且表示氢原子等。
CN106928218A公开了新型的可药用的吗啉衍生物(含氮的饱和杂环)的盐，包括其苹果酸盐、酒石酸盐、盐酸盐、醋酸盐和萘二磷酸盐，其中酒石酸盐有A晶型、B晶型和二水合物3种晶态盐型，苹果酸盐、盐酸盐、醋 酸盐各有1种晶态盐型，萘二磷酸盐为无定形。与已知的吗啉衍生物的游离碱相比，吗啉衍生物的盐具有一种或多种的改进性能，如具有较好的晶态，极大程度地提高了水溶解度、光稳定性及热稳定性等。上述的吗啉衍生物盐或其晶型，可以用于治疗和/或预防高血压。
发明内容
本发明所要解决的技术问题是针对现有技术中尚无特别有效的治疗慢性肾病的药物，而提供了一种含氮的饱和杂环化合物或其药学上可接受的盐在制备用于治疗和/或预防慢性肾病的药物中的应用。
背景技术中所述专利CN103562191B和专利申请CN106928218A的全部内容在此通过引用并入本说明书中。
在本发明的一个实施方式中，提供了一种由下式I所示的含氮的饱和杂环化合物或其药学上可接受的盐在制备治疗和/或预防慢性肾病的药物中的应用：
Figure PCTCN2021116338-appb-000002
在本发明的进一步的实施方式中，所述的药学上可接受的盐为盐酸盐、硫酸盐、磷酸盐、氢溴酸盐、乙酸盐、富马酸盐、草酸盐、柠檬酸盐、甲磺酸盐、苯磺酸盐、对甲苯磺酸盐、马来酸盐、苹果酸盐、酒石酸盐、醋酸盐或萘二磺酸盐。
在本发明的又进一步的实施方式中，所述的药学上可接受的盐为苹果酸盐、酒石酸盐、盐酸盐、醋酸盐或萘二磺酸盐。
在本发明的一个实施方式中，所述的药学上可接受的盐为式I化合物的苹果酸盐，其是式I化合物和苹果酸以1:1摩尔比形成的化合物，其结构式如下：
Figure PCTCN2021116338-appb-000003
在本发明的一个实施方式中，所述的苹果酸盐为结晶体，其X射线粉末衍射图谱在2θ为7.767°±0.2°、13.897°±0.2°、14.775°±0.2°、17.098°±0.2°、18.999°±0.2°、20.153±0.2°、20.960°±0.2°、21.423°±0.2°、26.348°±0.2°、27.892°±0.2°处具有特征峰。特别地，所述的苹果酸盐结晶体的X射线粉末衍射图谱还在2θ为5.598°±0.2°、7.357°±0.2°、10.395°±0.2°、11.108°±0.2°、16.037°±0.2°、16.523°±0.2°、19.410°±0.2°、22.645°±0.2°、26.630°±0.2°、26.891°±0.2°、27.380°±0.2°、31.056°±0.2°、33.306°±0.2°、33.775°±0.2°、39.231°±0.2°处具有特征峰。更特别地，所述式I化合物的苹果酸盐结晶体具有如图1所示的X射线粉末衍射图谱。
在本发明的一个实施方式中，所述的慢性肾病包括高血压合并肾病、高血压合并肾病并伴随糖代谢异常、慢性肾功能不全合并慢性心力衰竭或慢性肾病伴随糖代谢异常。
在本发明的一个实施方式中，所述的慢性肾病是指慢性肾病的G1、G2、G3a、G3b、G4期，优选为G2、G3a或G3b期。
在本发明的一个实施方式中，所述的高血压合并肾病中的高血压为1级高血压、2级高血压或3级高血压，优选为1级高血压或2级高血压。
在本发明的一个实施方式中，所述的药物的剂型包括片剂、胶囊剂、静脉注射剂、吸入剂、雾化剂、冻干剂、贴剂、凝胶剂、喷雾剂、或栓剂，优选为片剂。
在本发明的一个实施方式中，所述的药物为单元剂量。
在本发明的一个实施方式中，所述的药物的单元剂量中，含有25mg-200mg的如上式I所示的含氮的饱和杂环化合物或其药学上可接受的盐，例如25mg、50mg、100mg、150mg、200mg的如上式I所示的含氮的饱和杂环化合物或其药学上可接受的盐。
美国肾脏基金会(NKF)所属“肾病预后质量倡议”(KDOQI)工作组于2002年制定了CKD定义和分期标准。
慢性肾病定义为肾脏结构或功能异常>3个月。慢性肾病诊断标准出现下表1中任何一项指标，持续时间超过3个月。
表1：慢性肾病诊断标准
Figure PCTCN2021116338-appb-000004
注：至少满足1项：AER：尿白蛋白排泄率；ACR：尿白蛋白肌酐比值；GFR：肾小球滤过率
慢性肾病分期：慢性肾病根据肾小球滤过率(GFR)分为5期，见下表2。
表2：慢性肾病根据GFR分期
Figure PCTCN2021116338-appb-000005
高血压定义：未使用降压药物的情况下，非同日3次测量诊室血压，收缩压(SBP)≥140mmHg(1mmHg＝0.133kPa)和/或舒张压(DBP)≥90mmHg。
SBP≥140mmHg和DBP<90mmHg为单纯收缩期高血压。患者既往有高血压史，目前正在使用降压药物，血压虽低于140/90mmHg，仍应诊断高血压。
根据血压升高水平，进一步将高血压分为1、2和3级。血压水平分类和定义见下表3。
表3：血压水平分类和定义
Figure PCTCN2021116338-appb-000006
心力衰竭(心衰)是一种临床综合征，定义为由于任何心脏结构或功能异常导致心室充盈或射血能力受损的一组复杂临床综合征。其主要临床表现为呼吸困难和乏力(活动耐量受限)，以及液体潴留(肺淤血和外周水肿)。
依据左心室射血分数(left ventricular ejection fraction，LVEF)，将心衰分为射血分数降低的心衰(heart failure with reduced ejection fraction，HFrEF)、射血分数保留的心衰(heart failure with preserved ejection fraction，HFpEF)和射血分数中间值的心衰(heart failure with mid-range ejection fraction，HFmrEF)，3种心衰类型的定义见下表4：
表4：心力衰竭的分类与定义
Figure PCTCN2021116338-appb-000007
Figure PCTCN2021116338-appb-000008
注：HFrEF射血分数降低的心力衰竭；HFmrEF射血分数中间值的心力衰竭；HFpEF射血分数保留的心力衰竭； aB型利钠肽(BNP)>35ng/L和/或N末端B型利钠肽原(NT-proBNP)>125ng/L；b E/e′≥13、e′平均值(室间隔和游离壁)<9cm/s；-无
糖代谢异常包括糖尿病前期与糖尿病。
糖尿病前期即是糖调节受损，是指血糖值已超过正常值范围，但还未达到糖尿病的诊断标准，即正常人与糖尿病患者之间的中间状态。
糖尿病是一组常见的代谢性疾病，其特征是为高血糖、糖尿、葡萄糖耐量减低及胰岛素释放试验异常。
本发明的积极进步效果在于：含氮的饱和杂环化合物盐连续给药8周对慢性肾功能不全合并慢性心力衰竭恒河猴(中年/老年)有效性和安全性研究表明，本发明提供的含氮的饱和杂环化合物或其药学上可接受的盐可有效预防或治疗慢性肾病，所述的慢性肾病包括高血压合并肾病、高血压合并肾病并伴随糖脂代谢异常、慢性肾功能不全合并慢性心力衰竭或慢性肾病伴随糖脂代谢异常。同时本发明提供的含氮的饱和杂环化合物或其药学上可接受的盐安全性很好，未见与给药相关的不良事件。
附图说明
图1是本发明实施例中所用的式I化合物苹果酸盐的XRPD图谱。
图2是实施例3中静脉内(iv)和口服(po)给药后猴子体内式I化合物的浓度-时间图。其中，猴子的静脉内给药剂量为1mg/kg，口服给药剂量为1、3和9mg/kg。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
下述实施例中所用的式I化合物苹果酸盐是根据专利申请公开CN106928218A中实施例1所述的制备方法制备所得并确认结构；缬沙坦(Valsartan)通过商业途径购买；所用的恒河猴购买自四川普莱美行之生物科技有限公司(普莱美灵长类研究中心)。
缩略词
Figure PCTCN2021116338-appb-000009
Figure PCTCN2021116338-appb-000010
实施例1：式I化合物苹果酸盐连续给药8周对慢性肾功能不全合并慢性心力衰竭恒河猴(中年/老年)有效性和安全性研究
1.研究目的
评估式I化合物苹果酸盐连续给药8周对肾功能的改善，以及能够延缓或改善肾脏功能起效剂量、作用强度以及CVD事件的风险，为临床试验剂量设计、患者入选标准和安全性提供依据。主要疗效终点(ENDPOINT)：1.对肾功能改善：肾小球滤过率eGFR creat-cys，胱抑素C(CysC)，肌酐及UACR；2.对心脏功能改善：超声心动图指标分析心脏结构和收缩、舒张功能给药前后变化；3.安全性指标：血压，血钾，糖脂代谢相关指标等。
2.试验系统
2.1自发性慢性肾功能不全恒河猴
动物种属：Macaca mulatta(Rhesus Macaque)
等级：普通级。试验前检疫合格，内容包括体检、2次结核杆菌试验、寄生虫、沙门氏菌、致贺氏菌和B病毒检查。
动物标识：脖环上戴刻有阿拉伯数字的不锈钢号牌，胸口纹身。
供应单位：雅安普莱美生物科技有限公司。
生产许可证号：SCXK(川)2019-027。
动物合格证号：0016710，0016944，0016966。
2.2入选标准
23只雄性/雌性动物，年龄14-24岁(等同于40-70岁成年人)，体重雄性8.45-14.93kg，雌性1只：6.43kg；
糖脂代谢长期异常3年以上；糖代谢异常为空腹血糖(FPG)＞4.8mmol/l，vs年龄匹配对照组4.1±0.3mmol/l；
CKD-EPI评级G3a-G3b，肾小球滤过率eGFR:30～59ml/min/1.73m 2；或蛋白尿中重度增多(A2-A3)：尿白蛋白/肌酐比值(UACR)≥15mg/g-350mg/g(4-6小时尿液收集)，vs年龄匹配对照组3±2mg/g。
正常血压、1级或2级高血压(与临床患者标准一致)；
HFrEF：LVEF 30％-49％(正常值：50％-70％辛普森双平面法)或HFpEF(中度以上受损)：Ea<8或E/Ea>10(依据临床诊断标准)。
2.3排除标准
1)慢性肝病(包括已知活动性肝炎)和/或筛查丙氨酸转氨酶(ALT)或天冬氨酸转氨酶(AST)>3x正常上限(ULN)。
2)与血脂异常有关的排除标准：TC>7mmol/l；TG>3.5mmol/l。
3)任何可能会影响药效评价的其他疾病史。
下表5显示基线期-各剂量组受试慢性DKD肾功能不全恒河猴基础病因和特征。
表5：基线期-各剂量组受试慢性DKD肾功能不全恒河猴基础病因和特征
Figure PCTCN2021116338-appb-000011
Figure PCTCN2021116338-appb-000012
注：#257动物胸廓畸形无法采集心脏超声图像。
表5：基线期-各剂量组受试慢性DKD肾功能不全恒河猴基础病因和特征(续)
Figure PCTCN2021116338-appb-000013
Figure PCTCN2021116338-appb-000014
3.试验设计
3.1分组及剂量设计
本次试验分三个阶段纳入动物，第一阶段共两个组：式I化合物苹果酸盐5mg/kg组3只动物(#257，#4721，#287)，安慰剂组2只动物(#1029,#4713)，第二阶段式I化合物苹果酸盐5mg/kg组3只动物(#144，#5073，#6651)，Valsartan组3只动物(#2091，#4861，#6645)，安慰剂组2只动物(#6653，#231)，第三阶段式I化合物苹果酸盐2mg/kg组5只动物(#4829，#6501，#287，#4721，#2091)，式I化合物苹果酸盐1mg/kg组5只动物(#1567，#4861，#5453，#6653，#5073)，
其中第一阶段2例动物(#287和#4721)停药洗脱约9个月，第二阶段4例动物(#5073，#2091，#4861和#6653)停药洗脱约5个月后进入第三阶段基线期(基线期为1个月)。
详见下表6。
表6：组别设计信息
Figure PCTCN2021116338-appb-000015
备注：Valsartan组给药期第1～2周每天1次，第3～8周每天2次。
3.2给药信息
给药途径：口服给药。
给药频率：式I化合物苹果酸盐每天1次，Valsartan组给药期第1～2周每天1次，第3～8周每天2次。
给药量计算：以每次称量的体重计算接下来一周的给药量。
给药时间：08:00-09:00给药。
3.3主要药效指标
肾小球滤过率eGFR creat-cys：检测肌酐(Cr-P)，尿素氮(BUN)，胱抑素(CysC)，计算获eGFR值。给药前1次，给药后每两周检测1次。
eGFR male＝135×min(Cr/0.9,1) -0.207×max(Cr/0.9,1) -0.601×min(CysC/0.8,1) -0.375×max(CysC/0.8,1) -0.711×0.995 Age×3
eGFR female＝135×min(Cr/0.7,1) -0.284×max(Cr/0.7,1) -0.601×min(CysC/0.8,1) -0.375×max(CysC/0.8,1) -0.711×0.995 Age×3×0.969
UACR：4h/6h尿液收集，检测尿微量白蛋白(malb)和尿肌酐(Cr-U)，计算获得UACR值。给药前1次，给药结束1次。UACR＝MALB/Cr-U。
血压：麻醉后检测血压，包括收缩压(SBP)，舒张压(DBP)，平均血压(MBP)和心率(HR)。给药前1次，给药后4周和8周各1次。
心脏GE多普勒超声：1年内历史心超一次(入选动物)、给药前一次(基线期D-30天内)，给药后8周一次。
3.4次要药效指标
血钾，给药前2次，给药期每2周一次；
糖脂代谢及肝功：FPG，FRA，LDL-c，HDL-c，TG，TC，NT-proBNP，ALT，AST，TBIL等，给药前2次，给药期4周和8周各1次；
其他生化指标和血液学，给药前后各1次；
体重：给药前一次，给药期每周一次；
给药后每天观察24小时摄食、行为等变化。
3.5检测方法和设备
检测方法：见下表7和下表8。
血液学检测仪器：Siemens ADVIA 2120i Hematolagy Systems。
血生化和尿液指标检测仪器：Roche cobas6000 analyzer series C501模块检测，NT-proBNP采用ELISA试剂盒检测。
表7：生化检测项目
Figure PCTCN2021116338-appb-000016
表8：血液学检测项目
Figure PCTCN2021116338-appb-000017
3.6心脏多普勒超声
麻醉方法：采用15mg/kg盐酸氯胺酮肌肉注射麻醉，根据动物麻醉状态，兽医判定后进行补充麻醉，每次补充麻醉的剂量为初始剂量的1/2。
检测方法：动物取左侧卧位，采用6S-RS探头(频率2.7-8.0MHz)进行图像采集。
分析方法：图像采集保存后使用配套分析工作站EchoPAC Software在连续的3个心动周期内重复检测心脏收缩及舒张功能各项指标。
检测指标及超声技术：见下表9。
检测仪器：GE Vivid S5彩色多普勒超声诊断仪。
3.7血压检测
检测方法：肌肉注射15mg/kg盐酸氯胺酮对动物进行麻醉，麻醉后呈仰卧位，将左上臂毛发剃除干净，并按标准绑上合适大小的袖带，将血氧探头夹在动物手指或脚趾(左手除外)上，红色感光面在指肚一侧，使用自动模式连续检测3次动物血压，每次间隔1min。若三次以上血压的DBP、SBP和MBP差异不大(最高值与最低值差异小于15mmHg)，测定结束。
检测指标：检测指标包括收缩压(SBP)，舒张压(DBP)，平均血压(MBP)和心率(HR)。
检测仪器：GE B40i电生理监护仪。
表9：心脏超声检测指标
Figure PCTCN2021116338-appb-000018
Figure PCTCN2021116338-appb-000019
3.8临床观察
观察次数：每天观察1次。
观察方法：隔笼观察。
观察内容：注射部位、皮肤、被毛、眼、耳、鼻、口腔、胸部、腹部、泌尿生殖部、四肢等部位，以及呼吸、运动、泌尿、排便和行为改变等。
3.9摄食量测定
给料方法：上午8:00～9:00、10:00、下午2:00和下午4:00各给食一次，保证食盒中有食物，自由采食，第二天上午07:40-08:00撤去剩余饲料。
给料量：约250-500g/只/天，试验中保证食盒中24h有饲料，动物自由摄食。
摄食量测定方法：饲料采用半定量方法估算，每天记录给食量、丢弃量和食盒剩余量。摄食量＝给食量-丢弃量-食盒剩余量。
3.10体重测定
称重时间：当天喂食前。
测定方法：称重前动物禁食14-16h，动物在清醒状态下，进入转移笼后，用大动物称称重。
测定仪器：METTLER TOLEDO电子台秤。
4.0终止试验标准
a.发生严重不良事件。
b.发生严重感染。
5.结果与分析
5.1对UACR的影响
各组给药对蛋白尿(UACR)的影响见下表10。各组选择蛋白尿中重度增多，表现为基线尿白蛋白/肌酐比值(UACR)≥15mg/g-350mg/g(4-6小时尿液收集)的CKD恒河猴分析基线和给药8周(W8)的白蛋白/肌酐比值变化。
安慰剂组(n＝4)：与基线比，试验期结束W8的UACR平均升高30.64±34.50％；4例动物蛋白尿UACR在一定范围内稳定波动，未表现快速进展。
Valsartan组(n＝3，中重度蛋白尿增多纳入统计)：与基线比，给药W8的2例动物(#2091，#4861)UACR下降40％-70％，均呈现获益；1例动物基线的UACR为15mg/g，给药后W8的UACR为20.4％，获益不明显。由于入组动物数少，与安慰组比无统计学差异。
式I化合物苹果酸盐(5mg/kg每天一次(qd))组(n＝6，4/6只中重度蛋白尿增多纳入统计)：与基线比，给药后W8的UACR平均下降46.87±36.93％，与安慰剂组比呈显著降低(p＜0.05)。其中3例动物给药W8与基线比UACR下降60％-70％，均呈现获益；1例动物未见获益。
式I化合物苹果酸盐(2mg/kg qd)组(n＝5，3/5只中重度蛋白尿增多纳入统计)：与基线比，给药后W8的UACR平均下降66.00±17.42％，与安慰剂组比呈显著降低(p＜0.01)。其中3例动物给药W8与基线比UACR下降约50％-80％，均呈现获益。
式I化合物苹果酸盐(1mg/kg qd)组(n＝5，2/5只中重度蛋白尿增多纳入统计)：与基线比，1例动物给药后W8的UACR降低39％，1例获益不明显。
总之，式I化合物苹果酸盐的2-5mg/kg qd，给药8周能显著改善蛋白尿(UACR)。
表10：式I化合物苹果酸盐给药8周对自发性慢性肾病(CKD)恒河猴UACR的影响
Figure PCTCN2021116338-appb-000020
Figure PCTCN2021116338-appb-000021
注：1.UACR＝MALB/Cr-U
2.变化率＝(给药后时间点-基线值)/基线值*100％，负值为降低；“#”与安慰剂组比p＜0.05。
3.NA，UACR正常动物未纳入统计。
表10：式I化合物苹果酸盐给药8周对自发性慢性肾病(CKD)恒河猴UACR的影响(续)
Figure PCTCN2021116338-appb-000022
Figure PCTCN2021116338-appb-000023
注：1.UACR＝MALB/Cr-U
2.变化率＝(给药后时间点-基线值)/基线值*100％，负值为降低；“#”与安慰剂组比p＜0.05，“##”与安慰剂组比p＜0.01。
3.NA，UACR正常动物未纳入统计。
5.2对肾小球滤过率eGFR creat-cys影响
各组对eGFR的影响见下表11和下表12。各组入选eGFR creat-cys：30～59ml/min/1.73m 2，分析对基线和给药后W8的肾小球滤过率变化。
安慰剂组(n＝4)：与基线比，4例动物肾小球滤过率eGFR creat-cys在一定范围内稳定波动未表现快速进展。
Valsartan组(n＝3)：与基线比，给药后W8Valsartan组肾小球滤过率eGFR creat-cys平均增加6±2ml/min/1.73m 2，与安慰组变化值比呈极显著增加(p＜0.01)，显著改善了肾小球恶化，且疗效呈现时效性。
式I化合物苹果酸盐(5mg/kg qd)组(n＝6)：与基线比，给药后W8肾小球滤过率eGFR creat-cys平均增加6±6ml/min/1.73m 2，与安慰组变化值比呈显著增加(p＜0.05)，疗效呈现时效性。
式I化合物苹果酸盐(2mg/kg qd)组(n＝5)：与基线比，给药后W8肾小球滤过率eGFR creat-cys平均增加3±3ml/min/1.73m 2，与安慰组变化值比呈显著增加(p＜0.05)。
式I化合物苹果酸盐(1mg/kg qd)组(n＝5)：与基线比，给药后W8肾小球滤过率eGFR creat-cys平均增加3±4ml/min/1.73m 2，与安慰组变化值比无统计学意义上改变。
总之，式I化合物苹果酸盐的2-5mg/kg qd，给药8周能显著改善肾小球滤过率eGFR creat-cys。
表11：式I化合物苹果酸盐给药8周对自发性慢性肾病恒河猴eGFRcreat-cys的影响
Figure PCTCN2021116338-appb-000024
Figure PCTCN2021116338-appb-000025
Figure PCTCN2021116338-appb-000026
注：变化值＝8周-Baseline，负值为降低。“#”变化值安慰剂组比p＜0.05，“##”变化值与安慰剂组比p＜0.01
表12：式I化合物苹果酸盐给药8周自发性慢性肾病恒河猴eGFRcreat-cys变化值
Figure PCTCN2021116338-appb-000027
Figure PCTCN2021116338-appb-000028
注：变化值＝当前值-Baseline，负值为降低。
5.3对CysC，Cr-P和BUN影响
各组给药后对CysC，Cr-P和BUN的影响见下表13至表15。
表13：式I化合物苹果酸盐给药8周对自发性慢性肾病恒河猴Cr-P的影响
Figure PCTCN2021116338-appb-000029
Figure PCTCN2021116338-appb-000030
注：“*”与基线值Baseline比p＜0.05，“**”与基线值Baseline比p＜0.01。
表14：式I化合物苹果酸盐给药8周对自发性慢性肾病恒河猴CysC的影响
Figure PCTCN2021116338-appb-000031
Figure PCTCN2021116338-appb-000032
Figure PCTCN2021116338-appb-000033
注：“**”与Baseline比p＜0.01。
表15：式I化合物苹果酸盐给药8周对自发性慢性肾病恒河猴BUN的影响
Figure PCTCN2021116338-appb-000034
Figure PCTCN2021116338-appb-000035
5.4对血压的影响
各给药组给药对血压的影响见表16。灵长类的血压测试由于需要麻醉，因此需要控制心率在各个时间点的变异性才能统计。
安慰剂组(n＝4，2/4高血压)：与基线比，4例动物血压在一定范围内稳定波动。
Valsartan组(n＝3，1/3只高血压)：与基线比，给药后W8，SBP和DBP分别平均降低14±4mmHg和7±0mmHg，与安慰组变化值比呈极显著降低(p＜0.01)。
式I化合物苹果酸盐(5mg/kg qd)组(n＝6，4/6例高血压)：与基线比，给药后W8，SBP和DBP分别平均降低21±8mmHg和10±7mmHg，与安慰组变化值比呈极显著降低(p＜0.01)，疗效呈现时效性。
式I化合物苹果酸盐(2mg/kg qd)组(n＝5，2/5例高血压)：与基线比，给药后W8，SBP和DBP分别平均降低19±9mmHg和9±8mmHg，与安慰组变化值比呈极显著降低(p＜0.01)，疗效呈现时效性。
式I化合物苹果酸盐(1mg/kg qd)组(n＝5)：与基线比，给药后W8，SBP和DBP分别平均降低11±4mmHg和5±4mmHg，与安慰组变化值比呈显著降低(p＜0.05)。
总之，式I化合物苹果酸盐的1-5mg/kg qd，给药8周均呈现降低血压的活性。
表16：式I化合物苹果酸盐给药8周对自发性慢性肾病恒河猴血压的影响
Figure PCTCN2021116338-appb-000036
Figure PCTCN2021116338-appb-000037
注：“*”与基线值Baseline比p＜0.05，“**”与基线值Baseline比p＜0.01。“#”变化值与安慰剂组比p＜0.05，“##”变化值与安慰剂组比p＜0.01。NA，心率不稳定不纳入统计分析。
5.5对心功能的影响
各组对超声心动图指标的影响见表17。
式I化合物苹果酸盐各给药剂量组，Valsartan组和安慰剂组给药后心脏收缩和舒张功能未见明显不良反应。
另外，式I化合物苹果酸盐5mg/kg组2例表现为射血分数低下心功能不全(SD)恒河猴动物(#5073和#287)与基线比给药后8周LVEF％升高。但本品是否能有治疗慢性心衰作用需要后续更多的研究队列数据支持。
表17：式I化合物苹果酸盐给药8周对自发性慢性肾病恒河猴心脏功能主要指标的影响及疗效评价
Figure PCTCN2021116338-appb-000038
Figure PCTCN2021116338-appb-000039
备注：舒张功能异常的级别是在综合考虑了Ea、E/Ea、Ea/Aa以及其它功能指标后而定；当受试动物的舒张功能异常级别较基线有改善时视为“获益”，否则视为“无效”；#257动物胸廓畸形无法采集心脏超声图像。
5.6对心功能损伤血液Biomarker的影响
5.6.1对NT-proBNP的影响
式I化合物苹果酸盐对NT-proBNP的影响见表18。
与基线期比，给药8周后各给药组动物NT-proBNP水平未见明显改变。
表18：式I化合物苹果酸盐给药8周对自发性慢性肾病恒河猴NT-proBNP的影响
Figure PCTCN2021116338-appb-000040
Figure PCTCN2021116338-appb-000041
5.6.2对K +的影响
式I化合物苹果酸盐对K +的影响见表19。
与基线期比，给药8周后各给药组动物血清K +水平未见明显改变。
表19：式I化合物苹果酸盐给药8周对自发性慢性肾病恒河猴K +的影响
Figure PCTCN2021116338-appb-000042
Figure PCTCN2021116338-appb-000043
Figure PCTCN2021116338-appb-000044
5.7安全性耐受研究
给药期间式I化合物苹果酸盐组未见与给药相关的不良事件，肝功、肾功、摄食量、体重、血液学指标等未见明显改变。
6.结论
式I化合物苹果酸盐2-5mg/kg qd，给药8周能显著改善蛋白尿(UACR)，显著改善肾小球滤过率eGFR creat-cys，疗效与Valsartan组相当。
式I化合物苹果酸盐1-5mg/kg qd，给药8周均呈现降低血压的活性。同时在本试验条件下未见高血钾症的风险。
实施例2：式I化合物的临床前安全性评估
1.材料和方法
1.1.测试物质
式I化合物按照CN103562191B中的方法制备并确认结构。溶媒是0.5％的羧甲基纤维素钠(Sodium carboxymethyl cellulose，CMC-Na)。式I化合物在室温下保存，避免光照和潮湿。食蟹猴的安全性研究中，将测试物质以相关浓度悬浮于0.5％CMC-Na中。分析制备的悬浮液的均质性、浓度和一周稳定性，以确保正确施用报告的剂量。对活性药物成分(API)的六个月加速稳定性进行了测试。
1.2.动物饲养
3-4岁的健康雄性和雌性食蟹猴由海南新源生物技术有限公司(中国海南)提供。在治疗开始时，雄性体重范围为3.2–5.8kg，雌性体重范围为2.5–3.7kg。在研究之前，他们被安置了36天驯化期。每天早上一次，动物自由饮食和取水。
这项非临床实验研究按照中国食品药品监督管理局(CFDA)的指南进行。该实验是在国际实验室动物护理评估与鉴定协会(AAA LAC)批准的设施中进行的，并且根据《实验室动物护理和使用指南》(Nussberger等，2008)对动物进行饲养。
1.3.剂量和治疗时间表
使用基于体重的计算机随机程序选择食蟹猴(5/性别/组)，并通过鼻饲喂养式I化合物(剂量水平为0(对照组)、20、100、450mg/kg/天，体积为5ml/kg)。每周根据动物的体重调整个体剂量。对于所有组，在第28天随机选择2/3的动物并实施安乐死。在停药28天后对其余动物实施安乐死。
1.4.临床观察
从隔离期开始每天评估死亡率和临床体征。每天至少两次检查每只动物的行为、给药后的反应或疾病的发生。
观察包括但不限于以下内容：皮肤和毛皮的变化；眼睛和粘膜；呼吸，循环，自主，中枢神经系统和行为模式。分别在给药前(两次)，第一次给药后1和24小时，以及在第26天给药前和给药后1和24小时，测量直肠温度。每周测量体重。每天估算猴子的食物消耗量。使用便携式裂隙灯(YZ2)和直接检眼镜(GFJY-01B)对猴子进行检眼镜检查。
1.5.实验室测试
对于猴子研究，在隔离期(两次，第01天和第02天)，第14天，第28天和第56天，测量了以下参数，如下表20所示。(1)使用Bayer ADVIA2120(德国)和Sysmex CA-1500(日本)进行血液和凝血指数检查。(2)使用HITACHI 7080自动分析仪(日本)和Easylyte PLUS电解质分析仪(美国MEDICA)评估血清生化检查。(3)通过每个笼子下面的托盘收集每只动物的24小时合并尿液。使用Uritest-300(中国)进行尿液分析。(4)通过每个笼子下面的托盘收集每只猴子的24小时汇集的粪便。检测粪便潜血。(5)ACCESS 2化学发光免疫分析法用于心肌肌钙蛋白(TropI，非GLP)测试。
表20：在血液学，血清化学和尿液分析中评估的参数
Figure PCTCN2021116338-appb-000045
Figure PCTCN2021116338-appb-000046
1.6.常规心电图分析
在每个计划的时间(给药后第1天，给药后第01天，第02天，给药后第1天的第1和24小时，给药前，以及给药后第26天、第56天的第1小时，第24小时)进行常规ECG分析。ECG系统使用派生DII(ECG-6951E，中国上海)记录P波，R波，T波，PR间隔，Q-T间隔，QRS持续时间和心率。
1.7.尸检和组织病理学
在暴露阶段(第28天)和恢复阶段(第56天)结束时，通过目视检查对所有动物进行完整的尸检。修剪，称重并评估以下选定器官的绝对重量以及最终体重或脑重的百分比：大脑，心脏，肾脏，肾脏，肝脏，脾脏，胸腺，睾丸，附睾，子宫，卵巢，肾上腺和甲状腺(包括甲状旁腺)。将以下组织保 存在10％的中性福尔马林中：脑，垂体，甲状腺(包括甲状旁腺)，气管，心脏，胰腺，脾，肾上腺，前列腺，卵巢，子宫(包括子宫颈和输卵管)，阴道，睾丸，附睾，精囊，食道，十二指肠，空肠，回肠，盲肠，结肠，直肠，肠系膜淋巴结，淋巴集结，上颌下淋巴结，主动脉，眼睛，骨骼肌，坐骨神经，股骨(包括干骺端)，乳腺，胸骨，唾液腺，脊髓，膀胱，肺(包括支气管)，肝，肾，胃，骨髓(胸骨)，胸腺，胸骨，胆囊，任何明显的病变或肿块。尸检时肺组织被固定剂感染。所有保存的组织均进行包埋，切片，苏木精和曙红(HE)染色，并进行显微镜检查。涂片(猴胸骨)和成对包埋的胸骨切片均进行了骨髓细胞形态学检查。
1.8.毒代动力学
在给药后第1天和第26天的0小时、0.5小时、1.5小时、3小时、5小时、7小时和24小时采集血样。每次从静脉中抽取约1.0mL血液，并将其收集到EDTA-K 2抗凝管中。将血样放在冰上，然后在4℃下离心(3500rpm，5分钟)获得血浆样品，将其冷冻在约-80℃下直至分析。式I化合物的毒代动力学测定是在中国科学院上海药物研究所进行的。
1.9.统计分析
对于每种性别，使用统计软件SAS 9.3对体重、直肠温度、ECG、器官重量、血液学参数和血清生化数据进行分析。首先，使用Levene检验分析方差的均匀性。如果P>0.05，则采用单向方差分析。如果P<0.05，则使用Kruskal–Wallis检验。如果所得的ANOVA p值<0.05，则使用Dunnett T检验对每组进行比较。如果所得的Kruskal-Wallis检验p值<0.05，则在数据秩转换后执行Dunnett T检验。
2.结果
2.1.临床观察
与对照组相比，高剂量组的体重增加量略有下降(雌性为2.7±0.4对3.2±0.2；雄性为4.1±0.5对4.6±0.5)，无统计学意义。
与给药有关的早期死亡发生在第23天，一只高剂量雌性发生，临床体征包括活动不足，驼背，体温低下，腹泻，轻度肠鼓膜和胸腺萎缩。在其他动物中没有观察到与治疗相关的明显临床毒性迹象。
2.2.血液学与凝血
在所有接受治疗的雌性和大剂量雄性中均观察到NEUT％升高和LYMPH％降低。还观察到中高剂量动物的Fbg升高，高剂量雌性动物的APTT升高(表21)。
表21：式I化合物处理4周的猴子的血液学数据。
Figure PCTCN2021116338-appb-000047
Figure PCTCN2021116338-appb-000048
*p<0.05，与对照组相比，**p<0.01，与对照组相比，值为平均值±S.D。
2.3.血清生化
雄性雌性高剂量食蟹猴的血清化学参数CPK，BUN和CREA均显著增加，尤其是在第23天早期死亡的猴子中(第14天数据，BUN：34.99；CREA： 526)。高剂量雄性的CHOL明显降低。高剂量雄性的血清Na水平略有下降，但在第26天时有统计学意义。其他统计学上有意义的发现是单独发生的事件，被认为没有毒理学意义(表22)。
表22：式I化合物处理4周的猴子的血清化学成分
Figure PCTCN2021116338-appb-000049
Figure PCTCN2021116338-appb-000050
*p<0.05，与对照组相比，**p<0.01，与对照组相比，值为平均值±S.D。
2.4.尿液分析
在雄性或雌性猴子中检查的任何尿液分析参数均未报告有明显变化。
2.5.ECG参数
一只中剂量雌性在给药后第26天的1小时观察到QTc间隔延长(动物#303为296ms)。一只高剂量雄性在给药后第26天的1小时观察到窦性心律不齐，QRS间隔延长(动物#411为60ms)高剂量雌性在服药后第1天和第26天的1小时和也观察到QRS间隔延长(表23)。
表23：式I化合物处理4周的猴子的心电图数据
Figure PCTCN2021116338-appb-000051
Figure PCTCN2021116338-appb-000052
*p<0.05，与对照组相比，**p<0.01，与对照组相比，值为平均值±S.D。
2.6.尸检
2.6.1.总体病理和器官重量
没有猴子的宏观发现被认为与服用测试物质有关，但对于较早死亡的猴子而言，轻度的肠鼓膜和胸腺萎缩除外。高剂量猴子的肝脏和肾脏相对重量较高，胸腺相对重量较低，子宫的绝对或相对重量较低(表24)。
表24：式I化合物处理4周的食蟹猴的器官重量。
Figure PCTCN2021116338-appb-000053
Figure PCTCN2021116338-appb-000054
Figure PCTCN2021116338-appb-000055
Figure PCTCN2021116338-appb-000056
*p<0.05，与对照组相比，**p<0.01，与对照组相比，值为平均值±S.D。
2.7.毒代动力学
在20-450mg/kg剂量范围内全身暴露量(由化合物原型和主要代谢物的血浆AUC 0–24h和C max表示)的增加比例比剂量的增加比例大得多。连续28天给药后，未见积累。
实施例3：非临床药代动力学
1.材料和方法
1.1.材料
式I化合物按照CN103562191B中的方法制备并确认结构。
1.2.动物
食蟹猴(3/性别/组：体重3-5公斤，年龄5-6岁)来自苏州西山中科实验动物(中国上海)。研究前将动物驯化7天。将猴子单独关进笼子，放在可控温度(18–26℃)和湿度(50±20％)的动物房中，空气流量为10，每小时换新鲜空气。光照周期为12小时开和12小时关，每天早晨给猴子喂一次饲料和水。所有动物实验均按照AMMS动物护理和使用委员会制定的《动物护理和使用指南》进行。
2.式I化合物的静脉和口服给药
将式I化合物静脉或口服给予禁食过夜的食蟹猴。剂量分别为1mg/kg(静脉)，1mg/kg，3mg/kg，9mg/kg(口服)。在给药后0，5和15分钟时，分别在0.25、0.5、1、2、4、6、8、12和24小时从静脉采集血液样本(每个时间点0.2mL)。采集后，将所有血样立即以10,000×g离心5分钟，并将血浆保存在-75±10℃直至分析。通过蛋白质沉淀制备血浆样品。血浆中将式I化合物的浓度通过验证的液相色谱-串联质谱法测定。
药代动力学参数，例如血浆浓度-时间曲线下面积(AUC)，最大血浆浓度(C max)，达到C max的时间(T max)，半衰期(T 1/2)，全身血浆清除率(CL)，平均停留时间(MRT)和稳态时的分布体积(V ss)是通过WinNonlin v1.3(美国加利福尼亚州山景市的Pharsight)通过非房室分析计算得出的。对于异速 生长模型，动物的药代动力学数据是通过两室模型和Kinetica 5.1软件包(v.3.0；InnaPhase，费城，宾夕法尼亚州，美国)来模拟的。
3.结果
血浆药代动力学和生物利用度
表25和图2显示了在食蟹猴静脉和口服给药后的药代动力学数据。静脉注射后，药物浓度双指数下降，清除率为1.96L/h/kg，猴子的分布体积为1.86L/kg。口服后，式I化合物在1.0–1.30h达到C max，在血浆中的T 1/2消失为2.73–5.99h。从静脉内和口服给药后的AUC 0-24h计算得出，式I化合物在猴子中的口服生物利用度为3.3-11.3％，这表明涉及外排转运蛋白。
表25：
Figure PCTCN2021116338-appb-000057
iv后的CL清除率，iv后稳态下的V ss分布体积，MRT平均停留时间，T 1/2半衰期，C max最大血浆浓度，达到C max的T max时间，AUC 总血浆浓度-时间曲线下的总面积，CL/F口服后清除率，V ss/F口服后在稳态下的分布体积，F口服绝对生物利用度，iv静脉内静脉注射，po口服。

Claims (10)

  1. 由下式I所示的含氮的饱和杂环化合物或其药学上可接受的盐在制备治疗和/或预防慢性肾病的药物中的应用：
    Figure PCTCN2021116338-appb-100001
  2. 根据权利要求1所述的应用，其中，所述的药学上可接受的盐为盐酸盐、硫酸盐、磷酸盐、氢溴酸盐、乙酸盐、富马酸盐、草酸盐、柠檬酸盐、甲磺酸盐、苯磺酸盐、对甲苯磺酸盐、马来酸盐、苹果酸盐、酒石酸盐、醋酸盐或萘二磺酸盐。
  3. 根据权利要求1或2所述的应用，其中，所述的药学上可接受的盐为式I化合物的苹果酸盐，其是式I化合物和苹果酸以1:1摩尔比形成的化合物，其结构式如下：
    Figure PCTCN2021116338-appb-100002
  4. 根据权利要求3所述的应用，其中，所述的苹果酸盐为结晶体，其X射线粉末衍射图谱在2θ为7.767°±0.2°、13.897°±0.2°、14.775°±0.2°、17.098°±0.2°、18.999°±0.2°、20.153±0.2°、20.960°±0.2°、21.423°±0.2°、26.348°±0.2°、27.892°±0.2°处具有特征峰，
    特别地，所述的苹果酸盐结晶体的X射线粉末衍射图谱还在2θ为5.598°±0.2°、7.357°±0.2°、10.395°±0.2°、11.108°±0.2°、16.037°±0.2°、16.523°±0.2°、19.410°±0.2°、22.645°±0.2°、26.630°±0.2°、26.891°±0.2°、27.380°±0.2°、31.056°±0.2°、33.306°±0.2°、33.775°±0.2°、39.231°±0.2°处具有特征峰。
  5. 根据权利要求4所述的应用，其中，所述的式I化合物的苹果酸盐结晶体具有如图1所示的X射线粉末衍射图谱。
  6. 根据权利要求1-5中任一项所述的应用，其中，所述的慢性肾病为高血压合并肾病、高血压合并肾病并伴随糖代谢异常、慢性肾功能不全合并慢性心力衰竭或慢性肾病伴随糖代谢异常。
  7. 根据权利要求6所述的应用，其中，所述的高血压合并肾病中的高血压为1级高血压、2级高血压或3级高血压，优选为1级高血压或2级高血压。
  8. 根据权利要求1-7中任一项所述的应用，其中，所述的慢性肾病是指慢性肾病的G1、G2、G3a、G3b、G4期，优选为G2、G3a或G3b期。
  9. 根据权利要求1-8中任一项所述的应用，其中，所述药物的剂型包括片剂、胶囊剂、静脉注射剂、吸入剂、雾化剂、冻干剂、贴剂、凝胶剂、喷雾剂、或栓剂，优选为片剂。
  10. 根据权利要求1-9中任一项所述的应用，其中，所述药物为单元剂量，优选地，所述单元剂量中，含有25mg-200mg的由式I所示的含氮的饱和杂环化合物或其药学上可接受的盐，例如25mg、50mg、100mg、150mg、200mg的由式I所示的含氮的饱和杂环化合物或其药学上可接受的盐。
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CN103562191A (zh) 2011-03-16 2014-02-05 上海医药集团股份有限公司 含氮的饱和杂环化合物
JP2014074024A (ja) * 2012-09-14 2014-04-24 Mitsubishi Tanabe Pharma Corp 医薬組成物
CN106928218A (zh) 2015-12-29 2017-07-07 上海医药集团股份有限公司 吗啉衍生物的盐及其晶型、其制备方法及药物组合物、用途

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CN103562191A (zh) 2011-03-16 2014-02-05 上海医药集团股份有限公司 含氮的饱和杂环化合物
JP2014074024A (ja) * 2012-09-14 2014-04-24 Mitsubishi Tanabe Pharma Corp 医薬組成物
CN106928218A (zh) 2015-12-29 2017-07-07 上海医药集团股份有限公司 吗啉衍生物的盐及其晶型、其制备方法及药物组合物、用途

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