Enzastaurin crystalline forms and uses

Abstract

The present application relates to the crystal form of endoximide and its use. The present application first discloses the use of endoximide for preparing a drug for treating Cushing's disease. And first discloses the crystal form II of endoximide and its preparation method. Compared with the existing crystal form N2 and crystal form N21, the crystal form II has more significant therapeutic effect on Cushing's disease.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and more particularly, relates to a crystalline form of endoximstat and a preparation method and use thereof. BACKGROUND

[0002] Cushing syndrome is a group of syndromes caused by long-term excessive intake of glucocorticoids by the body, and can be divided into adrenocorticotropic hormone (ACTH)-dependent and non-dependent according to the cause. ACTH non-dependent is mainly caused by adrenal adenoma, adrenal adenocarcinoma, adrenal nodular hyperplasia and a small number of primary pigmented nodular adrenal cortical disease; ACTH-dependent accounts for 70-80% of Cushing syndrome, of which 60-70% is caused by pituitary adenoma, also known as Cushing's disease (CD). Excessive secretion of ACTH by tumors causes hypercortisolism, which in turn causes a series of multi-system disorder syndromes such as central obesity, hypertension, impaired glucose tolerance, lipid disorder, coagulation disorder, osteoporosis, mental abnormalities, etc., and this series of complications makes the mortality of patients 4 times higher than that of the general population.

[0003] Epidemiological surveys show that the incidence of Cushing's disease in the population is 2-3 per million per year. Cushing's disease is insidious in onset, rapid in development and difficult to control, which brings great challenges to clinical diagnosis and treatment, and also brings a heavy burden to the patient's family and even the society.

[0004] Untreated Cushing's disease has poor prognosis, and the morbidity and mortality are doubled due to severe cardiovascular complications and metabolic abnormalities, and the 5-year survival rate is only about 50%. In addition to hypertension and diabetes, sustained hypercortisolism can also cause hyperlipidemia, metabolic syndrome, coagulation dysfunction, osteoporosis, hypokalemia, depression, anxiety and cognitive impairment, etc., resulting in low quality of life for patients.

[0005] Timely and correct diagnosis and treatment can reduce the mortality of patients, improve the quality of life of patients, and avoid the occurrence of cardiovascular and cerebrovascular events, which is of great significance to patients with Cushing's syndrome.

[0006] In terms of treatment, there are three methods for treating Cushing's disease: surgery, radiotherapy and drug therapy.

[0007] Transsphenoidal surgery is considered the first choice of treatment, and the goal of transsphenoidal surgery is to completely remove the pituitary lesion, and to achieve normal cortisol levels without causing hypopituitarism. Due to the development of surgical microscopes and the wide application of neuro-navigation technology, 95% of pituitary adenomas can be removed by transsphenoidal surgery, which has the advantages of small trauma, fewer complications, rapid recovery, and low cost. Generally, transsphenoidal surgery for Cushing's disease can achieve a high remission rate. The common complications of transsphenoidal surgery include new-onset hypopituitarism (about 10% of patients), and permanent diabetes insipidus, cerebrospinal fluid leakage, and venous thromboembolism (VTE). For patients with poor postoperative efficacy, retranssphenoidal surgery or radiotherapy and drug therapy can be considered.

[0008] The drugs for treatment include two types:

[0009] A, drugs acting on the adrenal gland, which play a role by inhibiting cortisol synthesis, such as ketoconazole, metyrapone, and etomidate;

[0010] B, drugs mainly acting on the pituitary, which play a role by inhibiting secretion, and the representative drugs are pasireotide, cabergoline, cyproheptadine, and rosiglitazone. Recently, some scholars have pointed out that tyrosine kinase inhibitors (gefitinib) can be applied to the treatment of Cushing's disease.

[0011] After effective treatment, the disability rate and mortality rate of Cushing's disease patients can be significantly reduced, and then the survival rate level is basically the same as that of normal people.

[0012] However, the current treatment cannot effectively control Cushing's disease, or has a high safety risk, so new drugs for further treatment are urgently needed in clinical practice.

[0013] Aldehyde is a specific ligand for mineralocorticoid receptors (hereinafter also referred to as "MR") and is one of the mediators in the renin-angiotensin-aldosterone system (RAAS). Aldehyde was previously considered to be a mineralocorticoid that is mainly produced in the adrenal glands, acts on the distal tubules of the kidneys, and regulates the metabolism of sodium and water. However, in recent studies, it has been found that aldehyde is produced in various tissues such as the heart, blood vessels, and brain, and is widely distributed in cardiovascular tissues, and it has been recognized that aldehyde is not only a worsening factor for hypertension, but also a dangerous hormone that shows various disordering effects (cardiac fibrosis and necrosis, enhancement of the action of catecholamines, reduction of baroreceptor response, etc.) on cardiovascular tissues.

[0014] For cardiovascular diseases associated with aldosterone and its receptor, a means of blocking the action of the hormone is an effective treatment. MR antagonists (e.g., eplerenone or spironolactone) having affinity for MR and blocking the function of its receptor have been used for hypertension treatment. In addition, in large-scale clinical trials (RALES and EPHESUS trials), it was confirmed that the combination of MR antagonists with common therapeutic agents such as ACE inhibitors significantly reduced the hospitalization rate and mortality rate of patients with severe heart failure due to heart disease, and significantly improved the prognosis of patients with acute myocardial infarction.

[0015] On the other hand, MR antagonists (e.g., spironolactone or eplerenone) have a unique serious side effect (e.g., hyperkalemia), and in addition, for spironolactone, cases of gynecomastia, menstrual abnormalities, erectile dysfunction, and the like are not uncommon. Therefore, in the treatment of diseases associated with aldosterone, it is desirable to develop a compound with fewer side effects and higher safety. As an alternative (i.e., other means for blocking or attenuating the effects of aldosterone) from this perspective, aldosterone synthase inhibitors have been proposed.

[0016] Aldosterone synthase is a cytochrome P450 enzyme, and is known as an enzyme that catalyzes a series of reactions from 11-deoxycorticosterone (i.e., a precursor of aldosterone) to aldosterone. Aldosterone synthase is mainly expressed in the glomerular zone of the adrenal cortex, and the plasma aldosterone is regulated by the activity of this enzyme in the adrenal gland. In addition, expression of aldosterone has been confirmed in sites other than the adrenal gland, such as the cardiovascular system, kidney, adipose tissue, brain, and the like, and the locally produced aldosterone in each organ is associated with organ dysfunction.

[0017] It has been reported that aldosterone synthase inhibitors inhibit the production of aldosterone in studies using enzymes and cultured cells, and have an inhibitory effect on the production of aldosterone and a therapeutic effect in studies using various experimental animal models. In addition, it has been confirmed that aldosterone synthase inhibitors exhibit an effect of lowering the level of aldosterone in the plasma and urine and a hypotensive effect in patients with hypertension and primary aldosteronism. In summary, finding a means of blocking the biosynthetic pathway of aldosterone is a highly achievable approach for establishing an effective treatment for various diseases associated with aldosterone.

[0018] The aldosterone synthase is encoded by the aldosterone synthase gene (CYP11B2), which shares 95% of the nucleotide sequence with the 11-beta hydroxylase gene (CYP11B1), and they are arranged in tandem on human chromosome 8 (8q24) with only 40 kb in between, both of which are about 7 kb in total length, and both contain 9 exons and 8 introns arranged in the same order. The proteins encoded by them have 93% of the amino acid sequences completely identical. The CYP11B1 gene encodes 11-beta hydroxylase, which is involved in the hydroxylation reaction of 11-deoxycortisol to cortisol; at the same time, in the fascicular zone of the adrenal cortex, 11-deoxycortisol is finally converted to cortisol under the action of 11-beta hydroxylase. Therefore, the correlation of CYP11B2 and CYP11B1 genes with the pathogenesis of Cushing's disease has become the focus of research.

[0019] In 2013, FDA granted orphan drug designation to Osilodrostat for the treatment of Cushing's syndrome; in January 2020, EMA approved its marketing, and in March 2020, FDA approved its marketing.

[0020] Osilodrostat is a cortisol synthesis inhibitor. It inhibits 11-beta hydroxylase (CYP11B1), which is responsible for the last step of adrenal cortisol biosynthesis. In a Chinese hamster lung cell line V79-4 overexpressing human CYP11B1, adrenaline and adrenaline reductase, Osilodrostat dose-dependently inhibits the activity of human CYP11B1 with an IC50 value of 2.5 ± 0.1 nM (n = 4).

[0021] Japan Tobacco and its subsidiaries Akros Pharma and Torii developed Enarodustat (Enaroy; JTZ-951; SAL-0951), a small molecule hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) inhibitor with oral activity that stimulates endogenous EPO (erythropoietin) production for potential treatment of anemia associated with chronic kidney disease. Japan Tobacco and its subsidiary Torii are also investigating the drug for potential treatment of type II diabetes.

[0022] In December 2019, Shenzhen Sinolink Pharmaceuticals, a Chinese listed company, is researching Enarodustat in the Greater China region for the treatment of anemia associated with chronic kidney disease.

[0023] In June 2020, an IND was submitted in China. In August 2020, a clinical trial implicit permission was obtained for anemia associated with chronic kidney disease. In June 2023, marketing authorization was obtained in China.

[0024] In September 2020, Japan Tobacco obtained approval in Japan for the manufacture and sale of 2 mg and 4 mg tablets of enasidenib for the treatment of anemia associated with chronic kidney disease. It was launched in Japan in December 2020.

[0025] Enasidenib, chemical name: 2-[[7-oxo-5-(2-phenylethyl)-3H-[1,2,4]triazolo[1,5-a]pyridine-8- carbonyl]amino]acetic acid, molecular formula: C 17 H 16 N4O4, molecular weight: 340.34, white crystalline powder, melting point 187℃, chemical structure as follows:

[0026]

[0027] In the invention patent: triazolopyridine compounds and their effects as prolyl hydroxylase inhibitors and erythropoietin production inducers, publication number CN102471337A, enasidenib and its synthetic route are described, but no crystal form related diffraction data is given.

[0028] The described synthetic route is as follows:

[0029] Step 116-10

[0030]

[0031] The compound obtained in step 116-9 (0.050 g) and methanol (3 ml) were mixed, and then the mixture was heated to 60℃. The solution was cooled to room temperature and stirred for one day. The solid was collected by filtration to obtain the title compound (0.031 g, 61%).

[0032] 1H-NMR (DMSO-D6) δ: 3.12 (t, 2H, J = 7.9 Hz), 3.40 (t, 3H, J = 7.9 Hz), 4.22 (d, 2H, J = 5.2 Hz), 6.79 (s, 1H), 7.21-7.29 (m, 5H), 8.58 (s, 1H), 9.84 (t, 1H, J = 5.2 Hz), 12.97 (s, 1H), 14.22 (s, 1H).

[0033] However, the crystal form information of the obtained compound is not disclosed in this patent document.

[0034] Invention patent CN110214139A, a method for producing triazolopyridine compounds, discloses a synthesis method of enzalutamide and its pharmaceutical salts. Although the compound is disclosed as a crystal (this application is crystal form I), and the crystallization solvent is 2-propanol and water (volume ratio 330:83, specification 0260 paragraph), but also does not disclose its specific XRPD diffraction data.

[0035] Invention patent CN 115197210 A makes a thorough and detailed study of the crystal form of enzalutamide, and concludes that the crystal form of enzalutamide obtained in invention patent CN102471337A (specification 0986 paragraph) is N2, and the crystal form obtained in CN110214139A (specification 0260 paragraph) is N21. The XRPD diffraction data of crystal form N21 are disclosed as follows:

[0036] It has characteristic peaks at diffraction angles 2θ of 5.6±0.2°, 8.5±0.2°, 10.4±0.2°, 11.3±0.2°, 11.5±0.2°, 12.0±0.2°, 13.4±0.2°, 14.0±0.2°, 14.2±0.2°, 15.5±0.2°, 17.1±0.2°, 17.9±0.2°, 18.6±0.2°, 18.9±0.2°, 19.5±0.2°, 20.0±0.2°, 20.4±0.2°, 21.1±0.2°, 22.8±0.2°, 23.8±0.2°, 25.1±0.2°, 25.8±0.2°, 26.9±0.2°, 27.4±0.2°, 28.1±0.2°, 28.6±0.2°, 29.1±0.2°, 31.6±0.2°, 32.1±0.2°, 33.9±0.2°, 37.0±0.2° and 37.8±0.2°.

[0037] However, so far, no existing literature or patents have disclosed other medical uses of enzalutamide. SUMMARY

[0038] The inventors have studied the crystal form of enzalutamide, and this application first discloses a crystal form II of enzalutamide, which is confirmed by X-ray diffraction (XRPD) to have the strongest characteristic peak at a 2θ angle of 9.3° on the XRPD spectrum, with a relative intensity of 100%.

[0039] Further, the crystal form II of enzalutamide according to the application has a characteristic peak at a 2θ angle of about 23.8° on the XRPD spectrum, with a relative intensity of 33.0%.

[0040] Further, the enzalutamide crystal form II of the present application has characteristic peaks at positions of about 13.0°, 13.9°, 14.2°, 16.3°, 20.5°, 20.8°, 25.7°, 27.0° on the XRPD pattern with 2θ angles, and the relative intensity is greater than 10%.

[0041] Further, the enzalutamide crystal form II of the present application has characteristic peaks at positions of about 17.9°, 20.3°, 21.2°, 21.9°, 22.9°, 25.4°, 30.0°, 32.1° on the XRPD pattern with 2θ angles, and the relative intensity is greater than 5%.

[0042] Further, the enzalutamide crystal form II of the present application has the XRPD pattern as shown in the following figure. Figure 1

[0043] The XRPD data of the enzalutamide crystal form II of the present application is shown in Table 1.

[0044] The XRPD data of the enzalutamide crystal form II of the present application is shown in Table 1.

[0045] 2 theta (°) Intensity % 2 theta (°) Intensity % 9.3 100.0 25.4 8.1 23.8 33.0 21.9 7.6 13.0 14.6 20.3 6.8 16.3 13.7 22.9 6.6 13.9 13.6 21.2 6.1 20.5 13.6 32.1 6.0 27.0 13.4 30.0 5.4 14.2 12.2 17.9 5.3 25.7 11.8 30.9 4.7 20.8 11.5 .

[0046] In particular, due to the error of XRPD measurement, the above-mentioned 2θ angle may have an error of ±0.1°, or even ±0.2°.

[0047] The present application further discloses a preparation process of the crystal form II, as follows:

[0048] Step 1) adding enzalutamide into a mixed solvent, warming, and keeping reflux;

[0049] Step 2) cooling the system of step 1) to precipitate crystal form II;

[0050] Step 3) drying under reduced pressure to obtain solid crystal form II.

[0051] In the step 1), the solvent is a mixture of water and dimethyl sulfoxide, and the ratio of the mixture is water: dimethyl sulfoxide = 2-5:1.

[0052] In the step 1), the warming temperature is at least 80℃.

[0053] In the step 2), the cooling temperature is 60-65℃.

[0054] Finally, the present application discloses the use of enzalutamide crystal form II or other crystal forms for preparing a drug for treating Cushing's disease. The administration route is oral.

[0055] ​The enzalutamide acts by inhibiting aldosterone synthase or 11 beta-hydroxylase.

[0056] The crystalline form II of enzalutamide according to the present application needs to be further prepared into a specific formulation form. In particular solid, semi-solid and liquid dosage forms, in particular solid dosage forms, such as tablets, capsules. Such dosage forms can comprise at least one excipient selected from the group consisting of fillers (e.g. lactose), binders (e.g. microcrystalline cellulose), disintegrants (e.g. croscarmellose sodium), lubricants (e.g. magnesium stearate), surfactants.

[0057] Furthermore, the pharmaceutical composition can optionally comprise at least one further excipient selected from the group consisting of colorants, solvents, antimicrobials, flavorings and olfactory modulators. The tablets can be coated with conventional coatings containing, for example, polyvinyl alcohol or polyethylene glycol. The pharmaceutical composition can be prepared into almost any solid dosage form, such as tablets, capsules, powders, pellets or granules. The preferred dosage form is a tablet or a coated tablet. The tablet or coated tablet can preferably be prepared by mixing enzalutamide with at least one pharmaceutically acceptable excipient and tabletting the mixture, and then optionally coating. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 XRPD of the crystalline form II of enzalutamide in the present application. DETAILED DESCRIPTION

[0059] The beneficial effects of the present application are further illustrated by the following experiments. But the following examples are not intended to limit the present application, and equivalents or modifications made by those skilled in the art based on the present application without departing from the essential content of the present application are also within the scope of protection of the present application.

[0060] Comparative Example 1: Enzalutamide crystalline form N21 was prepared according to Chinese patent CN110214139A, specification 0260 paragraph, and enzalutamide crystalline form N2 was prepared according to Chinese patent CN102471337A.

[0061] Example 1 Preparation of enzalutamide crystalline form II

[0062] Enzalutamide 10 g was taken in a mixture solution of water (20 ml) and dimethyl sulfoxide (10 ml) and stirred at 80°C until the crystals dissolved. The solution was cooled to 60°C and stirred for 2 hours, and the crystals were precipitated. The precipitated crystals were collected by filtration. The obtained wet crystals were dried under reduced pressure to obtain enzalutamide crystals (8.9 g, yield 89%).

[0063] The XRPD test results thereof are shown in the attached Figure 1 It is crystalline form II.

[0064] Example 2 Preparation of crystalline form II of enzalutamide

[0065] Enzalutamide 10 g was taken in a mixture of water (30 ml) and dimethyl sulfoxide (10 ml) and stirred at 85°C till the crystals dissolved. The solution was cooled to 60°C and stirred for 2 hours and crystals were separated. The separated crystals were collected by filtration. The wet crystals obtained were dried under reduced pressure to get enzalutamide crystals (9.2 g, yield 92%). It was tested by XRPD as crystalline form II.

[0066] Example 3 Preparation of crystalline form II of enzalutamide

[0067] Enzalutamide 10 g was taken in a mixture of water (50 ml) and dimethyl sulfoxide (10 ml) and stirred at 90°C till the crystals dissolved. The solution was cooled to 65°C and stirred for 3 hours and crystals were separated. The separated crystals were collected by filtration. The wet crystals obtained were dried under reduced pressure to get enzalutamide crystals (8.1 g, yield 81%). It was tested by XRPD as crystalline form II.

[0068] Example 4 Preparation of crystalline form II of enzalutamide

[0069] Enzalutamide 10 g was taken in a mixture of water (50 ml) and dimethyl sulfoxide (10 ml) and stirred at 90°C till the crystals dissolved. The solution was cooled to 60°C and stirred for 3 hours and crystals were separated. The separated crystals were collected by filtration. The wet crystals obtained were dried under reduced pressure to get enzalutamide crystals (8.5 g, yield 85%). It was tested by XRPD as crystalline form II.

[0070] Example 5 In vitro inhibitory activity of enzalutamide crystals on aldosterone synthase and steroid 11 beta-hydroxylase (with reference to CN 102459223 B)

[0071] NCI-H295R cells were grown in 75 cm 2 culture vessels in Dulbecco's modified Eagle's Ham F-12 medium (DME / F12) supplemented with Ultroser SF serum, insulin, transferrin, selenite and antibiotics at 37°C in an atmosphere of 95% air-5% carbon dioxide. To form colonies, the cells were then transferred to 24-well culture vessels. They were incubated in this DME / F12 medium supplemented with 0.1% bovine serum instead of Ultroser SF for 24 hours. The test was started by incubating the cells in DME / F12 medium supplemented with 0.1% bovine serum and different crystalline forms of enzalutamide 0.5% sodium carboxymethylcellulose suspension without addition of cell stimulators. The concentration range of enzalutamide added was from 0.2 nanomolar to 20 millimolar.

[0072] According to the manufacturer's instructions, aldosterone and corticosterone secretion in the culture medium was determined by radioimmunoassay using specific monoclonal antibodies obtained commercially.

[0073] The inhibition of the release of certain steroid hormones can be used to determine the inhibition of the respective enzymes by the addition of the test compound.

[0074] The dose-dependent inhibition of the enzyme activity of the compounds can be calculated from the inhibition curves, characterized by the IC50, and the resulting IC50 data are shown in Table 2.

[0075] Table 2 Inhibition of aldosterone synthase and 11 β-hydroxylase by different crystal forms of enasidenib (mean, n = 3, in nmol / L)

[0076]

[0077]

[0078] As can be seen from the data in the above table, the crystal form II has a better inhibitory effect on aldosterone synthase and 11 β-hydroxylase than the existing enasidenib crystal forms N21 and N2.

[0079] Example 6: Activity of different crystal forms of enasidenib in olfactory bulbectomized rats (according to CN 103596573 B)

[0080] Bilateral olfactory bulbectomy (OBX) is an animal model of chronic depression, which demonstrates the key role of the hypothalamic-pituitary-adrenal (HPA) axis in maintaining homeostasis.

[0081] OBX disturbs the HPA axis and brings about behavioral and physiological changes in the model animals that are characteristic of chronic depression. After recovery from the operation, OBX animals show a dramatic increase in body weight, accompanied by other characteristic physiological changes, such as increased food intake, depression, elevated serum sodium concentration and corticosteroid levels. These symptoms are associated with Cushing's syndrome. That is, the OBX model realistically simulates the symptoms of Cushing's syndrome.

[0082] In order to investigate the activity of enasidenib, we used the animal model of bilateral olfactory bulbectomy (OBX) rats, which produces all the above-mentioned symptoms seen in Cushing's syndrome.

[0083] The experimental procedure was as follows:

[0084] Preparation of the test sample: The different crystal forms of enasidenib were ground to a particle size of approximately 20 um and suspended in a 0.5% aqueous solution of sodium carboxymethylcellulose at a concentration of 0.2 g / ml to produce a suspension.

[0085] Male Wistar rats were anesthetized with ketamine (80 mg / kg), and the olfactory bulb was surgically aspirated using a blunt-tipped subcutaneous injection needle connected to a water pump, without damaging the frontal lobe. The animals recovered in two weeks post-surgery.

[0086] Ennadustat was used for long-term treatment with different crystal forms and different dose levels for 14 days, administered once daily by gavage.

[0087] The model control group was given an equal volume of 0.5% sodium carboxymethyl cellulose aqueous solution.

[0088] The normal control group consisted of normal animals that had not undergone OBX surgery, were given a normal diet, and received no medication.

[0089] Treatment began 14 days before surgery (day 0). The weight of each group of animals was measured on the first 7 days and the first 14 days to determine the effect of the drug on the weight of the model animals.

[0090] At the start of treatment (day 0), day 7, and day 14, the food intake of each group of animals was recorded to determine the effect of the drug on the food intake of the model animals.

[0091] On the last day of treatment, blood was drawn to measure serum corticosteroids. Corticosteroids are the main glucocorticoids found in rodents and are equivalent to the cortisol hormone found in humans.

[0092] Table 3: Effects of different crystalline forms of endostatin on body weight in rats that have undergone olfactory bulbectomy ( n=3, unit: g)

[0093]

[0094] Compared with the normal control group, the OBX model animals showed significant weight gain. Ennadustat in different crystalline forms significantly inhibited OBX-induced weight gain at doses of 10, 20, and 40 mg / kg, with crystalline form II showing the most significant effect.

[0095] Table 4: Effects of different crystalline forms of endostatin on food intake in olfactory bulbectomized rats ( n=3 (in grams per day)

[0096] Time 1 day treatment 7 day treatment 14 day treatment Normal control group 17.3±0.4 18.4±0.1 18.8±0.5 Model control group 24.4±0.2 27.4±0.2 31.2±0.4 Enzastar N2 1 (10 mg / kg) 23.4±0.3 26.3±0.4 28.3±0.6 Enzastar N2 1 (20 mg / kg) 23.5±0.1 25.8±0.5 27.1±0.8 Enzastar N2 1 (40 mg / kg) 22.9±0.7 25.6±0.1 27.9±0.1 Enzastar N2 (10 mg / kg) 22.1±0.6 25.1±0.6 27.3±0.7 Enzastar N2 (20 mg / kg) 22.6±0.1 24.4±0.2 26.1±0.4 Enzastar N2 (40 mg / kg) 21.7±0.2 24.4±0.5 25.9±0.9 Enzastar II (10 mg / kg) 21.1±0.5 24.6±0.8 24.0±0.7 Enzastar II (20 mg / kg) 21.4±0.9 23.5±0.1 24.1±0.9 Enzastar II (40 mg / kg) 20.6±0.8 22.4±0.2 23.5±0.3 .

[0097] Food intake was monitored in all experimental animals 14 days after surgery. Animals in the OBX control group showed increased food intake compared to the normal control group. Treatment with different crystalline forms of endostat for 14 days significantly reduced food intake, bringing it closer to normal, with crystalline form II showing the best results.

[0098] Table 5: Effect of different crystalline forms of Epoetin Theta on serum sodium and corticosterone concentration in olfactory bulbectomized rats n = 3

[0099] Animal grouping Serum corticosterone level (ug / ml) Serum sodium concentration (mEq / l) Normal control group 18.7±0.5 13.1±0.7 Model control group 49.5±0.9 38.7±0.8 Enzastar N2 1 (10 mg / kg) 34.5±0.5 36.7±0.7 Enzastar N2 1 (20 mg / kg) 34.7±0.3 35.8±0.2 Enzastar N2 1 (40 mg / kg) 33.6±0.2 34.1±0.9 Enzastar N2 (10 mg / kg) 33.4±0.1 35.2±0.6 Enzastar N2 (20 mg / kg) 33.9±0.8 34.7±0.1 Enzastar N2 (40 mg / kg) 30.1±0.7 31.5±0.3 Enzastar II (10 mg / kg) 28.7±0.7 29.7±0.1 Enzastar II (20 mg / kg) 28.6±0.8 29.1±0.9 Enzastar II (40 mg / kg) 25.4±0.8 28.8±0.3

[0100] OBX model animals showed hypersecretion of corticosterone in blood. Animals treated with different crystalline forms of Epoetin Theta at all doses significantly reduced the level of corticosterone in blood. OBX induced an increase in serum aldosterone and further induced an increase in serum sodium concentration, which was significantly reduced by different crystalline forms of Epoetin Theta at different dose levels, especially with Epoetin Theta Form II, indicating that Epoetin Theta has potential as a therapeutic drug for Cushing's disease, especially Form II.

[0101] The above study shows the efficacy of different crystalline forms of Epoetin Theta in treating Cushing's syndrome conditions (i.e. weight gain, increased food intake, hypersecretion of corticosterone and increased serum sodium) induced by endogenous factors demonstrated in olfactory bulbectomized rats. Therefore, the different crystalline forms of Epoetin Theta are effective in treating and managing all other conditions of Cushing's syndrome and hypercortisolemia, especially Form II.​

Claims

1. The use of ennadustat in the preparation of a drug for treating Cushing's disease, characterized in that, The chemical structure of the ennadustat is as follows: 。 2. The use as described in claim 1, characterized in that, The crystal form of the described ennadustat is N21, with diffraction angles 2θ of 5.6±0.2°, 8.5±0.2°, 10.4±0.2°, 11.3±0.2°, 11.5±0.2°, 12.0±0.2°, 13.4±0.2°, 14.0±0.2°, 14.2±0.2°, 15.5±0.2°, 17.1±0.2°, 17.9±0.2°, 18.6±0.2°, 18.9±0.2°, and 19.5±0.2° in XRPD patterns. Characteristic peaks are observed at 20.0±0.2°, 20.4±0.2°, 21.1±0.2°, 22.8±0.2°, 23.8±0.2°, 25.1±0.2°, 25.8±0.2°, 26.9±0.2°, 27.4±0.2°, 28.1±0.2°, 28.6±0.2°, 29.1±0.2°, 31.6±0.2°, 32.1±0.2°, 33.9±0.2°, 37.0±0.2°, and 37.8±0.2°.

3. The use as described in claim 1, characterized in that, The crystal form of the stated ennadustat is crystal form II, and it exhibits characteristic peaks in the XRPD spectrum at diffraction angles 2θ of 9.3±0.2°, 23.8±0.2°, 13.0±0.2°, 16.3±0.2°, 13.9±0.2°, 20.5±0.2°, 27.0±0.2°, 14.2±0.2°, 25.7±0.2°, 20.8±0.2°, 25.4±0.2°, 21.9±0.2°, 20.3±0.2°, 22.9±0.2°, 21.2±0.2°, 32.1±0.2°, 30.0±0.2°, 17.9±0.2°, and 30.9±0.2°.

4. The use as described in claim 3, characterized in that, The crystal form II, as confirmed by X-ray diffraction, has the XRPD spectrum shown in Figure 1.

5. The use as described in any one of claims 3 and 4, characterized in that, The preparation method of the ennadustat crystal form II is as follows: Step 1) Add ennadustat to a mixed solvent of water and dimethyl sulfoxide, heat to 80°C or above, and keep under reflux; Step 2) Cool the system from Step 1) to 60-65℃ to precipitate crystal II; Step 3) Dry under reduced pressure to obtain solid crystal form II.

6. The use as described in any of claims 1-4, characterized in that, The drug is administered orally.

7. The use as described in any of claims 1-4, characterized in that, The drug works by inhibiting aldosterone synthase or 11β-hydroxylase.

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