WO2022179592A1

WO2022179592A1 - Combination therapeutic drug for acute myeloid leukemia

Info

Publication number: WO2022179592A1
Application number: PCT/CN2022/077828
Authority: WO
Inventors: 夏雪芳; 郝春颜; 范丽雪; 束云; 柴晓玲; 杨晓旭; 张笑
Original assignee: 石药集团中奇制药技术（石家庄）有限公司
Priority date: 2021-02-25
Filing date: 2022-02-25
Publication date: 2022-09-01
Also published as: CN114948983A

Abstract

Disclosed are a drug and method for treating acute myeloid leukemia by means of combined administration of a multi-target kinase inhibitor compound I or a pharmaceutically acceptable salt thereof with daunorubicin and cytarabine. The combination of compound I or the pharmaceutically acceptable salt thereof with daunorubicin and cytarabine can significantly inhibit the proliferation of human leukemia cells MV-4-11 and MOLM-13, produces a synergistic effect, and has a combined efficacy which is better than that of a scheme only using daunorubicin and cytarabine, and treated patients will have a longer survival period. The potential of the combination therapeutic scheme in prognosis improvement is much greater than the risk of potential toxicity thereof, and the scheme has good clinical application prospects.

Description

A combination therapy drug for acute myeloid leukemia

This application claims to enjoy the priority of the prior application submitted by the applicant to the State Intellectual Property Office of China on February 25, 2021, the patent application number is 202110212434.8, and the invention name is "a combination therapy drug for acute myeloid leukemia". The entire contents of this prior application are incorporated herein by reference.

technical field

The invention belongs to the field of medicine, and in particular relates to a combined therapeutic drug for treating acute myeloid leukemia.

Background technique

Acute myeloid leukemia (AML) is a hematological malignancy in which abnormal proliferation, differentiation and cloning of hematopoietic stem cells invade the bone marrow, blood and extramedullary tissues. Among common hematologic tumors, AML has the highest mortality rate, with a 5-year survival of approximately 24%. AML accounts for about 70% of adult leukemias. After treatment, 35% to 40% of adult AML patients aged 60 and below can achieve long-term survival, while only 5% to 15% of patients over 60 years old can achieve long-term survival. Elderly AML patients who tolerate intensive chemotherapy have a median survival time of only 5 to 10 months.

At present, there is no standard treatment regimen for newly diagnosed AML (non-APL) in China. Clinically recommended induction therapy regimen for newly diagnosed adult AML (non-APL) based on anthracycline combined with cytarabine, commonly used daunorubicin (IDA) or daunorubicin (DNR) The Ara-C/DNR ("7+3") regimen composed of cytarabine (Ara-C), the specific dose is determined according to the patient's condition. However, in subjects younger than 60 years with newly diagnosed AML, the standard "7+3" induction regimen is effective; however, in patients ≥60 years old, high-dose cytarabine showed age-related toxicity.

FMS-like tyrosine kinase (FLT3) is overexpressed in most AML cases. FLT3 is a member of the class III receptor tyrosine kinase (TK) family and is normally expressed on the surface of hematopoietic progenitor cells. FLT3 and its ligands play important roles in the proliferation, survival and differentiation of pluripotent stem cells. Furthermore, in AML cases, mutations in the juxtamembrane domain tyrosine kinase domain (TKD) around and near D835 in the activation loop and activated FLT3 with internal tandem repeats (ITD) accounted for 28% of AML cases, respectively- 34% and 11%-14%. These activating mutations in FLT3 are oncogenic and exhibit transforming activity in cells. Patients with FLT3-ITD mutations have been reported to have poorer prognosis and higher recurrence rates in clinical studies (R.M. Shallis, R. Wang, A. Davidoff, X. Ma, and A.M. Zeidan, "Epidemiology of acute myeloid leukemia: Recent progress and enduring challenges,"Blood Rev,vol.36,pp.70-87,Jul 2019), shorter duration of remission with initial therapy (6 months, 11.5 months in patients without FLT3-ITD mutation), disease free Survival was decreased (16% to 27% vs. 41% at 5 years in patients without the FLT3-ITD mutation), and OS was also decreased (15% to 31% vs. 5 years in patients without the FLT3-ITD mutation) OS was 42%). FLT3 has become an important target for AML treatment, and related drug research has attracted wide attention.

Studies have shown that, in addition to FLT3 mutations, KIT, CEBPA, NPM1, PHF6, ASXL1, DNMT3A, IDH1, IDH2, TET2, CBL, TP53, BCORL1, EZH2, MLL, SRSF2, SF3B1, U2AF1, ZRSR2, SF3A1, PRPF40B, U2AF2, SF1 and other gene abnormalities are also related to the onset, prognosis, low survival rate, and recurrence of AML.

Compound I is a novel multi-target protein kinase inhibitor, the main targets include FLT3, etc., and the structure is shown in the following formula (I):

WO 2011/147066A1 discloses the compound and its preparation method and medical use. PCT/CN2021/073285 further studies the pharmaceutically acceptable salts of compound I and their uses. The entire contents of the above-mentioned patents are incorporated herein by reference. In vitro and in vivo studies have shown that Compound I or its pharmaceutically acceptable salts have a strong growth inhibitory effect on FLT3-ITD mutant acute myeloid leukemia cell lines. However, whether compound I or its pharmaceutically acceptable salts combined with DNR and Ara-C in the treatment of newly diagnosed AML younger than 60 years old can further improve the efficacy, and whether it has advantages in safety and tolerance compared with similar drugs, it is not yet known. .

SUMMARY OF THE INVENTION

The present invention provides the use of compound I or a pharmaceutically acceptable salt thereof, daunorubicin and cytarabine in preparing a medicine for treating acute myeloid leukemia (AML). The structure of the compound I is shown in the following formula (I):

In some embodiments, the acute myeloid leukemia is preferably a FLT3 mutation-positive acute myeloid leukemia, more preferably an untreated FLT3 mutation-positive acute myeloid leukemia.

In some embodiments, the FLT3 mutation-positive comprises FLT3 internal tandem repeat (ITD) mutation-positive, FLT3 tyrosine kinase domain (TKD)/D835 mutation-positive, and/or FLT3 tyrosine kinase domain (TKD) /I836 mutation positive.

In some embodiments, the acute myeloid leukemia is FLT3 mutation-positive acute myeloid leukemia accompanied by one, two or more of the following gene mutations: KIT mutation, CEBPA mutation, NPM1 mutation, PHF6 Mutation, ASXL1 mutation, DNMT3A mutation, IDH1 mutation, IDH2 mutation, TET2 mutation, CBL mutation, TP53 mutation, BCORL1 mutation, EZH2 mutation, MLL mutation, SRSF2 mutation, SF3B1 mutation, U2AF1 mutation, ZRSR2 mutation, SF3A1 mutation, PRPF40B mutation, U2AF2 mutation, SF1 mutation, etc., preferably BCORL1 mutation, CEBPA mutation, CBL mutation, TP53 mutation.

In some embodiments, the acute myeloid leukemia is FLT3 mutation-negative acute myeloid leukemia. Further, the FLT3 mutation-negative acute myeloid leukemia is accompanied by one, two or more of the following gene mutations: KIT mutation, CEBPA mutation, NPM1 mutation, PHF6 mutation, ASXL1 mutation, DNMT3A mutation, IDH1 mutation , IDH2 mutation, TET2 mutation, CBL mutation, TP53 mutation, BCORL1 mutation, EZH2 mutation, MLL mutation, SRSF2 mutation, SF3B1 mutation, U2AF1 mutation, ZRSR2 mutation, SF3A1 mutation, PRPF40B mutation, U2AF2 mutation, SF1 mutation, etc. BCORL1 mutation is preferred , CEBPA mutation, CBL mutation, TP53 mutation.

The pharmaceutically acceptable salt of compound I is not particularly limited, and is prepared from the free base of compound I and acid. Those skilled in the art can select suitable salt forms by conventional methods. Specific examples of pharmaceutically acceptable salts of Compound 1 include, but are not limited to, the hydrochloride, mesylate, malate, tartrate, oxalate, succinate, acetate, sulfate, and the like of Compound 1; Hydrochloride, malate, tartrate, oxalate, succinate, acetate are preferred; hydrochloride is further preferred. The hydrochloride is more preferably trihydrochloride. The trihydrochloride includes the corresponding anhydrate, hydrate, solvate, etc., more preferably trihydrochloride pentahydrate.

In some embodiments, the molar ratio of cytarabine to daunorubicin is 1-100:1, preferably 1-25:1, more preferably 2-10:1, such as 5:1. The molar ratio of compound I or its pharmaceutically acceptable salt and daunorubicin is 100:1-1:100, preferably 2:1-1:70, more preferably 4:1-1:50 (with compound I content calculation).

Compound I or a pharmaceutically acceptable salt thereof, daunorubicin and cytarabine can be contained in the same pharmaceutical preparation, or can be separately prepared into clinically acceptable preparations, and the medicine can be prepared in a combined packaging manner. The clinically accepted preparations include oral preparations, injection preparations, topical preparations, external preparations and the like. In some embodiments, the medicament is in a single-dose or divided-dose form. The dosage form contains a therapeutically effective amount of daunorubicin, cytarabine, compound I or a pharmaceutically acceptable salt thereof.

When daunorubicin, cytarabine, compound I or a pharmaceutically acceptable salt thereof are prepared into clinically acceptable preparations, respectively, and the medicine is prepared in a combined packaging mode, the compound I or its pharmaceutically acceptable salts are prepared into the medicine. The formulation of the accepted salt is preferably an oral formulation, such as tablets, capsules, pills, etc.; each formulation unit contains about 0.001 mg to about 1000 mg of Compound I or a pharmaceutically acceptable salt thereof (calculated as Compound I), preferably about 1 mg to about 500 mg, or about 10 mg to about 400 mg, or about 15 mg to about 330 mg, or about 20 mg to about 300 mg, or about 20 mg to about 250 mg, or about 20 mg to about 210 mg, or about 20 mg to about 180 mg, or about 20 mg - about 160 mg, or about 20 mg to about 140 mg, or about 20 mg to about 120 mg, or about 20 mg to about 100 mg. Exemplary amounts are, for example, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 75 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 125 mg, about 130 mg, About 140 mg, about 150 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 250 mg, about 300 mg, about 310 mg, about 320 mg, about 360 mg, about 400 mg. The daunorubicin preparation is preferably daunorubicin hydrochloride for injection, such as daunorubicin hydrochloride lyophilized powder for injection; each preparation unit contains about 10 mg to about 30 mg of active ingredient, preferably about 20 mg. The cytarabine preparation is preferably cytarabine hydrochloride for injection, such as cytarabine hydrochloride lyophilized powder for injection; each preparation unit contains about 0.1g to about 0.5g of active ingredients, such as about 0.1g, about 0.3 g or about 0.5 g.

In some embodiments, the pharmaceutically acceptable salt of Compound I is the hydrochloride salt, preferably the trihydrochloride salt, more preferably the trihydrochloride pentahydrate. Each dosage unit contains about 1 mg to about 500 mg, or about 10 mg to about 400 mg, or about 15 mg to about 350 mg, or about 20 mg to about 310 mg, or about 25 mg to about 300 mg, or about 25 mg to about 250 mg, or about 50 mg to about 250 mg, or about 50 mg to about 200 mg, or about 50 mg to about 175 mg, or about 50 mg to about 160 mg, or about 50 mg to about 150 mg, or about 50 mg to about 125 mg, or about 50 mg to about 100 mg; exemplary amounts such as about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 160 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg, about 310 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg. The above compound I trihydrochloride is measured in anhydrous form.

In another aspect, the present invention provides the use of compound I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving the curative effect of daunorubicin and cytarabine in the treatment of acute myeloid leukemia.

In some embodiments, the acute myeloid leukemia is preferably FLT3 mutation-positive acute myeloid leukemia. More preferred is untreated FLT3 mutation-positive acute myeloid leukemia.

In some embodiments, the acute myeloid leukemia refers to FLT3 mutation-positive acute myeloid leukemia with one, two or more of the following gene mutations: KIT mutation, CEBPA mutation, NPM1 mutation, PHF6 mutation, ASXL1 mutation, DNMT3A mutation, IDH1 mutation, IDH2 mutation, TET2 mutation, CBL mutation, TP53 mutation, BCORL1 mutation, EZH2 mutation, MLL mutation, SRSF2 mutation, SF3B1 mutation, U2AF1 mutation, ZRSR2 mutation, SF3A1 mutation, PRPF40B mutation , U2AF2 mutation, SF1 mutation, etc., preferably BCORL1 mutation, CEBPA mutation, CBL mutation, TP53 mutation.

In some embodiments, the acute myeloid leukemia is FLT3 mutation-negative acute myeloid leukemia. Further, these FLT3 mutation-negative acute myeloid leukemias are accompanied by one, two or more of the following gene mutations: KIT mutation, CEBPA mutation, NPM1 mutation, PHF6 mutation, ASXL1 mutation, DNMT3A mutation, IDH1 mutation, IDH2 mutation, TET2 mutation, CBL mutation, TP53 mutation, BCORL1 mutation, EZH2 mutation, MLL mutation, SRSF2 mutation, SF3B1 mutation, U2AF1 mutation, ZRSR2 mutation, SF3A1 mutation, PRPF40B mutation, U2AF2 mutation, SF1 mutation, etc. BCORL1 mutation, CEBPA mutation, CBL mutation, TP53 mutation.

In some embodiments, the medicament is formulated into a clinically acceptable formulation, such as an oral formulation, an injection formulation, a topical formulation, a topical formulation, and the like.

In some embodiments, the medicament is in a single-dose or divided-dose form. The dosage form contains a therapeutically effective amount of Compound I or a pharmaceutically acceptable salt thereof.

In some embodiments, each formulation unit contains about 0.001 mg to about 1000 mg of Compound I or a pharmaceutically acceptable salt thereof (based on Compound I), preferably about 1 mg to about 500 mg, or about 10 mg to about 400 mg, or about 15 mg to about 330 mg, or about 20 mg to about 300 mg, or about 20 mg to about 250 mg, or about 20 mg to about 210 mg, or about 20 mg to about 180 mg, or about 20 mg to about 160 mg, or about 20 mg to about 140 mg, or about 20 mg to about 120 mg, or about 20 mg to about 100 mg. Exemplary amounts are, for example, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 75 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 125 mg, about 130 mg, About 140 mg, about 150 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 250 mg, about 300 mg, about 310 mg, about 320 mg, about 360 mg, about 400 mg.

The present invention also provides a method for treating acute myeloid leukemia, the method comprising administering to a subject or patient a therapeutically effective amount of daunorubicin, cytarabine and compound I or a pharmaceutically acceptable salt thereof. Daunorubicin, cytarabine and Compound I or a pharmaceutically acceptable salt thereof can be administered simultaneously or separately. The administration may be oral administration, injection administration, topical administration or in vitro administration. The therapeutically effective amount can treat or alleviate acute myeloid leukemia in the subject or patient.

In some embodiments, the acute myeloid leukemia refers to FLT3 mutation-positive acute myeloid leukemia with one, two or more of the following gene mutations: KIT mutation, CEBPA mutation, NPM1 mutation, PHF6 Mutation, ASXL1 mutation, DNMT3A mutation, IDH1 mutation, IDH2 mutation, TET2 mutation, CBL mutation, TP53 mutation, BCORL1 mutation, EZH2 mutation, MLL mutation, SRSF2 mutation, SF3B1 mutation, U2AF1 mutation, ZRSR2 mutation, SF3A1 mutation, PRPF40B mutation, U2AF2 mutation, SF1 mutation, etc., preferably BCORL1 mutation, CEBPA mutation, CBL mutation, TP53 mutation.

The pharmaceutically acceptable salt of compound I is not particularly limited, and is prepared from compound I free base plus acid. Those skilled in the art can select suitable salt forms by conventional methods. Specific examples of pharmaceutically acceptable salts of Compound 1 include, but are not limited to, the hydrochloride, mesylate, malate, tartrate, oxalate, succinate, acetate, sulfate, and the like of Compound 1; Hydrochloride, malate, tartrate, oxalate, succinate, acetate are preferred; hydrochloride is further preferred. The hydrochloride is more preferably trihydrochloride. The trihydrochloride includes the corresponding anhydrate, hydrate, solvate, etc., more preferably trihydrochloride pentahydrate.

In some embodiments, the method of treatment comprises:

(1) Induction therapy: one cycle every 28 days, up to two cycles. The treatments for each cycle are as follows:

Days 1-7: Cytarabine approximately 100 mg/m ² by continuous intravenous infusion (CIVI) for 7 days (168 h in total).

Days 1-3: daunorubicin about 60 mg/m ² or about 45 mg/m ² intravenously (IVP) or short infusion;

Days 8-21: Compound I or a pharmaceutically acceptable salt thereof about 20 mg/time to about 160 mg/time, orally, twice a day (BID) for 14 consecutive days. Preferably about 40 mg to about 140 mg per oral administration, more preferably about 40 mg to about 120 mg per oral administration. Examples of each oral dose include about 20 mg, or about 40 mg, or about 60 mg, or about 80 mg, or about 100 mg, or about 120 mg, or about 140 mg, or about 160 mg.

In some embodiments, Compound I is administered in the form of a hydrochloride salt, preferably trihydrochloride, more preferably trihydrochloride pentahydrate. Specifically: Days 8-21: Oral administration of Compound I trihydrochloride about 25 mg/time to about 200 mg/time, twice a day (BID), for 14 consecutive days. Preferably, from about 50 mg to about 175 mg, more preferably from about 50 mg to about 150 mg, per oral administration. Examples of each oral dose include about 50 mg, or about 75 mg, or about 100 mg, or about 125 mg, or about 150 mg, or about 175 mg, or about 200 mg. The dosage of the trihydrochloride salt is in anhydrous form.

(2) Consolidation therapy: one cycle every 28 days, up to 4 cycles. The treatments for each cycle are as follows:

Days 1, 3, and 5: cytarabine about 1g/m ² to about 5g/m ² is continuously infused, q12h. Preferably, cytarabine is administered at a dose of from about 1 g/m ² to about 4 g/m ² , more preferably from about 1.5 g/m ² to about 3 g/m ² . Specific examples of the administered dose are, for example, about 3 g/m ² or about 2 g/m ² or about 1.5 g/m ² .

(3) Maintenance therapy: one cycle every 28 days, up to 12 cycles. The treatments for each cycle are as follows:

Compound I or a pharmaceutically acceptable salt thereof is about 20 mg/time to about 160 mg/time, orally, twice a day (BID), daily. Preferably about 40 mg to about 140 mg per oral administration, more preferably about 40 mg to about 120 mg per oral administration. Examples of each oral dose include about 20 mg, or about 40 mg, or about 60 mg, or about 80 mg, or about 100 mg, or about 120 mg, or about 140 mg, or about 160 mg.

In some embodiments, Compound I is administered in the form of a hydrochloride salt, preferably trihydrochloride, more preferably trihydrochloride pentahydrate. Specifically: Oral administration of Compound I trihydrochloride about 25 mg/time to about 200 mg/time, twice a day (BID), daily. Preferably, from about 50 mg to about 175 mg, more preferably from about 50 mg to about 150 mg, per oral administration. Examples of each oral dose include about 50 mg, or about 75 mg, or about 100 mg, or about 125 mg, or about 150 mg, or about 175 mg, or about 200 mg. The dosage of the trihydrochloride salt is in anhydrous form.

The present invention also provides a method for improving the curative effect of daunorubicin and cytarabine in the treatment of acute myeloid leukemia, the method comprising: using compound I in combination with cytarabine and daunorubicin for treatment or a pharmaceutically acceptable salt thereof. Daunorubicin, cytarabine and Compound I or a pharmaceutically acceptable salt thereof can be administered simultaneously or separately. The administration may be oral administration, injection administration, topical administration or in vitro administration. The therapeutically effective amount can treat or alleviate acute myeloid leukemia in the subject or patient.

In some embodiments, the acute myeloid leukemia is FLT3 mutation-negative acute myeloid leukemia. Further, these FLT3 mutation-negative acute myeloid leukemias are accompanied by other gene mutations, including but not limited to KIT mutation, CEBPA mutation, NPM1 mutation, PHF6 mutation, ASXL1 mutation, DNMT3A mutation, IDH1 mutation, IDH2 mutation, TET2 mutation, CBL Mutation, TP53 mutation, BCORL1 mutation, EZH2 mutation, MLL mutation, SRSF2 mutation, SF3B1 mutation, U2AF1 mutation, ZRSR2 mutation, SF3A1 mutation, PRPF40B mutation, U2AF2 mutation, SF1 mutation, etc., preferably BCORL1 mutation, CEBPA mutation, CBL mutation, TP53 mutation.

In some embodiments, the method includes:

The present invention also provides a composition comprising (1) daunorubicin; (2) cytarabine; (3) compound I or a pharmaceutically acceptable salt thereof, which is used for the treatment of acute myeloid leukemia. Daunorubicin, cytarabine and Compound I or a pharmaceutically acceptable salt thereof can be administered simultaneously or separately. The administration may be oral administration, injection administration, topical administration or in vitro administration.

In some embodiments, the treatment is:

In some embodiments, Compound I is administered in the form of a hydrochloride salt, preferably trihydrochloride, more preferably trihydrochloride pentahydrate. Specifically: Oral administration of Compound I trihydrochloride about 25 mg/time to about 200 mg/time, twice a day (BID), daily. Preferably, from about 50 mg to about 175 mg, more preferably from about 50 mg to about 150 mg, per oral administration. Examples of each oral dose include about 50 mg, or about 75 mg, or about 100 mg, or about 125 mg, or about 150 mg or about 175 mg, or about 200 mg. The dosage of the trihydrochloride salt is in anhydrous form.

In some embodiments, the acute myeloid leukemia refers to FLT3 mutation-positive acute myeloid leukemia. More preferred is untreated FLT3 mutation-positive acute myeloid leukemia. In some embodiments, the FLT3 mutation-positive comprises FLT3 internal tandem repeat (ITD) mutation-positive, FLT3 tyrosine kinase domain (TKD)/D835 mutation-positive, and/or FLT3 tyrosine kinase domain (TKD) /I836 mutation positive.

Compound I or a pharmaceutically acceptable salt thereof, daunorubicin and cytarabine can be contained in the same pharmaceutical preparation, or can be separately prepared into clinically acceptable preparations, and the medicine can be prepared in a combined packaging manner. The clinically accepted preparations include oral preparations, injection preparations, topical preparations, external preparations and the like. In some embodiments, the medicament is in a single-dose or divided-dose form. The dosage form contains a therapeutically effective amount of daunorubicin, cytarabine, Compound I, or a pharmaceutically acceptable salt thereof.

On the other hand, the present invention also provides a medicament containing Compound I or a pharmaceutically acceptable salt thereof, which is used to improve the curative effect of cytarabine and daunorubicin in the treatment of acute myeloid leukemia. The improved method is: combined use of compound I or a pharmaceutically acceptable salt thereof on the basis of treatment with cytarabine and daunorubicin. Daunorubicin, cytarabine and Compound I or a pharmaceutically acceptable salt thereof may be administered simultaneously or separately. The administration may be oral administration, injection administration, topical administration or in vitro administration.

In some embodiments, the method of improvement is:

In some embodiments, the medicament is prepared into clinically acceptable formulations, such as oral formulations, injection formulations, topical formulations, topical formulations, etc., using conventional excipients and conventional preparation methods in the art. Oral formulations such as tablets, capsules, pills and the like are preferred.

The present invention also provides a kit, which can be used in the aforementioned method.

In some embodiments, the kit includes (1) daunorubicin; (2) cytarabine; (3) compound I or a pharmaceutically acceptable salt thereof.

In some embodiments, the kit comprises one or more containers comprising (1) daunorubicin; (2) cytarabine; (3) Compound I or a pharmaceutically acceptable of salt.

In some embodiments, the kit includes the composition: (1) daunorubicin; (2) cytarabine; (3) Compound I or a pharmaceutically acceptable salt thereof.

In some embodiments, the kit further includes instructions for use according to any of the methods described herein. The kit may also include instructions for selecting an individual suitable for treatment. The instructions provided in the kits of the invention are typically written instructions on a label or package insert (eg, a sheet of paper included in the kit), but machine-readable instructions (eg, instructions carried on a magnetic disk or compact disc) Also acceptable.

In some embodiments, daunorubicin, cytarabine, compound I, or a pharmaceutically acceptable salt thereof, are separately formulated into a clinically acceptable formulation and are present in the kit in a combined package. Wherein, the preparation containing Compound I or a pharmaceutically acceptable salt thereof is preferably an oral preparation, such as tablets, capsules, pills, etc.; each preparation unit contains about 0.001 mg to about 1000 mg of Compound I or its pharmaceutically acceptable salts Accepted salts (based on Compound I), preferably about 1 mg to about 500 mg, or about 10 mg to about 400 mg, or about 15 mg to about 330 mg, or about 20 mg to about 300 mg, or about 20 mg to about 250 mg, or about 20 mg to about 210 mg, or about 20 mg to about 180 mg, or about 20 mg to about 160 mg, or about 20 mg to about 140 mg, or about 20 mg to about 120 mg, or about 20 mg to about 100 mg. Exemplary amounts are, for example, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 75 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 125 mg, about 130 mg, About 140 mg, about 150 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 250 mg, about 300 mg, about 310 mg, about 320 mg, about 360 mg, about 400 mg. The daunorubicin preparation is preferably daunorubicin hydrochloride for injection, such as daunorubicin hydrochloride lyophilized powder for injection; each preparation unit contains about 10 mg to about 30 mg of active ingredient, preferably about 20 mg. The cytarabine preparation is preferably cytarabine hydrochloride for injection, such as cytarabine hydrochloride lyophilized powder for injection; each preparation unit contains about 0.1g to about 0.5g of active ingredients, such as about 0.1g, about 0.3 g or about 0.5 g.

In some embodiments, the (1) daunorubicin; (2) cytarabine; (3) Compound I, or a pharmaceutically acceptable salt thereof, may be present in separate containers or in a single container.

The kits of the present invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (eg, seled Mylar or plastic bags) and the like. The kit may optionally provide other components such as buffers and instructional information.

In the technical solution of the present application, "about" before a numerical value refers to ±10% of the numerical value, preferably ±5%, for example, ±10%, ±9%, ±8%, ±7%, ±5% of the numerical value 6%, ±5%, ±4%, ±3%, ±2% or ±1%.

Preclinical studies have shown that Compound I or a pharmaceutically acceptable salt thereof has a strong growth inhibitory effect on FLT3-ITD-mutated acute myeloid leukemia cells. Preliminary clinical studies have shown that Compound I or its pharmaceutically acceptable salts have a certain curative effect on FLT3-mutated relapsed/refractory AML, which is close to the early trial data of similar product Gilteritinib; Cardiac, liver, hematological adverse reactions, and the incidence of adverse reactions above grade 3 is low.

In order to evaluate the efficacy and safety of the "7+3" standard chemotherapy regimen of compound I or its pharmaceutically acceptable salts combined with daunorubicin and cytarabine in the treatment of human acute myeloid leukemia, the inventors of the present application developed a In vitro studies and clinical trials. The results showed that compound I combined with daunorubicin and cytarabine could significantly inhibit the proliferation of human leukemia cells MV-4-11 and MOLM-13, and produced a synergistic effect. The remission rate of compound I combined with "7+3" standard chemotherapy regimen is better than that of "7+3" standard chemotherapy regimen alone, and will bring longer survival period to patients. The potential of the combination therapy regimen to improve the prognosis is far greater than the risk of potential toxicity, and it has a good clinical application prospect.

Detailed ways

The following examples are for the purpose of better illustrating the content of the present invention, but the content of the present invention is not limited to the examples. Those skilled in the art make non-essential improvements and adjustments to the embodiments according to the above-mentioned contents of the invention, which still belong to the protection scope of the present invention.

Example 1 Inhibitory effect of compound I combined with daunorubicin and cytarabine on the proliferation of human leukemia cells MV-4-11 and MOLM-13

1. Research methods

1. Research purpose

Comparing the inhibitory effect of compound I combined with daunorubicin and cytarabine with compound I alone or only with daunorubicin and cytarabine on the proliferation of human leukemia cells MV-4-11 and MOLM-13, evaluating compounds Is there any synergistic effect of I combined with daunorubicin and cytarabine?

2. Trial drug

Compound I: provided by CSPC Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., Ltd. Weigh an appropriate amount of compound I, dissolve it completely with an appropriate amount of DMSO, and prepare a 10 mM stock solution; before use, it is gradiently diluted to 1.25nM, 2.5nM, 5nM, 10nM or 20nM with complete medium as the test solution. And take the complete medium as the test solution containing compound 10nM.

Daunorubicin, Cytarabine: Provided by CSPC Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., Ltd. Weigh an appropriate amount of cytarabine and daunorubicin, dissolve DMSO completely, and prepare a stock solution; before use, prepare a mixed drug solution according to the molar concentration ratio of cytarabine: daunorubicin = 5:1, and use it completely. The medium was serially diluted to 3.9nM, 7.8nM, 15.625nM, 31.25nM and 62.5nM (calculated as daunorubicin) as a mixed test solution. And take the complete medium as a mixed test solution containing cytarabine and daunorubicin at 0 nM.

3. Test method

Cells in logarithmic growth phase were seeded in 96-well plates (100 μL/well). Add 50 μL of compound I test solution and cytarabine and daunorubicin mixed test solution to each well to cross-act each drug concentration, and each action point has three duplicate wells, and set up corresponding blank wells (only medium) and normal wells (drug concentration of 0). After 72 hours of dosing and culture, add 20 μl of MTT to each well, remove the supernatant after culturing for 4 hours in the incubator, add 150 μL of DMSO per well, shake the microwells to mix, and use a microplate reader to detect the optical density value (OD) at a wavelength of 550 nm. ).

calculate:

Inhibition rate (%) = (well with normal OD value - well with OD value administration)/(well with normal OD value - blank well with OD value) × 100;

According to the inhibition rate of each concentration, use SPSS19.0 to calculate the IC50 value of the drug's half inhibitory concentration;

Combined action index CI = IC50 _(C1) /IC50 ₍₁₎ +IC50 _(C2) /IC50 ₍₂₎

Among them, IC50 ₍₁₎ is the concentration corresponding to the inhibition of half cell proliferation by compound I alone; IC50 ₍₂₎ is the concentration corresponding to half cell inhibition by only daunorubicin and cytarabine; IC50 _(C1) is the concentration of compound I corresponding to half cell inhibition after compound I combined with daunorubicin and cytarabine; IC50 _(C2) is the half of compound I produced by combined use of daunorubicin and cytarabine Cell inhibition corresponding to daunorubicin and cytarabine concentrations.

The CI value is calculated according to the above formula, and the meanings are as follows: CI>1.1 is an antagonistic effect; 0.9≤CI≤1.1 is an additive effect; CI<0.9 is a synergistic effect, and the smaller the CI value, the higher the synergistic effect of the two drugs.

2. Research results

The results showed that, for human leukemia cells MV-4-11 and MOLM-13, when compound I was used in combination with daunorubicin and cytarabine, the CI values were all less than 0.9 (see Table 1). It shows that compound I has synergistic effect in combination with daunorubicin and cytarabine.

Table 1 Synergistic effect of compound I combined with daunorubicin and cytarabine

Example 2 A single-arm, multi-center, open-label, dose-escalation/expansion phase I/II study of compound I or its pharmaceutically acceptable salt in combination with "7+3" standard chemotherapy in the treatment of FLT3-mutated newly diagnosed AML patients

1. Purpose of the test

To evaluate the safety, tolerability and efficacy of compound I or its pharmaceutically acceptable salt combined with "7+3" standard chemotherapy regimen in the treatment of newly diagnosed AML subjects with FLT3 mutation.

2. Experimental Design

This study is a single-arm, open-label, multicenter trial designed to evaluate oral administration of Compound I or a pharmaceutically acceptable salt thereof in combination with daunorubicin and cytarabine for the treatment of FLT3 in subjects with newly diagnosed FLT3-mutated AML. Safety, tolerability, and pharmacokinetics in subjects with newly diagnosed AML mutations and to explore optimal dosing regimens.

This study will adopt the "7+3" dosing regimen of compound I trihydrochloride combined with standard daunorubicin and cytarabine, which is divided into induction period, consolidation period and maintenance period. Phase I phase: 3+3 dose escalation, planned to enroll 9-18 subjects; Phase II phase: in the safe, tolerated and effective dose group, the number of cases in each group will be expanded to 20 cases. The specific experimental design is as follows:

2.1 Phase I (dose escalation)

Initially set up 3 climbing doses: 100mg, 125mg, 150mg; all are twice a day (BID).

Compound I trihydrochloride started to climb from the 100 mg BID dose group, and 3 subjects were enrolled first. If no DLT occurs during the first cycle of the induction period, continue with dose escalation. If there is 1 case of DLT in this dose group, 3 subjects should be added in this dose group; if no DLT occurs in these 3 subjects, enter the next dose group; if there is 1 case or more cases DLT, or if 2 or more patients in a dose cohort developed DLT, consider discontinuing dose escalation for that cohort.

Definition of DLT

Non-hematological toxicity DLT: Any ≥ grade 3 non-hematological toxicity or non-hematological toxicity that is definitely related, likely related, possibly related to the study drug occurring in the Phase I period from the first dose of the induction period to the end of the first induction period (within 42 days) Unexpected extramedullary toxicity. But exclude the following cases:

Hair loss, fatigue, anorexia, nausea, vomiting, electrolyte abnormalities;

Grade 3 diarrhea can be controlled after symptomatic treatment;

Grade 3 febrile neutropenia, with or without infection;

• Grade 3 infection.

Hematological toxicity DLT: Long-term myelosuppression, if there is no evidence of leukemia (blasts <5%), persistent myelosuppression after the initiation of the first induction phase therapy (including at least one of the following: ≥ Grade 4 leukopenia , ≥Grade 4 neutropenia, ≥Grade 4 thrombocytopenia), if not resolved within 42 days of the first induction phase treatment or before the start of the consolidation phase, will be considered a DLT.

When the DLT observation period for the last subject of Phase I dose escalation ends, an interim analysis will be conducted by mutual agreement between the investigator and the sponsor. In the mid-term, the safety, PK, and CRc rate at the end of the first or second induction period will be analyzed, and a safe and effective combination regimen will be recommended for the second phase.

2.2 Phase II (expansion of the number of cases)

In order to further fully determine the safety and PK characteristics of the dosing regimen of compound I trihydrochloride in combination, and to preliminarily confirm the effectiveness of the optimal drug dosing regimen, to provide a basis for the design of phase III clinical trials, in the effective and safe The effective dose group was expanded in the dosing schedule, and the number of cases in each group was expanded to 20, up to 40 cases. The DLT observation period is no longer set in the case number expansion stage.

3. Test population

3.1 Inclusion criteria:

1) Voluntarily test and sign the informed consent;

2) Male or female Chinese patients, aged ≥18 years and <60 years old;

3) Morphological diagnosis of primary AML with blast cells > 20% in accordance with the World Health Organization (WHO) 2016 classification;

4) Those with positive FLT3 mutation in the whole blood of subjects tested by the central laboratory, including FLT3 internal tandem repeat (ITD), FLT3 tyrosine kinase domain (TKD)/D835 or FLT3-TKD/I836 mutation positive;

5) ECOG score≤2;

6) Laboratory inspection:

a. Serum AST and ALT≤3×ULN; b. Total serum bilirubin≤2.5×ULN; c. Serum creatinine≤3×ULN or glomerular filtration rate>30mL/min.

7) Subjects can accept standard "7+3" induction chemotherapy regimen;

8) The subject can take the test drug orally;

9) Female/male subjects of childbearing age should take adequate non-drug contraceptive measures from the signing of the informed consent form until 180 days after the last dose, and should not donate sperm or eggs.

3.2 Exclusion criteria:

1) Confirmed acute promyelocytic leukemia (M3 type/APL), or BCR-ABL positive leukemia (ie, chronic myeloid leukemia blast crisis); received APL diagnostic workup and all-trans retinoic acid (ATRA) therapy However, subjects without APL were eligible for inclusion (ATRA treatment must be stopped 24h before induction chemotherapy was started).

2) Diagnosed with active malignant tumors other than AML;

3) AML secondary to other tumors treated with radiotherapy or chemotherapy;

4) AML with central nervous system involvement (defined as cases with clinical symptoms highly suspected of central involvement, or imaging evidence supporting the judgment);

5) Refractory hypokalemia or hypomagnesemia that is difficult to correct after symptomatic treatment and has recurred in the past;

6) Currently suffering from clinically significant graft-versus-host disease (GVHD) or receiving systemic cortisol hormone therapy for GVHD;

7) History of other malignant tumors in the past (except for the following cases: those without disease status for more than 5 years; non-melanoma skin cancer, cervical or breast carcinoma in situ [regardless of disease status] who have completed treatment; localized prostate cancer after radiotherapy or tumors that are expected to be cured after treatment, such as those without recurrence and progression after surgical treatment);

8) Patients in the screening period have clinically significant coagulation abnormalities, such as disseminated intravascular coagulation (DIC), hemophilia A, hemophilia B, and von Willebrand disease;

9) Major surgery on major organs has been performed within 4 weeks before entering the study (the definition of major surgery refers to the Class 3 and Class 4 surgeries specified in the “Administrative Measures for the Clinical Application of Medical Technology”); Full recovery during operation;

10) Received radiotherapy within 4 weeks before entering the study;

11) The subject has received prior therapy for AML, except for the following: a. Emergency leukocytosis; b. Hydroxyurea for emergency treatment of leukocytosis ≤ 10 days; c. Growth factor or cytokine support; d. Steroid use for allergic reactions or blood transfusion reactions;

12) Those who are currently suffering from New York Heart Association (NYHA) grade 3 or 4 congestive heart failure, or have a history of NYHA grade 3 or 4 congestive heart failure, unless echocardiography was performed within 1 month before entering the study, showing Left ventricular ejection fraction ≥ 45%, otherwise they are not allowed to participate in the study;

13) Currently suffering from bradycardia, the heart rate is less than 50 beats/min, except for subjects using pacemakers;

14) The average value of the QT interval (QTcF) corrected by the Fridericia formula was detected by three electrocardiograms during the screening period, male>450ms, female>470ms;

15) Diagnosis or suspicion of long QT syndrome during the screening period (including a family history of long QT syndrome);

16) History of second-degree (Mobita II) or third-degree atrioventricular block (except subjects using pacemakers);

17) History of uncontrolled angina pectoris or myocardial infarction within 6 months before screening;

18) Complete left bundle branch block in the screening period;

19) New clinically significant arrhythmias (except sinus tachycardia caused by anemia, infection and AML) or subjects with previous arrhythmias that require prolonged use of drugs that may prolong the QT interval;

20) Currently suffering from active or uncontrolled infection;

21) Hepatitis B surface antigen positive or previous history of hepatitis B, and HBV-DNA ≥ 2000IU/ml in the past 3 months; hepatitis C antibody positive, HCV-RNA positive in the past 3 months;

22) Those with positive anti-human immunodeficiency virus antibody or anti-Treponema pallidum specific antibody test;

23) The subject has taken a strong inducer or inhibitor of CYP2C8 and CYP3A4 enzymes within two weeks before taking the test drug;

24) Pregnant women (positive blood pregnancy test during screening) and lactating women;

25) There is a known history of immediate or delayed hypersensitivity reactions to similar drugs and excipients of the study drugs (daunorubicin, cytarabine) combined in this study.

26) Patients deemed by the investigator to be incapable of entering the study.

4. Drugs

(1) Compound I trihydrochloride: administered in the form of compound I trihydrochloride pentahydrate, provided by CSPC Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., Ltd. The drug doses described in this clinical study were calculated as the trihydrochloride anhydrate.

(2) Cytarabine: Cytarabine hydrochloride injection freeze-dried powder preparation, provided by CSPC Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., Ltd.

(3) Daunorubicin: Daunorubicin hydrochloride injection freeze-dried powder preparation, provided by CSPC Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., Ltd. 5. Treatment

5.1 Phase I Dosing Schedule

5.1.1 Remission induction therapy (up to 2 cycles, every 28 days)

(1) First induction therapy

Days 1-7: Cytarabine 100 mg/m ² continuous intravenous infusion (CIVI) for 7 days (total 168 h).

Days 1-3: daunorubicin 60mg/ ^m2 intravenously (IVP) or short infusion;

Days 8-21: Compound I trihydrochloride 100 mg/time or 125 mg/time or 150 mg/time, orally, twice a day (BID) for 14 consecutive days.

Efficacy evaluation after the first induction therapy: On the 28th day, the bone marrow and blood images were checked to evaluate the efficacy to determine the next treatment. If bone marrow aspirate is insufficient for evaluation, repeat bone marrow evaluation within one week. If CRc is obtained, enter into post-remission consolidation therapy; if CRc is not obtained, enter into the second induction therapy. In either case, a bone marrow examination to demonstrate CRc must be performed on or before day 42 after the first induction therapy.

(2) Second induction therapy

The treatment regimen is the same as the first induction treatment, unless the dose is reduced due to safety.

Efficacy evaluation after the second induction therapy: On the 28th day, the bone marrow and blood images were checked to evaluate the efficacy to determine the next treatment. If bone marrow aspirate is insufficient for evaluation, repeat bone marrow evaluation within one week. If CRc is obtained, consolidation therapy is performed after remission; if CRc is not obtained after the second induction therapy, the induction therapy fails, and the follow-up in the survival period is entered. In either case, a bone marrow examination to demonstrate CRc must be performed on or before day 42 after the second induction therapy. If CRc is achieved after 42 days, the investigator will decide whether to accept the consolidation phase treatment according to the benefit ratio of the subjects.

5.1.2 Consolidation therapy (up to 4 cycles, every 28 days)

Days 1, 3, and ⁵ : continuous intravenous infusion of high-dose cytarabine (HiDAC) 3g/m2, q12h.

Day 8-21: Oral compound I trihydrochloride, in principle, the dose is the same as the dose of the previous cycle, twice a day (BID), for 14 consecutive days.

For patients who can enter consolidation therapy, adverse events must be recovered to ≤ grade 1, and CRc must be achieved at the end of induction therapy. If the recovery is delayed up to day 56 due to chronic toxicity and hemogram recovery, the investigators will be based on the subject's experience. benefit ratio to decide whether to continue treatment.

According to the remission of the subjects, the investigator determines the number of cycles for the subjects to receive induction therapy and consolidation therapy.

5.1.3 Allogeneic hematopoietic stem cell transplantation (HSCT)

Subjects who obtain CRc after induction therapy, if they have a suitable bone marrow match, can undergo allogeneic hematopoietic stem cell transplantation (HSCT) without the need for group out. While the subjects are waiting for HSCT, they can receive consolidation therapy first, up to a maximum of 4 cycles. However, the trial drug should be discontinued prior to initiation of a conditioning regimen for HSCT, followed by a pre-HSCT treatment/end of treatment visit within 7 days of discontinuation.

5.1.4 Maintenance therapy (up to 12 cycles, every 28 days)

Oral compound I trihydrochloride, in principle, the dose is the same as the dose of the previous cycle, twice a day (BID), taken daily, unless the subject meets the criteria for discontinuing the drug or withdrawing from the study, the subject can receive up to the maintenance period treatment 12 cycles.

5.2 Phase II dosing schedule

Based on the results of the interim analysis in Phase I, an effective and safe combination dosing regimen will be determined.

5.3 Dose Adjustment and Administration

If an unexpected adverse event is encountered, in order to ensure the safety of the subject and make the subject eligible to continue to participate in the study, with the discussion and consent of the investigator and the sponsor, it is allowed to adjust the toxicity dose with the provided protocol or be The recommended supportive care deviates slightly. No dose adjustment is allowed during the DLT observation period, but the drug can be suspended. In principle, after adverse events occur in the study, the first choice is to adjust the dose of "7+3" chemotherapeutic drugs, and maintain compound I trihydrochloride to continue the administration. Dosage of salt.

(1) Daunorubicin: If the total bilirubin (TBIL) is >2 times ULN, the DNR can be down-regulated from 60 mg/m ² to 45 mg/m ² in the second induction period.

(2) High-dose cytarabine (HiDAC): ① neurotoxicity: the first occurrence of neurotoxicity adverse reactions of grade ≥ 2 during the consolidation period, HiDAC was discontinued during the remaining cycle, if the neurotoxicity was relieved and decreased to grade ≤ 1, The dose of HiDAC was then down-regulated from 3 g/m ² to 2 g/m ² in the next consolidation cycle. In the event of a second grade ≥2 neurotoxic adverse reaction, HiDAC will be permanently discontinued in this regimen. ②Hematological toxicity occurs: If ANC cannot reach 1.0×10 ⁹ /L and PLT cannot reach 100×10 ⁹ /L within 8 weeks of treatment in the consolidation phase, HiDAC will be lowered to 1.5 g/m ² in the next cycle. If ANC ≥ 1.0×10 ⁹ /L and PLT ≥ 100×10 ⁹ /L in one cycle, the consolidation phase treatment will be stopped.

(3) Compound I trihydrochloride: ① Induction period and consolidation period: Compound I trihydrochloride will not undergo dose adjustment due to adverse reactions, but the medication can be suspended. ②Maintenance period: the dose can be reduced due to ADR; after the dose reduction due to ADR, the dose can be gradually increased when ADR recovers. Compound I trihydrochloride dose adjustment gradient is as follows:

	①①	②②	③③	④④	⑤⑤	⑥⑥
早morning	150mg150mg	125mg125mg	100mg100mg	75mg75mg	50mg50mg	停药Withdrawal
晚Night	150mg150mg	125mg125mg	100mg100mg	75mg75mg	50mg50mg	停药Withdrawal

5.4 Concomitant treatment:

The use of CYP2C8 and CYP3A4 enzymes and strong inducers or inhibitors or substrates of P-gp should be avoided as much as possible in the trial, and the use of concomitant drugs known to prolong the QT or QTc interval should be avoided.

During the trial, in addition to hydroxyurea (QD, up to 2 weeks, for lowering white blood cell counts, usually allowed when white blood cell counts >10 x 10 ⁹ /L) and HSCT treatment plan (prophylactic intrathecal chemotherapy, cranial radiotherapy, and donor In addition to lymphocyte infusion), concomitant use of other AML treatments (including but not limited to chemotherapy, radiotherapy, surgery, immunotherapy or cell therapy, and traditional Chinese medicine) is prohibited. Concurrent participation in another interventional clinical trial is prohibited.

6. Pharmacokinetic studies

Blood collection time: (1) Induction period: C1D8 (before the first dose and 0.5h, 1h, 1.5h, 2h, 4h, 8h, 12h after the first dose), C1D15 (before the first dose), C1D18 (before the first dose), C1D21 ( Only administered once in the morning, before and 0.5h, 1h, 1.5h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, 96h); (2) Consolidation period: C1D8, C1D21 and C4D8, C4D21 Before the first dose; (3) Maintenance period: before the first dose of C8D1 and C12D1. The venous blood was collected, the blood concentration was detected, and the pharmacokinetic study was carried out.

7. Evaluation indicators

7.1 Safety and Tolerability

Adverse events, vital signs, laboratory tests, physical examinations, twelve-lead electrocardiograms (ECGs), ECOG scores, and the presence of DLT were included. Assessed according to definitions and methods routine in the art.

7.2 Pharmacokinetic characteristics

1) Induction period: AUC0-t, Cmax, Tmax, Ctrough, Cmax, ss, Tmax, ss, AUC0-inf, AUCss, t1/2, Ra(AUC), Ra(Cmax) of C1D8, C1D15, C1D18, C1D21 Wait;

2) Consolidation period: Ctrough of C1D8, C1D21 and C4D8, C4D21;

3) Maintenance period: Ctrough of C8D1 and C12D1.

Calculated according to the definitions and methods conventional in the art.

7.3 Efficacy evaluation indicators

Efficacy evaluation criteria refer to the revised [Cheson et al, "Revised Recommendations of the International Working Group for Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia", J Clin Oncol 21:4642-4649.2003 by American Society of Clinical Oncology.] and Criteria for the Diagnosis and Efficacy of Blood Disorders, 4th Edition.

1) CRc rate at the end of the induction period: the number of subjects with the best response at the end of the first or second induction period was CRc (CR+CRh+CRp+CRi) divided by the total number of cases in the protocol analysis set.

2) CR rate at the end of the induction period: the number of subjects with the best response at the end of the first or second induction period was divided by the total number of cases in the protocol analysis set.

3) Duration of remission (DOR): including CRc duration (CR+CRh+CRp+CRi), CR duration, CR/CRh duration, and CR/CRi duration. See revised [Cheson et al, 2003].

4) Overall survival (OS): defined as the time from the first administration to death from any cause.

5) Event-Free Survival (EFS): defined as the time from the first administration to the occurrence of treatment failure (the CRc was not reached at the end of the second induction treatment) or relapse or death due to any cause (the previous whichever occurs).

6) Leukemia-free survival (LFS): the time from first reaching CRc to relapse or death. For subjects with unknown recurrence or death, LFS was performed as of the date of the last recurrence-free disease assessment.

7) Hematopoietic stem cell transplantation rate: defined as the number of subjects undergoing hematopoietic stem cell transplantation divided by the total number of cases in the corresponding analysis set.

8. Statistical analysis

Continuous variables were compared between groups using t test or Wilcoxon rank sum test; categorical variables were compared between groups using chi-square test or Fisher's exact test.

Unless otherwise specified, the hypothesis test will use a two-sided test, with a test level of 0.05, and a 95% confidence interval for parameter estimation.

9. Test results

Compound I or its pharmaceutically acceptable salts (such as trihydrochloride) combined with the "7+3" standard chemotherapy regimen in the treatment of newly diagnosed AML with FLT3 mutation has a better response rate than the "7+3" standard chemotherapy regimen alone. Longer survival time for patients. This combination regimen is safe and tolerable, and the potential to improve outcomes far outweighs the risk of potential toxicity.

Example 3 A single-arm, multi-center, open-label, dose-escalation/expansion phase I/II study of compound I or its pharmaceutically acceptable salt combined with "7+3" standard chemotherapy in the treatment of newly diagnosed AML patients

The purpose of this study was to evaluate the safety, tolerability and efficacy of Compound I or its pharmaceutically acceptable salt combined with "7+3" standard chemotherapy regimen in the treatment of newly diagnosed AML subjects.

The inclusion criteria for the trial population were as follows:

1) Voluntarily test and sign the informed consent;

2) Male or female Chinese patients, aged ≥18 years and <60 years old;

4) ECOG score≤2;

5) Laboratory inspection:

6) Subjects can accept standard "7+3" induction chemotherapy regimen;

7) The subject can take the test drug orally;

8) Female/male subjects of childbearing age should take adequate non-drug contraceptive measures from the signing of the informed consent form until 180 days after the last administration, and should not donate sperm or eggs.

Except for the above-mentioned inclusion criteria of the experimental population, the experimental design, exclusion criteria of the experimental population, drugs, treatment methods, pharmacokinetic research methods, evaluation indicators, and statistical analysis methods are the same as those in Example 2.

The results of the study showed that the remission rate of compound I or its pharmaceutically acceptable salts (such as trihydrochloride) combined with the "7+3" standard chemotherapy regimen in the treatment of newly diagnosed AML was better than that of the "7+3" standard chemotherapy regimen alone, will give patients a longer survival period. This combination regimen is safe and tolerable, and the potential to improve outcomes far outweighs the risk of potential toxicity.

The following are typical cases for this study:

Case 1: acute myeloid leukemia (with BCORL1 mutation, poor prognosis)

The patient, male, 38 years old, was diagnosed with recurrent cough and fever for more than 1 month. Blood routine examination: WBC: 4.15×10 ⁹ /L, NE: 0.38×10 ⁹ /L, Hgb: 79g/L, Plt: 199×10 ⁹ /L; Bone marrow morphology: obvious hyperplasia, primitive naive monocytes 66%, considering AML-M5a; flow: acute myeloid leukemia (non-M3) immunophenotype. The patients were treated according to the phase I dosing regimen of the clinical trial (100 mg, BID dose group), and the efficacy was evaluated as CR (complete remission) after the first induction therapy. After two cycles of consolidation therapy, the patient underwent bone marrow transplantation.

Case 2: Acute myeloid leukemia (with CEBPA double mutation, CBL mutation)

The patient, a 49-year-old female, was admitted to the hospital mainly due to the discovery of thrombocytopenia for 1 week. Blood routine examination at admission: WBC: 6.88×10 ⁹ /L, NE: 0.65×10 ⁹ /L, Hgb: 135g/L, Plt: 64×10 ⁹ /L; Bone marrow morphology: acute myeloid leukemia (not M3) ; Flow: Consistent with the AML phenotype. The patients were treated according to the phase I dosing regimen of the clinical trial (100 mg, BID dose group), and the efficacy was evaluated as CR (complete remission) after the first induction therapy. Then enter the consolidation treatment.

The preparation of embodiment 4 compound I trihydrochloride pentahydrate

With reference to the method described in Example 90 in patent document WO2011/147066A1, 100 g of compound I represented by formula I was prepared.

Compound I (90g, 0.203mol) shown in formula I, 800ml of purified water and 400ml of acetone were added to the reactor, and after stirring and warming up to 40±5°C, (74g, 0.731mol) concentrated hydrochloric acid was added to the reactor, and the addition was complete. After concentrated hydrochloric acid, add 2L acetone, keep the temperature at 40±5℃, continue the reaction for 1 hour, then stir and cool down to 10±5℃ for 2 hours, filter with suction, and wash the filter cake with 300ml acetone to obtain yellow or light yellow hydrochloride 74.7g. ¹ H-NMR(600MHz,D2O)δ:1.556(d,6H),δ:2.896(s,3H),δ:3.058(t,2H),δ:3.187(t,2H),δ:3.586(d ,2H),δ:3.749(d,2H),δ:4.701(s,1H),δ:7.062(d,2H),δ:7.377(d,2H),δ:7.968(t,1H),δ : 8.086(s, 1H), δ: 8.431(d, 1H), δ: 8.636(d, 1H), δ: 9.171(s, 1H).

Calculate the free base content by HPLC, determine the moisture content, and then according to the ratio of hydrogen in 1H-NMR in nuclear magnetic resonance (see table below), it can be inferred that the base/acid/H ₂ O of the hydrochloride is 1:3:5. That is, in Example 4, the trihydrochloride·pentahydrate of Compound I was obtained.

Abbreviations and terms used in the present invention are as follows:

The embodiments of the present invention have been described above. However, the present invention is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

Use of compound I or a pharmaceutically acceptable salt thereof, daunorubicin and cytarabine in the preparation of a medicament for the treatment of acute myeloid leukemia (AML), the structure of said compound I is shown in the following formula (I):
The use according to claim 1, wherein the acute myeloid leukemia is preferably FLT3 mutation-positive acute myeloid leukemia, more preferably untreated FLT3 mutation-positive acute myeloid leukemia; preferably, the FLT3 Mutation positives include FLT3 internal tandem repeat (ITD) mutation positive, FLT3 tyrosine kinase domain (TKD)/D835 mutation positive, and/or FLT3 tyrosine kinase domain (TKD)/I836 mutation positive.
The use according to claim 1, wherein the acute myeloid leukemia is FLT3 mutation-negative acute myeloid leukemia.
The use according to any one of claims 1-3, wherein the acute myeloid leukemia is accompanied by one, two or more of the following gene mutations: KIT mutation, CEBPA mutation, NPM1 mutation, PHF6 mutation, ASXL1 mutation, DNMT3A mutation, IDH1 mutation, IDH2 mutation, TET2 mutation, CBL mutation, TP53 mutation, BCORL1 mutation, EZH2 mutation, MLL mutation, SRSF2 mutation, SF3B1 mutation, U2AF1 mutation, ZRSR2 mutation, SF3A1 mutation, PRPF40B mutation , U2AF2 mutation, SF1 mutation, etc., preferably BCORL1 mutation, CEBPA mutation, CBL mutation, TP53 mutation.
The use according to any one of claims 1-4, wherein the pharmaceutically acceptable salts of compound I include but are not limited to the hydrochloride, mesylate, malate, tartrate, Oxalate, succinate, acetate, sulfate, etc.; preferably hydrochloride, malate, tartrate, oxalate, succinate, acetate; more preferably hydrochloride; the hydrochloric acid The salt is further preferably trihydrochloride; the trihydrochloride includes the corresponding anhydrate, hydrate, solvate, etc., more preferably trihydrochloride pentahydrate.
The use according to any one of claims 1-5, characterized in that: Compound I or a pharmaceutically acceptable salt thereof, daunorubicin and cytarabine are contained in the same pharmaceutical preparation, or are prepared separately for clinical The received preparation is made into the medicine in a combined package; the preparations include oral preparations, injection preparations, topical preparations, external preparations, etc.; preferably, the medicine is in a single-dose dosage form or a divided-dose dosage form.
The use according to claim 6, characterized in that: daunorubicin, cytarabine, compound I or a pharmaceutically acceptable salt thereof are respectively prepared into clinically acceptable preparations, and the medicine is prepared in a combined packaging mode. ;

The preparation containing Compound I or a pharmaceutically acceptable salt thereof is preferably an oral preparation, such as tablets, capsules, pills, etc.; each preparation unit contains about 0.001 mg to about 1000 mg of Compound I or a pharmaceutically acceptable salt thereof. Salt (calculated as compound I), preferably about 1 mg to about 500 mg, or about 10 mg to about 400 mg, or about 15 mg to about 330 mg, or about 20 mg to about 300 mg, or about 20 mg to about 250 mg, or about 20 mg to about 210 mg, or about 20 mg to about 180 mg, or about 20 mg to about 160 mg, or about 20 mg to about 140 mg, or about 20 mg to about 120 mg, or about 20 mg to about 100 mg; exemplary amounts such as about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 75 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 125 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 250 mg, about 300 mg, about 310 mg, about 320 mg, about 360 mg, about 400 mg;

The daunorubicin preparation is preferably daunorubicin hydrochloride for injection, such as daunorubicin hydrochloride lyophilized powder for injection; each preparation unit contains about 10mg-about 30mg of active ingredient, preferably about 20mg;

The cytarabine preparation is preferably cytarabine hydrochloride for injection, such as cytarabine hydrochloride lyophilized powder for injection; each preparation unit contains about 0.1g to about 0.5g of active ingredients, such as about 0.1g, about 0.3 g or about 0.5 g.
The use according to claim 7, characterized in that: the pharmaceutically acceptable salt of compound I is hydrochloride, preferably trihydrochloride, more preferably trihydrochloride pentahydrate; each preparation unit contains the compound I trihydrochloride about 1 mg to about 500 mg, or about 10 mg to about 400 mg, or about 15 mg to about 350 mg, or about 20 mg to about 310 mg, or about 25 mg to about 300 mg, or about 25 mg to about 250 mg, or about 50 mg- about 250 mg, or about 50 mg to about 200 mg, or about 50 mg to about 175 mg, or about 50 mg to about 160 mg, or about 50 mg to about 150 mg, or about 50 mg to about 125 mg, or about 50 mg to about 100 mg; exemplary amounts such as about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 160 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg , about 310 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg; the above-mentioned compound I trihydrochloride is measured in the form of anhydrous.
Use of compound I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving the curative effect of daunorubicin and cytarabine in the treatment of acute myeloid leukemia, the structure of compound I is shown in the following formula (I):

The acute myeloid leukemia is preferably FLT3 mutation-positive acute myeloid leukemia, more preferably untreated FLT3 mutation-positive acute myeloid leukemia; preferably, the FLT3 mutation-positive includes FLT3 internal tandem repeat (ITD) mutation-positive, FLT3 tyrosine kinase domain (TKD)/D835 mutation positive, and/or FLT3 tyrosine kinase domain (TKD)/I836 mutation positive;

Alternatively, the acute myeloid leukemia is preferably FLT3 mutation-negative acute myeloid leukemia.
The use according to claim 9, characterized in that: the pharmaceutically acceptable salts of compound I include but are not limited to the hydrochloride, mesylate, malate, tartrate, oxalate, succinic acid of compound I salt, acetate, sulfate, etc.; preferably hydrochloride, malate, tartrate, oxalate, succinate, acetate; more preferably hydrochloride; further preferred hydrochloride is trihydrochloric acid Salt; the trihydrochloride includes the corresponding anhydrate, hydrate, solvate, etc., more preferably trihydrochloride pentahydrate.
The use according to claim 9, wherein the medicine is prepared into clinically acceptable preparations, such as oral preparations, injection preparations, topical preparations, external preparations and the like.
The use according to claim 9, characterized in that: the medicament is in a single-dose dosage form or a divided-dose dosage form; the dosage form contains a therapeutically effective amount of Compound I or a pharmaceutically acceptable salt thereof.
The use according to claim 11, wherein each preparation unit contains about 0.001 mg to about 1000 mg of compound I or a pharmaceutically acceptable salt thereof (calculated as compound I), preferably about 1 mg to about 500 mg, or about 10 mg to about 400 mg, or about 15 mg to about 330 mg, or about 20 mg to about 300 mg, or about 20 mg to about 250 mg, or about 20 mg to about 210 mg, or about 20 mg to about 180 mg, or about 20 mg to about 160 mg, or about 20 mg to about 140 mg, or about 20 mg to about 120 mg, or about 20 mg to about 100 mg; exemplary amounts such as about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 75 mg, about 80 mg , about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 125 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 250 mg, about 300 mg, about 310 mg, about 320mg, about 360mg, about 400mg.
The use according to claim 13, characterized in that: the pharmaceutically acceptable salt of compound I is hydrochloride, preferably trihydrochloride, more preferably trihydrochloride pentahydrate; each preparation unit contains the compound I trihydrochloride about 1 mg to about 500 mg, or about 10 mg to about 400 mg, or about 15 mg to about 350 mg, or about 20 mg to about 310 mg, or about 25 mg to about 300 mg, or about 25 mg to about 250 mg, or about 50 mg- about 250 mg, or about 50 mg to about 200 mg, or about 50 mg to about 175 mg, or about 50 mg to about 160 mg, or about 50 mg to about 150 mg, or about 50 mg to about 125 mg, or about 50 mg to about 100 mg; exemplary amounts such as about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 160 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg , about 310 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg; the above-mentioned compound I trihydrochloride is measured in the form of anhydrous.
A composition comprising (1) daunorubicin; (2) cytarabine; (3) compound I or a pharmaceutically acceptable salt thereof, wherein the structure of compound I is shown in the following formula (I):
The composition according to claim 15, wherein the molar ratio of cytarabine and daunorubicin is 1-100:1, and the molar ratio of compound I and daunorubicin is 100:1- 1:100.
A kit comprising (1) daunorubicin; (2) cytarabine; (3) compound I or a pharmaceutically acceptable salt thereof, wherein the structure of compound I is shown in the following formula (I):