CN114948983A

CN114948983A - Combined treatment medicine for acute myeloid leukemia

Info

Publication number: CN114948983A
Application number: CN202210184150.7A
Authority: CN
Inventors: 夏雪芳; 郝春颜; 范丽雪; 束云; 柴晓玲; 杨晓旭; 张笑
Original assignee: CSPC Zhongqi Pharmaceutical Technology Shijiazhuang Co Ltd
Current assignee: CSPC Zhongqi Pharmaceutical Technology Shijiazhuang Co Ltd
Priority date: 2021-02-25
Filing date: 2022-02-25
Publication date: 2022-08-30
Also published as: WO2022179592A1

Abstract

The invention provides a medicine and a method for treating acute myeloid leukemia by combined administration of a multi-target kinase inhibitor compound I or a pharmaceutically acceptable salt thereof, daunorubicin and cytarabine. The compound I or the pharmaceutically acceptable salt thereof can be combined with daunorubicin and cytarabine to obviously inhibit the proliferation of human leukemia cells MV-4-11 and MOLM-13 and generate a synergistic effect, and the combined curative effect is superior to that of a scheme only using the daunorubicin and the cytarabine, so that the compound I or the pharmaceutically acceptable salt thereof can bring a longer life cycle to a patient. The potential of the combination treatment scheme for improving prognosis is far greater than the risk of potential toxicity, and the clinical application prospect is good.

Description

Combined treatment medicine for acute myeloid leukemia

The applicant claims priority to a prior application entitled "a drug for the combination therapy of acute myeloid leukemia" filed on 25.2.2021 by the intellectual property office of the chinese patent application No. 202110212434.8. The entire content of this prior application is incorporated by reference into this application.

Technical Field

The invention belongs to the field of medicines, and particularly relates to a combined treatment medicine for treating acute myeloid leukemia.

Background

Acute Myeloid Leukemia (AML) is a hematological malignancy in which abnormally proliferating, differentiating, and cloned hematopoietic stem cells invade bone marrow, blood, and extramedullary tissues, and has heterogeneous characteristics. Among the common hematological tumors, AML mortality is highest with a 5-year survival of about 24%. AML accounts for about 70% of adult leukemia, and as a result of treatment, 35-40% of adult AML patients aged 60 years and younger can survive for a long time, while only 5-15% of patients aged 60 years and older can survive for a long time and cannot tolerate intensive chemotherapy, and the median survival time is only 5-10 months.

At present, no standard treatment scheme for diagnosing AML (non-APL) in the first time is available in China. Clinically recommended primary diagnostic adult AML (non-APL) induction treatment regimens are based on anthracyclines in combination with cytarabine, commonly used are Ara-C/DNR ("7 + 3") regimens consisting of either Idarubicin (IDA) or Daunorubicin (DNR) in combination with cytarabine (Ara-C), the specific dosage being determined by the patient's condition. However, for newly diagnosed AML subjects less than 60 years of age, the standard "7 + 3" induction treatment regimen is effective; but for patients aged 60 or older, age-related toxicity is shown if large doses of cytarabine are received.

In most cases of AML, there is overexpression of FMS-like tyrosine kinase (FLT 3). 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 an important role in the proliferation, survival and differentiation of pluripotent stem cells. Furthermore, in AML cases, the juxtamembrane domain Tyrosine Kinase Domain (TKD) mutation around and around D835 in the activation loop and activated FLT3 with internal tandem repeat (ITD) accounted for 28% -34% and 11% -14% of AML cases, respectively. These activating mutations in FLT3 are oncogenic and exhibit transforming activity in cells. Patients with the FLT3-ITD mutation were reported to have a poorer prognosis in clinical studies, higher relapse rates (r.m. shalis, r.wang, a.davidoff, x.ma, and a.m. zeidan, "epidemic of acid myelology leukamia: Recent progress and ending sessions," Blood Rev, vol.36, pp.70-87, Jul 2019), shorter remission times for initial treatment (6 months, 11.5 months for patients without the FLT3-ITD mutation), reduced disease-free survival (16% to 27%, while patients without the FLT3-ITD mutation had 41% disease-free 5 years), and also reduced OS (15% to 31%, while patients without the FLT3-ITD mutation had 42% OS 5 years). FLT3 has become an important target for AML treatment, and related drug research is of great interest.

Studies have shown that genetic abnormalities such as KIT, CEBPA, NPM1, PHF6, ASXL1, DNMT3A, IDH1, IDH2, TET2, CBL, TP53, BCORL1, EZH2, MLL, SRSF2, SF3B1, U2AF1, ZRSR2, SF3a1, PRPF40B, U2AF2, SF1, in addition to FLT3 mutations, are also associated with the onset, prognosis, low survival, relapse, etc. of AML.

The compound I is a novel multi-target protein kinase inhibitor, the main action targets comprise FLT3 and the like, and the structure is shown as the following formula (I):

WO2011/147066A1 discloses the compound and a preparation method and medical application thereof. PCT/CN2021/073285 further investigated pharmaceutically acceptable salts of compound I and their uses. All of the contents of the above patents are incorporated into the present invention. In vivo and in vitro studies show that the compound I or the pharmaceutically acceptable salt thereof has strong growth inhibition effect on FLT3-ITD mutant acute myelogenous leukemia cell strains. However, it is not known whether the new diagnosis of AML using compound i or its pharmaceutically acceptable salt in combination with DNR and Ara-C for treatment of AML <60 years of age can further improve the therapeutic effect, and whether it has an advantage in safety tolerance compared to the similar drugs.

Disclosure of Invention

The invention provides application of a compound I or pharmaceutically acceptable salts thereof, daunorubicin and cytarabine in preparation of a medicine for treating Acute Myeloid Leukemia (AML). The structure of the compound I is shown as the following formula (I):

in some embodiments, the acute myeloid leukemia is preferably acute myeloid leukemia positive for FLT3 mutation, more preferably acute myeloid leukemia positive for the untreated FLT3 mutation.

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 an acute myeloid leukemia negative for the FLT3 mutation. 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., 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. The appropriate salt form can be selected by one skilled in the art by routine methods. Specific examples of pharmaceutically acceptable salts of compound I include, but are not limited to, hydrochloride, mesylate, malate, tartrate, oxalate, succinate, acetate, sulfate, and the like of compound I; preferably hydrochloride, malate, tartrate, oxalate, succinate, acetate; the hydrochloride salt is more preferred. The hydrochloride is further preferably a trihydrochloride. The trihydrochloride includes the corresponding anhydrate, hydrate, solvate and the like, and the trihydrochloride pentahydrate is more preferable.

In some embodiments, the molar ratio of cytarabine to daunorubicin is 1 to 100:1, preferably 1 to 25:1, more preferably 2 to 10:1, for example 5: 1. The molar ratio of the compound I or the pharmaceutically acceptable salt thereof to the daunorubicin is 100:1 to 1:100, preferably 2:1 to 1:70, more preferably 4:1 to 1:50 (calculated as the content of the compound I).

The compound I or pharmaceutically acceptable salts thereof, daunorubicin and cytarabine can be contained in the same pharmaceutical preparation, or can be respectively prepared into clinically acceptable preparations, and the medicaments are prepared in a combined packaging mode. The clinically acceptable preparation includes oral preparation, injection preparation, topical preparation, external preparation, etc. In some embodiments, the medicament is in a single dose dosage form or a divided dose dosage 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 is prepared into clinically acceptable preparations respectively and the medicines are prepared in a combined packaging manner, the preparation containing the compound I or the pharmaceutically acceptable salt thereof is preferably an oral preparation, such as tablets, capsules, pills and the like; each unit of the preparation contains about 0.001mg to about 1000mg of Compound I or a pharmaceutically acceptable salt thereof (based on Compound I), preferably about 1mg to about 500mg, or about 10mg to about 400mg, or about 15mg to about 330mg, or about 20mg to about 300mg, or about 20mg to about 250mg, or about 20mg to about 210mg, or about 20mg to about 180mg, or about 20mg to about 160mg, or about 20mg to about 140mg, or about 20mg to about 120mg, or about 20mg to about 100 mg. Exemplary amounts are, for example, about 20mg, about 25mg, about 30mg, about 40mg, about 50mg, about 60mg, about 70mg, about 75mg, about 80mg, about 90mg, about 100mg, about 110mg, about 120mg, about 125mg, about 130mg, about 140mg, about 150mg, about 160mg, about 180mg, about 200mg, about 220mg, about 240mg, about 250mg, about 300mg, about 310mg, about 320mg, about 360mg, about 400 mg. The daunorubicin preparation is preferably daunorubicin hydrochloride for injection, such as daunorubicin hydrochloride freeze-dried powder for injection; each unit of the formulation contains from about 10mg to about 30mg, preferably about 20mg, of the active ingredient. The cytarabine hydrochloride preparation is preferably cytarabine hydrochloride for injection, such as cytarabine hydrochloride freeze-dried powder for injection; each unit of the formulation contains from about 0.1g to about 0.5g, for example about 0.1g, about 0.3g or about 0.5g, of active ingredient.

In some embodiments, the pharmaceutically acceptable salt of compound I is a hydrochloride salt, preferably a trihydrochloride salt, more preferably a trihydrochloride pentahydrate. Each unit of the formulation contains from about 1mg to about 500mg, or from about 10mg to about 400mg, or from about 15mg to about 350mg, or from about 20mg to about 310mg, or from about 25mg to about 300mg, or from about 25mg to about 250mg, or from about 50mg to about 200mg, or from about 50mg to about 175mg, or from about 50mg to about 160mg, or from about 50mg to about 150mg, or from about 50mg to about 125mg, or from about 50mg to about 100mg of the trihydrochloride of compound I; exemplary amounts are, for example, about 10mg, about 15mg, about 20mg, about 25mg, about 30mg, about 40mg, about 50mg, about 60mg, about 75mg, about 100mg, about 125mg, about 150mg, about 160mg, about 175mg, about 200mg, about 250mg, about 300mg, about 310mg, about 350mg, about 400mg, about 450mg, about 500 mg. The trihydrochloride salt of compound I above is metered in as an anhydrate.

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

In some embodiments, the acute myeloid leukemia is preferably acute myeloid leukemia positive for the FLT3 mutation. More preferred are acute myeloid leukemias that are positive for the untreated FLT3 mutation.

In some embodiments, the acute myeloid leukemia is acute myeloid leukemia positive for FLT3 mutation, 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 an acute myeloid leukemia negative for the FLT3 mutation. Further, these FLT3 mutation-negative acute myeloid leukemias are accompanied by one, two or more of the following genetic 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 medicament is formulated into a clinically acceptable formulation, such as an oral formulation, an injectable formulation, a topical formulation, an external preparation, and the like.

In some embodiments, 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.

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

The present invention also provides a method for treating acute myeloid leukemia, which comprises 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 may be administered simultaneously or separately. The administration may be oral, injection, topical or in vitro. The therapeutically effective amount can treat or ameliorate acute myeloid leukemia in the subject or patient.

In some embodiments, the method of treatment comprises:

(1) induction treatment: every 28 days for a period of at most two cycles. The treatment method for each cycle is as follows:

days 1-7: cytarabine approximately 100mg/m ² Intravenous infusion (CIVI) was continued for 7 days (168 h total).

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

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

In some embodiments, compound i is administered as the hydrochloride salt, preferably as the trihydrochloride salt, more preferably as the trihydrochloride pentahydrate. The method specifically comprises the following steps: day 8-21: compound I trihydrochloride is administered orally at a dose of about 25 mg/time to about 200 mg/time twice daily (BID) for 14 consecutive days. Preferably, about 50mg to about 175mg, more preferably about 50mg to about 150mg per oral dose. Examples of each oral dose include about 50mg, or about 75mg, or about 100mg, or about 125mg, or about 150mg, or about 175mg, or about 200 mg. The dosage of the trihydrochloride salt is in the form of an anhydrate.

(2) Consolidation treatment: every 28 days for a period of up to 4 cycles. The treatment method for each cycle is as follows:

days 1, 3, 5: cytarabine approximately 1g/m ² -about 5g/m ² Persistent sessile drop, q12 h. Preferably, cytarabine is administered in a dose of about 1g/m ² -about 4g/m ² More preferably about 1.5g/m ² -about 3g/m ² . Specific examples of the administration dose are about 3g/m ² Or about 2g/m ² Or about 1.5g/m ² 。

In some embodiments, compound i is administered as the hydrochloride salt, preferably as the trihydrochloride salt, more preferably as the trihydrochloride pentahydrate. The method specifically comprises the following steps: day 8-21: compound I trihydrochloride is administered orally at about 25 mg/time to about 200 mg/time twice daily (BID) for 14 consecutive days. Preferably, about 50mg to about 175mg, more preferably about 50mg to about 150mg per oral administration. Examples of each oral dose include about 50mg, or about 75mg, or about 100mg, or about 125mg, or about 150mg, or about 175mg, or about 200 mg. The dosage of the trihydrochloride salt is in the form of an anhydrate.

(3) Maintenance treatment: every 28 days for a period of 12 cycles at most. The treatment method for each cycle is as follows:

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

In some embodiments, compound i is administered as the hydrochloride salt, preferably as the trihydrochloride salt, more preferably as the trihydrochloride pentahydrate. The method specifically comprises the following steps: compound I trihydrochloride is administered orally at about 25 mg/time to about 200 mg/time twice daily (BID) daily. Preferably, about 50mg to about 175mg, more preferably about 50mg to about 150mg per oral administration. Examples of each oral dose include about 50mg, or about 75mg, or about 100mg, or about 125mg, or about 150mg, or about 175mg, or about 200 mg. The dosage of the trihydrochloride salt is in the form of an anhydrate.

The invention also provides a method for improving the curative effect of daunorubicin and cytarabine on treating acute myeloid leukemia, which comprises the following steps: compound I or a pharmaceutically acceptable salt thereof is used in combination with therapy 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, injection, topical or in vitro. The therapeutically effective amount can treat or ameliorate acute myeloid leukemia in the subject or patient.

In some embodiments, the acute myeloid leukemia is an acute myeloid leukemia negative for the FLT3 mutation. Further, these FLT3 mutation-negative acute myeloid leukemias are accompanied by other genetic 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 comprises:

Day 1-3: daunorubicin of about 60mg/m ² Or about 45mg/m ² Intravenous Injection (IVP) or short infusion;

In some embodiments, compound i is administered as the hydrochloride salt, preferably as the trihydrochloride salt, more preferably as the trihydrochloride pentahydrate. The method specifically comprises the following steps: day 8-21: compound I trihydrochloride is administered orally at a dose of about 25 mg/time to about 200 mg/time twice daily (BID) for 14 consecutive days. Preferably, about 50mg to about 175mg, more preferably about 50mg to about 150mg per oral administration. Examples of each oral dose include about 50mg, or about 75mg, or about 100mg, or about 125mg, or about 150mg, or about 175mg, or about 200 mg. The dosage of the trihydrochloride salt is in the form of an anhydrate.

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

In some embodiments, compound i is administered as the hydrochloride salt, preferably as the trihydrochloride salt, more preferably as the trihydrochloride pentahydrate. The method specifically comprises the following steps: compound I trihydrochloride is administered orally at a dose of about 25 mg/time to about 200 mg/time twice daily (BID) daily. Preferably, about 50mg to about 175mg, more preferably about 50mg to about 150mg per oral administration. Examples of each oral dose include about 50mg, or about 75mg, or about 100mg, or about 125mg, or about 150mg, or about 175mg, or about 200 mg. The dosage of the trihydrochloride salt is in the form of an anhydrate.

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

In some embodiments, the treatment is:

Day 1-3: daunorubicin about 60mg/m ² Or about 45mg/m ² Intravenous Injection (IVP) or short infusion;

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

In some embodiments, the acute myeloid leukemia is acute myeloid leukemia positive for FLT3 mutation. More preferred are acute myeloid leukemias that are positive for the untreated FLT3 mutation. 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.

The compound I or the pharmaceutically acceptable salt thereof, the daunorubicin and the cytarabine can be contained in the same pharmaceutical preparation, or can be respectively prepared into clinically acceptable preparations, and the medicaments are prepared in a combined packaging mode. The clinically acceptable preparation includes oral preparation, injection preparation, topical preparation, external preparation, etc. In some embodiments, the medicament is in a single dose dosage form or a divided dose dosage form. The dosage form contains a therapeutically effective amount of daunorubicin, cytarabine, compound I or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a medicament containing compound i or a pharmaceutically acceptable salt thereof for improving the therapeutic effect of cytarabine and daunorubicin on acute myeloid leukemia. The improved method comprises the following steps: compound I or a pharmaceutically acceptable salt thereof is used in combination with therapy 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, injection, topical or in vitro.

In some embodiments, the improvement method is:

In some embodiments, compound i is administered as the hydrochloride salt, preferably as the trihydrochloride salt, more preferably as the trihydrochloride pentahydrate. The method comprises the following specific steps: day 8-21: compound I trihydrochloride is administered orally at a dose of about 25 mg/time to about 200 mg/time twice daily (BID) for 14 consecutive days. Preferably, about 50mg to about 175mg, more preferably about 50mg to about 150mg per oral administration. Examples of each oral dose include about 50mg, or about 75mg, or about 100mg, or about 125mg, or about 150mg, or about 175mg, or about 200 mg. The dosage of the trihydrochloride salt is in the form of an anhydrate.

(3) Maintenance treatment: every 28 days for a period of up to 12 cycles. The treatment method for each cycle is as follows:

In some embodiments, the acute myeloid leukemia is an acute myeloid leukemia negative for the FLT3 mutation. Further, these FLT3 mutation-negative acute myeloid leukemias are accompanied by one, two or more of the following genetic 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 and the like, preferably BCORL1 mutation, CEBPA mutation, CBL mutation, TP53 mutation.

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

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

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

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

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

In some embodiments, the kit further comprises instructions for use according to any of the methods of the invention. The kit may further comprise 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 (e.g., a sheet of paper contained in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical disk) are also acceptable.

In some embodiments, daunorubicin, cytarabine, compound I, or a pharmaceutically acceptable salt thereof is separately prepared in a clinically acceptable formulation and presented in a combination package in the kit. Wherein, the preparation containing the compound I or the pharmaceutically acceptable salt thereof is preferably oral preparation, such as tablet, capsule, pill and the like; each unit of the preparation contains about 0.001mg to about 1000mg of Compound I or a pharmaceutically acceptable salt thereof (based on Compound I), preferably about 1mg to about 500mg, or about 10mg to about 400mg, or about 15mg to about 330mg, or about 20mg to about 300mg, or about 20mg to about 250mg, or about 20mg to about 210mg, or about 20mg to about 180mg, or about 20mg to about 160mg, or about 20mg to about 140mg, or about 20mg to about 120mg, or about 20mg to about 100 mg. Exemplary amounts are, for example, about 20mg, about 25mg, about 30mg, about 40mg, about 50mg, about 60mg, about 70mg, about 75mg, about 80mg, about 90mg, about 100mg, about 110mg, about 120mg, about 125mg, about 130mg, about 140mg, about 150mg, about 160mg, about 180mg, about 200mg, about 220mg, about 240mg, about 250mg, about 300mg, about 310mg, about 320mg, about 360mg, about 400 mg. The daunorubicin preparation is preferably daunorubicin hydrochloride for injection, such as daunorubicin hydrochloride freeze-dried powder for injection; each unit of the formulation contains from about 10mg to about 30mg, preferably about 20mg, of the active ingredient. The cytarabine hydrochloride preparation is preferably cytarabine hydrochloride for injection, such as cytarabine hydrochloride freeze-dried powder for injection; each unit of the formulation contains from about 0.1g to about 0.5g, for example about 0.1g, about 0.3g or about 0.5g, of active ingredient.

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

The kit of the invention is in a suitable package. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., selected Mylar or plastic bags), and the like. The kit may optionally provide other components, such as buffers and instructional information.

In the context of the present application, "about" preceding a numerical value means ± 10%, preferably ± 5% of the numerical value, for example ± 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or ± 1% of the numerical value.

Preclinical studies show that the compound I or the pharmaceutically acceptable salt thereof has a strong growth inhibition effect on FLT3-ITD mutant acute myeloid leukemia cells. Preliminary clinical research shows that the compound I or the pharmaceutically acceptable salt thereof has certain curative effect on FLT3 mutant relapsing/refractory AML and is close to early test data of a similar product Giltertinib; in the clinical research process, no serious adverse reactions of heart, liver and hematology appear, and the incidence rate of the adverse reactions above grade 3 is low.

In order to evaluate the efficacy and safety of compound i or its pharmaceutically acceptable salt in combination with the standard "7 + 3" chemotherapy regimen of daunorubicin and cytarabine for the treatment of acute myeloid leukemia in humans, the inventors of the present application conducted in vitro studies and clinical trials. The results show that the compound I combined with daunorubicin and cytarabine can obviously inhibit the proliferation of human leukemia cells MV-4-11 and MOLM-13 and generate a synergistic effect. The remission rate of the combination of the compound I and the standard chemotherapy scheme of 7+3 is better than that of the single standard chemotherapy scheme of 7+3, and the compound I brings longer survival period for patients. The potential of the combined treatment scheme for improving prognosis is far greater than the risk of potential toxicity, and the combined treatment scheme has good clinical application prospect.

Detailed Description

The following examples are given to better illustrate the present invention, but the present invention is not limited to the examples. Those skilled in the art can make insubstantial modifications and adaptations to the embodiments described above while remaining within the scope of the invention. EXAMPLE 1 inhibition of proliferation of human leukemia cells MV-4-11 and MOLM-13 by Compound I in combination with daunorubicin and cytarabine

First, research method

1. Purpose of study

Comparing the inhibitory effect of the combination of the compound I and the daunorubicin and the cytarabine with the compound I alone or the daunorubicin and the cytarabine alone on the proliferation of human leukemia cells MV-4-11 and MOLM-13, and evaluating whether the combination of the compound I and the daunorubicin and the cytarabine has a synergistic effect.

2. Test drug

A compound I: the Shi Yao group of the odd pharmaceutical technology (Shijiazhuang) Co. Weighing a proper amount of a compound I, completely dissolving the compound I with a proper amount of DMSO (dimethyl sulfoxide), and preparing a 10mM stock solution; test solutions were diluted with complete medium gradients to 1.25nM, 2.5nM, 5nM, 10nM or 20nM just before use. And the complete medium was taken as test solution containing 0nM of compound I.

Daunorubicin, cytarabine: the Shi Yao group of the odd pharmaceutical technology (Shijiazhuang) Co. Weighing proper amount of cytarabine and daunorubicin, completely dissolving in DMSO, and preparing stock solution; just before use, according to cytarabine: mixed solutions were prepared at a molar ratio of daunorubicin to 5:1, and were diluted to 3.9nM, 7.8nM, 15.625nM, 31.25nM, and 62.5nM (calculated as daunorubicin) in complete medium gradient as mixed test solutions. And the complete culture medium was taken as a mixed test solution containing 0nM of cytarabine and daunorubicin.

3. Test method

Cells in logarithmic growth phase were seeded in 96-well plates (100. mu.L/well). Each well is added with 50 mu L of compound I test solution and cytarabine and daunorubicin mixed test solution respectively, so that the concentration of each drug is crossed, each action point is provided with three multiple wells, and corresponding blank wells (only culture medium) and normal wells (the concentration of the drug is 0) are arranged. Adding medicine for culturing for 72 hours, adding 20 mu L of MTT into each hole, culturing in an incubator for 4 hours, removing supernatant, adding 150 mu L of DMSO into each hole, shaking and uniformly mixing micropores, and detecting Optical Density (OD) at the wavelength of 550nm by using an enzyme-labeling instrument.

And (3) calculating:

inhibition (%) × 100 (OD value normal well-OD value administration well)/(OD value normal well-OD value blank well);

calculating the half inhibition concentration IC50 value of the medicine by using SPSS19.0 according to the inhibition rate of each concentration;

combined action index CI-IC 50 _(C1) /IC50 ₍₁₎ +IC50 _(C2) /IC50 ₍₂₎

Wherein, IC50 ₍₁₎ Is the concentration corresponding to half of the cell proliferation inhibition produced by the single administration of the compound I; IC50 ₍₂₎ The concentration of daunorubicin and cytarabine which are used only produce half of the corresponding cell inhibition; IC50 _(C1) The concentration of the compound I corresponding to half cell inhibition generated after the compound I is combined with the daunorubicin and the cytarabine; IC50 _(C2) The concentrations of daunorubicin and cytarabine corresponding to half cell inhibition are generated after the compound I is combined with the daunorubicin and the cytarabine.

The CI value is calculated according to the formula, and the meaning is as follows: CI is more than 1.1, and antagonism is achieved; CI is more than or equal to 0.9 and less than or equal to 1.1, and the addition effect is achieved; CI <0.9 is synergistic, with lower CI values indicating higher synergy between the two drugs.

Second, research results

The results show that for human leukemia cells MV-4-11 and MOLM-13, compound I in combination with daunorubicin and cytarabine had CI values less than 0.9 (see Table 1). The compound I is proved to have synergistic effect when being combined with the daunorubicin and the cytarabine.

TABLE 1 synergistic effect of Compound I in combination with daunorubicin and cytarabine

EXAMPLE 2 Single arm, multicenter, open, dose escalation/expansion phase I/II study of New diagnostic AML patients with FLT3 mutation treated with Compound I or a pharmaceutically acceptable salt thereof in combination with "7 + 3" Standard chemotherapy

1. Purpose of the experiment

Novel diagnostic AML subjects for the treatment of the FLT3 mutation with compound i or a pharmaceutically acceptable salt thereof in combination with a "7 + 3" standard chemotherapeutic regimen were evaluated for safety, tolerability, efficacy.

2. Design of experiments

This study is a single-arm, open, multicenter trial, aimed primarily at evaluating the safety, tolerability and pharmacokinetic profile of newly diagnosed AML subjects with FLT3 mutations, orally administered compound i or a pharmaceutically acceptable salt thereof in combination with daunorubicin and cytarabine for the treatment of FLT3 mutations, and exploring optimal dosing regimens.

The study will use the compound i trihydrochloride in combination with the standard 7+3 dosing regimen of daunorubicin and cytarabine as divided into an induction phase, a consolidation phase and a maintenance phase. Planning stage I: 3+3 dose ramp-ups, scheduled to group 9-18 subjects; stage II: in the dose group, which is safely tolerated and effective, the number of cases per group extends to 20. The specific test design is as follows:

2.1 phase I (dose ramp)

Initially, 3 ramp doses were established: 100mg, 125mg, 150 mg; both were twice daily (BID).

Compound i trihydrochloride was ramped from the 100mg bid dose group into 3 subjects. If no DLT occurred during the first cycle of the induction period, dose escalation was continued. If there are 1 cases of DLT in the dose group, 3 subjects should be added to the dose group; if no DLT occurs in these 3 subjects, the next dose group is entered; if there are 1 more DLTs present, or if there are 2 more DLTs present in a dose group, then the dose ramp to the cohort is considered to be terminated.

Definition of DLT

Non-hematologic toxicity DLT: any grade 3 non-hematologic toxicity or non-expected extramedullary toxicity positively associated, likely associated with the study drug in phase I from after the first dose of the induction remission period to the end of the first induction period (within 42 days). But excluding the following cases:

alopecia, fatigue, anorexia, malignancy, vomiting, electrolyte abnormalities;

grade 3 diarrhea can be controlled after symptomatic treatment;

a reduction in grade 3 febrile central granulocytes with or without infection;

grade 3 infection.

Hematological toxicity DLT: long-term myelosuppression, if the persistent myelosuppression (at least including either the cases of leukopenia grade. gtoreq.4, neutropenia grade. gtoreq.4, thrombocytopenia grade. gtoreq.4) that occurs after the start of the first induction phase treatment without evidence of leukemia (< 5% primary cells), would be considered DLT if it is still not alleviated within 42 days of the first induction phase treatment or before the start of the consolidation phase.

After the end of the DLT observation period for the last 1 subjects in the I-phase dose ramp, one interim analysis will be performed by the investigator and the sponsor together. The mid-phase will be analyzed for safety, PK and CRc rate at the end of the first or second induction period, recommending a safe and effective combination regimen for phase ii.

2.2 phase II (case number expansion)

In order to further fully determine the safety and PK profile of the dosage regimen of the compound i trihydrochloride combination and to preliminarily confirm the effectiveness of the optimal dosage regimen of the drug, a basis is provided for phase iii clinical trial design, with an expansion of effective dose groups in an effective and safe dosage regimen, with an expansion of the number of cases per group to 20 cases and up to 40 cases. The DLT observation period is not set in the case number expansion stage.

3. Test population

3.1 inclusion criteria:

1) voluntarily receiving the test and signing an informed consent;

2) male or female Chinese patients, aged more than or equal to 18 years and less than 60 years;

3) morphological diagnosis primary AML > 20% primary cells according to the World Health Organization (WHO)2016 classification;

4) detecting in whole blood of the subject positive for the FLT3 mutation, including positive for FLT3 internal tandem repeat (ITD), FLT3 Tyrosine Kinase Domain (TKD)/D835 or FLT3-TKD/I836 mutations, by a central laboratory gene;

5) an ECOG score of less than or equal to 2;

6) laboratory examination:

a. the AST and ALT of the serum are less than or equal to 3 multiplied by ULN; b. total serum bilirubin is less than or equal to 2.5 × ULN; c. serum creatinine is less than or equal to 3 × ULN or glomerular filtration rate is more than 30 mL/min.

7) The subject can receive a standard "7 + 3" induction chemotherapy treatment regimen;

8) the subject is capable of orally administering the test agent;

9) female/male subjects in the fertile age must take adequate non-drug contraceptive measures from the start of signing an informed consent until 180 days after the last dose, and must not donate sperm or eggs.

3.2 exclusion criteria:

1) confirmed acute promyelocytic leukemia (M3/APL), or BCR-ABL positive leukemia (i.e., chronic myelocytic leukemia blast crisis); subjects who received APL diagnostic tests and all-trans retinoic acid (ATRA) treatment but found no APL were eligible for enrollment (ATRA treatment had to be discontinued 24h before initiation of induction chemotherapy).

2) Diagnosing with active malignancies other than AML;

3) AML secondary to radiotherapy or chemotherapy to treat other tumors;

4) centrally involved AML (defined as a case where clinical symptoms are highly suspected of central invasion, or where visual evidence supports the judgment);

5) intractable hypokalemia or hypomagnesemia which is not easy to correct by symptomatic treatment and has repeatedly occurred before;

6) (ii) presents with clinically significant Graft Versus Host Disease (GVHD) or is undergoing systemic cortisol therapy for GVHD;

7) there has been a history of other malignancies (except: disease-free state for more than 5 years; non-melanoma skin cancer, cervical or breast carcinoma in situ [ regardless of disease state ] that has completed treatment; localized prostate cancer is treated by radiotherapy or operation, and then tumors expected to heal after treatment such as relapse-free patients and progressors);

8) patients in the screening phase have clinically significant blood coagulation abnormalities, such as Disseminated Intravascular Coagulation (DIC), hemophilia a, hemophilia B, von willebrand disease;

9) major operative treatment of major organs was performed within 4 weeks before study (the definition of major operative treatment refers to 3-grade and 4-grade operations specified in "clinical practice and management method for medical technology"); or has not been fully recovered from any previous invasive operation;

10) radiotherapy was performed within 4 weeks before study entry;

11) subjects received prior therapy for AML, except for the following: a. emergency leukapheresis; b. the hydroxyurea is used for emergently treating the leukocytosis for less than or equal to 10 days; c. growth factor or cytokine support; d. steroids for allergic or transfusion reactions;

12) patients with New York Heart Association (NYHA) grade 3 or 4 congestive heart failure, or who had a history of NYHA3 or 4 congestive heart failure, were not enrolled in the study unless an echocardiographic examination within 1 month prior to study entry showed a left ventricular ejection fraction of 45% or greater;

13) bradycardia with heart rate less than 50 beats/minute, excluding subjects using pacemakers;

14) the average value (QTcF) of QT intervals after the correction of Fridericia formula is detected by electrocardiogram for three times in the screening period, wherein male time is more than 450ms, and female time is more than 470 ms;

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

16) a history of second degree (Mobita ii) or third degree atrioventricular block (except for subjects using a pacemaker);

17) a history of uncontrolled angina or myocardial infarction within 6 months prior to screening;

18) complete left bundle branch block exists in the screening period;

19) subjects who are newly experiencing clinically significant arrhythmias (except for anemia, infections and AML-induced sinus tachycardia) or who have experienced arrhythmias and who need to take drugs that may prolong the QT interval for extended periods of time;

20) present active or uncontrollable infection;

21) hepatitis B surface antigen is positive or has a history of hepatitis B, and HBV-DNA is accompanied by more than or equal to 2000IU/ml in the last 3 months; hepatitis C antibody positive patients, with HCV-RNA positive in about 3 months;

22) positive person detected by anti-HIV antibody or anti-treponema pallidum specific antibody;

23) the subjects used strong inducers or inhibitors of CYP2C8 and CYP3a4 enzymes within two weeks prior to taking the test drugs;

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

25) the analogous drug and adjuvant components of the study drugs (daunorubicin, cytarabine) combined in this study had a known history of immediate or delayed hypersensitivity.

26) The investigator considered the study-inaccessible patient.

4. Medicine

(1) Compound i trihydrochloride salt: the administration is in the form of the compound I trihydrochloride pentahydrate, provided by shijiazhuang, a fang pharmaceutical technology limited in shiyao. The drug doses in this clinical study were calculated as the trihydrochloride anhydrate.

(2) Cytarabine: lyophilized powder for injection of cytarabine hydrochloride is provided by Shijiazhuang pharmaceutical technology of Shiyao group, Inc.

(3) Daunorubicin: the daunorubicin hydrochloride freeze-dried powder preparation for injection is provided by Shijiazhuang Chizhongqi pharmaceutical technology (Shijiazhuang) Co.

5. Method of treatment

5.1 phase I phase dosing regimen

5.1.1 Induction remission therapy (2 cycles maximum, one cycle every 28 days)

(1) First induction therapy

Days 1-7: cytarabine 100mg/m ² Intravenous infusion (CIVI) was continued for 7 days (168 h total).

Days 1-3: daunorubicin 60mg/m ² Intravenous Injection (IVP) or short infusion;

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

Evaluation of efficacy after first induction treatment: bone marrow and hemograms were examined on day 28 to assess efficacy for further treatment. If bone marrow aspirate is insufficient for evaluation, the bone marrow evaluation is repeated over a week. If CRc is obtained, consolidation treatment is carried out after remission; if CRc is not obtained, the second induction treatment is carried out. In either case, bone marrow examination must be performed at or before day 42 after the first induction treatment to verify that CRc is reached.

(2) Second induction therapy

The treatment regimen is the same as the first induction treatment unless the dosage is down-regulated for safety.

Evaluation of efficacy after the second induction treatment: bone marrow and hemograms were examined on day 28 to assess efficacy for further treatment. If bone marrow aspirate is insufficient for evaluation, the bone marrow evaluation is repeated over a week. If CRc is obtained, consolidation treatment is carried out after remission; if CRc is not obtained after the second induction treatment, the induction treatment fails, and the survival period follow-up is carried out. In either case, bone marrow examination must be performed at or before day 42 after the second induction treatment to verify that CRc is reached. If CRc is reached after 42 days, the investigator decides whether to receive consolidation phase therapy based on the benefit ratio of the subject.

5.1.2 consolidation therapy (4 cycles maximum, one cycle every 28 days)

Days 1, 3, 5: high dose cytarabine (HiDAC)3g/m ² Persistent sessile drop, q12 h.

Day 8-21: compound i trihydrochloride was administered orally, in principle in the same dose as the previous cycle, twice daily (BID), for 14 consecutive days.

For patients who can enter consolidation therapy, adverse events need to recover to grade < 1 and CRc must be reached at the end of induction therapy, and if chronic toxicity and hemogram recovery are delayed up to day 56, the investigator decides whether to continue to receive therapy depending on the benefit ratio of the subject.

Based on the subject's remission, the investigator determines the number of cycles the subject received induction therapy and consolidation therapy.

5.1.3 allogeneic Hematopoietic Stem Cell Transplantation (HSCT)

Subjects who have acquired CRc after induction therapy, if appropriate for bone marrow commitment, can be allogeneic Hematopoietic Stem Cell Transplantation (HSCT) without need for export. While the subject is waiting for HSCT, the treatment may be first administered for a consolidation phase, up to 4 cycles. However, prior to starting the pretreatment regimen for HSCT, the test drug should be discontinued, and within 7 days after discontinuation, a treatment/treatment end visit prior to HSCT is performed followed by a follow-up visit.

5.1.4 maintenance therapy (12 cycles maximum, one cycle every 28 days)

Compound i trihydrochloride was administered orally, in principle at the same dose as the previous cycle, twice daily (BID), daily, unless subjects met the criteria for discontinuation of dosing or withdrawal from the study, and subjects received maintenance phase therapy for up to 12 cycles.

5.2 phase II phase dosing regimen

Based on the interim analysis of phase I, an effective and safe combination dosing regimen is determined.

5.3 dose adjustment and management

To ensure subject safety and qualify subjects for continued study if unexpected adverse events are encountered, slight deviations from the toxicity dose regimens provided or recommended support therapy are allowed, as discussed and agreed upon by the investigator and sponsor. Dose adjustments are not allowed during the DLT observation period, but medication can be suspended. In principle, following adverse events in the study, the dosage of the "7 + 3" chemotherapeutic agent was first adjusted to maintain continued administration of the compound i trihydrochloride, and optionally adjusted (including suspended) if not still alleviated.

(1) Daunorubicin: if Total Bilirubin (TBIL) > 2 times ULN, DNR can be varied from 60mg/m in the second induction period ² Down to 45mg/m ² 。

(2) High dose cytarabine (HiDAC): the neurotoxicity appears: during consolidation period, the adverse neurotoxic reaction with grade 2 or more occurs for the first time, HiDAC is stopped during the rest period, and if neurotoxicity is slowly reduced to grade 1 or less, the dose of HiDAC is changed from 3g/m in the next consolidation period ² Down-regulated to 2g/m ² . If the neurotoxic adverse reaction of level 2 or more occurs for the second time, HiDAC is stopped permanently in the scheme. Secondly, hematological toxicity occurs: if ANC can not reach 1.0X 10 within 8 weeks of consolidation phase treatment ⁹ L, PLT cannot reach 100X 10 ⁹ L, HiDAC will down-regulate to 1.5g/m in the next cycle ² If the ANC still can not be increased to be more than or equal to 1.0 multiplied by 10 in the next period ⁹ /L，PLT≥100×10 ⁹ at/L, the consolidation phase treatment will be stopped.

(3) Compound i trihydrochloride salt: induction period and consolidation period: the compound I trihydrochloride does not cause dosage adjustment due to adverse reactions, but can be taken temporarily. Maintenance period: dose reduction due to ADR; after the dose is reduced by ADR, the dose can be escalated when ADR is restored. Compound i trihydrochloride dose-adjusting gradient was as follows:

①

②

③

④

⑤

⑥

early stage

150mg

125mg

100mg

75mg

50mg

Medicine stopping

Night

150mg

125mg

100mg

75mg

50mg

Medicine for stopping drug taking

5.4 concomitant therapy:

strong inducers or inhibitors or substrates of CYP2C8 and CYP3a4 enzymes and P-gp should be avoided in the trial, as well as concomitant drugs known to prolong QT or QTc intervals.

During the experiment, hydroxyurea (QD, up to 2 weeks, for lowering the white blood cell count, usually allows the white blood cell count > 10X 10 ⁹ Use at/L) and HSCT treatment plan (prophylactic intrathecal chemotherapy, cranial radiotherapy and donor lymphocyte infusion), combined use of other AML treatments (including but not limited to chemotherapy) is prohibitedRadiotherapy, surgery, immunotherapy or cell therapy, chinese medicine). Simultaneous participation in another interventional clinical trial was prohibited.

6. Pharmacokinetic Studies

Blood sampling time: (1) an induction period: C1D8 (0.5 h, 1h, 1.5h, 2h, 4h, 8h, 12h before and after first dose), C1D15 (before first dose), C1D18 (before first dose), C1D21 (only once in the morning, 0.5h, 1h, 1.5h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, 96h before and after first dose); (2) a consolidation period: before the first dose of C1D8, C1D21, and C4D8, C4D 21; (3) a maintenance period: C8D1 and C12D1 prior to first dose. Collecting venous blood, detecting blood concentration, and carrying out pharmacokinetic study.

7. Evaluation index

7.1 safety and tolerability

Including adverse events, vital signs, laboratory examinations, physical examinations, twelve lead Electrocardiograms (ECGs), ECOG scores, and DLT occurrences. Evaluated according to the definitions and methods conventional in the art.

7.2 pharmacokinetic characteristics

1) An induction period: AUC0-t, Cmax, Tmax, Ctrough, Cmax, ss, Tmax, ss, AUC0-inf, AUCss, t1/2, Ra (AUC), Ra (Cmax), etc. of C1D8, C1D15, C1D18, C1D 21;

2) a consolidation period: ctrough for C1D8, C1D21, and C4D8, C4D 21;

3) a maintenance period: ctrough for C8D1 and C12D 1.

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

7.3 evaluation of therapeutic efficacy

Therapeutic efficacy evaluation Criteria reference is made to Revised [ Chesonet al, "reviewed Recommendations of the International Working Group for Diagnosis, Standardization of Response Criteria, Standardization outages, and Reporting Standards for Therapeutic Trials in enzyme bacterial leucoderma ], J Clin Oncol 21:4642-4649.2003by American Society of Clinical Oncology ] and the hematological diagnostic and Therapeutic efficacy Criteria, 4 th edition.

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

2) CR rate at the end of induction period: the number of subjects with optimal effect of CR 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 mitigation (DOR): including CRc duration (CR + CRh + CRp + CRi), CR duration, CR/CRh duration, and CR/CRi duration. Refer to revised [ Cheson et al,2003 ].

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

5) Event-Free survivor (EFS): defined as the time from the start of the first dose until the occurrence of a treatment failure (not reaching CRc by the end of the second induction treatment) or relapse or death due to any cause (whichever occurred first).

6) Leukemia-free survival (LFS): time to relapse or death from the first time CRc is reached. For subjects with unknown relapse or death, LFS was performed by the last day of relapse-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

Comparing between continuous variable groups by adopting a t test or a Wilcoxon rank sum test; and 4, comparing between the classification variable groups by using a chi-square test or Fisher exact probability method.

Unless otherwise stated, the hypothesis test will use a two-sided test with 0.05 as the test level and the confidence interval estimation of the parameters using 95% confidence intervals.

9. Test results

The remission rate of newly diagnosed AML with compound i or a pharmaceutically acceptable salt thereof (e.g., the trihydrochloride) in combination with a "7 + 3" standard chemotherapy regimen for FLT3 mutation, compared to the "7 + 3" standard chemotherapy regimen alone, will lead to longer survival of the patient. The combination treatment regimen is safe and tolerable, and the potential for improved prognosis is far greater than the risk of potential toxicity.

EXAMPLE 3 Single arm, multicenter, open, dose escalation/expansion phase I/II study of Compound I or a pharmaceutically acceptable salt thereof in combination with "7 + 3" Standard chemotherapy treatment of newly diagnosed AML patients

The study was conducted to evaluate the safety, tolerability, efficacy of compound i or its pharmaceutically acceptable salts in combination with "7 + 3" standard chemotherapy regimen in the treatment of newly diagnosed AML subjects.

The inclusion criteria for the test population were as follows:

1) voluntarily receiving the test and signing an informed consent;

3) morphological diagnosis primary AML > 20% of blasts conforming to World Health Organization (WHO)2016 classification;

4) an ECOG score of less than or equal to 2;

5) laboratory examination:

6) The subject may receive a standard "7 + 3" induction chemotherapy treatment regimen;

7) the subject is capable of orally administering the test agent;

8) female/male subjects in the fertile age must take adequate non-drug contraceptive measures from the start of signing an informed consent until 180 days after the last dose, and must not donate sperm or eggs.

The experimental design, exclusion criteria of the test population, drugs, treatment methods, pharmacokinetic study methods, evaluation indexes, statistical analysis methods, and the like of this study were the same as those of example 2, except for the above criteria for inclusion in the test population.

The results of the study show that the remission rate of compound i or a pharmaceutically acceptable salt thereof (e.g., the trihydrochloride) in combination with a "7 + 3" standard chemotherapy regimen for newly diagnosed AML is superior to that of the "7 + 3" standard chemotherapy regimen alone, which results in longer survival of the patient. The combination treatment regimen is safe and tolerable, and the potential for improved prognosis is far greater than the risk of potential toxicity.

The following are typical cases of the study:

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

The patients, male, are 38 years old, and have a diagnosis after 1 month due to repeated cough and fever. Blood-checking routine: WBC: 4.15X 10 ⁹ /L，NE：0.38×10 ⁹ /L，Hgb：79g/L，Plt：199×10 ⁹ L; bone marrow morphology: proliferation is obviously active, 66% of primary naive monocytes are considered, AML-M5 a; flow type: acute myeloid leukemia (non-M3) immunophenotype. Treatment was performed according to the phase I dosing regimen of clinical trial (100mg, BID dose group) and efficacy was assessed as CR (complete remission) after the first induction treatment. After two cycles of consolidation treatment, the patient was bone marrow transplanted.

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

The patient, female, was 49 years old, and was admitted to the hospital after 1 week of thrombocytopenia was found as the primary cause. Admission blood routine: WBC: 6.88X 10 ⁹ /L，NE：0.65×10 ⁹ /L，Hgb：135g/L，Plt：64×10 ⁹ L; bone marrow morphology: acute myeloid leukemia (non-M3); flow type: conforming to the AML phenotype. Treatment was performed according to the phase I dosing regimen of clinical trial (100mg, BID dose group) and efficacy was assessed as CR (complete remission) after the first induction treatment. And then proceed to consolidation therapy.

EXAMPLE 4 preparation of Compound I trihydrochloride pentahydrate

By a method described in example 90 of patent document WO2011/147066a1, 100g of compound I represented by formula I was prepared.

Adding a compound I (90g,0.203mol) shown in formula I, 800ml of purified water and 400ml of acetone into a reactor, stirring and heating to 40 +/-5 ℃, adding (74g,0.731mol) concentrated hydrochloric acid into the reactor, adding 2L of acetone after the concentrated hydrochloric acid is added, keeping the temperature at 40 +/-5 ℃, continuing to react for 1h, stirring and cooling to 10 +/-5 ℃ for crystallizationAfter 2h, the reaction mixture is filtered by suction, and the filter cake is washed by 300ml of acetone to obtain 74.7g of yellow or light yellow hydrochloride. 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)。

The base/acid/H of the hydrochloride salt can be inferred by calculating the free base content by HPLC, moisture determination, and the hydrogen ratio in 1H-NMR in nuclear magnetic resonance (see table below) ₂ O is 1:3: 5. In other words, the trihydrochloride pentahydrate of compound I was obtained in example 4.

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 embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The application of a compound I or a pharmaceutically acceptable salt thereof, daunorubicin and cytarabine in preparing a medicament for treating Acute Myeloid Leukemia (AML), wherein the compound I has a structure shown as the following formula (I):

2. use according to claim 1, characterized in that said acute myeloid leukemia is preferably acute myeloid leukemia positive for FLT3 mutation, more preferably acute myeloid leukemia positive for untreated FLT3 mutation; preferably, the FLT3 mutation positivity comprises FLT3 internal tandem repeat (ITD) mutation positivity, FLT3 Tyrosine Kinase Domain (TKD)/D835 mutation positivity, and/or FLT3 Tyrosine Kinase Domain (TKD)/I836 mutation positivity.

3. The use according to claim 1, wherein the acute myeloid leukemia is an acute myeloid leukemia negative for the FLT3 mutation.

4. Use according to any one of claims 1 to 3, wherein the acute myeloid leukaemia 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.

5. Use according to any one of claims 1 to 4, characterized in that: pharmaceutically acceptable salts of compound I include, but are not limited to, hydrochloride, mesylate, malate, tartrate, oxalate, succinate, acetate, sulfate, and the like, of compound I; preferably hydrochloride, malate, tartrate, oxalate, succinate, acetate; further preferred is the hydrochloride salt; the hydrochloride is further preferably a trihydrochloride; the trihydrochloride includes the corresponding anhydrate, hydrate, solvate and the like, and the trihydrochloride pentahydrate is more preferable.

6. Use according to any one of claims 1 to 5, characterized in that: the compound I or pharmaceutically acceptable salt thereof, daunorubicin and cytarabine are contained in the same pharmaceutical preparation, or are respectively prepared into clinically acceptable preparations, and the medicaments are prepared in a combined packaging mode; the preparation comprises oral preparation, injection preparation, local administration preparation, external preparation and the like; preferably, the medicament is in a single dose or divided dose form.

7. 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 medicaments are prepared in a combined packaging mode;

the preparation containing the compound I or the pharmaceutically acceptable salt thereof is preferably an oral preparation such as a tablet, a capsule, a pill and the like; each dosage unit contains about 0.001mg to about 1000mg of compound i or a pharmaceutically acceptable salt thereof (based on compound i), preferably about 1mg to about 500mg, or about 10mg to about 400mg, or about 15mg to about 330mg, or about 20mg to about 300mg, or about 20mg to about 250mg, or about 20mg to about 210mg, or about 20mg to about 180mg, or about 20mg to about 160mg, or about 20mg to about 140mg, or about 20mg to about 120mg, or about 20mg to about 100 mg; exemplary amounts are, for example, about 20mg, about 25mg, about 30mg, about 40mg, about 50mg, about 60mg, about 70mg, about 75mg, about 80mg, about 90mg, about 100mg, about 110mg, about 120mg, about 125mg, about 130mg, about 140mg, about 150mg, about 160mg, about 180mg, about 200mg, about 220mg, about 240mg, about 250mg, about 300mg, about 310mg, about 320mg, about 360mg, about 400 mg;

the daunorubicin preparation is preferably daunorubicin hydrochloride for injection, such as daunorubicin hydrochloride freeze-dried powder for injection; each formulation unit containing from about 10mg to about 30mg, preferably about 20mg, of active ingredient;

the cytarabine hydrochloride preparation is preferably cytarabine hydrochloride for injection, such as cytarabine hydrochloride freeze-dried powder for injection; each unit of the formulation contains from about 0.1g to about 0.5g, for example about 0.1g, about 0.3g or about 0.5g, of active ingredient.

8. Use according to claim 7, characterized in that: the pharmaceutically acceptable salt of compound I is the hydrochloride salt, preferably the trihydrochloride salt, more preferably the trihydrochloride pentahydrate; each unit of the formulation contains from about 1mg to about 500mg, or from about 10mg to about 400mg, or from about 15mg to about 350mg, or from about 20mg to about 310mg, or from about 25mg to about 300mg, or from about 25mg to about 250mg, or from about 50mg to about 200mg, or from about 50mg to about 175mg, or from about 50mg to about 160mg, or from about 50mg to about 150mg, or from about 50mg to about 125mg, or from about 50mg to about 100mg of the trihydrochloride of compound I; exemplary amounts are, for example, about 10mg, about 15mg, about 20mg, about 25mg, about 30mg, about 40mg, about 50mg, about 60mg, about 75mg, about 100mg, about 125mg, about 150mg, about 160mg, about 175mg, about 200mg, about 250mg, about 300mg, about 310mg, about 350mg, about 400mg, about 450mg, about 500 mg; the trihydrochloride salt of compound I above is metered in as an anhydrate.

9. The application of a compound I or a pharmaceutically acceptable salt thereof in preparing a medicament for improving the curative effect of daunorubicin and cytarabine on treating acute myeloid leukemia, wherein the structure of the compound I is shown as the following formula (I):

the acute myeloid leukemia is preferably acute myeloid leukemia positive for FLT3 mutation, more preferably acute myeloid leukemia positive for FLT3 mutation without treatment; preferably, the FLT3 mutation positivity comprises FLT3 internal tandem repeat (ITD) mutation positivity, FLT3 Tyrosine Kinase Domain (TKD)/D835 mutation positivity, and/or FLT3 Tyrosine Kinase Domain (TKD)/I836 mutation positivity;

alternatively, the acute myeloid leukemia is preferably acute myeloid leukemia negative for FLT3 mutation.

10. Use according to claim 9, characterized in that: pharmaceutically acceptable salts of compound I include, but are not limited to, hydrochloride, mesylate, malate, tartrate, oxalate, succinate, acetate, sulfate, and the like, of compound I; preferably hydrochloride, malate, tartrate, oxalate, succinate, acetate; further preferred is the hydrochloride salt; the hydrochloride is further preferably a trihydrochloride; the trihydrochloride includes the corresponding anhydrate, hydrate, solvate and the like, and the trihydrochloride pentahydrate is more preferable.

11. Use according to claim 9, characterized in that: the medicine is prepared into clinically acceptable preparations, such as oral preparations, injection preparations, topical preparations, external preparations and the like.

12. 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.

13. Use according to claim 11, characterized in that: each dosage unit contains from about 0.001mg to about 1000mg of compound I or a pharmaceutically acceptable salt thereof (based on compound I), preferably from about 1mg to about 500mg, or from about 10mg to about 400mg, or from about 15mg to about 330mg, or from about 20mg to about 300mg, or from about 20mg to about 250mg, or from about 20mg to about 210mg, or from about 20mg to about 180mg, or from about 20mg to about 160mg, or from about 20mg to about 140mg, or from about 20mg to about 120mg, or from about 20mg to about 100 mg; exemplary amounts are, for example, about 20mg, about 25mg, about 30mg, about 40mg, about 50mg, about 60mg, about 70mg, about 75mg, about 80mg, about 90mg, about 100mg, about 110mg, about 120mg, about 125mg, about 130mg, about 140mg, about 150mg, about 160mg, about 180mg, about 200mg, about 220mg, about 240mg, about 250mg, about 300mg, about 310mg, about 320mg, about 360mg, about 400 mg.

14. Use according to claim 13, characterized in that: the pharmaceutically acceptable salt of compound I is the hydrochloride salt, preferably the trihydrochloride salt, more preferably the trihydrochloride pentahydrate; each unit of the formulation contains from about 1mg to about 500mg, or from about 10mg to about 400mg, or from about 15mg to about 350mg, or from about 20mg to about 310mg, or from about 25mg to about 300mg, or from about 25mg to about 250mg, or from about 50mg to about 200mg, or from about 50mg to about 175mg, or from about 50mg to about 160mg, or from about 50mg to about 150mg, or from about 50mg to about 125mg, or from about 50mg to about 100mg of the trihydrochloride of compound I; exemplary amounts are, for example, about 10mg, about 15mg, about 20mg, about 25mg, about 30mg, about 40mg, about 50mg, about 60mg, about 75mg, about 100mg, about 125mg, about 150mg, about 160mg, about 175mg, about 200mg, about 250mg, about 300mg, about 310mg, about 350mg, about 400mg, about 450mg, about 500 mg; the trihydrochloride salt of compound I above is metered in as an anhydrate.

15. A composition comprising (1) daunorubicin; (2) cytarabine; (3) a compound I or a pharmaceutically acceptable salt thereof, wherein the compound I has a structure shown as the following formula (I):

16. the composition of claim 15, wherein the molar ratio of cytarabine to daunorubicin is 1 to 100:1 and the molar ratio of compound I to daunorubicin is 100:1 to 1: 100.

17. A kit comprising (1) daunorubicin; (2) cytarabine; (3) a compound I or a pharmaceutically acceptable salt thereof, wherein the compound I has a structure shown as the following formula (I):