WO2021228146A1

WO2021228146A1 - Use of lsd1 inhibitor

Info

Publication number: WO2021228146A1
Application number: PCT/CN2021/093362
Authority: WO
Inventors: 李家印; 林红梅
Original assignee: 石药集团中奇制药技术（石家庄）有限公司
Priority date: 2020-05-12
Filing date: 2021-05-12
Publication date: 2021-11-18
Also published as: CN115551501A; CN115551501B

Abstract

A use of a LSD1 inhibitor compound A or pharmaceutically acceptable salt thereof in the preparation of a medicine for preventing and/or treating a cell proliferative disease and a combination product containing the compound A or the pharmaceutically acceptable salt thereof and another targeting medicine or chemotherapeutic medicine. The cell proliferative disease is a cancer, particularly a small-cell lung cancer, preferably an extensive small-cell lung cancer or a relapsed and metastatic small-cell lung cancer, particularly a small-cell lung cancer which fails to be treated by or is intolerant to standard treatment.

Description

Use of LSD1 inhibitor

Technical field

This patent application relates to the field of medicine, in particular to the use of an LSD1 inhibitor in the preparation of drugs for the prevention and/or treatment of cell proliferative diseases, a combination product containing the LSD1 inhibitor and the prevention and/or treatment of cell proliferation Methods of sexual diseases.

Background technique

1. Small cell lung cancer

Tumor disease is one of the most common diseases in modern times and a common cause of unnatural death. Tumors include benign tumors and malignant tumors. The risk of malignant tumors is very high. Most of the malignant tumors are life-threatening. At present, the methods of treatment of malignant tumors include surgical resection, radiotherapy, drug chemotherapy, and targeted therapy. However, the existing treatment methods are difficult to produce good results for patients with various advanced tumors including small cell lung cancer (SCLC). Curative effect.

Lung cancer is called primary bronchial lung cancer, which is a malignant tumor derived from the trachea, bronchial mucosa or glands. Lung cancer is the most common malignant tumor in the world, and its morbidity and mortality rate ranks first among all kinds of cancer. In 2018, there were 2.09 million new cases of lung cancer worldwide, accounting for 11.6% of all new cancers, and 1.76 million deaths, accounting for 18.4% of all cancer deaths. Lung cancer is also the number one cancer in China in terms of morbidity and mortality, of which about 15% are small cell lung cancer (SCLC). Small cell lung cancer is a heterogeneous neuroendocrine tumor originating from the bronchial mucosal epithelium. There are more than 270,000 new cases of SCLC worldwide each year. Compared with non-small cell lung cancer, small cell lung cancer generally has a faster doubling time, a high proliferation ratio and widespread metastasis earlier. Most patients will have hematological metastasis, so the survival time of small cell lung cancer is significantly shorter than that of non-small cell lung cancer. AJCC Seventh Edition Tumor Staging Manual reported in 2010 the results of a meta-analysis of 2664 SCLC patients. The data showed that: the 5-year survival rate of stage I SCLC patients is about 50%; stage II is about 25%; stage III is reduced to 10% About; Stage IV is less than 3%. This result is similar to the statistical result of SCLC survival rate in my country.

Small cell lung cancer can be divided into limited stage and extensive stage. The median survival time of limited-stage SCLC receiving radiotherapy or chemotherapy is 16 to 24 months. However, more than two-thirds of patients are already in the extensive stage at the time of diagnosis, and can only be treated with a comprehensive treatment plan based on chemotherapy. The median survival period is usually 7 to 12 months, and the recurrence rate of extensive stage small cell lung cancer is very high. At present, comprehensive treatment based on chemotherapy is adopted for extensive-stage small cell lung cancer. Etoposide combined with platinum is the standard first-line chemotherapy. Irinotecan combined with platinum is also an effective first-line treatment for extensive-stage small cell lung cancer. Although small cell lung cancer is very sensitive to initial treatment, most patients with small cell lung cancer develop recurrence or drug resistance after first-line conventional chemotherapy. The median survival time of these patients after receiving follow-up chemotherapy is only 4 to 5 months. Clinical treatment programs have been tried repeatedly in the past 30 years, but they have not improved much. There are great unmet clinical needs in the treatment of relapsed or drug-resistant small cell lung cancer.

2. LSD1 inhibitor

Post-translational modifications of human histones include processes such as methylation, acetylation, phosphorylation, and ubiquitination. They are important regulatory means of epigenetics and affect gene expression by changing the structure of chromatin. The methylation status of histones is regulated by histone methyltransferase and histone demethylase. Lysine specific demethylase (LSD1, also known as KDM1A) is the first reported histone lysine demethylase, which regulates the methylation of histone lysine It is widely involved in transcriptional regulation and affects many physiological processes such as cell proliferation and differentiation, and the pluripotency of embryonic stem cells. In addition, LSD1 also regulates the methylation status of some non-histone substrates, including the tumor suppressor gene p53 and DNA methyltransferase 1 (DNMT1). The abnormal expression of LSD1 has been reported in a variety of tumors, which is closely related to the occurrence, development and poor prognosis of tumors.

Among them, the study found that the high expression of LSD1 was found in 98% of the samples of small cell lung cancer, which is involved in maintaining the expression of tumor stem cell-like genes in small cell lung cancer, which is one of the important reasons for the refractory and recurrence of the disease. LSD1 inhibitors can affect the expression of differentiation-related genes by regulating the monomethylation and dimethylation status of histones H3K4 and H3K9, promote the differentiation of cancer stem cells, inhibit tumor growth, and enhance the sensitivity of small cell lung cancer to chemotherapy. Therefore, LSD1 inhibitors have good application prospects for the treatment of poorly differentiated malignant tumors such as small cell lung cancer. However, at present, there are no LSD1 inhibitors on the market at home and abroad, and only small molecule drugs from many pharmaceutical companies are in the clinical stage, which are mainly used for the treatment of small cell lung cancer and other diseases. Among them, the development direction of the four drugs, INCB059872, ORY-1001, CC-90011 and GSK2879552, includes the indication of small cell lung cancer.

GSK2879552 is an irreversible LSD1 inhibitor developed by GlaxoSmithKline, which is intended for the treatment of small cell lung cancer. GSK2879552 started a phase I clinical study for the treatment of relapsed/refractory small cell lung cancer in February 2014. From February 4, 2014 to April 18, 2017, a total of 29 patients were enrolled, and a total of 22 patients completed the study; 7 patients withdrew from the study, mainly due to adverse events (AEs). Among them, 8 cases were taken to reduce the dose of the trial drug or to discontinue the treatment due to grade 3 or severe thrombocytopenia. 9 patients reported 12 serious adverse events (SAEs); 6 cases were considered treatment-related, of which the most common was encephalopathy (4 SAEs). Three patients died; one death was related to SAEs. The disease control rate at 16 weeks of evaluation was only 14% (4/29). According to the clinical Phase I results, the drug has now ceased to be developed. CC-90011 also found that the drug-related grade 3/4 adverse reaction was thrombocytopenia (9%) in the clinical phase I study (NCT02875223). The clinical research results of the above drugs suggest that LSD1 inhibitors have serious safety defects when used in small cell lung cancer, and thrombocytopenia is one of the main adverse reactions of LSD1 inhibitors. The LSD1 inhibitor drugs currently under development still need a long research journey from clinical application.

Compound A is a new type of LSD1 inhibitor. This compound and its preparation method are disclosed in Example 144 of WO2018137644A1. WO2020015745A1 discloses the dihydrochloride salt of compound A. However, it is still unclear whether compound A or its pharmaceutically acceptable salt is used in clinical practice, the important risk and exact efficacy, its use in specific diseases, and the combination of compound A or its pharmaceutically acceptable salt with other drugs Effect. Therefore, it is necessary to study the safety and effectiveness of compound A or its pharmaceutically acceptable salt in clinical application, in order to provide reference for clinical medication.

Summary of the invention

Based on the above-mentioned defects in the prior art, in the first aspect of the present invention, the purpose of the present invention is to provide compound A or a pharmaceutically acceptable salt thereof in the preparation of drugs for the prevention and/or treatment of cell proliferative diseases, especially cancer application. The structure of the compound A is shown in the following formula (I):

The cancer is selected from lung cancer, wherein the lung cancer is preferably small cell lung cancer.

In the second aspect of the present invention, the present invention also provides a medicine containing a preventive and/or therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof, which is used to prevent and/or treat cell proliferation. Diseases, especially cancer drugs. Further, the cancer is selected from lung cancer, wherein the lung cancer is preferably small cell lung cancer.

In the third aspect of the present invention, the present invention also provides a method for preventing and/or treating cell proliferative diseases, especially cancer. Further, the cancer is selected from lung cancer, wherein the lung cancer is preferably small cell lung cancer.

In the fourth aspect of the present invention, the present invention provides a combination product comprising Compound A or a pharmaceutically acceptable salt thereof, and another targeted drug or chemotherapeutic drug, the additional targeted drug or Chemotherapy drugs are selected from platinum drugs and topoisomerase inhibitors, including but not limited to cisplatin, carboplatin, lobaplatin, nedaplatin, cycloplatin, oxaliplatin, teniposide, etoposide, topo At least one of Tecan and Irinotecan. Preferably, the additional targeted drug or chemotherapeutic drug is selected from at least one of etoposide and cisplatin.

Description of the drawings

Figure 1 The effect of compound A on the expression of neuroendocrine-related genes ASCL1 and GRP.

Figure 2 Flow chart of Phase I clinical program.

Detailed description of the invention

definition

The term "pharmaceutically acceptable salt" as used herein refers to a salt of a free acid or a free base, preferably the hydrochloride.

The term "prevention" as used herein refers to when used for a disease or condition (e.g., cancer), when compared with a subject who has not been administered a compound or drug (e.g., a combination product claimed in this application), the compound or Drugs can reduce the frequency or delay the onset of medical symptoms in subjects.

The term "treatment" as used herein refers to alleviating, alleviating or ameliorating the symptoms of a disease or disorder, ameliorating the symptoms caused by potential metabolism, inhibiting the disease or symptom, such as preventing the development of the disease or disorder, alleviating the disease or disorder, or causing the disease Or the regression of the disease, the alleviation of the condition caused by the disease or disease, or the prevention of the symptoms of the disease or disease.

The term "cell proliferative disease" as used herein refers to a condition in which the growth rate of the cell population is lower than or higher than the expected rate under a given physiological state and condition.

The term "cancer" as used herein refers to a new organism or tumor caused by abnormal uncontrolled cell growth. Non-limiting examples include those exemplary cancers described in the detailed description of the invention. The term "cancer" includes diseases involving both premalignant cancer cells and malignant cancer cells.

The term "subject" as used herein is meant to include humans (e.g., patients) and animals (e.g., mice, rats, dogs, cats, rabbits, chickens, or monkeys, etc.).

The term "effective amount" or "preventively and/or therapeutically effective amount" as used herein refers to a sufficient amount (eg, dose) of a drug or compound administered, which will reduce the disease or condition being treated to a certain extent One or more symptoms. The result may be to shrink and/or alleviate the condition or cause of the disease or any other desired changes in the biological system. For example, an "effective amount" for therapeutic use is an amount that provides a compound or drug (for example, a combination product claimed in the present application) that significantly reduces the clinical symptoms of a disease or disorder without causing excessive toxic and side effects.

The term "dose" as used herein refers to the weight of the active substance per kilogram (kg) of the subject's body weight (e.g., milligrams (mg)), or the weight of the active substance taken by each subject at once (e.g., Milligrams (mg)).

The term "room temperature" as used herein refers to 25°C ± 5°C. At the same time, if the experiment temperature is not specified, it is room temperature.

As used herein, the term "about" refers to ±10% of the value modified by the term, more preferably ±5%, and most preferably ±2%. Therefore, those of ordinary skill in the art can clearly refer to the modified value. The numerical value determines the range of the term "about".

Herein, the compound A of the present invention or a pharmaceutically acceptable salt thereof can be prepared according to the methods disclosed in the prior art. For example, refer to Example 144 of WO2018137644A1 to prepare compound A, and refer to WO2020015745A1 to prepare compound A dihydrochloride. The full texts of WO2018137644A1 and WO2020015745A1 are incorporated into the present invention by reference.

In this article, in vitro studies of the present invention use Compound A as the test drug; in vivo pharmacodynamic studies, pharmacokinetic studies, toxicology studies and clinical studies are administered in the form of Compound A dihydrochloride. The drug doses are all calculated with compound A.

In this article, unless otherwise specified, the test materials used in the present invention (including but not limited to control drugs, test reagents, etc.) are all commercially available. The reference compound INCB059872 was prepared according to the method described in Example 6 of WO2017184934 (purity 99.33%).

In the first aspect of the present invention, the object of the present invention is to provide the use of compound A or a pharmaceutically acceptable salt thereof in the preparation of a medicine for preventing and/or treating cell proliferative diseases, especially cancer. The structure of the compound A is shown in the following formula (I):

The small cell lung cancer is preferably extensive-stage small cell lung cancer or relapsed and/or metastatic small cell lung cancer; further preferably, small cell lung cancer that has failed or is intolerant to standard treatments. The standard treatment plan for small cell lung cancer of the present invention includes but is not limited to: (1) Extensive-stage small cell lung cancer: Comprehensive treatment based on chemotherapy is recommended for extensive-stage small cell lung cancer. For those with local symptoms or brain metastases, it is recommended to be based on chemotherapy Combine radiotherapy or other treatment methods. The chemotherapy regimen recommends etoposide combined with cisplatin (EP), etoposide combined with carboplatin (EC), irinotecan combined with cisplatin (IP), irinotecan combined with carboplatin (IC), or etoposide combined with loplatin ( EL) program. (2) Relapsed small cell lung cancer: Topotecan, irinotecan, gemcitabine, temozolomide or paclitaxel are recommended for recurrence or progression within 3 months after first-line chemotherapy; Topotecan is recommended for recurrence or progression within 3 to 6 months Drug therapy such as Kang, irinotecan, gemcitabine, docetaxel, temozolomide or vinorelbine; patients who relapse or progress after 6 months can choose the initial treatment plan.

Further preferably, the small cell lung cancer of the present invention includes treatment with cisplatin, carboplatin, etoposide, lobaplatin, topotecan, irinotecan, gemcitabine, temozolomide, paclitaxel, docetaxel, vinorelbine, etc. Small cell lung cancer for which one or more of the drugs have failed or are intolerant to treatment.

More preferably, the small cell lung cancer of the present invention refers to small cell lung cancer with high expression of ASCL1 and/or GRP.

Further preferably, in the application of the present invention, the drug has good tolerance and no obvious adverse reactions.

Preferably, in the application of the present invention, the medicine contains a preventive and/or therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof. The preventive and/or therapeutic effective amount is preferably 0.001-1000 mg and the range between the following amounts: 0.01 mg, 0.1 mg, 0.5 mg, 1 mg, 1.5 mg, 3 mg, 6 mg, 9 mg, 11 mg, 12 mg, 13 mg, 20 mg , 30mg, 40mg, 50mg, 60mg, 70mg, 80mg, 90mg, 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg, 1000mg, including but not limited to: 0.01-1000mg, 0.1-1000mg, 0.1 -900mg, 0.1-800mg, 0.1-700mg, 0.1-600mg, 0.1-500mg, 0.1-400mg, 0.1-300mg, 0.1-200mg, 0.1-100mg, 0.1-90mg, 0.1-80mg, 0.1-70mg, 0.1-60mg , 0.1-50mg, 0.1-40mg, 0.1-30mg, 0.1-20mg, 0.1-15mg, 0.1-10mg, 0.5-1000mg, 0.5-900mg, 0.5-800mg, 0.5-700mg, 0.5-600mg, 0.5-500mg, 0.5 -400mg, 0.5-300mg, 0.5-200mg, 0.5-100mg, 0.5-90mg, 0.5-80mg, 0.5-70mg, 0.5-60mg, 0.5-50mg, 0.5-40mg, 0.5-30mg, 0.5-20mg, 0.5-15mg , 0.5-10mg, 0.5-13mg, 0.5-12mg, 0.5-11mg, 0.5-9mg, 0.5-6mg, 0.5-3mg, 0.5-1.5mg, 1.5-13mg, 1.5-12mg, 1.5-11mg, 1.5-9mg, 1.5-6mg, 1.5-3mg, 3-13mg, 3-12mg, 3-11mg, 3-9mg, 3-6mg, 6-13mg, 6-12mg, 6-11mg, 6-9mg, 9-13mg, 9- 12mg, 9-11mg, 11-13mg, 11-12mg, 12-13mg. It can be administered in a single dose or in divided doses. The medicine is prepared into clinically accepted preparations, such as oral preparations, injection preparations, topical administration preparations or external preparations and the like.

Further, in addition to Compound A or a pharmaceutically acceptable salt thereof as an active ingredient, the medicament also contains at least one pharmaceutically acceptable carrier, excipient and/or diluent. The medicament can be prepared at a suitable dosage level in a conventional solid or liquid carrier or diluent and a conventional pharmaceutically prepared auxiliary agent in a known manner. The preferred formulations are suitable for oral administration. The forms of administration include, for example, pills, tablets, film tablets, coated tablets, capsules, powders and depots.

In addition, the present invention also includes preparations for parenteral administration, including dermal, intradermal, intradermal, intravascular, intravenous, intramuscular, intraperitoneal, intranasal, intravaginal, intrabuccal, transdermal, rectal, For subcutaneous, sublingual, topical or transdermal administration, in addition to typical vehicles and/or diluents, the formulation also contains Compound A and/or a pharmaceutically acceptable salt thereof as active ingredients.

The compounds of the present invention can be administered as a monotherapy or together with other active agents, especially targeted drugs, chemotherapeutics or anti-tumor antibodies. In addition, they can also be used in conjunction with surgery and/or irradiation.

Further, in the application of the present invention, Compound A or a pharmaceutically acceptable salt thereof can be used in combination with one or more of other targeted drugs or chemotherapeutic drugs. The other targeted drugs or chemotherapeutic drugs refer to targeted drugs or chemotherapeutic drugs that are clinically used to treat tumor-related diseases, such as platinum drugs and topoisomerase inhibitors, including but not limited to cisplatin, carboplatin, and chemotherapeutics. At least one of platinum, nedaplatin, cycloplatin, oxaliplatin, teniposide, etoposide, topotecan, and irinotecan. More preferably, the targeted drug or chemotherapeutic drug is selected from at least one of etoposide and cisplatin.

The present invention provides a medicament for the prevention and/or treatment of small cell lung cancer, which is characterized in that it contains a preventive and/or therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof, and optionally, pharmaceutically acceptable Accepted excipients, carriers and/or diluents. The medicine is prepared into clinically accepted preparations, such as oral preparations, injection preparations, topical administration preparations or external preparations. The drug may also contain one or more of other targeted drugs or chemotherapeutic drugs. The other targeted drugs or chemotherapeutic drugs refer to targeted drugs or chemotherapeutic drugs that are clinically used to treat tumor-related diseases, such as platinum drugs and topoisomerase inhibitors, including but not limited to cisplatin, carboplatin, and chemotherapeutics. At least one of platinum, nedaplatin, cycloplatin, oxaliplatin, teniposide, etoposide, topotecan, and irinotecan. More preferably, the targeted drug or chemotherapeutic drug is selected from at least one of etoposide and cisplatin.

Further, the medicament of the present invention is as described in the first aspect of the present invention.

In the third aspect of the present invention, another object of the present invention also includes providing a method for preventing and/or treating small cell lung cancer, the method comprising administering to a subject a preventive and/or therapeutically effective amount of Compound A Or a pharmaceutically acceptable salt thereof. The administration may be oral administration, injection administration, topical administration or in vitro administration. Correspondingly, compound A or a pharmaceutically acceptable salt thereof is prepared into clinically accepted preparations and then administered. The preparations include oral preparations, injection preparations, topical administration preparations, topical preparations, and the like.

The method also includes providing a safe and effective dose and dose frequency of Compound A or a pharmaceutically acceptable salt thereof for the treatment of small cell lung cancer.

The preventive and/or therapeutically effective amount can treat or alleviate the tumor disease in the subject. A suitable dosage range of Compound A or a pharmaceutically acceptable salt thereof is from about 0.001 mg/kg to about 1000 mg/kg; preferably, from about 0.01 mg/kg to about 100 mg/kg. Preferably, the dose of compound A or its pharmaceutically acceptable salt per administration is 0.001mg-1000mg; further preferably, the dose per administration is 0.01mg-100mg, or 0.1mg-50mg, most preferably 0.5-25mg . It is administered in a single dose or in divided doses; it can be administered continuously or at intervals. Exemplarily, the dosage and dosage frequency are: once a week, 0.5-13mg, 0.5-12mg, 0.5-11mg, 0.5-9mg, 0.5-6mg, 0.5-3mg, 0.5- 1.5mg, 1.5-13mg, 1.5-12mg, 1.5-11mg, 1.5-9mg, 1.5-6mg, 1.5-3mg, 3-13mg, 3-12mg, 3-11mg, 3-9mg, 3-6mg, 6-13mg , 6-12mg, 6-11mg, 6-9mg, 9-13mg, 9-12mg, 9-11mg, 11-13mg, 11-12mg, 12-13mg, for example, 0.5, 1.5, 3, 6, 9, 11 , 12, 13 mg, etc., preferably 6 mg to 9 mg.

The dosage of Compound A or its pharmaceutically acceptable salt of the present invention is all based on Compound A.

In the fourth aspect of the present invention, the present invention provides a combination product comprising Compound A or a pharmaceutically acceptable salt thereof, and another targeted drug or chemotherapeutic drug, the additional targeted drug or Chemotherapy drugs are selected from platinum drugs and topoisomerase inhibitors, including but not limited to cisplatin, carboplatin, lobaplatin, nedaplatin, cycloplatin, oxaliplatin, teniposide, etoposide, topo At least one of Tecan and Irinotecan. More preferably, the additional targeted drug or chemotherapeutic drug is selected from at least one of etoposide and cisplatin. Still more preferably, the present invention provides a combination product comprising Compound A or a pharmaceutically acceptable salt thereof and cisplatin, or Compound A or a pharmaceutically acceptable salt thereof and cisplatin and etoposide.

The combination product is in the form of a pharmaceutical composition. In some embodiments, in the combination product, the compound A or a pharmaceutically acceptable salt thereof and another targeted drug or chemotherapeutic drug are each in the form of a separate preparation. Further, the compound A or a pharmaceutically acceptable salt thereof and another targeted drug or chemotherapeutic drug can be administered simultaneously or sequentially.

The combination product contains a preventive and/or therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof, wherein the preventive and/or therapeutically effective amount is preferably 0.001-1000 mg, and the range between the following amounts: 0.01 mg, 0.1mg, 0.5mg, 1mg, 1.5mg, 3mg, 6mg, 9mg, 11mg, 12mg, 13mg, 20mg, 30mg, 40mg, 50mg, 60mg, 70mg, 80mg, 90mg, 100mg, 200mg, 300mg, 400mg, 500mg , 600mg, 700mg, 800mg, 900mg, 1000mg, including but not limited to: 0.01-1000mg, 0.1-1000mg, 0.1-900mg, 0.1-800mg, 0.1-700mg, 0.1-600mg, 0.1-500mg, 0.1-400mg, 0.1 -300mg, 0.1-200mg, 0.1-100mg, 0.1-90mg, 0.1-80mg, 0.1-70mg, 0.1-60mg, 0.1-50mg, 0.1-40mg, 0.1-30mg, 0.1-20mg, 0.1-10mg, 0.5-1000mg , 0.5-900mg, 0.5-800mg, 0.5-700mg, 0.5-600mg, 0.5-500mg, 0.5-400mg, 0.5-300mg, 0.5-200mg, 0.5-100mg, 0.5-90mg, 0.5-80mg, 0.5-70mg, 0.5 -60mg, 0.5-50mg, 0.5-40mg, 0.5-30mg, 0.5-20mg, 0.5-15mg, 0.5-10mg, 0.5-13mg, 0.5-12mg, 0.5-11mg, 0.5-9mg, 0.5-6mg, 0.5-3mg , 0.5-1.5mg, 1.5-13mg, 1.5-12mg, 1.5-11mg, 1.5-9mg, 1.5-6mg, 1.5-3mg, 3-13mg, 3-12mg, 3-11mg, 3-9mg, 3-6mg, 6-13mg, 6-12mg, 6-11mg, 6-9mg, 9-13mg, 9-12mg, 9-11mg, 11-13mg, 11-12mg, 12-13mg. The combined product is prepared into clinically accepted preparations, such as oral preparations, injection preparations, topical administration preparations or topical preparations and the like.

Further, the combination product further comprises at least one pharmaceutically acceptable carrier, excipient and/or diluent. The combination product can be prepared in a known manner in a conventional solid or liquid carrier or diluent and a conventional pharmaceutically-prepared adjuvant at a suitable dosage level. The preferred formulations are suitable for oral administration. The forms of administration include, for example, pills, tablets, film tablets, coated tablets, capsules, powders, and reservoirs.

Further, the combination product is used to prevent and/or treat cell proliferative diseases, especially cancer. Further, the cancer is selected from lung cancer, wherein the lung cancer is preferably small cell lung cancer.

In order to evaluate the efficacy and safety of compound A or its pharmaceutically acceptable salt in the treatment of small cell lung cancer, the inventors of this application carried out LSD1 in vitro activity inhibition test, in vitro cultured tumor cell proliferation test, in vivo tumor suppression test, pharmacokinetics Tests, safety toxicology tests, and clinical phase I studies have shown that compound A has a clear mechanism of action, can significantly inhibit the activity of LSD1 protein, and has a significant effect on inhibiting tumor growth in small cell lung cancer cell culture in vitro and in vivo transplantation tumor models. Toxicology research Show that the drug is safe and tolerable. In the clinical phase I study, compound A can safely and effectively treat extensive-stage small cell lung cancer. It can be seen that compound A can effectively treat small cell lung cancer with good clinical safety. In addition, the combination of compound A and cisplatin or cisplatin + etoposide can significantly enhance the anti-tumor effect, produce a synergistic effect, and provide support for clinical combined treatment programs.

Detailed ways

The examples listed below are for better describing 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 based on the above-mentioned content of the invention, which still belongs to the protection scope of the present invention.

1. In vitro research

1. The inhibitory effect of compound A on LSD1

The enzyme used in this test is human LSD1, the standard substrate is histone H3(1-21) K4me2 peptide (10μM), and INCB059872 is used as the reference compound. The enzyme fluorescence coupling method is adopted by horseradish peroxidase (HPR ) And the fluorescent reagent Amplex Red to detect the activity of the compound by detecting _{the H 2} O ₂ generated by the combination of LSD1 and FAD to produce demethylation activity. Dilute 3 times from 10 μM, and test the IC50 value of the compound and the control compound INCB059872 at 10 concentrations. The compound is pre-incubated with the enzyme for 30 minutes before adding the substrate to start the reaction. Fluorescence detector: EnVision, excitation wavelength: Ex/Em=535/590nM.

The specific test conditions are as follows:

LSD1 buffer composition: 50mM Tris-HCl, pH 7.5, 0.05% CHAPS, 1% DMSO.

Reaction time: 1 hour at room temperature

reaction process:

1. Add the enzyme to the freshly prepared buffer.

2. Use nano-upgraded Acoustic Technology (Echo 550, LabCyte Inc. Sunnyvale, CA) to add the compound's DMSO solution to the enzyme mixture, and incubate at room temperature for 30 minutes.

3. Add the substrate to the freshly prepared buffer.

4. Incubate for 1 hour at room temperature.

5. Prepare the test mixture.

6. Use Perkin Elmer Envision to read the data.

7. Use Excel and GraphPad Prism software to analyze the data.

The results show that: as shown in Table 1, compound A has a significant inhibitory effect on LSD1, IC50=8.0 nM, and its inhibitory effect is better than the reference compound INCB059872 (IC50=12.5 nM).

Table 1 Inhibitory effect of compound A on LSD1

2. Compound A inhibits the proliferation of small cell lung cancer cell lines NCI-H1417 and NCI-H526

In this experiment, the Cell Titer Glo experimental method was used to analyze the effects of LSD1 inhibitor compound A and the reference compound INCB059872 on the proliferation of small cell lung cancer cell lines NCI-H1417 and NCI-H526.

Experimental materials: RPMI 1640 medium, fetal bovine serum, penicillin streptomycin double antibody, L-glutamine, Promega CellTiter-Glo kit, dimethyl sulfoxide (DMSO). Envision multi-label analyzer (PerkinElmer 2104).

experimental method:

Cell culture medium for experiment: Add 10% fetal bovine serum, 1% penicillin/streptomycin double antibody, and 1% L-glutamine to RPMI1640 cell culture medium at 4°C for later use.

After the suspended cells are evenly pipetted, count them with a Vi-cell cytometer, prepare 3000 cells/90μL of NCI-H1417 cell suspension with culture medium, and add it to a 96-well cell culture plate, 90μL/well. The reference compound INCB059872 and the test compound A were diluted with DMSO into a 10mM stock solution, and then using this concentration as the starting concentration, the compound was diluted 3 times with DMSO 9 times to obtain 10 concentrations, which were diluted with culture medium. The compound was diluted 100 times, and at the same time, the same volume of DMSO as the compound solution was diluted 100 times in the medium, and 10 μL of the diluted mixture was transferred to the corresponding cell plate (ie 10-fold dilution), and the DMSO dilution was Transfer to F and G rows of the control group. The final concentration of the compound was 10 μM as the initial concentration, and 10 concentrations were diluted in 3 folds, mixed and centrifuged, and placed in _{a cell incubator containing 5% CO 2} at 37° C. for 10 days. Take out the 96-well cell culture plate, add CTG reagent, 50μL/well, mix and centrifuge, and incubate at room temperature for 10 minutes. Use Envison multi-marker analyzer to read.

Use the same method to test against NCI-H526 cells.

Data analysis: NCI-H1417 cell test, using the equation (1-(sample value-minimum)/(maximum-minimum))*100 to convert the original data into the inhibition rate, and calculate the IC50 using a four-parameter dose-response Model, the formula is Y=Bottom+(Top-Bottom)/(1+10^((logEC50-x)*HillSlope)). The NCI-H526 cell test uses the formula: Vsample/Vvehicle control x100% to calculate the cell survival rate. Among them, Vsample is the reading of the drug treatment group, and Vvehicle control is the average value of the solvent control group. Using GraphPad Prism 5.0 software, a non-linear regression model was used to draw the S-type dose-survival rate curve and calculate the IC50 value.

The results show that: as shown in Table 2, compound A has a significant proliferation inhibitory effect on NCI-H1417, IC50=207.8nM, and its inhibitory activity is better than the reference compound INCB059872 (IC50=421.0nM). Compound A has a significant proliferation inhibitory effect on NCI-H526, IC50=610.6nM, which is significantly better than the reference compound INCB059872 (IC50>1000.0nM).

Table 2 Compound A inhibits the proliferation of NCI-H1417 and NCI-H526

3. The effect of compound A on the expression of neuroendocrine-related genes ASCL1 and GRP

Small cell lung cancer is a neuroendocrine tumor of the lung. About 75% of small cell lung cancer patients express ASCL1. ASCL1 is a neuroendocrine-related transcription factor and a potential target for the treatment of highly malignant neuroendocrine lung cancer. Gastrin-releasing peptide (GRP) is a gastrointestinal hormone that is widely distributed in the nervous system, gastrointestinal tract and lungs. Small cell lung cancer cells can secrete GRP, and the detection of GRP expression is helpful for disease monitoring.

In this experiment, real-time quantitative PCR was used to determine the changes in the expression of ASCL1 and GRP in NCI-H1417 cells after drug treatment. Real-time fluorescent quantitative PCR adopts the SYBR Green method. 5μL of SYBR Green is added to the qPCR reaction system. SYBR Green binds to double-stranded DNA and emits fluorescence. The PCR product amplification amount is detected by detecting the fluorescence intensity of SYBR Green in the reaction system. The Ct values of the target gene and the internal reference gene were analyzed, and the ΔΔCT method was used to calculate the expression of the target gene relative to the control group. Based on this data, statistical analysis was performed to evaluate differences between groups. The comparison between two groups was analyzed by T-test, and the comparison between three or more groups was analyzed by one-way ANOVA.

Test results: Compared with the control group, the mRNA expression levels of ASCL1 and GRP in the compound A and INCB059872 added group decreased, and the mRNA expression of ASCL1 and GRP in the compound A added group decreased in a dose-dependent manner. See Figure 1 for details.

2. In vivo drug efficacy research

1. In vivo pharmacodynamic study of compound A in a mouse model of small cell lung cancer NCI-H1417 cell subcutaneous xenotransplantation

Test method and procedure:

Cell culture: Human small cell lung cancer NCI-H1417 (ATCC, Manassas, Virginia, catalog number: CRL-5869) cells are cultured in a monolayer in vitro, and the culture conditions are RPMI-1640 medium plus 10% fetal bovine serum, 37 Cultivation and passage at 5% CO _2. When the cell saturation is 80%-90%, the cells are collected by trypsin-EDTA digestion, counted, adjusted to 10×10 ⁷ cells/mL and resuspended in PBS.

Cell inoculation: 0.2mL (10×10 ⁶ cells) NCI-H1417 cells (with Matrigel, volume 1:1) were subcutaneously inoculated on the right back of each mouse, and the average tumor volume reached about 100-150mm ³ and randomly grouped , Start the administration.

Daily observation of experimental animals: Monitor the health and death of animals every day. Routine inspections include observation of tumor growth and drug treatment's influence on the daily behavior of animals, such as behavioral activities, food and water intake (visual inspection only), and weight changes (weekly Measure your body weight twice), physical signs or other abnormalities. Based on the number of animals in each group, the number of animal deaths and side effects in the group were recorded.

Tumor measurement: Measure the diameter of the tumor with a vernier caliper twice a week. The calculation formula of tumor volume is: V=0.5a×b ² , a and b represent the long diameter and short diameter of the tumor, respectively. The anti-tumor efficacy of the compound was evaluated by TGI (%) or relative tumor growth rate T/C (%). Relative tumor proliferation rate T/C(%)= _TRTV / _CRTV ×100% ( _TRTV : treatment group RTV; C _RTV : negative control group RTV). Calculate the relative tumor volume (RTV) according to the results of tumor measurement. The calculation formula is RTV=Vt/V0, where V0 is the tumor volume measured during group administration (ie D0), and Vt corresponds to a certain time in the mouse For the tumor volume at the time of measurement, the _{data of T RTV} and C _{RTV are} taken on the same day. TGI (%) reflects the tumor growth inhibition rate. TGI(%)=[(1-(Average tumor volume at the end of a certain treatment group-average tumor volume at the beginning of the treatment group)/(Average tumor volume at the end of treatment in the solvent control group-when the solvent control group starts treatment Average tumor volume)]×100%.

After the experiment is over, the tumor weight is detected, and the _{percentage of T weight} /C _{weight is} calculated. T _weight and C _weight represent the tumor weight of the administration group and the vehicle control group, respectively.

The test also observed the distribution of the drug in the lung tissue and blood. PK sampling and analysis: After the last administration, the animal's whole blood, plasma, lung and tumor tissues were collected for in vitro PK/PD experiments.

Statistical analysis: Including the mean and standard error (SEM) of the tumor volume at each time point in each group. The treatment group showed the best treatment effect at the end of the trial (35th day after administration), so statistical analysis was performed based on this data to evaluate the difference between the groups. The comparison between three or more groups is analyzed by one-way ANOVA. In this experiment, the F values of RTV and tumor weight statistics are significantly different, and the Games-Howell method is used to test. Use SPSS 17.0 for all data analysis. p<0.05 considered a significant difference.

Using King’s formula to evaluate the combined effect of compound A and cisplatin, the specific method is as follows: Q=E(a+b)/[E(a)+E(b)-E(a)*E(b) ](E(a+b): tumor inhibition rate after a and b combined administration; E(a): tumor inhibition rate when a single administration; E(b): b single administration Tumor inhibition rate. Q<0.85 has an antagonistic effect, 0.85≤Q≤1.15 has an additive effect, and Q>1.15 has a synergistic effect.

The results show that: as shown in Table 3, in the small cell lung cancer NCI-H1417 cell subcutaneous xenograft mouse model, compound A alone has significant tumor inhibition at doses of 1 mg/kg, 1.5 mg/kg and 3 mg/kg It is effective and dose-dependent. TGI is 81.9%, 99.2% and 115.6%, and T/C is 40.6%, 27.0% and 15.1%.

When compound A (1mg/kg, PO QD or 3mg/kg, PO BIW) and cisplatin (1mg/kg, IP BIW) were combined treatment, compared with the solvent control group and the cisplatin group, it had a significant anti-tumor effect. The TGI is 130.1% and 134.5%, and the calculated Q values are 1.34 and 1.31, respectively. It can be seen that compound A and cisplatin have a synergistic effect in inhibiting tumors.

Table 3 Inhibitory effect of compound A on NCI-H1417 cell subcutaneous xenograft tumor

The pharmacokinetic data after the last administration showed that the drug concentration ratio of compound A (1mg/kg) in lung tissues at 4h and 24h was 125 and 140, respectively, indicating that the drug can be enriched in lung tissue and is expected to be effective in the treatment of lung cancer. Certain advantages.

Compound A showed good tolerance at all doses, and no animal died in all treatment groups. In the NCI-H1417 in vivo efficacy test, the effect of the drug on blood cells was also investigated. The three dose groups of compound A were used alone or in combination with cisplatin. The blood routine was tested 35 days after administration, and there were no key blood routine indicators such as white blood cells, red blood cells, platelets, monocytes, neutrophils, and reticulocytes. Significant impact, showing good hematological safety.

2. The in vivo pharmacodynamic study test method and procedure of compound A in a mouse model of small cell lung cancer NCI-H526 cell subcutaneous xenotransplantation:

Cell culture: human small cell lung cancer NCI-H526 (ATCC, Manassas, Virginia, catalog number: CRL-5811) cells, suspension culture in vitro, culture condition is RPMI-1640 medium plus 10% fetal bovine serum, 100U /mL penicillin and 100μg/mL streptomycin, culture and subculture _{at 37°C with 5% CO 2.} When the cell saturation is 80%-90%, count, adjust 10×10 ⁷ cells/mL and resuspend in PBS.

Cell inoculation: Take 5 BABL/c nude mice and inoculate 0.1 mL (5×10 ⁶ cells) NCI-H526 cells in the subcutaneous position of the right back of each animal. When the tumor volume is close to 500 mm ³ , remove the tumor for use Organize vaccination. After removing the necrotic tissue, the tumor tissue was cut into ^{small pieces (20-30mm 3} ), and subcutaneously inoculated on the right back of each mouse to wait for the tumor to grow. When the average tumor volume reached about 90mm ³ , they were randomly divided into groups and the drug was started.

Statistical analysis: Including the mean and standard error (SEM) of the tumor volume at each time point in each group. The treatment group showed the best treatment effect at the end of the trial (on the 21st day after administration), so statistical analysis was performed based on this data to evaluate the differences between the groups. The comparison between three or more groups is analyzed by one-way ANOVA. In this experiment, the F values of RTV and tumor weight statistics are significantly different, and the Games-Howell method is used to test. Use SPSS 17.0 for all data analysis. p<0.05 considered a significant difference.

The King’s formula was used to evaluate the combined effects of compound A with cisplatin and etoposide. The specific method is as follows: Q=E(a+b)/[E(a)+E(b)-E(a)* E(b)] (where E(a+b): the tumor inhibition rate after the combined administration of a and b; E(a): the tumor inhibition rate when a single drug is administered; E(b): b alone Tumor inhibition rate during administration. Q<0.85 has an antagonistic effect, 0.85≤Q≤1.15 has an additive effect, and Q>1.15 has a synergistic effect.

The results show that: as shown in Table 4, in the small cell lung cancer NCI-H526 cell subcutaneous xenograft mouse model, the compound A (1.5 mg/kg) single administration group and the INCB059872 (1.5 mg/kg) single administration group The average tumor volume of the tumors was 1,427 and 1,571mm ³ , respectively, and there was no significant tumor suppressor effect. Compared with the solvent control group, the combined administration of Cisplatin and Etoposide had a certain inhibitory effect. The average tumor volume was 999 mm ³ , the T/C value was 50.15%, and the TGI value was 53.33%. When compound A (1.5mg/kg, PO QD or 3mg/kg, PO BIW) is used in combination with cisplatin (Cisplatin) plus etoposide (Etoposide), compared with the solvent control group and chemotherapy combination administration, it has Significant anti-tumor effect (T/C = 11.38% and 19.01%, TGI = 90.36% and 80.46%). The animals in all the administration groups were well tolerated, and there was no significant weight loss or animal death. When compound A (1.5mg/kg, PO QD) is used in combination with cisplatin (Cisplatin) + etoposide (Etoposide), the Q value is 1.33. It can be seen that compound A, cisplatin and etoposide in combination therapy can inhibit tumors Synergies have been produced in this respect.

Table 4 Inhibitory effect of compound A on NCI-H526 cell subcutaneous xenograft tumor

3. Pharmacokinetic research

1. The pharmacokinetic study of compound A in rodent SD rats

The pharmacokinetic properties of Compound A in male and female SD rats include: (1) 0.5 mg/kg single intravenous injection study; (2) 0.5, 1.5 and 5 mg/kg intragastric dose escalation studies; (3) ) Study on repeated intragastric administration once every 3 days, 1.5 mg/kg each time, 3 consecutive times.

experiment method:

Twenty-four SD rats were divided into 4 groups, 3 males and 3 females, according to their similar body weights. The animals of the first group were given 0.5 mg/kg compound A by a single intravenous injection, and the vehicle was 10% HP-β-CD (β-cyclodextrin aqueous solution). The animals of group 2 and group 4 were given 0.5 and 5 mg/kg compound A by single gavage, respectively. The animals in the third group were given 3 consecutive intragastric administrations, once every 3 days, 1.5 mg/kg compound A each time. The first group of animals collected plasma samples before and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36 and 48 hours after administration. Plasma samples were collected from animals in groups 2 and 4 before administration and 0.25, 1, 2, 4, 6, 8, 12, 24, 36, and 48 hours after administration, respectively. Group 3 animals were collected plasma samples before the first and third doses, 0.25, 1, 2, 4, 6, 8, 12, 24, 36, and 48 hours after the dose and before the second dose. The concentration of compound A in the plasma sample was determined using the LC-MS/MS method.

The results showed: (1) After a single intravenous injection of 0.5 mg/kg, the plasma clearance (CL) of compound A in male and female SD rats was 88.6±6.29 mL/min/kg, and the steady-state apparent volume of distribution ( Vdss) is 63.7±10.4L/kg, elimination half-life (T1/2) is 8.71±0.532h, and exposure (AUC0-last) is 263±19.7nM·h.

(2) After a single intragastric administration of 0.5 mg/kg compound A in male and female SD rats, the bioavailability was 29.9%. After a single intragastric administration of 0.5, 1.5 and 5 mg/kg compound A in female and male SD rats, the AUC0-last values were 78.6±21.0, 315±60.4 and 1470±227 nM·h, respectively, and the peak concentration (Cmax) was 5.24, respectively ±1.56, 22.1±3.95 and 102±24.1nM, the time to peak (Tmax) appeared at 5.33±2.07h, 3.33±2.07h and 3.50±2.74h after administration, respectively.

(3) After the SD rats were given intragastrically once every 3 days, 1.5mg/kg each time, 3 consecutive times, there was no drug accumulation in the system exposure. Within the dose range of 0.5 to 5 mg/kg, the AUC0-last and Cmax of male and female rats both increased proportionally with the increase in dose, and there was no significant gender difference in each dose group.

2. Study on the pharmacokinetics of compound A in beagle dogs

2.1 Pharmacokinetic study of compound A after intravenous injection and oral administration in female and male beagle dogs

Studies on the pharmacokinetic properties of compound A in female and male beagle dogs include (1) 0.1 mg/kg single intravenous injection study; (2) 0.1, 0.3 and 0.6 mg/kg oral dose escalation studies; (3) 0.3 mg The oral administration study was repeated once a week at the dose of /kg for 3 consecutive weeks.

experiment method:

Divide 24 beagles into 4 groups, each with 6 dogs, half male and half female. Animals in group 1 were given a single intravenous injection of 0.1 mg/kg of compound A, and animals in groups 2 and 4 were given a single oral administration of 0.1 and 0.6 mg/kg of compound A solution, respectively. Group 3 animals were orally administered for 3 consecutive weeks, once a week, with 0.3 mg/kg of compound A solution orally each time. The solvent of the intravenous injection group was 10% HP-β-CD (β-cyclodextrin) aqueous solution. The vehicle of the oral group was 0.5% MC (methyl cellulose) aqueous solution. Animals in

groups

1, 2 and 4 are 0.0833 before and after administration (only group 1, i.e. intravenous injection group), 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 48, Plasma samples were collected at 72, 96, 120, and 168 hours. Animals in group 3 were administered 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72, 96, 120, 168 before and after administration in the first and third weeks (the second Plasma samples were collected one hour before administration. The concentration of compound A in the plasma sample was determined by the LC-MS/MS method.

The results showed that: (1) After a single intravenous injection of 0.1 mg/kg of compound A in female and male beagle dogs, the CL was 17.0 ± 10.3 mL/min/kg, the Vdss was 45.1 ± 25.2 L/kg, and T1/2 and 0 o’clock The area under the plasma concentration curve (AUC0-inf) at the infinite time point was 31.9±3.32h and 338±127nM·h, respectively. After a single oral administration of 0.1 mg/kg compound A solution in female and male beagle dogs, the exposure AUC0-inf was 248±88.0 nM·h, and the bioavailability was 73.4%.

(2) After a single oral administration of 0.1, 0.3, and 0.6 mg/kg of compound A solution in female and male beagle dogs, the AUC0-last was 234±83.2, 900±248 and 1490±432 nM·h, respectively, and the Cmax was 5.88± 1.96, 21.1±7.48 and 30.5±13.1nM, Tmax appeared at 9.33±2.07, 7.33±2.42 and 9.00±2.45h after administration, respectively. Within the dose range of 0.1 to 0.6 mg/kg, the systemic exposure (Cmax and AUC0-last) of male and female beagle dogs showed a dose-related linear increase, and there was no obvious gender difference.

(3) After oral administration of 0.3 mg/kg compound A solution once a week for 3 consecutive weeks, compared with the first week, the systemic exposure (Cmax and AUC0-last) of male and female beagle dogs did not change significantly.

2.2 Pharmacokinetics of compound A after intravenous injection and oral administration in female and male beagle dogs

The purpose of this experiment was to study the pharmacokinetic properties of a single oral 0.2mg/kg compound A capsule in male and female beagle dogs, to investigate the bioavailability after oral administration, and to compare the differences between male and female.

experiment method:

Three male and three female beagle dogs were selected, each of which was orally administered 1 capsule of compound A (each capsule contains 2mg of active ingredient compound A), 0.25, 0.5, 1, 2 before and after administration , 4, 6, 8, 12, 24, 48, 72, 96, 120, 168 hours to collect plasma samples. The concentration of compound A in the plasma sample was determined by the LC-MS/MS method.

After a single oral administration of 0.2 mg/kg compound A capsules in female and male beagle dogs, the AUC0-inf and Cmax were 689±334h·nM and 12.8±5.87nM, respectively. The peak time appeared at 11.3±1.63h after administration. Bioavailability The degree is 101%. The ratio of female to male AUC0-last was 1.75, and there was no significant gender difference in system exposure.

4. Toxicology Research

1. Safety pharmacology test

Single oral administration of compound A in SD rats at 0.5mg/kg, 1.5mg/kg and 5mg/kg doses, each dose group did not see obvious drug correlation with the rats' nervous system function-related indicators within the 24h observation range Influence.

A single oral administration of compound A 0.075mg/kg, 0.25mg/kg and 0.75mg/kg groups in beagle dogs had no obvious drug-related effects on the cardiovascular system and respiratory system.

2. The effect of compound A on hERG potassium channel current

CHO (Chinese Hamster Ovary) cells stably expressing hERG potassium channels were used, cisapride was used as a positive control compound, and electrophysiological manual patch clamp technology was used to investigate the inhibitory effect of compound A on hERG potassium channels. The inhibitory percentage of compound A on hERG potassium current is 55.8% after being administered at a concentration of 40 μM for 1 minute. Under these conditions, the compound has an IC50 value of 34.5 μM for inhibiting hERG potassium current, and the risk of cardiovascular side effects is low.

This test uses cisapride as a positive control compound. According to the literature, the IC50 value of cisapride inhibition of hERG potassium current ranges from 5 to 66 nM. The IC50 value obtained from cisapride detection on the same day in this test is 32.6 nM, indicating that the test system is stable and reliable, and the test results precise.

3. Acute toxicity test

A single oral administration of compound A 6, 20, and 50 mg/kg in SD rats to recover the toxicity test for 14 days, the maximum tolerated dose (MTD) is 50 mg/kg, and the changes related to the test substance are mainly skin erythema, redness and swelling And extramedullary hematopoiesis in the red pulp of the spleen.

Beagle dogs were given a single intragastric administration of Compound A 0.3mg/kg, 1mg/kg and 3mg/kg to recover for 21 days with toxicokinetic experiment, the maximum tolerated dose (MTD) was 3mg/kg (AUC0-120h: ♂4778.05) ng·h/mL, ♀6647.67ng·h/mL), the changes related to the test substance are mainly manifested as skin toxicity and gastrointestinal reactions, accompanied by weight loss and poor food intake. Hematology can see white blood cells and their classification, Changes in red blood cell-related indicators, platelet-related indicators, as well as lymphopenia with small thymus and extramedullary hematopoiesis in red marrow with enlarged spleen.

4. Long-term toxicity test

4.1 Compound A is used in the long-term toxicity test of SD rats

SD rats were administered by oral gavage repeatedly 9 times (twice a week, on the 1st and 4th day respectively), administered 0.6, 2, and 6 mg/kg of compound A. The drug was stopped and recovered for 4 weeks. No toxicity was seen in both males and females. Response dose (NOAEL) is 0.6mg/kg, male maximum tolerated dose (MTD) is 2mg/kg (D29: AUC0-48h: ♂75.56ng·h/mL), female maximum tolerated dose (MTD) is 6mg /kg (D29: AUC0-48h: ♀383.60ng·h/mL).

The toxic changes related to the test substance are mainly manifested as erythema, swelling and crusting. The blood system shows changes in white blood cell classification, erythroid indicators, and platelet-related indicators. Histopathology shows lymphopenia (thymus, spleen) and increased extramedullary hematopoiesis ( Spleen, liver), multiple organs/tissue hemorrhage (testicular interstitium, epididymal interstitium, eyeball, stomach, ovarian corpus luteum, lung alveoli, thymus, pancreas); liver bile duct hyperplasia, bone marrow (sternum and femur) bone new bones can also be seen Increased metaphyseal bone trabeculae decreased and megakaryocytes increased, ovarian atresia follicles increased and new corpus luteum decreased, lung macrophage aggregation and fibrosis, pancreatic acinar atrophy/necrosis, and peripancreatic fibrosis. The drug was stopped and recovered for 4 weeks. The above changes showed a complete recovery or a trend of recovery, and no other toxic reactions related to the test product were seen. It is basically consistent with the toxicity of the same target drug.

4.2 Compound A used in long-term toxicity test of beagle dogs

Beagle dogs were given 5 times by oral gavage (administered on the first day of the week) 0.075, 0.25, 0.75 mg/kg compound A was administered, and the drug was stopped and recovered for 4 weeks. The NOAEL dose was 0.075 mg /kg, the highest non-serious toxic dose (HNSTD) is 0.25mg/kg (D29: AUC0-120h: ♂126.53ng·h/mL, ♀150.65ng·h/mL), and the minimum lethal dose (MLD) is 0.75mg/ kg (D29: AUC0-120h: ♂1826.43ng·h/mL, ♀701.55ng·h/mL).

The toxic changes related to the test substance are mainly manifested as gastrointestinal toxicity, malaise and reduced activity. The blood system can see changes in white blood cell classification, red blood cell-related indicators, and platelet-related indicators. Drug-related histopathological changes are seen in hematopoietic-lymphoid organs/ Tissues, digestive tract, adrenal glands, lungs, liver, muscles. The drug was stopped and recovered for 4 weeks. The above changes showed a complete recovery or a trend of recovery, and no other drug-related toxic reactions were seen. It is basically consistent with the toxicity of the same target drug.

In vivo drug efficacy tests in mice showed that compound A 1-3 mg/kg has a significant inhibitory effect on small cell lung cancer. The equivalent human dose is about 5.35 mg-16.05 mg (based on 60 kg body weight). Based on the above-mentioned pharmacodynamic and toxicological research results, the human equivalent dose is calculated by converting the effective dose of mouse 1mg/kg and the long-term toxicity test MTD dose (male 2mg/kg, female 6mg/kg) into the human equivalent to calculate the safety of compound A The window is 4.5 times for males and 13.6 times for females, indicating that compound A is well tolerated.

5. Clinical Phase I Study of Compound A

1. Scheme design

1.1 Test procedure

This trial is an open, dose escalation and dose expansion phase I clinical study for patients with extensive-stage small cell lung cancer. It aims to evaluate the safety, tolerability, and pharmacokinetics of compound A in patients with extensive-stage small cell lung cancer. Characteristics and preliminary anti-tumor activity. The anti-tumor activity was carried out in accordance with the Evaluation Criteria for Efficacy of Solid Tumors (RECIST) V1.1.

This study is conducted in two stages, the first stage (Stage I) is a dose escalation study, and the second stage (Stage II) is a dose expansion study.

Stage I: Dose escalation study

The initial dose of compound A was set at 0.5 mg/week, and five dose groups were initially planned: 0.5, 1.5, 3, 6, and 9 mg. The preset maximum escalating dose is 9 mg/week. This study will follow the "3+3" dose escalation plan.

After completing the core dosing period, evaluate the safety, tolerability, pharmacokinetic characteristics and anti-tumor activity of the subject. If the subject is well tolerated and I agree, the treatment can be continued until the disease progresses, or untolerable toxicity occurs, or the study is terminated for other reasons.

Stage II: Dose amplification research

According to the safety, tolerability, and effectiveness data obtained in the dose escalation study, the target dose group dose expansion study and the exploration study of different dosing schedules can be carried out when necessary. Sponsors and investigators will continue to conduct safety evaluations, and based on the available data of previous dose levels, determine the drug dose level and dosing schedule during the expansion period.

In the above two phases of Phase I clinical trials, a total of 34-71 patients with extensive-stage small cell lung cancer are planned to be included.

1.2 Setting of increasing dose group

The initial dose of compound A was set at 0.5 mg, and the preset highest escalating dose was 9 mg. On the premise of ensuring safety, avoid too many subjects from being exposed to invalid doses, and closely monitor the safety indicators of subjects. This study adopts an increasing proportion of 200%, 100%, 100%, and 50%. The slope plan is designed as 0.5, 1.5, 3, 6, 9 mg.

If the dose is increased to the preset highest dose group, the safety and tolerability of the dose group are still good, the investigator and the sponsor can discuss whether to try a higher dose, for example, adjust the dose in 20% or 30% increments . See Table 5 for the specific dose group settings of the dose escalation phase:

Table 5 Dose group settings in the dose escalation phase

分组Grouping	剂量 dose	递增幅度Increment		受试者Subject
11	0.5mg0.5mg	--	11
22	1.5mg1.5mg	200％200%	3+33+3
33	3mg3mg	100％100%	3+33+3
44	6mg6mg	100％100%	3+33+3
55	9mg9mg	50％50%	3+33+3

6 (if necessary)

To be determined

3+3

1.3 The principle of dose escalation

The dose escalation will start from the initial dose (0.5mg), and only 1 patient will be evaluated in the initial dose group. From the second dose group (1.5mg), follow the "3+3 principle", and each dose group will be included in the group 3~ 6 subjects. After all subjects have completed a certain dose of the drug test, the sponsor and the investigator jointly decide whether to increase to the next dose level.

After the initial dose group is completed, whether to conduct subsequent dose group trials is judged according to the following rules:

(1) If the patient has an AE of grade 2 or below (non-DLT), it can be increased to the next dose group;

(2) If the patient has an AE of grade 3 or above (non-DLT), the investigator judges that it is related to the drug, and it needs to be expanded to 3 cases for continued observation;

(3) If the patient has DLT related to the study drug, 5 patients (6 in total) should be re-enrolled in this dose group, and the safety tolerability will continue to be observed. If ≥2/6 patients develop DLT, consider reducing the dose or discontinuing the study;

In each subsequent dose group, after each dose group is completed, whether to conduct the next dose group test is judged according to the following rules:

(1) If none of the 3 patients in a certain dose group has DLT, then increase to the next dose group;

(2) If 2 or more of the 3 patients in a certain dose group have DLT, it will be decremented to the previous dose group;

(3) If 1 out of 3 patients in a certain dose group has DLT, add 3 more patients in this dose group. If 1/6 patients have DLT, then increase to the next dose group, if there is ≥2/6 If the patient develops DLT, it will decrease to the previous dose group;

(4) When decreasing to the previous dose group, if there are only 3 patients in this dose group, 3 more patients will be added. If there are 6 patients in this dose group, the dose escalation test is over, and this dose is MTD.

MTD definition: Refers to the current dose group after ≥33% of subjects in the high-dose group have DLT, and 0/6 or 1/6 of the current dose group has DLT, then the current dose group is defined as MTD.

If MTD is not found in all planned dose groups, the sponsor will further discuss with the investigator whether to conduct a higher dose group study.

1.4 Case supplement

Considering that the core trial period (cycle 1) of the study may fall off of cases and lead to insufficient evaluable cases (such as early disease progression), each dose group may be enrolled in 3 subjects at an early stage On the basis of this, additional new subjects were enrolled. The principle of case supplement is as follows:

Subjects who have signed the informed consent form but have not received research intervention can be replaced. For subjects who have signed an informed consent form and have received research interventions, but subsequently discontinued the research, substitutes will be allowed if they meet the following conditions:

1) During the dose escalation test, the subject failed to complete the core treatment period observation due to non-DLT reasons;

2) Non-DLT causes the dose in the first cycle to be less than 75% of the planned dose.

1.5 Intrapatient dose escalation

Subjects who have completed the treatment and observation of the core trial period are judged by the investigator that the subject has benefit and can tolerate the study drug treatment, and can enter the subsequent treatment period according to the subject's wishes. The same subject entering the subsequent treatment period is not allowed to increase to the next higher dose group.

2. Entry criteria

2.1 Selection criteria

Subjects eligible to participate in this study must meet the following selection criteria:

①Male or female, aged 18 to 70 years (until the time of obtaining informed consent).

②The diagnosis of the disease is clear:

The selection criteria for the first part of the trial: patients with small cell lung cancer diagnosed histologically, who have failed or are intolerant to the standard treatment, and have at least one measurable lesion according to the RECIST1.1 standard;

The selection criteria for the second part of the trial: patients with small cell lung cancer diagnosed histologically, who have failed or are intolerant to standard therapies (etoposide combined with platinum regimens have progressed or become intolerant after at least one treatment) , And at least one focus can be measured according to the RECIST1.1 standard.

③ECOG score: 0 or 1 point.

④Recovered from the toxicity of the previous treatment, except for hair loss and pigmentation, (determined as 0-1 grade according to CTCAE 5.0).

⑤The expected survival time is more than 3 months.

⑥ The function of major organs meets the following criteria in Table 6 within 7 days before treatment (have not received blood transfusion within 14 days before study drug administration):

Table 6 Criteria for main organ function of selected subjects

⑦Eligible patients (male and female) with fertility must agree to use reliable contraceptive methods (hormonal or barrier method or abstinence) with their partners during the trial period and at least 6 months after the last medication; female patients of childbearing age are selected The blood pregnancy test within the first 7 days must be negative.

⑧ Subjects voluntarily joined the study, signed an informed consent form, and had good compliance.

2.2 Exclusion criteria

Subjects can be excluded from any of the following items and not included in the study:

① Patients with primary malignant tumors other than small cell lung cancer, except for skin basal cell carcinoma and cervical carcinoma in situ that have been cured for more than 2 years.

②Patients with central nervous system metastasis (such as brain metastasis or meningeal metastasis) confirmed by imaging evaluation;

③Patients with uncontrollable pleural effusion, pericardial effusion or ascites that require repeated drainage.

④Receive any of the following anti-tumor treatments within 4 weeks before the first use of the study drug, including: chemotherapy, radiotherapy (except for local radiotherapy to relieve symptoms), biological therapy, targeted therapy, and immunotherapy.

⑤ Those who have used any LSD1 inhibitor therapy.

⑥Unable to swallow the drug orally, or there is a condition that has been judged by the investigator to seriously affect the absorption of the gastrointestinal tract.

⑦Suffering from clinically significant cardiovascular diseases, including but not limited to:

--Severe arrhythmia, or need to take anti-arrhythmic drugs (excluding anticoagulant drugs, blood coagulation indicators related to taking anticoagulant drugs must meet the selection criteria);

- Patients with prolonged QT/QTc interval of ECG at baseline (QTc>480ms, Fridericia formula: QTc=QT/RR0.33);

-Any of the following cardiovascular events occurred within 6 months before the first administration, including: acute coronary syndrome, congestive heart failure, stroke or other cardiovascular events of grade 3 or above;

--Heart failure, New York College of Cardiology (NYHA) grade II and above;

- Detected by echocardiography, the left ventricular ejection fraction is less than 60%.

⑧Patients who have a history of other serious systemic diseases, who are judged by the investigator to be unsuitable to participate in clinical trials.

⑨A history of serious autoimmune diseases and immunodeficiency as judged by the researcher, including a positive HIV test, or other acquired congenital immunodeficiency diseases, or a history of organ transplantation.

⑩Hepatitis B virus HBsAg positive, hepatitis C virus antibody positive or syphilis antibody positive.

Those who have used a strong inhibitor or inducer of CYP3A4 liver metabolic enzymes within 2 weeks before the first administration of Compound A and still need to continue to use such drugs (see Appendix C for details).

People with mental disorders or poor compliance.

Women who are pregnant or breastfeeding.

Those who participated in other clinical trials and used study drugs as subjects within 4 weeks before participating in this trial.

The researcher believes that the subjects are not suitable for participating in this clinical study for other reasons.

3. Research results:

At present, 1 subject has been enrolled in the 0.5 mg dose group and 3 subjects have been enrolled in the 1.5 mg dose group, all of which have completed the enrollment and DLT observation period. One subject in the 3mg dose group has been enrolled and completed the DLT observation period. All subjects had no DLT, no SAE, and no adverse events of special concern.

One subject in the 0.5 mg dose group took the drug for 1 cycle, and the anti-tumor efficacy was evaluated as SD, and the efficacy of the drug was evaluated at the end of the treatment for 2 cycles. PD. There were 13 adverse events during the treatment period, all of which were judged to have nothing to do with the study drug.

In the 1.5 mg dose group, 3 subjects in the 1.5 mg dose group took the drug for 1 cycle to evaluate the efficacy of PD. 33 adverse events occurred during the treatment period, of which 8 adverse events were determined to be related to the study drug, including increased GGT (2 times), dyspnea, Fever (2 times), increased LDH, increased ALP, increased D-Di, and CTCAE grades were all 1-2.

Subjects in the 3.0 mg dose group have completed a cycle of medication, and the efficacy has been evaluated for SD, and the treatment is currently continuing. The CTCAE grades of adverse events that occurred during the study were all grade 1-2, and they were all judged to have nothing to do with the study drug.

4. Typical cases

4.1 Case 1

Female, 63 years old, was diagnosed with small cell lung cancer on January 22, 2020, with metastases in the liver and lymph nodes. At the first diagnosis, the TNM stage was IVB stage, and the VALG stage was extensive stage. Receive 3 cycles of etoposide + carboplatin combined chemotherapy from February 19 to April 7, 2020, and receive 3 cycles of etoposide monotherapy from May 19 to June 3, the best curative effect is PR . Irinotecan+cisplatin second-line chemotherapy was started on August 12, 2020, and the best curative effect was PD. In September 2020, the informed consent form was signed. At the time of screening, the TNM staging was the IVB stage, and the VALG staging was the extensive stage. From September 23 to November 4, 2020, take Compound A 0.5 mg once a week. The anti-tumor efficacy evaluation of C1D28 on October 18, 2020 was SD, and the anti-tumor efficacy evaluation of the last visit on November 27, 2020 was PD. Adverse events that occurred during the study included decreased lymphocyte count, hypoalbuminemia, fatigue, hyponatremia, expectorant cough, hypokalemia, weight loss, bilateral rib pain, hyperglycemia, and decreased white blood cell count , Decrease in neutrophil count and platelet count. The CTCAE grade of all adverse events was 1-2, and they were judged not to be related to the study drug.

4.2 Case 2

A 63-year-old male was diagnosed with small cell lung cancer in October 2017, with metastatic lymph nodes. The TNM staging at the first diagnosis was unknown, and the VALG staging was unknown. On October 10, 2017, a right lower lobectomy and systemic lymph node dissection were performed. Received etoposide + cisplatin combined postoperative adjuvant chemotherapy from November 6, 2017 to March 1, 2018, with the best effect SD. Received whole brain radiotherapy from March 30 to April 4, 2018. Received etoposide + cisplatin combined chemotherapy from April 20th to August 17th, 2019, the best curative effect was PR, and the disease progressed on November 21st, 2019. Received second-line etoposide single-agent chemotherapy from December 6, 2019 to February 9, 2020. The best efficacy is unknown. The disease progressed on March 4, 2020. Received irinotecan single-agent third-line chemotherapy from March 11, 2020 to April 15, 2020, and the best efficacy is unknown. From May 13th to June 17th, 2020, received radiation therapy behind the esophagus.

The informed consent form will be signed on February 24, 2021. At the time of screening, the TNM staging was the IVB stage, and the VALG staging was the extensive stage. From March 3, 2021, take Compound A 3mg once a week. The anti-tumor efficacy evaluation of C1D28 on March 30, 2021 is SD, and follow-up treatment and efficacy evaluation are in progress. Adverse events that occurred during the study included double pneumonia, right emphysema, right pleural effusion, liver cyst, decreased lymphocyte count, increased GGT, increased LDH, sinus tachycardia, insomnia, and weight loss. The CTCAE grade of all adverse events was 1-2, and they were judged not to be related to the study drug.

List of acronyms

Claims

Application of compound A or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and/or treatment of cell proliferative diseases, the structure of said compound A is shown in the following formula (I):
The application according to claim 1, wherein the cell proliferative disease is cancer, preferably the cancer is lung cancer, more preferably the lung cancer is small cell lung cancer.
The application according to claim 2, wherein the small cell lung cancer is extensive-stage small cell lung cancer; or, the small cell lung cancer is recurring and/or metastatic small cell lung cancer; or, the small cell lung cancer It is small cell lung cancer that has failed standard treatment or is intolerant to standard treatment; or, said small cell lung cancer is cisplatin, carboplatin, etoposide, lobaplatin, topotecan, irinotecan, gemcitabine, temozolomide , Paclitaxel, docetaxel, vinorelbine and other drugs for small cell lung cancer that have failed or are intolerant to treatment; or, the small cell lung cancer is a small cell with high ASCL1 and/or GRP expression Lung cancer.
The use according to any one of claims 1 to 3, wherein the medicine contains a preventive and/or therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof, and optionally, a pharmaceutically acceptable The excipients, carriers and/or diluents; the preventive and/or therapeutic effective amount is 0.001-1000 mg.
The application according to any one of claims 1 to 3, wherein the medicine is made into a clinically accepted preparation, preferably an oral preparation, an injection preparation, a topical administration preparation or an external preparation.
The application according to any one of claims 1 to 3, wherein the medicine also contains one or more of other targeted drugs or chemotherapeutic drugs, and the other targeted drugs or chemotherapeutic drugs are selected from Platinum drugs and topoisomerase inhibitors.
The application according to any one of claims 1 to 3, characterized in that the dosage of the drug is 0.01 mg-100 mg per administration.
A combination product comprising Compound A or a pharmaceutically acceptable salt thereof, and another targeted drug or chemotherapeutic drug, the additional targeted drug or chemotherapeutic drug selected from platinum drugs and topoisomerism Enzyme inhibitor, the structure of the compound A is shown in the following formula (I):
The combination product according to claim 8, wherein the combination product is in the form of a composition, or the compound A or a pharmaceutically acceptable salt thereof and another targeted drug or chemotherapeutic drug are each separately Formulations.
The use of the combination product according to any one of claims 8-9 in the preparation of a medicament for the prevention and/or treatment of cell proliferative diseases.