CN106265660B - Application of A674563 in acute leukemia carrying FLT3 mutant gene - Google Patents

Abstract

The present invention relates to the use of A674563 in the preparation of a medicament for treating patients with acute myeloid leukemia carrying a FLT3 mutant gene, wherein the patients suffering from said leukemia may or may not have resistance associated with high expression of FL ligands Medicinal properties. The present invention also relates to a non-therapeutic method for inhibiting acute myeloid leukemia cells carrying a FLT3 mutant gene, comprising contacting said cells with A674563.

Description

Application of A674563 in acute leukemia carrying FLT3 mutant gene

technical field

The invention relates to the field of medicine, in particular to a new application of A-674563 (A674563).

Background technique

Acute myelocytic leukemia (AML) or acute nonlymphocytic leukemia (ANLL) includes all acute leukemias of non-lymphocyte origin. It is a kind of disease formed by the karyotype mutation of pluripotent stem cells or slightly differentiated precursor cells, and it is a clonal malignant disease of the hematopoietic system. Epidemiological surveys show that environmental, occupational and genetic factors are closely related to the pathogenesis of AML. The incidence rate is higher in developed countries than in developing countries, and higher in western countries than in eastern countries. The annual incidence rate around the world is 2.25/100,000 population, and the incidence rate increases with age. It begins to rise significantly at the age of 50, reaches a peak at the age of 60-69, and the incidence rate is 1/100,000 under the age of 30. /100,000. Therefore, AML is actually a disease of middle-aged and elderly people, accounting for 80% to 90% of acute leukemia in adults, but only 15% to 20% of acute leukemia in children. At the same time, the incidence of men is higher than that of women.

FLT3 (Fms-like tyrosine kinase 3) is FMS-like tyrosine kinase 3, which belongs to the type III receptor tyrosine kinase (receptor tyrosine kinase III, RTK III) family member together with c-Kit, c-FMS and PDGFR. Its protein structure includes an extracellular domain composed of five immunoglobulin (Ig)-like domains, a transmembrane domain, a juxtamembrane domain (JM), and two intracellular domains separated by a kinase insertion domain. The amino acid kinase (TK) domain (S.D. Lyman et al., Oncogene, 1993, 8, 815-822). After FLT3 binds to its ligand FL (FLT3ligand, FL), the tyrosine residues undergo autophosphorylation, and conduct activation signals through various cytoplasmic proteins to provide proliferation stimulation, and play a role in the self-renewal, proliferation, and differentiation of hematopoietic stem cells. makes an important impact. FLT3 kinase abnormality plays an important role in the occurrence and development of AML, which may be achieved through the following two mechanisms: one is through FLT3 gene mutations, such as internal tandem duplication (ITD) mutations in the FLT3 gene, which have been extensively studied in the past ten years It was found that about one-third of AML patients have FLT3ITD mutations, which lead to elongation of the juxtamembrane domain, changes in the spatial conformation of the kinase, autophosphorylation and activation of downstream abnormal signaling in a ligand-independent manner, and promote leukemia malignancy Clonal proliferation and maintenance. FLT3 mutation was first discovered in AML cells as early as 1996, and its mutation type is internal tandem duplication (FLT3-ITD). In recent years, many studies have confirmed that the activating mutation of FLT3 plays a very important pathological role in the occurrence and progression of AML. Another mechanism is that AML cells overexpress FLT3, and the ligand FL co-expressed with it leads to the continuous activation of the kinase in the form of autocrine, paracrine and other forms of strengthening the normal signal transduction of wild-type FLT3 (Kusec R, Jaksic O, Ostojic S , et a1. Moreon prognostic significance of FLT3/ITD size in acute myeloid leukemia (AML). Blood, 2006, 108: 405-406. Kuchenbaner F, Kern W, Sehoeh C, et a1. Detailed analysis of expression levels in acute myeloid leukemia. Haematologica, 2005, 90: 1617-1625).

AML patients with FLT3-ITD activating mutations usually have unique clinical features such as high peripheral blood white blood cell count, poor clinical prognosis, and easy recurrence, and because the detection method of FLT3 activating mutations is simple and easy, more and more researchers Committed to developing FLT3 into a routine detection method for AML to guide the treatment and prognosis of AML patients and as a detection method for minimal residual leukemia, and to use it as another new target for chemotherapy drugs in leukemia patients.

It has been confirmed that there are two main types of activating mutations in FLT3: one is the internal tandem duplication (ITD) of exons 14 and 15 in the juxtamembrane domain; the other is the tyrosine kinase domain (tyrosine kinase domain). , TKD) exon 20 point mutation (point mutation, PM), mostly located in its activation loop, the most common is the 835th aspartic acid (ASP 835) residue mutation, such as D835Y. Both mutations result in constitutive activation of the FLT3 tyrosine kinase. Under physiological conditions, the expression of FLT3 is limited to CD34+ hematopoietic stem cells, and its ligand is FL. When there is no ligand, the juxtamembrane domain inhibits the formation and activation of FLT3 dimers. When the ligand FL and FLT3 are stimulated Dimerization of the latter leads to changes in the conformation of the kinase domain, phosphorylation of tyrosine residues, activation of substrate proteins such as RAS-GAP, PLC-γ, PI3K, STAT5 and ERK1/2, Through a series of intracellular signal transmission, the cell proliferation signal is transduced into the nucleus, resulting in the proliferation and activation of hematopoietic stem cells. Evidence has shown that both internal tandem duplication of the proximal membrane region of FLT3 and point mutations in the activation loop lead to constitutive activation of the FLT3 kinase and activation of downstream target molecules, including STAT5 and the RAS/MAPK pathway. These two activating mutations of FLT3 can cause autophosphorylation of FLT3, leading to ligand-independent constitutive activation of FLT3, further activating its downstream abnormal signal transduction, thereby promoting proliferation and inhibiting apoptosis , so that the clinical prognosis of leukemia patients with this mutant phenotype is poor.

At present, the targeted inhibition of FLT3 gene mutation has become a research hotspot, mainly for the development of small molecule tyrosine kinase inhibitors, which inhibit its activity by competing with FLT3 tyrosine kinase for the ATP binding site. At present, the kinase inhibitors that inhibit FLT3 have entered the clinic include AC220, TCS359 and so on. However, some AML patients treated with these drugs were found to develop resistance to the therapeutic drugs later in the treatment, which was caused by the high expression of FL (FLT3 Ligand), a ligand co-expressed with FLT3.

A674563 is an inhibitor of protein kinase AKT (also known as protein kinase B: PKB), which can be used alone or in combination with other therapeutic agents for the treatment of autoimmune diseases or disorders, heteroimmune diseases or disorders , cancers include lymphomas and inflammatory diseases or conditions. At present, there is no relevant report on the use of A674563 in the treatment of acute myeloid leukemia carrying the FLT3 mutant gene, and there is no report on the use of A674563 in the treatment of patients with FLT3 mutant gene carrying drug resistance associated with high expression of FL ligands related reports of patients with acute myeloid leukemia.

SUMMARY OF THE INVENTION

In view of the foregoing technical problems, the inventors conducted extensive research. As a result, the inventors unexpectedly found that A674563, an AKT kinase inhibitor, is effective in treating acute myeloid leukemia carrying the FLT3 mutant gene, and can be used to treat acute myeloid leukemia with drug resistance associated with high expression of the FL ligand. Acute myeloid leukemia patients with FLT3 mutant gene. Specifically, the inventors found that A674563 has a strong inhibitory effect on acute myeloid leukemia cells carrying FLT3-ITD and/or FLT3-D835Y mutant genes, such as MOLM-13, MOLM-14, MV-4-11, etc. effects (GI50 were 0.1 μM, 0.22 μM, 0.12 μM respectively); at the same time, in the drug resistance experiment of adding FLT3 ligand FL to stimulate cells, A674563 had no effect on acute myeloid cells carrying FLT3-ITD mutant and wild-type FLT3 genes. Cellular leukemia cells such as MOLM-13 and MOLM-14 cells also play a strong inhibitory effect. Therefore, A674563 is suitable for clinical treatment of acute myeloid leukemia patients carrying FLT3 mutant genes, especially FLT3-ITD and/or FLT3-D835Y mutant genes. Further, the patient may have drug resistance associated with high expression of FL ligand.

In one aspect, the present invention relates to the use of A674563 in the preparation of medicines for treating acute myeloid leukemia patients carrying FLT3 mutant genes (especially carrying FLT3-ITD and/or FLT3-D835Y mutant genes). Further, the patient may have drug resistance associated with high expression of FL ligand.

During the course of treatment, the drug can be used alone or in combination with one or more other therapeutic agents according to the situation. The drug comprising A674563 can be administered to the acute myeloid leukemia patient carrying the FLT3 mutant gene by at least one of injection, oral administration, inhalation, rectal administration, and transdermal administration. Additional therapeutic agents may be selected from the following drugs: immunosuppressants (e.g., tacrolimus, cyclosporine, rapamycin, methotrexate, cyclophosphamide, azathioprine, mercaptopurine, mycophenolate mofetil or FTY720), glucocorticoids (e.g., prednisone, cortisone acetate, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, fludoxyprednisolone, beclox metasone, fludrocortisone acetate, deoxycorticosterone acetate, aldosterone), NSAIDs (e.g., salicylates, arylalkanoic acids, 2-arylpropionic acids, N-arylanthranilic acids , oxicams, coxicams, or sulfanilides), allergy vaccines, antihistamines, antileukotrienes, beta-agonists, theophylline, anticholinergics, or other selective kinase inhibitors (such as mTOR inhibitors agent, c-Met inhibitor) or her2 antibody-drug. In addition, other therapeutic agents can also be rapamycin (Rapamycin), crizotinib (Crizotinib), tamoxifen, raloxifene, anastrozole, exemestane, letrozole, Herceptin ^TM (trastuzumab), Gleevec ^TM (imatinib), paclitaxel ^TM (paclitaxel), cyclophosphamide, lovastatin, minocycline (Minosine), cytarabine, 5-fluorouracil ( 5-FU), methotrexate (MTX), taxotere ^{TM (docetaxel), Noredex TM (} goserelin), vincristine, vinblastine, nocodazole, tinib ^Poside , Etoposide, Gemcitabine, Epothilone, Novibine, Camptothecin, Daunonibicin, Dactinomycin, Mitoxantrone, Amsacrine , Doxorubicin (Adriamycin), Epirubicin or Idarubicin. Alternatively, the other therapeutic agent may also be a cytokine such as G-CSF (granulocyte colony stimulating factor). Alternatively, other therapeutic agents can also be used in combination in the same treatment regimen, such as, but not limited to, CMF regimen (cyclophosphamide, methotrexate, and 5-fluorouracil), CAF regimen (cyclophosphamide, doxorubicin, and 5-fluorouracil), -fluorouracil), AC regimen (doxorubicin and cyclophosphamide), FEC regimen (5-fluorouracil, epirubicin and cyclophosphamide), ACT or ATC regimen (doxorubicin, cyclophosphamide and Paclitaxel) or CMFP regimen (cyclophosphamide, methotrexate, 5-fluorouracil, and prednisone).

In another aspect, the present invention also relates to a pharmaceutical composition comprising A674563 and a pharmaceutically acceptable carrier or adjuvant. The composition may further comprise one or more other therapeutic agents. Other therapeutic agents mentioned herein are as defined above.

In yet another aspect, the present invention relates to a method of using A674563 to treat an acute myeloid leukemia patient carrying a FLT3 mutant gene, wherein the patient may or may not have drug resistance associated with high expression of FL ligand. During the course of treatment, A674563 can be administered to acute myeloid leukemia patients carrying FLT3 mutant genes (especially carrying FLT3-ITD and/or FLT3-D835Y mutant genes) by injection, oral administration, inhalation, rectal or transdermally, Wherein the patient may or may not have drug resistance associated with high expression of FL ligand. An effective amount of A674563 can also be used alone or in combination with one or more other therapeutic agents according to the situation. Other therapeutic agents mentioned are as defined above. Chemotherapy with A674563 may also be administered in combination with radiation therapy during the course of treatment.

In yet another aspect, the present invention relates to a method of inhibiting acute myeloid leukemia cells carrying a FLT3 mutant gene comprising contacting said cells with A674563. The methods may be used for therapeutic or non-therapeutic purposes. Further, the cells may not have been or have been treated with FLT3 inhibitors. According to the present invention, the cells are preferably one or more selected from MOLM-13, MOLM-14, MV-4-11. Further preferably, the cells are contacted with A674563 at an effective concentration of at least 0.01 μM, more preferably the amount of A674563 is at least 0.1 μM, further preferably 0.2-10 μM, eg 0.2-3 μM.

In yet another aspect, the present invention relates to the use of A674563 in the preparation of a medicament for inhibiting acute myeloid leukemia cells carrying a FLT3 mutant gene. Specifically, the cell carries the FLT3-ITD mutant gene or the LT3-D835Y mutant gene, and the cell may be one or more selected from MOLM-13, MOLM-14 and MV-4-11.

Description of drawings

Figures 1a-1c show the effects of various inhibitors on FLT3 relatively closely related proteins and related signaling pathways in MOLM-14, MOLM-13, and MV-4-11 cells, in which 1a: different concentrations of A674563 and Effects of control inhibitors on FLT3 relatively closely related proteins and related signaling pathways; 1b: Comparison of the effects of A674563 on FLT3 relatively closely related proteins and related signaling pathways with or without FL ligand addition; 1c : Comparison results of the effects of control MLN518 on FLT3 relatively closely related proteins and related signaling pathways with or without FL ligand addition.

Figure 2 shows the effect of A674563 on FLT3 autophosphorylation in the BaF3 cell line at the isogenic locus.

Figure 3 shows the effects of A674563 on the apoptosis of MOLM-13, MOLM-14, and MV-4-11, respectively.

Figures 4a-4c show the effect of A674563 on the cell cycle distribution of MOLM-13(a), MOLM-14(b), MV-4-11(c), respectively.

Detailed ways

Before further describing the present invention, some terms are explained for a better understanding of the present invention.

definition

"A674563" is an inhibitor of protein kinase B (PKB/AKT). As a small molecule PKB/AKT inhibitor, A674563 has the structure shown in the following formula (I):

FLT3 (Fms-like tyrosine kinase 3) is FMS-like tyrosine kinase 3.

FLT3-ITD mutant gene or FLT3-ITD mutant gene refers to the FLT3internal tandem duplication (FLT3-ITD) mutation, which has the highest incidence in acute myeloid leukemia (AML) and is associated with prognosis mutation.

FLT3-D835Y mutant gene or FLT3-D835Y mutant gene refers to a point mutation in exon 20 of the tyrosine kinase domain (TKD): the 835th aspartic acid (Asp835) residue is mutated to tyrosine ( Tyr), abbreviated as D835Y.

As used herein, the term "administering" or "administering" includes the means by which a compound is introduced into a subject to achieve its intended function and effect. Examples of administration routes that can be used include injection (subcutaneous injection, intravenous injection, parenteral injection, intraperitoneal injection, intrathecal injection), oral, inhalation, rectal, transdermal, and the like. Pharmaceutical formulations can be administered in forms suitable for each route of administration.

As used herein, the term "pharmaceutically acceptable" means, within the scope of sound medical judgment, suitable for use in contact with tissues of humans and other mammals without undue toxicity, irritation, allergic response, etc., and with a reasonable interest /risk ratio commensurate components.

As used herein, the term "effective amount" includes an amount effective, in dosages and for periods of time necessary, to achieve the desired result (eg, sufficient to treat a disease or condition described herein). An effective amount of a compound of the invention can vary depending on factors such as the disease state, age, and weight of the subject, and the ability of the compound to elicit a desired response in the cell or in the subject, among others. Dosage regimens may be adjusted to provide the optimum therapeutic response.

"Drug resistance" refers to the tolerance of microorganisms, parasites, and tumor cells to the effects of chemotherapy drugs. Once drug resistance occurs, the chemotherapy effects of drugs will be significantly reduced. Drug resistance can be divided into acquired drug resistance and natural drug resistance according to the cause of its occurrence. For anti-tumor drugs, the insensitivity of tumor cells to anti-malignant drugs, that is, drug resistance, is an important reason for the failure of cancer chemotherapy, and it is also a problem that needs to be solved urgently in cancer chemotherapy. The drug resistance involved in the present invention generally refers to the drug resistance developed by AML patients carrying FLT3 mutant gene after being treated with AML drugs other than A674563 (such as FLT3 inhibitors).

In an embodiment of the present invention, according to the present invention, subjects suffering from AML carrying the FLT3 mutant gene (including subjects who develop drug resistance in the previous treatment for AML carrying the FLT3 mutant gene) When administering A674563 for treatment, the amount of a given drug depends on many factors, such as the specific dosing regimen, the type of disease or condition and its severity, the uniqueness of the subject or host in need of treatment (e.g., body weight, sex, age, , whether the patient has previously experienced other treatment (such as treatment with a FLT3 inhibitor), etc.), however, depending on the specific situation, including, for example, the specific drug used, the route of administration, the condition being treated, and the subject of the treatment The subject or host, and the dosage to be administered can be routinely determined by methods known in the art. In general, for dosages used in the treatment of adults, the administered dosage is typically in the range of 0.02-5000 mg/day, for example about 1-1500 mg/day. The desired dose may conveniently be presented as one dose, or as divided doses administered simultaneously (or within a short period of time) or at appropriate intervals, for example as two, three, four or more divided doses per day. Those skilled in the art can understand that, although the above dosage range is given, the effective amount of A674563 can be adjusted appropriately according to the condition of the patient and combined with the doctor's diagnosis.

As mentioned above, the AML subjects carrying the FLT3 mutant gene mentioned in the present invention may be subjects who have previously experienced AML treatment, or subjects who have not previously experienced relevant treatment. In some embodiments, the subject has previously experienced treatment with a FLT3 inhibitor and developed resistance to the previous treatment (or Phase I treatment). In other embodiments, the subject has previously experienced treatment with a FLT3 inhibitor and has not developed drug resistance to the previous treatment (or Phase I treatment). In the above-mentioned embodiments, A674563 can be used to treat these patients according to the present invention.

As used herein, "GI50" refers to the drug concentration required to inhibit 50% of cell growth, that is, the drug concentration at which the growth of 50% of cells (such as cancer cells) is inhibited or controlled.

As used herein, "IC50", also known as the half-inhibitory concentration, refers to the amount, concentration, or concentration of a particular inhibitor that achieves 50% inhibition of the maximal effect (e.g., inhibition of PKB/AKT kinase activity) in an assay measuring an effect. dose.

As used herein, "EC50" refers to the determination of a dose, concentration or amount of a compound that elicits a dose-dependent response that is 50% of the maximal expression of a specific response induced, stimulated or potentiated by a particular assay compound.

Application of the invention

In some embodiments of the present invention, A674563 is administered according to the present invention to treat acute myeloid leukemia carrying a FLT3 mutant gene, wherein patients suffering from said leukemia may or may not have FL ligand-associated high expression drug resistance. Treatment can consist of a single treatment or a series of treatments.

In some embodiments of the present invention, a dose of A674563 can be administered to the patient every day, every other day or every week for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or several years. It can be understood that the dose of A674563 administered to a patient can be appropriately increased or decreased during treatment according to the patient's condition and treatment needs.

In some embodiments of the invention, A674563 and one or more other therapeutic agents may be administered to a subject simultaneously or sequentially. Alternatively, in some embodiments of the present invention, a pharmaceutical composition formulated to include A674563 and a pharmaceutically acceptable carrier or adjuvant, and optionally one or more other therapeutic agents.

A674563 for use in the above treatments may be formulated in suitable pharmaceutical formulations for oral administration (e.g., in liquid form in a solvent such as aqueous or non-aqueous liquid, or in a solid carrier), rectal administration , parenteral, intracisternal, intraperitoneal, topical (by powder, ointment, lotion, gel, drops, transdermal or transdermal patch), oral , in transbronchial form or as an oral or nasal spray, etc. Specifically, A674563 can be formulated into, for example, solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained-release preparations or elixirs, etc. for oral administration. In the embodiment of injection administration, A674563 can also be formulated into a suitable injection.

Administration can be performed daily, weekly, monthly, every other month, quarterly, or on any other dosing schedule as a single dose injection or infusion, in multiple doses, or in continuous dosage form. Administration of the formulations of the invention may be intermittent to the subject, or at a gradual, continuous, constant or controlled rate. Furthermore, the time of day the dosage form is administered and the number of times per day the dosage form is administered can vary. In some embodiments of the invention, the formulation of A674563 administered to a patient may range from 0.02-5000 mg/day, eg, about 1-1500 mg/day. The desired dose may conveniently be presented as one dose, or as divided doses administered simultaneously (or within a short period of time) or at appropriate intervals, for example as two, three, four or more divided doses per day.

The pharmaceutical composition containing A674563 for the above treatment can be formulated into dosage forms such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained-release preparations or elixirs. The composition may contain 0.02-5000 mg of A674563, but the effective dosage of the active ingredient used may vary according to the specific dosage regimen used, the route of administration, and the severity of the condition to be treated. The skilled artisan will appreciate that the effective dose for each patient may vary depending on the severity of the disease, individual genetic variation or metabolic rate. Generally, however, the desired results are obtained when the compounds of the present invention are administered at a daily dosage of about 0.5-1000 mg, optionally in divided doses 2-4 times a day or in sustained release form. A total daily dosage of about 1-1000 mg, preferably about 2-500 mg is planned.

The present invention will be illustrated below through embodiments and in conjunction with the accompanying drawings. It should be understood that the illustrated embodiments and drawings are only used to help the understanding of the present invention, but not to limit the present invention.

Example

Experimental materials: In the examples, different concentrations of AKT inhibitor A674563 were used to carry out experiments of the present invention, and PKB/AKT inhibitor CCT128930, PKB/AKT inhibitor GDC0068, PKB/AKT inhibitor GSK690693, PKB/AKT inhibitor MK2206 were used , FLT3 inhibitor MLN518, and FLT3 inhibitor TCS359 were used as controls (these drugs were purchased from HaoYuan Chemexpress Company (Shanghai)).

Example 1: Effect of A674563 on proliferation of cancer cells

The selectivity of A674563 to inhibit the proliferation of cancer cells was evaluated by testing the effect of A674563 on the growth of cancer cells.

In this embodiment, acute myeloid leukemia cells HL-60 (expressing wild-type FLT3 gene), acute promyelocytic leukemia cell line NB-4 (Lu+) (expressing wild-type FLT3 gene), human acute myeloid leukemia cells OCI-AML-3 (expressing FLT3 A680V mutant gene), human acute myeloid leukemia cell line MOLM-14 (expressing FLT3-ITD mutant gene and wild-type FLT3 gene), human acute myeloid leukemia cell line MOLM-13 (express FLT3-ITD mutant gene and wild-type FLT3 gene), human acute myeloid leukemia cell line MV-4-11 (express FLT3-ITD mutant gene), MDS-RAEB (myelodysplastic syndrome-increased blast type) cell line SKM-1 (expressing wild-type FLT3 gene), human acute myeloid leukemia cell line U-937 (expressing wild-type FLT3 gene), mouse cell BaF3. The above cells were purchased from ATCC. Mouse TEL-FLT3-BaF3 (stably expressing FLT3WT kinase) cells, mouse BaF3-FLT3-ITD (stably expressing FLT3ITD mutated activated kinase) cells, mouse BaF3-FLT3-D835Y (stably expressed FLT3-D835Y mutated activated kinase) ) cells, mouse BaF3-FLT3-ITD-D835Y (activated kinases stably expressing FLT3-ITD and FLT3-D835Y mutations) cells, mouse BaF3-FLT3-ITD-F691L (stable expression of FLT3-ITD and FLT3-F691L mutations Activated kinase) cells were all constructed by our laboratory, and the construction method was as follows: respectively amplify the sequences of human FLT3, FLT3-ITD, FLT3-D835Y, FLT3-ITD+FLT3-D835Y, FLT3-ITD+FLT3-F691L kinase regions by PCR, and The amplified fragments were respectively inserted into the pMSCV-Puro vector (purchased from Clontech, U.S.) with N-terminal TEL or TPR fragments, and Murine Embryonic StemCell Virus (MESV) mouse embryonic stem cell virus (virus packaging vector pMSCV- Puro was purchased from Clontech, the United States) and stably transferred to mouse BaF3 cells. The above-constructed mouse BaF3 cell culture medium was RPMI 1640 plus 10% FBS (calf embryo serum) and 1% P/S (double antibody) , and IL-3 growth factor was withdrawn, and finally the cell lines expressing FLT3-ITD, FLT3-D835Y, FLT3, FLT3-ITD+FLT3-D835Y, FLT3-ITD+FLT3-F691L transfer protein were obtained.

In the examples, different concentrations (0.000508 μM, 0.00152 μM, 0.00457 μM, 0.0137 μM, 0.0411 μM, 0.123 μM, 0.370 μM, 1.11 μM, 3.33 μM, 10 μM in DMSO) of A674563 were used alone or simultaneously with 1 ng/mL, 5ng/mL or 10ng/mL of FLT3 ligand FL (purchased from CST Cell Signing Technology, the United States), was added to the above cells, and then the cells were incubated at 37 degrees in the incubator for 72 hours, and Cell Titer- (Promega, USA) Chemiautoluminescence Cell Viability Detection Kit, through the quantitative determination of ATP in living cells to detect the number of living cells. The obtained results are shown in Table 1 (without addition of FLT3 ligand FL) and Table 2 (with addition of FLT3 ligand FL) below, respectively.

As shown in Table 1, it was found that A674563 was effective against human acute myeloid leukemia cell line MOLM-14 (expressing FLT3-ITD mutant gene and wild-type FLT3 gene), human acute myeloid leukemia cell line MOLM-13 (expressing FLT3-ITD mutant type gene and wild-type FLT3 gene), human acute myeloid leukemia cell line MV-4-11 (expressing FLT3-ITD mutant gene) had obvious inhibitory effect on the proliferation of three cancer cell lines, and A674563 had a significant inhibitory effect on cell line MOLM- 13. The GI50 of MOLM-14 and MV-4-11 are 0.1μM, 0.22μM, 0.12μM respectively, but they have no obvious inhibitory effect on the proliferation of other cancer cells carrying only wild-type FLT3 gene. At the same time, A674563 also had a significant effect on the proliferation of FLT3-ITD transfer protein-dependent cells (mouse BaF3-FLT3-ITD cells) and FLT3-D835Y transfer protein-dependent cells (mouse BaF3-FLT3-D835Y cells) constructed in our laboratory. Strong inhibitory effect, GI50 were 0.088μM, 0.075μM. In contrast, A674563 had little inhibitory effect on the proliferation of FLT3 WT transferin-dependent cells (TEL-FLT3-BaF3 cells, GI50=1.0 μM).

Table 1. Determination results of the inhibitory effect of A674563 on cancer cells

Continuation of Table 1. A674563 Determination Results of Inhibitory Effect on Cancer Cells

In the experiment of adding FLT3 ligand FL (as shown in Table 2 below), we can clearly see that in human acute myeloid leukemia cell lines MOLM-13, MOLM- After adding 1ng/ml of FLT3 ligand FL in 14, the FLT3 inhibitor TCS359 was obviously inactivated; another FLT3 inhibitor MLN518 was effective in human acute myeloid leukemia cell line MOLM- 13. In MOLM-14, as the amount of FLT3 ligand FL added increases, its activity also decreases; in contrast, in human acute myeloid leukemia cell lines carrying FLT3-ITD mutant gene and wild-type FLT3 gene In MOLM-13 and MOLM-14, with the increase of the amount of FLT3 ligand FL added, there was almost no change in the activity of A674563. In the human acute myeloid leukemia cell line MV-4-11 carrying only the FLT3 mutant gene, whether adding FL has basically no effect on the cytostatic activity of A674563.

The results in Table 2 indicate that the activity of A674563 is largely unaffected by the presence of the FL ligand. Therefore, even in the case of acute myeloid leukemia cells carrying the FLT3 mutant gene developing drug resistance due to increased secretion of the ligand FL, A674563 can exert a significant inhibitory effect on it. This means that A674563 can be applied to the treatment of acute myeloid leukemia carrying FLT3 mutant gene with drug resistance associated with high expression of FL ligand.

Table 2. The results of the determination of the inhibitory effect of A674563 on cancer cells in the case of adding different concentrations of FL

In summary, the results of Example 1 show that A674563 has a strong inhibitory effect on the proliferation of acute myeloid leukemia cells carrying FLT3 mutant genes, especially acute myeloid leukemia cells carrying FLT3-ITD and FLT3-D835Y mutant genes Cell proliferation has a very strong inhibitory effect. The results of Example 1 also indicate that A674563 can be used to treat acute myeloid leukemia carrying the FLT3 mutant gene that is highly expressed in FL ligands and causes drug resistance.

Example 2: Effects of A674563 on Upstream and Downstream Signaling Pathways in Cells

In human acute myeloid leukemia cell line MV-4-11 (expressing FLT3-ITD mutant gene), human acute myeloid leukemia cell line MOLM-13 (expressing FLT3-ITD mutant gene and wild-type FLT3 gene) and human In the acute myeloid leukemia cell line MOLM-14 (expressing FLT3-ITD mutant gene and wild-type FLT3 gene), the effects of A674563 on FLT3 protein kinase, PKB /AKT kinase and the phosphorylation of STAT5, ErK, GSK3β, FOX01, PRAS40, p70S6K, and 4EBP1 proteins closely related to FLT3 kinase and PKB/AKT kinase. At the same time, we also detected the effect of A674563 on the phosphorylation of protein C-Myc and transcription factor NF-κB subunit p65 (Fig. 1a). With different concentrations of 0 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM (in DMSO) of A674563 and 1 μM (in DMSO) of PKB/AKT inhibitor GSK690693, PKB/AKT inhibitor MK2206, FLT3 inhibitor TCS359 Treat three strains of acute myeloid leukemia cells carrying the FLT3-ITD mutant gene, MOLM-13, MOLM-14, and MV-4-11 (Fig. 1a) for 4 hours, and at the same time, use only 1% FBS (fetal calf serum) The medium (purchased from Corning Incorporated, USA) was used to starve the cells for 4 hours, and the cell samples were collected. The effects of compounds on the phosphorylation of STAT5, ErK, GSK3β, FOX01, PRAS40, p70S6K, 4EBP1, C-Myc, NF-κB p65, and AKT proteins in these three cell lines were determined (see Figure 1a).

As shown in Figure 1a, in the three human acute myeloid leukemia cell lines MOLM-13, MOLM-14, and MV-4-11, A674563 had a strong inhibitory effect on the phosphorylation of the downstream protein STAT5 of FLT3-ITD in the cells, And it has obvious degradation effect on protein C-Myc which is closely related to FLT3 protein kinase. In the same experiment, the control compound PKB/AKT inhibitor GSK690693 and PKB/AKT inhibitor MK2206 had no effect on the phosphorylation of FLT3 kinase, and had no significant effect on the phosphorylation of the downstream protein STAT5 of FLT3-ITD. The related protein C-Myc was also not significantly degraded. Another control compound, FLT3 inhibitor TCS359, can strongly inhibit the phosphorylation of STAT5, a protein closely related to FLT3-ITD, and can also significantly degrade the protein C-Myc, which is closely related to FLT3 protein kinase. This indicates that A674563 can inhibit the phosphorylation of protein STAT5 downstream of the protein kinase FLT3 signaling pathway in cells, thereby inhibiting the cell proliferation of acute myeloid leukemia cell lines carrying the FLT3-ITD gene or carrying the FLT3-ITD gene and the FLT3 WT gene.

In the experiment of adding FLT3 ligand FL (as shown in Figure 1b, 1c, using MLN518 as a control), the results of 2-hour stimulation with 10 ng/ml FL were compared with the results of no stimulation. Signaling pathways are affected differently. In the human acute myeloid leukemia cell line MOLM-14 carrying FLT3-ITD mutant gene and wild-type FLT3 gene, before and after adding 10 ng/ml of FLT3 ligand FL, A674563 (as shown in Figure 1b) can significantly inhibit Phosphorylation of STAT5, and a significant degradation of C-Myc, a protein closely related to FLT3 protein kinase, indicates that the effect of A674563 on signaling pathways in cells is not affected by the presence of FL ligands. Therefore, even in the case of acute myeloid leukemia cells carrying FLT3 mutant gene drug resistance due to increased secretion of ligand FL co-expressed with FLT3, A674563 has a significant effect on its signaling pathway. In the same experiment, the control compound FLT3 inhibitor MLN518 (as shown in Figure 1c) could strongly inhibit the phosphorylation of STAT5, a protein closely related to FLT3-ITD, when 10 ng/ml of FLT3 ligand FL was not added. The protein C-Myc, which is closely related to protein kinase, also has obvious degradation effect. However, after adding 10ng/ml of FLT3 ligand FL, the degradation effect of MLN518 on the protein C-Myc closely related to FLT3 protein kinase was significantly weakened, indicating that the control compound FLT3 inhibitor MLN518 is resistant to the increased secretion of ligand FL. The effect of the signal pathway of the cell line is weakened. In contrast, in the human acute myeloid leukemia cell line MV-4-11 carrying only the FLT3 mutant gene, because there is no wild-type FLT3 gene, even if FL is added, there is no high expression of FL ligand associated drug resistance. The experimental results showed that in the MV-4-11 cell line, A674563 (Fig. 1b) had basically the same effect on the signaling pathway of the cell line before and after adding FL. The effect of the control compound FLT3 inhibitor MLN518 (Fig. 1c) on the signaling pathway of the cell lines is also basically the same.

The test results of adding FLT3 ligand FL further prove from the level of signaling pathways that the effect of A674563 on signaling pathways in cells is not affected by the presence of FL ligands, so A674563 can be used to treat diseases associated with high expression of FL ligands. Drug-resistant acute myeloid leukemia with FLT3 mutations.

Example 3: Effect of A674563 on FLT3 autophosphorylation in the BaF3 cell line at the isogenic locus

Mouse TEL-FLT3-BaF3 (activating kinase stably expressing wild-type FLT3) cells, mouse BaF3-FLT3-ITD (activating kinase stably expressing FLT3 ITD mutation) cells, mouse BaF3-FLT3-D835Y (stably expressing FLT3D835Y mutant activated kinase) cells, mouse BaF3-FLT3-ITD-D835Y (activated kinase stably expressing FLT3-ITD+D835Y mutation) cells, mouse BaF3-FLT3-ITD-F691L (stable activated kinase) cells (see Example 1 for the construction method of these cells). The effect of A674563 on the autophosphorylation of FLT3 and/or FLT3-ITD mutant protein kinases in cells was tested in the above cell lines (Figure 2). With different concentrations of 0 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM (in DMSO) A674563, 0.05 μM (in DMSO) FLT3 kinase inhibitor AC220 (purchased from Hao Yuan Chemexpress Company (Shanghai)) respectively BaF3 cell lines treated with different isogenic loci (TEL-FLT3-BaF3, BaF3-FLT3-ITD, BaF3-FLT3-D835Y, BaF3-FLT3-ITD-D835Y, BaF3-FLT3-ITD-F691L), harvested after 4 hours Cells, the effect of A674563 on the autophosphorylation of FLT3 kinase was detected by Western Blot (Figure 2).

The determination data of the influence of A674563 on the FLT3 autophosphorylation in the BaF3 cell line of the isogenic locus is shown in Table 3: in the BaF3 cell lines of all the isogenic loci in this embodiment, A674563 has an effect on the protein in the cell line The autophosphorylation of the kinase FLT3 has certain effects, especially in mouse BaF3-FLT3-ITD (activating kinase stably expressing FLT3 ITD mutation) and mouse BaF3-FLT3-D835Y (activating kinase stably expressing FLT3 D835Y mutation) cells strains, their EC50 were 0.085 μM and 0.045 μM (see Table 3). In the same experiment, the control FLT3 kinase inhibitor AC220 also had a very obvious inhibitory effect on the autophosphorylation of protein kinase FLT3 (see Figure 2). Example 3 shows that A674563 is able to inhibit the autophosphorylation of the protein kinase FLT3 in the BaF3 cell line at different isogenic loci.

Table 3. Determination data for the effect of A674563 on FLT3 autophosphorylation in the BaF3 cell line at the isogenic locus

Cell line name A674563 p-FLT3: EC50(μM) TEL-FLT3-BaF3 0.87 BaF3-FLT3-ITD 0.085 BaF3-FLT3-D835Y 0.045 BaF3-FLT3-ITD-F691L 0.4

p-FLT3: phosphorylated FLT3.

Example 4: Effect of A674563 on apoptosis in cells

In order to prove whether the cell death after treatment was caused by apoptosis or necrosis, acute myeloid leukemia cells MOLM-13, MOLM-14, MV- In 4-11 cells, the effect of A674563 on DNA repair enzyme polyadenosine diphosphate-ribose polymerase PARP and cysteine-containing aspartic acid proteolytic enzyme Caspase 3 proteins closely related to cell apoptosis was detected. The effect of shearing. With different concentrations of 0 μM, 0.3 μM, 1 μM, 3 μM (in DMSO) A674563, 1 μM (in DMSO) FLT3 kinase inhibitor TCS359, 1 μM (in DMSO) PKB/AKT kinase inhibitor MK2206, 1 μM (in DMSO) The PKB/AKT kinase inhibitor GSK690693 in DMSO was used to treat MOLM-13, MOLM-14, and MV-4-11 cells respectively, and the cells were collected after 24 hours. Western Blot was used to detect the effects of different concentrations of drugs on the shearing proteins of DNA repair enzyme polyadenosine diphosphate-ribose polymerase PARP and cysteine-containing aspartic acid proteolytic enzyme Caspase 3.

The experimental results are shown in Figure 3: For MOLM-13 and MV-4-11 cells, when the concentration of A674563 is 0.3 μM, the DNA repair enzyme polyadenosine diphosphate-ribose can be seen very obviously after 24 hours of treatment. Cleavage by the polymerase PARP and, in particular, by the cysteine-containing aspartate proteolytic enzyme Caspase 3. In the same experiment, when the drug concentration of the control FLT3 kinase inhibitor TCS359, PKB/AKT kinase inhibitor MK2206, and PKB/AKT kinase inhibitor GSK690693 was 1 μM, the above phenomenon was not obvious after 24 hours of action. For MOLM-14 cells, when the concentration of A674563 is 1 μM, after 24 hours of action, it can be seen that the DNA repair enzyme polyadenosine diphosphate-ribose polymerase PARP is cleaved, and the particularly obvious cysteine-containing Cleavage of the aspartate proteolytic enzyme Caspase 3. In the same experiment, when the drug concentration of the control FLT3 kinase inhibitor TCS359, PKB/AKT kinase inhibitor MK2206, and PKB/AKT kinase inhibitor GSK690693 was 1 μM, no obvious DNA repair enzyme polyadenosine was observed after 24 hours of action. Cleavage of phosphate-ribose polymerase PARP and cleavage of cysteine-containing aspartate proteolytic enzyme Caspase 3. Therefore, the results of Example 4 indicated that A674563 caused apoptosis (not necrosis) of acute myeloid leukemia cells carrying the FLT3-ITD mutant gene.

Example 5: Effect of A674563 on cell cycle in cells

In order to study in which growth cycle cells are arrested after drug administration, acute myeloid leukemia cells MOLM-13, MOLM-14, MV-4-11 carrying FLT3-ITD mutant gene or FLT3-ITD mutant gene + FLT3 WT gene Among the cell lines, the effect of A674563 on the cell cycle distribution of these cell lines was tested. With different concentrations of 0 μM, 0.3 μM, 3 μM (in DMSO) of A674563, 1 μM (in DMSO) of the FLT3 kinase inhibitor TCS359, 1 μM (in DMSO) of the PKB/AKT kinase inhibitor MK2206, 1 μM (in DMSO) The PKB/AKT kinase inhibitor GSK690693 in middle) acted on acute myeloid leukemia cells MOLM-13, MOLM-14, MV-4-11. After acting for 24 hours, the cells were collected, washed twice with 1×PBS buffer, and The washed cells were fixed with 75% ethanol at -20°C for 24 hours, washed twice with 1×PBS buffer, and 0.5 mL of 1×PBS buffer and 0.5 mL of PI staining solution (purchased from BD Bioscience, USA) were added to the cells The cells were placed in the dark at 37°C and stained for 15 minutes in the dark, and the cell cycle distribution was detected by flow cytometry (BD FACS Calibur).

The experimental results are shown in Figure 4: in the acute myeloid leukemia cell line MOLM-13, as the drug concentration of A674563 increased from 0 μM to 3 μM, the proportion of cells in the G0-G1 phase captured increased from 49.7% to 59.76%; When TCS359, MK2206, and GSK690693 were treated with 1 μM, the proportions of cells in the G0-G1 phase that could be captured were 62.02%, 76.81%, and 70.07%, respectively (Fig. 4a). For acute myeloid leukemia cell MOLM-14, as the drug concentration of A674563 increased from 0.3 μM to 3 μM, the proportion of cells in the G0-G1 phase captured increased from 47.08% to 53.87%; compared with TCS359, MK2206, GSK690693 when the cells were treated at 1 μM The proportions of cells in the G0-G1 phase that could be captured were: 55.04%, 63.9%, and 54.52% (Fig. 4b). For acute myeloid leukemia cells MV-4-11, as the drug concentration of A674563 increased to 3 μM, the proportion of cells in the G0-G1 phase captured increased to 68.69%; compared with TCS359, MK2206, GSK690693, the G0 captured when the cells were treated with 1 μM The proportions of cells in the -G1 phase were: 73.62%, 64.92%, and 60.48%, respectively (Fig. 4c). Example 5 proves that A674563 has a certain influence on the cell cycle distribution of acute myeloid leukemia cell lines carrying FLT3 WT gene + FLT3-ITD mutant gene or FLT3-ITD mutant gene.

Claims (10)

1. The use of A674563 in the preparation of medicines for treating acute myeloid leukemia patients carrying the FLT3 mutant gene, wherein A674563 has a structure shown in the following formula (I): 2. The use according to claim 1, wherein the patient has drug resistance caused by high expression of FL ligand. 3. The use according to claim 1, wherein the FLT3 mutant gene is at least one selected from FLT3-ITD and FLT3-D835Y mutant genes. 4. A non-therapeutic method for suppressing acute myeloid leukemia cells carrying the FLT3 mutant gene, comprising contacting the cells with A674563, wherein A674563 has a structure shown in the following formula (I): 5. The method according to claim 4, wherein the acute myeloid leukemia cells carrying the FLT3 mutant gene are one or more selected from MOLM-13, MOLM-14 and MV-4-11. 6. The method of claim 4 or 5, wherein the cells are contacted with A674563 at a concentration of at least 0.01 [mu]M. 7. The use of A674563 in the preparation of medicines for inhibiting acute myeloid leukemia cells carrying the FLT3 mutant gene, wherein A674563 has a structure shown in the following formula (I): 8. The use of A674563 in the preparation of drugs for inhibiting acute myeloid leukemia cells carrying the FLT3-ITD mutant gene, wherein A674563 has the structure shown in the following formula (I): 9. The use according to claim 8, wherein the acute myeloid leukemia cells carrying the FLT3-ITD mutant gene are selected from one or more of MOLM-13, MOLM-14 and MV-4-11 . 10. The use of A674563 in the preparation of drugs for inhibiting acute myeloid leukemia cells carrying the FLT3-D835Y mutant gene, wherein A674563 has the structure shown in the following formula (I):