CN104873515B - The application of acute leukemia FLT3 ITD being mutated for Buddhist nun according to Shandong - Google Patents

Abstract

The present invention discloses a new application of ibrutinib (PCI32765), specifically, the present invention finds that ibrutinib can be used to treat acute myeloid leukemia, especially acute myeloid leukemia carrying FLT3/ITD mutant gene .

Description

Application of ibrutinib in acute leukemia with FLT3-ITD mutation

technical field

The invention relates to the field of medicine, in particular to a new application of ibrutinib (PCI32765).

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, is 1/100,000 under the age of 30, and is as high as 17/10 over the age of 75. Ten thousand. 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 five immunoglobulin (Ig)-like domains consisting of a foreign preservation domain, a transmembrane domain, a membrane juxtaposition 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). FLT3 mutation was first discovered in AML cells in 1996, and its mutation type is an 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. 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 FLT3 activation mutations: internal tandem duplication (internal tandem duplication, ITD) and point mutation in the activation loop (point mutation in the activation loop, Tkd point mutation). 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, such as AC220 and so on.

Ibrutinib (Ibrutinib, also known as PCI32765) is an inhibitor of Bruton's tyrosine kinase (Bruton'styrosine kinase, BTK), which can be used alone or in combination with other therapeutic agents for Treatment of autoimmune diseases or disorders, heteroimmune diseases or disorders, cancers including lymphomas, and inflammatory diseases or disorders. At present, there is no relevant report on the use of ibrutinib (PCI32765) in the treatment of acute myeloid leukemia carrying the FLT3/ITD mutant gene.

Contents of the invention

In view of the foregoing technical problems, the inventors conducted extensive research. As a result, the inventors unexpectedly discovered that ibrutinib (PCI32765), a Bruton's tyrosine kinase inhibitor, can effectively treat acute myeloid leukemia carrying the FLT3/ITD mutant gene. More specifically, the inventors found that ibrutinib had a strong inhibitory effect on acute myeloid leukemia cells carrying FLT3/ITD mutant genes such as MOLM-13, MOLM-14, and MV-4-11 (IC50 respectively 0.47μM, 0.66μM, 0.33μM), while ibrutinib had no obvious inhibitory effect on acute myeloid leukemia cells carrying FLT3WT (wild type) and FLT3 A680V mutant genes. Therefore, ibrutinib can be applied to the clinical treatment of acute myeloid leukemia carrying FLT3/ITD mutant gene.

In one aspect, the present invention relates to the use of ibrutinib (PCI32765) in the preparation of medicines for treating acute myeloid leukemia cancer patients carrying FLT3/ITD mutant genes.

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 ibrutinib may be administered to the acute myeloid leukemia patient carrying the FLT3/ITD 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 (such as tacrolimus, cyclosporine, rapamycin, methotrexate, cyclophosphamide, azathioprine, mercaptopurine, mycophenolate mofetil or FTY720), glucocorticoids (eg, prednisone, cortisone acetate, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, hydroxyprednisolone, beclomethasone , fludrocortisone acetate, deoxycorticosterone acetate, aldosterone), NSAIDs (such as salicylates, aryl alkanoic acids, 2-aryl propionic acids, N-aryl anthranilic acids, oxicams, coxicams, or sulfanilides), allergy vaccines, antihistamines, antileukotrienes, beta-agonists, theophylline, anticholinergics, or other selective kinase inhibitors (such as mTOR inhibitors , c-Met inhibitor) or her2 antibody-drug. In addition, other therapeutic agents mentioned may also be Rapamycin, Crizotinib, Tamoxifen, Raloxifene, Anastrozole, Exemestane, Letrozole , HerceptinTM (trastuzumab), ^{GleevecTM (} imatinib), paclitaxelTM ⁽ paclitaxel), cyclophosphamide, lovastatin, minocycline (Minosine), cytarabine, 5-Fluorouracil (5-FU), methotrexate (MTX), TaxotereTM ⁽ docetaxel), ^NuoladTM (goserelin), vincristine, vinblastine, Nocoda Azole, Teniposide, 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 may be, for example but not limited to, CMF (cyclophosphamide, methotrexate, and 5-fluorouracil), CAF (cyclophosphamide, doxorubicin, and 5-fluorouracil), AC (sub- doxorubicin and cyclophosphamide), FEC (5-fluorouracil, epirubicin and cyclophosphamide), ACT or ATC (driamycin, cyclophosphamide and paclitaxel) or CMFP (cyclophosphamide, formazan aminopterin, 5-fluorouracil, and prednisone).

In another aspect, the present invention also relates to a pharmaceutical composition comprising ibrutinib (PCI32765) 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 for treating acute myeloid leukemia patients carrying a FLT3/ITD mutant gene using ibrutinib. During the course of treatment, ibrutinib can be administered to acute myeloid leukemia patients carrying the FLT3/ITD mutant gene by injection, oral, inhalation, rectal or transdermal. An effective amount of ibrutinib 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 ibrutinib (PCI32765) can also be administered in combination with radiotherapy 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/ITD mutant gene, comprising contacting said cells with ibrutinib. According to the present invention, the acute myeloid leukemia cells carrying the FLT3/ITD mutant gene are preferably one or more selected from MOLM-13, MOLM-14, MV-4-11, PL-21 and MUTZ-11. Further preferably, the cells are contacted with ibrutinib at an effective concentration of at least 0.05 μM, more preferably at least 0.1 μM, further preferably 0.3-10 μM, eg 0.3-3 μM.

Description of drawings

Figure 1 shows the effects of PCI32765 on the BTK signaling pathway in MOLM-14 (1a) and OCI-AML-3 (1b) cells, respectively.

Figure 2 shows the effects of PCI32765 on FLT3/ITD relatively closely related proteins and related signaling pathways on MOLM-13(2a), MOLM-14(2b) and MV-4-11(2c) cells, respectively.

Figure 3 shows proteins that are relatively closely related to FLT3/ITD in cells.

Figure 4 shows the effect of PCI32765 on the apoptosis of MOLM-13 (4a), MOLM-14 (4b), MV-4-11 (4c), OCI-AML-3 (4d) and NOMO-1 (4e) respectively influences.

Figure 5 shows the effect of PCI32765 on the cell cycle distribution of MOLM-14 (5a), MV-4-11 (5b), MOLM-13 (5c), OCI-AML-3 (5d) and NOMO-1 (5e) respectively influences.

detailed description

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

definition

"Ibrutinib (also known as PCI32765)" is an inhibitor of Bruton's tyrosine kinase (BTK). As a small molecule BTK inhibitor, it can covalently bind to the cysteine residue in the active center of BTK, thereby inhibiting its activity. Ibrutinib has a 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 FLT3 internal tandem duplication (FLT3/ITD) mutation, which has the highest incidence rate in acute myeloid leukemia (AML) and is associated with Prognostic-associated mutations.

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. Dosage regimens may be adjusted to provide the optimum therapeutic response.

In an embodiment of the present invention, when a subject suffering from acute myeloid leukemia carrying a FLT3/ITD mutant gene is treated with ibrutinib according to the present invention, the amount of a given drug depends on many factors, Such as the particular 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), however, depending on the particular surrounding circumstances including, for example, the particular drug already employed, the administration of The route, condition to be treated, and subject or host to be treated, and 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 dose range is given, the effective dose of ibrutinib can be adjusted appropriately according to the condition of the patient and combined with the diagnosis of the physician.

As used herein, "IC50", also known as the median inhibitory concentration, refers to the amount, concentration or dose of a particular inhibitor that achieves 50% inhibition of the maximal effect (eg, inhibition of BTK kinase activity) in an assay measuring an effect.

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

Application of the invention

In some embodiments of the present invention, ibrutinib is administered according to the present invention to treat acute myeloid leukemia carrying a FLT3/ITD mutant gene. Treatment can consist of a single treatment or a series of treatments.

In some embodiments of the present invention, a certain dose of ibrutinib can be administered to the patient every day, every other day or every week, and last for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months Months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or several years. It can be understood that the dose of ibrutinib 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, ibrutinib 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 ibrutinib and a pharmaceutically acceptable carrier or adjuvant, and optionally a or various other therapeutic agents.

Ibrutinib for the treatment of acute myeloid leukemia carrying a FLT3/ITD mutant gene can be formulated into a suitable pharmaceutical formulation for oral administration (e.g., in liquid form in a solvent such as aqueous or non-aqueous liquid, or in a solid carrier), rectally, parenterally, intracisternally, intraperitoneally, topically (by powder, ointment, lotion, gel, drops, transdermal patch or transdermal patch), orally, in transbronchial form or as a buccal or nasal spray, etc. Specifically, ibrutinib can be formulated into, for example, solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained-release preparations or elixirs for oral administration. In the embodiment of injection administration, ibrutinib 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 ibrutinib administered to a patient may be in the range of 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 comprising ibrutinib for the treatment of acute myeloid leukemia carrying FLT3/ITD mutant gene can be formulated into solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release Dosage forms such as preparations or elixirs. The composition may contain 0.02-5000 mg of ibrutinib, 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 disease 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: AVL-292, CGI1746 and AC220 were used as controls in the examples. AVL-292 is a highly selective BTK inhibitor through covalent binding, and its IC50 is less than 0.5nM (Robak T, Robak E, "Tyrosinekinase inhibitors as potential drugs for B-cell lymphoid malignancies and autoimmune disorders", Expert. Opin. Investig. Drugs, 2012, 21(7):921-947). CGI1746 is a novel highly selective small molecule BTK kinase inhibitor with an IC50 of 1.9nM (Di Paolo JA, et al., "Specific Btk inhibition suppresses B cell-and myeloid cell-mediated arthritis", Nat.Chem.Biol. , 2011, 7(1):41-50). AC220 is a FLT3 kinase inhibitor that has entered clinical practice. Ibrutinib (PCI32765), AVL292, CGI1746 and AC220 were purchased from Hao Yuan Chemexpress Company (Shanghai).

Example 1: Effect of Ibrutinib (PCI32765) on Cancer Cell Proliferation

The selectivity of ibrutinib (PCI32765) to inhibit cancer cell proliferation was further evaluated by testing the effect of ibrutinib (PCI32765) on cancer cell growth. In this embodiment, acute myeloid leukemia cells MOLM-16 (expressing wild-type FLT3 gene), acute myeloid leukemia cells HL-60 (expressing wild-type FLT3 gene), acute myeloid leukemia cells KASUMI-1 (expressing wild-type FLT3 gene) were selected. FLT3 gene), acute promyelocytic leukemia cell line NB-4 (Lu+) (expressing wild-type FLT3 gene), human acute myeloid leukemia cell line OCI-AML-2 (expressing FLT3 A680V mutant gene), human acute myeloid leukemia cell line leukemia cell line 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 monocytic leukemia cell line MV-4-11 (express FLT3/ITD mutant gene), human acute myeloid leukemia cell line NOMO -1 (expressing wild-type FLT3 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), human acute erythroleukemia cell line HEL (expressing wild-type FLT3 gene), human acute megakaryoblastic leukemia CMK (expressing wild-type FLT3 gene), human peripheral lymphocyte JVM-2 infected by human Epstein-Barr virus, Human Burkitt's lymphoma cell Namalwa, human mantle cell lymphoma (mantel cell lymphoma, MCL) cell REC-1, human mantle cell lymphoma (mantel cell lymphoma, MCL) cell Z-138, human diffuse tissue lymphoma cell SU-DHL -2. Human diffuse large B-cell lymphoma cell line TMD8, human B-cell chronic lymphocytic leukemia cell line MEC-1, human B-cell chronic lymphocytic leukemia cell line MEC-2, human B-cell leukemia cell line NALM -6. Human acute T lymphocytic leukemia cell line JURKAT and blood cancer cell line K562. The above cells were purchased from ATCC.

In the example, 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 ibrutinib (PCI32765) were added to In the above cells, and incubated for 72 hours, with 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 experimental results are shown in Table 1. It was found that ibrutinib (PCI32765) was only effective against the human acute myeloid leukemia cell line MOLM-14 expressing the FLT3/ITD mutant gene (expressing the FLT3/ITD mutant gene and the wild-type FLT3 gene). , human acute myeloid leukemia cell line MOLM-13 (expressing FLT3/ITD mutant gene and wild-type FLT3 gene), human acute monocytic leukemia cell line MV-4-11 (expressing FLT3/ITD mutant gene) The proliferation of the three cancer cell lines had a strong inhibitory effect. The IC50 of ibrutinib on the cell lines MOLM-13, MOLM-14 and MV-4-11 were 0.47μM, 0.66μM and 0.33μM, respectively, while that on The proliferation of other cancer cells has no inhibitory effect (the IC50 value of the half inhibitory concentration is greater than 2 μM). The results of Example 1 support the selectivity of ibrutinib (PCI32765) for the treatment of acute myeloid leukemia carrying the FLT3/ITD mutant gene.

Table 1. Measurement data of PCI32765 on cancer cells

cell line cell type FLT3 type PCI32765IC50(μM) MOLM-16 AML-M0 FLT3WT 4.4 HL-60 AML-M2 FLT3WT 2.1 KASUMI-1 AML-M2 FLT3WT 3.0 NB-4 AML-M3 FLT3WT 7.5 OCI-AML-2 AML-M4 FLT3 A680V >10 OCI-AML-3 AML-M4 FLT3 A680V >10 MOLM-13 AML-M5a FLT3/ITD, FLT3WT 0.47 MOLM-14 AML-M5a FLT3/ITD, FLT3WT 0.66 MV-4-11 AML-M5 FLT3/ITD 0.33 NOMO-1 AML-M5 FLT3WT >10 SKM-1 AML-M5 FLT3WT 8.6 U-937 AML-M5 FLT3WT 8.5 HEL AML-M6 FLT3WT >10 CMK AML-M7 FLT3WT >10 JVM-2 MCL -- >10

Namalwa Burkitt lymphoma -- >10 REC-1 MCL -- >10 Z-138 MCL -- >10 SU-DHL-2 ABC DLBCL -- >10 TMD8 ABC DLBCL -- >10 MEC-1 B-CLL -- >10 MEC-2 B-CLL -- >10 NALM-6 ALL -- >10 JURKAT ALL -- >10 K562 CML -- >10

Example 2: Effects of ibrutinib (PCI32765) on BTK upstream and downstream signaling pathways in cells

On two lines of acute myeloid leukemia cells MOLM-14 (expressing FLT3/ITD mutant gene and wild-type FLT3 gene) and acute myeloid leukemia cell line OCI-AML-3 (expressing FLT3 A680V mutant gene), by determining A number of cellular biochemical and functional endpoints evaluated the effects of ibrutinib (PCI32765) on BTK kinases and protein kinases closely related to BTK kinases PLCγ, AKT, ErK, and GSK3β in cells. With different concentrations of 0 μM, 0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM (in DMSO) of ibrutinib (PCI32765), 1 μM (in DMSO) of AVL-292, and 1 μM (in DMSO) Middle) CGI1746 treated acute myeloid leukemia cell MOLM-14 (expressing FLT3/ITD mutant gene and wild-type FLT3 gene) and acute myeloid leukemia cell OCI-AML-3 (expressing FLT3 A680V mutant gene) for 4 hours, And stimulated with human immunoglobulin IgM for 10min, collected samples. The effect of compounds on phosphorylation of BTKY223, PLCγY1217, AKT308, AKT S473, Erk 42/44 in these two cell lines was determined (Figure 1).

The experimental results are shown in Figure 1: whether in acute myeloid leukemia cell MOLM-14 (expressing FLT3/ITD mutant gene and wild-type FLT3 gene), or in acute myeloid leukemia cell OCI-AML-3 (expressing FLT3 A680V In mutant gene) cells, although ibrutinib (PCI32765) can inhibit the phosphorylation of BTK, it has no effect on the phosphorylation of PLCγ downstream of BTK (Figure 1), and has no effect on the phosphorylation of AKT and Erk . This shows that ibrutinib (PCI32765) does not affect the cell proliferation of the acute myeloid leukemia cell line MOLM-14 carrying the FLT3/ITD mutant gene by inhibiting the phosphorylation of protein kinase BTK.

Example 3: Effects of ibrutinib (PCI32765) on FLT3 upstream and downstream signaling pathways in cells

Ibrutinib was tested in three lines of acute myeloid leukemia cells MOLM-13, MOLM-14, and MV-4-11 carrying the FLT3/ITD mutant gene by measuring a number of cellular biochemical and functional endpoints. The effect of Ni (PCI32765) on the phosphorylation of FLT3/ITD mutant protein kinase in cells and the downstream STAT5 protein phosphorylation of closely related signaling pathways (Figure 3), and the phosphorylation of other related protein kinases ERK and AKT , At the same time, we also detected the effect on protein C-Myc and transcription factor NF-κB subunit p65 and its phosphorylation. With different concentrations of 0 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM (in DMSO) ibrutinib (PCI32765), 0.1 μM (in DMSO) FLT3 kinase inhibitor AC220, 1 μM (in DMSO) BTK kinase inhibitor AVL-292, BTK kinase inhibitor CGI1746 at 1 μM (in DMSO) treated three acute myeloid leukemia cells carrying the FLT3/ITD mutant gene, MOLM-13, MOLM-14, and MV-4-11, respectively Samples were collected for 4 hours while cells were starved for 4 hours in a medium containing only 1% FBS (fetal bovine serum). The effects of compounds on STAT5, C-Myc, ERK, NF-κB p65, AKT protein and phosphorylation in these three cell lines were determined ( FIG. 2 ).

The experimental results are shown in Figure 2: in the MOLM-14 cell line, ibrutinib (PCI32765) can strongly inhibit the phosphorylation of protein kinase FLT3, and its EC50 is 0.039 μM. In MOLM-13 and MV-4-11 cell lines, ibrutinib (PCI32765) can also significantly inhibit the phosphorylation of protein kinase FLT3, with EC50 of 0.32 μM and 0.36 μM, respectively (Figure 2). In addition, in three lines of acute myeloid leukemia cells MOLM-13, MOLM-14, and MV-4-11 carrying the FLT3/ITD mutant gene, ibrutinib (PCI32765) inhibited the expression of FLT3/ITD downstream proteins in the cells. Phosphorylation of STAT5 has a strong inhibitory effect, and its EC50 is 0.32μM, 0.11μM, 0.91μM, respectively, and has obvious degradation effect on protein C-Myc closely related to FLT3 protein kinase. In the same experiment, the control BTK kinase inhibitors AVL-292 and CGI1746 did not inhibit the phosphorylation of protein kinase FLT3 at a concentration of 1 μM, and CGI1746 had no effect on the downstream protein STAT5 of FLT3/ITD and the protein C-Myc in cells . The control FLT3/ITD kinase inhibitor AC220 can strongly inhibit the phosphorylation of protein kinase FLT3 and the protein STAT5 closely related to FLT3/ITD and the degradation of protein C-Myc. Example 3 shows that the BTK kinase inhibitor ibrutinib (PCI32765) can inhibit the phosphorylation of protein kinase FLT3, affect the phosphorylation of protein STAT5 downstream of the signaling pathway of protein kinase FLT3 in cells, and then inhibit the expression of FLT3/ITD mutant gene. Cell proliferation of acute myeloid leukemia cell lines.

Example 4: Effect of ibrutinib (PCI32765) on cell apoptosis

In order to prove whether the cell death after treatment is through apoptosis or necrosis, acute myeloid leukemia cells MOLM-13, MOLM-14, MV-4-11 carrying the FLT3/ITD mutant gene and acute myeloid leukemia cells carrying the FLT3A680V mutant gene In leukemia cells OCI-AML-3 and acute myeloid leukemia cells NOMO-1 carrying wild-type FLT3 gene, the effect of ibrutinib (PCI32765) on the DNA repair enzyme polyadenylation closely related to cell apoptosis was detected. Effects of glycoside diphosphate-ribose polymerase PARP and cysteine-containing aspartic acid proteolytic enzyme Caspase 3 on protein cleavage. With different concentrations of 0 μM, 0.3 μM, 1 μM, 3 μM, 10 μM (in DMSO) ibrutinib (PCI32765), 1 μM (in DMSO) FLT3 kinase inhibitor AC220, 1 μM BTK kinase inhibitor AVL-292, 1μM (in DMSO) BTK kinase inhibitor CGI1746 treated MOLM-13, MOLM-14, MV-4-11, OCI-AML-3, NOMO-1 respectively, and then after 12 hours, 24 hours, 48 hours Collect cells. 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 at different time periods.

The experimental results are shown in Figure 4: For the acute myeloid leukemia cell line MOLM-13 carrying the FLT3/ITD mutant gene, when the drug concentration of ibrutinib (PCI32765) was 3 μM, it could be seen obviously after 12 hours of action. The cleavage of the DNA repair enzyme polyadenosine diphosphate-ribose polymerase PARP, and the cleavage of the partial cysteine-containing aspartic acid proteolytic enzyme Caspase 3, the same use of 1 μM FLT3 kinase inhibitor AC220 also The same phenomenon can be observed, while using 1 μM BTK kinase inhibitor AVL-292, 1 μM BTK kinase inhibitor CGI1746 can not see any DNA repair enzyme poly ADP-ribose polymerase PARP or cysteine-containing Cleavage of the aspartate proteolytic enzyme Caspase 3. For the acute myeloid leukemia cell MOLM-14 carrying the FLT3/ITD mutant gene, when ibrutinib (PCI32765) was administered at a concentration of 10 μM, after 12 and 24 hours of treatment, obvious DNA repair enzyme polyadenylation could be seen. Cleavage of glycoside diphosphate-ribose polymerase PARP. For acute myeloid leukemia cells MV-4-11 carrying the FLT3/ITD mutant gene, when ibrutinib (PCI32765) was administered at a concentration of 3 μM, obvious DNA repair enzymes could be seen after 12 and 24 hours of treatment respectively Cleavage of polyadenosine diphosphate-ribose polymerase PARP, and partial cleavage of cysteine-containing aspartic acid proteolytic enzyme Caspase 3. However, in the acute myeloid leukemia cells OCI-AML-3 carrying the FLT3A680V mutant gene, even if the concentration of ibrutinib (PCI32765) was 10 μM, no DNA could be seen after 12, 24 and 48 hours of treatment respectively. Cleavage by the repair enzyme polyadenosine diphosphate-ribose polymerase PARP, and by the cysteine-containing aspartic acid proteolytic enzyme Caspase 3. In the acute myeloid leukemia cell NOMO-1 carrying the FLT3 wild-type gene, even if the concentration of ibrutinib (PCI32765) was 10 μM, after 12 and 24 hours of treatment, the DNA repair enzyme polyadenylation could not be seen. The cleavage of the glycoside diphosphate-ribose polymerase PARP, and the cleavage of the cysteine-containing aspartic acid proteolytic enzyme Caspase 3; after 48 hours of action, some DNA repair enzymes polyadenosine diphosphate can be seen - Cleavage by ribose polymerase PARP. Example 4 proves that ibrutinib (PCI32765) can induce apoptosis of acute myeloid leukemia cells carrying FLT3/ITD mutant gene.

Example 5: Effect of ibrutinib (PCI32765) on cell cycle on 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 the FLT3/ITD mutant gene and acute myeloid leukemia cells carrying the FLT3 A680V mutant gene In the leukemia cell line OCI-AML-3, the effect of ibrutinib (PCI32765) on the cell cycle distribution of these cell lines was tested. With different concentrations of 0 μM, 0.5 μM, 1 μM (in DMSO) ibrutinib (PCI32765), 1 μM (in DMSO) of the BTK kinase inhibitor CGI1746 and 0.01 μM (in DMSO) of the FLT3 kinase inhibitor AC220 Act on three strains of acute myeloid leukemia cells MOLM-13, MOLM-14, MV-4-11 carrying the FLT3/ITD mutation gene and an acute random leukemia cell line OCI-AML-3 carrying the FLT3A680V mutation gene, the effect After 24 hours, the cells were collected, washed twice with 1XPBS buffer, fixed with 75% ethanol at -20°C for 24 hours, washed twice with 1XPBS buffer, added with 0.5 mL of 1XPBS buffer and 0.5 mL of PI staining solution (purchased from USA BD Bioscience) into the cells and placed the cells in the dark at 37°C for 15 minutes in the dark, and stained with a flow cytometer (BD FACS Calibur) to detect the cell cycle distribution.

The experimental results are shown in Figure 5: in the acute myeloid leukemia cell line MOLM-14 carrying the FLT3/ITD mutant gene, as the drug concentration of ibrutinib (PCI32765) increased from 0.5 μM to 1 μM, the captured G0 -The cells in G1 phase also increased from 54.47% to 58.13%; the selective and strong FLT3 kinase inhibitor AC220 can capture 80.15% of the cells in G0-G1 phase at 0.01 μM; while 1 μM BTK kinase inhibitor CGI1746 captures The cells in the G0-G1 phase were only comparable to those of the control (only the same amount of DMSO was added without any drug) (Fig. 5a). For the acute myeloid leukemia cell MV-4-11 carrying the FLT3/ITD mutation gene, as the drug concentration of ibrutinib (PCI32765) increased from 0.5 μM to 1 μM, the captured cells in the G0-G1 phase also increased from 64.94% to 71.75%; the selective strong FLT3 kinase inhibitor AC220 can capture G0-G1 phase cells at 0.01 μM as high as 97.29%; while the G0-G1 phase cells captured by 1 μM BTK kinase inhibitor CGI1746 are only as good as the control quite (Fig. 5b). For the acute myeloid leukemia cell MOLM-13 carrying the FLT3/ITD mutant gene, as the drug concentration of ibrutinib (PCI32765) increased from 0.5 μM to 1 μM, the captured cells in the G0-G1 phase also increased from 59.40% to 67.41 %; the selective strong FLT3 kinase inhibitor AC220 can capture 73.53% of the cells in the G0-G1 phase at 0.01 μM; while the cells in the G0-G1 phase captured by the 1 μM BTK kinase inhibitor CGI1746 are only comparable to the control ( Figure 5c). For the acute myeloid leukemia cells OCI-AML-3 carrying the FLT3 A680V mutant gene, the G0-G1 phase cell deficiency captured by ibrutinib (PCI32765) or the BTK kinase inhibitor CGI1746 was comparable to that of the control. For acute myeloid leukemia cells OCI-AML-3 carrying the FLT3 A680V mutant gene, neither ibrutinib (PCI32765) nor the selective FLT3 kinase inhibitor AC220 had any significant effect on the distribution of the cell cycle. This proves from another aspect that ibrutinib (PCI32765) has a significant effect on the acute myeloid leukemia cells carrying the FLT3/ITD mutation gene, and also has a significant effect on the distribution of the cell cycle (Fig. 5d). For acute myeloid leukemia cells NOMO-1 carrying the FLT3 wild-type gene, neither ibrutinib (PCI32765) nor the selective FLT3 kinase inhibitor AC220 had any significant effect on the distribution of the cell cycle. This further proves that ibrutinib (PCI32765) has a significant effect on the acute myeloid leukemia cells carrying the FLT3/ITD mutation gene, and also has a significant effect on the distribution of the cell cycle (Fig. 5e).

Claims (7)

1. preparing for treating the white blood of acute myeloid for carrying FLT3/ITD mutated genes for Buddhist nun (PCI32765) according to Shandong Purposes in the medicine of patient.

2. purposes according to claim 1, wherein be administered alone for Buddhist nun according to Shandong or with one or more other treatment Agent is administered in combination.

3. it is a kind of suppress carry FLT3/ITD mutated genes Acute Myeloid Leukemia Cells Contributing non-treatment method, including by The cell with contact for Buddhist nun according to Shandong.

4. method according to claim 3, wherein the white blood of acute myeloid of the carrying FLT3/ITD mutated genes Sick cell is one or more in MOLM-13, MOLM-14, MV-4-11, PL-21 and MUTZ-11.

5. the method according to claim 3 or 4, wherein the cell and valid density are at least 0.05 μM replacing according to Shandong Buddhist nun contacts.

6. the medicine of the Acute Myeloid Leukemia Cells Contributing of FLT3/ITD mutated genes is carried for suppression in preparation for Buddhist nun according to Shandong Purposes in thing.

7. purposes according to claim 6, wherein the white blood of acute myeloid of the carrying FLT3/ITD mutated genes Sick cell is one or more in MOLM-13, MOLM-14, MV-4-11, PL-21 and MUTZ-11.