WO2016065674A1 - 依鲁替尼对flt3-itd突变的急性白血病的应用 - Google Patents

依鲁替尼对flt3-itd突变的急性白血病的应用 Download PDF

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WO2016065674A1
WO2016065674A1 PCT/CN2014/091375 CN2014091375W WO2016065674A1 WO 2016065674 A1 WO2016065674 A1 WO 2016065674A1 CN 2014091375 W CN2014091375 W CN 2014091375W WO 2016065674 A1 WO2016065674 A1 WO 2016065674A1
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flt3
ibrutinib
acute myeloid
myeloid leukemia
mutant gene
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PCT/CN2014/091375
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French (fr)
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刘青松
刘静
吴宏
胡晨
王傲莉
王文超
陈程
李希祥
赵铮
王黎
王蓓蕾
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中国科学院合肥物质科学研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine

Definitions

  • This invention relates to the field of medicine, and in particular to a new use of Ibrutinib (PCI32765).
  • Acute myelocytic leukemia (AML) or acute non-lymphocytic leukemia (ANLL) includes all non-lymphocytic-derived acute leukemias. It is a type of disease caused by mutations in pluripotent stem cells or karyotypes of slightly differentiated precursor cells, and 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 in developed countries is higher than in developing countries, and Western countries are higher than those in Eastern countries. The annual incidence rate is 2.25/100,000 people around the world. The incidence rate increases with age, and starts to increase obviously at 50 years old.
  • AML is actually a middle-aged and geriatric disease, accounting for 80% to 90% of adult acute leukemia, but only 15% to 20% of children with acute leukemia. At the same time, male incidence is higher than female.
  • FLT3 Fms-like tyrosine kinase 3
  • RTK III receptor tyrosine kinase III
  • RTK III type III receptor tyrosine kinase III
  • JM proximal membrane region
  • TK region Aminokinase (TK) region (SDLyman et al, Oncogene, 1993, 8, 815-822).
  • FLT3 mutations were first discovered in AML cells, and the mutation type was internal tandem repeat (FLT3/ITD).
  • FLT3/ITD internal tandem repeat
  • AML patients with FLT3/ITD activating mutations usually have unique clinical features such as high peripheral blood leukocyte count, poor clinical prognosis, and recurrence, and because the detection method of FLT3 activating mutation is simple and easy, more and more researchers Committed to the development of FLT3 as a routine detection of AML to guide the treatment and prognosis of patients with AML and as a means of detection of minimal residual leukemia, and as a new target for chemotherapy drugs in leukemia patients.
  • FLT3 gene mutation has become a research hotspot, mainly for the development of small molecule tyrosine kinase inhibitors, which inhibits the activity by competing with the FLT3 tyrosine kinase for ATP binding sites.
  • the kinase inhibitors that have entered the clinical inhibition of FLT3 are AC220 and the like.
  • Ibrutinib (also known as PCI32765) is an inhibitor of Bruton's tyrosine kinase (BTK), which can be used alone or in combination with other therapeutic agents.
  • BTK Bruton's tyrosine kinase
  • PCI32765 ibrutinib
  • Ibrutinib (PCI32765), a Bruton's tyrosine kinase inhibitor, is effective for treating acute myeloid leukemia carrying the FLT3/ITD mutant gene.
  • ibrutinib has a strong inhibitory effect on acute myeloid leukemia cells carrying the FLT3/ITD mutant gene such as MOLM-13, MOLM-14, and MV-4-11 (IC50, respectively) It was 0.47 ⁇ M, 0.66 ⁇ M, 0.33 ⁇ M), and ibrutinib had no significant inhibitory effect on acute myeloid leukemia cells carrying the FLT3WT (wild type) and FLT3 A680V mutant genes. Therefore, Ibrutinib can be used for the clinical treatment of acute myeloid leukemia carrying the FLT3/ITD mutant gene.
  • the invention relates to the use of ibrutinib (PCI32765) for the manufacture of a medicament for the treatment of an acute myeloid leukemia patient carrying a FLT3/ITD mutant gene.
  • the drug may be used alone or in combination with one or more other therapeutic agents, as appropriate.
  • the drug comprising ibrutinib can be administered to an acute myeloid leukemia patient carrying the FLT3/ITD mutant gene by at least one of injection, oral, inhalation, rectal and transdermal administration.
  • Other therapeutic agents may be selected from the group consisting of immunosuppressive agents (eg, tacrolimus, cyclosporin, rapamycin, methotrexate, cyclophosphamide, azathioprine, guanidine, mycophenolate mofetil or FTY720), glucocorticoids (eg prednisone, cortisone acetate, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, hydroxyprednisolone, beclomethasone , fludrocortisone acetate, deoxycorticosterone acetate, aldosterone), non-steroidal anti-inflammatory drugs (eg salicylate, aryl alkanoic acid, 2-arylpropionic acid, N-aryl anthranilic acid, Oxicam, oxicam or thioanilide, allergy vaccine, antihistamine, anti-leukotriene, beta-agonist
  • therapeutic agents mentioned may also be rapamycin, crizotinib, tamoxifen, raloxifene, anastrozole, exemestane, letrozole.
  • Herceptin TM (trastuzumab), Gleevec TM (imatinib), taxol TM (paclitaxel), cyclophosphamide, lovastatin, Miele tetracycline (Minosine), cytarabine, 5-fluorouracil (5-FU), methotrexate (MTX), taxotere TM (docetaxel), Zoladex TM (goserelin), vincristine, vinblastine, nocodazole oxazole, teniposide, etoposide, GEMZAR (TM) (gemcitabine), epothilone (epothilone), the promise of this CD, camptothecin, daunorubicin (Daunonibic
  • the other therapeutic agent may also be a cytokine such as G-CSF (granulocyte colony stimulating factor).
  • other therapeutic agents may be, for example but not limited to, CMF (cyclophosphamide, methotrexate and 5-fluorouracil), CAF (cyclophosphamide, doxorubicin and 5-fluorouracil), AC (Asia) Deriamycin and cyclophosphamide), FEC (5-fluorouracil, epirubicin and cyclophosphamide), ACT or ATC (adriamycin, cyclophosphamide and paclitaxel) or CMFP (cyclophosphamide, A Aminopterin, 5-fluorouracil and prednisone).
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising Ibrutinib (PCI 32765) and a pharmaceutically acceptable carrier or adjuvant.
  • the composition may further comprise one or more additional therapeutic agents.
  • additional therapeutic agents mentioned herein are as defined above.
  • the invention relates to a method of treating an acute myeloid leukemia patient carrying a FLT3/ITD mutant gene using ibrutinib.
  • Ibrutinib can be administered to the FLT3/ITD mutant gene by injection, oral, inhalation, rectal or transdermally during treatment.
  • An effective amount of Ibrutinib can also be used alone or in combination with one or more other therapeutic agents, as appropriate. Other therapeutic agents mentioned are as defined above.
  • Chemotherapy with ibrutinib (PCI32765) may also be administered in combination with radiation therapy during the course of treatment.
  • 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.
  • the acute myeloid leukemia cell carrying the FLT3/ITD mutant gene is preferably one or more selected from the group consisting of MOLM-13, MOLM-14, MV-4-11, PL-21 and MUTZ-11.
  • the cells are contacted with Ibrutinib at an effective concentration of at least 0.05 ⁇ M, more preferably the amount of Ibrutinib is at least 0.1 ⁇ M, further preferably 0.3-10 ⁇ M, such as 0.3-3 ⁇ M.
  • Figure 1 shows the effect of PCI32765 on BTK signaling pathways on MOLM-14 (1a) and OCI-AML-3 (1b) cells, respectively.
  • Figure 2 shows the effect 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 in cells that are relatively closely related to FLT3/ITD.
  • Figure 4 shows apoptosis of PCI32765 for MOLM-13 (4a), MOLM-14 (4b), MV-4-11 (4c), OCI-AML-3 (4d) and NOMO-1 (4e), respectively. influences.
  • Figure 5 shows the cell cycle distribution of PCI32765 for MOLM-14 (5a), MV-4-11 (5b), MOLM-13 (5c), OCI-AML-3 (5d) and NOMO-1 (5e), respectively. influences.
  • Ibrutinib (also known as PCI32765) is a Bruton tyrosine Inhibitor of kinase (Bruton's tyrosine kinase, BTK). As a small molecule BTK inhibitor, it can covalently bind to the cysteine residue of the BTK active center, thereby inhibiting its activity. Ibrutinib has the structure shown by the following formula (I):
  • FLT3 (Fms-like tyrosine kinase 3) is FMS-like tyrosine kinase 3.
  • the FLT3/ITD mutant gene or the FLT3/ITD mutant gene refers to the FLT3 internal tandem duplication (FLT3/ITD) mutation, which is the highest incidence and prognosis in acute myeloid leukaemia (AML). Related mutations.
  • administering or “administering” as used herein includes a route to introduce a compound into a subject to achieve its intended function and effect.
  • administration route which can be used include injection (subcutaneous injection, intravenous injection, parenteral injection, intraperitoneal injection, intrathecal injection), oral administration, inhalation, rectal and transdermal, and the like.
  • the pharmaceutical preparation can be administered in a form suitable for various administration routes.
  • 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 reasonable benefits. / risk ratio commensurate components.
  • an effective amount includes an amount effective to achieve a desired result (eg, sufficient to treat a disease or condition described herein) in terms of dosage and necessary time period.
  • An effective amount of a compound of the invention may vary depending on, for example, the subject's disease state, age, and body weight, and the ability of the compound to elicit a desired response in the cell or in the subject.
  • the dosage regimen can be adjusted to provide the optimal therapeutic response.
  • the amount of a given drug depends on a number of factors.
  • the condition, as well as the subject or host being treated, can be routinely determined by methods known in the art.
  • the dosage administered will typically range from 0.02 to 5000 mg/day, for example from about 1 to 1500 mg per day, for dosages used in adult treatment.
  • the desired dose may conveniently be presented as a single dose, or concurrently (or in a short period of time) or in divided doses at appropriate intervals, such as two, three, four or more divided doses per day. It will be understood by those skilled in the art that, although the above dosage ranges are given, the effective amount of Ibrutinib can be suitably adjusted depending on the condition of the patient and in connection with the physician's diagnosis.
  • IC50 is also referred to as the half-inhibitory concentration, which refers to the amount, concentration, or dose of a particular inhibitor at which 50% inhibition of maximum effect (eg, inhibition of BTK kinase activity) is achieved in an assay that measures an effect.
  • EC50 refers to determining the dose, concentration, or amount of a compound that results in a dose-dependent response to 50% of the maximum expression of a particular reaction that induces, stimulates, or potentiates a particular assay compound.
  • Ibrutinib is administered according to the invention to treat acute myeloid leukemia carrying a FLT3/ITD mutant gene.
  • Treatment can include a single treatment or a series of treatments.
  • a dose of Ibrutinib can be administered to the patient daily, every other day, or weekly 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 years. It will be appreciated that the dose of ibrutinib administered to the patient may be suitably increased or decreased during the treatment depending on the condition of the patient and the need for treatment.
  • the subject can be administered ibrutinib and one or more additional therapeutic agents simultaneously or sequentially.
  • a pharmaceutical composition can be administered to a subject, the composition being formulated to comprise ibrutinib and a pharmaceutically acceptable carrier or adjuvant, and optionally a Or a variety of other therapeutic agents.
  • Ibrutinib for the treatment of acute myeloid leukemia carrying the FLT3/ITD mutant gene can be formulated into a suitable pharmaceutical formulation for oral administration (for example, in a liquid form dissolved in a solvent such as aqueous or non-aqueous Liquid, or in a solid carrier), rectal, parenteral, intracisternal, intraperitoneal, topical (through powder) Agents, ointments, lotions, gels, drops, transdermal patches or transdermal patches), buccal administration, transbronchial form or as an oral spray or nasal spray.
  • a suitable pharmaceutical formulation for oral administration for example, in a liquid form dissolved in a solvent such as aqueous or non-aqueous Liquid, or in a solid carrier
  • parenteral intracisternal
  • intraperitoneal intraperitoneal
  • buccal administration transbron
  • ibrutinib can be formulated, for example, as a solution, suspension, tablet, dispersible tablet, pill, capsule, powder, sustained release preparation or elixir for oral administration.
  • ibrutinib can also be formulated into a suitable injection.
  • Administration can be by single dose injection or infusion, multiple doses, or in a continuous dosage form daily, weekly, monthly, every month, quarterly, or by any other administration schedule.
  • Administration of the formulations of the invention may be intermittent to the subject, or at a progressive, continuous, constant or controlled rate.
  • the time of administration of the dosage form during the day and the number of times the dosage form is administered daily may vary.
  • the Ibrutinib formulation administered to the patient may range from 0.02 to 5000 mg/day, such as from about 1 to 1500 mg/day.
  • the desired dose may conveniently be presented as a single dose, or concurrently (or in a short period of time) or in divided doses at appropriate intervals, such as two, three, four or more divided doses per day.
  • the pharmaceutical composition comprising Ibrutinib for treating acute myeloid leukemia carrying the FLT3/ITD mutant gene can be formulated into a solution, a suspension, a tablet, a dispersible tablet, a pill, a capsule, a powder, and a sustained release. Formulations such as preparations or elixirs.
  • the composition may contain from 0.02 to 5000 mg of Ibrutinib, but the effective dose of the active ingredient employed may vary depending on the particular dosage regimen employed, the route of administration, and the severity of the condition being treated. The skilled artisan will appreciate that the effective dosage for each patient will vary depending on the severity of the disease, the genetic variation of the individual, or the rate of metabolism.
  • the desired results are generally obtained when the compound of the present invention is administered in a daily dose of about 0.5 to 1000 mg, optionally in 2-4 divided doses a day or in a sustained release form.
  • the total daily dose is planned to be about 1-1000 mg, preferably about 2-500 mg.
  • AVL-292 is a highly selective BTK inhibitor with covalent attachment with an IC50 of less than 0.5 nM (Robak T, Robak E, "Tyrosine kinase inhibitors as potential drugs for B-cell lymphoid malignancies and autoimmune disorders", Expert. Opin. Investig. Drugs, 2012, 21(7): 921-947).
  • CGI1746 is new A highly selective small molecule BTK kinase inhibitor with an IC50 of 1.9 nM (Di Paolo JA, et al., "Specific Btk inhibition suppresses B cell-and myeloid cell-mediated arthritis", Nat. Chem.
  • AC220 is a FLT3 kinase inhibitor that has entered the clinic.
  • Ibrutinib PCI32765
  • AVL292 AVL292, CGI1746 and AC220 were purchased from Hao Yuan Chemexpress (Shanghai).
  • the selectivity of Ibrutinib (PCI32765) for inhibiting cancer cell proliferation was further evaluated by testing the effect of Ibrutinib (PCI32765) on cancer cell growth.
  • acute myeloid leukemia cells MOLM-16 (expressing wild-type FLT3 gene), acute myeloid leukemia cell HL-60 (expressing wild-type FLT3 gene), and acute myeloid leukemia cell KASUMI-1 (expressing wild type) 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 marrow 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 (expressing FLT3/ITD mutant gene and wild-type FLT3 gene), human acute monocytic leukemia cell line MV-4-11 (expressing FLT3/ITD mutant gene), human acute myeloid leukemia cell line NOMO -1 (expressing wild-type FLT3 gene), MDS-RAEB (myelodysplastic syndrome-primordial cell type) cell line SKM-1 (expressing wild-type FLT3 gene), human acute myeloid leukemia cell line U-937 (expression Wild type FL
  • Ibrutinib (PCI32765) was added to different concentrations (0.000508 ⁇ M, 0.00152 ⁇ M, 0.00457 ⁇ M, 0.0137 ⁇ M, 0.0411 ⁇ M, 0.123 ⁇ M, 0.370 ⁇ M, 1.11 ⁇ M, 3.33 ⁇ M, 10 ⁇ M in DMSO).
  • PCI32765 PCI32765
  • the IC50 of the half-inhibitory concentration of ibrutinib on the cell lines MOLM-13, MOLM-14 and MV-4-11 is 0.47 ⁇ M, 0.66 ⁇ M, 0.33 ⁇ M, respectively.
  • the proliferation of other cancer cells was not inhibited (the IC50 values of the half inhibitory concentrations were all greater than 2 ⁇ M).
  • the results of Example 1 support the selectivity of Ibrutinib (PCI32765) for acute myeloid white blood treatment with the FLT3/ITD mutant gene.
  • Example 2 Ibrutinib (PCI32765) transmits BTK upstream and downstream signals in cells Road influence
  • Ibrutinib (PCI32765), 1 ⁇ M (in DMSO), AVL-292, and 1 ⁇ M (in DMSO) at different concentrations of 0 ⁇ M, 0.3125 ⁇ M, 0.625 ⁇ M, 1.25 ⁇ M, 2.5 ⁇ M, 5 ⁇ M, 10 ⁇ M (in DMSO)
  • CGI1746 treated acute myeloid leukemia cells 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.
  • the samples were collected by stimulation with human immunoglobulin IgM for 10 min.
  • ibrutinib 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 the protein kinase BTK.
  • Example 3 Ibrutinib (PCI32765) in the cells upstream and downstream of FLT3 signal Pathway impact
  • Iruti was tested in three cells, the 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 endpoints and functional endpoints. Effects of Ni (PCI32765) on phosphorylation of FLT3/ITD mutant protein kinases in cells and its closely related signaling pathway downstream of STAT5 protein phosphorylation (Fig. 3), other related protein kinases ERK, AKT phosphorylation At the same time, we also examined the effects of the protein C-Myc and the transcription factor NF- ⁇ B subunit p65 and its phosphorylation.
  • Ibrutinib (PCI32765), 0.1 ⁇ M (in DMSO), FLT3 kinase inhibitor AC220, 1 ⁇ M (in DMSO) at different concentrations of 0 ⁇ M, 0.03 ⁇ M, 0.1 ⁇ M, 0.3 ⁇ M, 1 ⁇ M (in DMSO) BTK kinase inhibitor AVL-292, 1 ⁇ M (in DMSO) BTK kinase inhibitor CGI1746 treated MOLM-13, MOLM-14, MV-4-11 three acute myeloid leukemia cells carrying the FLT3/ITD mutant gene The cells were collected for 4 hours while being starved for 4 hours with medium containing only 1% FBS (calf serum). The effects of the compounds on STAT5, C-Myc, ERK, NF- ⁇ B p65, AKT protein and phosphorylation in these three cells were determined (Fig. 2).
  • Ibrutinib (PCI32765) is a downstream protein of FLT3/ITD in cells.
  • the phosphorylation of STAT5 has a strong inhibitory effect, and its EC50 is 0.32 ⁇ M, 0.11 ⁇ M, and 0.91 ⁇ M, respectively, and has a significant degradation effect on the protein C-Myc, which is closely related to FLT3 protein kinase.
  • control BTK kinase inhibitors AVL-292 and CGI1746 did not inhibit the phosphorylation of protein kinase FLT3 at a concentration of 1 ⁇ M.
  • CGI1746 had no effect on the FLT3/ITD downstream protein STAT5 and protein C-Myc in the cells.
  • the control FLT3/ITD kinase inhibitor AC220 strongly inhibited the phosphorylation of the protein kinase FLT3 and the protein STAT5, which is closely related to FLT3/ITD, and the degradation protein C-Myc.
  • Example 3 demonstrates that the BTK kinase inhibitor ibrutinib (PCI32765) inhibits phosphorylation of the protein kinase FLT3, affects phosphorylation of the protein STAT5 downstream of the protein kinase FLT3 signaling pathway, and thereby inhibits the expression of the FLT3/ITD mutant gene.
  • PCI32765 BTK kinase inhibitor
  • STAT5 downstream of the protein kinase FLT3 signaling pathway
  • Ibrutinib (PCI32765), 1 ⁇ M (in DMSO), FLT3 kinase inhibitor AC220, 1 ⁇ M BTK kinase inhibitor AVL-292, at different concentrations of 0 ⁇ M, 0.3 ⁇ M, 1 ⁇ M, 3 ⁇ M, 10 ⁇ M (in DMSO), 1 ⁇ M (in DMSO) of BTK kinase inhibitor CGI1746 treated MOLM-13, MOLM-14, MV-4-11, OCI-AML-3, NOMO-1, respectively, then 12 hours, 24 hours, 48 hours later Collect cells.
  • Western Blot was used to detect the effects of different concentrations of the drug on the DNA repair enzyme polyadenylation diphosphate-ribose polymerase PARP and cysteine-containing aspartate proteolytic enzyme Caspase 3 at different time intervals.
  • the cells were collected, washed twice with 1X PBS buffer, fixed with 75% ethanol at -20 °C for 24 hours, washed twice with 1X PBS buffer, and added with 0.5 mL of 1X PBS buffer and 0.5 mL of PI staining solution. From the BD Bioscience of the United States to the cells and the cells were stained in the dark at 37 ° C for 15 minutes, and the cell cycle distribution was measured by flow cytometry (BD FACS Calibur).
  • the cell concentration of the captured G0-G1 phase increased from 64.94% as the drug concentration of Ibrutinib (PCI32765) increased from 0.5 ⁇ M to 1 ⁇ M. Up to 71.75%; the highly selective FLT3 kinase inhibitor AC220 can capture up to 97.29% of cells in G0-G1 phase at 0.01 ⁇ M; whereas the G0-G1 phase of cells captured by 1 ⁇ M BTK kinase inhibitor CGI1746 only and control Quite ( Figure 5b).
  • the cell deficiency of the G0-G1 phase captured by either ibrutinib (PCI32765) or the BTK kinase inhibitor CGI1746 was comparable to the control.
  • ibrutinib (PCI32765) or the selective FLT3 kinase inhibitor AC220 had a significant effect on cell cycle distribution.
  • ibrutinib (PCI32765) has a significant effect on acute myeloid leukemia cells carrying the FLT3/ITD mutant gene and also has a significant effect on cell cycle distribution (Fig. 5d).
  • ibrutinib (PCI32765)
  • the selective FLT3 kinase inhibitor AC220 had a significant effect on cell cycle distribution.
  • ibrutinib (PCI32765) has a significant effect on acute myeloid leukemia cells carrying the FLT3/ITD mutant gene and also has a significant effect on cell cycle distribution (Fig. 5e).

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Abstract

公开了依鲁替尼（PCI32765）的一种新用途，具体地发现依鲁替尼可以用于治疗急性髓细胞白血病，特别是携带FLT3/ITD突变型基因的急性髓细胞白血病。

Description

依鲁替尼对FLT3-ITD突变的急性白血病的应用 技术领域
本发明涉及医药领域，特别是涉及依鲁替尼(PCI32765)的一种新用途。
背景技术
急性髓细胞白血病(acute myelocytic leukemia，AML)或急性非淋巴细胞白血病(ANLL)包括所有非淋巴细胞来源的急性白血病。它是多能干细胞或已轻度分化的前体细胞核型发生突变所形成的一类疾病，是造血系统的克隆性恶性疾病。流行病学调查显示，环境、职业及遗传因素与AML的发病关系密切。发达国家的发病率高于发展中国家，西方国家高于东方国家。世界各地年发病率为2.25/10万人口，随年龄增加而发病率增高，50岁开始明显上升，60～69岁达高峰，30岁以下为1/10万、75岁以上则高达17/10万。因此，AML实际是一种中、老年病，占成人急性白血病的80％～90％，但仅占儿童急性白血病的15％～20％。同时，男性发病高于女性。
FLT3(Fms-like tyrosine kinase 3)即FMS样酪氨酸激酶3，与c-Kit、c-FMS和PDGFR同属于III型受体酪氨酸激酶(receptor tyrosine kinase III,RTK III)家族成员，其蛋白结构包括5个免疫球蛋白(Ig)样结构域组成的保外区，1个跨膜区，1个近膜区(JM)，以及胞内由激酶插入区分隔而成的2个酪氨酸激酶(TK)区(S.D.Lyman等,Oncogene,1993,8,815-822)。1996年首先在AML细胞中发现了FLT3突变，其突变类型是内部串联重复(FLT3/ITD)。近年来，许多研究已经证实FLT3的激活突变在AML的发生及疾病的进展中起到十分重要的病理作用。具有FLT3/ITD激活突变的AML患者通常具有外周血白细胞计数高，临床预后较差，易复发等独特的临床特征，并且由于FLT3激活突变的检测方法简单易行，故越来越多的研究者致力于将FLT3发展成为AML的常规检测手段用来指导AML患者的治疗和预后的判断以及作为微小残留白血病的检测手段，并将其作为白血病患者化疗药物的又一新的靶点。
现已证实FLT3的激活突变主要有两种：内部串联重复 (internal tandem duplication,ITD)和活化环中的点突变(point mutation in the activation loop,Tkd点突变)。FLT3的这两种激活突变均能引起FLT3发生自动磷酸化进而导致FLT3发生配体非依赖性的组成性激活，进一步激活其下游异常的信号转导，从而起到促进增殖和抑制凋亡的作用，使得具有此突变表型的白血病患者临床预后较差。
目前对FLT3基因突变的靶向抑制成为研究热点，主要为开发小分子酪氨酸激酶抑制剂，通过与FLT3酪氨酸激酶竞争ATP结合位点而抑制其活性。目前已经进入临床的抑制FLT3的激酶抑制剂有AC220等。
依鲁替尼(Ibrutinib，也称为PCI32765)是一种布鲁顿酪氨酸激酶(Bruton's tyrosine kinase，BTK)的抑制剂，其可以以单独使用或者与其它治疗剂联合使用的方式用于治疗自身免疫性疾病或病症、异种免疫性疾病或病症、癌症包括淋巴瘤以及炎性疾病或病症。目前，还未见关于使用依鲁替尼(PCI32765)治疗携带FLT3/ITD突变型基因的急性髓细胞白血病的相关报道。
发明内容
鉴于前述的技术问题，发明人进行了广泛的研究。结果，发明人意外地发现，依鲁替尼(PCI32765)这种布鲁顿酪氨酸激酶抑制剂能有效地治疗携带FLT3/ITD突变型基因的急性髓细胞白血病。更具体地，发明人发现，依鲁替尼对携带FLT3/ITD突变型基因的急性髓细胞白血病细胞如MOLM-13、MOLM-14、MV-4-11起到较强的抑制作用(IC50分别为0.47μM、0.66μM、0.33μM)，而依鲁替尼对携带FLT3WT(野生型)及FLT3 A680V突变型基因的急性髓细胞白血病细胞都没有明显的抑制作用。因此依鲁替尼可适用于临床治疗携带FLT3/ITD突变型基因的急性髓细胞白血病。
一方面，本发明涉及依鲁替尼(PCI32765)在制备用于治疗携带FLT3/ITD突变型基因的急性髓细胞白血病癌患者的药物中的用途。
在治疗过程中，该药物可以根据情况单独或与一种或多种其它的治疗剂组合使用。可以通过注射、口服、吸入、直肠和经皮施用中的 至少一种将包含依鲁替尼的药物施用给携带FLT3/ITD突变基因的急性髓细胞白血病患者。其它的治疗剂可以选自以下药物：免疫抑制剂(例如他克莫司、环抱菌素、雷帕霉素、甲氨蝶岭、环磷酰胺、硫唑嘌呤、巯嘌呤、麦考酚酯或FTY720)、糖皮质激素类药(例如泼尼松、醋酸可的松、泼尼松龙、甲泼尼龙、地塞米松、倍他米松、曲安西龙、氢羟强的松龙、倍氯米松、醋酸氟氢可的松、醋酸脱氧皮质酮、醛固酮)、非甾体抗炎药(例如水杨酸盐、芳基烷酸、2-芳基丙酸、N-芳基邻氨基苯甲酸、昔康类、考昔类或硫酰替苯胺)、变态反应疫苗、抗组胺药、抗白三烯药、β-激动剂、茶碱、抗胆碱药或其它选择性激酶抑制剂(例如mTOR抑制剂、c-Met抑制剂)或her2抗体-药物。另外，所提及的其它治疗剂还可以是雷帕霉素(Rapamycin)、克唑替尼(Crizotinib)、他莫昔芬、雷洛昔芬、阿那曲唑、依西美坦、来曲唑、赫赛汀TM(曲妥珠单抗)、格列卫TM(伊马替尼)、紫杉醇TM(紫杉醇)、环磷酰胺、洛伐他汀、美诺四环素(Minosine)、阿糖胞苷、5-氟尿嘧啶(5-FU)、甲氨蝶呤(MTX)、紫杉特尔TM(多西他赛)、诺雷德TM(戈舍瑞林)、长春新碱、长春碱、诺考达唑、替尼泊苷、依托泊苷、健择TM(吉西他滨)、埃博霉素(Epothilone)、诺唯本、喜树碱、柔红霉素(Daunonibicin)、更生霉素、米托蒽醌、安吖啶、多柔比星(亚德里亚霉素)、表柔比星或伊达比星。或者，其它治疗剂也可以是细胞因子例如G-CSF(粒细胞集落刺激因子)。或者，其它治疗剂也可以是，例如但不限于，CMF(环磷酰胺、甲氨蝶呤和5-氟尿嘧啶)、CAF(环磷酰胺、亚德里亚霉素和5-氟尿嘧啶)、AC(亚德里亚霉素和环磷酰胺)、FEC(5-氟尿嘧啶、表柔比星和环磷酰胺)、ACT或ATC(亚德里亚霉素、环磷酰胺和紫杉醇)或CMFP(环磷酰胺、甲氨蝶呤、5-氟尿嘧啶和泼尼松)。
在另一方面，本发明还涉及一种药物组合物，其包含依鲁替尼(PCI32765)和药学上可接受的载体或助剂。该组合物还可以进一步包含一种或多种其它的治疗剂。这里提及的其它治疗剂如上文所限定。
在又一方面，本发明涉及使用依鲁替尼治疗携带FLT3/ITD突变型基因的急性髓细胞白血病患者的方法。在治疗过程中，可以通过注射、口服、吸入、直肠或经皮将依鲁替尼施用给携带FLT3/ITD突变型基因 的急性髓细胞白血病患者。还可以根据情况将有效量的依鲁替尼单独或与一种或多种其它的治疗剂组合使用。所提及的其它的治疗剂如上文所限定。在治疗过程中，还可以将使用依鲁替尼(PCI32765)的化学疗法联合放射疗法进行施用。
在又一方面，本发明涉及一种抑制携带FLT3/ITD突变型基因的急性髓细胞白血病细胞的方法，包括将所述细胞与依鲁替尼相接触。根据本发明，携带FLT3/ITD突变型基因的急性髓细胞白血病细胞优选是选自MOLM-13、MOLM-14、MV-4-11、PL-21和MUTZ-11中的一种或多种。进一步优选地，将所述细胞与有效浓度至少为0.05μM的依鲁替尼相接触，更优选依鲁替尼的量为至少0.1μM，进一步优选0.3-10μM，例如0.3-3μM。
附图说明
图1示出PCI32765分别在MOLM-14(1a)和OCI-AML-3(1b)细胞上对BTK信号通路的影响。
图2示出PCI32765分别在MOLM-13(2a)、MOLM-14(2b)和MV-4-11(2c)细胞上对FLT3/ITD相对密切相关的蛋白及相关信号通路的影响。
图3示出细胞中与FLT3/ITD相对密切相关的蛋白。
图4示出PCI32765分别对MOLM-13(4a)、MOLM-14(4b)、MV-4-11(4c)、OCI-AML-3(4d)和NOMO-1(4e)的细胞凋亡的影响。
图5示出PCI32765分别对MOLM-14(5a)、MV-4-11(5b)、MOLM-13(5c)、OCI-AML-3(5d)和NOMO-1(5e)的细胞周期分布的影响。
具体实施方式
在对本发明进一步说明之前，为了更好地理解本发明，对一些术语进行说明。
定义
“依鲁替尼(Ibrutinib，也称为PCI32765)”是一种布鲁顿酪氨酸 激酶(Bruton's tyrosine kinase，BTK)的抑制剂。作为一种小分子BTK抑制剂，它能够与BTK活性中心的半胱氨酸残基共价结合，从而抑制其活性。依鲁替尼具有以下式(I)所示的结构：
Figure PCTCN2014091375-appb-000001
FLT3(Fms-like tyrosine kinase 3)即FMS样酪氨酸激酶3。FLT3/ITD突变基因或FLT3/ITD突变型基因是指跨膜区内部串联重复(FLT3internal tandem duplication，FLT3/ITD)突变，是急性髓系白血病(acute myeloid leukaemia，AML)中发生率最高且与预后相关的突变。
本文使用的术语“给药”或“施用”包括将化合物引入受试者中以实现其预定功能和作用的途径。可以使用的给药途径的例子包括注射(皮下注射、静脉注射、肠胃外注射、腹膜内注射、鞘内注射)、口服、吸入、直肠和经皮等。可以通过适于各种给药途径的形式来施用药物制剂。
本文使用的术语“药学可接受的”是指，在合理的医学判断的范围内，适合用于与人和其他哺乳动物的组织接触而没有过度毒性、刺激、过敏反应等，并且与合理的利益/风险比相称的组分。
本文使用的术语“有效量”包括就剂量和必要的时间周期而言，有效达到所期望的结果(例如，足以治疗本文中描述的疾病或病症)的量。本发明化合物的有效量可以根据例如如下的因素而不同：受试者的疾病状态、年龄、和体重、及化合物在细胞中或在受试者中引起期望的响应的能力。可以调节给药方案以提供最佳的治疗反应。
在本发明的实施方式中，在根据本发明对患有携带FLT3/ITD突变型基因的急性髓细胞白血病的受试者施用依鲁替尼进行治疗时，给定药物的量取决于诸多因素，如具体的给药方案、疾病或病症类型及其严重性、需要治疗的受治疗者或宿主的独特性(例如体重)，但是，根据特定的周围情况，包括例如已采用的具体药物、给药途径、治疗的 病症、以及治疗的受治疗者或宿主，施用剂量可由本领域已知的方法常规决定。通常，就成人治疗使用的剂量而言，施用剂量典型地在0.02-5000mg/天，例如约1-1500mg/天的范围。该所需剂量可以方便地被表现为一剂、或同时给药的(或在短时间内)或在适当的间隔的分剂量，例如每天二、三、四剂或更多分剂。本领域技术人员可以理解的是，尽管给出了上述剂量范围，但依鲁替尼的有效量可根据患者的情况并结合医师诊断而适当调节。
本文使用的“IC50”又称半数抑制浓度，其是指在测量某种效应的分析中获得最大效应的50％抑制(例如对BTK激酶活性的抑制)时特定抑制剂的量、浓度或剂量。
本文使用的“EC50”是指测定化合物的剂量、浓度或量，其引起特定测定化合物诱导、刺激或加强的特定反应的50％的最大表达的剂量依赖反应。
本发明的应用
在本发明的一些实施方式中，根据本发明施用依鲁替尼来治疗携带FLT3/ITD突变型基因的急性髓细胞白血病。治疗可以包括单一治疗，也可以包括系列治疗。
在本发明的一些实施方式中，可以对患者每天、隔日或每周施用一定剂量的依鲁替尼，并持续1个月、2个月、3个月、4个月、5个月、6个月、7个月、8个月、9个月、10个月、11个月、1年或数年。可以理解的是，对患者施用的依鲁替尼的剂量可以在治疗期间根据患者情况和治疗需求而适当增加或降低。
在本发明的一些实施方式中，可以同时或顺序地对受试者施用依鲁替尼和一种或多种其它的治疗剂。或者，在本发明的一些实施方式中，可以对受试者施用一种药物组合物，该组合物配制为包含依鲁替尼和药学上可接受的载体或助剂，以及任选的一种或多种其它的治疗剂。
用于治疗携带FLT3/ITD突变型基因的急性髓细胞白血病的依鲁替尼可以被配制成合适的药物制剂，以便用于口服给药(例如，以溶于溶剂的液体形式比如含水或不含水的液体、或在固体载体内)、经直肠给药、肠胃外给药、脑池内给药、腹膜内给药、局部给药(通过粉 剂、软膏剂、洗剂、凝胶剂、滴剂、透皮贴剂或经皮贴剂)、口腔给药、经支气管形式或作为口腔喷剂或鼻喷剂等。具体而言，依鲁替尼可以被配制成，例如，用于口服给药的溶液、混悬剂、片剂、分散片、丸剂、胶囊剂、粉剂、缓释制剂或酏剂等。在注射给药的实施方式中，依鲁替尼也可以配制成合适的注射剂。
可以每日、每周、每月、隔月、每季度或按任何其他给药日程以单剂量注射或输注、多剂量、或以连续剂型进行给药。本发明制剂的给药可以对受试者是间歇性的，或者处于渐进、连续、恒定或受控的速度。此外，一天中施用剂型的时间和每日施用剂型的次数可以不同。在本发明的一些实施方式中，施用给患者的依鲁替尼制剂可以在0.02-5000mg/天，例如约1-1500mg/天的范围。该所需剂量可以方便地被表现为一剂、或同时给药的(或在短时间内)或在适当的间隔的分剂量，例如每天二、三、四剂或更多分剂。
用于治疗携带FLT3/ITD突变型基因的急性髓细胞白血病的包含依鲁替尼的药物组合物可以被配制成溶液、混悬剂、片剂、分散片、丸剂、胶囊剂、粉剂、缓释制剂或酏剂等剂型。该组合物可以含有0.02-5000mg的依鲁替尼，但所使用活性成分的有效剂量可以根据具体使用的给药方案、给药途径、和被治疗的病症的严重程度不同而改变。技术人员可以理解，用于各个患者的有效剂量可依疾病严重程度、个体遗传变异或代谢速率不同而变化。然而，通常以约0.5-1000mg的每天剂量，任选一天2-4次分剂量或以缓释形式给予本发明化合物时，得到期望的结果。计划每天总剂量约1-1000mg，可优选约2-500mg。
下面将通过实施例并结合附图来说明本发明。应理解，示出的实施例和附图仅用于帮助理解本发明，但不构成对本发明的限制。
实施例
实验材料：在实施例中使用AVL-292、CGI1746和AC220作为对照。AVL-292是通过共价结合的具有高度选择性的BTK抑制剂，其IC50小于0.5nM(Robak T,Robak E,“Tyrosine kinase inhibitors as potential drugs for B-cell lymphoid malignancies and autoimmune disorders”,Expert.Opin.Investig.Drugs,2012,21(7):921-947)。CGI1746是新型的 高度选择性的小分子BTK激酶抑制剂，其IC50为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是目前已经进入临床的FLT3激酶抑制剂。依鲁替尼(PCI32765)及AVL292、CGI1746和AC220均购自Hao Yuan Chemexpress公司(上海)。
实施例1：依鲁替尼(PCI32765)对癌细胞增殖的影响
通过测试依鲁替尼(PCI32765)对癌细胞生长的影响，进一步评估依鲁替尼(PCI32765)抑制癌细胞增殖的选择性。本实施例中选用了急性髓性白血病细胞MOLM-16(表达野生型FLT3基因)、急性髓性白血病细胞HL-60(表达野生型FLT3基因)、急性髓性白血病细胞KASUMI-1(表达野生型FLT3基因)、急性早幼粒细胞白血病细胞株NB-4(Lu+)(表达野生型FLT3基因)、人急性髓性白血病细胞株OCI-AML-2(表达FLT3 A680V突变型基因)、人急性髓性白血病细胞株OCI-AML-3(表达FLT3 A680V突变型基因)、人急性髓性白血病细胞株MOLM-14(表达FLT3/ITD突变型基因及野生型FLT3基因)、人急性髓性白血病细胞株MOLM-13(表达FLT3/ITD突变型基因及野生型FLT3基因)、人急性单核细胞性白血病细胞株MV-4-11(表达FLT3/ITD突变型基因)、人急性髓性白血病细胞株NOMO-1(表达野生型FLT3基因)、MDS-RAEB(骨髓增生异常综合征-原始细胞增多型)细胞株SKM-1(表达野生型FLT3基因)、人急性髓性白血病细胞株U-937(表达野生型FLT3基因)、人急性红白细胞白血病细胞株HEL(表达野生型FLT3基因)、人急性巨核性细胞白血病CMK(表达野生型FLT3基因)、人EB病毒感染的人外周淋巴细胞JVM-2、人Burkitt's淋巴瘤细胞Namalwa、人套细胞淋巴瘤(mantel cell lymphoma，MCL)细胞REC-1、人套细胞淋巴瘤(mantel cell lymphoma，MCL)细胞Z-138、人弥漫性组织淋巴瘤细胞SU-DHL-2、人弥漫性大B细胞淋巴瘤细胞系TMD8、人B细胞慢性淋巴细胞白血病细胞株MEC-1、人B细胞慢性淋巴细胞白血病细胞株MEC-2、人B-细胞性白血病细胞株NALM-6、人急性T淋巴细胞白血病细胞株JURKAT和血癌细胞株K562。以上细胞均购自ATCC。
在实施例中将不同浓度(0.000508μM、0.00152μM、0.00457μM、 0.0137μM、0.0411μM、0.123μM、0.370μM、1.11μM、3.33μM、10μM于DMSO中)的依鲁替尼(PCI32765)加入到上述细胞中，并孵育72小时，用Cell
Figure PCTCN2014091375-appb-000002
(Promega，美国)化学自发光法细胞活力检测试剂盒，通过对活细胞中的ATP进行定量测定来检测活细胞数目。
实验结果如表1所示，发现依鲁替尼(PCI32765)仅对表达FLT3/ITD突变型基因的人急性髓性白血病细胞株MOLM-14(表达FLT3/ITD突变型基因及野生型FLT3基因)、人急性髓性白血病细胞株MOLM-13(表达FLT3/ITD突变型基因及野生型FLT3基因)、人急性单核细胞性白血病细胞株MV-4-11(表达FLT3/ITD突变型基因)这三株癌细胞的增殖具有强烈的抑制作用，依鲁替尼对细胞株MOLM-13、MOLM-14及MV-4-11的半数抑制浓度IC50分别为0.47μM、0.66μM、0.33μM，而对其它的癌细胞的增殖均无抑制作用(半数抑制浓度IC50值均大于2μM)。实施例1的结果支持了依鲁替尼(PCI32765)对于携带FLT3/ITD突变型基因的急性髓细胞白血治疗的选择性。
表1.PCI32765在癌细胞上的测定数据
细胞系 细胞类型 FLT3类型 PCI32765IC50(μM)
MOLM-16 AML-M0 FLT3 WT 4.4
HL-60 AML-M2 FLT3 WT 2.1
KASUMI-1 AML-M2 FLT3 WT 3.0
NB-4 AML-M3 FLT3 WT 7.5
OCI-AML-2 AML-M4 FLT3 A680V >10
OCI-AML-3 AML-M4 FLT3 A680V >10
MOLM-13 AML-M5a FLT3/ITD,FLT3 WT 0.47
MOLM-14 AML-M5a FLT3/ITD,FLT3 WT 0.66
MV-4-11 AML-M5 FLT3/ITD 0.33
NOMO-1 AML-M5 FLT3 WT >10
SKM-1 AML-M5 FLT3 WT 8.6
U-937 AML-M5 FLT3 WT 8.5
HEL AML-M6 FLT3 WT >10
CMK AML-M7 FLT3 WT >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
实施例2：依鲁替尼(PCI32765)在细胞中对BTK上下游信号通 路的影响
在急性髓性白血病细胞MOLM-14(表达FLT3/ITD突变型基因及野生型FLT3基因)以及急性髓性白血病细胞OCI-AML-3(表达FLT3A680V突变型基因)这两株细胞上，通过测定许多细胞生物化学终点和功能性终点，评估了依鲁替尼(PCI32765)对细胞中BTK激酶以及与BTK激酶密切相关的蛋白激酶PLCγ、AKT、ErK、GSK3β的影响。用不同浓度0μM、0.3125μM、0.625μM、1.25μM、2.5μM、5μM、10μM(于DMSO中)的依鲁替尼(PCI32765)，1μM(于DMSO中)的AVL-292，以及1μM(于DMSO中)的CGI1746分别处理急性髓性白血病细胞MOLM-14(表达FLT3/ITD突变型基因及野生型FLT3基因)、急性髓性白血病细胞OCI-AML-3(表达FLT3 A680V突变型基因)4小时，并用人免疫球蛋白IgM刺激10min，收集样品。测定化合物对这两个细胞系中BTKY223、PLCγY1217、AKT308、AKT S473、Erk 42/44磷酸化的影响(图1)。
实验结果如图1所示：无论在急性髓性白血病细胞MOLM-14(表达FLT3/ITD突变型基因及野生型FLT3基因)中，还是在急性髓性白血病细胞OCI-AML-3(表达FLT3 A680V突变型基因)细胞中，依鲁替尼(PCI32765)虽然能够抑制BTK的磷酸化，但是对BTK下游的PLCγ的磷酸化毫无影响(图1)，且对AKT、Erk的磷酸化也没有影响。这说明依鲁替尼(PCI32765)并不是通过抑制蛋白激酶BTK的磷酸化来影响携带FLT3/ITD突变型基因的急性髓性白血病细胞株MOLM-14的细胞增殖。
实施例3：依鲁替尼(PCI32765)在细胞中对FLT3上下游信号 通路的影响
在携带FLT3/ITD突变型基因的急性髓性白血病细胞MOLM-13、MOLM-14、MV-4-11这三株细胞中，通过测定许多细胞生物化学终点和功能性终点，测试了依鲁替尼(PCI32765)对细胞中的FLT3/ITD突变型的蛋白激酶的磷酸化及其密切相关的信号通路下游STAT5蛋白磷酸化的影响(图3)、其它相关的蛋白激酶ERK、AKT磷酸化的影响，同时我们还检测了对蛋白C-Myc以及转录因子NF-κB亚单位p65及其磷酸化的影响。用不同浓度0μM、0.03μM、0.1μM、0.3μM、1μM(于DMSO中)的依鲁替尼(PCI32765)、0.1μM(于DMSO中)的FLT3激酶抑制剂AC220，1μM(于DMSO中)的BTK激酶抑制剂AVL-292，1μM(于DMSO中)的BTK激酶抑制剂CGI1746分别处理MOLM-13、MOLM-14、MV-4-11三株携带FLT3/ITD突变型基因的急性髓性白血病细胞4小时，同时用仅含1％FBS(小牛血清)的培养基细胞饥饿4小时，收集样品。测定化合物对这三株细胞中STAT5、C-Myc、ERK、NF-κB p65、AKT蛋白及磷酸化的影响(图2)。
实验结果如图2所示：在MOLM-14细胞系中，依鲁替尼(PCI32765)能够强烈地抑制蛋白激酶FLT3的磷酸化，其EC50是0.039μM。在MOLM-13及MV-4-11细胞系中，依鲁替尼(PCI32765)也能够明显地抑制蛋白激酶FLT3的磷酸化，其EC50分别是0.32μM、0.36μM(图2)。此外，在携带FLT3/ITD突变型基因的急性髓性白血病细胞MOLM-13、MOLM-14、MV-4-11这三株细胞中，依鲁替尼(PCI32765)对细胞中FLT3/ITD下游蛋白STAT5的磷酸化有强烈的抑制作用，其EC50分别是0.32μM、0.11μM、0.91μM，且对与FLT3蛋白激酶密切相关的蛋白C-Myc有明显的降解作用。同样的实验中，对照BTK激酶抑制剂AVL-292、CGI1746在浓度为1μM时对蛋白激酶FLT3的磷酸化没有任何抑制，CGI1746对细胞中FLT3/ITD下游蛋白STAT5以及蛋白C-Myc也没有任何影响。而对照FLT3/ITD激酶抑制剂AC220能够强烈地抑制蛋白激酶FLT3及与FLT3/ITD密切相关的蛋白STAT5的磷酸化以及降解蛋白C-Myc。实施例3表明BTK激酶抑制剂依鲁替尼(PCI32765)能够抑制蛋白激酶FLT3的磷酸化，影响细胞中蛋白激酶FLT3的信号通路下游蛋白STAT5的磷酸化，进而抑制携带FLT3/ITD突变型基因的急性髓性白血病细胞株的细胞增殖。
实施例4：依鲁替尼(PCI32765)在细胞上对细胞凋亡的影响
为了证明用药以后细胞的死亡是通过凋亡还是坏死，在携带FLT3/ITD突变型基因的急性髓性白血病细胞MOLM-13、MOLM-14、MV-4-11及携带FLT3 A680V突变型基因的急性髓性白血病细胞OCI-AML-3、携带野生型FLT3基因的急性髓性白血病细胞NOMO-1中，检测了依鲁替尼(PCI32765)在细胞中对与细胞凋亡密切相关的DNA修复酶聚腺苷二磷酸-核糖聚合酶PARP、含半胱氨酸的天冬氨酸蛋白水解酶Caspase 3蛋白剪切的影响。用不同浓度0μM、0.3μM、1μM、3μM、10μM(于DMSO中)的依鲁替尼(PCI32765)、1μM(于DMSO中)的FLT3激酶抑制剂AC220、1μM的BTK激酶抑制剂AVL-292、1μM(于DMSO中)的BTK激酶抑制剂CGI1746分别处理MOLM-13、MOLM-14、MV-4-11、OCI-AML-3、NOMO-1，然后分别在12小时、24小时、48小时后收集细胞。用Western Blot检测不同浓度的药在不同时间段对DNA修复酶聚腺苷二磷酸-核糖聚合酶PARP和含半胱氨酸的天冬氨酸蛋白水解酶Caspase 3的剪切蛋白的影响。
实验结果如图4所示：对于携带FLT3/ITD突变型基因的急性髓性白血病细胞株MOLM-13，当依鲁替尼(PCI32765)用药浓度为3μM时，作用12小时后就能够看到明显的DNA修复酶聚腺苷二磷酸-核糖聚合酶PARP的剪切，以及部分含半胱氨酸的天冬氨酸蛋白水解酶Caspase 3的剪切，同样地使用1μM的FLT3激酶抑制剂AC220也能观测到同样的现象，而使用1μM的BTK激酶抑制剂AVL-292、1μM的BTK激酶抑制剂CGI1746则看不到任何DNA修复酶聚腺苷二磷酸-核糖聚合酶PARP或含半胱氨酸的天冬氨酸蛋白水解酶Caspase 3的剪切。对于携带FLT3/ITD突变型基因的急性髓性白血病细胞MOLM-14，当依鲁替尼(PCI32765)用药浓度为10μM时，分别作用12和24小时后，能够看到明显的DNA修复酶聚腺苷二磷酸-核糖聚合酶PARP的剪切。对于携带FLT3/ITD突变型基因的急性髓性白血病细胞MV-4-11，当依鲁替尼(PCI32765)用药浓度为3μM时，分别作用12和24小时后，能够看到明显的DNA修复酶聚腺苷二磷酸-核糖聚合酶PARP的剪切，以及部分含半胱氨酸的天冬氨酸蛋白水解酶Caspase 3的剪切。然而在携带FLT3 A680V突变型基因的急性髓性白血病细胞 OCI-AML-3中，即使依鲁替尼(PCI32765)的用药浓度为10μM，分别作用12、24和48小时后，也均没看到DNA修复酶聚腺苷二磷酸-核糖聚合酶PARP的剪切，以及含半胱氨酸的天冬氨酸蛋白水解酶Caspase 3的剪切。在携带FLT3野生型基因的急性髓性白血病细胞NOMO-1中，即使依鲁替尼(PCI32765)的用药浓度为10μM，分别作用12和24小时后，也均不能够看到DNA修复酶聚腺苷二磷酸-核糖聚合酶PARP的剪切，以及含半胱氨酸的天冬氨酸蛋白水解酶Caspase 3的剪切；作用48小时后，能够看到有部分DNA修复酶聚腺苷二磷酸-核糖聚合酶PARP的剪切。实施例4证明了依鲁替尼(PCI32765)能够引起携带FLT3/ITD突变型基因的急性髓性白血病细胞的凋亡。
实施例5：依鲁替尼(PCI32765)在细胞上对细胞周期的影响
为了研究用药后细胞被阻止在哪个生长周期，在携带FLT3/ITD突变型基因的急性髓性白血病细胞MOLM-13、MOLM-14、MV-4-11及携带FLT3 A680V突变型基因的急性髓性白血病细胞OCI-AML-3细胞株中，测试了依鲁替尼(PCI32765)对这些细胞株的细胞周期分布的影响。用不同浓度的0μM、0.5μM、1μM(于DMSO中)的依鲁替尼(PCI32765)，1μM(于DMSO中)的BTK激酶抑制剂CGI1746以及0.01μM(于DMSO中)的FLT3激酶抑制剂AC220作用于三株携带FLT3/ITD突变基因的急性髓性白血病细胞MOLM-13、MOLM-14、MV-4-11以及一株携带FLT3 A680V突变型基因的急性随性白血病细胞OCI-AML-3，作用24小时后，收集细胞，1XPBS缓冲液洗涤两次，75％的乙醇于-20℃固定24小时，1XPBS缓冲液再洗涤两次，加0.5mL 1XPBS缓冲液和0.5mL的PI染色液(购自美国BD Bioscience)到细胞中并将细胞放置于黑暗避光37℃染色15分钟，用流式细胞仪(BD FACS Calibur)检测细胞周期分布。
实验结果如图5所示：在携带FLT3/ITD突变型基因的急性髓性白血病细胞株MOLM-14中，随着依鲁替尼(PCI32765)的药物浓度从0.5μM增加到1μM，捕获的G0-G1期的细胞也从54.47％增加到58.13％；选择性较强的FLT3激酶抑制剂AC220在0.01μM时能捕获的G0-G1期的细胞就高达80.15％；而1μM BTK激酶抑制剂CGI1746 捕获的G0-G1期的细胞仅跟对照(只加等量的DMSO，不加任何药物)相当(图5a)。对于携带FLT3/ITD突变基因的急性髓性白血病细胞MV-4-11，随着依鲁替尼(PCI32765)药物浓度从0.5μM增加到1μM，捕获的G0-G1期的细胞也从64.94％增加到71.75％；选择性较强的FLT3激酶抑制剂AC220在0.01μM时能捕获的G0-G1期的细胞就高达97.29％；而1μM BTK激酶抑制剂CGI1746捕获的G0-G1期的细胞仅和对照相当(图5b)。对于携带FLT3/ITD突变基因的急性髓性白血病细胞MOLM-13，随着依鲁替尼(PCI32765)药物浓度从0.5μM增加到1μM，捕获的G0-G1期的细胞也从59.40％增加到67.41％；选择性较强的FLT3激酶抑制剂AC220在0.01μM时能捕获的G0-G1期的细胞就高达73.53％；而1μM BTK激酶抑制剂CGI1746捕获的G0-G1期的细胞仅和对照相当(图5c)。对于携带FLT3 A680V突变型基因的急性髓性白血病细胞OCI-AML-3，无论依鲁替尼(PCI32765)还是BTK激酶抑制剂CGI1746捕获的G0-G1期的细胞缺均和对照相当。对于携带FLT3 A680V突变型基因的急性髓性白血病细胞OCI-AML-3，无论是依鲁替尼(PCI32765)还是选择性较强的FLT3激酶抑制剂AC220对细胞周期的分布均没有明显的影响。这从另外一个方面证明了依鲁替尼(PCI32765)对于携带FLT3/ITD突变基因的急性髓性白血病细胞有明显的作用，并且对细胞周期的分布也有明显的影响(图5d)。对于携带FLT3野生型基因的急性髓性白血病细胞NOMO-1，无论是依鲁替尼(PCI32765)还是选择性较强的FLT3激酶抑制剂AC220对细胞周期的分布均没有明显的影响。这更加证明了依鲁替尼(PCI32765)对于携带FLT3/ITD突变基因的急性髓性白血病细胞有明显的作用，并且对细胞周期的分布也有明显的影响(图5e)。

Claims (10)

  1. 依鲁替尼(PCI32765)在制备用于治疗携带FLT3/ITD突变型基因的急性髓细胞白血病患者的药物中的用途。
  2. 根据权利要求1所述的用途，其中依鲁替尼单独施用或者与一种或多种其它的治疗剂组合施用。
  3. 一种药物组合物，包括依鲁替尼和药学上可接受的载体或助剂，以及任选的一种或多种其它治疗剂。
  4. 一种抑制携带FLT3/ITD突变型基因的急性髓细胞白血病细胞的方法，包括将所述细胞与依鲁替尼相接触。
  5. 根据权利要求4所述的方法，其中所述携带FLT3/ITD突变型基因的急性髓细胞白血病细胞是选自MOLM-13、MOLM-14、MV-4-11、PL-21和MUTZ-11中的一种或多种。
  6. 根据权利要求4或5所述的方法，其中将所述细胞与有效浓度至少为0.05μM的依鲁替尼相接触。
  7. 依鲁替尼在制备用于抑制携带FLT3/ITD突变型基因的急性髓细胞白血病细胞的药物中的用途。
  8. 根据权利要求7所述的用途，其中所述携带FLT3/ITD突变型基因的急性髓细胞白血病细胞是选自MOLM-13、MOLM-14、MV-4-11、PL-21和MUTZ-11中的一种或多种。
  9. 一种用于治疗患有或易患有携带FLT3/ITD突变型基因的急性髓细胞白血病的受试者的方法，包括对所述受试者施用治疗有效量的依鲁替尼(PCI32765)。
  10. 根据权利要求9所述的方法，通过注射、口服、吸入、直肠或经皮将依鲁替尼施用给所述受试者。
PCT/CN2014/091375 2014-10-30 2014-11-18 依鲁替尼对flt3-itd突变的急性白血病的应用 WO2016065674A1 (zh)

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