WO2022228323A1 - Application of ly3009120 in preparation of drugs for treatment of myeloproliferative neoplasms - Google Patents

Application of ly3009120 in preparation of drugs for treatment of myeloproliferative neoplasms Download PDF

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WO2022228323A1
WO2022228323A1 PCT/CN2022/088677 CN2022088677W WO2022228323A1 WO 2022228323 A1 WO2022228323 A1 WO 2022228323A1 CN 2022088677 W CN2022088677 W CN 2022088677W WO 2022228323 A1 WO2022228323 A1 WO 2022228323A1
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drug
resistant
myelofibrosis
myeloproliferative
cells
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PCT/CN2022/088677
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French (fr)
Chinese (zh)
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邓沱
胡婉钰
吾甫尔
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中南大学湘雅二医院
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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

  • the invention relates to the technical field of medicine, in particular to the application of LY3009120 in the preparation of a medicine for treating myeloproliferative tumors.
  • Myeloproliferative neoplasmas refer to a group of neoplastic diseases caused by the clonal proliferation of relatively mature one-line or multi-lineage myeloid cells. The clinical manifestations are the proliferation of one or more blood cells, accompanied by enlargement of the liver, spleen or lymph nodes.
  • WHO World Health Organization
  • Myeloproliferative neoplasms are clonal hematopoietic stem cell diseases.
  • the main disease-driving gene mutations include JAK2/V617F, CALR, and MPL mutations.
  • JAK2/V617F mutation is the most common type, which can be found in 95% of PV and 50-60% of patients. ET and 55-65% of PMF patients.
  • Activation of the gene leads to the activation of the JAK-STAT pathway, which leads to the occurrence of diseases.
  • RUX Ruxolitinib
  • the commonly used drugs for the treatment of myeloproliferative tumors included hydroxyurea and polyethylene glycol-recombinant interferon- ⁇ 2a. Hydroxyurea only relieves symptoms but does not inhibit clonal hematopoiesis, and long-term use may increase the risk of myelodysplastic syndrome and acute myeloid leukemia. The use of interferon is limited due to its high toxicity and side effects.
  • Ruxolitinib as a JAK1/JAK2 inhibitor, is approved by the FDA for first-line treatment of intermediate and high-risk myelofibrosis (MF), and as a second-line drug for hydroxyurea (Hydroxyurea, HU) resistant or intolerant. PV patients. Phase II and Phase III clinical trial results suggest that RUX can reduce spleen volume and reduce symptoms in patients with intermediate and high-risk MF and PV compared with optimal therapy. However, there are also many problems in the use of ruxolitinib. The results of the COMFORT and RESPONSE clinical trials showed that MF patients treated with ruxolitinib had more severe anemia.
  • type I JAK inhibitors such as RUX can induce the occurrence of drug resistance.
  • RUX type I JAK inhibitors
  • MF patients who received treatment for 1 year more than 40% of the patients developed drug resistance, and several JAKs were also found in clinical studies.
  • the US FDA approved Fedratinib a novel oral JAK2 selective inhibitor
  • the FDA also issued a boxed warning about the risk of encephalopathy, including Wernicke's encephalopathy, with Fedratinib.
  • bone marrow transplantation is mainly used to treat high-risk myelofibrosis patients, but the timing of bone marrow transplantation for patients with other types of myeloproliferative tumors needs further study and research to confirm. Bone marrow transplants are expensive and, in the current medical environment, are not an option for most patients.
  • ruxolitinib is a milestone drug in the treatment of myeloproliferative tumors
  • the current application of ruxolitinib is narrow, and myelosuppression is a common side effect that limits its application in the main indication MF.
  • Ruxolitinib cannot reduce the load of mutated genes, which means that ruxolitinib treatment cannot achieve molecular remission of the disease and cannot fundamentally treat myeloproliferative tumors.
  • the limited treatment drugs are a major challenge.
  • the purpose of the present invention is to provide an effective, safe and reliable method for myeloproliferative tumor patients, especially myeloproliferative tumor patients resistant to ruxolitinib, aiming at the problems existing in the current treatment of myeloproliferative tumors. medicine.
  • the present invention provides the application of LY3009120 in the preparation of medicaments for preventing and/or treating myeloproliferative tumors.
  • LY3009120 is a potent pan-Raf inhibitor that inhibits all RAF isoforms, as well as homodimers and heterodimers of BRAF and CRAF. In in vitro and in vivo experiments, LY3009120 exhibits very low inverse activation and exhibits activity against BRAF- or RAS-mutated cells. Results of a phase I clinical trial (NCT02014116) in advanced or metastatic melanoma, non-small cell lung and colorectal cancer showed that the recommended phase II dose (RP2D) of LY3009120 was 300 mg twice daily It is taken orally and has better safety and lower toxic and side effects.
  • the molecular formula of LY3009120 is C 23 H 29 FN 6 O, the molecular weight is 424.51, and the structural formula is shown in formula I.
  • myeloproliferative neoplasms include, but are not limited to, polycythemia vera, essential thrombocythemia, or myelofibrosis, which is primary myelofibrosis, myelofibrosis secondary to polycythemia vera Myelofibrosis or myelofibrosis secondary to essential thrombocythemia.
  • the present invention also provides the application of LY3009120 in the preparation of medicines for preventing and/or treating drug-resistant myeloproliferative tumors.
  • drug-resistant myeloproliferative tumors include but are not limited to ruxolitinib-resistant myeloproliferative tumors.
  • drug-resistant myeloproliferative neoplasms include, but are not limited to, drug-resistant polycythemia vera, drug-resistant essential thrombocythemia, or drug-resistant myelofibrosis
  • the bone marrow Fibrosis is primary myelofibrosis, myelofibrosis secondary to polycythemia vera, or myelofibrosis secondary to essential thrombocythemia.
  • the present invention also provides the application of LY3009120 in the preparation of drugs for inhibiting the proliferation of HEL and/or SET2 cells.
  • the present invention also provides the application of LY3009120 in the preparation of a drug for inhibiting the proliferation of drug-resistant HEL and/or SET2 cells.
  • the present invention also provides the application of LY3009120 in the preparation of medicines for promoting apoptosis of HEL and/or SET2 cells.
  • the present invention also provides the application of LY3009120 in the preparation of a drug for promoting apoptosis of drug-resistant HEL and/or SET2 cells.
  • the medicine also contains pharmaceutically acceptable excipients.
  • the dosage form of the drug includes, but is not limited to, an oral preparation or an injection preparation.
  • the present invention also provides a method for preventing and/or treating myeloproliferative tumors or drug-resistant myeloproliferative tumors, characterized in that LY3009120 is administered.
  • the myeloproliferative tumor includes, but is not limited to, polycythemia vera, essential thrombocythemia, or myelofibrosis, which is primary myelofibrosis, secondary to red blood cells vera Myelofibrosis of hyperplasia or myelofibrosis secondary to essential thrombocythemia;
  • the drug-resistant myeloproliferative tumors include, but are not limited to, ruxolitinib-resistant myeloproliferative tumors;
  • the drug-resistant myeloproliferative tumor includes but is not limited to drug-resistant polycythemia vera, drug-resistant essential thrombocythemia or drug-resistant myelofibrosis, so
  • the myelofibrosis is primary myelofibrosis, myelofibrosis secondary to polycythemia vera, or myelofibrosis secondary to essential thrombocythemia.
  • the myeloproliferative tumor is polycythemia vera, essential thrombocythemia or myelofibrosis and ruxolitinib-resistant myeloproliferative tumor, and the myelofibrosis is primary myelofibrosis, secondary myelofibrosis.
  • Myelofibrosis arising from polycythemia vera or myelofibrosis secondary to essential thrombocythemia.
  • LY3009120 in the treatment of myeloproliferative tumors provides a new treatment approach for the majority of patients with myeloproliferative tumors, and provides more choices for clinicians and patients.
  • LY3009120 can provide continued oral drug therapy and avoid bone marrow transplantation.
  • LY3009120 can be chemically synthesized, and the cost is lower than that of biological preparations. And it has passed the Phase I clinical trial, and is expected to pass the Phase II and Phase III clinical trials smoothly. It will be used for clinical treatment in the future, and has a good clinical application prospect.
  • Figure 1 shows the results of Example 1: the establishment of ruxolitinib-resistant myeloproliferative tumor cell models HEL RE and SET2 RE ; a is the successful construction of the HEL RE model; b is the successful construction of the SET2 RE model;
  • Figure 2 shows the results of Example 2: LY3009120 treated two kinds of myeloproliferative tumor cells, and the cell proliferation was detected by CellTiter-Lumi TM luminescence method; a is the HEL cell proliferation result; b is the SET2 cell proliferation result;
  • Figure 3 shows the results of Example 3: LY3009120 treated two myeloproliferative tumor cells, and the results of flow detection of apoptosis after AnnexinV-PI staining; a is the result of HEL cell apoptosis; b is the result of SET2 cell apoptosis ;
  • Figure 4 shows the results of Example 4: LY3009120 treated two ruxolitinib-resistant myeloproliferative tumor cells, and the cell proliferation was detected by CellTiter-Lumi TM luminescence method; a is the HEL RE cell proliferation result; b is SET2 RE cell proliferation result graph;
  • Figure 5 shows the results of Example 5: LY3009120 treated two ruxolitinib-resistant myeloproliferative tumor cells, and the results of flow detection of apoptosis after AnnexinV-PI staining; a is the result of HEL RE cell apoptosis; b is the result of apoptosis of SET2 RE cells.
  • the invention discloses the application of LY3009120 in the preparation of a drug for the treatment of myeloproliferative tumors, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention.
  • the method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.
  • the inhibitory effect of LY3009120 on myeloproliferative tumor cells was clarified by a cell line model.
  • the present invention is based on two commonly used human myeloproliferative tumor cell lines containing the JAK2-V617F mutation, namely HEL cells (Human erythroleukemia cell line, human erythroleukemia cell line) and SET2 cell (essential platelet hyperplasia cells), and established their respective ruxolitinib-resistant cell models HEL RE and SET2 RE , respectively.
  • HEL cells Human erythroleukemia cell line, human erythroleukemia cell line
  • SET2 cell essential platelet hyperplasia cells
  • the present invention treats two myeloproliferative tumor cell lines (HEL and SET2) with increasing concentrations of LY3009120, and cell proliferation is detected by CellTiter-Lumi TM luminescence assay. The results showed that LY3009120 could successfully inhibit the proliferation of HEL and SET2 cells.
  • the present invention treats two myeloproliferative tumor cell lines (HEL and SET2) with increasing concentrations of LY3009120, and the apoptosis of the cells is detected by flow cytometry after AnnexinV-PI staining. The results showed that LY3009120 could promote the apoptosis of HEL and SET2 cells.
  • LY3009120 can be used for the treatment of myeloproliferative tumor diseases.
  • the present invention uses increasing concentrations of LY3009120 to treat ruxolitinib-resistant myeloproliferative tumor cells (HEL RE and SET2 RE ), and the cell proliferation is detected by CellTiter-Lumi TM luminescence method.
  • HEL RE and SET2 RE ruxolitinib-resistant myeloproliferative tumor cells
  • LY3009120 could successfully inhibit the proliferation of HEL RE and SET2 RE cells.
  • the present invention uses increasing concentrations of LY3009120 to treat ruxolitinib-resistant myeloproliferative tumor cells (HEL RE and SET2 RE ), and the apoptosis of the cells is detected by flow cytometry after AnnexinV-PI staining. The results showed that LY3009120 could promote the apoptosis of HEL RE and SET2 RE cells.
  • LY3009120 can be used for the treatment of ruxolitinib-resistant myeloproliferative tumor diseases.
  • the present invention provides the application of LY3009120 in the preparation of medicines for inhibiting the proliferation of HEL and SET2 cells and promoting the apoptosis of HEL and SET2 cells.
  • the present invention provides the application of LY3009120 in the preparation of a medicament for the treatment of myeloproliferative tumors.
  • the myeloproliferative tumor is polycythemia vera, essential thrombocythemia, and myelofibrosis (including primary myelofibrosis, myelofibrosis secondary to polycythemia vera, and myelofibrosis secondary to primary polycythemia vera).
  • myelofibrosis with thrombocythemia and drug-resistant myeloproliferative neoplasms.
  • the drug-resistant myeloproliferative tumor is a ruxolitinib-resistant myeloproliferative tumor.
  • the drug-resistant myeloproliferative neoplasm is drug-resistant polycythemia vera, drug-resistant myelofibrosis (including primary myelofibrosis, secondary to erythrocyte vera) hypertrophic myelofibrosis and myelofibrosis secondary to essential thrombocythemia) and drug-resistant essential thrombocythemia.
  • the drug is LY3009120.
  • the medicine also includes pharmaceutically acceptable excipients.
  • the drug can be any dosage form in the current pharmaceutical field, including oral preparations or injection preparations.
  • Each pharmaceutical dosage form can be prepared by selecting appropriate acceptable excipients according to the actual needs of the dosage form, which belongs to the conventional dosage form preparation technology in the art. Such as capsules, tablets, injection powder and so on.
  • the reagents or instruments used in the present invention can be purchased from the market.
  • Example 1 Establishment of two common ruxolitinib-resistant cell models (HEL RE , SET2 RE ).
  • HEL Human erythroleukemia cell line
  • ruxolitinib-resistant HEL cells SET2 cells
  • ruxolitinib-resistant SET2 cells were cultured in 20% heat-inactivated fetal bovine serum (Gibco) and 1% penicillin/ Streptomycin in RPMI medium (Gibco).
  • the HEL model with resistance to ruxolitinib was constructed by adding ruxolitinib at a concentration lower than the original cell IC50, and slowly increasing to a high concentration to maintain the cells from being killed.
  • Our starting concentration was 0.1 ⁇ M, and the drug was added as soon as the cells proliferated.
  • the dosing gradient was 1.25-fold increments, and the final concentration was 2.0 ⁇ M.
  • Stable resistant cells were obtained after 4-6 weeks.
  • Another ruxolitinib resistance model is SET2 RE , or SET2-resistant model.
  • the construction method is to start adding ruxolitinib at a concentration lower than the original cell IC50, and slowly increase it to a high concentration to keep the cells from being killed.
  • Our starting concentration was 0.03 ⁇ M, and the drug was added as soon as the cells proliferated.
  • the dosing gradient was 1.25-fold increments, and the final concentration was 0.5 ⁇ M. Stable resistant cells were obtained after
  • Ruxolitinib and LY3009120 were purchased from Selleck Company, dissolved in DMSO, the concentration of the stock solution was 10 mM, frozen at -80 °C, and the working solution was diluted with RPMI medium to the specified times before treating the cells.
  • Ruxolitinib is specifically ruxolitinib phosphate.
  • the above cell lines were cultured in a system of 3000 cells/100 ⁇ L per well, and increasing concentrations of ruxolitinib were added (HEL cell concentration gradient: 0, 0.1, 1, 2.5, 5 ⁇ M; SET2 cell concentration Gradient: 0, 0.01, 0.1, 1, 2.5, 5 ⁇ M), DMSO rounded up to equal volume. Set up 4 parallel replicate groups, and set up 3 blank wells (culture medium wells without cells). Cell proliferation was detected by CellTiter-Lumi TM luminescence method (Biyuntian) after 48 hours. Multi-plate reader reading, IC50 calculated by GraphPadprism.
  • cell proliferation rate (Luminescence value of drug-added group-average Luminescence value of blank well)/(Luminescence value of DMSO control group-average Luminescence value of blank well) ⁇ 100%.
  • the method to evaluate whether the model is successfully constructed is to compare the IC50 of drug-resistant cells and original cells, and the ratio is the drug-resistance index.
  • the IC50 of HEL cells was 2.31 (1.42-4.36) ⁇ M
  • the IC50 of HEL RE cells was 44.7 (16.0-370) ⁇ M
  • the drug resistance index was 19.0, indicating the successful construction of the drug resistance model HEL RE
  • the IC50 of SET2 cells was 0.048 (0.035-0.065) ⁇ M
  • the IC50 of SET2 RE cells was 34.3 (16.0–108) ⁇ M
  • the drug resistance index was 714, indicating the successful construction of the drug resistance model SET2 RE .
  • results of the proliferation rate in the figure are shown as the mean ⁇ standard deviation, the comparison of the proliferation rate between the two groups in the figure is by t test (***p ⁇ 0.001), and the IC50 is shown as the mean. Results are shown as means (95% confidence interval) in the analysis.
  • Example 2 LY3009120 can inhibit the proliferation of myeloproliferative tumor cells
  • the method was to treat HEL original cell line and SET2 original cell line with increasing concentrations of ruxolitinib and LY3009120 respectively, and detect the cell proliferation by CellTiter-Lumi TM luminescence method. Proliferation, the method is the same as that of Example 1, and the results are shown in Figure 2.
  • Figure 2a reflects that in HEL cells, the drug concentrations (mean proliferation % ⁇ SD) of the ruxolitinib-treated group were: 0 ⁇ M (100 ⁇ 2.40) (not shown in the figure), 0.1 ⁇ M (77.9 ⁇ 2.43), 1 ⁇ M (43.2 ⁇ 3.75), 2.5 ⁇ M (50.6 ⁇ 2.19), 5 ⁇ M (47.0 ⁇ 1.15); LY3009120 treatment group drug concentration (average proliferation rate): 0 ⁇ M (100 ⁇ 7.65) (not shown in the figure), 0.1 ⁇ M (61.7 ⁇ 5.66), 1 ⁇ M (39.5 ⁇ 1.39), 2.5 ⁇ M (15.5 ⁇ 1.05), 5 ⁇ M (6.19 ⁇ 1.02).
  • Figure 2b reflects that in SET2 cells, the drug concentrations (mean proliferation rate) of the ruxolitinib-treated group were: 0 ⁇ M (100 ⁇ 5.51) (not shown in the figure), 0.01 ⁇ M (80.7 ⁇ 9.07), 0.1 ⁇ M (30.1 ⁇ 1.95), 1 ⁇ M (12.6 ⁇ 1.23), 2.5 ⁇ M (12.1 ⁇ 1.13), 5 ⁇ M (12.2 ⁇ 0.74); LY3009120 treatment group drug concentration (average proliferation rate): 0 ⁇ M (100 ⁇ 4.72) (not shown in the figure), 0.01 ⁇ M (99.4 ⁇ 6.46), 0.1 ⁇ M (44.4 ⁇ 3.86), 1 ⁇ M (23.2 ⁇ 1.03), 2.5 ⁇ M (22.2 ⁇ 2.75), 5 ⁇ M (11.8 ⁇ 1.75).
  • Example 3 LY3009120 can promote the apoptosis of myeloproliferative tumor cells
  • HEL original cell line and SET2 original cell line were treated with ruxolitinib and LY3009120 for 24 hours (concentration: 0, 0.1, 0.5, 1 ⁇ M), respectively, and DMSO was supplemented to an equal amount.
  • Three parallel replicate groups were set up, and the apoptosis of cells was detected by flow cytometry after AnnexinV and PI staining.
  • apoptosis rate ratio of early apoptotic cells (Annexin V + /PI - ) + ratio of late apoptotic cells and necrotic cells (Annexin V + /PI + ).
  • the drug concentrations (mean apoptotic rate % ⁇ SD) of HEL cells treated with ruxolitinib were: 0 ⁇ M (4.37 ⁇ 0.318), 0.1 ⁇ M (5.59 ⁇ 0.097), 0.5 ⁇ M (6.63 ⁇ 1.12), 1 ⁇ M (6.68 ⁇ 0.304); the drug concentration (apoptosis rate) in the LY3009120 treatment group was 0 ⁇ M (4.37 ⁇ 0.318), 0.1 ⁇ M (5.96 ⁇ 0.036), 0.5 ⁇ M (7.30 ⁇ 0.358), 1 ⁇ M (8.08 ⁇ 0.560); in Figure 3b , the drug concentration (average apoptosis rate) of ruxolitinib treatment group in SET2 cells was: 0 ⁇ M (5.47 ⁇ 0.894), 0.1 ⁇ M (14.8 ⁇ 0.397), 0.5 ⁇ M (19.3 ⁇ 2.64), 1 ⁇ M (23.1 ⁇ 2.33); LY3009120 The drug concentration (apoptosis rate) in the treatment
  • Example 4 LY3009120 can inhibit the proliferation of drug-resistant myeloproliferative tumor cells.
  • the method was to treat HEL RE and SET2 RE with increasing concentrations of ruxolitinib and LY3009120, respectively, and detect the cell proliferation by CellTiter-Lumi TM luminescence method.
  • the method is the same as in Example 1, and the results are shown in Figure 4.
  • Figure 4a reflects that in HEL RE cells, the drug concentrations (mean proliferation % ⁇ SD) of the ruxolitinib treatment group were: 0 ⁇ M (100 ⁇ 1.73) (not shown in the figure), 0.1 ⁇ M (103.17 ⁇ 1.9), 1 ⁇ M (91.14 ⁇ 1.63), 2.5 ⁇ M (91.42 ⁇ 1.7), 5 ⁇ M (84.15 ⁇ 1.38); LY3009120 treatment group drug concentration (average proliferation rate): 0 ⁇ M (100 ⁇ 2.91) (not shown in the figure), 0.1 ⁇ M ( 88.11 ⁇ 13.93), 1 ⁇ M (14.55 ⁇ 1.91), 2.5 ⁇ M (11.43 ⁇ 0.62), 5 ⁇ M (11.65 ⁇ 0.42).
  • Figure 4b reflects that in SET RE cells, the drug concentrations (mean proliferation rate) of the ruxolitinib-treated group were: 0 ⁇ M (100 ⁇ 0.38) (not shown in the figure), 0.01 ⁇ M (88.19 ⁇ 4.11), 0.1 ⁇ M (82.21 ⁇ M) ⁇ 5.1), 1 ⁇ M (71.18 ⁇ 2.37), 2.5 ⁇ M (68.34 ⁇ 3.79), 5 ⁇ M (60.17 ⁇ 3.4); LY3009120 treatment group drug concentration (average proliferation rate): 0 ⁇ M (100 ⁇ 1.13) (not shown in the figure) , 0.01 ⁇ M (88.27 ⁇ 5.18), 0.1 ⁇ M (29.97 ⁇ 1.05), 1 ⁇ M (12.33 ⁇ 1.3), 2.5 ⁇ M (11.67 ⁇ 0.99), 5 ⁇ M (6.96 ⁇ 0.68).
  • the comparison of the proliferation rate between the two groups in the figure was performed by t test (***p ⁇ 0.001). This suggests that LY3009120 can inhibit the proliferation
  • Example 5 LY3009120 can promote the apoptosis of drug-resistant myeloproliferative tumor cells
  • the method was to treat HEL RE and SET2 RE with increasing concentrations of ruxolitinib and LY3009120, respectively, and to detect the apoptosis of cells after staining with AnnexinV and PI. .
  • the method is the same as in Example 3, and the results are shown in Figure 5.
  • the drug concentrations (mean apoptotic rate % ⁇ SD) of HEL RE cells treated with ruxolitinib were: 0 ⁇ M (2.14 ⁇ 0.111), 0.1 ⁇ M (1.67 ⁇ 0.128), 0.5 ⁇ M (2.04 ⁇ 0.128), 1 ⁇ M (1.88 ⁇ 0.238); the drug concentration (apoptosis rate) in LY3009120 treatment group was 0 ⁇ M (2.14 ⁇ 0.111), 0.1 ⁇ M (3.6 ⁇ 0.259), 0.5 ⁇ M (14.7 ⁇ 1.12), 1 ⁇ M (16.4 ⁇ 0.786).
  • the drug concentrations (average apoptosis rate) of ruxolitinib-treated SET2 RE cells were: 0 ⁇ M (1.37 ⁇ 0.156), 0.1 ⁇ M (0.927 ⁇ 0.184), 0.5 ⁇ M (1.13 ⁇ 0.232), 1 ⁇ M (1.78 ⁇ 0.184) 0.261); the drug concentration (apoptosis rate) of LY3009120 treatment group was 0 ⁇ M (1.37 ⁇ 0.156), 0.1 ⁇ M (1.96 ⁇ 0.155), 0.5 ⁇ M (9.19 ⁇ 1.74), 1 ⁇ M (10.5 ⁇ 1.56).
  • the comparison of the apoptosis rate between the two groups in the figure was performed by t test (**p ⁇ 0.01, ***p ⁇ 0.001). This indicates that LY3009120 can promote the apoptosis of ruxolitinib-resistant myeloproliferative tumor cells, and the effect increases with the increase of drug concentration.

Abstract

The present invention relates to the technical field of medicine, and particularly relates to an application of LY3009120 in the preparation of drugs for the treatment of myeloproliferative neoplasms. Myeloproliferative neoplasms include polycythaemia vera, primary thrombocythaemia, and myelofibrosis, and in particular ruxolitinib-resistant myeloproliferative neoplasms.

Description

LY3009120在制备治疗骨髓增殖性肿瘤的药物中的应用Application of LY3009120 in the preparation of drugs for the treatment of myeloproliferative tumors
本申请要求于2021年04月26日提交中国专利局、申请号为202110455195.9、发明名称为“LY3009120在制备治疗骨髓增殖性肿瘤的药物中的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims the priority of the Chinese patent application with the application number 202110455195.9 and the invention name "Application of LY3009120 in the preparation of drugs for the treatment of myeloproliferative tumors", which was submitted to the China Patent Office on April 26, 2021, the entire contents of which are approved by Reference is incorporated in this application.
技术领域technical field
本发明涉及医药技术领域，特别涉及LY3009120在制备治疗骨髓增殖性肿瘤的药物中的应用。The invention relates to the technical field of medicine, in particular to the application of LY3009120 in the preparation of a medicine for treating myeloproliferative tumors.
背景技术Background technique
骨髓增殖性肿瘤(Myeloproliferative neoplasmas，MPNs)是指分化相对成熟的一系或多系骨髓细胞克隆性增殖所致的一组肿瘤性疾病。在临床表现为一种或多种血细胞增生，伴肝、脾或***肿大。2016年世界卫生组织(WHO)对骨髓肿瘤进行分类修订，将真性红细胞增多症(polycythemia vera,PV)、原发性骨髓纤维化(primary myelofibrosis，PMF)、原发性血小板增多症(essential thrombocythemia，ET)归入费城阴性经典骨髓增殖性肿瘤(Philadelphia-negative classical骨髓增殖性肿瘤)的范畴。骨髓增殖性肿瘤为克隆性造血干细胞疾病，主要的疾病驱动基因突变包括JAK2/V617F、CALR、MPL突变，其中JAK2/V617F突变是最常见的类型，可见于95％的PV、50-60％的ET和55-65％的PMF患者。基因的活化带来JAK-STAT通路的激活从而导致疾病的发生。全球每年新增骨髓增殖性肿瘤患者约20万，给医疗卫生***带来沉重的负担。Myeloproliferative neoplasmas (MPNs) refer to a group of neoplastic diseases caused by the clonal proliferation of relatively mature one-line or multi-lineage myeloid cells. The clinical manifestations are the proliferation of one or more blood cells, accompanied by enlargement of the liver, spleen or lymph nodes. In 2016, the World Health Organization (WHO) revised the classification of bone marrow tumors, including polycythemia vera (PV), primary myelofibrosis (PMF), essential thrombocythemia, ET) is classified as Philadelphia-negative classic myeloproliferative tumor. Myeloproliferative neoplasms are clonal hematopoietic stem cell diseases. The main disease-driving gene mutations include JAK2/V617F, CALR, and MPL mutations. Among them, JAK2/V617F mutation is the most common type, which can be found in 95% of PV and 50-60% of patients. ET and 55-65% of PMF patients. Activation of the gene leads to the activation of the JAK-STAT pathway, which leads to the occurrence of diseases. There are about 200,000 new myeloproliferative tumors in the world every year, which brings a heavy burden to the medical and health system.
在芦可替尼(Ruxolitinib，RUX)面世之前，骨髓增殖性肿瘤的常用治疗药物包括羟基脲及聚乙二醇-重组干扰素-α2a。羟基脲只能缓解症状却不能抑制克隆性造血，长期使用可能会增加骨髓增生异常综合征和急性髓系白血病的风险。而干扰素则因存在较高的毒副反应限制了其使用。芦可替尼作为JAK1/JAK2抑制剂，被FDA批准一线用于对中、高危骨髓纤维化(myelofibrosis,MF)，并作为二线药物用于羟基脲(Hydroxyurea， HU)耐药或不能耐受的PV患者。二期、三期临床试验结果提示，与最佳疗法相比，RUX能够减少中、高危MF和PV患者的脾脏体积和减轻症状。但是芦可替尼在使用过程中也存在许多问题，COMFORT和RESPONSE临床试验结果显示，接受芦可替尼治疗的MF患者贫血更重。更严重的是，长时间使用RUX等I型JAK抑制剂可诱导耐药的发生，在接受治疗1年的MF病人中，超过40％病人出现耐药，在临床研究中也发现了几种JAK抑制剂之间的交叉耐药。2019年8月，美国FDA批准了新型口服JAK2选择性抑制剂Fedratinib
Figure PCTCN2022088677-appb-000001
用于成人中、高危原发性或继发性(PV后或ET后)MF，其中包括以前接受过芦可替尼治疗的患者。FDA同时予以黑框警示Fedratinib可能引起脑病，包括韦尼克脑病的风险。为了进一步评估Fedratinib的有效性及安全性，新的多中心IIIb期临床实验(NCT03755518)正在进行。目前的JAK抑制剂不能显著减少突变等位基因负荷，因此其治疗潜力有限。骨髓移植是唯一治愈骨髓增殖性肿瘤的方法，但是仍然有一些问题需要解决。移植方式和方案的选择还不确定，选择同种异体移植还是单倍体同种移植尚不清楚。此外，当选择移植时，必须考虑到移植相关死亡率和骨髓增殖性肿瘤的长期性。目前骨髓移植主要用于治疗高危的骨髓纤维化患者，但其他类型的骨髓增殖性肿瘤患者选择骨髓移植的时机需要进一步探讨和研究证实。骨髓移植费用昂贵，在目前的医疗环境下，对大多数患者来说无法选择该治疗手段。 Before the introduction of Ruxolitinib (RUX), the commonly used drugs for the treatment of myeloproliferative tumors included hydroxyurea and polyethylene glycol-recombinant interferon-α2a. Hydroxyurea only relieves symptoms but does not inhibit clonal hematopoiesis, and long-term use may increase the risk of myelodysplastic syndrome and acute myeloid leukemia. The use of interferon is limited due to its high toxicity and side effects. Ruxolitinib, as a JAK1/JAK2 inhibitor, is approved by the FDA for first-line treatment of intermediate and high-risk myelofibrosis (MF), and as a second-line drug for hydroxyurea (Hydroxyurea, HU) resistant or intolerant. PV patients. Phase II and Phase III clinical trial results suggest that RUX can reduce spleen volume and reduce symptoms in patients with intermediate and high-risk MF and PV compared with optimal therapy. However, there are also many problems in the use of ruxolitinib. The results of the COMFORT and RESPONSE clinical trials showed that MF patients treated with ruxolitinib had more severe anemia. More seriously, long-term use of type I JAK inhibitors such as RUX can induce the occurrence of drug resistance. Among MF patients who received treatment for 1 year, more than 40% of the patients developed drug resistance, and several JAKs were also found in clinical studies. Cross-resistance between inhibitors. In August 2019, the US FDA approved Fedratinib, a novel oral JAK2 selective inhibitor
Figure PCTCN2022088677-appb-000001
For adults with intermediate- and high-risk primary or secondary (post-PV or post-ET) MF, including patients previously treated with ruxolitinib. The FDA also issued a boxed warning about the risk of encephalopathy, including Wernicke's encephalopathy, with Fedratinib. To further evaluate the efficacy and safety of Fedratinib, a new multicenter Phase IIIb clinical trial (NCT03755518) is underway. Current JAK inhibitors do not significantly reduce mutant allele burden and thus have limited therapeutic potential. Bone marrow transplantation is the only cure for myeloproliferative tumors, but there are still some problems to be solved. The choice of transplantation modality and protocol is uncertain, and the choice of allogeneic or haploidentical transplantation is unclear. In addition, transplantation-related mortality and the long-term nature of myeloproliferative tumors must be considered when selecting transplantation. At present, bone marrow transplantation is mainly used to treat high-risk myelofibrosis patients, but the timing of bone marrow transplantation for patients with other types of myeloproliferative tumors needs further study and research to confirm. Bone marrow transplants are expensive and, in the current medical environment, are not an option for most patients.
尽管芦可替尼是骨髓增殖性肿瘤治疗的里程碑药物，但芦可替尼目前适用的范围较窄，骨髓抑制作为常见的副反应限制了其在主要适应症MF中的应用。芦可替尼不能减少突变基因的负荷，这意味着芦可替尼治疗不能使疾病达到分子水平的缓解，无法从根本上治疗骨髓增殖性肿瘤。尤其是在出现芦可替尼耐药后，治疗药物有限是目前面临的重大挑战。Although ruxolitinib is a milestone drug in the treatment of myeloproliferative tumors, the current application of ruxolitinib is narrow, and myelosuppression is a common side effect that limits its application in the main indication MF. Ruxolitinib cannot reduce the load of mutated genes, which means that ruxolitinib treatment cannot achieve molecular remission of the disease and cannot fundamentally treat myeloproliferative tumors. Especially after the emergence of ruxolitinib resistance, the limited treatment drugs are a major challenge.
发明内容SUMMARY OF THE INVENTION
有鉴于此，本发明的目的在于针对目前骨髓增殖性肿瘤治疗中存在的问题，对骨髓增殖性肿瘤病人尤其是对芦可替尼耐药的骨髓增殖性肿瘤病人提供一种有效、安全、可靠的药物。In view of this, the purpose of the present invention is to provide an effective, safe and reliable method for myeloproliferative tumor patients, especially myeloproliferative tumor patients resistant to ruxolitinib, aiming at the problems existing in the current treatment of myeloproliferative tumors. medicine.
为了实现上述发明目的，本发明提供以下技术方案：In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:
本发明提供了LY3009120在制备预防和/或治疗骨髓增殖性肿瘤的药物中的应用。The present invention provides the application of LY3009120 in the preparation of medicaments for preventing and/or treating myeloproliferative tumors.
LY3009120是一种有效的泛Raf抑制剂，能够抑制所有的RAF亚型，以及抑制BRAF和CRAF的同二聚体和异二聚体。在体外和体内的实验中，LY3009120出现极低的反向活化，并表现出抗BRAF-或RAS-突变细胞的活性。在进展或转移性的黑色素瘤、非小细胞肺和结直肠癌中进行的I期临床试验(NCT02014116)结果显示，LY3009120推荐的II期剂量(recommend phase II dose，RP2D)为300mg，每日两次口服，同时具有较好的安全性和较低毒副作用。LY3009120的分子式为C 23H 29FN 6O，分子量424.51，结构式如式I所示。 LY3009120 is a potent pan-Raf inhibitor that inhibits all RAF isoforms, as well as homodimers and heterodimers of BRAF and CRAF. In in vitro and in vivo experiments, LY3009120 exhibits very low inverse activation and exhibits activity against BRAF- or RAS-mutated cells. Results of a phase I clinical trial (NCT02014116) in advanced or metastatic melanoma, non-small cell lung and colorectal cancer showed that the recommended phase II dose (RP2D) of LY3009120 was 300 mg twice daily It is taken orally and has better safety and lower toxic and side effects. The molecular formula of LY3009120 is C 23 H 29 FN 6 O, the molecular weight is 424.51, and the structural formula is shown in formula I.
Figure PCTCN2022088677-appb-000002
Figure PCTCN2022088677-appb-000002
作为优选，骨髓增殖性肿瘤包括但不限于真性红细胞增多症、原发性血小板增多症或骨髓纤维化，所述骨髓纤维化为原发性骨髓纤维化、继发于真性红细胞增多症的骨髓纤维化或继发于原发性血小板增多症的骨髓纤维化。Preferably, myeloproliferative neoplasms include, but are not limited to, polycythemia vera, essential thrombocythemia, or myelofibrosis, which is primary myelofibrosis, myelofibrosis secondary to polycythemia vera Myelofibrosis or myelofibrosis secondary to essential thrombocythemia.
本发明还提供了LY3009120在制备预防和/或治疗具有耐药性的骨髓增殖性肿瘤的药物中的应用。The present invention also provides the application of LY3009120 in the preparation of medicines for preventing and/or treating drug-resistant myeloproliferative tumors.
在本发明提供的实施例中，具有耐药性的骨髓增殖性肿瘤包括但不限于具有芦可替尼耐药性的骨髓增殖性肿瘤。In the embodiments provided by the present invention, drug-resistant myeloproliferative tumors include but are not limited to ruxolitinib-resistant myeloproliferative tumors.
作为优选，具有耐药性的骨髓增殖性肿瘤包括但不限于具有耐药性的真性红细胞增多症、具有耐药性的原发性血小板增多症或具有耐药性的骨髓纤维化，所述骨髓纤维化为原发性骨髓纤维化、继发于真性红细胞增多症的骨髓纤维化或继发于原发性血小板增多症的骨髓纤维化。Preferably, drug-resistant myeloproliferative neoplasms include, but are not limited to, drug-resistant polycythemia vera, drug-resistant essential thrombocythemia, or drug-resistant myelofibrosis, the bone marrow Fibrosis is primary myelofibrosis, myelofibrosis secondary to polycythemia vera, or myelofibrosis secondary to essential thrombocythemia.
本发明还提供了LY3009120在制备抑制HEL和/或SET2细胞的增殖的药物中的应用。The present invention also provides the application of LY3009120 in the preparation of drugs for inhibiting the proliferation of HEL and/or SET2 cells.
本发明还提供了LY3009120在制备抑制具有耐药性的HEL和/或SET2细胞的增殖的药物中的应用。The present invention also provides the application of LY3009120 in the preparation of a drug for inhibiting the proliferation of drug-resistant HEL and/or SET2 cells.
本发明还提供了LY3009120在制备促进HEL和/或SET2细胞的凋亡的药物中的应用。The present invention also provides the application of LY3009120 in the preparation of medicines for promoting apoptosis of HEL and/or SET2 cells.
本发明还提供了LY3009120在制备促进具有耐药性的HEL和/或SET2细胞的凋亡的药物中的应用。The present invention also provides the application of LY3009120 in the preparation of a drug for promoting apoptosis of drug-resistant HEL and/or SET2 cells.
作为优选，药物还包含药学上可接受的辅料。Preferably, the medicine also contains pharmaceutically acceptable excipients.
作为优选，药物的剂型包括但不限于口服制剂或注射制剂。Preferably, the dosage form of the drug includes, but is not limited to, an oral preparation or an injection preparation.
此外，本发明还提供了预防和/或治疗骨髓增殖性肿瘤或具有耐药性的骨髓增殖性肿瘤的方法，其特征在于，施用LY3009120。In addition, the present invention also provides a method for preventing and/or treating myeloproliferative tumors or drug-resistant myeloproliferative tumors, characterized in that LY3009120 is administered.
在一些实施例中，所述骨髓增殖性肿瘤包括但不限于真性红细胞增多症、原发性血小板增多症或骨髓纤维化，所述骨髓纤维化为原发性骨髓纤维化、继发于真性红细胞增多症的骨髓纤维化或继发于原发性血小板增多症的骨髓纤维化；In some embodiments, the myeloproliferative tumor includes, but is not limited to, polycythemia vera, essential thrombocythemia, or myelofibrosis, which is primary myelofibrosis, secondary to red blood cells vera Myelofibrosis of hyperplasia or myelofibrosis secondary to essential thrombocythemia;
在一些实施例中所述具有耐药性的骨髓增殖性肿瘤包括但不限于具有芦可替尼耐药性的骨髓增殖性肿瘤；In some embodiments, the drug-resistant myeloproliferative tumors include, but are not limited to, ruxolitinib-resistant myeloproliferative tumors;
作为优选，所述具有耐药性的骨髓增殖性肿瘤包括但不限于具有耐药性的真性红细胞增多症、具有耐药性的原发性血小板增多症或具有耐药性的骨髓纤维化，所述骨髓纤维化为原发性骨髓纤维化、继发于真性红细胞增多症的骨髓纤维化或继发于原发性血小板增多症的骨髓纤维化。Preferably, the drug-resistant myeloproliferative tumor includes but is not limited to drug-resistant polycythemia vera, drug-resistant essential thrombocythemia or drug-resistant myelofibrosis, so The myelofibrosis is primary myelofibrosis, myelofibrosis secondary to polycythemia vera, or myelofibrosis secondary to essential thrombocythemia.
由上述技术方案可知，本发明提供了LY3009120在制备治疗骨髓增殖性肿瘤的药物中的应用。所述骨髓增殖性肿瘤为真性红细胞增多症、原发性血小板增多症或骨髓纤维化以及芦可替尼耐药性的骨髓增殖性肿瘤，所述骨髓纤维化为原发性骨髓纤维化、继发于真性红细胞增多症的骨髓纤维化或继发于原发性血小板增多症的骨髓纤维化。It can be known from the above technical solutions that the present invention provides the application of LY3009120 in the preparation of a drug for the treatment of myeloproliferative tumors. The myeloproliferative tumor is polycythemia vera, essential thrombocythemia or myelofibrosis and ruxolitinib-resistant myeloproliferative tumor, and the myelofibrosis is primary myelofibrosis, secondary myelofibrosis. Myelofibrosis arising from polycythemia vera or myelofibrosis secondary to essential thrombocythemia.
本发明具有的技术效果为：The technical effect that the present invention has is:
LY3009120用于骨髓增殖性肿瘤的治疗给广大骨髓增殖性肿瘤的患 者提供了新的治疗途径，给临床医生及患者提供了更多选择。对于芦可替尼耐药的骨髓增殖性肿瘤患者，LY3009120能为患者提供继续的口服药物治疗，免于接受骨髓移植。LY3009120可以化学合成，成本较生物制剂要低。且已经通过I期临床试验，有望顺利通过II、III期临床试验，将来用于临床治疗，具有较好的临床应用前景。The use of LY3009120 in the treatment of myeloproliferative tumors provides a new treatment approach for the majority of patients with myeloproliferative tumors, and provides more choices for clinicians and patients. For patients with ruxolitinib-resistant myeloproliferative tumors, LY3009120 can provide continued oral drug therapy and avoid bone marrow transplantation. LY3009120 can be chemically synthesized, and the cost is lower than that of biological preparations. And it has passed the Phase I clinical trial, and is expected to pass the Phase II and Phase III clinical trials smoothly. It will be used for clinical treatment in the future, and has a good clinical application prospect.
附图说明Description of drawings
图1示实施例1：芦可替尼耐药的骨髓增殖性肿瘤细胞模型HEL RE和SET2 RE的建立结果图；a为HEL RE模型成功构建；b为SET2 RE模型的成功构建； Figure 1 shows the results of Example 1: the establishment of ruxolitinib-resistant myeloproliferative tumor cell models HEL RE and SET2 RE ; a is the successful construction of the HEL RE model; b is the successful construction of the SET2 RE model;
图2示实施例2：LY3009120处理两种骨髓增殖性肿瘤细胞，通过CellTiter-Lumi TM发光法检测细胞增殖的结果图；a为HEL细胞增殖结果图；b为SET2细胞增殖结果图； Figure 2 shows the results of Example 2: LY3009120 treated two kinds of myeloproliferative tumor cells, and the cell proliferation was detected by CellTiter-Lumi TM luminescence method; a is the HEL cell proliferation result; b is the SET2 cell proliferation result;
图3示实施例3：LY3009120处理两种骨髓增殖性肿瘤细胞，使用AnnexinV-PI染色后流式检测细胞凋亡的结果图；a为HEL细胞凋亡结果图；b为SET2细胞凋亡结果图；Figure 3 shows the results of Example 3: LY3009120 treated two myeloproliferative tumor cells, and the results of flow detection of apoptosis after AnnexinV-PI staining; a is the result of HEL cell apoptosis; b is the result of SET2 cell apoptosis ;
图4示实施例4：LY3009120处理两种芦可替尼耐药的骨髓增殖性肿瘤细胞，通过CellTiter-Lumi TM发光法检测细胞增殖的结果图；a为HEL RE细胞增殖结果图；b为SET2 RE细胞增殖结果图； Figure 4 shows the results of Example 4: LY3009120 treated two ruxolitinib-resistant myeloproliferative tumor cells, and the cell proliferation was detected by CellTiter-Lumi TM luminescence method; a is the HEL RE cell proliferation result; b is SET2 RE cell proliferation result graph;
图5示实施例5：LY3009120处理两种芦可替尼耐药的骨髓增殖性肿瘤细胞，使用AnnexinV-PI染色后流式检测细胞凋亡的结果图；a为HEL RE细胞凋亡结果图；b为SET2 RE细胞凋亡结果图。 Figure 5 shows the results of Example 5: LY3009120 treated two ruxolitinib-resistant myeloproliferative tumor cells, and the results of flow detection of apoptosis after AnnexinV-PI staining; a is the result of HEL RE cell apoptosis; b is the result of apoptosis of SET2 RE cells.
具体实施方式Detailed ways
本发明公开了LY3009120在制备治疗骨髓增殖性肿瘤的药物中的应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本 文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。The invention discloses the application of LY3009120 in the preparation of a drug for the treatment of myeloproliferative tumors, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.
在本发明中，通过细胞系模型来明确LY3009120对骨髓增殖性肿瘤细胞(耐药与非耐药)的抑制作用。In the present invention, the inhibitory effect of LY3009120 on myeloproliferative tumor cells (drug-resistant and non-drug-resistant) was clarified by a cell line model.
在一些实施方案中，本发明基于常用的两种含JAK2-V617F突变的人源骨髓增殖性肿瘤细胞系，即HEL细胞(Human erythroleukemia cell line，人红白血病细胞)和SET2细胞(原发性血小板增多症细胞)，分别建立了各自芦可替尼耐药细胞模型HEL RE和SET2 RE。耐药模型的构建方法为使用低于
Figure PCTCN2022088677-appb-000003
细胞IC50浓度开始加芦可替尼，缓慢递增至高浓度，维持细胞不被杀灭，通过比较IC50来验证模型的构建是否成功。 In some embodiments, the present invention is based on two commonly used human myeloproliferative tumor cell lines containing the JAK2-V617F mutation, namely HEL cells (Human erythroleukemia cell line, human erythroleukemia cell line) and SET2 cell (essential platelet hyperplasia cells), and established their respective ruxolitinib-resistant cell models HEL RE and SET2 RE , respectively. The resistance model was constructed using methods below
Figure PCTCN2022088677-appb-000003
The IC50 concentration of cells was initially added with ruxolitinib, and the concentration was slowly increased to a high concentration to keep the cells from being killed. The successful model construction was verified by comparing IC50.
在一些实施方案中，本发明使用递增浓度的LY3009120处理两个骨髓增殖性肿瘤细胞株(HEL和SET2)，通过CellTiter-Lumi TM发光法检测细胞的增殖。结果显示，LY3009120可成功抑制HEL和SET2细胞的增殖。 In some embodiments, the present invention treats two myeloproliferative tumor cell lines (HEL and SET2) with increasing concentrations of LY3009120, and cell proliferation is detected by CellTiter-Lumi ™ luminescence assay. The results showed that LY3009120 could successfully inhibit the proliferation of HEL and SET2 cells.
在一些实施方案中，本发明使用递增浓度的LY3009120处理两个骨髓增殖性肿瘤细胞株(HEL和SET2)，使用AnnexinV-PI染色后流式检测细胞的凋亡情况。结果显示，LY3009120可促进HEL和SET2细胞的凋亡。In some embodiments, the present invention treats two myeloproliferative tumor cell lines (HEL and SET2) with increasing concentrations of LY3009120, and the apoptosis of the cells is detected by flow cytometry after AnnexinV-PI staining. The results showed that LY3009120 could promote the apoptosis of HEL and SET2 cells.
由此可见，LY3009120可用于治疗骨髓增殖性肿瘤疾病。Thus, LY3009120 can be used for the treatment of myeloproliferative tumor diseases.
进一步的，在一些实施方案中，本发明使用递增浓度的LY3009120处理芦可替尼耐药的骨髓增殖性肿瘤细胞(HEL RE和SET2 RE)，通过CellTiter-Lumi TM发光法检测细胞的增殖。结果显示，LY3009120可成功抑制HEL RE和SET2 RE细胞的增殖。 Further, in some embodiments, the present invention uses increasing concentrations of LY3009120 to treat ruxolitinib-resistant myeloproliferative tumor cells (HEL RE and SET2 RE ), and the cell proliferation is detected by CellTiter-Lumi ™ luminescence method. The results showed that LY3009120 could successfully inhibit the proliferation of HEL RE and SET2 RE cells.
在一些实施方案中，本发明使用递增浓度的LY3009120处理芦可替尼耐药的骨髓增殖性肿瘤细胞(HEL RE和SET2 RE)，使用AnnexinV-PI染色后流式检测细胞的凋亡情况。结果显示，LY3009120可促进HEL RE和SET2 RE细胞的凋亡。 In some embodiments, the present invention uses increasing concentrations of LY3009120 to treat ruxolitinib-resistant myeloproliferative tumor cells (HEL RE and SET2 RE ), and the apoptosis of the cells is detected by flow cytometry after AnnexinV-PI staining. The results showed that LY3009120 could promote the apoptosis of HEL RE and SET2 RE cells.
由此可见，LY3009120可用于治疗芦可替尼耐药的骨髓增殖性肿瘤疾病。It can be seen that LY3009120 can be used for the treatment of ruxolitinib-resistant myeloproliferative tumor diseases.
进一步的，本发明提供了LY3009120在制备抑制HEL和SET2细胞的增殖、促进HEL和SET2细胞的凋亡的药物中的应用。Further, the present invention provides the application of LY3009120 in the preparation of medicines for inhibiting the proliferation of HEL and SET2 cells and promoting the apoptosis of HEL and SET2 cells.
综上所述，本发明提供了LY3009120在制备治疗骨髓增殖性肿瘤的药物中的应用。To sum up, the present invention provides the application of LY3009120 in the preparation of a medicament for the treatment of myeloproliferative tumors.
进一步的，所述骨髓增殖性肿瘤为真性红细胞增多症、原发性血小板增多症和骨髓纤维化(包括原发性骨髓纤维化、继发于真性红细胞增多症的骨髓纤维化和继发于原发性血小板增多症的骨髓纤维化)和具有耐药性的骨髓增殖性肿瘤。Further, the myeloproliferative tumor is polycythemia vera, essential thrombocythemia, and myelofibrosis (including primary myelofibrosis, myelofibrosis secondary to polycythemia vera, and myelofibrosis secondary to primary polycythemia vera). myelofibrosis with thrombocythemia) and drug-resistant myeloproliferative neoplasms.
在一些实施方案中，所述具有耐药性的骨髓增殖性肿瘤为芦可替尼耐药性的骨髓增殖性肿瘤。In some embodiments, the drug-resistant myeloproliferative tumor is a ruxolitinib-resistant myeloproliferative tumor.
在一些实施方案中，所述具有耐药性的骨髓增殖性肿瘤为具有耐药性的真性红细胞增多症、具有耐药性的骨髓纤维化(包括原发性骨髓纤维化、继发于真性红细胞增多症的骨髓纤维化和继发于原发性血小板增多症的骨髓纤维化)以及具有耐药性的原发性血小板增多症。In some embodiments, the drug-resistant myeloproliferative neoplasm is drug-resistant polycythemia vera, drug-resistant myelofibrosis (including primary myelofibrosis, secondary to erythrocyte vera) hypertrophic myelofibrosis and myelofibrosis secondary to essential thrombocythemia) and drug-resistant essential thrombocythemia.
其中，所述药物为LY3009120。Wherein, the drug is LY3009120.
进一步的，所述药物还包括药学上可接受的辅料。Further, the medicine also includes pharmaceutically acceptable excipients.
所述药物可以为当前药品领域任何剂型，包括口服制剂或注射制剂。The drug can be any dosage form in the current pharmaceutical field, including oral preparations or injection preparations.
各药物剂型可根据该剂型实际需要选取合适的可接受辅料来制备，这属于本领域常规的剂型制备技术。如制成胶囊剂、片剂、注射粉剂等。Each pharmaceutical dosage form can be prepared by selecting appropriate acceptable excipients according to the actual needs of the dosage form, which belongs to the conventional dosage form preparation technology in the art. Such as capsules, tablets, injection powder and so on.
本发明中所用试剂或仪器均可由市场购得。The reagents or instruments used in the present invention can be purchased from the market.
下面结合实施例，进一步阐述本发明：Below in conjunction with embodiment, the present invention is further elaborated:
实施例1、两种常见芦可替尼耐药细胞模型的建立(HEL RE，SET2 RE)。 Example 1. Establishment of two common ruxolitinib-resistant cell models (HEL RE , SET2 RE ).
一、材料与方法1. Materials and methods
1、细胞系1. Cell line
HEL(Human erythroleukemia cell line)、芦可替尼耐药的HEL细胞、SET2细胞及芦可替尼耐药的SET2细胞均培养于含20％热灭活胎牛血清(Gibco)及1％青霉素/链霉素的RPMI培养基(Gibco)。HEL (Human erythroleukemia cell line), ruxolitinib-resistant HEL cells, SET2 cells and ruxolitinib-resistant SET2 cells were cultured in 20% heat-inactivated fetal bovine serum (Gibco) and 1% penicillin/ Streptomycin in RPMI medium (Gibco).
芦可替尼耐药的HEL模型即HEL RE模型，构建方法为使用低于原始细胞IC50浓度开始加芦可替尼，缓慢递增至高浓度，维持细胞不被杀灭。我们的起始浓度为0.1μM，细胞出现增殖就加药，加药梯度为1.25倍递增， 终浓度为2.0μM。4-6周后获得稳定的耐药细胞。另外一种芦可替尼耐药模型为SET2 RE即SET2-resistant模型，构建方法为使用低于原始细胞IC50浓度开始加芦可替尼，缓慢递增至高浓度，维持细胞不被杀灭。我们的起始浓度为0.03μM，细胞出现增殖就加药，加药梯度为1.25倍递增，终浓度为0.5μM。4-6周后获得稳定的耐药细胞。 The HEL model with resistance to ruxolitinib, the HEL RE model, was constructed by adding ruxolitinib at a concentration lower than the original cell IC50, and slowly increasing to a high concentration to maintain the cells from being killed. Our starting concentration was 0.1 μM, and the drug was added as soon as the cells proliferated. The dosing gradient was 1.25-fold increments, and the final concentration was 2.0 μM. Stable resistant cells were obtained after 4-6 weeks. Another ruxolitinib resistance model is SET2 RE , or SET2-resistant model. The construction method is to start adding ruxolitinib at a concentration lower than the original cell IC50, and slowly increase it to a high concentration to keep the cells from being killed. Our starting concentration was 0.03 μM, and the drug was added as soon as the cells proliferated. The dosing gradient was 1.25-fold increments, and the final concentration was 0.5 μM. Stable resistant cells were obtained after 4-6 weeks.
2、抑制剂2. Inhibitors
芦可替尼及LY3009120均购自Selleck公司，溶于DMSO，母液浓度为10mM，冻存于-80℃，工作液采用RPMI培养基稀释至指定倍数后处理细胞。芦可替尼具体为磷酸芦可替尼。Ruxolitinib and LY3009120 were purchased from Selleck Company, dissolved in DMSO, the concentration of the stock solution was 10 mM, frozen at -80 °C, and the working solution was diluted with RPMI medium to the specified times before treating the cells. Ruxolitinib is specifically ruxolitinib phosphate.
3、体外抑制试验3. In vitro inhibition test
为了检测抑制剂的抗增殖效应，上述细胞系以每孔3000个细胞/100μL体系培养，加入递增浓度的芦可替尼(HEL细胞浓度梯度：0，0.1，1，2.5，5μM；SET2细胞浓度梯度：0，0.01，0.1，1，2.5，5μM)，DMSO补齐至等量。设4个平行重复组，并设3个空白孔(不含细胞的培养液孔)。48小时后通过CellTiter-Lumi TM发光法(碧云天)检测细胞的增殖。多功能酶标仪读数，IC50通过GraphPadprism计算得来。 In order to test the anti-proliferative effect of the inhibitor, the above cell lines were cultured in a system of 3000 cells/100 μL per well, and increasing concentrations of ruxolitinib were added (HEL cell concentration gradient: 0, 0.1, 1, 2.5, 5 μM; SET2 cell concentration Gradient: 0, 0.01, 0.1, 1, 2.5, 5 μM), DMSO rounded up to equal volume. Set up 4 parallel replicate groups, and set up 3 blank wells (culture medium wells without cells). Cell proliferation was detected by CellTiter-Lumi ™ luminescence method (Biyuntian) after 48 hours. Multi-plate reader reading, IC50 calculated by GraphPadprism.
细胞增殖率计算公式：细胞增殖率＝(加药组Luminescence值-空白孔平均Luminescence值)/(DMSO对照组Luminescence值-空白孔平均Luminescence值)×100％。The calculation formula of cell proliferation rate: cell proliferation rate=(Luminescence value of drug-added group-average Luminescence value of blank well)/(Luminescence value of DMSO control group-average Luminescence value of blank well)×100%.
评价模型是否构建成功的方法是比较耐药细胞与原始细胞的IC50，比值为耐药指数，大于3即为构建成功。The method to evaluate whether the model is successfully constructed is to compare the IC50 of drug-resistant cells and original cells, and the ratio is the drug-resistance index.
二、结果分析2. Analysis of the results
图1a中，HEL细胞IC50为2.31(1.42-4.36)μM，HEL RE细胞IC50为44.7(16.0-370)μM，其耐药指数为19.0，提示耐药模型HEL RE的成功构建。图1b中，SET2细胞IC50为0.048(0.035-0.065)μM，SET2 RE细胞IC50为34.3(16.0–108)μM，其耐药指数为714，提示耐药模型SET2 RE的成功构建。图中增殖率结果显示为平均数±标准差，图中两组增殖率的比较采用t检验(***p<0.001)，IC50显示为均数。结果分析中显示为均数(95％可信区间)。 In Figure 1a, the IC50 of HEL cells was 2.31 (1.42-4.36) μM, the IC50 of HEL RE cells was 44.7 (16.0-370) μM, and the drug resistance index was 19.0, indicating the successful construction of the drug resistance model HEL RE . In Figure 1b, the IC50 of SET2 cells was 0.048 (0.035-0.065) μM, and the IC50 of SET2 RE cells was 34.3 (16.0–108) μM, and the drug resistance index was 714, indicating the successful construction of the drug resistance model SET2 RE . The results of the proliferation rate in the figure are shown as the mean ± standard deviation, the comparison of the proliferation rate between the two groups in the figure is by t test (***p<0.001), and the IC50 is shown as the mean. Results are shown as means (95% confidence interval) in the analysis.
实施例2、LY3009120可抑制骨髓增殖性肿瘤细胞的增殖Example 2. LY3009120 can inhibit the proliferation of myeloproliferative tumor cells
为了检测LY3009120对骨髓增殖性肿瘤耐药细胞增殖能力的影响，方法为分别使用递增浓度的芦可替尼及LY3009120处理HEL原始细胞株及SET2原始细胞株，通过CellTiter-Lumi TM发光法检测细胞的增殖，方法同实施例1，结果见图2。 In order to detect the effect of LY3009120 on the proliferation ability of myeloproliferative tumor-resistant cells, the method was to treat HEL original cell line and SET2 original cell line with increasing concentrations of ruxolitinib and LY3009120 respectively, and detect the cell proliferation by CellTiter-Lumi TM luminescence method. Proliferation, the method is the same as that of Example 1, and the results are shown in Figure 2.
图2a反映了在HEL细胞中，芦可替尼处理组药物浓度(平均增殖率％±标准差)为：0μM(100±2.40)(图中未显示)、0.1μM(77.9±2.43)、1μM(43.2±3.75)、2.5μM(50.6±2.19)、5μM(47.0±1.15)；LY3009120处理组药物浓度(平均增殖率)为：0μM(100±7.65)(图中未显示)、0.1μM(61.7±5.66)、1μM(39.5±1.39)、2.5μM(15.5±1.05)、5μM(6.19±1.02)。图2b反映了在SET2细胞中，芦可替尼处理组药物浓度(平均增殖率)为：0μM(100±5.51)(图中未显示)、0.01μM(80.7±9.07)、0.1μM(30.1±1.95)、1μM(12.6±1.23)、2.5μM(12.1±1.13)、5μM(12.2±0.74)；LY3009120处理组药物浓度(平均增殖率)为：0μM(100±4.72)(图中未显示)、0.01μM(99.4±6.46)、0.1μM(44.4±3.86)、1μM(23.2±1.03)、2.5μM(22.2±2.75)、5μM(11.8±1.75)。图中两组增殖率的比较采用t检验(**p<0.01,***p<0.001)。这提示LY3009120可抑制HEL细胞及SET2细胞的增殖，该效应随药物浓度增加而增加，其抑制效果与芦可替尼相当。Figure 2a reflects that in HEL cells, the drug concentrations (mean proliferation % ± SD) of the ruxolitinib-treated group were: 0 μM (100 ± 2.40) (not shown in the figure), 0.1 μM (77.9 ± 2.43), 1 μM (43.2±3.75), 2.5μM (50.6±2.19), 5μM (47.0±1.15); LY3009120 treatment group drug concentration (average proliferation rate): 0μM (100±7.65) (not shown in the figure), 0.1μM (61.7 ±5.66), 1 μM (39.5 ± 1.39), 2.5 μM (15.5 ± 1.05), 5 μM (6.19 ± 1.02). Figure 2b reflects that in SET2 cells, the drug concentrations (mean proliferation rate) of the ruxolitinib-treated group were: 0 μM (100 ± 5.51) (not shown in the figure), 0.01 μM (80.7 ± 9.07), 0.1 μM (30.1 ± 1.95), 1 μM (12.6 ± 1.23), 2.5 μM (12.1 ± 1.13), 5 μM (12.2 ± 0.74); LY3009120 treatment group drug concentration (average proliferation rate): 0 μM (100 ± 4.72) (not shown in the figure), 0.01 μM (99.4±6.46), 0.1 μM (44.4±3.86), 1 μM (23.2±1.03), 2.5 μM (22.2±2.75), 5 μM (11.8±1.75). The comparison of the proliferation rate between the two groups in the figure was performed by t test (**p<0.01, ***p<0.001). This suggests that LY3009120 can inhibit the proliferation of HEL cells and SET2 cells, and the effect increases with the increase of drug concentration, and its inhibitory effect is comparable to that of ruxolitinib.
实施例3、LY3009120可促进骨髓增殖性肿瘤细胞的凋亡Example 3. LY3009120 can promote the apoptosis of myeloproliferative tumor cells
一、材料与方法1. Materials and methods
1、细胞系与抑制剂同实施例1。1. Cell lines and inhibitors are the same as in Example 1.
2、细胞凋亡检测2. Apoptosis detection
为了检测抑制剂的促凋亡效应，分别使用芦可替尼及LY3009120处理HEL原始细胞株及SET2原始细胞株24小时(浓度：0、0.1、0.5、1μM)，补齐DMSO至等量。设3个平行重复组，使用AnnexinV和PI染色后流式细胞术检测细胞的凋亡情况。In order to detect the pro-apoptotic effect of the inhibitor, HEL original cell line and SET2 original cell line were treated with ruxolitinib and LY3009120 for 24 hours (concentration: 0, 0.1, 0.5, 1 μM), respectively, and DMSO was supplemented to an equal amount. Three parallel replicate groups were set up, and the apoptosis of cells was detected by flow cytometry after AnnexinV and PI staining.
细胞凋亡率计算公式：细胞凋亡率＝早期凋亡细胞比率(Annexin  V +/PI -)+晚期凋亡细胞及坏死细胞比率(AnnexinV +/PI +)。 The calculation formula of apoptosis rate: apoptosis rate = ratio of early apoptotic cells (Annexin V + /PI - ) + ratio of late apoptotic cells and necrotic cells (Annexin V + /PI + ).
二、结果分析2. Analysis of the results
图3a中，HEL细胞芦可替尼处理组药物浓度(平均凋亡率％±标准差)为：0μM(4.37±0.318)、0.1μM(5.59±0.097)、0.5μM(6.63±1.12)、1μM(6.68±0.304)；LY3009120处理组药物浓度(凋亡率)为0μM(4.37±0.318)、0.1μM(5.96±0.036)、0.5μM(7.30±0.358)、1μM(8.08±0.560)；图3b中，SET2细胞芦可替尼处理组药物浓度(平均凋亡率)为：0μM(5.47±0.894)、0.1μM(14.8±0.397)、0.5μM(19.3±2.64)、1μM(23.1±2.33)；LY3009120处理组药物浓度(凋亡率)为0μM(5.47±0.894)、0.1μM(8.13±0.494)、0.5μM(17.3±3.08)、1μM(17.1±1.97)。图中两组凋亡率的比较采用t检验(*p<0.05,**p<0.01,***p<0.001)。这提示LY3009120能促进骨髓增殖性肿瘤细胞的凋亡，这种效应随浓度的上升而增强，其促凋亡效果与芦可替尼相当。In Figure 3a, the drug concentrations (mean apoptotic rate %±SD) of HEL cells treated with ruxolitinib were: 0 μM (4.37±0.318), 0.1 μM (5.59±0.097), 0.5 μM (6.63±1.12), 1 μM (6.68±0.304); the drug concentration (apoptosis rate) in the LY3009120 treatment group was 0 μM (4.37±0.318), 0.1 μM (5.96±0.036), 0.5 μM (7.30±0.358), 1 μM (8.08±0.560); in Figure 3b , the drug concentration (average apoptosis rate) of ruxolitinib treatment group in SET2 cells was: 0 μM (5.47±0.894), 0.1 μM (14.8±0.397), 0.5 μM (19.3±2.64), 1 μM (23.1±2.33); LY3009120 The drug concentration (apoptosis rate) in the treatment group was 0 μM (5.47±0.894), 0.1 μM (8.13±0.494), 0.5 μM (17.3±3.08), and 1 μM (17.1±1.97). The comparison of apoptosis rate between the two groups in the figure was performed by t test (*p<0.05, **p<0.01, ***p<0.001). This suggests that LY3009120 can promote the apoptosis of myeloproliferative tumor cells, and this effect increases with increasing concentration, and its pro-apoptotic effect is comparable to that of ruxolitinib.
实施例4、LY3009120可抑制耐药骨髓增殖性肿瘤细胞的增殖。Example 4. LY3009120 can inhibit the proliferation of drug-resistant myeloproliferative tumor cells.
为了检测LY3009120对骨髓增殖性肿瘤耐药细胞增殖能力的影响，方法为分别使用递增浓度的芦可替尼及LY3009120处理HEL RE及SET2 RE，通过CellTiter-Lumi TM发光法检测细胞的增殖。方法同实施例1，结果见图4。 In order to detect the effect of LY3009120 on the proliferation ability of myeloproliferative tumor drug-resistant cells, the method was to treat HEL RE and SET2 RE with increasing concentrations of ruxolitinib and LY3009120, respectively, and detect the cell proliferation by CellTiter-Lumi TM luminescence method. The method is the same as in Example 1, and the results are shown in Figure 4.
图4a反映了在HEL RE细胞中，芦可替尼处理组药物浓度(平均增殖率％±标准差)为：0μM(100±1.73)(图中未显示)、0.1μM(103.17±1.9)、1μM(91.14±1.63)、2.5μM(91.42±1.7)、5μM(84.15±1.38)；LY3009120处理组药物浓度(平均增殖率)为：0μM(100±2.91)(图中未显示)、0.1μM(88.11±13.93)、1μM(14.55±1.91)、2.5μM(11.43±0.62)、5μM(11.65±0.42)。图4b反映了在SET RE细胞中，芦可替尼处理组药物浓度(平均增殖率)为：0μM(100±0.38)(图中未显示)、0.01μM(88.19±4.11)、0.1μM(82.21±5.1)、1μM(71.18±2.37)、2.5μM(68.34±3.79)、5μM(60.17±3.4)；LY3009120处理组药物浓度(平均增殖率)为：0μM(100±1.13)(图中未显示)、0.01μM(88.27±5.18)、0.1μM(29.97±1.05)、1μM (12.33±1.3)、2.5μM(11.67±0.99)、5μM(6.96±0.68)。图中两组增殖率的比较采用t检验(***p<0.001)。这提示LY3009120可抑制耐药骨髓增殖性肿瘤细胞的增殖，该效应随药物浓度增加而增加。 Figure 4a reflects that in HEL RE cells, the drug concentrations (mean proliferation % ± SD) of the ruxolitinib treatment group were: 0 μM (100 ± 1.73) (not shown in the figure), 0.1 μM (103.17 ± 1.9), 1μM (91.14±1.63), 2.5μM (91.42±1.7), 5μM (84.15±1.38); LY3009120 treatment group drug concentration (average proliferation rate): 0μM (100±2.91) (not shown in the figure), 0.1μM ( 88.11±13.93), 1 μM (14.55±1.91), 2.5 μM (11.43±0.62), 5 μM (11.65±0.42). Figure 4b reflects that in SET RE cells, the drug concentrations (mean proliferation rate) of the ruxolitinib-treated group were: 0 μM (100 ± 0.38) (not shown in the figure), 0.01 μM (88.19 ± 4.11), 0.1 μM (82.21 μM) ±5.1), 1 μM (71.18 ± 2.37), 2.5 μM (68.34 ± 3.79), 5 μM (60.17 ± 3.4); LY3009120 treatment group drug concentration (average proliferation rate): 0 μM (100 ± 1.13) (not shown in the figure) , 0.01μM (88.27±5.18), 0.1μM (29.97±1.05), 1μM (12.33±1.3), 2.5μM (11.67±0.99), 5μM (6.96±0.68). The comparison of the proliferation rate between the two groups in the figure was performed by t test (***p<0.001). This suggests that LY3009120 can inhibit the proliferation of drug-resistant myeloproliferative tumor cells, and this effect increases with increasing drug concentration.
实施例5、LY3009120可促进耐药骨髓增殖性肿瘤细胞的凋亡Example 5. LY3009120 can promote the apoptosis of drug-resistant myeloproliferative tumor cells
为了检测LY3009120对骨髓增殖性肿瘤耐药细胞存活能力的影响，方法为分别使用递增浓度的芦可替尼及LY3009120处理HEL RE及SET2 RE，使用AnnexinV和PI染色后流式检测细胞的凋亡情况。方法同实施例3，结果见图5。 In order to detect the effect of LY3009120 on the survival of myeloproliferative tumor-resistant cells, the method was to treat HEL RE and SET2 RE with increasing concentrations of ruxolitinib and LY3009120, respectively, and to detect the apoptosis of cells after staining with AnnexinV and PI. . The method is the same as in Example 3, and the results are shown in Figure 5.
图5a中，HEL RE细胞芦可替尼处理组药物浓度(平均凋亡率％±标准差)为：0μM(2.14±0.111)、0.1μM(1.67±0.128)、0.5μM(2.04±0.128)、1μM(1.88±0.238)；LY3009120处理组药物浓度(凋亡率)为0μM(2.14±0.111)、0.1μM(3.6±0.259)、0.5μM(14.7±1.12)、1μM(16.4±0.786)。图5b中，SET2 RE细胞芦可替尼处理组药物浓度(平均凋亡率)为：0μM(1.37±0.156)、0.1μM(0.927±0.184)、0.5μM(1.13±0.232)、1μM(1.78±0.261)；LY3009120处理组药物浓度(凋亡率)为0μM(1.37±0.156)、0.1μM(1.96±0.155)、0.5μM(9.19±1.74)、1μM(10.5±1.56)。图中两组凋亡率的比较采用t检验(**p<0.01,***p<0.001)。这说明LY3009120可促进芦可替尼耐药的骨髓增殖性肿瘤细胞的的凋亡，该效应随药物浓度的增加而增加。 In Figure 5a, the drug concentrations (mean apoptotic rate %±SD) of HEL RE cells treated with ruxolitinib were: 0 μM (2.14±0.111), 0.1 μM (1.67±0.128), 0.5 μM (2.04±0.128), 1μM (1.88±0.238); the drug concentration (apoptosis rate) in LY3009120 treatment group was 0μM (2.14±0.111), 0.1μM (3.6±0.259), 0.5μM (14.7±1.12), 1μM (16.4±0.786). In Figure 5b, the drug concentrations (average apoptosis rate) of ruxolitinib-treated SET2 RE cells were: 0 μM (1.37±0.156), 0.1 μM (0.927±0.184), 0.5 μM (1.13±0.232), 1 μM (1.78±0.184) 0.261); the drug concentration (apoptosis rate) of LY3009120 treatment group was 0μM (1.37±0.156), 0.1μM (1.96±0.155), 0.5μM (9.19±1.74), 1μM (10.5±1.56). The comparison of the apoptosis rate between the two groups in the figure was performed by t test (**p<0.01, ***p<0.001). This indicates that LY3009120 can promote the apoptosis of ruxolitinib-resistant myeloproliferative tumor cells, and the effect increases with the increase of drug concentration.
以上对本发明所提供的LY3009120在制备治疗骨髓增殖性肿瘤的药物中的应用。进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。The above is about the application of the LY3009120 provided by the present invention in the preparation of a medicament for the treatment of myeloproliferative tumors. described in detail. The principles and implementations of the present invention are described herein by using specific examples, and the descriptions of the above embodiments are only used to help understand the method and the core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can also be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims (12)

  1. LY3009120在制备预防和/或治疗骨髓增殖性肿瘤的药物中的应用。Application of LY3009120 in the preparation of medicaments for preventing and/or treating myeloproliferative tumors.
  2. 根据权利要求1所述的应用，其特征在于，所述骨髓增殖性肿瘤包括但不限于真性红细胞增多症、原发性血小板增多症或骨髓纤维化，所述骨髓纤维化为原发性骨髓纤维化、继发于真性红细胞增多症的骨髓纤维化或继发于原发性血小板增多症的骨髓纤维化。The use according to claim 1, wherein the myeloproliferative tumor includes but is not limited to polycythemia vera, essential thrombocythemia or myelofibrosis, and the myelofibrosis is primary myelofibrosis fibrosis, myelofibrosis secondary to polycythemia vera, or myelofibrosis secondary to essential thrombocythemia.
  3. LY3009120在制备预防和/或治疗具有耐药性的骨髓增殖性肿瘤的药物中的应用。Application of LY3009120 in the preparation of a medicament for preventing and/or treating drug-resistant myeloproliferative tumors.
  4. 根据权利要求3所述的应用，其特征在于，所述具有耐药性的骨髓增殖性肿瘤包括但不限于具有芦可替尼耐药性的骨髓增殖性肿瘤。The use according to claim 3, wherein the drug-resistant myeloproliferative tumors include but are not limited to ruxolitinib-resistant myeloproliferative tumors.
  5. 根据权利要求3所述的应用，其特征在于，所述具有耐药性的骨髓增殖性肿瘤包括但不限于具有耐药性的真性红细胞增多症、具有耐药性的原发性血小板增多症或具有耐药性的骨髓纤维化，所述骨髓纤维化为原发性骨髓纤维化、继发于真性红细胞增多症的骨髓纤维化或继发于原发性血小板增多症的骨髓纤维化。The use according to claim 3, wherein the drug-resistant myeloproliferative tumor includes but is not limited to drug-resistant polycythemia vera, drug-resistant essential thrombocythemia or Drug-resistant myelofibrosis, which is primary myelofibrosis, myelofibrosis secondary to polycythemia vera, or myelofibrosis secondary to essential thrombocythemia.
  6. LY3009120在制备抑制HEL和/或SET2细胞的增殖的药物中的应用。Application of LY3009120 in the preparation of a drug for inhibiting the proliferation of HEL and/or SET2 cells.
  7. LY3009120在制备抑制具有耐药性的HEL和/或SET2细胞的增殖的药物中的应用。Use of LY3009120 in the preparation of a drug for inhibiting the proliferation of drug-resistant HEL and/or SET2 cells.
  8. LY3009120在制备促进HEL和/或SET2细胞的凋亡的药物中的应用。Application of LY3009120 in the preparation of medicines for promoting apoptosis of HEL and/or SET2 cells.
  9. LY3009120在制备促进具有耐药性的HEL和/或SET2细胞的凋亡的药物中的应用。Application of LY3009120 in the preparation of a drug for promoting apoptosis of drug-resistant HEL and/or SET2 cells.
  10. 根据权利要求1至9中任一项所述的应用，其特征在于，所述药物还包含药学上可接受的辅料；所述药物的剂型为口服制剂或注射制剂。The application according to any one of claims 1 to 9, wherein the medicine further comprises a pharmaceutically acceptable adjuvant; and the dosage form of the medicine is an oral preparation or an injection preparation.
  11. 预防和/或治疗骨髓增殖性肿瘤或具有耐药性的骨髓增殖性肿瘤 的方法，其特征在于，施用LY3009120。A method for preventing and/or treating myeloproliferative tumors or drug-resistant myeloproliferative tumors, characterized by administering LY3009120.
  12. 如权利要求11所述的方法，其特征在于，所述骨髓增殖性肿瘤包括但不限于真性红细胞增多症、原发性血小板增多症或骨髓纤维化，所述骨髓纤维化为原发性骨髓纤维化、继发于真性红细胞增多症的骨髓纤维化或继发于原发性血小板增多症的骨髓纤维化；The method of claim 11, wherein the myeloproliferative tumor includes but is not limited to polycythemia vera, essential thrombocythemia, or myelofibrosis, and the myelofibrosis is primary myelofibrosis myelofibrosis secondary to polycythemia vera or myelofibrosis secondary to essential thrombocythemia;
    所述具有耐药性的骨髓增殖性肿瘤包括但不限于具有芦可替尼耐药性的骨髓增殖性肿瘤；The drug-resistant myeloproliferative tumors include, but are not limited to, ruxolitinib-resistant myeloproliferative tumors;
    作为优选，所述具有耐药性的骨髓增殖性肿瘤包括但不限于具有耐药性的真性红细胞增多症、具有耐药性的原发性血小板增多症或具有耐药性的骨髓纤维化，所述骨髓纤维化为原发性骨髓纤维化、继发于真性红细胞增多症的骨髓纤维化或继发于原发性血小板增多症的骨髓纤维化。Preferably, the drug-resistant myeloproliferative tumor includes but is not limited to drug-resistant polycythemia vera, drug-resistant essential thrombocythemia or drug-resistant myelofibrosis, so The myelofibrosis is primary myelofibrosis, myelofibrosis secondary to polycythemia vera, or myelofibrosis secondary to essential thrombocythemia.
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