CN115054695A - Application of MEK/ERK signal pathway inhibitor in preparation of medicine for treating myeloproliferative tumors - Google Patents
Application of MEK/ERK signal pathway inhibitor in preparation of medicine for treating myeloproliferative tumors Download PDFInfo
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- CN115054695A CN115054695A CN202210846544.4A CN202210846544A CN115054695A CN 115054695 A CN115054695 A CN 115054695A CN 202210846544 A CN202210846544 A CN 202210846544A CN 115054695 A CN115054695 A CN 115054695A
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- mek
- apoptosis
- myeloproliferative
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Abstract
The invention relates to the technical field of medicines, in particular to application of an MEK/ERK signal pathway inhibitor in preparing a medicine for treating myeloproliferative tumors. The MEK/ERK signal channel inhibitor used for treating the myeloproliferative tumor provides a new treatment approach for patients with the myeloproliferative tumor, and provides more choices for clinicians and patients. For patients with an RCT-resistant myeloproliferative tumor, MEK/ERK signaling pathway inhibitors can provide continued oral drug therapy to the patient, free of bone marrow transplantation. MEK/ERK signaling pathway inhibitors can be chemically synthesized at lower cost than biologies. The inhibitors in the application document pass through a first-stage clinical experiment, have fewer side effects and are well tolerated by clinical patients.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to application of an MEK/ERK signal pathway inhibitor in preparing a medicine for treating myeloproliferative tumors.
Background
Myeloproliferative neoplasms (MPNs) refer to a group of neoplastic diseases caused by clonal proliferation of one or more lines of myeloid cells that are relatively mature in differentiation. Clinically, it is manifested by one or more kinds of blood cell hyperplasia accompanied by enlargement of liver, spleen or lymph nodes. The 2016 World Health Organization (WHO) classified and revised bone marrow tumors, classified and revised Polycythemia Vera (PV), Primary Myelofibrosis (PMF), and primary thrombocythemia (ET) into the category of Philadelphia-negative classical myeloproliferative tumors (Philadelphia-negative classical myeloproliferative tumors). Myeloproliferative tumors are clonal hematopoietic stem cell diseases, and the major disease-driving gene mutations include JAK2/V617F, CALR, MPL mutations, of which JAK2/V617F mutations are the most common type, found in 95% of PV, 50-60% of ET, and 55-65% of PMF patients. Activation of the gene leads to activation of the JAK-STAT pathway leading to disease. About 20 ten thousand of myeloproliferative tumor patients are newly increased every year around the world, and a heavy burden is brought to a medical health system.
Prior to the advent of Ruxolitinib (RUX), common treatments for myeloproliferative tumors included hydroxyurea and polyethylene glycol-recombinant interferon-alpha 2 a. 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. Interferon has a high toxic side effect, which limits its use. Lucigenin is approved by the FDA as a JAK1/JAK2 inhibitor for primary use in Myelofibrosis (MF) at moderate and high risk, and as a secondary drug for Hydroxyurea (HU) resistant or intolerant PV patients. Results of second and third phase clinical trials suggest that RUX can reduce spleen volume and alleviate symptoms in middle and high risk MF and PV patients compared to optimal therapy. But do notThere are many problems with the use of lucentitinib, and the results of the COMFORT and RESPONSE clinical trials show that MF patients receiving lucentitinib are more anemia-prone. More seriously, prolonged use of type I JAK inhibitors such as RUX induces drug resistance, more than 40% of MF patients treated for 1 year, and cross-drug resistance between several JAK inhibitors has also been discovered in clinical studies. 8 months 2019, a novel oral JAK2 selective inhibitor Fedratinib was approved by FDA in the united states
For use in adult intermediate, high risk primary or secondary (post-PV or post-ET) MF, including patients previously treated with luccotinib. The FDA also alerts the black box that Fedratinib may be causing encephalopathy, including the risk of weirnike encephalopathy. 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 therefore 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 the mode and protocol of transplantation is uncertain and it is unclear whether allogeneic or haploid allografts are selected. Furthermore, when selecting transplantation, the graft-related mortality and the long-term nature of myeloproliferative tumors must be considered. Currently, bone marrow transplantation is mainly used for treating high-risk myelofibrosis patients, but the timing of bone marrow transplantation for other types of myeloproliferative tumor patients needs further discussion and research confirmation. Bone marrow transplantation is expensive and in the current medical environment, the treatment option is not available to most patients.
Although luccotinib is a milestone drug for myeloproliferative tumor therapy, luccotinib is currently applied in a narrow range, and myelosuppression, as a common side effect, limits its application in the major indication MF. The inability of lucentitinib to reduce the load of the mutated gene means that lucentitinib treatment does not achieve molecular level relief of the disease and is not able to fundamentally treat myeloproliferative tumors. Especially after the emergence of drug resistance to luccotinib, the limitation of therapeutic drugs is a major challenge at present.
Disclosure of Invention
In view of the above, the present invention aims to provide an effective, safe and reliable medicine for myeloproliferative tumor patients, especially for drug-resistant myeloproliferative tumor patients of luctinib, aiming at the problems existing in the current myeloproliferative tumor treatment.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of MEK/ERK signal pathway inhibitor in preparing a medicament for treating myeloproliferative tumors.
Preferably, the myeloproliferative tumor is a myeloproliferative tumor resistant to a chemotherapeutic drug.
Preferably, the chemotherapeutic drug is a chemotherapeutic drug for treating myeloproliferative tumors, and the chemotherapeutic drug comprises at least one of the group consisting of luccotinib and fexolitinib.
Preferably, the myeloproliferative tumor is polycythemia vera, essential thrombocythemia, or myelofibrosis, and the myelofibrosis is at least one of essential myelofibrosis, myelofibrosis secondary to polycythemia vera, and myelofibrosis secondary to essential thrombocythemia.
Preferably, the MEK/ERK signaling pathway inhibitor is at least one of Trematinib, Cobimetinib, Binimetinib, Selumetinib, Mirdamentib, Refametinib, TIC10, Ulixertinib, PD184352, Pimasertib.
Trametinib (GSK1120212, JTP-74057, Mekinist) is a high specificity, effective MEK1/2 inhibitor, which is approved by the U.S. FDA to be marketed in 2013, and is suitable for treating adult patients with non-resectable melanoma or metastatic melanoma after surgery carrying BRAF V600E or V600K mutation. 1/8 days 2014, the FDA approved dabrafenib and trametinib in combination for BRAF V600E/K mutant metastatic melanoma patients. 5.1.2018, FDA approved the combination dabrafenib/trametinib as adjuvant therapy. Surgery was performed according to the results of the COMBI-AD3 phase studyThe post-resection BRAF V600E mutant melanoma stage III makes it the first oral chemotherapy regimen to prevent lymph node positivity, cancer recurrence of BRAF mutant melanoma. Trametinib has a molecular formula of C 26 H 23 FIN 5 O 4 The molecular weight is 615.39, and the structural formula is shown in formula I.
Cobimetinib (GDC-0973, RG7420) is a potent and highly selective MEK1 inhibitor marketed as FDA approved on 10/11/2015 for treatment with vemurafenib in combination with BRAF V600E or V600K mutant migratory melanoma. Currently, NCT03695380 is being recruited in a phase I clinical trial for ovarian tumor treatment. The molecular formula of Cobimetinib is C 21 H 21 F 3 IN 3 O 2 Molecular weight is 531.31, and structural formula is shown in formula II.
Binimetinib is a mitogen-activated extracellular signal-regulated kinase 1(MEK1) and the reversible inhibitor of cytokines MEK2 activity. MEK proteins are upstream regulatory factor pathways of extracellular signal-related kinases (ERKs). In vitro, Binimetinib inhibits extracellular signal-related kinase (ERK) phosphorylation immunoassays in cells as well as the viability and MEK-dependent phosphorylation of BRAF-mutant human melanoma cells. Binimetinib also inhibited ERK phosphorylation and tumor growth xenograft models in BRAF mutant mice. FDA approved Binimetinib in combination with Encorafenib for the treatment of patients with unresectable or metastatic melanoma with BRAF V600E or V600K mutations. The molecular formula of Binimetinib is C 17 H 15 BrF 2 N 4 O 3 The molecular weight is 441.23, and the structural formula is shown in formula III.
Selumetinib is also called AZD6244 and Y-142886, and is a high-efficiency and non-ATP competitive MEK1/2 and ERK1/2 inhibitor. Selumetinib also has significant efficacy in a variety of tumor models, significantly inhibiting ERK activity, inhibiting tumor growth, and inhibiting lung metastasis. On 10/4/2020, the U.S. FDA announces approval of koselugo (selumetinib) capsules for treatment of type I neurofibroma (NF1) pediatric patients aged 2 and older. This is the first NF1 treatment approved by the FDA. The medicine has the following indications: is used for treating children patients with symptomatic and inoperable plexiform neurofibromas. The molecular formula of Selumetinib is C 17 H 15 BrClFN 4 O 3 The molecular weight is 457.68, and the structural formula is shown in formula IV.
Mirdamatinib is an oral small molecule inhibitor of MEK1 and MEK 2. The European Committee (EC) of Mirdamentinib and the U.S. FDA have granted Mirdamentib (formerly PD-0325901) orphan drug eligibility to treat neurofibromatosis type 1 (NF 1). The molecular formula of Mirdamentinib is C 16 H 14 F 3 IN 2 O 4 The molecular weight is 482.19, and the structural formula is shown in formula V.
Refetinib (RDEA119) is a potent, non-ATP-competitive, highly selective inhibitor of MEK1 and MEK2 with an IC50 of 19nM and 47nM, respectively. Refetinib is an oral MEK inhibitor that has anti-tumor activity when used in combination with Sorafenib in the treatment of RAS-mutated hepatocellular carcinoma (HCC) patients. CLIN CANCER RES A paper is published to report the efficacy of a single drug of refetinib and the combination of refetinib and Sorafenib in treating patients with unresectable or metastatic HCC due to RAS mutations. Refetinib is currently undergoing phase II clinical studies in advanced biliary tract cancer. Refametinib has a molecular formula of C 19 H 20 F 3 IN 2 O 5 S, molecular weight 572.34, knotThe formula is shown in formula VI.
TIC10(ONC201) inhibits Akt and ERK activity, induces TNF-related apoptosis-inducing ligand (TRAIL) by FoxO3a, can penetrate blood brain barrier, has superior stability, and improved pharmacokinetic properties. TIC10 caused significant and long-term expression of TRAIL on the cell surface of tumor cells. In HCT116 p 53-/-cells, TIC10 also caused TRAIL-mediated apoptosis. In addition, TIC10 simultaneously inactivates Akt and ERK, resulting in nuclear translocation of Foxo3a and subsequent upregulation of TRAIL. In tumor xenograft mice, TIC10 had a TRAIL-dependent anti-tumor effect, causing tumor-specific cell death through TRAIL-mediated direct and bystander effects. The molecule is found to be effective on various solid tumors, clinical phase 1 and phase 2 experiments are carried out, and the clinical experiments are difficult to advance due to disputes of compound patents at present. TIC10 having the formula C 24 H 26 N 4 O, molecular weight of 386.49, and structural formula shown in formula VII.
Ulixertinib (BVD-523, VRT752271) is a potent reversible ERK1/ERK2 inhibitor that inhibits IC50 of ERK2<0.3nM, can be administered orally. Ulixertinib works well in clinical trials on patients with advanced solid tumors. The molecular formula of Ulixertinib is C 21 H 22 Cl 2 N 4 O 2 Molecular weight is 433.33, and structural formula is shown in formula VIII.
PD184352(CI-1040) is an ATP-noncompetitive inhibitor of MEK1/2 with an IC50 of 17nM and 100-fold greater selectivity for MEK1/2 than MEK5 in cellular assays. PD184352(CI-1040) can be selectivelyInducing apoptosis. PD184352 is the MEK inhibitor that first entered clinical trials, and has been discontinued in phase ii clinical trials because of its poor solubility, short half-life, low oral bioavailability, large individual variability, and the like. PD184352 has the molecular formula of C 17 H 14 ClF 2 IN 2 O 2 The molecular weight is 478.67, and the structural formula is shown in formula IX.
Pimasertib (AS-703026, MSC1936369B, SAR 245509) is a highly selective ATP-noncompetitive orally available MEK1/2 allosteric inhibitor with an IC50 of 5nM to 2. mu.M in MM cell lines. Pimasertib has performed 10 more phase 1/2 clinical trials in approximately 900 patients with various tumor types. Pimasertib is used in combination with DAY101 for the treatment of patients ≧ 12 years old and suffering from recurrent, progressive or refractory solid tumors and MAPK pathway abnormalities. The molecular formula of Pimasertib is C 15 H 15 FIN 3 O 3 Molecular weight is 431.20, and structural formula is shown in formula X.
Preferably, the treatment comprises inhibiting proliferation and/or promoting apoptosis of the tumor cells.
The invention also provides application of the MEK/ERK signal pathway inhibitor in preparing a medicament for inhibiting proliferation of HEL cells and/or promoting apoptosis of the HEL cells.
Wherein the HEL cells comprise non-drug-resistant HEL cells and/or drug-resistant HEL cells.
Experiments show that, for non-drug-resistant HEL cells, Trematinib, Cobimetinib, Binimetinib, Selumetinib, Mirdamentib, Refametinib, Ulixertinib, PD184352 and Pimasertib can not promote apoptosis of the HEL cells, and only TIC10 can promote apoptosis. Wherein, Trematinib and TIC10 can inhibit the proliferation of HEL cells, Pimasertib has weaker effect of inhibiting the proliferation of HEL cells, and other inhibitors have no inhibiting effect.
For drug-resistant HEL cells, Trematinib, Cobimetinib, Binimetinib, Selumetinib, Mirdatinib, Refametinib, TIC10, Ulixetinib, PD184352 and Pimasertib can inhibit the proliferation of drug-resistant HEL cells, wherein only Refametinib can promote the apoptosis of drug-resistant HEL cells. It is proved that the curative effect of the Refametinib on the drug-resistant myeloproliferative tumor is more obvious.
The results show that the MEK/ERK signal pathway inhibitor has strong inhibition effect on the proliferation of drug-resistant HEL cells, and partial inhibitors can promote the apoptosis of the cells. This indicates that MEK/ERK signaling pathway inhibitors have significant therapeutic effects on drug-resistant myeloproliferative tumors.
Preferably, the medicine also comprises other medicinal components for treating the myeloproliferative tumors and pharmaceutically acceptable auxiliary materials.
Preferably, the dosage form of the medicament is an oral preparation or an injection preparation.
According to the technical scheme, the invention provides the application of the MEK/ERK signal pathway inhibitor in preparing the medicine for treating the myeloproliferative tumor. The myeloproliferative tumor is polycythemia vera, essential thrombocythemia or myelofibrosis and the myeloproliferative tumor resistant to lucentine, and the myeloproliferative tumor is the primary myelofibrosis, the myelofibrosis secondary to the polycythemia vera or the myelofibrosis secondary to the essential thrombocythemia. The invention has the technical effects that:
the MEK/ERK signal pathway inhibitor used for treating the myeloproliferative tumor provides a new treatment approach for a large number of myeloproliferative tumor patients, and provides more choices for clinicians and patients. For patients with an RCT-resistant myeloproliferative tumor, MEK/ERK signaling pathway inhibitors can provide continued oral drug therapy to the patient, free of bone marrow transplantation. MEK/ERK signaling pathway inhibitors can be chemically synthesized at lower cost than biologies. And the MEK/ERK signal pathway inhibitors in the application pass phase I clinical tests, and part of the MEK/ERK signal pathway inhibitors pass phase II and III clinical tests smoothly, so that the MEK/ERK signal pathway inhibitors can be used for clinical treatment in the future, and have better clinical application prospects.
Drawings
Fig. 1 illustrates example 1: rcotinib-resistant myeloproliferative tumor cell model HEL RE Establishing a result graph;
fig. 2 illustrates example 2: MEK/ERK signaling pathway inhibitor treatment of myeloproliferative tumor cells by CellTiter-Lumi TM A result graph of cell proliferation detected by a luminescence method; a is a Trematinib processed HEL cell proliferation result graph; b is a graph of proliferation results of Cobimetinib-treated HEL cells; c is a graph of proliferation results of Binimetiib-treated HEL cells; d is a graph of proliferation results of Selumetinib-treated HEL cells; e is a graph of proliferation results of HEL cells treated with Mirdamentinib; f is a graph of proliferation results of Refametinib-treated HEL cells; g is a graph of proliferation results of TIC 10-treated HEL cells; h is a proliferation result graph of the HEL cells treated by Ulixertinib; i is a graph of proliferation results of HEL cells treated by PD 184352; j is a graph of proliferation results of Pimasertib-treated HEL cells;
figure 3 illustrates example 3: graph of the results of flow-based detection of apoptosis after treatment of myeloproliferative tumor cells with MEK/ERK signaling pathway inhibitors, using annexin V-PI staining; a is a Trematinib treatment HEL cell apoptosis result graph; b is a graph of the apoptosis results of Cobimetinib-treated HEL cells; c is a result chart of apoptosis of BInimetib-treated HEL cells; d is the apoptosis result chart of HEL cells treated by Selumetinib; e is the result chart of HEL cell apoptosis treated by Mirdatinib; f is the apoptosis result chart of Refametinib-treated HEL cells; g is a graph of the result of HEL apoptosis treated by TIC 10; h is a result chart of apoptosis of HEL cells treated by Ulixertinib; i is a graph of the result of HEL apoptosis treated by PD 184352; j is a result graph of Pimasertib treatment HEL cell apoptosis;
fig. 4 illustrates example 4: MEK/ERK signaling pathway inhibitor treatment of lucertinib-resistant myeloproliferative tumor cells by CellTiter-Lumi TM A result graph of cell proliferation detected by a luminescence method; a Trematinib treatment of HEL RE Cell proliferation result graph; b is Cobimetinib treatment of HEL RE Cell proliferation result graph; c Binimetib treatment of HEL RE Cell proliferation result graph; d is Selumetinib treatment HEL RE Cell proliferation result graph; e is the treatment of HEL with Mirdamentinib RE A cell proliferation result graph; f is Refmetinib-treated HEL RE A cell proliferation result graph; g is TIC10 treated HEL RE Cell proliferation result graph; h treatment of HEL for Ulixertinib RE Cell proliferation result graph; i handling HEL for PD184352 RE Cell proliferation result graph; j is Pimasertib Process HEL RE Cell proliferation result graph;
fig. 5 illustrates example 5: a result graph of flow detection of apoptosis after MEK/ERK signal pathway inhibitor treatment of lucentin-resistant myeloproliferative tumor cells using Annexin V-PI staining; a is Trematinib processing HEL RE A graph of apoptosis results; b is Cobimetinib treatment of HEL RE A graph of apoptosis results; c Binimetib treatment of HEL RE A graph of apoptosis results; d is Selumetinib treatment HEL RE A graph of apoptosis results; e is the treatment of HEL by Mirdastatin RE A graph of apoptosis results; f is Refametinib treatment HEL RE A graph of apoptosis results; g is TIC10 processing HEL RE A graph of apoptosis results; h treatment of HEL for Ulixertinib RE A graph of apoptosis results; i handling HEL for PD184352 RE A graph of apoptosis results; j is Pimasertib Process HEL RE Apoptosis results.
Detailed Description
The invention discloses application of MEK/ERK signal pathway inhibitor in preparing a medicament for treating myeloproliferative tumors, and the method can be realized by appropriately improving process parameters by taking the contents into consideration by the technical personnel in the field. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
In the present invention, the inhibitory effect of MEK/ERK signaling pathway inhibitors on myeloproliferative tumor cells (resistant versus non-resistant) is defined by a cell line model.
In some embodiments, the present invention establishes a ricochenib resistant cell model HEL based on two commonly used Human myeloproliferative tumor cell lines, namely HEL cells (Human erythroleukemia cells), containing JAK2-V617F mutations RE . The drug resistance model is constructed by using a drug resistance lower than that of the drug resistance model
The concentration of cell IC50 begins to be added with incarnib, slowly increases to a high concentration, maintains the cells not to be killed, and verifies whether the construction of the model is successful by comparing IC 50.
In some embodiments, the invention uses increasing concentrations of MEK/ERK signaling pathway inhibitors to treat myeloproliferative tumor cell lines by CellTiter-Lumi TM Proliferation of cells was detected by luminescence. The results show that partial MEK/ERK signaling pathway inhibitors can successfully inhibit proliferation of HEL cells.
In some embodiments, the invention uses increasing concentrations of MEK/ERK signaling pathway inhibitors to treat myeloproliferative tumor cell lines and flow-tests for apoptosis using Annexin V-PI staining. The results show that partial inhibitors of the MEK/ERK signaling pathway can promote apoptosis in HEL cells.
It follows that a partial inhibitor of the MEK/ERK signaling pathway may be useful in the treatment of myeloproliferative neoplastic diseases.
Further, in some embodiments, the invention uses increasing concentrations of MEK/ERK signaling pathway inhibitors to treat RCA-resistant myeloproliferative tumor cells with CellTiter-Lumi TM Proliferation of cells was detected by luminescence. The results show that inhibitors of MEK/ERK signaling pathway inhibit HEL RE And (4) cell proliferation.
In some embodiments, the present invention treats an incarnib-resistant myeloproliferative tumor cell with increasing concentrations of a MEK/ERK signaling pathway inhibitor and flow-detects apoptosis of the cell after staining with annexin v-PI. The results show that partial MEK/ERK signaling pathway inhibitors can promote HEL RE Apoptosis of the cell.
Thus, MEK/ERK signaling pathway inhibitors are useful for treating RCT-resistant myeloproliferative neoplastic diseases.
Furthermore, the invention provides application of a part of MEK/ERK signal pathway inhibitors in preparing medicines for inhibiting proliferation of HEL cells and promoting apoptosis of HEL cells.
In conclusion, the invention provides the application of the MEK/ERK signaling pathway inhibitor in preparing the medicine for treating myeloproliferative tumors.
Further, the myeloproliferative tumors are polycythemia vera, essential thrombocythemia and myelofibrosis (including primary myelofibrosis, myelofibrosis secondary to polycythemia vera and myelofibrosis secondary to essential thrombocythemia) and myeloproliferative tumors with drug resistance.
In some embodiments, the drug-resistant myeloproliferative tumor is an luctinib-resistant myeloproliferative tumor.
In some embodiments, the myeloproliferative tumor with drug resistance is polycythemia vera with drug resistance, myelofibrosis with drug resistance (including primary myelofibrosis, myelofibrosis secondary to polycythemia vera, and myelofibrosis secondary to primary thrombocythemia), and primary thrombocythemia with drug resistance.
Wherein the medicine is Trematinib, Cobimetinib, Binimetinib, Selumetinib, Mirdatiniib, Refametinib, TIC10, Ulixetinib, PD184352, Pimasertib.
Furthermore, the medicine also comprises pharmaceutically acceptable auxiliary materials.
The medicine can be in any dosage form in the current medicine field, including oral preparations or injection preparations.
Each pharmaceutical dosage form can be prepared by selecting proper acceptable auxiliary materials according to the actual needs of the dosage form, which belongs to the conventional preparation technology of the dosage form in the field. Such as capsule, tablet, injection powder, etc.
The reagents or apparatus used in the present invention are commercially available.
The invention is further illustrated by the following examples:
example 1 establishment of two common Lucotinib resistant cell models (HEL) RE )。
Materials and methods
1. Cell lines
HEL (human erythroleukamia cell line), and RCA resistant HEL cells were cultured in RPMI medium (Gibco) containing 20% heat-inactivated fetal bovine serum (Gibco) and 1% penicillin/streptomycin.
HEL model of drug resistance of luccotinib, HEL RE The model is constructed by starting to add incarnib at a concentration lower than that of the original cell IC50, and slowly increasing to a high concentration to maintain the cells from being killed. Our initial concentration was 0.1. mu.M, the drug was added when cell proliferation occurred, the drug gradient was increased 1.25 times, and the final concentration was 2.0. mu.M. After 4-6 weeks, stable drug-resistant cells were obtained.
2. Inhibitors
The cells were treated with the luccotinib and MEK/ERK signaling pathway inhibitors purchased from Selleck in DMSO at a stock concentration of 10mM, frozen at-80 ℃ and working solution diluted to the indicated fold in RPMI medium. The luccotinib is specifically luccotinib phosphate.
3. In vitro inhibition assay
To test the antiproliferative effect of the inhibitors, the above cell lines were cultured in a system of 3000 cells/100. mu.L per well, increasing concentrations of lucentitinib (HEL cell gradient: 0, 0.1, 0.3, 1, 3, 10. mu.M; or 0, 0.001, 0.003, 0.01, 0.03, 0.1. mu.M) were added, and DMSO was replenished to equal amounts. Set 4 replicate groups in parallel and set 3 blank wells (cell-free medium wells). After 48 hours, the mixture was passed through CellTiter-Lumi TM The proliferation of the cells was detected by luminescence (Biyun day). Multifunctional microplate reader readings, IC50 were calculated by GraphPadprism.
The cell proliferation rate calculation formula is as follows: the cell proliferation rate (luminescences value in dosing group-mean luminescences value in blank wells)/(luminescences value in DMSO control group-mean luminescences value in blank wells) x 100%.
The method for evaluating whether the model is successfully constructed is to compare the IC50 of the drug-resistant cells and the original cells, wherein the ratio is the drug-resistant index, and if the ratio is more than 3, the model is successfully constructed.
Second, result analysis
In FIG. 1, HEL cell IC50 was 0.801. mu.M, HEL RE The cell IC50 was 12.27. mu.M, and the drug resistance index was 15.3, suggesting a drug resistance model HEL RE Successful construction of the same. Proliferation rate results are shown as mean ± sd, and the proliferation rates of the two groups are compared using t-test (. about.. about.p)<0.001), IC50 is shown as mean.
Example 2 partial MEK/ERK Signaling pathway inhibitors inhibit the proliferation of myeloproliferative tumor cells
To examine the effect of MEK/ERK signaling pathway inhibitors on the proliferative capacity of myeloproliferative tumor resistant cells, HEL primary cell lines were treated with increasing concentrations of each of reecotinib and MEK/ERK signaling pathway inhibitors, respectively, by CellTiter-Lumi TM Proliferation of cells was detected by luminescence in the same manner as in example 1, and the results are shown in FIG. 2.
Figure 2 reflects the drug concentrations in HEL cells for the lucentitinib-treated groups (% mean proliferation ± standard deviation): 0 μ M (100. + -. 1.47) (not shown), 0.1 μ M (81.0. + -. 1.35), 0.3 μ M (54.9. + -. 4.35), 1 μ M (42.7. + -. 4.05), 3 μ M (34.7. + -. 1.14), 10 μ M (29.4. + -. 0.497); figure 2a reflects Trematinib treatment group drug concentrations (mean proliferation rate) in HEL cells as: 0. mu.M (100. + -. 9.58) (not shown), 0.0001. mu.M (93.91. + -. 2.37), 0.0003. mu.M (98.17. + -. 3.44), 0.01. mu.M (84.82. + -. 6.67), 0.03. mu.M (83.96. + -. 9.48), 0.1. mu.M (85.99. + -. 6.9). This suggests that Trematinib has a weaker effect than that of ruckerib in inhibiting the proliferation of HEL cells. Figure 2b reflects Cobimetinib treatment group drug concentrations (mean proliferation rate) in HEL cells as: 0. mu.M (100. + -. 6.6) (not shown), 0.001. mu.M (98.53. + -. 4.64), 0.003. mu.M (105.51. + -. 7.14), 0.01. mu.M (99.78. + -. 5.17), 0.03. mu.M (99.66. + -. 1.93), 0.1. mu.M (96.59. + -. 8.57). This suggests that Cobimetinib cannot inhibit proliferation of HEL cells. Figure 2c reflects Binimetinib treatment group drug concentrations (mean proliferation rate) as: 0. mu.M (100. + -. 2.65) (not shown), 0.1. mu.M (111. + -. 13.9), 0.3. mu.M (105. + -. 17.2), 1. mu.M (115. + -. 22.9), 3. mu.M (107. + -. 14.4), 10. mu.M (92.4. + -. 2.44). This suggests that Binimetinib cannot inhibit proliferation of HEL cells. Figure 2d reflects the Selumetinib treatment group drug concentrations (mean proliferation rates) in HEL cells as: 0 μ M (100. + -. 1.62) (not shown), 0.1 μ M (107. + -. 17.6), 0.3 μ M (117. + -. 16.3), 1 μ M (110. + -. 26.3), 3 μ M (103. + -. 14.5), 10 μ M (88.8. + -. 2.69). This suggests that Selumetinib cannot inhibit proliferation of HEL cells. Figure 2e reflects mirdamatinib-treated group drug concentrations (mean proliferation rates) in HEL cells as: 0 μ M (100. + -. 1.73) (not shown), 0.1 μ M (107. + -. 17.6), 0.3 μ M (117. + -. 16.3), 1 μ M (110. + -. 26.3), 3 μ M (103. + -. 14.5), 10 μ M (88.8. + -. 2.69). This suggests that mirdomatinib cannot inhibit proliferation of HEL cells. Figure 2f reflects in HEL cells that the refetinib-treated group drug concentrations (mean proliferation rates) are: 0 μ M (100. + -. 2.63) (not shown), 0.1 μ M (96.0. + -. 7.44), 0.3 μ M (99.8. + -. 4.36), 1 μ M (93.4. + -. 4.31), 3 μ M (89.1. + -. 7.23), 10 μ M (84.6. + -. 8.89). This suggests that refetinib cannot inhibit proliferation of HEL cells. Figure 2g reflects in HEL cells, TIC10 treatment group drug concentrations (mean proliferation rate) are: 0 μ M (100. + -. 4.31) (not shown), 0.1 μ M (113. + -. 15.0), 0.3 μ M (116. + -. 14.9), 1 μ M (80.3. + -. 3.43), 3 μ M (19.6. + -. 2.64), 10 μ M (11.1. + -. 7.15). This suggests that TIC10 can inhibit proliferation of HEL cells, and that the effect increases with increasing drug concentration, and that the inhibitory effect increases with increasing concentration. Figure 2h reflects in HEL cells that the Ulixertinib treatment group drug concentrations (mean proliferation rate) are: 0 μ M (100. + -. 2.58) (not shown), 0.1 μ M (98.9. + -. 8.68), 0.3 μ M (109. + -. 5.36), 1 μ M (96.1. + -. 5.13), 3 μ M (100. + -. 4.61), 10 μ M (95.2. + -. 5.83). This suggests that Ulixertinib cannot inhibit proliferation of HEL cells. Figure 2i reflects in HEL cells, the PD184352 treatment group drug concentrations (mean proliferation rate) are: 0. mu.M (100. + -. 4.46) (not shown), 0.1. mu.M (105. + -. 16.7), 0.3. mu.M (118. + -. 21.2), 1. mu.M (103. + -. 22.7), 3. mu.M (99.0. + -. 20.3), 10. mu.M (82.3. + -. 6.50). This suggests that PD184352 could not inhibit proliferation of HEL cells. Figure 2j reflects in HEL cells, the pimasetib treatment group drug concentration (mean proliferation rate) is: 0 μ M (100. + -. 3.07) (not shown), 0.1 μ M (89.0. + -. 1.42), 0.3 μ M (95.0. + -. 7.62), 1 μ M (86.9. + -. 1.71), 3 μ M (82.8. + -. 2.13), 10 μ M (77.3. + -. 2.52). This suggests that pimasetib has a weaker inhibitory effect on HEL cell proliferation than does lucentin. The two groups of proliferation rates in the graph were compared using t-test (p <0.05, p <0.01, p < 0.001).
Example 3 partial MEK/ERK Signaling pathway inhibitors promote apoptosis in myeloproliferative tumor cells
Materials and methods
1. The cell line and inhibitor were as in example 1.
2. Apoptosis detection
To examine the pro-apoptotic effects of the inhibitors, HEL primary cell lines were treated with either lucentitinib or MEK/ERK signaling pathway inhibitors for 24 hours (concentrations: 0, 0.1, 0.3, 1, 3, 10. mu.M), respectively, and supplemented with DMSO to equal amounts. 3 parallel repeats were set and flow cytometry was used to detect apoptosis following annexin V and PI staining.
The apoptosis rate calculation formula is as follows: apoptosis rate (annexin v) is the rate of early apoptotic cells + /PI - ) + late apoptotic and necrotic cell ratio (annexin V) + /PI + )。
Second, result analysis
In fig. 3, the drug concentrations (mean% apoptosis ± standard deviation) of Trematinib, Cobimetinib, TIC10, Ulixertinib, PD184352, pimarstib control HEL cell luccotinib-treated groups were: 0 μ M (7.26. + -. 0.360), 0.1 μ M (7.39. + -. 0.0600), 0.3 μ M (7.40. + -. 0.820), 1 μ M (8.58. + -. 0.490), 3 μ M (7.12. + -. 0.180), 10 μ M (6.04. + -. 0.700); binimetinib, Selumetinib, mirdometinib, refetinib-controlled HEL cell lucentinib treatment group drug concentrations (mean apoptosis rate% ± standard deviation) were: 0 μ M (2.30. + -. 0.720), 0.1 μ M (2.30. + -. 0.210), 0.3 μ M (1.90. + -. 0.360), 1 μ M (2.4. + -. 0.50), 3 μ M (2.00. + -. 0.480), 10 μ M (2.30. + -. 0.720). Figure 3a reflects Trematinib treatment group drug concentrations (mean apoptosis rate) in HEL cells as: 0 μ M (6.94. + -. 0.690) (not shown), 0.1 μ M (7.09. + -. 1.35), 0.3 μ M (6.32. + -. 0.780), 1 μ M (6.11. + -. 1.19), 3 μ M (6.83. + -. 1.83), 10 μ M (6.41. + -. 0.240). This suggests that Trematinib could not promote apoptosis in HEL cells. Figure 3b reflects Cobimetinib treatment group drug concentrations (mean apoptosis rate) in HEL cells as: 0 μ M (5.09. + -. 0.060) (not shown), 0.1 μ M (5.35. + -. 0.500), 0.3 μ M (6.53. + -. 0.370), 1 μ M (6.31. + -. 0.670), 3 μ M (6.61. + -. 1.490), 10 μ M (5.64. + -. 0.470). This suggests that Cobimetinib cannot promote apoptosis in HEL cells. In FIG. 3c, the Binimetinib-treated group drug concentration (apoptosis rate) was 0. mu.M (1.20. + -. 0.320) (not shown), 0.1. mu.M (1.21. + -. 0.110), 0.3. mu.M (1.55. + -. 0.310), 1. mu.M (1.73. + -. 0.06), 3. mu.M (1.39. + -. 0.29), 10. mu.M (2.19. + -. 0.07), which suggests that Binimetinib could not promote apoptosis of myeloproliferative tumor cells. In fig. 3d, the drug concentrations (mean apoptosis rates) in HEL cell Selumetinib-treated groups were: 0 μ M (1.91. + -. 0.51) (not shown), 0.1 μ M (2.24. + -. 0.0580), 0.3 μ M (2.14. + -. 0.748), 1 μ M (2.45. + -. 0.473), 3 μ M (1.91. + -. 0.225), 10 μ M (2.40. + -. 0.260), suggesting that Selumetinib is unable to promote apoptosis in myeloproliferative tumor cells. Figure 3e reflects mirdamatinib-treated group drug concentrations (mean apoptosis rate) in HEL cells as: 0 μ M (1.46. + -. 0.146) (not shown), 0.1 μ M (1.99. + -. 0.367), 0.3 μ M (2.23. + -. 0.432), 1 μ M (2.45. + -. 0.473), 3 μ M (1.91. + -. 0.225), 10 μ M (2.40. + -. 0.260). This suggests that mirdomatinib does not promote apoptosis in HEL cells. Figure 3f reflects in HEL cells that the refetinib-treated group drug concentrations (mean apoptosis rate) are: 0 μ M (2.08. + -. 0.467) (not shown), 0.1 μ M (2.78. + -. 1.02), 0.3 μ M (2.21. + -. 0.369), 1 μ M (2.06. + -. 0.457), 3 μ M (2.39. + -. 0.713), 10 μ M (1.79. + -. 0.745). This suggests that refetinib does not promote apoptosis in HEL cells. Figure 3g reflects in HEL cells TIC10 treatment group drug concentrations (mean apoptosis rate) as: 0 μ M (3.69. + -. 0.192) (not shown), 0.1 μ M (3.19. + -. 0.516), 0.3 μ M (5.63. + -. 2.50), 1 μ M (4.39. + -. 0.2880), 3 μ M (5.16. + -. 0.266), 10 μ M (9.43. + -. 0.445). This suggests that TIC10 promotes apoptosis of HEL cells, and its effect increases with increasing concentration. Figure 3h reflects in HEL cells, Ulixertinib treatment group drug concentrations (mean apoptosis rate) are: 0 μ M (6.00. + -. 0.717) (not shown), 0.1 μ M (5.56. + -. 1.22), 0.3 μ M (6.22. + -. 1.86), 1 μ M (5.72. + -. 0.619), 3 μ M (5.99. + -. 1.73), 10 μ M (6.12. + -. 0.903). This suggests that Ulixertinib cannot promote apoptosis in HEL cells. Figure 3i reflects in HEL cells, the PD184352 treatment group drug concentrations (mean apoptosis rate) are: 0 μ M (3.86. + -. 0.198) (not shown), 0.1 μ M (4.65. + -. 0.959), 0.3 μ M (4.90. + -. 0.494), 1 μ M (4.72. + -. 0.161), 3 μ M (4.19. + -. 0.709), 10 μ M (4.77. + -. 0.902). This suggests that PD184352 could not promote apoptosis of HEL cells. Figure 3j reflects in HEL cells that pimarstib treatment group drug concentrations (mean apoptosis rate) are: 0 μ M (3.98. + -. 0.115) (not shown), 0.1 μ M (4.61. + -. 0.535), 0.3 μ M (5.74. + -. 1.68), 1 μ M (1.37. + -. 0.290), 3 μ M (6.37. + -. 0.272), 10 μ M (4.89. + -. 0.182). This suggests that Pimasertib cannot promote apoptosis in HEL cells. The two groups of the graph used t test for apoptosis rate (p <0.05, p <0.01, p < 0.001).
Example 4 inhibitors of the MEK/ERK signaling pathway inhibit the proliferation of drug-resistant myeloproliferative tumor cells.
To examine the effect of MEK/ERK signaling pathway inhibitors on the proliferative capacity of myeloproliferative tumor resistant cells, HEL was treated with increasing concentrations of each of reecotinib and MEK/ERK signaling pathway inhibitors RE By CellTiter-Lumi TM Proliferation of cells was detected by luminescence. The procedure is as in example 1, and the results are shown in FIG. 4.
FIG. 4 is reflected in HEL RE In cells, the drug concentrations (mean proliferation rate ± standard deviation) of the luccotinib-treated groups were: 0 μ M (100. + -. 3.79) (not shown), 0.1 μ M (105. + -. 2.08), 0.3 μ M (105. + -. 3.60), 1 μ M (104. + -. 9.46), 3 μ M (109. + -. 4.40), 10 μ M (96.0. + -. 3.69); FIG. 4a is reflected in HEL RE In cells, the concentration of Trematinib-treated group drugs (mean proliferation rate) was: 0. mu.M (100. + -. 2.27) (not shown), 0.001. mu.M (99.38. + -. 2.75), 0.003. mu.M (94.51. + -. 3.41), 0.01. mu.M (67.59. + -. 6.26), 0.03. mu.M (51.61. + -. 2.5), 0.1. mu.M (40.45. + -. 4.5). This suggests that Trematinib can effectively inhibit HEL RE And (4) proliferation of the cells. FIG. 4b is reflected in HEL RE In cells, the Cobimetinib treatment group drug concentrations (mean proliferation rate) were: 0 μ M (100. + -. 2.22) (not shown), 0.001 μ M (94.82. + -. 3.95), 0.003 μ M (101.27. + -. 4.23), 0.01 μ M (98.06. + -. 2.53), 0.03 μ M (83.06. + -. 2.48), 0.1 μ M (58.69. + -. 3.51). This suggests that Cobimetinib is effective in inhibiting HEL RE And (4) proliferation of the cells. Figure 4c reflects Binimetinib treatment group drug concentrations (mean proliferation rate) as: 0 μ M (100. + -. 4.10) (not shown), 0.1 μ M (67.9. + -. 3.44), 0.3 μ M (53.8. + -. 1.41), 1 μ M (50.4. + -. 3.27), 3 μ M (43.1. + -. 2.18), 10 μ M (37.6. + -. 1.24). This suggests that Binimetinib can inhibit HEL RE The proliferation and inhibition rate of the cells are increased along with the increase of the concentration of the medicine. FIG. 4d is reflected in HEL RE In cells, the drug concentrations (mean proliferation rates) of the Selumetinib treatment group were: 0 μ M (100. + -. 1.45) (not shown), 0.1 μ M (77.1. + -. 1.86), 0.3 μ M (62.0. + -. 3.90), 1 μ M (54.9. + -. 1.63), 3 μ M (48.2. + -. 2.63), 10 μ M (41.0. + -. 3.57). This suggests that Selumetinib inhibits HEL RE The proliferation and inhibition rate of the cells are increased along with the increase of the concentration of the medicine. FIG. 4e reflects in HEL RE In cells, the mirdomatinib treatment group drug concentrations (mean proliferation rate) were: 0 μ M (100. + -. 1.70) (not shown), 0.1 μ M (54.8. + -. 2.74), 0.3 μ M (57.9. + -. 4.50), 1 μ M (44.2. + -. 3.38), 3 μ M (39.6. + -. 0.380), 10 μ M (32.5. + -. 3.73). This suggests that Mirdastatin inhibits HEL RE The proliferation and inhibition rate of the cells are increased along with the increase of the concentration of the medicine. FIG. 4f is reflected in HEL RE In cells, the refastib treatment group drug concentrations (mean proliferation rate) were: 0 μ M (100. + -. 3.85) (not shown), 0.1 μ M (66.0. + -. 3.12), 0.3 μ M (61.2. + -. 2.05), 1 μ M (50.6. + -. 2.09), 3 μ M (43.3. + -. 1.23), 10 μ M (36.6. + -. 2.83). This suggests that refetinib inhibits HEL RE The proliferation and inhibition rate of the cells are increased along with the increase of the concentration of the medicine. FIG. 4g is reflected in HEL RE In cells, the TIC10 treatment group drug concentrations (mean proliferation rate) were: 0 μ M (100. + -. 6.41) (not shown), 0.1 μ M (101. + -. 7.19), 0.3 μ M (105. + -. 3.69), 1 μ M (62.5. + -. 5.82), 3 μ M (31.3. + -. 1.45), 10 μ M (19.6. + -. 1.01). This suggests that TIC10 inhibits HEL RE Proliferation of cells, the effect increasing with increasing drug concentration. FIG. 4h is reflected in HEL RE In cells, the drug concentrations (mean proliferation rates) in the Ulixertinib-treated groups were: 0 μ M (100. + -. 1.65) (not shown), 0.1 μ M (95.6. + -. 5.27), 0.3 μ M (91.4. + -. 3.49), 1 μ M (58.5. + -. 14.1), 3 μ M (48.4. + -. 7.37), 10 μ M (37.5. + -. 6.71). This suggests that Ulixertinib can inhibit HEL RE Proliferation of cells, the effect increasing with increasing drug concentration. FIG. 4i is reflected in HEL RE In cells, the PD184352 treatment group drug concentrations (mean proliferation rate) were: 0 μ M (100. + -. 4.73) (not shown), 0.1 μ M (81.8. + -. 1.27), 0.3 μ M (79.6. + -. 3.79), 1 μ M (52.2. + -. 1.24), 3 μ M (42.7. + -. 0.720), 10 μ M (33.9. + -. 0.700). This suggests that PD184352 could inhibit HEL RE The proliferation of the cells is carried out,this effect increases with increasing drug concentration. FIG. 4j is reflected in HEL RE In cells, the pimarsertib treatment group drug concentrations (mean proliferation rate) were: 0 μ M (100. + -. 3.71) (not shown), 0.1 μ M (59.3. + -. 1.78), 0.3 μ M (53.8. + -. 0.410), 1 μ M (42.2. + -. 3.46), 3 μ M (36.5. + -. 1.66), 10 μ M (30.3. + -. 2.22). This suggests that Pimasertib can inhibit HEL RE Proliferation of cells, the effect increasing with increasing drug concentration. Comparison of proliferation rates in the two groups in the graph by t-test (. about.p)<0.05,**p<0.01,***p<0.001)。
Example 5 partial MEK/ERK Signaling pathway inhibitors promote apoptosis in drug-resistant myeloproliferative tumor cells
To examine the effect of MEK/ERK signaling pathway inhibitors on the survival of myeloproliferative tumor resistant cells, HEL was treated with increasing concentrations of each of reecotinib and MEK/ERK signaling pathway inhibitors RE The apoptosis of the cells was detected by flow-through after staining with annexin v and PI. The procedure is as in example 3, and the results are shown in FIG. 5.
In FIG. 5, Trematinib, Cobimetinib control HEL RE The drug concentration (mean apoptosis rate% ± standard deviation) of the cell luccotinib treatment group was: 0 μ M (8.36. + -. 0.636), 0.1 μ M (12.29. + -. 0.642), 0.3 μ M (13.14. + -. 1.241), 1 μ M (10.82. + -. 1.245), 3 μ M (9.74. + -. 0.255), 10 μ M (10.07. + -. 0.804); binimetinib, Selumetinib, Mirdamentib, Refametinib-controlled HEL RE The drug concentration (mean apoptosis rate ± standard deviation) of the cell rucotinib treatment group was: 0 μ M (0.300. + -. 0.0240), 0.1 μ M (0.290. + -. 0.0800), 0.3 μ M (0.330. + -. 0.0430), 1 μ M (0.290. + -. 0.0800), 3 μ M (0.280. + -. 0.0560), 10 μ M (0.360. + -. 0.0260). TIC10, Ulixertinib, PD184352, Pimasertib-control HEL RE The drug concentration (mean apoptosis rate% ± standard deviation) of the cell luccotinib treatment group was: 0 μ M (2.15. + -. 1.01), 0.1 μ M (2.73. + -. 0.748), 0.3 μ M (2.11. + -. 0.989), 1 μ M (2.73. + -. 0.748), 3 μ M (2.09. + -. 0.982), 10 μ M (1.900. + -. 0.751); FIG. 5a is reflected in HEL RE In cells, the Trematinib treatment group drug concentrations (mean apoptosis rate) were: 0 μ M (7.13. + -. 1.46) (not shown), 0.1 μ M (9.42. + -. 2.48), 0.3 μ M (6.49. + -. 1.58), 1 μ M (9.56. + -. 1.39), 3 μ M (8.01. + -. 2.04), 10 μ M (7.72. + -. 0.167). This suggests Trematinib fail to promote apoptosis of drug-resistant myeloproliferative tumor cells. FIG. 5b is reflected in HEL RE In cells, the Cobimetinib treatment group drug concentrations (mean apoptosis rate) were: 0 μ M (4.87. + -. 1.37) (not shown), 0.1 μ M (3.53. + -. 1.10), 0.3 μ M (1.88. + -. 0.160), 1 μ M (4.09. + -. 1.46), 3 μ M (2.90. + -. 0.344), 10 μ M (2.36. + -. 0.414). This suggests that Cobimetinib cannot promote HEL RE Apoptosis of the cell. In FIG. 5c, the concentration (apoptosis rate) of Binimetinib-treated drugs was 0. mu.M (0.250. + -. 0.0620) (not shown), 0.1. mu.M (0.220. + -. 0.0300), 0.3. mu.M (0.160. + -. 0.0410), 1. mu.M (0.270. + -. 0.0360), 3. mu.M (0.240. + -. 0.0180), and 10. mu.M (0.220. + -. 0.111), indicating that Binimetinib could not promote apoptosis of drug-resistant myeloproliferative tumor cells. In FIG. 5d, HEL RE The drug concentrations (mean apoptosis rate) of the cell Selumetinib-treated groups were: 0 μ M (0.290. + -. 0.110) (not shown), 0.1 μ M (0.230. + -. 0.0160), 0.3 μ M (0.270. + -. 0.0790), 1 μ M (0.220. + -. 0.0400), 3 μ M (0.310. + -. 0.0290), 10 μ M (0.320. + -. 0.00800), suggesting that Selumetinib is unable to promote apoptosis in drug-resistant myeloproliferative tumor cells. FIG. 5e is reflected in HEL RE In cells, the mirdomatinib treatment group drug concentrations (mean apoptosis rate) were: 0 μ M (0.270. + -. 0.0250) (not shown), 0.1 μ M (0.220. + -. 0.0180), 0.3 μ M (0.370. + -. 0.0640), 1 μ M (0.410. + -. 0.0340), 3 μ M (0.140. + -. 0.141), 10 μ M (0.330. + -. 0.0220). This suggests that mirdomatinib cannot promote apoptosis in drug-resistant myeloproliferative tumor cells. FIG. 5f is reflected in HEL RE In cells, the refastib treatment group drug concentrations (mean apoptosis rate) were: 0. mu.M (0.320. + -. 0.0670) (not shown), 0.1. mu.M (0.320. + -. 0.0590), 0.3. mu.M (0.330. + -. 0.0660), 1. mu.M (0.330. + -. 0.0710), 3. mu.M (0.560. + -. 0.0420), 10. mu.M (0.800. + -. 0.0810). This suggests that refastib promotes apoptosis of drug-resistant myeloproliferative tumor cells, and the effect is enhanced with the increase of concentration. FIG. 5g is reflected in HEL RE In cells, TIC10 treatment group drug concentrations (mean apoptosis rate) were: 0 μ M (2.450. + -. 0.169) (not shown), 0.1 μ M (2.35. + -. 0.286), 0.3 μ M (1.89. + -. 0.253), 1 μ M (2.03. + -. 0.271), 3 μ M (2.29. + -. 0.210), 10 μ M (2.80. + -. 0.684). This suggests that TIC10 is unable to promote apoptosis in drug resistant myeloproliferative tumor cells. FIG. 5h is reflected in HEL RE In cells, Ulixertinib treatment group drug concentrations (mean apoptosis rate) were: 0 muM (2.62. + -. 0.210) (not shown), 0.1. mu.M (2.28. + -. 0.450), 0.3. mu.M (2.53. + -. 0.382), 1. mu.M (2.39. + -. 0.399), 3. mu.M (3.11. + -. 0.891), 10. mu.M (3.29. + -. 0.284). This suggests that Ulixertinib cannot promote apoptosis of drug-resistant myeloproliferative tumor cells. FIG. 5i is reflected in HEL RE In cells, the PD184352 treatment group drug concentrations (mean apoptosis rate) were: 0 μ M (1.07. + -. 0.532) (not shown), 0.1 μ M (0.650. + -. 0.157), 0.3 μ M (0.990. + -. 0.153), 1 μ M (0.870. + -. 0.0560), 3 μ M (0.970. + -. 0.0600), 10 μ M (1.46. + -. 0.0250). This suggests that PD184352 could not promote apoptosis of drug-resistant myeloproliferative tumor cells. FIG. 5j is reflected in HEL RE In cells, the pimarsertib-treated group drug concentrations (mean apoptosis rate) were: 0 μ M (1.55. + -. 0.372) (not shown), 0.1 μ M (1.45. + -. 0.528), 0.3 μ M (1.63. + -. 0.284), 1 μ M (1.43. + -. 0.189), 3 μ M (1.60. + -. 0.217), 10 μ M (1.77. + -. 0.167). This suggests that pimasetib does not promote apoptosis in drug-resistant myeloproliferative tumor cells. Comparison of apoptosis rates in the two panels in the graph by t-test (. about.p)<0.05,**p<0.01,***p<0.001)。
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (10)
- Use of an inhibitor of the MEK/ERK signalling pathway in the manufacture of a medicament for the treatment of a myeloproliferative tumour.
- 2. The use of claim 1, wherein the myeloproliferative neoplasm is resistant to chemotherapeutic agents.
- 3. The use according to claim 2, wherein the chemotherapeutic agent is a chemotherapeutic agent for the treatment of myeloproliferative tumors, the chemotherapeutic agent comprising luccotinib and/or fexolitinib.
- 4. The use of claim 1, wherein the myeloproliferative neoplasm is polycythemia vera, primary thrombocythemia, or myelofibrosis, which is at least one of primary myelofibrosis, myelofibrosis secondary to polycythemia vera, and myelofibrosis secondary to primary thrombocythemia.
- 5. The use according to claim 1, wherein the MEK/ERK signaling pathway inhibitor is at least one of Trematinib, Cobimetinib, Binimetinib, Selumetinib, mirdometinib, refetinib, TIC10, Ulixertinib, PD184352, pimasetinib.
- 6. The use of claim 1, wherein the treatment comprises inhibiting proliferation and/or promoting apoptosis of tumor cells.
- Use of a MEK/ERK signalling pathway inhibitor in the manufacture of a medicament for inhibiting proliferation and/or promoting apoptosis of HEL cells.
- 8. The use of claim 7, wherein said HEL cells comprise non-drug resistant HEL cells and/or drug resistant HEL cells.
- 9. The use according to any one of claims 1 to 8, wherein the medicament further comprises other pharmaceutical ingredients for the treatment of myeloproliferative tumors and pharmaceutically acceptable excipients.
- 10. The use according to claim 9, wherein the medicament is in the form of an oral or injectable formulation.
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| WO2015095831A1 (en) * | 2013-12-20 | 2015-06-25 | Biomed Valley Discoveries, Inc. | Cancer treatments using combinations of mtor and erk inhibitors |
| CN111870600A (en) * | 2020-07-13 | 2020-11-03 | 中南大学湘雅二医院 | New application of sorafenib, regorafenib and analogues or derivatives thereof |
| WO2022074600A1 (en) * | 2020-10-08 | 2022-04-14 | Novartis Ag | Use of an erk inhibitor for the treatment of myelofibrosis |
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| WO2015051252A1 (en) * | 2013-10-03 | 2015-04-09 | Duke University | Compositions and methods for treating cancer with jak2 activity |
| WO2015066432A1 (en) * | 2013-11-01 | 2015-05-07 | Foundation Medicine, Inc. | Method of treating post-myeloproliferative neoplasms (mpns) and post-mpn acute myeloid leukemia |
| CN112843055B (en) * | 2021-01-15 | 2022-04-12 | 徐州医科大学 | Pharmaceutical composition and application thereof in preparation of medicines for treating myeloproliferative diseases of targeted calreticulin mutation type |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015095831A1 (en) * | 2013-12-20 | 2015-06-25 | Biomed Valley Discoveries, Inc. | Cancer treatments using combinations of mtor and erk inhibitors |
| CN111870600A (en) * | 2020-07-13 | 2020-11-03 | 中南大学湘雅二医院 | New application of sorafenib, regorafenib and analogues or derivatives thereof |
| WO2022074600A1 (en) * | 2020-10-08 | 2022-04-14 | Novartis Ag | Use of an erk inhibitor for the treatment of myelofibrosis |
| TW202227077A (en) * | 2020-10-08 | 2022-07-16 | 瑞士商諾華公司 | Use of an erk inhibitor for the treatment of myelofibrosis |
Non-Patent Citations (1)
| Title |
|---|
| SIMONA STIVALA等: "Targeting compensatory MEK/ERK activation increases JAK inhibitor efficacy in myeloproliferative neoplasms", 《THE JOURNAL OF CLINICAL INVESTIGATION》, vol. 129, no. 4, pages 1596 - 1611, XP055869761, DOI: 10.1172/JCI98785 * |
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| CN115054695B (en) | 2024-06-25 |
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