Use of egfr inhibitors in the preparation of a medicament for reversing venetoclax resistance in acute myeloid leukemia

Abstract

The application relates to the field of biological medicine, and particularly relates to application of an EGFR inhibitor in preparation of a medicine for reversing acute myeloid leukemia venetoclax drug resistance. Experimental results show that combination of osimertinib and venetoclax can significantly reduce tumor load of a venetoclax drug resistance model mouse, and obviously prolong survival time. It is shown that the combination can effectively reverse the venetoclax drug resistance, and provides a new way for the acute myeloid leukemia venetoclax drug resistance.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of EGFR inhibitors in the preparation of drugs to reverse Venecella resistance in acute myeloid leukemia. Background Technology

[0002] Venecella, an FDA-approved small molecule inhibitor of BCL2, has been widely used in the treatment of acute myeloid leukemia (AML) in combination with demethylating agents or low-dose cytarabine, with an objective response rate as high as 70%. However, veneclade resistance usually occurs within one year, and there is currently no effective salvage therapy strategy. The median survival time after drug resistance is only 2.4 months.

[0003] Combining MCL1 inhibitors with veneclade is a crucial approach to reversing veneclade resistance in AML. Currently developed small molecule MCL1 inhibitors, including S64315, VU661013, AZD5991, AMG-397, AMG-176, ABBV-467, PRT-1419, and A-1210477, are mostly in preclinical research. It is noteworthy that the development of MCL1 small molecule inhibitors has frequently encountered obstacles. For example, AZD5991 and ABBV-467, which had already entered clinical trials, were halted by the FDA due to cardiotoxicity and other reasons. Considering the high investment and risk of new drug development, this study proposes a "drug repurposing" approach, employing an efficient, economical, and low-risk drug development strategy to provide a drug that can effectively overcome AML veneclade resistance, aiming to offer a potential treatment strategy for reversing AML veneclade resistance in clinical practice. Summary of the Invention

[0004] In view of this, the present invention provides the use of EGFR inhibitors in the preparation of medicaments for reversing Venecella resistance in acute myeloid leukemia.

[0005] This invention provides the application of EGFR inhibitors in the preparation of drugs to reverse drug resistance in leukemia.

[0006] In this invention, the leukemia is acute myeloid leukemia.

[0007] In this invention, the drug resistance refers to resistance to BCL2 small molecule inhibitors. In some embodiments, the BCL2 small molecule inhibitor includes veneclade.

[0008] In this invention, the EGFR inhibitor includes at least one of osimertinib, omamolinib, dacomitinib, afatinib, and ametinib.

[0009] In a specific embodiment of the present invention, the constructed veneclade-resistant AML cell lines MV-4-11 VR and MOLM13 VR were used to screen for drugs that could reverse veneclade resistance in AML. The results showed that several EGFR inhibitors, such as osimertinib, omamotinib, dacomitinib, afatinib, and ametinib, exhibited significant synergistic effects with veneclade. Among these, osimertinib had the highest synergistic index (synergistic index = 24.425), while icotinib, gefitinib, and erlotinib showed no synergistic effect with veneclade.

[0010] Both in vitro colony formation assays and flow cytometry studies showed that the combination of osimertinib and veneclade significantly increased apoptosis in veneclade-resistant AML cells. In vivo experiments demonstrated that the combination of osimertinib and veneclade significantly reduced tumor burden in veneclade-resistant mouse models, significantly prolonged survival time, reduced splenomegaly, and prevented or reduced liver and spleen damage.

[0011] Based on the above results, the present invention provides a composition containing an EGFR inhibitor and Venecla, and the use of the composition in the preparation of a medicament for reversing (or preventing) acute myeloid leukemia.

[0012] This invention also provides a remedy for reversing Venecella resistance in acute myeloid leukemia, including EGFR inhibitors.

[0013] Compositions for reversing (or preventing) acute myeloid leukemia, including EGFR inhibitors and veneclade.

[0014] In the composition of the present invention, the EGFR inhibitor includes at least one of osimertinib, omamolinib, dacomitinib, afatinib, and ametinib.

[0015] In some embodiments, the composition comprises osimertinib and veneclade. Preferably, the mass ratio of osimertinib to veneclade is 1:2.

[0016] The present invention also provides the use of the composition in the preparation of a medicament for reversing (or preventing) acute myeloid leukemia.

[0017] The present invention also provides a medicament for reversing (or preventing) acute myeloid leukemia, comprising the composition described above.

[0018] The medicament described in this invention also includes pharmaceutically acceptable excipients. This invention does not impose any particular limitation on the specific types of excipients. Those skilled in the art can select suitable types according to actual circumstances and needs, and formulate suitable dosage forms according to methods commonly used in the art. The dosage forms include, but are not limited to, tablets, granules, powders, pills, injections, suspensions, etc.

[0019] The present invention also provides a method for reversing (or preventing) Venecella resistance in acute myeloid leukemia, comprising: administering an EGFR inhibitor to the human body.

[0020] The present invention also provides a method for reversing (or preventing) acute myeloid leukemia, comprising: administering to a human the composition, drug, or composition comprising an EGFR inhibitor and veneclade, or a drug thereof.

[0021] The types of leukemia, drug-resistant drugs, and EGFR inhibitors mentioned above are as described above and will not be repeated here.

[0022] In this invention, the composition and drug can be used preventively or as a therapeutic agent, and this invention does not limit this use.

[0023] In this invention, the order of use of the EGFR inhibitor and veneclade in the composition or drug is not particularly restricted. They can be used simultaneously or sequentially, i.e., veneclade can be used first followed by the EGFR inhibitor, or the EGFR inhibitor can be used first followed by veneclade. The specific timing and method of use can be selected according to the actual situation, and this invention does not impose any special restrictions on them.

[0024] This invention addresses the slow progress in the development of MCL1 small molecule inhibitors. Taking a "drug repurposing" approach, it screened several EGFR inhibitors, including osimertinib, to overcome veneclade resistance in AML. Osimertinib, widely used in the treatment of non-small cell lung cancer (NSCLC) with guaranteed safety and controllability, was chosen as the primary candidate for animal studies. The results showed that the combination of osimertinib and veneclade significantly prolonged survival in mice without significant toxic side effects. Furthermore, the cellular concentration of osimertinib used was comparable to the reported blood concentrations in the literature (DOI: 10.1016 / j.lungcan.2017.07.007), and these blood concentrations showed no toxic side effects on patients. In conclusion, the combination of osimertinib and veneclade has good clinical translational value and is expected to become a treatment strategy for overcoming veneclade resistance in AML patients. Attached Figure Description

[0025] Figure 1 Construction of vinecrate-resistant AML cell lines; A shows the sensitivity test of 11 common AML cell lines to vinecrate; B shows the sensitivity test of MV-4-11 VR and MOLM13 VR to vinecrate.

[0026] Figure 2Screening for FDA-approved drugs revealed that osimertinib can overcome AML veneclade resistance; A is the drug screening flowchart; B shows the synergy index of eight EGFR inhibitors combined with veneclade, with a synergy index greater than 10 indicating a synergistic effect, -10 to 10 indicating an additive effect, and less than 10 indicating an antagonistic effect; C-D show the synergistic effect of osimertinib combined with veneclade in two veneclade-resistant cell lines (MV-4-11 VR and MOLM13 VR); E-G show the effect of simertinib combined with veneclade on the colony-forming ability of the two veneclade-resistant cell lines (MV-4-11 VR and MOLM13 VR); HK shows the effect of flow cytometry on apoptosis of the two veneclade-resistant cell lines (MV-4-11 VR and MOLM13 VR); ***p<0.001;

[0027] Figure 3 The combination therapy of osimertinib and veneclade inhibits the proliferation of veneclade-resistant AML cells in vivo; A is the flowchart of the animal experiment; B shows the survival status of mice after drug treatment; C-D show the proportion of human CD45 positive cells in bone marrow; E-F show photographs and weighing of mouse spleens; G-H show immunohistochemistry and HE staining of mouse liver and spleen; I shows the change in mouse body weight; J and M represent the serum levels of alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, and creatinine in mice, respectively; ***p<0.001;

[0028] Figure 4 The study aimed to significantly promote apoptosis in primary cells from resistant patients using osimertinib combined with veneclax. A represents flow cytometry analysis of apoptosis in primary cells from veneclax-resistant patients after treatment with osimertinib, veneclax, or the combination therapy. B represents statistical analysis of apoptosis in 6 primary samples. **p<0.01. Detailed Implementation

[0029] This invention provides the application of an EGFR inhibitor in the preparation of a drug for treating veneclade-resistant retrograde myeloid leukemia. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0030] In this document, the terms "including", "comprising", and "having" describe both closed-loop technical solutions consisting of the listed features and open-loop technical solutions that include the listed features.

[0031] The term “and / or” as used herein includes any and all combinations of one or more of the related listed items.

[0032] In this document, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when a range refers to an integer, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0033] Experiments showed that the combination of osimertinib and veneclade significantly reduced the tumor burden and prolonged the survival time of veneclade-resistant mouse models. This indicates that the combination can effectively prevent and treat veneclade resistance. Based on this, the present invention provides the application of EGFR inhibitors in the preparation of drugs for preventing and treating drug-resistant leukemia.

[0034] This invention successfully constructed a veneclade-resistant AML cell line using a concentration gradient approach. After two rounds of screening using an FDA-approved drug library, the first round used a 10 μM concentration for general screening, identifying 236 FDA-approved drugs that could significantly kill veneclade-resistant AML cells. These included several EGFR inhibitors (osimertinib, afatinib, ametinib, dacomitinib, omamotinib, icotinib, gefitinib, and erlotinib). A second round of screening was then conducted using concentration gradients (EGFR inhibitors: 0, 200, 500, 1000 nM; VEN: 0, 20, 50, 100, 200 nM). The results showed that osimertinib, omamotinib, dacomitinib, afatinib, and ametinib exhibited significant synergistic effects with veneclade, with osimertinib showing the highest synergistic index (synergistic index = 24.425). Icotinib, gefitinib, and erlotinib, however, showed no synergistic effect with veneclade. Colony formation assays also showed that the combination of osimertinib and veneclade significantly killed veneclade-resistant AML cells. Furthermore, using a mouse model constructed with veneclade-resistant cells, the in vivo killing effect of the combination of osimertinib and veneclade on resistant cells was evaluated. The results showed that the combination of osimertinib and veneclade significantly reduced tumor burden and prolonged survival time in the resistant mouse model. Flow cytometry analysis of primary samples from veneclade-resistant patients revealed that the combination of osimertinib and veneclade significantly increased apoptosis in primary cells. Based on these results, it can be concluded that the strategy of using osimertinib in combination with veneclade can effectively overcome veneclade resistance in AML.

[0035] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0036] The present invention will be further illustrated below with reference to the embodiments:

[0037] Example 1

[0038] (1) Construction of Veneclare-resistant cell lines

[0039] We selected 11 common AML cell lines (MV-4-11, MOLM13, HL-60, NB4, U937, KG-1a, Kasumi-1, THP-1, HEL, K562, and OCI-AML3) and tested their sensitivity to Veneclare using CCK-8 assay. Figure 1 In section A, relatively sensitive MV-4-11 and MOLM13 cells were selected to construct veneclade-resistant cell lines (denoted as MV-4-11 VR and MOLM13 VR, respectively) through a concentration gradient escalation method. Their tolerance to veneclade was increased by approximately 80-fold and 100-fold, respectively. Figure 1 (B)

[0040] (2) Screening for drugs that can reverse AML veneclade resistance through the FDA-approved drug library.

[0041] We used a constructed veneclade-resistant AML cell line and conducted two rounds of drug screening. The first round involved a broad screening at a 10 μM concentration, which identified 236 FDA-approved drugs that significantly killed veneclade-resistant AML cells. These included several EGFR inhibitors (osimertinib, afatinib, ametinib, dacomitinib, omamolinib, icotinib, gefitinib, and erlotinib). We then set up concentration gradients (EGFR inhibitors: 0, 200, 500, 1000 nM; VEN: 0, 20, 50, 100, 200 nM) for the second round of screening. Figure 2 (A) Using the ZIP model calculated by the online software SynerFinder (https: / / synergyfinder.fimm.fi / ), the study found that osimertinib, omamotinib, dacomitinib, afatinib, and ametinib had significant synergistic effects with veneclax. Among them, osimertinib and veneclax had the highest synergistic index (synergistic index = 24.425), while icotinib, gefitinib, and erlotinib showed no synergistic effect with veneclax. Figure 2 (BD). Colony formation assays also showed that osimertinib combined with veneclade significantly killed veneclade-resistant AML cells ( Figure 2 In addition, flow cytometry analysis using Annexin V / PI staining revealed that the combination of osimertinib and veneclade significantly increased apoptosis in MV-4-11 VR and MOLM13 VR cells. Figure 2 (Hong Kong).

[0042] (3) Investigating the effect of osimertinib combined with veneclax on reversing AML veneclax in a mouse model

[0043] To evaluate the safety and synergistic effects of osimertinib and veneclax in vivo, we established a xenograft AML mouse model using MOLM13 VR cells. First, 24 hours after irradiation with 2 Gy X-rays, MOLM13 VR cells were intravenously transplanted into NOD / SCID mice via the tail vein. Seven days post-transplantation, mice were administered osimertinib (20 mg / kg / day), veneclax (40 mg / kg / day), or a combination thereof via intraperitoneal injection (Figure 3A). Kaplan-Meier overall survival analysis showed that the combination therapy of osimertinib and veneclax significantly prolonged the survival time of MOLM13 VR cell transplanted mice (Figure 3B). Flow cytometry analysis of human CD45-positive leukemia cells in the bone marrow revealed that the AML burden in the combination therapy group was significantly lower than that in the osimertinib or veneclax monotherapy groups (Figure 3C, D). The combination therapy reduced the mean proportion of human CD45-positive leukemia cells from 71.52% to 19.10%. Spleen weight analysis and photographs showed that, compared with the solvent control group, the combination of osimertinib and veneclax significantly reduced splenomegaly in mice treated with osimertinib or veneclax alone (Figure 3, E, F). Immunohistochemical staining (IHC) of paraffin sections of spleen and liver with human CD45 antibody showed that the proportion of CD45-positive cells in the spleen of mice treated with osimertinib or veneclax alone was significantly higher, while the proportion of CD45-positive cells in the spleen of mice treated with the combination of osimertinib and veneclax was significantly lower, and no CD45 expression was detected in the liver (Figure 3, G). Hematoxylin-eosin (HE) staining was also performed. The degree of splenic structural damage in mice treated with the combination of osimertinib and veneclax was less than that in mice treated with osimertinib or veneclax alone and in the solvent control group, while no significant liver damage was observed. Figure 3 (H).

[0044] Furthermore, the weight changes in the solvent control group and all treatment groups were similar (Figure 3, I), and there were no significant differences in serum aspartate aminotransferase, alanine aminotransferase, creatinine, and blood urea nitrogen levels (Figure 3, JM), indicating that the combination of osimertinib and veneclade had no obvious toxicity.

[0045] (4) Primary cell assay in patients with veneclade-resistant AML: efficacy of osimertinib combined with veneclade in reversing veneclade resistance in AML

[0046] Bone marrow aspiration specimens were collected from six patients with veneclade-resistant AML. After density gradient centrifugation, mononuclear cells were collected and treated with osimertinib, veneclade, or a combination of both drugs. Flow cytometry was used to detect cell apoptosis. The results showed that the combination of osimertinib and veneclade significantly promoted cell apoptosis. Figure 4 (A and B in the middle).

[0047] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of an EGFR inhibitor in the preparation of a drug for reversing the drug resistance of venetoclax in acute myeloid leukemia; the EGFR inhibitor comprises at least one of osimertinib, olmutinib, dacomitinib, afatinib, amatinib.

2. Use of a composition in the preparation of a drug for reversing acute myeloid leukemia; the composition comprises an EGFR inhibitor and venetoclax; the EGFR inhibitor comprises at least one of osimertinib, olmutinib, dacomitinib, afatinib, amatinib. The composition consists of osimertinib and venetoclax.

3. Use according to claim 2, characterized in that, The mass ratio of the osimertinib and venetoclax is 1:

2.

4. Use according to claim 3, characterized in that, The drug comprises the composition and adjuvants.

5. Use according to claim 2, characterized in that, The dosage form of the drug comprises tablets, granules, powders, pills, injections or suspensions.

6. Use according to claim 2, characterized in that, ​

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