Combined medicine for treating leukemia and application thereof

Abstract

The invention relates to a combined medicine for treating leukemia and application of the combined medicine, and belongs to the technical field of medicines. The medicine is prepared from the following components: momotinib CYT387 and vonakula. In the embodiment of the invention, the high-concentration Vuniclara can induce the cancer cells to generate SGs, so that the cancer cells are resistant to drugs; the drug molecule CYT387 can effectively target G3BP2, significantly inhibit the formation of SGs and promote the depolymerization of SGs, and the drug molecule CYT387 can cooperatively inhibit the proliferation of leukemia cells and enhance the anti-acute leukemia effect of Venoclara when being combined with Venoclara; the application finds that CYT387 for targeted inhibition of SGs can significantly improve the sensitivity of various acute leukemia cell strains to Vickers, possibly fills the blank that no drug for targeted inhibition of G3BPs exists clinically, and provides a theoretical basis and a candidate scheme for development of novel drug resistance reversal drugs.

Description

Technical Field

[0001] Embodiments of the present disclosure relate to the field of pharmaceutical technology, and in particular, to a combination drug for treating leukemia and its application. Background Art

[0002] Acute leukemia is a malignant hematological tumor that seriously threatens human health, and its incidence shows an upward trend globally. Currently, the main treatment strategies clinically adopted include comprehensive treatment plans such as traditional chemotherapy, molecular targeted therapy, immunotherapy, and hematopoietic stem cell transplantation. Compared with the high cost of immunotherapy and the high risk of hematopoietic stem cell transplantation, chemotherapy and targeted therapy are still the main means of clinical treatment of acute leukemia due to their wide range of applicable populations and mature treatment plans. However, with the in-depth treatment, the problem of leukemia cells developing drug resistance to chemotherapy drugs and targeted drugs has become increasingly prominent. Drug resistance caused by different reasons leads to a decline in the treatment effect of patients and an increase in the recurrence rate, seriously affecting the long-term survival of patients. According to statistics, the 5-year overall survival rate of acute leukemia patients is still less than 40%, and drug resistance recurrence is one of the main reasons for treatment failure. Therefore, in view of the problem of leukemia cell drug resistance, further exploring and discovering new molecular targets and developing new drugs remains an important direction in current medical research.

[0003] Therefore, it is necessary to improve one or more problems existing in the above-mentioned related technical solutions.

[0004] It should be noted that this part aims to provide background or context for the technical solutions of the present disclosure stated in the claims. The description herein is not admitted to be prior art merely because it is included in this part. Summary of the Invention

[0005] The purpose of the embodiments of the present disclosure is to provide a combination drug for treating leukemia and its application, thereby at least to some extent overcoming one or more problems caused by the limitations and defects of the related art.

[0006] The embodiments of the present disclosure first provide a combination drug for treating leukemia, and the drug includes: Momelotinib CYT387 and Venetoclax.

[0007] In the embodiments of the present disclosure, the Momelotinib CYT387 is in the form of capsules or tablets, and the Venetoclax is in the form of capsules or tablets.

[0008] The embodiments of the present disclosure secondly provide an application of the combination drug in the preparation of a drug for treating leukemia, using the combination drug described in any one of the above embodiments as a drug for treating leukemia.

[0009] In the embodiments of the present disclosure, the leukemia is acute leukemia.

[0010] In the embodiments of the present disclosure, the leukemia is acute lymphoblastic leukemia or acute myeloid leukemia.

[0011] The technical solutions provided by the embodiments of the present disclosure may include the following beneficial effects:

[0012] (1) The drug molecule CYT387 can effectively target G3BP2, significantly inhibit the formation of stress granules (SGs) and promote the disassembly of SGs. When used in combination with venetoclax, it has a good synergistic inhibitory effect on leukemia cells. It may fill the gap in clinically available drugs that target and inhibit G3BPs, providing a theoretical basis and candidate solutions for the development of new drug resistance reversal drugs, and having important scientific significance and clinical application prospects.

[0013] (2) CYT387 can target the SGs protein G3BP2 and promote the disassembly of SGs, thereby synergistically enhancing the anti-acute leukemia effect of venetoclax. This discovery reveals a new target and new mechanism of CYT387, providing experimental evidence and theoretical support for its clinical application and the development of new uses.

[0014] (3) The present invention discovers that high concentrations of venetoclax can also induce cancer cells to produce SGs, and this application discovers that CYT387, which targets and inhibits SGs, can significantly improve the sensitivity of various leukemia cell lines to venetoclax. These results indicate that venetoclax may cause cell drug resistance by inducing tumor cells to produce SGs, and combining it with drugs that inhibit the formation of SGs can improve the drug resistance situation of venetoclax. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings herein are incorporated into the specification and form a part of the specification, showing embodiments consistent with the present disclosure, and are used together with the specification to explain the principles of the present disclosure. Obviously, the accompanying drawings in the following description are only some embodiments of the present disclosure, and those of ordinary skill in the art can obtain other drawings based on these drawings without creative efforts.

[0016] Figure 1 It is a result diagram of the specific induction of the degradation of G3BPs protein in MDA-MB-231 cells by CYT387 in the present invention;

[0017] Figure 2 It is a molecular docking simulation diagram of CYT387 with G3BP1-NTF2L (A) and G3BP2-NTF2L (B) respectively in the present invention;

[0018] Figure 3 It is a detection result diagram of the binding of CYT387 to the in vitro purified His-G3BP2-NTF2L protein in the present invention;

[0019] Figure 4 This is the result graph of CYT387 in the present invention inhibiting the formation of stress granules (SGs) induced by NaAsO2;

[0020] Figure 5 This is the result graph of CYT387 in the present invention promoting the disassembly of SGs induced by NaAsO2;

[0021] Figure 6 This is the result graph of high - concentration venetoclax (ABT - 199) inducing SGs in cells in the present invention;

[0022] Figure 7 This is the result graph of CYT387 in the present invention significantly enhancing the pro - apoptotic effect of venetoclax in OCI - AML - 2, U937, MOLT - 4, and CCRF - CEM cells;

[0023] Figure 8 This is the result graph of CYT387 in the present invention significantly enhancing the anti - proliferative effect of venetoclax in OCI - AML - 2, U937, MOLT - 4, and CCRF - CEM cells;

[0024] Figure 9 This is the result graph of knocking down G3BPs enhancing the inhibitory effect of venetoclax on the proliferation of MOLT - 4 cells in the present invention. Detailed implementation manners

[0025] Example embodiments will now be described more fully with reference to the accompanying drawings. However, the example embodiments can be implemented in various forms and should not be construed as limited to the examples set forth herein; rather, these embodiments are provided so that this disclosure will be more thorough and complete, and will fully convey the concept of the example embodiments to those skilled in the art. The features, structures, or characteristics described can be combined in any suitable manner in one or more embodiments.

[0026] In addition, the accompanying drawings are only schematic illustrations of the embodiments of the present disclosure and are not necessarily drawn to scale. The same reference numerals in the drawings denote the same or similar parts, and thus repeated descriptions thereof will be omitted. The experimental methods in the examples are conventional methods unless otherwise specified. The experimental materials used in the examples are conventional biochemical reagents unless otherwise specified.

[0027] In this example embodiment, a combined drug for treating leukemia is provided. The drug includes momelotinib CYT387 and venetoclax. Among them, the momelotinib CYT387 is in the form of capsules or tablets, and the venetoclax is in the form of capsules or tablets.

[0028] Among them, CYT387 (Momelotinib) is an inhibitor of tyrosine protein kinases JAK1 / JAK2 and tyrosine kinase receptor ALK2, and was first approved by the US Food and Drug Administration (FDA) in September 2023 for the treatment of myelofibrosis. CYT387 can inhibit KRAS-driven tumor growth and overcome KRAS-mediated immune tolerance responses by targeting TBK1 / IKKε, indicating that CYT387 is a multi-target drug and has the potential to treat malignant tumors. However, in the related technologies, there is no disclosure of the technology of combining it with Venetoclax for the treatment of leukemia, especially for the treatment of acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML).

[0029] It should be noted here that generally, the drugs used to treat ALL and AML are very different, and the drugs between the two cannot be used interchangeably. In most cases, the drugs for AML cannot be used for ALL. There are significant differences in the cell origin, molecular mechanism and drug sensitivity between the two.

[0030] First of all, there are differences in the cell origin and targets between ALL and AML. ALL targets lymphoid progenitor cells (such as CD19, CD22, BCR-ABL fusion gene, etc.), while AML targets myeloid progenitor cells (such as CD33, FLT3, IDH mutations, etc.).

[0031] Secondly, the core chemotherapy drugs for ALL and AML are different. The core drug for ALL, such as asparaginase, is ineffective for AML (myeloid cells can synthesize asparagine by themselves). The first-choice drug for AML, such as cytarabine, has a significant effect on AML, but has limited efficacy for ALL.

[0032] Then, the targeted therapies for ALL and AML each have specificities. The targeted drug for ALL (such as BCR-ABL TKI) is only effective for Ph+ALL, and BCR-ABL is rare in AML (except in the blast crisis of CML). The targeted drugs for AML, such as FLT3 / IDH inhibitors, are only effective for AML carrying the corresponding mutations, and ALL usually does not have these mutations.

[0033] Therefore, the targets of ALL and AML are different, and their responses to drugs are different. The drugs and targets for AML cannot be routinely used for the treatment of ALL.

[0034] Venetoclax is mainly used for the treatment of acute myeloid leukemia and chronic lymphocytic leukemia. The combination drug of this application broadens its application to the treatment of acute lymphoblastic leukemia.

[0035] The R & D process, mechanism of action and test effects of the combination drug of this application will be described below.

[0036] Ras-GAP SH3 domain binding proteins (G3BPs) are a class of RNA-binding proteins in mammals, including G3BP1 and G3BP2. G3BPs are highly expressed in a variety of solid tumors such as breast cancer, head and neck cancer, colon cancer, and lung cancer and promote their occurrence and development, but their role in leukemia has rarely been reported. G3BPs promote tumorigenesis and development through various pathways. For example, by participating in the regulation of various survival-related signaling pathways related to carcinogenesis and metastasis, including the NF-κB, Ras, P53 signaling pathways, JAK2 / STAT3 signaling pathway, and ubiquitin-proteasome system, etc., to promote the growth of tumor cells. It can also promote tumor cell immune escape by stabilizing the expression of PD-L1 and downregulating the MHC-I antigen complex. In particular, G3BPs can act as a regulatable switch to trigger phase separation to form stress granules (SGs). G3BP1 and G3BP2 are necessary conditions for the formation of SGs, and at the same time, the ubiquitination and degradation of G3BP1 are also necessary for the disassembly of SGs. SGs are a protective mechanism produced by cells under stress conditions. All studies to date have shown that compared with normal cells, cancer cells have an enhanced ability to form SGs and a high degree of dependence on SGs for survival under adverse conditions and chemotherapy resistance. Therefore, reducing the expression of G3BPs and the formation of SGs through various pathways and small molecule inhibitors targeting G3BPs all show good anti-tumor effects and the effect of making drug-resistant tumor cells sensitive to chemotherapy drugs again. Research has shown that G3BPs are a very promising anti-cancer target, but currently, there are no drugs targeting G3BPs that have been verified clinically.

[0037] This application provides the use of G3BP1 and G3BP2 proteins / genes as anti-acute leukemia cell drug resistance targets.

[0038] Among them, the G3BP1 gene is Ensembl: ENSG00000145907, and the G3BP2 gene is Ensembl: ENSG00000138757.

[0039] The G3BP1 protein is UniProtKB-Q13283, and its amino acid sequence is: MVMEKPSPLLVGREFVRQYYTLLNQAPDMLHRFYGKNSSYVHGGLDSNGKPADAVYGQKEIHRKVMSQNFTNCHTKIRHVDAHATLNDGVVVQVMGLLSNNNQALRRFMQTFVLAPEGSVANKFYVHNDIFRYQDEVFGGFVTEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQAVVSNDMEEHLEEPVAEPEPDPEPEPEQEPVSEIQEEKPEPVLEETAPEDAQKSSSPAPADIAQTVQEDLRTFSWASVTSKNLPPSGAVPVTGIPPHVVKVPASQPRPESKPESQIPPQRPQRDQRVREQRINIPPQRGPRPIREAGEQGDIEPRRMVRHPDSHQLFIGNLPHEVDKSELKDFFQSYGNVVELRINSGGKLPNFGFVVFDDSEPVQKVLSNRPIMFRGEVRLNVEEKKTRAAREGDRRDNRLRGPGGPRGGLGGGMRGPPRGGMVQKPGFGVGRGLAPRQ;The G3BP2 protein is UniProtKB-Q9UN86, and its amino acid sequence is: MVMEKPSPLLVGREFVRQYYTLLNKAPEYLHRFYGRNSSYVHGGVDA SGKPQEAVYGQNDIHHKVLSLNFSECHTKIRHVDAHATLSDGVVVQVMGLLSNSGQPERKFMQTFVLAPEGSVPNKFYVHNDMFRYEDEVFGDSEPELDEESEDEVEEEQEERQPSPEPVQENANSGYYEAHPVTNGIEEPLEESSHEPEPEPESETKTEELKPQVEEKNLEELEEKSTTPPPAEPVSLPQEPPKAFSWASVTSKNLPPSGTVSSSGIPPHVKAPVSQPRVEAKPEVQSQPPRVREQRPRERPGFPPRGPRPGRGDMEQNDSDNRRIIRYPDSHQLFVGNLPHDIDENELKEFFMSFGNVVELRINTKGVGGKLPNFGFVVFDDSEPVQRILIAKPIMFRGEVRLNVEEKKTRAARERETRGGGDDRRDIRRNDRGPGGPRGIVGGGMMRDRDGRGPPPRGGMAQKLGSGRGTGQMEGRFTGQRR.;

[0040] The basic structure of G3BPs proteins consists of a highly conserved nuclear transport factor 2-like domain (NTF2L) at the N-terminus, a mostly disordered and variable middle region, and an RNA-binding domain (RBD) at the C-terminus. The NTF2L domain is a highly conserved region in the G3BPs protein sequence, and the NTF2L regions of G3BP1 and G3BP2 are highly similar. The dimerization of G3BP1 through the NTF2L domain is essential for the assembly of SGs. Disrupting the dimerization of G3BP1 and the ubiquitination of the NTF2L region can both cause the formed SGs to decompose rapidly.

[0041] In the present invention, the NTF2L domain of G3BPs is used as a target, and through computer-aided drug virtual screening technology based on specific targets, 196 hit molecules that may bind to G3BPs are screened out from a bioactive molecule library containing clinical drug molecules. Then, the effects of these compounds on the protein level of G3BPs are detected in the MDA-MB-231 cell line with high expression of G3BPs.

[0042] Figure 1After MDA-MB-231 cells were treated with different compounds (10 μM) for 24 hours, the protein levels of G3BPs were detected by Western Blot (A); After MDA-MB-231 cells were treated with different concentrations of CYT387 for 24 hours (B) and with 10 μM CYT387 for different times (C), the protein levels of G3BPs were detected. The results showed that CYT387 could specifically induce the degradation of G3BPs protein in MDA-MB-231 cells.

[0043] Figure 2 : The crystal structure of CYT387 with G3BP1-NTF2L 7XHF (A) or the crystal structure of G3BP2-NTF2L 5DRV was used for molecular docking with the Glide module of the software, and data processing was performed with Maestro.PyMOL (version 2.3.0) software.

[0044] Figure 3 : According to the principle that the binding of small molecules to proteins will change the thermal stability of proteins, it was detected by fluorescence-labeled differential scanning fluorimetry (FL-DSF) that CYT387 would increase the thermal stability of His-G3BP2-NTF2L protein, and the increase in its TM value would tend to saturate with the increase in compound concentration, indicating that CYT387 could directly bind to His-G3BP2-NTF2L protein, and the binding constant was 2.46 ± 0.75 μM (A); The binding of small molecules to proteins can quench the fluorescence of proteins, and it was detected by fluorescence titration that the binding constant of CYT387 to His-G3BP2-NTF2L protein was 8.65 ± 1.69 μM (B).

[0045] Figure 4 : After MDA-MB-231 cells were pretreated with DMSO or 10 μM CYT387 for 30 minutes, then the strong inducer of SGs, NaAsO2 (500 μM), was added and treated for 30 minutes. The cells were fixed with 4% paraformaldehyde, and immunofluorescence staining experiments were performed with G3BP1 and G3BP2 antibodies and DAPI dye. The results showed that the SGs induced in the CYT387 pretreatment group were significantly reduced.

[0046] Figure 5 : After MDA-MB-231 cells were treated with NaAsO2 (500 μM) for 30 minutes, then DMSO or 10 μM CYT387 was added and treated for 30 minutes. The cells were fixed with 4% paraformaldehyde, and immunofluorescence staining experiments were performed with G3BP1 and G3BP2 antibodies and DAPI dye. The results showed that the addition of CYT387 could significantly depolymerize the already formed SGs.

[0047] Figure 6 : Immunofluorescence staining experiments were performed to detect the generation of stress granules (SGs) in MDA-MB-231 and HeLa cells after treatment with different concentrations of venetoclax for different durations. The results showed that short-term treatment with high concentrations (50 μM or 100 μM, comparable to the concentration that induces SGs formation by docetaxel) of venetoclax or long-term treatment with lower concentrations could significantly induce the generation of SGs in cells.

[0048] Figure 7 : OCI-AML-2, U937, MOLT-4, and CCRF-CEM cells were treated with the corresponding concentrations of the compound alone and in combination for 24 hours, and then the apoptosis rate of the cells was detected using a cell apoptosis detection kit, and the apoptosis of each group was statistically analyzed. The results showed that the pro-apoptotic effect of the combination of CYT387 and venetoclax was greater than the sum of the pro-apoptotic effects of the two drugs alone, indicating strong synergy between the two drugs.

[0049] Figure 8 : U937, OCI-AML-2, MOLT-4, and CCRF-CEM cells were treated with different concentration combinations of CYT387 and venetoclax for 48 hours, and the cell viability was detected using a CCK-8 kit. Then, the obtained results were used to calculate the synergy coefficient of the drug combination using the Synergyfinder R package (version 2.4.13). A synergy coefficient greater than 0 indicates synergy. The results showed that the synergy coefficients of the combination of CYT387 and venetoclax in U937, OCI-AML-2, MOLT-4, and CCRF-CEM cells were 7.1, 8.33, 9.76, and 8.49, respectively, and all were statistically significant, further demonstrating the effectiveness of the combination of the two drugs.

[0050] Figure 9 : MOLT-4 cells infected with lentiviruses packaged with shRNA-negative control (shNC) and shG3BP1 + shG3BP2 (shG3BPs) were treated with different concentrations of ABT-199 for 48 hours, and the cell viability was detected using a CCK-8 kit. The results showed that knockdown of the expression of G3BPs could significantly increase the inhibitory effect of venetoclax on the proliferation of MOLT-4 cells.

[0051] After the above-mentioned experiments and tests, it was found in this application that the drug molecule CYT387 can specifically induce the degradation of G3BP1 and G3BP2 proteins. The concentration gradient and time gradient experiments further demonstrated the degradation effect of CYT387 on G3BPs. The results of molecular docking simulations showed that CYT387 can bind to the active sites F124 and N122 of the G3BP1-NTF2L protein, and can also bind to F124 of the G3BP2-NTF2L protein, and both are hydrogen bond interactions with relatively strong forces. The results of in vitro purified protein and small molecule binding experiments further verified the binding of CYT387 to G3BP2-NTF2L. G3BPs are key molecules that regulate the formation and disassembly of SGs. It was found in this invention that when cells were pretreated with CYT387 for 30 minutes and then with the strong inducer of SGs, NaAsO2, it was difficult to induce the formation of SGs. However, when cells were first induced to produce SGs with NaAsO2 and then treated with CYT387 for 30 minutes, it could significantly promote the disassembly of the already formed SGs, indicating that CYT387 can both inhibit the formation of SGs and promote the disassembly of SGs. It was also found in this invention that high concentrations of venetoclax can induce the production of SGs in cancer cells. The combination of CYT387 and venetoclax can significantly enhance the inhibitory effect of venetoclax on the proliferation of acute myeloid leukemia cell line OCI-AML-2 and acute lymphoblastic leukemia cell lines U937, MOLT-4, and CCRF-CEM. Moreover, knocking down G3BPs can also significantly enhance the sensitivity of MOLT-4 cells to venetoclax. These experimental results indicate that the drug combination of CYT387 targeting G3BPs and venetoclax is an effective and innovative treatment plan for acute leukemia.

[0052] In summary, the beneficial effects of this application are as follows:

[0053] (1) The drug molecule CYT387 can effectively target G3BP2, significantly inhibit the formation of SGs and promote the disassembly of SGs. The combination of CYT387 and venetoclax has a good synergistic inhibitory effect on leukemia cells, which may fill the gap in clinically available drugs that target and inhibit G3BPs, provide a theoretical basis and candidate plan for the development of new drug resistance reversal drugs, and has important scientific significance and clinical application prospects.

[0054] (2) CYT387 can target the SGs protein G3BP2 and promote the disassembly of SGs, thereby synergistically enhancing the anti-acute leukemia effect of venetoclax. This discovery reveals a new target and new mechanism of CYT387, providing experimental basis and theoretical support for its clinical application and the development of new uses.

[0055] (3) It has been found in the present invention that high concentrations of venetoclax can also induce the formation of stress granules (SGs) in cancer cells, and it has been discovered in this application that CYT387, which targets and inhibits SGs, can significantly enhance the sensitivity of various leukemia cell lines to venetoclax. These results indicate that venetoclax may render cells drug-resistant by inducing the formation of SGs in tumor cells, and combining it with drugs that inhibit the formation of SGs can improve the drug-resistant situation of venetoclax.

[0056] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present disclosure. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine the different embodiments or examples described in this specification.

[0057] After considering the specification and practicing the invention disclosed herein, those skilled in the art will readily conceive of other embodiments of the present disclosure. This application is intended to cover any variations, uses, or adaptations of the present disclosure, which follow the general principles of the present disclosure and include the common general knowledge or conventional technical means in the technical field not disclosed in the present disclosure. The specification and examples are only regarded as exemplary, and the true scope and spirit of the present disclosure are pointed out by the appended claims.

Claims

1. A combined drug for treating leukemia, characterized in that, The drugs include momelotinib CYT387 and venetoclax.

2. The combination drug for treating leukemia according to claim 1, characterized in that, The momelotinib CYT387 is in the form of capsules or tablets, and the venetoclax is in the form of capsules or tablets.

3. Use of a combination drug in the preparation of a drug for treating leukemia, characterized in that, The combined drug described in claim 1 or 2 is used as a drug for treating leukemia.

4. Use of the combined drug according to claim 3 in the preparation of a drug for treating leukemia, characterized in that, The leukemia is acute leukemia.

5. Use of the combined drug according to claim 4 in the preparation of a drug for treating leukemia, characterized in that, The leukemia is acute lymphoblastic leukemia or acute myeloid leukemia.

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