A combined preparation for ph+ all and use thereof

Abstract

This invention belongs to the field of biomedicine, specifically relating to a combination drug composition for Ph+ALL and its application. This invention discloses a combination drug composition for Ph+ALL, characterized by comprising a BCL-2 inhibitor, a demethylating agent, and a TKI. The technical solution of this invention expands the chemotherapy-free treatment modality for Ph+ALL, not only enabling patients to achieve rapid and deep remission while observing no unacceptable treatment-related toxicities, but also significantly improving the quality of life of treatment-naïve patients, reducing treatment-related complications, decreasing the consumption of blood products, shortening hospital stays, and significantly reducing medical insurance expenditures.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a combination drug composition for Ph+ALL and its application. Background Technology

[0002] Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) is the most common type of ALL in adults, accounting for approximately 25%-30%. Ph+ALL shows a trend towards onset in middle-aged and elderly individuals, accounting for more than 50% of B-ALL patients over 50 years of age. Based on molecular biology, Ph+ALL is a high-risk subtype with a poor prognosis. Traditional chemotherapy is not only ineffective but also carries significant treatment-related toxicities due to multi-drug combination regimens, resulting in long-term survival rates of less than 20%. Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) has long been considered the only cure for this disease.

[0003] Ph chromosome 20 is formed by the translocation of the C-ABL proto-oncogene from the long arm of chromosome 9 to the breakpoint cluster region (BCR) of the long arm of chromosome 22, creating the BCR-ABL fusion gene. The fusion protein encoded by the BCR-ABL fusion gene has enhanced tyrosine kinase activity, leading to uncontrolled cell growth and promoting the development of leukemia. Since 2000, the introduction of BCR-ABL-targeting tyrosine kinase inhibitors (TKIs) into the overall treatment of Ph+ALL has significantly improved treatment outcomes. The treatment strategy for Ph+ALL has shifted from the traditional multi-drug combination chemotherapy model to a model based on TKIs combined with standard or reduced-dose chemotherapy. Under the current treatment model, the complete remission (CR) rate for Ph+ALL patients can reach 90%-100%, and the 5-year overall survival (OS) has increased to 45%-70%. Despite the significant efficacy of TKIs combined with chemotherapy, frequent relapses and drug resistance remain pressing issues in clinical treatment. Data from MD Anderson Cancer Center in the United States shows that 25% of patients experience morphological relapse 15.9 months after the median first remission. 75% of relapsed patients receive TKI+ salvage chemotherapy. Although 84% of patients can achieve remission again, the 1-year overall survival (OS) is only 41%, and the 2-year OS is less than 20%. Even Allo-HSCT does not change the survival outcome. At the same time, TKI combined with chemotherapy also faces problems such as a high risk of treatment-related complications and poor tolerance in elderly patients. Poor treatment adherence or repeated discontinuation due to toxicity can also increase the risk of disease progression. Therefore, there is an unmet clinical need to explore new, effective, low-toxicity, or even chemotherapy-free treatment strategies to overcome the limitations of existing treatments, enable more Ph+ALL patients to achieve deep molecular remission in the early stages, reduce relapse, and even achieve long-term survival without the need for Allo-HSCT. Summary of the Invention

[0004] The purpose of this invention is to provide a combination drug composition for Ph+ALL and its application, which effectively promotes the clinical need for low-toxicity and rapid deep remission of Ph+ALL.

[0005] Therefore, the present invention discloses a combination drug composition for Ph+ALL, characterized in that it comprises a BCL-2 inhibitor drug, a demethylating drug and a TKI drug.

[0006] This invention utilizes a three-drug combination regimen consisting of a BCL-2 inhibitor, a demethylating agent, and a TKI to achieve early and deep molecular remission in Ph+AL patients, especially newly diagnosed Ph+ALL patients, through the synergistic effect of the drugs.

[0007] In some preferred embodiments, the Ph+ALL is preferably a newly diagnosed Ph+ALL.

[0008] In some preferred embodiments, the BCL-2 inhibitor is venetoclax, navittoclax, ombatoc, S55746, APG-2575, ABT-737, AMG176, AZD5991 or APG-1252, with venetoclax (V) being the most preferred.

[0009] In some preferred embodiments, the demethylating agent is azacitidine or decitabine, with azacitidine (A) being the most preferred.

[0010] In some preferred embodiments, the TKI drug is preferably a second-generation TKI, such as flumatinib, dasatinib, nilotinib, ladotinib, and bosutinib, with flumatinib (F) being the most preferred.

[0011] In some preferred embodiments, the combined pharmaceutical composition is a combination of three separate formulations, more preferably, the three separate formulations are administered simultaneously or sequentially.

[0012] In some preferred embodiments, the mass ratio of venetoclax, azacitidine, and flumatinib is 21–80:15–22:112–168.

[0013] In a preferred embodiment, venetoclax, azacitidine, and flumatinib are all packaged separately, with 21 to 79 tablets of venetoclax (100mg / tablet), 15 to 22 vials of azacitidine (100mg / vial for injection), and 56 to 84 tablets of flumatinib (200mg / tablet) to meet the needs of a single course of treatment.

[0014] Secondly, the present invention provides the use of the combined pharmaceutical composition as described herein in the preparation of a medicament for treating Ph+ALL.

[0015] Thirdly, the present invention provides the use of the combined pharmaceutical composition as described herein in the preparation of a Ph+ALL cell proliferation inhibitor.

[0016] This article discloses a method for treating Ph+ALL disease, the method comprising administering a therapeutically effective amount of a combination of pharmaceutical compositions to a subject in need.

[0017] This invention expands the chemotherapy-free treatment modality for Ph+ALL, enabling patients to achieve rapid and deep remission. Compared with other historical treatment regimens, the three-drug combination regimen of this invention has a higher CR rate (11 / 11, 100%) and early complete molecular remission (CMR) rate (9 / 10, 90%; median CMR time, 21 days). Simultaneously, no unacceptable treatment-related toxicities were observed. Furthermore, it significantly improves the quality of life for treatment-naïve patients, reduces treatment-related complications, decreases blood product consumption, shortens hospital stays, and significantly reduces medical insurance expenditures. In the future, Ph+ALL is expected to become a disease with a good prognosis, similar to acute promyelocytic leukemia (APL) and chronic myeloid leukemia (CML), no longer relying on transplantation for long-term survival, and may even be treatable at home or in day clinics. Moreover, to date, there are no reports of using the three-drug combination of VAF for the treatment of Ph+ALL. Attached Figure Description

[0018] Figure 1 Vinecoxib, azacitidine, and flumatinib on the IC50 of SUPB15 cells 50 Situation diagram;

[0019] Figure 2 A schematic diagram comparing the inhibition rates of SUPB15 cells by single and combined applications of venetoc, azacitidine, and flumatinib.

[0020] Figure 3 A schematic diagram illustrating the significant synergistic effect of the three drugs combined with CompuSyn.

[0021] Figure 4 A schematic diagram showing the results of quantitative analysis of bone marrow blast cells and fusion genes in Case 1 treated with the VAF regimen of this invention. Detailed Implementation

[0022] List of abbreviations, English translations, and key term definitions

[0023] 1. Ph, Philadelphia chromosome

[0024] 2. ALL, Acute lymphoblastic leukemia

[0025] 3. TKI, Tyrosine Kinase Inhibitor

[0026] 4. CMR, Complete molecular response

[0027] 5. OS, Overall Survival

[0028] 6、CR,Complete remission

[0029] 7、MMR,Major molecular response

[0030] 8、CCyR,Complete cytogenetic response

[0031] 9、Allo-HSCT,Allogeneic haematopoietic stem cell transplant

[0032] 10、BCR,Breakpoint cluster region

[0033] 11、BCL-2,B-cell lymphoma-2

[0034] The term "BCL-2 protein inhibitor" refers to an inhibitor of the anti-apoptotic BCL-2 protein. The BCL-2 protein family primarily regulates endogenous apoptosis, specifically the mitochondrial / cytochrome c-mediated apoptosis pathway. Currently, 27 members of the BCL-2 family have been identified, which can be divided into antagonistic and pro-apoptotic categories based on their function. There are six antagonistic apoptosis proteins: BCL-XL, BCL-2, BCL-W, MCL-1, BCL-B, and BFL-1, which inhibit the apoptosis pathway. Pro-apoptotic proteins fall into two categories: one is the final executor of mitochondrial damage, namely BAX and BAK. When these two are activated, they can form oligomers that act on the outer mitochondrial membrane. After the outer mitochondrial membrane is damaged, cytochrome c is released, thereby activating caspases to complete the apoptosis process. BCL-2 and other antagonistic apoptosis proteins inhibit apoptosis by directly binding to BAX and BAK. Another class of pro-apoptotic proteins is the BH3-only subclass, which is further divided into activators (including BID and BIM, which can directly activate BAX / BAK) and sensitizers (BAD, BIK, NOXA, HRK, PUMA, and BMF, etc.). BH3-only proteins are also natural antagonists of anti-apoptotic proteins such as BCL-2. Upon receiving stress signals (such as DNA damage, oxidative stress, etc.), BH3-only proteins can activate activators, which then directly activate BAX / BAK, thereby activating the caspase cascade apoptosis response. At the same time, they activate sensitizers to competitively bind to anti-apoptotic proteins. BCL-2 protein inhibitors include, but are not limited to, venetoclax, navitoclax, obatoclax, S55746, APG-2575, ABT-737, AMG176, AZD5991, and APG-1252.

[0035] Demethylating drugs, both in vivo and in vitro, can restore tumor suppressor genes to their normal demethylated state by inhibiting DNA methyltransferase 1 (DNMT1), reactivating genes that have been inactivated due to DNA hypermethylation, and enabling cells to return to normal terminal differentiation, senescence, or apoptosis; representative drugs are azacitidine and decitabine.

[0036] The term "tyrosine kinase inhibitor" (TKI) selectively blocks the binding site of ATP to the BCR-ABL kinase, effectively inhibiting the phosphorylation of tyrosine residues in the BCR-ABL kinase substrate, thus inactivating the enzyme and preventing a series of signal transductions, leading to apoptosis in BCR-ABL-positive cells. Based on the different binding sites of TKIs to the BCR-ABL protein, they can be classified into three types of inhibitors. Bosutinib and dasatinib bind to the ATP site of BCR-ABL using a "DFG-in" conformation and are classified as type I inhibitors; imatinib, nilotinib, flumatinib, and ponatinib bind to the ATP site of BCR-ABL using a "DFG-out" conformation to prevent substrate phosphorylation and are classified as type II inhibitors; GNF-2 and asciminib bind to the myristoyl pocket of BCR-ABL, allosterically inhibiting tyrosine kinase activity and are classified as type IV inhibitors, also called allosteric inhibitors.

[0037] When applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, the terms “administration,” “administering,” “treating,” and “treatment” herein refer to contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and an animal, human, subject, cell, tissue, organ, or biological fluid. Cell treatment includes contact between the reagent and the cell, as well as contact between the reagent and a fluid, wherein the fluid contacts the cell. The terms “administration” and “treatment” also mean in vitro and ex vivo treatments, such as treatment of cells by means of a reagent, diagnostic agent, conjugated compound, or by means of another cell. The term “subject” herein includes any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit), and most preferably a human.

[0038] The term "effective amount" or "therapeutic effective amount" refers to the amount of an active ingredient, such as a compound, sufficient to affect such treatment of a disease, condition, or symptom when administered to a subject for the treatment of a disease or at least one clinical symptom of a disease or condition. "Therapeutic effective amount" can vary depending on the compound, the disease, condition, and / or the symptoms of the disease or condition, the severity of the symptoms, the age of the subject being treated, and / or the weight of the subject being treated. An appropriate amount in any given situation will be obvious to those skilled in the art or can be determined by routine experiments. In some embodiments, "therapeutic effective amount" is the amount of at least one compound disclosed herein and / or at least one stereoisomer thereof and / or at least one pharmaceutically acceptable salt thereof that effectively "treats" the subject's disease or condition as defined above. In the case of combination therapy, "therapeutic effective amount" refers to the total amount of the combination of substances used to effectively treat the disease, condition, or symptom.

[0039] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0040] Example 1:

[0041] Experimental methods:

[0042] 1. Obtaining and culturing Ph+ALL cell lines.

[0043] The SUPB15 cell line was purchased from BeNa Culture Collection (BNCC). The cell line was cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% neomycin / penicillin / streptomycin at 37°C in an incubator containing 5% CO2.

[0044] 2. Drug source

[0045] Venetoclax, azacitidine, and flumatinib were purchased from Selleck Chemicals.

[0046] 3. CCK-8 cytotoxicity assay to detect the IC50 values ​​of venetoclax, azacitidine, and flumatinib in Ph+ ALL cell lines. 50 value.

[0047] SUPB15 cells were added to 96-well plates and then treated with different concentrations of venetoc, azacitidine, and flumatinib for 48 h. Complete culture medium without cells was used as a blank control. At the test sites, 10 μl of CCK-8 was added to each well, and the cells were incubated at 37°C and 5% CO2 for approximately 5 h. The OD (optical density) was then measured at 450 nm using an ELx800 automated microplate reader (BioTek Instrument, USA).

[0048] 4. CompuSyn drug combination therapy

[0049] Through the CompuSyn platform, based on IC 50 SUPB15 cells were treated with different concentrations of venetoc, azacitidine, and flumatinib, either alone or in combination. After 48 hours of treatment, the effects of each drug combination on SUPB15 cells were examined using a CCK-8 cytotoxicity assay. The drug synergy index (CI) for different combinations was calculated using this platform.

[0050] Experimental results

[0051] 1. The IC50 values ​​of venetoclax, azacitidine, and flumatinib on SUPB15 cells 50 Condition

[0052] like Figure 1 As shown, venetoclax, azacitidine, and flumatinib have IC50 effects on SUPB15 cells. 50 The values ​​were: 8.306±1.06 nM, 2.178±0.1786 μM, and 668.1±132 nM (three independent replicate experiments, IC50). 50 (represented by Mean±SD).

[0053] 2. CompuSyn's drug combination studies have demonstrated that the combination of venetoclax, azacitidine, and flumatinib has a significant synergistic effect.

[0054] Using the CompuSyn platform, the synergistic effects of venetoclax, azacitidine, and flumatinib in SUPB15 cells were investigated, and a significant synergistic effect among the three drugs was found.

[0055] like Figure 2 As shown, the inhibition rate of the three drugs on SUPB15 cells was less than 50% when used alone, while the inhibition rate of the three drugs combined could reach more than 80%.

[0056] like Figure 3 As shown, the CI-Fa curves demonstrate that the CI values ​​among the three drugs are all less than 1, indicating a significant synergistic effect.

[0057] Example 2:

[0058] Eleven newly diagnosed adult patients with Ph+ ALL were treated with the three-drug combination regimen of this invention. The median age of the patients was 41 (range 19-59) years, and all patients had an ECOG score ≤3. Among these patients, 6 patients had the p190 BCR-ABL fusion gene transcript type, and 5 patients had the p210 type. None of the patients had ABL1 kinase domain mutations at initial diagnosis. Among the 4 patients who could be fully evaluated, 2 patients had an additional abnormality that led to poor prognosis: IKZF1 deletion.

[0059] A three-drug combination chemotherapy-free regimen (VAF)

[0060] Venetoclax 100-400 mg, once daily, orally, for days 1-21;

[0061] Azacitidine 50-75 mg / m 2 Days 1-7; once daily, subcutaneous injection;

[0062] Flumatinib 400-600mg, once daily orally, starting from day 4;

[0063] Following one cycle of induction therapy, all 11 patients achieved complete remission. Ten patients achieved molecular responses (complete molecular response (CMR) and major molecular response (MMR)), with 8 achieving CMR, 2 achieving MMR, and 1 achieving only a complete genetic response (CCyR) due to an IKZF1 deletion abnormality. Following a second cycle of triple therapy, the two patients who achieved only MMR in the first cycle further achieved CMR. The fusion gene quantification in the CCyR patient with additional IKZF1 abnormalities also showed a further decrease. One patient dropped out during the second cycle due to severe COVID-19 infection. The median time from induction to achieving CMR was 21 days (range 14–70 days). The novel regimen was generally well-tolerated, with no treatment-related deaths during the induction phase. The most common reported hematological adverse event was febrile neutropenia (grade 1–2, 3 out of 11 [27.3%]). The median recovery time for neutropenia and thrombocytopenia was 11 days and 5 days, respectively. The median transfusion amounts for erythrocytes and platelets were 5 U and 15 U, respectively. Non-hematologic toxicities were mild and manageable, mainly including gastrointestinal reactions, abnormal liver enzymes, and fatigue. The median hospital stay for the first and second cycles was 22 days and 7 days, respectively. All patients received their second cycle of treatment in a day ward. The median follow-up was 6 months, and the median overall survival (OS) had not yet been reached. The 6-month OS was 100%, and no relapses occurred in any of the 11 patients, with a projected 6-month relapse-free survival of 100%. In this study, compared with other historical treatment regimens, the three-drug combination regimen of this invention had a higher CR rate (11 / 11, 100%) and an early CMR rate (9 / 10, 90%; median CMR time, 21 days), while no unacceptable treatment-related toxicities were observed.

[0064] Example 3:

[0065] Brief introduction of typical clinical cases:

[0066] A 55-year-old male patient denied any history of chronic diseases, family history of genetic disorders, or history of infectious diseases. He experienced rib pain without any obvious cause in the early morning of November 26, 2022, and presented to our outpatient clinic. A blood test showed a white blood cell count of 2.36 × 10⁻⁶. 9 / L, hemoglobin 80g / L, platelets 17×10 9 / L. Bone marrow morphology: ALL possible; Leukemia: 72.9% immature cells, B lymphocytes with myeloid expression, targeted transcriptome sequencing: BCR-ABL(ela2) P190+; BCR-ABL FISH 87%+; normal karyotype; NGS: Class I negative, germline possible (ARID1A\ARID5B\TYK2), BCR-ABL(ela2) quantification: 97.2%; Gene chip: IKZF1 deletion, chromosome 3 fragmentation. A definitive diagnosis of Ph+ ALL with a poor prognosis is made.

[0067] Physical examination: The patient is alert and in good spirits, appears anemic, and there is no enlargement of superficial lymph nodes. Two mucosal ulcers are observed in the oral cavity. There is tenderness upon sternal palpation. Lung auscultation reveals clear breath sounds without rales. The heart rhythm is regular and no pathological murmurs are heard. The abdomen is soft, without tenderness or rebound tenderness. The liver and spleen are not palpable below the costal margin. There is no edema in the lower extremities.

[0068] Treatment: VAF regimen started on December 2, 2022.

[0069] Efficacy assessment: Bone marrow aspiration on day 14 of induction and days 12-15: Bone marrow morphology in remission, MRD: 1.3*10-3, BCR-ABL FISH negative, BCR-ABL fusion gene quantification negative. Figure 4 This indicates that the patient has achieved complete molecular remission (CMR).

[0070] Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A combination composition for Ph+ ALL, comprising, It comprises a BCL-2 inhibitor drug, a demethylation drug and a TKI drug; the BCL-2 inhibitor drug is venetoclax; the demethylation drug is azacitidine; the TKI drug is a second-generation TKI, and the second-generation TKI is forasatinib.

2. The combination composition according to claim 1, wherein The combination drug composition is a combination of three separate preparations.

3. Use of the combination drug composition of claim 1 in the preparation of a medicament for treating Ph+ ALL.

4. Use of the combination drug composition of claim 1 in the preparation of a Ph+ ALL cell proliferation inhibitor.

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