A combined pharmaceutical composition and its application

Abstract

This application relates to the field of pharmaceutical technology, and more particularly to a combination pharmaceutical composition and its use. The pharmaceutical composition comprises a CCR2 / 5 dual receptor antagonist and a Bruton's tyrosine kinase inhibitor. The combination of LF0376 and zanubrutinib, or LF0376 and ibrutinib, demonstrates a significant tumor-suppressing effect on B-cell lymphoma compared to either agent alone.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and in particular to a combination pharmaceutical composition and its application. Background Art

[0002] Lymphoma is a common malignant tumor in my country, with an annual incidence of approximately 75,400 cases, an incidence rate of 4.75 per 100,000 people, and 40,500 deaths, with a mortality rate of 2.64 per 100,000 people. There are significant disparities between regions and between urban and rural areas. Lymphoma can be divided into Hodgkin's lymphoma and non-Hodgkin's lymphoma, each with complex pathological types, diverse treatments, and distinct prognoses. Therefore, diagnosis and treatment generally require the participation of a multidisciplinary team. Among non-Hodgkin's lymphomas, diffuse large B-cell lymphoma (DLBCL) accounts for about 30% to 40%. Its standard treatment is immunotherapy combined with chemotherapy (R-CHOP). About 50% to 60% of patients can be cured by current standard treatment, but 40% to 50% of patients still become refractory or relapse after treatment (about 15% to 25% of patients are primary refractory (progression during or after treatment), about 20% to 30% of patients relapse after complete remission (CR), and 5% of patients are in partial remission (PR). For the above-mentioned relapsed / refractory patients, Blood stem cell transplantation and CART are the preferred treatments, but due to the high cost and conditions of transplantation, their target population is limited. Other treatments, primarily chemotherapy, have limited efficacy and significant toxic side effects. Furthermore, young high-risk or moderate-high-risk patients, "double hit" with simultaneous MYC and BCL2 rearrangements, "triple hit" with BCL6 rearrangements, "double expression" of both MYC and BCL2, and non-germinal center B-cell-like lymphomas all have poor prognoses. Currently, there are no effective treatments, creating unmet clinical needs.

[0003] CCR2 and CCR5 are both G protein-coupled receptors that recognize chemokines and share 73% sequence homology. The primary ligand for CCR2 is the chemokine CCL2, but it also recognizes CCL7, CCL8, CCL12, and CCL13. CCR5 has high affinity for several chemokines, including CCL3, CCL4, CCL5, CCL3L1, CCL8, and CCL11. CCR2 is primarily expressed on monocytes, NK cells, and T cells, recruiting these cells to sites of inflammation under inflammatory conditions. In most cases, the CCR2-CCL2 axis primarily plays a proinflammatory role, but CCR2 expressed on Treg cells plays an anti-inflammatory role. CCR5 is more widely expressed, including on T cells, macrophages, granulocytes, dendritic cells, microglia, and even epidermal cells.

[0004] In various diseases involving inflammatory responses, such as viral infections and liver fibrosis, both CCR2 and CCR5 mediate the migration and infiltration of lymphocytes, monocytes, and macrophages, contributing to the development and progression of the disease. Studies have shown that CCR2 is more relevant to the migration and infiltration of monocytes and macrophages, while CCR5 has a greater effect on lymphocytes. Therefore, while CCR2 and CCR5 share functional similarities, their emphasis is different.

[0005] There are currently no studies demonstrating a specific link between CCR2 / 5 dual receptor antagonists and B-cell lymphoma.

[0006] Bruton tyrosine kinase (BTK) is a key kinase in the BCR signaling pathway. BTK is expressed in myeloid cells such as B lymphocytes, basophils, and monocytes. Based on its outstanding efficacy in multiple clinical trials, the first-generation BTK inhibitor ibrutinib has been approved by the FDA for seven indications, including small lymphocytic lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, Waldenstrom's macroglobulinemia, graft-versus-host disease, and marginal zone lymphoma. The first three of these indications have already been approved in China. Zanubrutinib, a second-generation, highly selective BTK inhibitor independently developed in China, has also been approved for adult patients with mantle cell lymphoma, Waldenstrom's macroglobulinemia, relapsed / refractory marginal zone lymphoma, chronic lymphocytic leukemia (CLL), or small lymphocytic lymphoma (SLL). Although BTK inhibitors are not approved for DLBCL, they are recommended for this indication by the CSCO guidelines based on clinical practice. Summary of the Invention

[0007] In view of the shortcomings of the prior art described above, the present application aims to provide a combination pharmaceutical composition and its application to address the problems of the prior art. This study explores the potential of combining a CCR2 / 5 dual receptor antagonist with a BTK inhibitor in the treatment of B-cell lymphoma, particularly diffuse large B-cell lymphoma, to explore new avenues for the treatment of B-cell lymphoma.

[0008] To achieve the above-mentioned and other related objectives, the present application provides, in a first aspect, the use of a CCR2 / 5 dual receptor antagonist in the preparation of a drug for treating or preventing B-cell lymphoma.

[0009] In any embodiment of the present application, the CCR2 / 5 dual receptor antagonist is selected from a combination of one or more of LF0376, BMS-813160, and Cenicriviroc; preferably, the CCR2 / 5 dual receptor antagonist is selected from LF0376.

[0010] The second aspect of the present application provides a pharmaceutical composition comprising a CCR2 / 5 dual receptor antagonist and a Bruton's tyrosine kinase inhibitor.

[0011] In any embodiment of the present application, the Bruton's tyrosine kinase inhibitor is selected from zanubrutinib, ibrutinib, spebrutinib, acalabrutinib, olmutinib, poseltinib, tirabrutinib, evobrutinib, fenebrutinib, vecabrutinib, ARQ-531, BMS-986195, BMS-986142, CGI-1746, GDC-0834, RN-486, JNJ-642646 81. a combination of one or more of DTRMWXHS-12, CT-1530, AC0058TA, ICP-022, WXFL10230486, SHR1459, PRN-1008, PRN-473, PRN-2246, LOU-064, LOXO-305, ABBV-105, PCI-45292, TAK-020, M-7583, BIIB-068, BMS-935177, CNX-774, TAS-5315, TGH-663, and LFM-A13.

[0012] In any embodiment of the present application, the Bruton's tyrosine kinase inhibitor is selected from zanubrutinib or ibrutinib.

[0013] In any embodiment of the present application, the pharmaceutical composition comprises LF0376 and ibrutinib; preferably, the weight ratio of LF0376 to ibrutinib is 0.5 to 10:1.

[0014] In any embodiment of the present application, the pharmaceutical composition comprises LF0376 and zanubrutinib; preferably, the weight ratio of LF0376 to zanubrutinib is 0.8 to 40:1.

[0015] The third aspect of the present application provides the use of the pharmaceutical composition in the preparation of drugs for treating or preventing cancer.

[0016] In any embodiment of the present application, the cancer is selected from one or more combinations of B-cell lymphoma, bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, central nervous system cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastrointestinal cancer, external genital cancer, urogenital tract cancer, head cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, muscle tissue cancer, cervical cancer, oral or nasal mucosal cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, spleen cancer, small intestine cancer, large intestine cancer, stomach cancer, testicular cancer and thyroid cancer.

[0017] In any embodiment of the present application, the B-cell lymphoma is diffuse large B-cell lymphoma.

[0018] The fourth aspect of the present application provides a drug for treating cancer, comprising the CCR2 / 5 dual receptor antagonist in the aforementioned use, or the aforementioned pharmaceutical composition, and pharmaceutically acceptable excipients and / or carriers.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] 1. This application discloses for the first time that LF0376 has a significant tumor-suppressing effect on B-cell lymphoma.

[0021] 2. The combination of LF0376 and Zebutinib has a significant tumor inhibitory effect on B-cell lymphoma compared with single drug use.

[0022] 3. The combination of LF0376 and ibrutinib has a significant tumor inhibitory effect on B-cell lymphoma compared with single drug use. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Shown is the experimental design diagram for the combination of LF0376 and zanubrutinib.

[0024] Figure 2 The figure shows the tumor inhibition curve of the experiment of LF0376 (200 mg) combined with Zanubrutinib in Example 1. Each data point is represented by mean ± sem.

[0025] Figure 3 The tumor weight after 27 days of treatment in the experiment of LF0376 (200 mg) combined with Zanubrutinib in Example 1 is shown. Each data point is represented by mean ± sem.

[0026] Figure 4 Shown are the percentage (%) changes in body weight compared to the baseline of mice during the treatment period of Example 1. Each data point is expressed as mean ± sem.

[0027] Figure 5 Shown is the experimental design diagram for the combination of LF0376 and ibrutinib.

[0028] Figure 6 The tumor inhibition curve of the LF0376 (100 mg) combined with Ibrutinib experiment in Example 1 is shown. Each data point is represented by mean ± sem.

[0029] Figure 7The tumor weight after 27 days of treatment in the LF0376 (100 mg) combined with Ibrutinib experiment in Example 1 is shown. Each data point is represented by mean ± sem.

[0030] Figure 8 Shown are the percentage (%) changes in body weight compared to the baseline of mice during the treatment period of Example 1. Each data point is expressed as mean ± sem.

[0031] Figure 9 Shown are the tumor volume change curves of all experimental groups in Example 2.

[0032] Figure 10 Shown are the tumor volume change curves of the Vehicle group, LF0376 monotherapy group, Ibrutinib monotherapy group, and LF0376+Ibrutinib group in Example 2.

[0033] Figure 11 Shown are the tumor volume change curves of the Vehicle group, LF0376 monotherapy group, Zanubrutinib monotherapy group, and LF0376+Zanubrutinib group in Example 2.

[0034] Figure 12 Shown are the tumor weights of each experimental group at the end of the experiment in Example 2 (day 28).

[0035] Figure 13 Shown are the tumor weights of the Vehicle group, LF0376 monotherapy group, Ibrutinib monotherapy group, and LF0376+Ibrutinib group at the end of the experiment in Example 2 (day 28).

[0036] Figure 14 Shown are the tumor weights of the Vehicle group, LF0376 single-drug group, Zanubrutinib single-drug group, and LF0376+Zanubrutinib group at the end of the experiment in Example 2 (day 28).

[0037] Figure 15 Shown is the average RCBW (%) curve of tumor-bearing mice during the treatment with the test drug in Example 2.

[0038] Figure 16 Shown is the average body weight change curve of tumor-bearing mice during the test drug treatment in Example 2.

[0039] Figure 17 The graph shows the changes in tumor volume during treatment in each group of mice. Note: Data points represent the mean tumor volume of each group, and error bars represent SEM.

[0040] Figure 18 Shown are graphs showing changes in individual tumor volume during treatment in each group of mice.

[0041] Figure 19 The data are shown as graphs of tumor weights of mice in each PG-D13 group. Note: Data points represent mouse tumor weights, the midline represents the mean tumor weight of each group, and the error bars represent the SEM.

[0042] Figure 20 Shown are the mean body weights of mice in each group during treatment. Note: Data points represent the mean body weight of each group. Error bars represent SEM.

[0043] Figure 21 Body weight changes of mice in each group compared with baseline during treatment. Note: Data points represent the mean of body weight change rate of each group, and error bars represent SEM. DETAILED DESCRIPTION

[0044] In order to make the invention objectives, technical solutions and beneficial effects of this application clearer, the present application is further described below with reference to the following examples. It should be understood that the following examples are only used to illustrate this application and are not intended to limit the scope of this application. Unless otherwise specified, the test methods used in the following examples are all conventional methods. People familiar with this technology can easily understand other advantages and effects of this application from the content disclosed in this description.

[0045] After extensive exploration and research, the inventors of this application discovered a combination pharmaceutical composition and its application, and completed this application on this basis.

[0046] In one aspect, the present application provides the use of a CCR2 / 5 dual receptor antagonist in the preparation of a drug for treating or preventing B-cell lymphoma.

[0047] In the pharmaceutical composition provided in the present application, the CCR2 / 5 dual receptor antagonist is selected from a combination of one or more of LF0376, BMS-813160, and Cenicriviroc. CCR2 / 5 is a cell surface chemokine receptor 2 and 5, a member of the G protein-coupled receptor superfamily, expressed on the surface of a variety of cells, and exerts biological effects by binding to its specific ligand. Preferably, the CCR2 / 5 dual receptor antagonist is selected from LF0376, also known as an azabenzo eight-membered ring compound, and other code names are WXFL40050414 or WXSH0376. In a preferred embodiment of the present application, LF0376 is produced by Shanghai WuXi AppTec New Drug Development Co., Ltd. on behalf of the applicant, with a batch number of ES16578-17-P1, a molecular weight of 739.98, a purity of 97.28%, and is stored at room temperature. This application discloses for the first time the specific connection between the CCR2 / 5 dual receptor antagonist (LF0376) and B-cell lymphoma, and the use of LF0376 has a significant tumor-suppressing effect on B-cell lymphoma.

[0048] On the other hand, the present application provides a pharmaceutical composition comprising a CCR2 / 5 dual receptor antagonist and a Bruton's tyrosine kinase inhibitor.

[0049] In some embodiments, the composition of the pharmaceutical composition is formulated in a clinically acceptable dosage, which depends on the species, weight, age, and type of tumor being treated, the condition of the individual case, or its severity. A physician, clinician, or veterinarian skilled in the art can readily determine the effective dosage of the pharmaceutical composition required to prevent, treat, or inhibit the progression of a disorder or disease.

[0050] In the pharmaceutical composition provided herein, the Bruton's tyrosine kinase inhibitor is selected from zanubrutinib, ibrutinib, spebrutinib, acalabrutinib, olmutinib, poseltinib, tirabrutinib, evobrutinib, fenebrutinib, vecabrutinib, ARQ-531, BMS-986195, BMS-986142, CGI-1746, GDC-0834, RN-486, JNJ-6426468 1. A combination of one or more of DTRMWXHS-12, CT-1530, AC0058TA, ICP-022, WXFL10230486, SHR1459, PRN-1008, PRN-473, PRN-2246, LOU-064, LOXO-305, ABBV-105, PCI-45292, TAK-020, M-7583, BIIB-068, BMS-935177, CNX-774, TAS-5315, TGH-663, and LFM-A13. Bruton's tyrosine kinase inhibitors (BTK) are important signaling molecules in the B cell receptor pathway. They are expressed at various developmental stages of B lymphocytes and participate in regulating B cell proliferation, differentiation, and apoptosis, playing a crucial role in the survival and spread of malignant B cells.

[0051] In the pharmaceutical composition provided herein, the Bruton's tyrosine kinase inhibitor is selected from Zanubrutinib or Ibrutinib. Zanubrutinib is a multi-target kinase inhibitor and the first approved BTK small molecule inhibitor. It inhibits the activity of the BTK enzyme by forming a covalent bond with the cysteine ​​in the BTK active site. Ibrutinib belongs to the second generation of BTK inhibitors, which irreversibly inactivates the enzyme by covalently binding to tyrosine kinase. In a preferred embodiment of the present application, Zanubrutinib is produced by MCE and the article number is Lot#79640. Ibrutinib is produced by MCE and the article number is Lot#114225.

[0052] The pharmaceutical composition provided herein comprises LF0376 and ibrutinib, and the weight ratio of the two is 0.5 to 10:1. In a specific embodiment of the present application, the weight ratio of LF0376 to ibrutinib can be, for example, 0.5 to 1:1, 1 to 2.5:1, 2.5 to 5:1, 5 to 8:1, or 8 to 10:1. More specifically, the weight ratio of LF0376 to ibrutinib can be 5:1. In some embodiments, the pharmacological effects of LF0376 and ibrutinib have a synergistic effect in the treatment of B-cell lymphoma.

[0053] The pharmaceutical composition provided herein comprises LF0376 and zanubrutinib, with the weight ratio of the two being 0.8 to 40:1. In a specific embodiment of the present application, the weight ratio of LF0376 to zanubrutinib can be, for example, 0.8 to 1:1, 1 to 5:1, 5 to 10:1, 10 to 20:1, 20 to 30:1, 30 to 40:1, or 40 to 50:1. More specifically, the weight ratio of LF0376 to zanubrutinib can be 40:1. In some embodiments, the pharmacological effects of LF0376 and zanubrutinib are synergistic in the treatment of B-cell lymphoma.

[0054] In the pharmaceutical compositions provided herein, the administration is simultaneous or sequential. The therapeutically effective dose of the pharmaceutical composition depends on the species, weight, age and type of tumor being treated, the condition of the individual case or its severity. Doctors, clinicians or veterinarians who are proficient in the treatment techniques of the relevant fields can easily determine the use of the aforementioned pharmaceutical compositions in the preparation of drugs for treating or preventing cancer by the active ingredients required to prevent, treat or inhibit the development of disorders or diseases. The term "treatment" as used herein includes treatment that slows down, alleviates or relieves at least one symptom in the subject or achieves delayed disease development. For example, treatment can be to reduce one or more symptoms of a disease or completely eliminate a disease, such as cancer. Within the meaning of the present disclosure, the term "treatment" also refers to blocking, delaying the onset of the disease (i.e., the stage before the clinical manifestation of the disease) and / or reducing the risk of disease development or worsening. The term "prevention" as used herein includes preventing at least one symptom that is related to or caused by the state, disease or disorder being prevented.

[0055] In the application provided in the present application, the cancer is selected from one or more combinations of B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, slow cell lymphoma, bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, central nervous system cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastrointestinal cancer, external genital cancer, urogenital tract cancer, head cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, muscle tissue cancer, cervical cancer, oral or nasal mucosal cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, spleen cancer, small intestine cancer, colorectal cancer, stomach cancer, testicular cancer and thyroid cancer.

[0056] In the application provided in this application, the B cell lymphoma is diffuse large B cell lymphoma.

[0057] On the other hand, the present application provides a drug for treating cancer, comprising the CCR2 / 5 dual receptor antagonist in the aforementioned use, or the aforementioned pharmaceutical composition, and a pharmaceutically acceptable excipient and / or carrier. The term "pharmaceutically acceptable" as used herein refers to compounds, therapeutic agents (such as antibodies), materials, compositions and / or dosage forms that are suitable for contact with warm-blooded animals such as mammals or human tissues within the scope of reasonable judgment, without excessive toxicity, irritation, allergic reactions and other problems or complications, and with a reasonable benefit / benefit ratio. The terms "carrier" or "excipient" as used herein include any and all solvents, dispersion media, coating agents, surfactants, antioxidants, preservatives (such as antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, etc., and combinations thereof, and must have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to the patient to be treated. The carrier can be inert or it can itself have pharmaceutical benefits. Some carriers may be listed in more than one category, e.g., vegetable oils may be used as lubricants in some formulations and as diluents in others. Exemplary pharmaceutical carriers include sugars, starches, cellulose, malt, gelatin, talc, and vegetable oils. Optional active agents may be included in the pharmaceutical compositions that do not substantially affect the activity of the compounds of the invention.

[0058] The present application is further described below by way of examples, but the scope of the present application is not limited thereby.

[0059] The main reagents used in the following examples are shown in Table 1.

[0060] Table 1 List of main reagents

[0061]

[0062] The main instrument information used in the following examples is shown in Table 2.

[0063] Table 2 Instrument information

[0064]

[0065]

[0066] The information of the test drugs used in the following examples is as follows:

[0067] 1. LF0376

[0068] Provider: Wuxi Lingfang Biopharmaceutical Technology Co., Ltd. Lot: CR-C-210219013-FP22001

[0069] Purity: 98.6%

[0070] Description of properties: Powder Molecular weight: Salt: 835.09; Free base: 738.38 Correction factor: 1.15

[0071] Saved in: RT

[0072] The drug solution was stored at 4°C during administration.

[0073] 2. Ibrutinib

[0074] Provider: MCELot:136640, HY-10997

[0075] Purity: 99.46%

[0076] Correction factor: NA

[0077] Description of properties: Powder

[0078] Store at: 4°C

[0079] The drug solution was stored at 4°C during administration.

[0080] 3. Zanubrutinib

[0081] Provided by: MCE

[0082] Lot:79640,HY-101474A

[0083] Purity: 99.08%

[0084] Correction factor: NA

[0085] Description of properties: Powder

[0086] Store at: 4°C

[0087] The drug solution was stored at 4°C during administration.

[0088] Example 1 In vivo efficacy evaluation of test drugs on PDX tumor transplant models

[0089] 1. LF0376 combined with Zanubrutinib (ZAN)

[0090] 1.1 Compound preparation

[0091] The 200mg / kg LF0376 group was prepared using 5% DMSO + 95% (10% HP-β-CD). The specific preparation method was as follows: Weigh 210mg of LF0376 and add 0.53ml of DMSO. Vortex and shake to obtain a clear yellow solution. Then, add 9.975ml (10% HP-β-CD) and vortex to obtain a homogenous suspension. Adjust the suspension to pH 3 to obtain a clear solution. After preparation, aliquot 3.3ml into 5ml EP tubes and store at 4°C. Prepare once every three days.

[0092] Zanubrutinib is prepared using 10% DMSO + 40% PEG300 + 5% Tween 80 + 45% saline. The stock solution is prepared by adding 60mg of zanubrutinib to 6ml of DMSO. Aliquot 0.4ml into 5ml EP tubes. Upon use, add 1.6ml of PEG300, 0.2ml of Tween 80, and 1.8ml of saline in that order. Store at 4°C. Reconstitute every three days.

[0093] 1.2 Construction of PDX tumor transplantation model

[0094] Forty female NOG mice were subaxillary inoculated with patient-derived PDX. NOG mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Tumor inoculation occurred at 6-7 weeks of age. Animal husbandry and care conditions: Animals were housed under SPF animal husbandry conditions, with cages, bedding, feed, and drinking water sterilized by high temperature and high pressure. Cages were placed in the intravitreal cavity (IVC), with 3-5 animals per cage. The animal room temperature was maintained at 21-25°C, with a relative humidity of 40%-70%. PDX were derived from patients with diffuse large B-cell lymphoma of the non-germinal center (non-GCB) subtype. Immunohistochemical results of the PDX model showed: CD10-, MUM-1 (100%+), Bcl6 (80%+), CD20+, Bcl2+, CD3-, and Ki67 (90%+).

[0095] On the 30th day after inoculation, the rats were regrouped according to the tumor volume, with 8 rats in each group, and a total of 32 rats in 4 groups. The mean tumor volume of each group reached 133.47±1.75mm 3 , which is Day 0. The remaining mice were treated at the end of the experiment or when the tumor burden reached 1500 mm 3 Each group of mice was given the drug according to the set dose. The tumor volume was measured every 3 days. Each group was given different doses and drugs, see Table 3 and Figure 1 .

[0096] Table 3 Experimental dosage and grouping

[0097]

[0098] Note: The vehicle control group received 5% DMSO + 95% (10% HP-β-CD). The vehicle and LF0376 were administered starting on Day 0 (Days of injection) twice daily with an 8-hour interval. Zanubrutinib was administered orally once daily.

[0099] 1.3 Indicator Observation

[0100] 1.3.1 Tumor volume

[0101] Tumor volume was measured every 3 days. The formula for calculating tumor volume (TV) is: TV = 1 / 2 × a × b 2 Where a and b represent the long and short diameters of the tumor mass, respectively.

[0102] 1.3.2 Weight

[0103] The body weight of the animals was measured every 3 days.

[0104] 1.4 Data Analysis

[0105] All data are expressed as mean ± sem.

[0106] Statistical analysis was performed using PRISM software, one-way ANOVA was used for data analysis, and Tukey's method was used for comparison between groups.

[0107] 1.5 Experimental Results

[0108] This phase is a study on the combination of LF0376 and zanubrutinib, with a total of 27 days of administration.

[0109] like Figure 2 Observation of the overall tumor inhibition curve showed that the LF0376 monotherapy group, the zanubrutinib group, and the LF0376 combined with zanubrutinib group could inhibit tumor growth compared with the control group, among which the tumor inhibition effect of the combination group was the most significant.

[0110] like Figure 3 The tumors were weighed 27 days after administration, and it was found that the tumor weight of the LF0376 combined with Zanubrutinib group was significantly smaller than that of the control group and the two single-drug groups.

[0111] like Figure 4 During treatment, mice in the control group experienced a significant increase in weight, consistent with the natural process of tumor growth and weight gain. Following treatment, the LF0376 monotherapy group, the zanubrutinib group, and the LF0376 combined with zanubrutinib group significantly reduced tumor volume and weight compared to the control group, with none exceeding the weight loss range. This suggests that both the combination therapy and monotherapy were well tolerated and safe in mice.

[0112] 2. LF0376 combined with ibrutinib (IBR)

[0113] 2.1 Compound preparation

[0114] The 100mg / kg LF0376 group was prepared using 5% DMSO + 95% (10% HP-β-CD). The specific preparation method was as follows: Weigh 210mg of LF0376 and add 1.05ml of DMSO. Vortex and shake to obtain a clear yellow solution. Then, add 19.95ml (10% HP-β-CD) and vortex to obtain a homogenous suspension. Adjust the suspension to pH 3 to obtain a clear solution. After preparation, aliquot 3.3ml into 5ml EP tubes and store at 4°C. Prepare once every three days.

[0115] Ibrutinib is prepared using 10% DMSO + 40% PEG-300 + 5% Tween-80 + 45% saline. The preparation method is as follows: 240 mg of ibrutinib is added to 6 ml of DMSO to prepare the stock solution. Then, aliquot 0.4 ml into 5 ml EP tubes. Upon use, add 1.6 ml of PEG-300, 0.2 ml of Tween-80, and 1.8 ml of saline in that order. Store at 4°C. Reconstitute every three days.

[0116] 2.2 Construction of PDX tumor transplantation model

[0117] Forty female NOG mice were subaxillary inoculated with patient-derived PDX. NOG mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Tumor inoculation occurred at 6-7 weeks of age. Animal husbandry and care conditions: Animals were housed under SPF animal husbandry conditions, with cages, bedding, feed, and drinking water sterilized by high temperature and high pressure. Cages were placed in the intravitreal cavity (IVC), with 3-5 animals per cage. The animal room temperature was maintained at 21-25°C, with a relative humidity of 40%-70%. PDX were derived from patients with diffuse large B-cell lymphoma of the non-germinal center (non-GCB) subtype. Immunohistochemical results of the PDX model showed: CD10-, MUM-1 (100%+), Bcl6 (80%+), CD20+, Bcl2+, CD3-, and Ki67 (90%+).

[0118] On the 24th day after inoculation, the mice were regrouped according to tumor volume, with 8 mice in each group, for a total of 4 groups. The mean tumor volume of each group reached 108.72±0.33mm 3 , which is day 0. The remaining mice were treated at the end of the experiment or when the tumor burden reached 1500 mm 3 The mice were then killed. The mice were given the prescribed doses for each group. The tumor volume and body weight were measured every 3 days. Each group was given different doses and drugs, as shown in Table 4 and Figure 5 .

[0119] Table 4 Experimental dosage and grouping

[0120]

[0121] Note: The vehicle control group received 5% DMSO + 95% (10% HP-β-CD). The vehicle and LF0376 were administered starting on Day 0 (Days of injection) twice daily, with an 8-hour interval between doses. Ibrutinib was administered orally once daily.

[0122] 2.3 Indicator Observation

[0123] Same as 1.3.

[0124] 2.4 Data Analysis

[0125] Same as 1.4.

[0126] 2.5 Experimental Results

[0127] This phase is a study of the combination of LF0376 and ibrutinib, with a total of 27 days of administration.

[0128] like Figure 6 Observation of the overall tumor inhibition curve showed that the LF0376 monotherapy group, ibrutinib group, and LF0376 combined with ibrutinib group could inhibit tumor growth compared with the control group, among which the combined group had the most significant tumor inhibition effect.

[0129] like Figure 7 The tumors were weighed 27 days after administration, and it was found that the tumor weight in the LF0376 combined with ibrutinib group was significantly smaller than that in the control group and the two single-drug groups.

[0130] like Figure 8 During treatment, mice in the control group experienced a significant increase in weight, consistent with the natural process of tumor growth and weight gain. Following treatment, the LF0376 monotherapy group, the ibrutinib group, and the LF0376 combined with ibrutinib group significantly reduced tumor volume and weight compared to the control group, with none exceeding the recommended weight loss range. This suggests that both the combination therapy and monotherapy were well tolerated and safe in mice.

[0131] Example 2 In vivo efficacy evaluation of the test drug on the human DLBCL xenograft tumor model LD1-0026-361717

[0132] This example mainly tests the growth inhibitory effect or complete cure ability of the test drugs LF0376 alone, Ibrutinib alone, Zanubrutinib alone, and LF0376 combined with Ibrutinib or Zanubrutinib on the human DLBCL in vivo transplanted tumor model LD1-0026-361717.

[0133] 1. Compound preparation

[0134] See Table 5 for details:

[0135] Table 5 Preparation of the test drugs

[0136]

[0137] 2. In vivo transplanted tumor model

[0138] The human-derived DLBCL tumor tissue of LD1-0026-361717 was passaged to FP3+2 generation for this efficacy experiment.

[0139] Basic information of the LD1-0026-361717 PDX model can be seen in Table 6.

[0140] Table 6 Basic information of the model

[0141]

[0142] 95 NU / NU mice, female, weighing 18 - 21 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (SCXK(Beijing)2021-0006), certificate number: 110011231108918138. Experimental animal use license number: SYXK(Shaanxi)2023-008. Feeding environment: SPF level. The animals were acclimated and observed for 3 days before the start of the experiment.

[0143] The tumor mass of the in vivo transplanted tumor of human-derived DLBCL of LD1-0026-361717 at FP3+1 generation was cut into tumor tissues with a size of approximately 3 mm × 3 mm × 3 mm (about 45 - 60 mg), and inoculated subcutaneously in NU / NU mice. The mice were observed after inoculation and the growth of the tumor was monitored, and the mice with tumors were grouped and given drugs on the 28th day after inoculation when the average tumor volume was 137.11 ± 6.36 mm 3 at this time, and the day of grouped drug administration was defined as day 0. Specific grouping information and drug administration plan can be seen in Table 7.

[0144] Table 7 Drug administration and treatment

[0145]

[0146] Note: Drug administration volume: Adjust the drug administration volume according to the body weight of the tumor-bearing mice (0.2 mL / ​​​​​​​​Tumor volume was measured twice a week using a vernier caliper and calculated using the formula V = 0.5a × b 2 , a and b represent the long diameter and wide diameter of the tumor, respectively.

[0150] 3.2 Relative tumor proliferation rate T / C (%)

[0151] The calculation formula is as follows: T / C% = T RTV / C RTV ×100%(T RTV :RTV in treatment group; C RTV : RTV of the Vehicle group). The relative tumor volume (RTV) was calculated based on the tumor measurement results. The calculation formula is RTV=V t / V0, where V0 is the average tumor volume measured at the time of group administration (i.e., d0), V t is the average tumor volume at a certain measurement, T RTV with C RTV Get data for the same day.

[0152] 3.3 Tumor growth inhibition rate TGI (%)

[0153] TGI (%) = [1-(T i -T0) / (V i -V0)]×100%, T i is the average tumor volume of the compound group after the start of administration, T0 is the average tumor volume of the compound group at the first administration, V0 is the average tumor volume of the vehicle group at the first administration, and V i The average tumor volume of the Vehicle group after the start of drug administration. If TGI (%)>50%, it is considered that the compound has a significant effect of inhibiting tumor growth.

[0154] 3.4 weight

[0155] The body weight of the mice was measured every day. The relative change ratio of the body weight after administration was calculated: RCBW (%) = (BW i –BW0) / BW0×100, BW i BW0 is the body weight after the start of drug administration, and BW1 is the body weight at the first drug administration.

[0156] 4. Data Analysis

[0157] All data are expressed as Mean ± SEM, SEM = SD / SQRT (n), n = the number of animals in the experimental group.

[0158] 5. Experimental results

[0159] like Figure 9 、 Figure 10 、 Figure 11 As shown in Table 8, on day 28, the average tumor volume in the Vehicle group was 1470.55±411.29 mm 3 The average tumor volumes of the LF0376 monotherapy group, Ibrutinib monotherapy group, Zanubrutinib monotherapy group, LF0376+Ibrutinib group, and LF0376+Zanubrutinib group were 1219.51±254.70mm, respectively. 3 、906.95±185.57mm 3 、972.72±342.44mm 3 、566.26±159.12mm 3 and 752.64±212.66mm 3 The TGI (%) was 18.60%, 42.24%, 37.33%, 67.86%, and 53.86%, respectively; the T / C (%) was 84.86%, 61.94%, 66.29%, 38.45%, and 51.21%, respectively. Both the LF0376+ibrutinib and LF0376+zanubrutinib groups showed significant tumor growth inhibition in the human DLBCL PDX model LD1-0026-361717 compared to the vehicle group. The LF0376+ibrutinib and LF0376+zanubrutinib groups exhibited stronger tumor growth inhibition than the LF0376 monotherapy group and the ibrutinib / zanubrutinib monotherapy group, respectively, indicating that LF0376 can enhance the anti-tumor effects of ibrutinib or zanubrutinib.

[0160] Table 8 Summary of the antitumor efficacy evaluation of the test drugs in the LD1-0026-361717 human DLBCL subcutaneous xenograft tumor model

[0161]

[0162] like Figure 12 、 Figure 13 、 Figure 14As shown in Table 8, on day 28, the average tumor weights of the Vehicle group, LF0376 monotherapy group, Ibrutinib monotherapy group, Zanubrutinib monotherapy group, LF0376+Ibrutinib group, and LF0376+Zanubrutinib group were 1.239±0.375 g, 1.024±0.218 g, 0.739±0.162 g, 0.785±0.297 g, 0.475±0.134 g, and 0.596±0.184 g, respectively. The tumor weight results were basically consistent with the tumor volume results.

[0163] like Figure 15 、 Figure 16 As shown in Table 9, on day 28, the RCBW (%) of the Vehicle group, LF0376 monotherapy group, Ibrutinib monotherapy group, Zanubrutinib monotherapy group, LF0376+Ibrutinib group, and LF0376+Zanubrutinib group were 11.91±2.20%, 12.00±1.28%, 5.48±1.16%, 9.32±1.80%, 4.82±1.64%, and 5.33±1.33%, respectively.

[0164] Table 9 RCBW (%) of animals treated with the test drugs in the LD1-0026-361717 human DLBCL subcutaneous xenograft tumor model

[0165]

[0166]

[0167] 6. Experimental Conclusion

[0168] LF0376 alone, ibrutinib alone, and zanubrutinib alone did not demonstrate significant antitumor activity in this model at current dose levels. However, LF0376 combined with ibrutinib and LF0376 combined with zanubrutinib demonstrated significant antitumor activity in this model, suggesting that LF0376 significantly enhances the antitumor effects of ibrutinib and zanubrutinib. During treatment with the test drug, no animals in any group experienced significant weight loss, indicating a favorable safety profile for the test drug.

[0169] Example 3 Evaluation of the efficacy of the test drug in the Balb / c mouse A20 tumor model

[0170] The purpose of this example is to evaluate the efficacy and mechanism of the test drug in the Balb / c mouse A20 tumor model (mouse B cell lymphoma).

[0171] 1. Compound preparation

[0172] See Table 10 for details

[0173] Table 10 Preparation of test drugs

[0174]

[0175] Note: *Dosage concentration is based on active ingredient.

[0176] 2. Animal Model

[0177] Animal information is shown in Table 11:

[0178] Table 11 Experimental Animal Information

[0179]

[0180] The animals were isolated and bred for adaptation before experimental treatment.

[0181] A20 cells were cultured and expanded in vitro, and cells in the logarithmic growth phase were collected and resuspended in PBS at a cell suspension concentration of 5×10 6 / mL.

[0182] The cell suspension was injected subcutaneously into the right side of Balb / c mice using a 1 mL syringe. Each animal was injected with 100 μL of the suspension. The number of cells to be inoculated was 5 × 10 5 / Only.

[0183] The average tumor volume was 60.75±0.48mm 3 When the tumors were collected, animals with large or small tumors or irregular tumor shapes were eliminated. The animals were randomly divided into 6 groups with 6 animals in each group. The day of grouping was defined as PG-D0.

[0184] Drug administration was initiated according to the grouping scheme. All groups were administered via oral gavage, with a dose volume of 5 mL / kg per group, and the frequency of administration was once or twice daily. During the experiment, the animals were weighed and the tumor volume was measured three times a week.

[0185] At the experimental endpoint of PG-D13, all mice were euthanized, and tumor tissues were removed, weighed, and photographed. Tumor volume inhibition (TGI) and animal body weight change (BWC%) were calculated based on tumor volume and body weight measured at PG-D12.

[0186] The dosing regimen for mice is shown in Table 12:

[0187] Table 12 Dosage regimen for each group of mice

[0188]

[0189] Note: G: Group; po: oral administration;

[0190] 1. The day of grouping was defined as PG-D0, and drug administration began on PG-D0;

[0191] 2. Dosage volume: adjusted according to the weight of the mouse;

[0192] 3. After the experiment, the mice will be processed according to customer needs.

[0193] Animal grouping and identification:

[0194] When the average tumor volume reached approximately 60.75 ± 0.48 mm 3 At 4 hr, mice were randomly divided into 6 groups of 6 mice each based on tumor volume. Detailed information is shown in Table 13 below: The cages were clearly labeled with cage cards, including animal number, sex, strain, date of receipt, treatment, study number, and group number.

[0195] Table 13 Experimental animal grouping information

[0196]

[0197] 3. Indicator observation

[0198] 3.1 Tumor volume (TV)

[0199] TV=1 / 2×a×b 2

[0200] Wherein: a represents the long diameter of the tumor; b represents the short diameter of the tumor.

[0201] 3.2 Tumor volume inhibition rate (TGI)

[0202] TGI=[1-(TV D t Treatment group-TV D 0 treatment group) / (TV D t Control group-TV D 0 control group)]×100%

[0203] TV D t Experimental group: tumor volume of the treatment group on day t after drug administration

[0204] TV D 0 Experimental group: tumor volume of the treatment group at the time of drug administration

[0205] TV D t Control group: tumor volume of the control group on day t after administration

[0206] TV D0 Control group: the tumor volume of the control group at the time of group drug administration.

[0207] 3.3 Animal body weight change rate (BWC%)

[0208] Weight change rate = (BW D t -BW D 0) / BW D 0×100%

[0209] BW D t : Animal body weight on day t after administration

[0210] BW D 0: Animal body weight at the time of grouping and drug administration.

[0211] 3.4 Tumor Weight Inhibition Rate (IR)

[0212] IR=(W C -W T ) / W C ×100%

[0213] Among them, W C W represents the tumor weight of the control group; T Indicates tumor weight in treatment group.

[0214] 4. Data Analysis

[0215] 5.1 Mouse tumor volume

[0216] like Figure 17 、 Figure 18 As shown in Tables 14 and 15, the average tumor volume of the PG-D12 control group was 687.68±46.59mm 3 The average tumor volumes of the LF0376 monotherapy group, Ibrutinib monotherapy group, Zanubrutinib monotherapy group, LF0376+Ibrutinib group, and LF0376+Zanubrutinib group were 551.12±40.66mm, respectively. 3 、640.57±48.47mm 3 、651.25±39.04mm 3 、497.63±57.96mm 3 and 454.45±46.55mm 3, TGI were 21.74%, 7.47%, 5.79%, 30.30%, and 37.12%, respectively. Both the LF0376+ibrutinib and LF0376+zanubrutinib groups showed significant tumor growth inhibition compared to the control group. The LF0376+ibrutinib and LF0376+zanubrutinib groups each demonstrated more significant tumor inhibition compared to the LF0376 monotherapy group and the ibrutinib / zanubrutinib monotherapy group, respectively.

[0217] Table 14 Average tumor volume (mm) of mice in each group during treatment 3 )

[0218]

[0219] Note: Data are expressed as Mean ± SEM.

[0220] Table 15 TGI (%) of mice in each experimental group of PG-D12

[0221]

[0222] 5.2 Tumor weight in mice

[0223] like Figure 19 As shown in Table 16, for PG-D13, the average tumor weight in the control group was 1.8387±0.2200 g, while the average tumor weights in the LF0376 monotherapy group, the ibrutinib monotherapy group, the zanubrutinib monotherapy group, the LF0376+ibrutinib group, and the LF0376+zanubrutinib group were 1.1853±0.1562 g, 1.5809±0.1216 g, 1.4855±0.1700 g, 1.3331±0.1810 g, and 1.0855±0.1081 g, respectively. The IRs were 35.54%, 14.02%, 19.21%, 27.50%, and 40.96%, respectively. The tumor weight results were generally consistent with the tumor volume results.

[0224] Table 16 Average tumor weight of mice in each group of PG-D13 (g)

[0225]

[0226] Note: n / n means: actual number of samples / original number of samples.

[0227] 5.3 Mouse body weight

[0228] like Figure 20As shown in Table 17, the average body weights of PG-D12, Group 1, Group 2, Group 3, Group 4, Group 5 and Group 6 mice were 25.97±1.13 g, 24.73±0.89 g, 26.33±1.20 g, 25.68±0.69 g, 24.52±1.06 g and 24.80±0.32 g, respectively.

[0229] like Figure 21 As shown in Table 18, PG-D12 treatment increased the average body weight of mice in Groups 1, 2, 3, 4, 5, and 6, with rates of change of 9.6±1.4%, 5.3±1.4%, 9.6±2.5%, 7.9±3.0%, 5.2±2.8%, and 5.8±1.8%, respectively. The body weight change rates of mice in the experimental groups during treatment ranged from -10.8% to 21.6%. No mice required discontinuation of treatment due to significant weight loss or poor condition.

[0230] Table 17 Average body weight of mice in each group during treatment (g)

[0231]

[0232]

[0233] Note: Data are expressed as Mean ± SEM.

[0234] Table 18 Body weight change rate of mice in each group during treatment compared with baseline (%)

[0235]

[0236] Note: The body weight change rate was calculated based on the body weight on PG-D0. Data are expressed as Mean ± SEM.

[0237] 6. Experimental Conclusion

[0238] The test drug showed good safety during treatment. LF0376 alone had a modest tumor-suppressing effect on A20, while the combined effects of LF0376 + ibrutinib and LF0376 + zanubrutinib were even more pronounced, suggesting that LF0376 can significantly enhance the anti-tumor effects of ibrutinib and zanubrutinib.

[0239] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit this application. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical concepts disclosed herein shall be covered by the claims of this application.

Claims

1. A pharmaceutical composition, characterized in that Includes LF0376 and ibrutinib, or LF0376 and zanubrutinib.

2. The pharmaceutical composition according to claim 1, wherein The weight ratio of LF0376 to ibrutinib is 0.5-10:

1.

3. The pharmaceutical composition according to claim 1, wherein The weight ratio of LF0376 to zanubrutinib is 0.8-40:

1.

4. Use of the pharmaceutical composition according to any one of claims 1 to 3 in the preparation of a drug for treating or preventing cancer, wherein the cancer is B-cell lymphoma.

5. The use according to claim 4, characterized in that The B-cell lymphoma is diffuse large B-cell lymphoma.

6. A drug for treating cancer, comprising the pharmaceutical composition according to any one of claims 1 to 3, and pharmaceutically acceptable excipients and / or carriers.

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