WO2024240025A1 - Combined pharmaceutical composition and use thereof - Google Patents

Abstract

A combined pharmaceutical composition and the use thereof. The pharmaceutical composition comprises a CCR2/5 dual-receptor antagonist and a Bruton's tyrosine kinase inhibitor. The combination of LF0376 and zebutinib and the combination of LF0376 and ibrutinib have a significant anti-tumor effect against B cell lymphoma than administration alone.

Description

A combined pharmaceutical composition and its application Technical Field

The present application relates to the field of medical technology, and in particular to a combined pharmaceutical composition and its application.

Background Art

Lymphoma is a common malignant tumor in my country, with an annual incidence of about 75,400 people, an incidence rate of 4.75/100,000, a death rate of 40,500, and a mortality rate of 2.64/100,000. There are also significant differences between regions and between urban and rural areas. Lymphoma can be divided into Hodgkin's lymphoma and non-Hodgkin's lymphoma, which have complex pathological types, diverse treatment methods, and different prognoses. Therefore, multidisciplinary teams are generally required in the diagnosis and treatment process. 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%-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 relapsed/refractory patients, Blood stem cell transplantation and CART are the first-choice treatment methods, but due to the high conditions and price of transplantation, the target population is limited, while the remaining treatment methods are mainly chemotherapy, which has limited efficacy and large toxic side effects. In addition, young high-risk or medium-high-risk patients, "double hit" with simultaneous rearrangement of MYC and BCL2, "triple hit" with rearrangement of BCL6, "double expression" with both MYC and BCL2, and non-germinal center B-cell-like subtype lymphoma all indicate a poor prognosis. There is currently no effective treatment measure, and there is an unmet clinical need.

Both CCR2 and CCR5 are G protein-coupled receptors that can recognize chemokines and have 73% sequence homology. The main ligand of CCR2 is the chemokine CCL2, and it can also recognize CCL7, CCL8, CCL12 and CCL13; CCR5 has a high affinity for multiple chemokines such as CCL3, CCL4, CCL5, CCL3L1, CCL8, CCL11, etc. CCR2 is mainly expressed on monocytes, NK cells and T cells, and these cells can be recruited to the inflammatory site under inflammatory conditions. In most cases, the CCR2-CCL2 axis mainly plays a pro-inflammatory role, but CCR2 expressed on Treg cells plays an anti-inflammatory role. CCR5 is more widely expressed, including T cells, macrophages, granulocytes, DCs, microglia and even epidermal cells.

In the course of many diseases, such as viral infection and liver fibrosis, which involve inflammatory responses, both CCR2 and CCR5 mediate the migration and infiltration of lymphocytes, monocytes, and macrophages, promoting the occurrence and development 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, although CCR2 and CCR5 have similarities in function, their emphasis is not the same.

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

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

Summary of the invention

In view of the shortcomings of the prior art described above, the purpose of this application is to provide a combined pharmaceutical composition and its application to solve the problems in the prior art. The combination of CCR2/5 dual receptor antagonist and BTK inhibitor is used to treat B cell lymphoma, especially diffuse large B cell lymphoma. This study provides a new direction for exploring the combination of the two in the treatment of B cell lymphoma.

To achieve the above-mentioned purpose and other related purposes, the first aspect of 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.

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.

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.

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.

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

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

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

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

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.

In any embodiment of the present application, the B cell lymphoma is diffuse large B cell lymphoma.

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.

Compared with the prior art, the beneficial effects of this application are:

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

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

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

FIG1 shows the experimental design diagram for the combination therapy of LF0376 and Zanubrutinib.

Figure 2 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.

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

Figure 4 shows the percentage (%) of body weight change compared to the baseline of mice during the treatment period of Example 1. Each data point is represented by mean±sem.

FIG5 shows the experimental design diagram of the combination therapy of LF0376 and ibrutinib.

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

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

Figure 8 shows the percentage (%) of body weight change compared to the baseline of mice during the treatment period of Example 1. Each data point is represented by mean±sem.

FIG. 9 shows the tumor volume change curves of all experimental groups in Example 2.

FIG. 10 shows the tumor volume change curves of the Vehicle group, LF0376 monotherapy group, Ibrutinib monotherapy group and LF0376+Ibrutinib group in Example 2.

FIG. 11 shows the tumor volume change curves of the Vehicle group, LF0376 monotherapy group, Zanubrutinib monotherapy group and LF0376+Zanubrutinib group in Example 2.

FIG. 12 shows the tumor weight of each experimental group at the end of the experiment in Example 2 (day 28).

FIG13 shows the tumor weights of the Vehicle group, the LF0376 monotherapy group, the Ibrutinib monotherapy group, and the LF0376+Ibrutinib group at the end of the experiment in Example 2 (Day 28).

FIG. 14 shows the tumor weights of the Vehicle group, the LF0376 monotherapy group, the Zanubrutinib monotherapy group, and the LF0376+Zanubrutinib group at the end of the experiment in Example 2 (Day 28).

FIG. 15 shows the average RCBW (%) curve of tumor-bearing mice during the treatment with the test drug in Example 2.

FIG. 16 shows the average body weight change curve of tumor-bearing mice during the treatment with the test drug in Example 2.

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

FIG. 18 shows the changes in individual tumor volumes of mice in each group during treatment.

Figure 19 shows the tumor weight of each group of PG-D13 mice. Note: Data points represent the tumor weight of mice, the midline represents the mean of the tumor weight of each group, and the error bars represent SEM.

Figure 20 shows the average body weight of each group of mice during treatment. Note: Data points represent the mean of body weight of each group. Error bars represent SEM.

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

DETAILED DESCRIPTION

In order to make the invention purpose, technical scheme and beneficial effects of the present application clearer, the present application is further described below in conjunction with the examples. It should be understood that the following examples are only used to explain the present application and are not used to limit the scope of the application. The test methods used in the following examples are conventional methods unless otherwise specified, and people familiar with this technology can easily understand other advantages and effects of the present application from the contents disclosed in this description.

The inventors of this application have discovered a combination drug composition and its application after extensive research and exploration. Completed this application.

On the one hand, 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.

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 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.

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.

In some embodiments, the composition of the pharmaceutical composition is a clinically acceptable dose, depending on the species, weight, age and type of tumor to be treated, the condition of the individual case or its severity. A doctor, clinician or veterinarian who has the relevant field of treatment technology can easily determine the effective dose of the pharmaceutical composition required to prevent, treat or inhibit the development of a disorder or disease.

In the pharmaceutical composition provided in 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-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 inhibitor (BTK) is an important signaling molecule in the B cell receptor pathway. It is expressed at various developmental stages of B lymphocytes, participates in regulating the proliferation, differentiation, and apoptosis of B cells, and plays an important role in the survival and spread of malignant B cells.

In the pharmaceutical composition provided in the present application, 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 item number is Lot#79640. Ibrutinib is produced by MCE The product number is Lot#114225.

In the pharmaceutical composition provided in the present application, the pharmaceutical composition is 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 and 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 and ibrutinib can be 5:1. In some embodiments, the efficacy of LF0376 and ibrutinib has a synergistic effect in the treatment of B-cell lymphoma.

In the pharmaceutical composition provided in the present application, the pharmaceutical composition is LF0376 and zanubrutinib, and the weight ratio of the two is 0.8-40:1. In a specific embodiment of the present application, the weight ratio of LF0376 and zanubrutinib can be, for example, 0.8-1:1, 1-5:1, 5-10:1, 10-20:1, 20-30:1, 30-40:1, or 40-50:1. More specifically, the weight ratio of LF0376 and zanubrutinib can be 40:1. In some embodiments, the efficacy of LF0376 and zanubrutinib has a synergistic effect in the treatment of B-cell lymphoma.

In the pharmaceutical composition provided in the present application, the mode of administration is simultaneous or sequential administration. The therapeutically effective dose of the pharmaceutical composition depends on the species, weight, age and tumor type treated, the condition of the individual case or its severity. Doctors, clinicians or veterinarians who master the treatment techniques in the relevant field can easily determine the use of the aforementioned pharmaceutical composition in the preparation of a drug for treating or preventing cancer by the active ingredients required for preventing, treating or inhibiting the development of disorders or diseases. The term "treatment" 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 the disease or completely eliminate the disease, such as cancer. Within the meaning of the present disclosure, the term "treatment" also refers to blocking, delaying the occurrence of the disease (i.e., the stage before the clinical manifestation of the disease) and/or reducing the risk of disease development or deterioration. The term "prevention" used herein includes preventing at least one symptom, which is related to or caused by the prevented state, disease or disorder.

In the application provided by 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, large intestine cancer, stomach cancer, testicular cancer and thyroid cancer.

In the application provided by the present application, the B cell lymphoma is diffuse large B cell lymphoma.

On the other hand, the present application provides a drug for treating cancer, including the CCR2/5 dual receptor antagonist in the aforementioned use, or the aforementioned pharmaceutical composition, and pharmaceutically acceptable excipients and/or carriers. The term "pharmaceutically acceptable" as used herein refers to those 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 a reasonable judgment range without excessive toxicity, irritation, allergic reactions and other problems or complications, and have 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 (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, adhesives, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, etc., and combinations thereof, and must have sufficiently high purity and very low toxicity to make them suitable for being given to patients to be treated. The carrier can be inert or it can itself have a medicinal benefit. Some carriers can be listed in more than one category, such as: vegetable oil can be used as a lubricant in some preparations and as a diluent in other preparations. Exemplary pharmaceutical carriers include sugar, starch, cellulose, malt, gelatin, talcum and vegetable oil. Optional activating agents can be included in the pharmaceutical composition, which does not substantially affect the activity of the compound of the present invention.

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

The main reagent information used in the following examples is shown in Table 1.

Table 1 List of main reagents

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

Table 2 Instrument information

The information of the tested drugs used in the following examples is as follows:

1. LF0376

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

Purity: 98.6%

Description: Powder

Molecular weight: Salt: 835.09; Free base: 738.38

Correction factor: 1.15

Saved in: RT

The drug solution was kept at 4°C during administration.

2. Ibrutinib

Provided by: MCE

Lot:136640,HY-10997

Purity: 99.46%

Correction factor: NA

Description: Powder

Store at: 4℃

The drug solution was kept at 4°C during administration.

3. Zanubrutinib

Provided by: MCE

Lot:79640,HY-101474A

Purity: 99.08%

Correction factor: NA

Description: Powder

Store at: 4℃

The drug solution was kept at 4°C during administration.

Example 1 In vivo efficacy evaluation of the test drug on PDX tumor transplantation model

1. LF0376 combined with Zanubrutinib (ZAN)

1.1 Compound preparation

The LF0376 200mg/kg group was prepared with 5% DMSO + 95% (10% HP-β-CD). The specific preparation method is: weigh 210mg LF0376, add 0.53ml DMSO, and vortex to obtain a clear yellow solution. Then add 9.975ml (10% HP-B-CD) and vortex to obtain a uniform suspension. The suspension is adjusted to pH = 3 to obtain a clear solution. After preparation, it is divided into 5ml EP tubes, 3.3mL per tube, and stored at 4℃. Prepare once every three days.

Zanubrutinib is prepared with 10% DMSO + 40% PEG300 + 5% Tween 80 + 45% saline. The specific preparation method is: 60mg Zanubrutinib is added to 6ml DMSO to prepare the mother solution, and then divided into 5ml EP tubes at 0.4ml per tube. When using, add 1.6ml PEG300, 0.2ml Tween 80, and 1.8ml saline in order, and store at 4℃. Prepare once every three days.

1.2 Construction of PDX tumor transplantation model

40 female NOG mice were inoculated with patient PDX in the axilla. NOG mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. They were 6-7 weeks old at the time of tumor inoculation. Feeding and management conditions: The mice were raised according to SPF animal feeding conditions, and the cages, bedding, feed and drinking water were sterilized by high temperature and high pressure. The cages were placed in the IVC, with 3-5 animals per cage. The indoor temperature of the animal room was 21-25°C, and the relative humidity was 40%-70%. PDX came from patients with diffuse large B-cell lymphoma and belonged to the non-germinal center (non-GCB) subtype. Immunohistochemical results of the PDX model: CD10-, MUM-1 (100%+), Bcl6 (80%+), CD20+, Bcl2+, CD3-, Ki67 (90%+).

On the 30th day after inoculation, mice were regrouped according to tumor volume, with 8 mice in each group, a total of 4 groups, a total of 32 mice, and the mean tumor volume of each group reached 133.47±1.75mm ³ , which was Di 0 day. The remaining mice were killed after the end of the experiment or when the tumor load reached 1500mm ^3. Each group of mice was administered according to the dose set for each group. The tumor volume was measured every 3 days. Each group was given different doses and drugs, see Table 3 and Figure 1 for details.

Table 3 Experimental dosage and grouping

Note: The vehicle control group was given 5% DMSO + 95% (10% HP-β-CD). The vehicle and LF0376 were administered starting at Di0 (Di: Days of injection), twice a day, with an interval of 8 hours. Zanubrutinib was administered by gavage once a day.

1.3 Indicator Observation

1.3.1 Tumor volume

The tumor volume was measured every 3 days. The calculation formula of 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.

1.3.2 Weight

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

1.4 Data Analysis

All data are expressed as mean ± sem.

PRISM software was used for statistical analysis, one-way ANOVA was used for data analysis, and Tukey’s method was used for inter-group comparison.

1.5 Experimental Results

This phase is a study on the combination of LF0376 and Zebutinib, with a total of 27 days of medication.

As shown in Figure 2, by observing the overall tumor inhibition curve, it can be seen that the LF0376 monotherapy group, the zanubrutinib group, and the LF0376 combined with zanubrutinib group can inhibit tumor growth compared with the control group, among which the tumor inhibition effect of the combination group was the most significant.

As shown in Figure 3, the tumors were weighed 27 days after administration, and it was found that the tumor weight in the LF0376 combined with zanubrutinib group was significantly smaller than that in the control group and the two single-drug groups.

As shown in Figure 4, the weight of mice in the control group increased significantly during treatment, which is consistent with the natural law that the tumor grows larger and the mice become heavier. After medication, the LF0376 monotherapy group, the zanubrutinib group, and the LF0376 combined with zanubrutinib group significantly reduced the tumor volume of mice compared with the control group, thereby reducing the weight of mice, and all did not exceed the range of mouse weight loss, indicating that both combined medication and monotherapy were well tolerated and safe in mice.

2. LF0376 combined with ibrutinib (IBR)

2.1 Compound preparation

The LF0376 100mg/kg group was prepared with 5% DMSO + 95% (10% HP-β-CD). The specific preparation method is: weigh 210mg LF0376, add 1.05ml DMSO, and vortex to obtain a clear yellow solution. Then add 19.95ml (10% HP-B-CD) and vortex to obtain a uniform suspension. The suspension is adjusted to pH = 3 to obtain a clear solution. After preparation, it is divided into 5ml EP tubes, 3.3mL per tube, and stored at 4℃. Prepare once every three days.

Ibrutinib is prepared with 10% DMSO + 40% PEG300 + 5% Tween 80 + 45% saline. The specific preparation method is: 240mg Ibrutinib is added to 6ml DMSO to prepare the mother solution, and then divided into 5ml EP tubes at 0.4ml per tube. When using, follow the order Add 1.6 ml PEG300, 0.2 ml Tween 80, and 1.8 ml normal saline and store at 4°C. Prepare once every three days.

2.2 Construction of PDX tumor transplantation model

40 female NOG mice were inoculated with patient PDX in the axilla. NOG mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. They were 6-7 weeks old at the time of tumor inoculation. Feeding and management conditions: The mice were raised according to SPF animal feeding conditions, and the cages, bedding, feed and drinking water were sterilized by high temperature and high pressure. The cages were placed in the IVC, with 3-5 animals per cage. The indoor temperature of the animal room was 21-25°C, and the relative humidity was 40%-70%. PDX came from patients with diffuse large B-cell lymphoma and belonged to the non-germinal center (non-GCB) subtype. Immunohistochemical results of the PDX model: CD10-, MUM-1 (100%+), Bcl6 (80%+), CD20+, Bcl2+, CD3-, Ki67 (90%+).

On the 24th day after inoculation, 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 was day 0. The remaining mice were killed after the end of the experiment or when the tumor burden reached 1500mm ^3. Mice were administered according to the dose set for each group. Tumor volume was measured once every 3 days, and body weight was weighed. Different doses and drugs were given to each group, see Table 4 and Figure 5 for details.

Table 4 Experimental dosage and grouping

Note: The vehicle control group was given 5% DMSO + 95% (10% HP-β-CD). The vehicle and LF0376 were administered starting at Di 0 (Di: Days ofinjection), twice a day, with an interval of 8 hours. Ibrutinib was administered by gavage once a day.

2.3 Indicator Observation

Same as 1.3.

2.4 Data Analysis

Same as 1.4.

2.5 Experimental Results

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

As shown in Figure 6, by observing the overall tumor inhibition curve, it can be seen that the LF0376 monotherapy group, the ibrutinib group, and the LF0376 combined with ibrutinib group can inhibit tumor growth compared with the control group, among which the combined group had the most significant tumor inhibition effect.

As shown in 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.

As shown in Figure 8, the weight of mice in the control group increased significantly during treatment, which is consistent with the natural law that the tumor grows larger and the mice become heavier. After medication, the LF0376 monotherapy group, the ibrutinib group, and the LF0376 combined with ibrutinib group significantly reduced the tumor volume of mice compared with the control group, thereby reducing the weight of mice, and all did not exceed the range of mouse weight loss, indicating that both combined medication and monotherapy were well tolerated and safe in mice.

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

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.

1. Compound preparation

See Table 5 for details:

Table 5 Preparation of test drugs

2. In vivo transplanted tumor model

LD1-0026-361717 human DLBCL tumor tissue, passed to FP3+2 generation, was used for this efficacy experiment.

For basic information of the LD1-0026-361717PDX model, please see Table 6.

Table 6 Basic information of the model

95 NU/NU mice, female, weighing 18-21 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (SCXK (Beijing) 2021-0006), with a certificate number of 110011231108918138. Laboratory animal use license number: SYXK (Shaanxi) 2023-008. Breeding environment: SPF grade. The animals were adapted for observation for 3 days before the start of the experiment.

The tumor mass of the FP3+1 generation LD1-0026-361717 human DLBCL in vivo transplanted tumor was cut into tumor tissues of approximately 3 mm × 3 mm × 3 mm (approximately 45-60 mg) and inoculated subcutaneously into NU/NU mice. The mice after inoculation were observed and the growth of the tumor was monitored. When the average tumor volume of the tumor-bearing mice was 137.11±6.36 mm ³ on the 28th day of inoculation, the grouping and administration were performed, and the day of grouping and administration was defined as day 0. Specific grouping information and dosing regimen are detailed in Table 7.

Table 7 Drug administration and treatment

Note: Dosing volume: adjust the dosing volume according to the weight of tumor-bearing mice (0.2mL/20g); PO: intragastric administration; QD: once a day; BID: twice a day, with an interval of about 8 hours.

3. Indicator observation

3.1 Tumor volume

Tumor volume was measured twice a week using a vernier caliper. The formula for calculating tumor volume was V = 0.5a × b ² , where a and b represented the long diameter and wide diameter of the tumor, respectively.

3.2 Relative tumor proliferation rate T/C (%)

The calculation formula is as follows: T/C%= _TRTV / _CRTV ×100% ( _TRTV : RTV of treatment group; _CRTV : RTV of vehicle group). The relative tumor volume (RTV) is calculated according to the results of tumor measurement. The calculation formula is RTV= _Vt / _V0 , where _V0 is the average tumor volume measured at the time of group administration (i.e., _d0 ), _Vt is the average tumor volume at a certain measurement, and _TRTV and _CRTV are obtained from the same day.

3.3 Tumor growth inhibition rate TGI (%)

TGI (%) = [1-(T _i -T ₀ )/(V _i -V ₀ )] × 100%, T _i is the average tumor volume of the compound group after the start of administration, T ₀ is the average tumor volume of the compound group at the first administration, V ₀ is the average tumor volume of the Vehicle group at the first administration, and _Vi is the average tumor volume of the Vehicle group after the start of administration. If TGI (%) > 50%, it is considered that the compound exhibits a significant inhibitory effect on tumor growth.

3.4 Weight

The weight of mice was measured every day. The relative change ratio of body weight after administration was calculated: RCBW (%) = (BW _i - BW ₀ )/BW ₀ × 100, where BW _i is the body weight after the start of administration and BW ₀ is the body weight at the first administration.

4. Data Analysis

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

5. Experimental results

As shown in Figures 9, 10, 11 and Table 8, on day 28, the average tumor volume of the Vehicle group was 1470.55±411.29 mm ³ , and the average tumor volumes of the LF0376 monotherapy group, the Ibrutinib monotherapy group, the Zanubrutinib monotherapy group, the LF0376+Ibrutinib group and the LF0376+Zanubrutinib group were 1219.51±254.70 mm ³ , 906.95±185.57 mm ³ , 972.72±342.44 mm ³ , 566.26±159.12 mm ³ and 752.64±212.66 mm ^{3 ,} respectively. ; TGI (%) were 18.60%, 42.24%, 37.33%, 67.86% and 53.86% respectively; T/C (%) were 84.86%, 61.94%, 66.29%, 38.45% and 51.21% respectively; LF0376+Ibrutinib group and LF0376+Zanubrutinib group showed significant inhibitory effect on tumor growth in human DLBCL PDX model LD1-0026-361717 compared with Vehicle group. LF0376+Ibrutinib group and LF0376+Zanubrutinib group showed stronger inhibitory effect on tumor growth compared with LF0376 monotherapy group and Ibrutinib/Zanubrutinib monotherapy group, indicating that LF0376 can enhance the anti-tumor effect of Ibrutinib or Zanubrutinib.

Table 8 Summary of the anti-tumor efficacy evaluation of the tested drugs in the LD1-0026-361717 human DLBCL subcutaneous xenograft tumor model

As shown in Figures 12, 13, 14 and 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, and the tumor weight results were basically consistent with the tumor volume results.

As shown in Figures 15, 16 and 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.

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

6. Experimental conclusion

LF0376 alone, Ibrutinib alone, and Zanubrutinib alone did not show significant anti-tumor effects in this model at the current dose level; LF0376 combined with Ibrutinib and LF0376 combined with Zanubrutinib showed significant anti-tumor effects in this model, indicating that LF0376 can significantly enhance the anti-tumor effects of Ibrutinib and Zanubrutinib. During the treatment with the test drug, no animals in each group showed significant weight loss, indicating that the test drug was safe.

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

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

1. Compound preparation

See Table 10 for details

Table 10 Preparation of test drugs

Note: *Dosage concentration is based on active ingredient.

2. Animal Model

Animal information is shown in Table 11:

Table 11 Experimental Animal Information

The animals were isolated and adaptively raised before experimental treatment.

A20 cells were cultured and expanded in vitro, and cells in the logarithmic growth phase were collected and resuspended in PBS. The concentration of the cell suspension was 5×10 ⁶ /mL.

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 cell suspension. The number of cells inoculated was 5×10 ⁵ /mouse.

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

Drug administration was started according to the grouping scheme. Each group was administered by gavage. The administration volume of each group was 5 mL/kg. The administration frequency was once or twice a day. During the experiment, the animal body weight was weighed and the tumor volume was measured three times a week.

The experimental endpoint was PG-D13, all mice were euthanized, tumor tissues were removed, weighed and photographed. Based on the tumor volume and body weight measured at PG-D12, the tumor volume inhibition rate (TGI) and animal body weight change rate (BWC%) were calculated respectively.

The dosing regimen for mice is shown in Table 12:

Table 12 Dosage schedule for each group of mice

Note: G: Group; p.o.: intragastric administration;

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

2. Dosing volume: adjusted according to the weight of the mouse;

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

Animal grouping and identification:

When the mean tumor volume reached approximately 60.75 ± 0.48 mm ³ , the mice were randomly divided into 6 groups of 6 mice each according to the tumor volume. Detailed information is shown in Table 13 below: The cages were clearly marked with cage cards, including the animal number, sex, strain, date of receipt, treatment, study number, and group number.

Table 13 Experimental Animal Grouping Information

3. Indicator observation

3.1 Tumor volume (TV)

TV = 1/2 × a × b ²

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

3.2 Tumor volume inhibition rate (TGI)

TGI = [1-(TV _Dt treatment group-TV _D0 treatment group)/(TV _Dt control group-TV _D0 control group)] × 100%

TV _Dt experimental group: tumor volume of the treatment group on day t after drug administration

TV _D0 experimental group: tumor volume of the treatment group at the time of group administration

TV _Dt control group: tumor volume of the control group on day t after drug administration

TV _D0 control group: the tumor volume of the control group at the time of group drug administration.

3.3 Animal body weight change rate (BWC%)

Weight change rate = (BW _Dt - BW _D0 )/BW _D0 × 100%

BW _Dt : Animal body weight t days after administration

BW _D0 : animal body weight at the time of group dosing.

3.4 Tumor weight inhibition rate (IR)

IR＝(W _C - _WT )/W _C ×100％

Wherein, W _C represents the tumor weight of the control group; _WT represents the tumor weight of the treatment group.

4. Data Analysis

5.1 Mouse tumor volume

As shown in Figures 17 and 18, Tables 14 and 15, for PG-D12, the average tumor volume of the control group was 687.68±46.59 mm ³ , and the average tumor volumes of the LF0376 monotherapy group, the Ibrutinib monotherapy group, the Zanubrutinib monotherapy group, the LF0376+Ibrutinib group, and the LF0376+Zanubrutinib group were 551.12±40.66 mm ³ , 640.57±48.47 mm ³ , 651.25±39.04 mm ³ , 497.63±57.96 mm ³ , and 454.45±46.55 mm ³ , respectively, and the TGIs were 21.74%, 7.47%, 5.79%, 30.30%, and 37.12%, respectively. Compared with the control group, the LF0376+Ibrutinib group and the LF0376+Zanubrutinib group showed significant tumor growth inhibition. Compared with the LF0376 monotherapy group and the Ibrutinib/Zanubrutinib monotherapy group, the LF0376+Ibrutinib group and the LF0376+Zanubrutinib group showed more significant tumor inhibition.

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

Note: Data are expressed as Mean ± SEM.

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

5.2 Tumor weight in mice

As shown in Figure 19 and Table 16, for PG-D13, the average tumor weight of the control group was 1.8387±0.2200 g, and the average tumor weights of the LF0376 monotherapy group, Ibrutinib monotherapy group, Zanubrutinib monotherapy group, LF0376+Ibrutinib group, and 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, and the IRs were 35.54%, 14.02%, 19.21%, 27.50%, and 40.96%, respectively. The tumor weight results were basically consistent with the tumor volume results.

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

Note: n/n means: actual number of samples/original number of samples.

5.3 Mouse body weight

As shown in Figure 20 and 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±1.13g, 24.73±0.89g, 26.33±1.20g, 25.68±0.69g, 24.52±1.06g and 24.80±0.32g, respectively.

As shown in Figure 21 and Table 18, the average weight of mice in Group 1, Group 2, Group 3, Group 4, Group 5 and Group 6 increased with PG-D12, and the weight change rates were 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 weight change rates of mice in the experimental groups during the treatment period ranged from -10.8% to 21.6%. No mice were suspended from the drug due to significant weight loss or poor condition.

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

Note: Data are expressed as Mean ± SEM.

Table 18 Weight change rate of mice in each group during treatment compared with the baseline (%)

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

6. Experimental conclusion

The safety of the test drug was good during the treatment. LF0376 alone had a certain anti-tumor effect on A20, and the combined anti-tumor effect of LF0376+Ibrutinib and LF0376+Zanubrutinib was more significant, indicating that LF0376 can significantly enhance the anti-tumor effect of Ibrutinib and Zanubrutinib.

The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present application. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed by the present invention shall still be covered by the claims of the present application.

Claims (10)

Use of a CCR2/5 dual receptor antagonist in the preparation of a drug for treating or preventing B-cell lymphoma. The use according to claim 1, characterized in that 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. A pharmaceutical composition, characterized in that it comprises the CCR2/5 dual receptor antagonist for use according to any one of claims 1 to 2, and a Bruton's tyrosine kinase inhibitor. The pharmaceutical composition of claim 3, wherein 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-64 A combination of one or more of 264681, 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. The pharmaceutical composition according to claim 4, characterized in that the Bruton's tyrosine kinase inhibitor is selected from zanubrutinib or ibrutinib. The pharmaceutical composition according to claim 5, characterized in that the pharmaceutical composition comprises LF0376 and ibrutinib; preferably, the weight ratio of LF0376 to ibrutinib is 0.5 to 10:1. The pharmaceutical composition according to claim 5, characterized in that the pharmaceutical composition comprises LF0376 and zanubrutinib; preferably, the weight ratio of LF0376 to zanubrutinib is 0.8 to 40:1. Use of the pharmaceutical composition according to any one of claims 3 to 7 in the preparation of a drug for treating or preventing cancer, wherein 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, large intestine cancer, stomach cancer, testicular cancer and thyroid cancer. The use according to claim 8, characterized in that the B cell lymphoma is diffuse large B cell lymphoma. A drug for treating cancer, comprising the CCR2/5 dual receptor antagonist for use as described in any one of claims 1 to 2, or the pharmaceutical composition as described in any one of claims 3 to 7, and pharmaceutically acceptable excipients and/or carriers.