CN118987211A - Combined medicine composition and application thereof - Google Patents

Abstract

The present application relates to the field of medical technology, and in particular to a combined pharmaceutical composition and its application. The pharmaceutical composition comprises a CCR2/5 dual receptor antagonist and a Bruton's tyrosine kinase inhibitor. The combined use of LF0376 and zanubrutinib, and the combined use of LF0376 and ibrutinib have a significant tumor inhibition effect on B-cell lymphoma compared with single use of the drug.

Description

Combined medicine composition and application thereof

Technical Field

The application relates to the technical field of medicines, in particular to a combined medicine composition and application thereof.

Background

Lymphoma is a common malignant tumor in China, the number of incidents per year is about 7.54 ten thousand, the incidence rate is 4.75/10 ten thousand, the death rate is 4.05 ten thousand, the death rate is 2.64/10 ten thousand, and the differences among regions and urban and rural areas are obvious. Lymphomas can be classified into hodgkin lymphomas and non-hodgkin lymphomas, and are complex in pathological type, diverse in treatment methods, and quite different in prognosis, so that multidisciplinary team participation is generally required in diagnosis and treatment. In non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL) accounts for about 30-40%, the standard treatment is immune combined chemotherapy (R-CHOP), about 50% -60% of patients can be cured by the current standard treatment, but there is still a problem that 40-50% of patients are refractory or recur after treatment (about 15% -25% of patients are primary refractory (treatment period or treatment progress), about 20% -30% of patients recur after Complete Remission (CR), and 5% of patients are in Partial Remission (PR). For the recurrent/refractory patients, hematopoietic stem cell transplantation and the first-push treatment method of CART are limited due to transplantation conditions and high price, and other treatment methods mainly use chemotherapy, have the problems of curative effect limitation and great toxic and side effects, in addition, young high-risk or medium-high-risk patients, MYC and BCL2 are simultaneously rearranged "double", and BCL 6-expressing double-center B-cell-type lymphomas are not satisfied, and the current clinical requirements of clinical treatment do not satisfy the clinical requirements of the recurrences.

Both CCR2 and CCR5 are G protein-coupled receptors, recognizing chemokines, with 73% sequence homology. The primary ligand for CCR2 is the chemokine CCL2, which in addition recognizes CCL7, CCL8, CCL12 and CCL13; CCR5 has a high affinity for multiple chemokines such as CCL3, CCL4, CCL5, CCL3L1, CCL8, CCL11, etc. CCR2 is expressed primarily on monocytes, NK cells and T cells, which can be recruited to sites of inflammation under inflammatory conditions. In most cases, the CCR2-CCL2 axis plays a major proinflammatory role, but CCR2 expressed on Treg cells plays an anti-inflammatory role. CCR5 expression is more extensive and includes T cells, macrophages, granulocytes, DCs, microglia, even epidermal cells, etc.

In many disease processes, such as viral infection and liver fibrosis, including inflammatory diseases, CCR2 and CCR5 mediate migration and infiltration of lymphocytes, monocytes, macrophages, promoting disease development. Studies have shown that CCR2 is more involved in migration and infiltration of monocytes and macrophages and CCR5 has a greater effect on lymphocytes. Thus, CCR2 and CCR5 are functionally identical, but not of equal importance.

There is no current study showing a specific link between CCR2/5 dual receptor antagonists and B cell lymphomas.

Bruton's tyrosine kinase (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. The first generation BTK inhibitor Ibrutinib (ibutenib) based on its prominent therapeutic efficacy in a number of clinical trials has been approved by the FDA for 7 indications, including small lymphocytic linban tumor, mantle cell lymphoma, chronic lymphocytic leukemia, fahrenheit macroglobulinemia, graft versus host disease and marginal zone lymphoma, with the first three having been approved in China; the second-generation highly selective BTK inhibitor Zanubrutinib (zebutinib) is an autonomous development of new anticancer drugs in china, and is also successfully batched in adult mantle cell lymphoma, fahrenheit macroglobulinemia, recurrent/refractory marginal zone lymphoma, and Chronic Lymphocytic Leukemia (CLL) or Small Lymphocytic Lymphoma (SLL). Although BTK inhibitors are not batched in DLBCL, they are recommended for this indication by the CSCO guidelines based on clinical practice.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, an object of the present application is to provide a pharmaceutical composition for combination and application thereof, which are used for solving the problems of the prior art. The CCR2/5 dual receptor antagonist and the BTK inhibitor are combined, so that a new direction is provided for exploring the combined use of the two in the treatment of B cell lymphoma, especially diffuse large B cell lymphoma.

To achieve the above and other related objects, a first aspect of the present application provides the use of a CCR2/5 dual receptor antagonist for the manufacture of a medicament for the treatment or prevention of B cell lymphoma.

In any embodiment of the application, the CCR2/5 dual receptor antagonist is selected from the group consisting of one or more of LF0376, BMS-813160, cenicriviroc; preferably, the CCR2/5 dual receptor antagonist is selected from LF0376.

In a second aspect, the application provides a pharmaceutical composition comprising a CCR2/5 dual receptor antagonist and a bruton's tyrosine kinase inhibitor.

In any embodiment of the application, the bruton's tyrosine kinase inhibitor is selected from the group consisting of one or more of zebutinib, ibutenib 、spebrutinib、acalabrutinib、olmutinib、poseltinib、tirabrutinib、evobrutinib、fenebrutinib、vecabrutinib、ARQ-531、BMS-986195、BMS-986142、CGI-1746、GDC-0834、RN-486、JNJ-64264681、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、LFM-A13.

In any embodiment of the application, the bruton's tyrosine kinase inhibitor is selected from zebutinib or ibutenib.

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

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

The third aspect of the application provides the use of the pharmaceutical composition in the preparation of a medicament for the treatment or prophylaxis of cancer.

In any embodiment of the application, the cancer is selected from the group consisting 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, gall bladder cancer, gastrointestinal cancer, external genitalia cancer, genitourinary tract cancer, head cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, muscle tissue cancer, neck cancer, oral or nasal mucosa 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 application, the B-cell lymphoma is diffuse large B-cell lymphoma.

In a fourth aspect, the application provides a medicament for the treatment of cancer comprising a CCR2/5 dual receptor antagonist for the aforementioned use, or a pharmaceutical composition as described above, together with pharmaceutically acceptable excipients and/or carriers.

Compared with the prior art, the application has the beneficial effects that:

1. the application discloses that LF0376 has obvious tumor inhibiting effect on B cell lymphoma for the first time.

2. The combination of LF0376 and zebutinib has obvious tumor inhibiting effect on B cell lymphoma compared with single administration.

3. The combination of LF0376 and ibutenib has obvious tumor inhibiting effect on B cell lymphoma compared with single administration.

Drawings

Fig. 1 shows a graph of the experimental design for the combination of LF0376 and zebutinib.

FIG. 2 shows the tumor inhibition curve of the LF0376 (200 mg) combination Zanubrutinib experiment of example 1. Each data point is represented by mean±sem.

FIG. 3 shows tumor weights 27 days after treatment for the LF0376 (200 mg) of example 1 in combination with Zanubrutinib experiments. Each data point is represented by mean±sem.

Fig. 4 shows the percent (%) change in body weight from baseline in mice during treatment of example 1. Each data point is represented by mean±sem.

Fig. 5 shows a design of experiments for the combination of LF0376 and ibutenib.

FIG. 6 shows the tumor inhibition curve of the LF0376 (100 mg) combination Ibrutinib experiment of example 1. Each data point is represented by mean±sem.

FIG. 7 shows tumor weights 27 days after treatment for the LF0376 (100 mg) combination Ibrutinib experiment of example 1. Each data point is represented by mean±sem.

Fig. 8 shows the percent (%) change in body weight from baseline in mice during treatment of example 1. Each data point is represented by mean±sem.

Fig. 9 shows the tumor volume change curves for all experimental groups of example 2.

Fig. 10 shows tumor volume change curves for example 2Vehicle, LF0376, ibrutinib, and LF0376+ Ibrutinib.

Fig. 11 shows tumor volume change curves for example 2Vehicle, LF0376, zanubrutinib, and LF0376+ Zanubrutinib.

Figure 12 shows the tumor weights for each experimental group at the end of the experiment of example 2 (day 28).

Figure 13 shows tumor weights for the Vehicle group, LF0376 single drug group, ibrutinib single drug group, and LF0376+ Ibrutinib group at the end of the experiment of example 2 (day 28).

Figure 14 shows tumor weights for the Vehicle group, LF0376 single drug group, zanubrutinib single drug group, and LF0376+ Zanubrutinib group at the end of the experiment of example 2 (day 28).

Figure 15 shows the average RCBW (%) curve of tumor-bearing mice during treatment with the drug of example 2.

FIG. 16 shows the average body weight change profile of tumor-bearing mice during treatment with the drug of example 2.

Fig. 17 shows a graph of tumor volume change during treatment for each group of mice. And (3) injection: data points represent Mean for each set of tumor volumes, error bars represent SEM.

Figure 18 shows a graph of individual tumor volume changes during treatment for each group of mice.

FIG. 19 shows tumor weight patterns of mice of each group of PG-D13. And (3) injection: data points represent mouse tumor weights, midlines represent Mean of each group of tumor weights, error bars represent SEM.

Figure 20 shows a graph of average body weight during treatment of each group of mice. And (3) injection: data points represent Mean for each group of body weights. Error bars represent SEM.

Figure 21 changes in body weight from baseline during treatment in mice of each group. And (3) injection: data points represent Mean for each group of body weight change rates, error bars represent SEM.

Detailed Description

In order to make the objects, technical solutions and advantageous effects of the present application clearer, the present application will be further described with reference to examples. It is to be understood that the following examples are illustrative of the present application and are not intended to limit the scope of the application. The test methods used in the following examples are conventional, and other advantages and effects of the present application will be readily apparent to those skilled in the art from the disclosure herein.

The inventor of the present application has found a combination pharmaceutical composition and its application through a great deal of research and study, and completed the present application on the basis of this.

In one aspect, the application provides the use of a CCR2/5 dual receptor antagonist in the manufacture of a medicament for the treatment or prophylaxis of B cell lymphoma.

In the pharmaceutical composition provided by the application, the CCR2/5 dual receptor antagonist is selected from one or more of LF0376, BMS-813160 and Cenicriviroc. CCR2/5 is a cell surface chemokine receptor 2 and 5, which belongs to a G protein coupled receptor superfamily member, expressed on the surface of a variety of cells and binds to its specific ligand for biological action. Preferably, the CCR2/5 dual receptor antagonist is selected from LF0376, also known as an azabenzooctacyclic compound, other code numbers WXFL40050414 or WXSH0376. In a preferred embodiment of the application, LF0376 is manufactured by Shanghai Minkangde New drug development Co., ltd., lot number ES16578-17-P1, molecular weight 739.98, purity 97.28%, and stored at ambient temperature. The application discloses a specific connection between a CCR2/5 dual receptor antagonist (LF 0376) and B cell lymphoma for the first time, and the LF0376 has obvious tumor inhibiting effect on the B cell lymphoma.

In another aspect, the application provides a pharmaceutical composition comprising a CCR2/5 dual receptor antagonist and a bruton's tyrosine kinase inhibitor.

In some embodiments, the pharmaceutical composition is formulated as a clinically acceptable dose, depending on the species, weight, age and tumor type being treated, the condition of the individual case, or severity thereof. A physician, clinician or veterinarian in possession of the relevant art of treatment can readily determine the effective dosage of the pharmaceutical composition required to prevent, treat or inhibit the development of the disorder or disease.

In the pharmaceutical composition provided by the application, the Bruton tyrosine kinase inhibitor is selected from one or a combination of a plurality of zebutinib and ibutenib 、spebrutinib、acalabrutinib、olmutinib、poseltinib、tirabrutinib、evobrutinib、fenebrutinib、vecabrutinib、ARQ-531、BMS-986195、BMS-986142、CGI-1746、GDC-0834、RN-486、JNJ-64264681、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、LFM-A13. Bruton tyrosine kinase inhibitors (BTKs) are important signal molecules of B cell receptor pathways, are expressed in each development stage of B lymphocytes, participate in regulating proliferation, differentiation and apoptosis of the B cells, and play an important role in survival and diffusion of malignant B cells.

In the pharmaceutical composition provided by the application, the bruton's tyrosine kinase inhibitor is selected from zebutinib (Zanubrutinib) or ibutenib (Ibrutinib). Zanubrutinib is a multi-target kinase inhibitor, the first approved small molecule BTK inhibitor, that inhibits BTK enzyme activity by forming a covalent bond with a cysteine in the BTK active site. Ibrutinib are second generation BTK inhibitors that irreversibly inactivate enzymes by covalent binding to tyrosine kinases. In a preferred embodiment of the application Zanubrutinib is produced by MCE under the designation Lot #79640.Ibrutinib is produced by MCE under the trade designation Lot #114225.

In the pharmaceutical composition provided by the application, the pharmaceutical composition is LF0376 and ibutenib, and the weight ratio of the two is 0.5-10: 1. in a specific embodiment of the present application, the weight ratio of LF0376 to ibutenib may be, for example, 0.5 to 1: 1. 1 to 2.5: 1. 2.5 to 5:1. 5-8: 1. or 8-10: 1, etc. More specifically, the weight ratio of LF0376 and ibutenib may be 5:1. in some embodiments, the potency of LF0376 and ibutenib have a synergistic effect in the treatment of B-cell lymphomas.

In the pharmaceutical composition provided by the application, the pharmaceutical composition is LF0376 and zebutinib, 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 to zebutinib may be, for example, 0.8 to 1: 1. 1 to 5: 1. 5-10: 1. 10-20: 1. 20-30: 1. 30-40: 1. or 40-50: 1, etc. More specifically, the weight ratio of LF0376 and zebutinib may be 40:1. in some embodiments, the potency of LF0376 and zebutinib has a synergistic effect in the treatment of B-cell lymphomas.

In the pharmaceutical composition provided by the application, the administration mode is simultaneous or sequential administration. The therapeutically effective dose of the pharmaceutical composition depends on the species, weight, age and tumor type being treated, the disease condition of the individual case or its severity of the subject. The doctor, clinician or veterinarian in possession of the relevant art of treatment can readily ascertain that each of the active ingredients required to prevent, treat or inhibit the progression of a disorder or disease has the use of the aforementioned pharmaceutical compositions in the manufacture of a medicament for the treatment or prophylaxis of cancer. The term "treatment" as used herein includes treatment that slows, alleviates or alleviates at least one symptom in a subject or achieves a delay in disease progression. For example, the treatment may be to reduce one or more symptoms of the disease or to completely eliminate the disease, such as cancer. Within the meaning of the present disclosure, the term "treatment" also refers to blocking, delaying the onset of, i.e. the pre-clinical characterization of, the disease and/or reducing the risk of developing or worsening the disease. The term "preventing" as used herein includes preventing at least one symptom associated with or caused by the state, disease or disorder being prevented.

In the present application, there is provided a use wherein the cancer is selected from the group consisting 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, gall bladder cancer, gastrointestinal cancer, external genitalia cancer, genitourinary cancer, head cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, muscle tissue cancer, neck cancer, oral or nasal mucosa 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 application, the B cell lymphoma is diffuse large B cell lymphoma.

In another aspect, the application provides a medicament for treating cancer, comprising a CCR2/5 dual receptor antagonist for the aforementioned use, or a pharmaceutical composition for the aforementioned use, and pharmaceutically acceptable excipients and/or carriers. The term "pharmaceutically acceptable" as used herein refers to those compounds, therapeutic agents (e.g., antibodies), materials, compositions and/or dosage forms which are, within the scope of sound judgment, suitable for use in contact with the tissues of warm-blooded animals such as mammals or humans without undue toxicity, irritation, allergic response and other problem or complication and are commensurate with a reasonable benefit/benefit ratio. The term "carrier" or "adjuvant" as used herein includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonic, absorption delaying agents, salts, preservatives, medicaments, pharmaceutical stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof, and must be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to a patient to be treated. The carrier may be inert or it may have a pharmaceutical benefit itself. Some vectors may be listed in more than one category, such as: vegetable oils may be used as lubricants in some formulations and as diluents in other formulations. Exemplary pharmaceutically acceptable carriers include sugar, starch, cellulose, malt, gelatin, talc and vegetable oils. Alternative active agents may be included in the pharmaceutical compositions that do not substantially affect the activity of the compounds of the present application.

The application is further illustrated by the following examples, which are not intended to limit the scope of the application.

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

TABLE 1 list of main reagents

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

Table 2 instrument information

The test drug information used in the following examples is as follows:

1、LF0376

A provider: tin-free Ling formula biological medicine science and technology Co Ltd Lot CR-C-210219013-FP22001

Purity: 98.6%

Description of properties and the like: molecular weight of powder: salt: 835.09; free base: 738.38 correction factors: 1.15

And (3) storing in: RT (reverse transcription) method

The medicinal solution is stored at 4deg.C during administration

2、Ibrutinib

A provider: MCELot:136640, HY-10997

Purity: 99.46%

Correction factors: NA (NA)

Description of properties and the like: powder

And (3) storing in: 4 DEG C

The medicinal solution is stored at 4deg.C during administration

3、Zanubrutinib

A provider: MCE (MCE)

Lot:79640，HY-101474A

Purity: 99.08%

Correction factors: NA (NA)

Description of properties and the like: powder

And (3) storing in: 4 DEG C

The medicinal solution is stored at 4deg.C during administration

EXAMPLE 1 evaluation of in vivo efficacy of test drugs on PDX tumor transplantation model

1. LF0376 in combination with zebutinib (Zanubrutinib, ZAN)

1.1 Compound formulation

LF0376 200mg/kg was formulated with 5% DMSO+95% (10% HP-beta-CD). The specific configuration method comprises the following steps: 210mg of LF0376 were weighed and added to 0.53ml of DMSO and vortexed to give a clear yellow solution. Then 9.975ml (10% hp-B-CD) of vortex was added and the suspension was adjusted to ph=3 to give a clear solution. After preparation, the mixture was dispensed into 5ml EP tubes, each of which was stored at 3.3mL and 4 ℃. It is formulated every three days.

Zanubrutinib was prepared with 10% dmso+40% peg300+5% tween 80+45% saline. The specific configuration method comprises the following steps: 60mg Zanubrutinib ml DMSO is added to prepare mother liquor, and then each tube is divided into 0.4ml and filled into 5ml EP tubes, 1.6ml PEG300,0.2ml Tween 80 and 1.8ml physiological saline are sequentially added when the EP tubes are used, and the EP tubes are preserved at 4 ℃. It is administered once every three days.

1.2PDX tumor transplantation model construction

Axillary inoculation of 40 female NOG mice contained patient PDX. NOG mice were purchased from beijing velariwa laboratory animal technologies limited. The tumor is 6-7 weeks old. Feeding management conditions: raising according to SPF-level animal raising condition, and sterilizing squirrel cage, padding, feed and drinking water at high temperature and high pressure. The squirrel cage is placed in IVC, each cage (3-5) animals. The temperature in the animal room is 21-25 ℃ and the relative humidity is 40-70%. PDX is from diffuse large B-cell lymphoma patients and belongs to the non-germinal center (non-GCB) subtype. Immunohistochemical results for PDX model: CD10-, MUM-1 (100% +), bcl6 (80% +), CD20+, bcl2+, CD3-, ki67 (90% +).

On day 30 after inoculation, the tumors are regrouped according to tumor volumes, 8 tumors in each group, and the total number of tumors in each group is 4, and the average tumor volume of each group reaches 133.47 +/-1.75 mm ³, which is Di 0 day. The remaining mice were sacrificed at the end of the experiment or after tumor burden reached 1500mm ³. Each group of mice was dosed at the dose set for each group. Tumor volume was measured once for 3 days. Each group was given a different dose and drug, see table 3 and figure 1 for details.

Table 3 experimental dosing and grouping

Note that: vehicle control group was given 5% DMSO+95% (10% HP-beta-CD). Vehicle and LF0376 were dosed twice daily beginning at Di 0 (Di: days of injection) with 8 hours between dosing intervals. Zanubrutinib is administered by intragastric administration once daily.

1.3 Index Observation

1.3.1 Tumor volumes

Tumor volume was measured once for 3 days. The calculation formula of Tumor Volume (TV) is: tv=1/2× a×b ². Wherein a and b respectively represent the long diameter and the short diameter of the tumor mass.

1.3.2 Body weight

Animal body weight was measured once a3 day measurement.

1.4 Data analysis

All data are expressed as mean ± sem.

Statistical analysis was performed using PRISM software, data analysis was performed using one-way ANOVA, and group comparisons were performed using Tukey's method.

1.5 Experimental results

This stage was a combination study of LF0376 and zebutinib administered for a total of 27 days.

As shown in fig. 2, observing the overall tumor inhibition curve, the LF0376 single drug group, the zebutinib group, and the LF0376 combined zebutinib group can inhibit tumor growth compared with the control group, wherein the tumor inhibition effect of the combined group is most remarkable.

As shown in fig. 3, tumors were weighed 27 days after dosing and LF0376 in combination with zebutinib group were found to weigh significantly less than the control group and 2 single drug groups.

As shown in fig. 4, the mice of the control group had significantly increased body weight during the treatment period, which was consistent with the natural laws of tumor enlargement and weight gain. After the administration, the tumor volume of the mice is obviously slowed down by the LF0376 single-drug group, the Zybutinib group and the LF0376 combined Zybutinib group relative to the control group, so that the weight of the mice is reduced, and the weight reduction range value of the mice is not exceeded, which indicates that the combined administration and the single-drug treatment are well tolerated in the mice and the safety is good.

2. LF0376 combined with ibutinib (Ibrutinib, IBR)

2.1 Compound formulation

LF0376 100mg/kg was formulated with 5% DMSO+95% (10% HP-beta-CD). The specific configuration method comprises the following steps: 210mg of LF0376 were weighed and 1.05ml of DMSO was added and vortexed to give a clear yellow solution. A further 19.95ml (10% HP-B-CD) of vortex was added and the suspension was adjusted to pH=3 to give a clear solution. After preparation, the mixture was dispensed into 5ml EP tubes, each of which was stored at 3.3mL and 4 ℃. It is formulated every three days.

Ibrutinib was prepared with 10% dmso+40% peg300+5% tween 80+45% saline. The specific configuration method comprises the following steps: 240mg Ibrutinib ml DMSO is added to prepare mother liquor, and then each tube is divided into 0.4ml and filled into 5ml EP tubes, 1.6ml PEG300,0.2ml Tween 80 and 1.8ml physiological saline are sequentially added when the EP tubes are used, and the EP tubes are preserved at 4 ℃. It is administered once every three days.

2.2PDX tumor transplantation model construction

Axillary inoculation of 40 female NOG mice contained patient PDX. NOG mice were purchased from beijing velariwa laboratory animal technologies limited. The tumor is 6-7 weeks old. Feeding management conditions: raising according to SPF-level animal raising condition, and sterilizing squirrel cage, padding, feed and drinking water at high temperature and high pressure. The squirrel cage is placed in IVC, each cage (3-5) animals. The temperature in the animal room is 21-25 ℃ and the relative humidity is 40-70%. PDX is from diffuse large B-cell lymphoma patients and belongs to the non-germinal center (non-GCB) subtype. Immunohistochemical results for PDX model: CD10-, MUM-1 (100% +), bcl6 (80% +), CD20+, bcl2+, CD3-, ki67 (90% +).

On day 24 post inoculation, the tumors were regrouped by tumor volume, 8 per group, 4 total groups. The average tumor volume of each group reaches 108.72 +/-0.33 mm ³, which is day 0. The remaining mice were sacrificed at the end of the experiment or after tumor burden reached 1500mm ³. Mice were dosed in the doses set for each group. Tumor volume was measured once every 3 days and body weight was measured. Each group was given a different dose and drug, see table 4 and fig. 5 for details.

Table 4 experimental dosing and grouping

Note that: vehicle control group was given 5% DMSO+95% (10% HP-beta-CD). Vehicle and LF0376 were dosed twice daily beginning at Di 0 (Di: days of injection) with 8 hours between dosing intervals. Ibrutinib is administered by intragastric administration once daily.

2.3 Index Observation

The same as 1.3.

2.4 Data analysis

Same as 1.4.

2.5 Experimental results

This phase was a combination study of LF0376 and ibutenib, administered for a total of 27 days.

As shown in fig. 6, the overall tumor inhibition curves can be observed, and the LF0376 single drug group, the ibutenib group and the LF0376 combined ibutenib group can inhibit tumor growth compared with the control group, wherein the combined group has the most remarkable tumor inhibition effect.

As shown in fig. 7, tumors were weighed 27 days after dosing and LF0376 combined with ibutenib group tumors were found to weigh significantly less than the control and 2 single groups.

As shown in fig. 8, the mice of the control group had significantly increased body weight during the treatment period, which was consistent with the natural laws of tumor enlargement and weight gain. After the administration, the tumor volume of the mice is obviously slowed down by the LF0376 single-drug group, the ibutinib group and the LF0376 combined ibutinib group relative to the control group, so that the weight of the mice is reduced, and the weight reduction range value of the mice is not exceeded, which indicates that the combined administration and the single-drug treatment are well tolerated in the mice and the safety is good.

EXAMPLE 2 evaluation of in vivo efficacy of test drug on model LD1-0026-361717 of human DLBCL xenograft tumor

This example is mainly to test the growth inhibition or complete cure of the tested drug LF0376 alone, ibrutinib alone, zanubrutinib alone, and LF0376 in combination with Ibrutinib or Zanubrutinib on a model LD1-0026-361717 of human DLBCL in vivo engraftment.

1. Compound formulation

See table 5 for details:

Table 5 test drug formulation

2. In vivo tumor implantation model

LD1-0026-361717 humanized DLBCL tumor tissue is transferred to Fp3+2 to substitute for the drug effect experiment.

The basic information of the LD1-0026-361717PDX model is shown in Table 6.

TABLE 6 model basic information

95 NU/NU mice, female, weighing 18-21 g, purchased from Beijing Vitrenlhua laboratory animal technologies Co., ltd (SCXK (Beijing) 2021-0006), eligibility number: 110011231108918138. experimental animal use license number: SYXK (shan) 2023-008. Feeding environment: SPF stage. Animals were acclimatized for 3 days prior to starting the experiment.

Tumor pieces of the human DLBCL in vivo engrafting tumor of Fp3+1 generation LD1-0026-361717 were cut into tumor tissue of about 3mm by 3mm (about 45 to 60 mg) and inoculated subcutaneously in NU/NU mice. Post-inoculation mice were observed and tumor growth monitored, and group dosing was performed at day 28 of inoculation at an average tumor volume of 137.11 ±6.36mm ³ for tumor-bearing mice, the day of group dosing being defined as day 0. Specific grouping information and dosing schedules are detailed in table 7.

Table 7 administration and treatment

Note that: dosing volume: adjusting the administration volume (0.2 mL/20 g) according to the weight of the tumor-bearing mice; PO: gastric lavage administration; QD: dosing once per day; BID: dosing was 2 times per day, at intervals of about 8 hours.

3. Index observation

3.1 Tumor volume

The tumor volume was calculated using a vernier caliper twice weekly, with the formula v=0.5a×b ², a, b representing the long and wide diameters of the tumor, respectively.

3.2 Relative tumor proliferation Rate T/C (%)

The calculation formula is as follows: T/C% = T _RTV/C_RTV×100％(T_RTV: treatment group RTV; c _RTV: vehicle group RTV). The relative tumor volume (relative tumor volume, RTV) was calculated from the results of the tumor measurements, with the calculation formula rtv=v _t/V₀, where V ₀ is the average tumor volume measured at the time of group administration (i.e. d ₀), V _t is the average tumor volume at a certain time of measurement, and T _RTV and C _RTV are taken on the same day.

3.3 Tumor growth inhibition TGI (%)

TGI (%) = [1- (T _i-T₀)/(V_i-V₀)]×100％,T_i) is the average tumor volume after the start of the administration of the compound group, T ₀ is the average tumor volume when the compound group is first administered, V ₀ is the average tumor volume when the Vehicle group is first administered, V _i is the average tumor volume after the Vehicle group is started.

3.4 Body weight

Measured daily. The relative change ratio of the body weight of the mice after administration was also calculated: RCBW (%) = (BW _i–BW₀)/BW₀×100,BW_i is the body weight after the start of administration, 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 experimental group.

5. Experimental results

As shown in fig. 9, 10, 11 and table 8, on day 28, the average tumor volume of the vecle group was 1470.55 ± 411.29mm ³, the average tumor volumes of the LF0376 single drug group, ibrutinib single drug group, zanubrutinib single drug group, the LF0376+ Ibrutinib group and the LF0376+ Zanubrutinib group were 1219.51±254.70mm³、906.95±185.57mm³、972.72±342.44mm³、566.26±159.12mm³ and 752.64 ± 212.66mm ³, respectively; TGI (%) was 18.60%, 42.24%, 37.33%, 67.86% and 53.86%, respectively; T/C (%) was 84.86%, 61.94%, 66.29%, 38.45% and 51.21%, respectively; both the LF0376+ Ibrutinib group and the LF0376+ Zanubrutinib group showed significant tumor growth inhibition effect on the human DLBCL PDX model LD1-0026-361717 compared with the Vehicle group. The LF0376+ Ibrutinib group and the LF0376+ Zanubrutinib group respectively show stronger tumor growth inhibition effect compared with the LF0376 single drug group and the Ibrutinib/Zanubrutinib single drug group, which indicates that the LF0376 can enhance the antitumor effect of Ibrutinib or Zanubrutinib.

TABLE 8 evaluation and summary of anti-tumor efficacy of test drugs on LD1-0026-361717 human DLBCL subcutaneous xenograft tumor model

As shown in fig. 12, 13, 14 and table 8, on day 28, the average tumor weights of the Vehicle group, LF0376 single drug group, ibrutinib single drug group, zanubrutinib single drug group, LF0376+ Ibrutinib group, LF0376+ Zanubrutinib group were 1.239±0.375g, 1.024±0.218g, 0.739±0.162g, 0.785±0.297g, 0.475±0.134g and 0.596±0.184g, respectively, and the tumor weight results were substantially consistent with the tumor volume results.

As shown in fig. 15, 16 and table 9, day 28, vehicle group, LF0376 single drug group, ibrutinib single drug group, zanubrutinib single drug group, LF0376+ Ibrutinib group and LF0376+ Zanubrutinib group RCBW (%) 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 human drug action on LD1-0026-361717 DLBCL subcutaneous xenograft tumor animal RCBW on model (%)

6. Conclusion of the experiment

LF0376 single drug, ibrutinib single drug and Zanubrutinib single drug do not show significant anti-tumor effect on the model at the current dose level; the combination of LF0376, ibrutinib and LF0376, zanubrutinib showed significant anti-tumor effects for this model, suggesting that LF0376 could significantly enhance the anti-tumor effects of Ibrutinib and Zanubrutinib. During the treatment period of the tested medicines, the weight of each group of animals does not drop obviously, which indicates that the safety of the tested medicines is good.

EXAMPLE 3 evaluation of the efficacy of the test drug on Balb/c mouse A20 tumor model

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

1. Compound formulation

See Table 10 for details

Table 10 test drug formulation

Note that: * The administration concentration is calculated by the active ingredient.

2. Animal model

Animal information is shown in table 11:

table 11 table of experimental animal information

Animals were kept isolated and acclimatized prior to experimental treatment.

A20 cells were expanded by in vitro culture, and cells in logarithmic growth phase were harvested and resuspended in PBS at a cell suspension concentration of 5X 10 ⁶/mL.

Cell suspension was injected subcutaneously into the right side of Balb/c mice with a 1mL syringe, 100 μl per animal, number of vaccinated cells: 5X 10 ⁵/min.

When the average tumor volume is 60.75+/-0.48 mm ³, eliminating animals with oversized, undersized or irregular tumor shape, and dividing the animals into 6 groups by adopting a random block method, wherein each group comprises 6 animals; the day of the grouping is defined as PG-D0.

The administration was started according to a split schedule, each group was administered by gavage, each group was administered at a volume of 5mL/kg, and the frequency of administration was once or twice daily. During the experiment, animal weights were weighed and tumor volumes were measured three times per week.

The end point of the experiment was PG-D13, all mice were euthanized, tumor tissue was peeled off, weighed and photographed. Tumor volume inhibition (TGI) and animal weight change (BWC%) were calculated from the tumor volume and body weight measured by PG-D12, respectively.

The dosing regimen for mice is as shown in table 12:

Table 12 table of dosing schedules for mice of each group

Note that: g, group; gastric lavage administration;

1. The day of grouping was defined as PG-D0 days, and administration was started at PG-D0;

2. dosing volume: adjusting according to the weight of the mice;

3. mice were treated according to customer demand after the experiment was completed.

Grouping and identifying animals:

When the average tumor volume reached approximately 60.75±0.48mm ³, the mice were randomly divided into 6 groups of 6 according to tumor volume. The detailed information is shown in table 13 below: feeder cages were specifically marked with cage cards, including animal number, sex, strain, date received, treatment, study number, and group number.

TABLE 13 grouping information table for experimental animals

3. Index observation

3.1 Tumor Volume (TV)

TV＝1/2×a×b²

Wherein: a represents the tumor long diameter; b represents the tumor minor diameter.

3.2 Tumor volume inhibition (TGI)

Tgi= [1- (TV _{D t} treatment group-TV _{D 0} treatment group)/(TV _{D t} control group-TV _{D 0} control group) ] ×100%

TV _{D t} experimental group: tumor volume in treatment group on day t after dosing

TV _{D 0} experimental group: tumor volume in treatment groups at time of group administration

TV _{D t} control group: tumor volume of control group on day t after administration

TV _{D 0} control group: tumor volume of control group at the time of group administration.

3.3 Animal weight Change Rate (BWC%)

Body weight change rate= (BW _{D t}-BW_{D 0})/BW_{D 0} ×100%

BW _{D t}: animal body weight t days after dosing

BW _{D 0}: animals body weight at the time of group administration.

3.4 Tumor weight Inhibition Rate (IR)

IR＝(W_C-W_T)/W_C×100％

Wherein W _C represents the tumor weight of the control group; w _T represents the treatment group tumor weight.

4. Data analysis

5.1 Tumor volume in mice

As shown in fig. 17, 18, table 14 and table 15, the average tumor volumes of the control group were 687.68 ± 46.59mm ³, the LF0376 single drug group, the Ibrutinib single drug group, the Zanubrutinib single drug group, the LF0376+ Ibrutinib group and the LF0376+ Zanubrutinib group were 551.12 ±40.66mm ³、640.57±48.47mm³、651.25±39.04mm³、497.63±57.96mm³ and 454.45 ±46.55mm ³, respectively, and the TGI was 21.74%, 7.47%, 5.79%, 30.30% and 37.12%, respectively. Both the LF0376+ Ibrutinib group and the LF0376+ Zanubrutinib group showed a significant effect of inhibiting tumor growth compared to the control group. The LF0376+ Ibrutinib group and the LF0376+ Zanubrutinib group each exhibited more pronounced tumor suppression than the LF0376 and Ibrutinib/Zanubrutinib single groups, respectively.

Table 14 average tumor volume (mm ³) during treatment for each group of mice

Note that: data are expressed in mean±sem.

TABLE 15PG-D12 experiments TGI (%)

5.2 Tumor weight of mice

As shown in FIG. 19 and Table 16, the average tumor weights of PG-D13, control group, LF0376, ibrutinib, zanubrutinib, LF0376+ Ibrutinib and LF0376+ Zanubrutinib were 1.8387.+ -. 0.2200g, each 1.1853.+ -. 0.1562g, 1.5809.+ -. 0.1216g, 1.4855.+ -. 0.1700g, 1.3331.+ -. 0.1810g and 1.0855.+ -. 0.1081g, each 35.54%, 14.02%, 19.21%, 27.50% and 40.96% respectively. Tumor weight results were substantially consistent with tumor volume results.

Table 16 average tumor weights (g) of tumors in mice of each group of PG-D13

Note that: n/n represents: actual number of samples/original number of samples.

5.3 Mouse body weight

As shown in FIG. 20 and Table 17, PG-D12, group 1, group 2, group 3, group 4, group 5 and Group6 mice had average weights of 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 FIG. 21 and Table 18, PG-D12, group 1, group 2, group 3, group 4, group 5 and Group6 mice all had average body weights increased at rates 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 weight change rate of the mice in the experimental group during the treatment period is between-10.8 percent and 21.6 percent. No drug suspension caused by obvious weight reduction or poor state of the mice.

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

Note that: data are expressed in mean±sem.

TABLE 18 rate of body weight change (%)

Note that: the rate of change of body weight was calculated from the body weight of PG-D0. Data are expressed in mean±sem.

6. Conclusion of the experiment

The safety of the tested medicine during the treatment period is good. The single LF0376 has a certain tumor inhibiting effect on A20, and the combined tumor inhibiting effect of the two LF0376+ Ibrutinib and the LF0376+ Zanubrutinib is more obvious, which suggests that the LF0376 can obviously enhance the anti-tumor effect of Ibrutinib and Zanubrutinib.

The above embodiments are merely illustrative of the principles of the present application and its effectiveness, and are not intended to limit the application. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the application. Accordingly, it is intended that all equivalent modifications and variations of the application be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

Claims (10)

1. Use of CCR2/5 dual receptor antagonists in the preparation of drugs for treating or preventing B-cell lymphoma. 2. 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. 3. 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. 4. The pharmaceutical composition according to claim 3, characterized in that 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-6 A combination of one or more of 4264681, 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. 5. The pharmaceutical composition according to claim 4, characterized in that the Bruton's tyrosine kinase inhibitor is selected from zanubrutinib or ibrutinib. 6. 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 0.5-10:1. 7. 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 0.8-40:1. 8. 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. 9. The use according to claim 8, characterized in that the B cell lymphoma is diffuse large B cell lymphoma. 10. A drug for treating cancer, comprising the CCR2/5 dual receptor antagonist for use as claimed in any one of claims 1 to 2, or the pharmaceutical composition as claimed in any one of claims 3 to 7, and pharmaceutically acceptable excipients and/or carriers.