WO2021169974A1 - Combined pharmaceutical composition for resisting double-hit lymphomas, and use thereof - Google Patents

Abstract

Disclosed are a combined pharmaceutical composition for resisting double-hit lymphomas, and the use thereof. The combined pharmaceutical composition comprises the drug Venetoclax and the drug Chiauranib. The drug Venetoclax and the drug Chiauranib are creatively combined to be used as the combined pharmaceutical composition for resisting double-hit lymphomas. It has been found that the combined pharmaceutical composition has significant killing effects on various DHL cell strains, and presents concentration dependence and time dependence; the research result of in vivo tumor formation experiments also proves that the the combined pharmaceutical composition can inhibit the growth of DHL cells in vivo, alleviate tumor load and invasion degree, and does not have obvious toxic side effects. The combined pharmaceutical composition has a higher double-hit lymphoma resistance activity than Venetoclax or Chiauranib on its own, and can more effectively inhibit the growth of in vivo tumors, and a new strategy and new thought are provided for the treatment of double-hit lymphomas.

Description

A combined pharmaceutical composition for resisting double-hit lymphoma and its application Technical field

The application belongs to the field of biomedicine, and specifically relates to a combined pharmaceutical composition for anti-double-hit lymphoma and its application.

Background technique

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin’s lymphoma (NHL), accounting for approximately 25% of NHL cases. BCL2, BCL6 and MYC are the most common mutant genes in DLBCL. Double-hit lymphomas (DHL) are a group of high-grade B-cell lymphomas with simultaneous chromosomal translocations of MYC and BCL2 or BCL6. BCL2 and MYC gene translocations are the most common, accounting for 62 of all DHL. %about. In 2008, the World Health Organization (WHO) classified DHL as an unclassified B cell with morphological and immunophenotypic characteristics that are between diffuse large B-cell lymphoma (BLBCL) and Burkitt lymphoma (BL) Lymphoma (BCLU). Although the incidence of DHL is low, accounting for only about 2% of B-cell lymphoma, its clinical behavior is highly aggressive and the effect of chemotherapy is poor. Therefore, strengthening the understanding of DHL plays an important role in the diagnosis and treatment of lymphoma. DHL is not sensitive to traditional chemotherapy. Whether it is an enhanced regimen or a regimen containing rituximab, the effect is unsatisfactory, with a median overall survival of 0.2-1.5 years. The incidence is low, and there is a lack of large-scale clinical studies at home and abroad. There is currently no standard treatment plan. The treatment plan includes CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone), R-CHOP (rituximab, Cyclophosphamide, vincristine, doxorubicin, prednisone), R-Hyper CVAD (rituximab, cyclophosphamide, vincristine, doxorubicin, dexamethasone), high-dose chemotherapy Combining hematopoietic stem cell transplantation, palliative treatment, etc., which chemotherapy regimen is better is still controversial.

The current clinical treatment plan does not have much effect on DHL. Therefore, it is very meaningful to develop a strategy that can effectively treat DHL.

Summary of the invention

This application provides a combined pharmaceutical composition for anti-double-hit lymphoma and its application.

In the first aspect, this application provides a combined pharmaceutical composition for anti-double-hit lymphoma. The combined pharmaceutical composition includes the drug Venetoclax and the drug Chiauranib.

Venetoclax (ABT-199) is a highly effective, selective and orally active Bcl-2 inhibitor that can be combined with obinutuzumab to treat untreated adult patients with chronic lymphocytic leukemia (CLL). Chiauranib (CS2164, Cioroni) is a highly selective inhibitor of AuroraB/VEGFR/PDGFR/c-Kit/CSF1R targets. It can simultaneously inhibit tumor angiogenesis, inhibit tumor cell mitosis, and regulate tumor microenvironment. Play a comprehensive anti-tumor effect, and at the same time have better animal pharmacodynamic activity and good safety than drugs of the same mechanism. It is currently in clinical stage I/II (ovarian cancer).

This application creatively uses the drug Venetoclax and the drug Chiauranib as a combined drug composition for anti-double-strike lymphoma, which has a significant killing effect on a variety of DHL cell lines (such as TMD8, MCA, LY19, etc.), and presents a concentration And time-dependent, the results of the in vivo tumor formation experiment also proved that it can inhibit the growth of DHL cells in the body, reduce the tumor burden and the degree of infiltration, and has no obvious side effects.

In this application, the dosage form of the combined pharmaceutical composition includes any pharmaceutically acceptable dosage form. For example, tablets, powders, suspensions, capsules, injections, sprays, solutions, enemas, emulsions, films, suppositories, patches, nasal drops or pills, etc.

Preferably, the combined pharmaceutical composition further includes any one or a combination of at least two of the pharmaceutically acceptable pharmaceutical excipients.

The combination pharmaceutical composition described in the present application can be administered alone or in combination with adjuvants to form an appropriate dosage form for administration. The adjuvants include diluents, binders, wetting agents, disintegrants, emulsifiers, and adjuvants. Any one or a combination of at least two of solvents, solubilizers, osmotic pressure regulators, surfactants, pH regulators, antioxidants, bacteriostatic agents, or buffers. The at least two combinations, such as the combination of a diluent and a binder, a combination of a wetting agent and a disintegrant, a combination of a solubilizer and an osmotic pressure regulator, etc., other arbitrary combinations will not be repeated here.

In a specific embodiment, the combined pharmaceutical composition is a single compound preparation.

In another specific embodiment, the combined pharmaceutical composition is a combination of two separate formulations, Venetoclax formulation and Chiauranib formulation. In this embodiment, the two separate formulations are administered simultaneously. Alternatively, the two separate formulations are administered sequentially.

The combined pharmaceutical composition may be in the form of a single compound preparation, or a combination of two separate preparations; when it is a combination of two separate preparations, the mode of administration may be simultaneous administration or sequential administration For example, Venetoclax can be administered first and Chiauranib can be administered after a period of time, or Chiauranib can be administered first and Venetoclax can be administered after a period of time, or the two can be administered alternately.

In the present application, the administration route of the combined pharmaceutical composition includes intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, or transdermal administration.

Preferably, the combined pharmaceutical composition is a combined pharmaceutical composition loaded on a pharmaceutical carrier.

Preferably, the pharmaceutical carrier includes liposomes, micelles, dendrimers, microspheres or microcapsules.

In the second aspect, the present application provides an application of the combination pharmaceutical composition as described above in the preparation of an anti-double-strike lymphoma drug.

In the third aspect, this application provides an application of the above-mentioned combination pharmaceutical composition in the preparation of a double-hit lymphoma cell proliferation inhibitor.

In the fourth aspect, the present application provides an application of the combination pharmaceutical composition as described above in the preparation of a double-hit lymphoma cell apoptosis inducer.

The combination pharmaceutical composition involved in this application induces cell apoptosis by activating the mitochondrial-mediated intrinsic pathway, and the mitochondrial membrane potential after treatment with the combination pharmaceutical composition is significantly reduced, and it also affects Bcl-2, Bcl-xL, and BAX. , The expression level of PUMA and PARP.

In a fifth aspect, the present application provides an application of the above-mentioned combination pharmaceutical composition in preparing double-hit lymphoma cells PI3K-AKT-mTOR signaling pathway or DNA damage repair pathway inhibitor.

The application found that the anti-double-hit lymphoma of the combined pharmaceutical composition may be related to the inhibition of the PI3K-AKT-mTOR pathway or the DNA damage repair pathway.

In the sixth aspect, this application provides a novel combination therapy against double-hit lymphoma, the combination therapy being a combination therapy of Venetoclax and Chiauranib. This therapy has stronger anti-double-strike lymphoma activity than single Venetoclax therapy or Chiauranib therapy, and can more effectively inhibit the growth of tumors in vivo, providing new strategies and ideas for the treatment of double-strike lymphoma.

Compared with the prior art, this application has the following beneficial effects:

This application creatively uses the drug Venetoclax and the drug Chiauranib as a combined drug composition for anti-double-strike lymphoma, which has a significant killing effect on a variety of DHL cell lines (such as TMD8, MCA, LY19, etc.), and presents a concentration And time-dependent, the results of the in vivo tumor formation experiment also proved that it can inhibit the growth of DHL cells in the body, reduce the tumor burden and the degree of infiltration, and has no obvious side effects.

Description of the drawings

Figure 1 is the result of CCK8 method detecting the inhibitory effect of drugs on the proliferation of MCA cells for 24 hours;

Figure 2 is the result of CCK8 method detecting the inhibitory effect of drugs on the proliferation of TMD8 cells for 24 hours;

Figure 3 is a graph showing the results of the CCK8 method detecting the inhibitory effect of the drug on the proliferation of MCA cells for 48 hours;

Figure 4 is the result of CCK8 method detecting the inhibitory effect of drugs on the proliferation of TMD8 cells for 48 hours;

Figure 5 is a graph showing the results of MCA cell apoptosis induced by Annexin V/PI kit after 24 hours of drug action;

Figure 6 is a graph showing the results of the Annexin V/PI kit detecting the apoptosis of TMD8 cells after 24 hours of drug action;

Figure 7 is a graph showing the results of the Annexin V/PI kit detecting the apoptosis of MCA cells after 48 hours of drug action;

Fig. 8 is a graph showing the result of apoptosis of TMD8 cells induced by Annexin V/PI kit after 48 hours of drug action;

Figure 9 is a diagram of each group of mice and their removed tumor bodies in Example 3;

Figure 10 is a graph of changes in tumors of each group of mice (a is a graph of changes in tumor volume, b is a graph of changes in tumor weight);

Figure 11 is a graph showing changes in body weight of mice in each group;

Figure 12 is a graph showing the results of HE staining of tumor tissues in each group of mice;

Figure 13 is a graph showing the effect of each group of drugs on the expression level of PI3K-AKT-mTOR signal pathway after 24 hours of action on MCA cells;

Figure 14 is a graph showing the effect of each group of drugs on the expression level of PI3K-AKT-mTOR signal pathway after acting on LY19 cells for 24 hours;

Figure 15 is a graph showing the statistical results of mitochondrial membrane potential of each group of drugs acting on MCA cells for 24 hours;

Figure 16 is a graph showing the effect of each group of drugs on the expression levels of Bcl-2, Bcl-xL, BAX, PUMA and PARP after acting on MCA cells for 24 hours;

Figure 17 is a graph showing the effect of each group of drugs on the expression levels of Bcl-2, Bcl-xL, BAX, PUMA and PARP after acting on LY19 cells for 24 hours;

Figure 18 is a graph showing the effect of each group of drugs on the expression levels of γH2A.X and Rad51 proteins after acting on MCA cells for 24 hours;

Figure 19 is a graph showing the effect of each group of drugs on the expression levels of γH2A.X and Rad51 proteins after acting on LY19 cells for 24 hours.

Detailed ways

The technical solutions of the present application will be further explained through specific implementations below. It should be understood by those skilled in the art that the described embodiments are merely to help understand the application and should not be regarded as specific limitations to the application.

Example 1

Inhibitory effect of combined pharmaceutical composition on the proliferation of DHL cell line

Take 2×10 ⁴ logarithmic growth phase DHL cell lines TMD8 and MCA inoculated into 96-well plates, and set up control group (DMSO), different concentrations of Venetoclax group (nm level), different concentrations of Chiauranib group (μm level), Venetoclax After combined treatment with Chiauranib group for 24h and 48h, CCK8 kit was used to detect the proliferation of DHL cells in different experimental groups, and compusyn software was used to make the combination index (CI) after the two drugs were combined. A CI less than 1 indicates a synergistic effect, and a CI greater than 1 means antagonistic effect, CI equal to 1 means superimposing effect, the smaller the CI value, the stronger the synergistic effect of the two drugs on cytotoxicity. The results are shown in Figure 1 (MCA 24h), Figure 2 (TMD8 24h), Figure 3 (MCA 48h) and Figure 4 (TMD8 48h). It can be seen from the figure that the combination pharmaceutical composition involved in this application has a significant inhibitory effect on DHL The effect of cell proliferation is time- and concentration-dependent.

The combination index value (CI) of Chiauranib and Venetoclax in the combination pharmaceutical composition involved in this application is shown in Table 1 and Table 2. From the data in Table 1 and Table 2, it can be seen that the combination of the two drugs has a certain synergistic effect, especially After 48h of drug treatment.

Table 1

Table 2

Example 2

Apoptosis-inducing effect of combined pharmaceutical composition on DHL cell line

Inoculate 2×10 ⁵ logarithmic growth phase DHL cell lines TMD8 and MCA in 24-well plates, and set the control group (DMSO), different concentrations of Venetoclax group (nm level), different concentrations of Chiauranib group (μm level), Venetoclax After treatment with Chiauranib for 24h and 48h, Annexin V/PI kit was used to detect the apoptosis of DHL cells. The results are shown in Figure 5 (MCA 24h), Figure 6 (TMD8 24h), Figure 7 (MCA 48h) and Figure 8 (TMD8 48h). The histogram in the figure can be seen from the figure: the combination pharmaceutical composition involved in the application It has an obvious effect of inducing DHL cell apoptosis, which is time- and concentration-dependent.

Example 3

It is verified from the animal level that the combined pharmaceutical composition has the effect of killing DHL in the body

1. Experimental method

1) Use DHL cell line to construct nude mouse tumor-bearing model

SPF-grade nude mice were purchased from Shanghai Slack, 4-6 weeks old, half male and half male. All operations on the mice were performed in a sterile laminar flow chamber. Suspend MCA cells in 0.2mL medium containing 0.5% FBS (5×10 ⁶ cells per 0.2mL), and inoculate them under the skin of the right forelimb of mice. When the tumor volume grows to 75-150mm ³ , the body can be started Medication experiment.

2) In vivo experiment

Set up the control group (reagent is normal saline), Venetoclax group (20μg/g/day), Chiauranib group (40μg/g/day), Venetoclax combined with Chiauranib group, administered daily for two weeks, monitoring mice every two days Body weight and tumor size.

3) After the medication, the mice were directly euthanized and the tumors were taken and photographed, as shown in Figure 9 (it can be seen from the figure that the combined pharmaceutical composition involved in this application can significantly inhibit the growth of tumors in the body), and used for calculation Weight and pathological section.

2. Experimental results

1) The changes of mouse tumors are shown in Figure 10 (a is the tumor volume change, b is the tumor weight change). It can be seen from the figure that the combination pharmaceutical composition involved in this application can significantly inhibit DHL cells in Growth in the body.

2) The change of the weight of the mouse is shown in Figure 11, which shows that the combined pharmaceutical composition involved in the present application has no obvious toxic and side effects on the mouse in vivo, and does not change the weight of the mouse.

3) The HE staining results of mouse tumors are shown in Figure 12 (200×). It can be seen from the figure that the combined pharmaceutical composition involved in this application can reduce tumor burden and degree of invasion.

Example 4

Using the WB method to study the mechanism of the combined drug composition killing DHL cells

1) Take the MCA and LY19 cells in the logarithmic growth phase, and set up the control group (reagent is DMSO), Venetoclax group (20μM), Chiauranib group (10μM), Venetoclax (20μM) combined with Chiauranib (10μM) 4 groups, after 24 hours of treatment Harvest the cells, and then extract the protein for western blot to detect the expression level of PI3K-AKT-mTOR signaling pathway. The results are shown in Figure 13 (MCA) and Figure 14 (LY19). It can be seen from the figure that the combined pharmaceutical composition involved in the application inhibits the PI3K-AKT-mTOR signal pathway after acting on LY19 cells and MCA cells for 24 hours to achieve drug killing effect.

2) Take the MCA and LY19 cells in the logarithmic growth phase, and set up the control group (reagent is DMSO), Venetoclax group (20μM), Chiauranib group (10μM), Venetoclax (20μM) combined with Chiauranib (10μM) 4 groups, after 24 hours of treatment The cells were harvested and stained with JC-1 to measure the mitochondrial membrane potential by flow cytometry. The results are shown in Figure 15. Then the protein was extracted and used for western blot to detect the expression level of mitochondrial-mediated intrinsic pathway-related proteins. The results are shown in Figure 16 (MCA) and Figure 17 (LY19). It can be seen from Figures 15-17 that the mitochondrial membrane potential is significantly reduced after treatment with the combined pharmaceutical composition involved in the present application, and apoptosis is induced by activating the intrinsic pathway mediated by mitochondria.

3) Take the MCA and LY19 cells in logarithmic growth phase, set up the control group (reagent is DMSO), Venetoclax group (20μM), Chiauranib group (10μM), Venetoclax (20μM) combined with Chiauranib (10μM) 4 groups, after 24h treatment Harvest the cells, and then extract the protein for western blot to detect the expression level of γH2A.X and Rad51 protein. The results are shown in Figure 18 (MCA) and Figure 19 (LY19). It can be seen from the figure that the combined pharmaceutical composition involved in this application acts on LY19 cells and MCA cells for 24 hours to induce cell apoptosis by inducing DNA damage.

The applicant declares that this application uses the above-mentioned examples to illustrate a combination pharmaceutical composition and its application for anti-double-strike lymphoma of this application, but this application is not limited to the above-mentioned examples, which does not mean that this application must It can be implemented only by relying on the above-mentioned embodiments. Those skilled in the art should understand that any improvement to this application, the equivalent replacement of each raw material of the product of this application, the addition of auxiliary components, the selection of specific methods, etc., fall within the scope of protection and disclosure of this application.

The preferred embodiments of the present application are described in detail above. However, the present application is not limited to the specific details in the foregoing embodiments. Within the scope of the technical concept of the present application, a variety of simple modifications can be made to the technical solution of the present application. These simple modifications All belong to the protection scope of this application.

In addition, it should be noted that the various specific technical features described in the above-mentioned specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this application provides various possible The combination method will not be explained separately.

Claims (12)

A combined pharmaceutical composition for anti-double-hit lymphoma, which includes the medicine Venetoclax and the medicine Chiauranib. The combination pharmaceutical composition of claim 1, wherein the dosage form of the combination pharmaceutical composition includes any pharmaceutically acceptable dosage form. The combination pharmaceutical composition according to claim 1 or 2, wherein the combination pharmaceutical composition further comprises any one or a combination of at least two of pharmaceutically acceptable pharmaceutical excipients. The combined pharmaceutical composition according to any one of claims 1 to 3, wherein the combined pharmaceutical composition is a single compound preparation. The combination pharmaceutical composition according to any one of claims 1 to 3, wherein the combination pharmaceutical composition is a combination of two separate preparations, Venetoclax preparation and Chiauranib preparation. The combination pharmaceutical composition according to claim 5, wherein the two separate formulations are administered simultaneously; or, the two separate formulations are administered sequentially. The combination pharmaceutical composition according to any one of claims 1 to 6, wherein the combination pharmaceutical composition is a combination pharmaceutical composition loaded on a pharmaceutical carrier. The combination pharmaceutical composition according to claim 7, wherein the pharmaceutical carrier comprises liposomes, micelles, dendrimers, microspheres or microcapsules. The use of the combined pharmaceutical composition according to any one of claims 1-8 in the preparation of an anti-double-strike lymphoma drug. The use of the combined pharmaceutical composition according to any one of claims 1-8 in the preparation of a double-hit lymphoma cell proliferation inhibitor. The use of the combined pharmaceutical composition according to any one of claims 1-8 in the preparation of a double-hit lymphoma cell apoptosis inducer. The use of the combined pharmaceutical composition according to any one of claims 1-8 in the preparation of double-hit lymphoma cells PI3K-AKT-mTOR signaling pathway or DNA damage repair pathway inhibitor.

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