TW202131919A - United medicine composition for resisting double-hit lymphomas and application of united medicine composition for resisting double-hit lymphomas - Google Patents

Abstract

The invention relates to a united medicine composition for resisting double-hit lymphomas and application of the united medicine composition for resisting double-hit lymphomas. The united medicine composition comprises a medicine Venetoclax and a medicine Chiauranib. According to the united medicine composition disclosed by the invention, the medicine Venetoclax and the medicine Chiauranib are creatively united to be used as the united medicine composition for resisting double-hit lymphomas; the inventor finds that the united medicine composition for resisting double-hit lymphomas has significant killing effects on varied DHL cell strains, and presents concentration dependence and time dependence; the research result of in vitro tumor formation experiment also proves that the united medicine composition for resisting double-hit lymphomas can restrain the growth of DHL cells in vivo, alleviates tumor load and immersion degree, and does not have obvious toxic and side effects; and the united medicine composition for resisting double-hit lymphomas has higher activity for resisting double-hit lymphomas than single Venetoclax or single Chiauranib, and can more effectively restrain the growth of in vivo tumors, and a new strategy and thought are provided for the treatment of the resisting double-hit lymphomas.

Description

Combined medicine composition for resisting double-hit lymphoma and application thereof

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

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin 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) is 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 morphological, immunophenotypic and other aspects of the characteristics of the B cell 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 combined with 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.

The invention provides a combined drug composition for resisting double-hit lymphoma and its application.

In the first aspect, the present invention provides a combined drug composition for anti-double-hit lymphoma, and the combined drug 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).

The present invention 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 in vivo tumor formation experiments 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 the present invention, the dosage form of the combination 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.

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

The combination pharmaceutical composition of the present invention can be administered alone or in combination with adjuvants to make an appropriate dosage form for administration. The adjuvants include diluents, binders, wetting agents, disintegrants, emulsifiers, and cosolvents. Any one or a combination of at least two of, solubilizer, osmotic pressure regulator, surfactant, pH regulator, antioxidant, bacteriostatic agent or buffer. 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 can 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 can be simultaneous 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 invention, 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.

Ideally, the combination drug composition is a combination drug composition loaded on a pharmaceutical carrier.

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

In the second aspect, the present invention provides an application of the above-mentioned combination drug composition in the preparation of anti-double-strike lymphoma drugs.

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

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

The combination drug composition involved in the present invention induces cell apoptosis by initiating the mitochondrial-mediated intrinsic pathway, and the mitochondrial membrane potential after treatment with the combination drug composition is significantly reduced, and it also affects Bcl-2, Bcl-xL, and BAX. , PUMA and PARP expression levels.

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

The present invention finds that the anti-double-hit lymphoma of the combined drug composition may be related to the inhibition of the PI3K-AKT-mTOR pathway or the DNA damage repair pathway.

In the sixth aspect, the present invention provides a novel anti-double-hit lymphoma combination therapy, which is 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, the present invention has the following effects:

The present invention 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 in vivo tumor formation experiments 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.

[Figure 1] The result of CCK8 method for detecting the inhibitory effect of drugs on MCA cell proliferation for 24 hours.

[Figure 2] CCK8 method to detect the effect of drugs on the proliferation of TMD8 cells for 24 hours.

[Figure 3] CCK8 method detects the 48-hour inhibitory effect of the drug on the proliferation of MCA cells.

[Figure 4] CCK8 method detects the 48-hour inhibitory effect of the drug on the proliferation of TMD8 cells.

[Figure 5] The Annexin V/PI kit detects the induced apoptosis of MCA cells after 24 hours of drug action.

[Figure 6] The results of Annexin V/PI kit detection of TMD8 cell apoptosis after 24 hours of drug action.

[Figure 7] The Annexin V/PI kit detects the apoptosis of MCA cells after 48 hours of drug action.

[Figure 8] The Annexin V/PI kit detects the apoptosis of TMD8 cells after 48 hours of drug action.

[Fig. 9] A diagram of each group of mice and their removed tumor bodies in Example 3.

[Figure 10] is a graph of changes in tumors in each group of mice (a is a graph of changes in tumor volume, and b is a graph of changes in tumor weight).

[Figure 11] A graph of changes in body weight of mice in each group.

[Figure 12] The results of HE staining of tumor tissues in each group of mice.

[Figure 13] The results of 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] Results of 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] The statistical results of the mitochondrial membrane potential of each group of drugs acting on MCA cells for 24 hours.

[Figure 16] Results of 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] Results of 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] The results of the effects of each group of drugs on the expression levels of γH2A.X and Rad51 proteins after 24 hours of action on MCA cells.

[Figure 19] The results of 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.

The technical means of the present invention will be further explained below through specific implementations. Those with ordinary knowledge in the technical field should understand that the embodiments are only to help understand the present invention and should not be regarded as specific limitations to the present invention.

[Examples]

Example 1

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

Inoculate 2×10 ⁴ logarithmic growth phase DHL cell lines TMD8 and MCA in 96-well plates, and set up control group (DMSO), different concentrations of Venetoclax group (nm level), different concentrations of Chiauranib group (μm level), Venetoclax combined After 24 hours and 48 hours of the Chiauranib group, the CCK8 kit was used to detect the proliferation of DHL cells in the different experimental groups, and the 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. 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 24 hours), Figure 2 (TMD8 24 hours), Figure 3 (MCA 48 hours) and Figure 4 (TMD8 48 hours). It can be seen from the figures that: the combination drug composition involved in the present invention It can significantly inhibit the proliferation of DHL cells in a time and concentration-dependent manner.

The combination index value (CI) of Chiauranib and Venetoclax in the combination drug composition involved in the present invention 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 48 hours of drug treatment.

Table 1

Table 2

Example 2

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

Take 2×10 ⁵ logarithmic growth phase DHL cell lines TMD8 and MCA inoculated into 24-well plates, and set up control group (DMSO), different concentrations of Venetoclax group (nm level), different concentrations of Chiauranib group (μm level), Venetoclax combined After 24 hours and 48 hours of treatment in Chiauranib group, Annexin V/PI kit was used to detect the apoptosis of DHL cells. The results are shown in Figure 5 (MCA 24 hours), Figure 6 (TMD8 24 hours), Figure 7 (MCA 48 hours) and Figure 8 (TMD8 48 hours). The combined drug composition 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 combination drug 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

The control group (reagent is normal saline), the Venetoclax group (20μg/g/day), the Chiauranib group (40μg/g/day), the Venetoclax combined with Chiauranib group were set up respectively, and the drugs were administered daily for two weeks, with monitoring every two days. The weight of the mouse and the size of the tumor.

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 combination drug composition involved in the present invention 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 change in tumor volume, b is the change in tumor weight). It can be seen from the figure that the combination drug composition of the present invention can significantly inhibit the growth of DHL cells. Growth in the body.

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

3) The results of HE staining of mouse tumors are shown in Figure 12 (200×). From the figure, it can be seen that the combined drug composition of the present invention can reduce tumor burden and degree of invasion.

Example 4

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

1) Take MCA and LY19 cells in logarithmic growth phase and set up control group (reagent is DMSO), Venetoclax group (20μM), Chiauranib group (10μM), Venetoclax (20μM) combined with Chiauranib (10μM) 4 groups, respectively, 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 signal pathway. The results are shown in Figure 13 (MCA) and Figure 14 (LY19). It can be seen that the combined drug composition of the present invention 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 the control group (the reagent is DMSO), the Venetoclax group (20μM), the Chiauranib group (10μM), and the 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 invention, and apoptosis is induced by starting the mitochondrial-mediated intrinsic pathway.

3) Take MCA and LY19 cells in logarithmic growth phase, set up control group (reagent is DMSO), Venetoclax group (20μM), Chiauranib group (10μM), Venetoclax (20μM) combined with Chiauranib (10μM) 4 groups, respectively, after 24 hours of 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 drug composition of the present invention acts on LY19 cells and MCA cells for 24 hours to induce cell apoptosis by inducing DNA damage.

The inventor declares that the present invention uses the above-mentioned embodiments to illustrate a combination drug composition of the present invention for combating double-strike lymphoma and its application, but the present invention is not limited to the above-mentioned embodiments, which does not mean that the present invention must rely on The above-mentioned embodiments can be implemented. Those with ordinary knowledge in the technical field should understand that any improvement of the present invention, the equal replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., fall under the protection of the present invention. Within the scope and public scope.

The ideal embodiments of the present invention are described in detail above, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, many simple modifications can be made to the technical means of the present invention, all of which are simple modifications. It belongs to the protection scope of the present invention.

In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention makes various possible combinations. The method will not be explained separately.

Claims (10)

A combined drug composition for anti-double-hit lymphoma, which is characterized in that it includes the drug Venetoclax and the drug Chiauranib. The combination pharmaceutical composition according to 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; ideally, the two separate preparations Simultaneous application; alternatively, the two separate formulations are applied sequentially. The combination drug composition according to any one of claims 1 to 5, wherein the combination drug composition is a combination drug composition loaded on a pharmaceutical carrier; Ideally, the pharmaceutical carrier includes liposomes, micelles, dendrimers, microspheres or microcapsules. The application of the combined drug composition according to any one of claims 1 to 6 in the preparation of a drug for anti-double-strike lymphoma. Application of the combined pharmaceutical composition according to any one of claims 1 to 6 in the preparation of a double-strike lymphoma cell proliferation inhibitor. Application of the combined pharmaceutical composition according to any one of claims 1 to 6 in the preparation of a double-strike lymphoma cell apoptosis inducer. The combination pharmaceutical composition according to any one of claims 1 to 6 is used in the preparation of a double-hit lymphoma cell PI3K-AKT-mTOR signaling pathway or DNA damage repair pathway inhibitor Applications.

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