阅读说明：本技术 一种治疗双打击淋巴瘤的药物及其应用 (Medicine for treating double-attack lymphoma and application thereof ) 是由 徐兵 陈钦伟 王善春 邓漫漫 李志峰 查洁 于 2021-03-03 设计创作，主要内容包括：本发明涉及一种治疗双打击淋巴瘤的药物及其应用,所述治疗双打击淋巴瘤的药物包括安罗替尼。本发明创造性地发现安罗替尼可以作为治疗双打击淋巴瘤的药物进行使用,其对多株双打击淋巴瘤细胞的增殖具有抑制作用,能够诱导多株双打击淋巴瘤细胞凋亡并呈浓度及时间依赖性。同时还能下调VEGFR2、PDGFRb、FGFR1蛋白的磷酸化水平,降低c-MYC、BCL-2蛋白的表达水平,并抑制其主要功能通路PI3K/Akt的激活。本发明为研究双打击淋巴瘤的治疗策略提供了理论依据,为制备新的治疗双打击淋巴瘤的药物提供了一个嵌入点。(The invention relates to a medicine for treating double-stroke lymphoma and application thereof. The invention creatively discovers that the nilotinib can be used as a medicine for treating the double-hit lymphoma, has an inhibiting effect on the proliferation of multiple double-hit lymphoma cells, can induce the apoptosis of the multiple double-hit lymphoma cells, and has concentration and time dependence. Meanwhile, the phosphorylation levels of VEGFR2, PDGFRb and FGFR1 proteins can be reduced, the expression levels of c-MYC and BCL-2 proteins are reduced, and the activation of a main functional pathway PI3K/Akt is inhibited. The invention provides a theoretical basis for researching the treatment strategy of the double-hit lymphoma and provides an insertion point for preparing a novel medicine for treating the double-hit lymphoma.)

1. A medicament for treating double-stroke lymphoma, wherein the medicament for treating double-stroke lymphoma comprises nilotinib.

2. The medicament of claim 1, wherein the medicament inhibits proliferation of double-hit lymphoma cells;

preferably, the drug induces apoptosis of double-hit lymphoma cells.

3. The medicament of claim 1 or 2, wherein the medicament down-regulates the phosphorylation levels of VEGFR2, PDGFRb, FGFR1 proteins;

preferably, the medicament reduces the expression level of c-MYC, BCL-2 protein;

preferably, the drug inhibits the activation of the PI3K/Akt pathway.

4. Application of nilotinib in preparation of medicine for treating double-stroke lymphoma.

5. Application of nilotinib in preparation of drugs for inhibiting proliferation of double-hit lymphoma cells.

6. Application of nilotinib in preparation of drugs for inducing apoptosis of double-hit lymphoma cells.

7. Application of nilotinib in preparation of drugs for inhibiting phosphorylation of VEGFR2, PDGFRb and FGFR1 proteins.

8. Application of nilotinib in preparation of c-MYC and BCL-2 protein inhibitors.

9. Application of nilotinib in preparation of PI3K/Akt pathway inhibitors.

10. The use of any one of claims 4 to 9, wherein the medicament is in a dosage form selected from the group consisting of a suspension, granules, a capsule, a powder, a tablet, an emulsion, a solution, a drop pill, an injection, a suppository, an enema, an aerosol, a patch or a drop;

preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials;

preferably, the auxiliary materials comprise any one or a combination of at least two of a carrier, a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a coloring agent, a pH regulator, an antioxidant, a bacteriostatic agent or a buffering agent.

Technical Field

The invention belongs to the technical field of biological medicines, relates to a new application of an anluotinib medicine, and particularly relates to a medicine for treating double-stroke lymphoma and application thereof.

Background

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) are a group of high-grade B-cell lymphomas with concurrent chromosomal translocations of MYC and BCL2 or BCL6, with BCL2 and MYC gene translocations being the most common, accounting for about 62% of all DHLs. The World Health Organization (WHO) classification in 2008 classified DHL as a B-cell lymphoma (BCLU) that could not be classified with morphological, immunophenotypic, etc. features intermediate between diffuse large B-cell lymphoma (BLBCL) and Burkitt Lymphoma (BL). Although the incidence rate of DHL is low and only accounts for about 2 percent of B cell lymphoma, the clinical behavior of DHL is highly invasive and the chemotherapy effect is poor, so that the enhancement of the understanding of DHL plays an important role in the diagnosis and treatment of lymphoma. DHL is not sensitive to traditional chemotherapy, either in a booster regimen or a regimen containing rituximab, with an unsatisfactory effect, with a median overall survival of 0.2-1.5 years. The incidence of diseases is low, large-scale clinical research at home and abroad is lacked, and at present, no standard treatment scheme exists, and the treatment scheme comprises CHOP (cyclophosphamide, adriamycin, vincristine and prednisone), R-CHOP (rituximab, cyclophosphamide, vincristine, doxorubicin and prednisone), R-Hyper CVAD (rituximab, cyclophosphamide, vincristine, doxorubicin and dexamethasone), high-dose chemotherapy combined with hematopoietic stem cell transplantation, palliative therapy and the like, and what chemotherapy scheme is better is still controversial.

Current clinical treatment regimens do not have much effect on DHL. Therefore, it would be of great interest to develop a strategy that could effectively treat DHL.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a new application of an anluotinib medicament, and particularly provides a medicament for treating double-hit lymphoma and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a medicament for treating double stroke lymphoma, wherein the medicament for treating double stroke lymphoma comprises nilotinib.

Anrotinib (Anlotinib) is an anti-tumor angiogenesis drug applied to solid tumor treatment, the results of domestic II and III phase clinical trials show that the Anrotinib has good clinical effectiveness on various solid tumors including NSCLC, renal clear cell carcinoma, colon cancer and the like, and no relevant report on Anrotinib research on lymphoma, especially double-hit lymphoma is found. It is well known to those skilled in the art that lymphoma is a type of tumor distinct from solid tumors, and is a group of highly heterogeneous malignant tumors, up to tens of pathological subtypes, originating in lymph nodes affected by the lymphohematopoietic system, bone marrow, liver, spleen and systemic tissues and organs. Double-hit lymphoma (DHL) is more specific, and is a type of high-grade B-cell lymphoma accompanied by MYC and BCL2 or BCL6 rearrangement, which is detected by FISH technology, and the pathology of which is intermediate between activated B-cell large-cell lymphoma and germinal center large-cell lymphoma. DHL is highly invasive, often manifested as high Ki-67 index, central nervous system and extranodal invasion, insensitive to conventional chemotherapy regimens, patients with median OS generally no more than 1.5 years, short remission, easy recurrence, median OS after recurrence of only 8.6 months, very poor prognosis. Even if a strong chemotherapy or autologous hematopoietic stem cell transplantation scheme is applied, the prognosis of a patient cannot be obviously improved, and the method is a difficult point in the field of lymphoma treatment. Theoretically, targeted drugs such as Bcl-2 inhibitors and BET inhibitors for negatively regulating c-MYC transcription should have good curative effects, but current researches show that the Bcl-2 inhibitors and the BET inhibitors are not ideal in curative effects on DHL, and further researches show that MYC genes are protooncogenes of 'uniduggle' and drugs for directly inhibiting MYC genes, such as BET inhibitors, are easy to cause drug resistance problems.

As is well known to those skilled in the art, angiogenesis plays an essential role in the development of solid tumors, and many leading-edge preclinical studies in recent years have shown that anti-angiogenic drugs have a certain in vitro and in vivo anti-tumor effect on a variety of malignant lymphomas. However, due to the high genetic and clonal heterogeneity of lymphomas, anti-vascular therapy is not effective for all lymphomas. Research progress is currently limited to low invasive lymphomas such as hodgkin lymphoma, and patients with indolent lymphomas still cannot achieve curative effects, can only prolong survival time, and have not ideal effects for high invasive lymphomas and the like, and cannot enter conventional first-line treatment. A study on treatment of B-cell non-hodgkin lymphoma with or without R-CHOP regimen with VEGF mab showed that PFS was not significantly prolonged and toxic side effects were increased in patients with VEGF mab.

The invention creatively discovers that the nilotinib can be used as a medicine for treating double-hit lymphoma, has an inhibiting effect on the proliferation of multiple double-hit lymphoma cells (LR, MCA, Will2, TMD-8, DB), can induce the apoptosis of the multiple double-hit lymphoma cells, and is concentration-dependent and time-dependent. Meanwhile, the phosphorylation levels of VEGFR2, PDGFRb and FGFR1 proteins can be reduced, the expression levels of c-MYC and BCL-2 proteins are reduced, and the activation of a main functional pathway PI3K/Akt is inhibited. The invention provides a theoretical basis for researching the treatment strategy of the double-hit lymphoma and provides an insertion point for preparing a novel medicine for treating the double-hit lymphoma.

In the present invention, the drug inhibits the proliferation of double-hit lymphoma cells.

In the present invention, the drug induces apoptosis of double-hit lymphoma cells.

In the present invention, the drug down-regulates the phosphorylation levels of VEGFR2, PDGFRb, FGFR1 proteins.

In the present invention, the drug reduces the expression level of c-MYC, BCL-2 protein.

In the present invention, the drug inhibits the activation of the PI3K/Akt pathway.

In a second aspect, the invention provides the use of erlotinib in the manufacture of a medicament for the treatment of double-hit lymphoma.

In a third aspect, the invention provides an application of erlotinib in preparing a medicament for inhibiting proliferation of double-hit lymphoma cells.

In a fourth aspect, the invention provides an application of erlotinib in preparing a medicament for inducing apoptosis of double-click lymphoma cells.

In a fifth aspect, the invention provides an application of erlotinib in preparing a medicament for inhibiting VEGFR2, PDGFRb and FGFR1 protein phosphorylation.

In a sixth aspect, the invention provides application of erlotinib in preparing c-MYC and BCL-2 protein inhibitors.

In a seventh aspect, the invention provides an application of erlotinib in preparing a PI3K/Akt pathway inhibitor.

In the invention, the dosage form of the medicament comprises any one of suspension, granules, capsules, powder, tablets, emulsions, solutions, dripping pills, injections, suppositories, enemas, aerosols, patches or drops.

Preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials.

Preferably, the auxiliary materials comprise any one or a combination of at least two of a carrier, a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a coloring agent, a pH regulator, an antioxidant, a bacteriostatic agent or a buffering agent. The combination of at least two of the above-mentioned components, such as the combination of diluent and excipient, the combination of binder and wetting agent, the combination of emulsifier and cosolvent, etc., can be selected in any combination manner, and will not be described in detail herein.

Compared with the prior art, the invention has the following beneficial effects:

the invention creatively discovers that the nilotinib can be used as a medicine for treating double-hit lymphoma, has an inhibiting effect on the proliferation of multiple double-hit lymphoma cells (LR, MCA, Will2, TMD-8, DB), can induce the apoptosis of the multiple double-hit lymphoma cells, and is concentration-dependent and time-dependent. Meanwhile, the phosphorylation levels of VEGFR2, PDGFRb and FGFR1 proteins can be reduced, the expression levels of c-MYC and BCL-2 proteins are reduced, and the activation of a main functional pathway PI3K/Akt is inhibited. The invention provides a theoretical basis for researching the treatment strategy of the double-hit lymphoma and provides an insertion point for preparing a novel medicine for treating the double-hit lymphoma.

Drawings

FIG. 1 is a statistical chart showing the results of detecting the inhibition of the proliferation of a plurality of DHL cell lines (LR, MCA, Will2, TMD-8, DB) by Anlotinib with 48h treatment time by the CCK8 method;

FIG. 2 is a statistical chart showing the results of detecting the inhibition of the proliferation of a plurality of DHL cell lines (LR, MCA, Will2, TMD-8, DB) by Anlotinib with 72h treatment time by the CCK8 method;

FIG. 3 is a classic flow-scattering density plot of Angotinib induced apoptosis of DHL cells;

FIG. 4 is a histogram of the statistics of the apoptosis rates of groups of DHL cells induced by Anlotinib;

FIG. 5 is a WB assay of the protein levels of Anlotinib as measured 48h on DHL cell lines;

FIG. 6 is a WB assay showing the activation of PI3K/AKT pathway after 48h of the action of Anlotinib on DHL cell lines;

FIG. 7 is a graph showing the results of mRNA expression level detection of c-MYC after the DHL cell strain is treated with the allotinib by the qPCR method;

FIG. 8 is a graph showing the results of mRNA expression levels of Mcl-1 after the DHL cell strain is treated with the allotinib by the qPCR method.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The procedures, conditions, reagents, test methods and the like for carrying out the present invention are those generally known in the art and are not specifically limited except for the contents specifically mentioned below. The test methods in each example, in which the specific conditions are not specified, are generally carried out under the conventional conditions or under the conditions recommended by the manufacturer.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, but in the event of conflict, the present specification, including definitions, will control.

The nilotinib used in the examples described below was provided by Ningdanqing pharmaceutical industry group, with a production lot number of 420044-201901, and a specification of 100 mg/count.

DHL cell lines (LR, MCA, Will2, TMD-8, DB) were given by professor Helxu Hua, university of Duke, USA.

Example 1

Evaluation of inhibition of proliferation of DHL cell line by Arotinib

The operation method comprises the following steps: taking DHL cell strain (LR, MCA, wil 2, TMD-8, DB) in logarithmic growth phase at 2 × 10⁴The cells were inoculated in 96-well plates overnight, and after the cells were well grown, RPMI-1640 complete medium containing different concentrations of anitinib (0, 1, 2, 4, 8. mu.M) was added, and the culture was stopped for 48h and 72h, respectively, and a control group was set. Then, the proliferation of the cells was detected by using a CCK8 kit. The results are shown in FIGS. 1 and 2. As can be seen from the figure: the activity of DHL cells decreased with increasing treatment concentration in 48h and 72h of Arotinib treatment, and the half-inhibitory rate of each cell decreased in 72h (3.776. mu.M, 2.965. mu.M, 3.546. mu.M, 1.353. mu.M, 3.663. mu.M) compared to 48h (5.784. mu.M, 8.213. mu.M, 3.305. mu.M, 1.693. mu.M, 7.486. mu.M), suggesting that the antiproliferative effect of Arotinib on DHL cell lines is time-and concentration-dependent.

Example 2

Evaluation of Antrodib induced apoptosis in DHL cell line

The operation method comprises the following steps: taking DHL cell strain (LR, MCA, TMD-8) in logarithmic growth phase at 2 × 10⁵Each well was inoculated in a 24-well plate overnight, and after the cells grew well, RPMI-1640 complete medium containing different concentrations of anitinib (0, 1, 2, 4, 8. mu.M) was added, and the culture was stopped after 48 hours of culture, and a control group was set. Then, the Annexin V/PI kit is used for detecting the apoptosis conditions of cells of different experimental groups. The classical flow scatter density chart is shown in fig. 3, the statistic of the apoptosis rate of each group is shown in fig. 4, and the following can be known from fig. 3 and fig. 4: after the anitinib treatment, the proportion of apoptotic cells (AnnexinV positive) is increased along with the increase of the drug concentration, which indicates that the anitinib can induce the DHL cells to undergo apoptosis.

Example 3

Effect of Arotinib on the phosphorylation levels of VEGFR2, PDGFRb, FGFR1 proteins, and on c-MYC, Bcl-2, Mcl-1, PARP expression

The operation method comprises the following steps: taking DHL cell strain (TMD-8) at logarithmic growth phase at 5X 10⁶Each well was inoculated into a 10cm dish, and RPMI-1640 medium containing different concentrations of apratinib (0, 1, 2. mu.M) was added thereto, and the culture was stopped after 48 hours. Then the protein is extracted and used for western blot to detect the phosphorylation levels of VEGFR2, PDGFRb and FGFR1 proteins and the expression of c-MYC, Bcl-2, Mcl-1 and PARP, and the result is shown in figure 5, and the following results are shown in the figure: after drug treatment, the phosphorylation levels of the DHL cells on the apratinib targets such as VEGFR2, PDGFRb and FGFR1 proteins are all reduced with the increase of the concentration, the apoptosis marker protein PARP is increased with the increase of the drug concentration, and the downstream lysis band is also gradually enhanced, which proves the anti-tumor and apoptosis-inducing effects of the apratinib on the DHL cells. In addition, the survival-dependent proteins c-MYC, Bcl-2, etc. related to the DHL characteristics are also significantly reduced at the protein level.

Example 4

Activation of PI3K/AKT pathway by Arotinib

The operation method comprises the following steps: taking DHL cell strain (TMD-8) at logarithmic growth phase at 5X 10⁶Each well was inoculated into 10cm dishes, and RPMI-1640 complete medium containing different concentrations of anitinib (0, 1, 2. mu.M) was added thereto, and the culture was stopped after 48 hours. Then the protein is extracted and used for detecting the phosphorylation level of the PI3K/AKT pathway by western blot, and the result is shown in figure 6, and the graph shows that: after the treatment of the medicine, the phosphorylation levels of the c-MYC upstream regulation pathway PI3K and AKT protein are both obviously reduced along with the increase of the treatment concentration, and the background protein is not obviously changed. FOXO3a transcription factor downstream of PI3K/AKT and its target gene Bim are not changed significantly, suggesting that the Arotinib can inhibit the activation of the PI3K/AKT pathway of DHL cell strain and is not dependent on FOXO3 a.

Example 5

Effect of Arotinib on mRNA expression levels of c-MYC and Mcl-1

The operation method comprises the following steps: taking DHL cell strain (LR, MCA, TMD-8) at logarithmic growth phase at 5 × 10⁵Separate inoculation of wellsAfter the cells grew well overnight in 6-well plates, complete RPMI-1640 medium containing different concentrations of anitinib (0, 2, 4. mu.M) was added and the culture was stopped after 24h of each culture. The mRNA expression levels of c-MYC and Mcl-1 were then determined using q-PCR. The results are shown in FIGS. 7 and 8, which show that: after 24h of drug treatment, the mRNA level of DHL cells (LR, MCA, TMD-8) in the 4 μ M group decreased to a different extent compared to that in the untreated group, suggesting that the apratinib could down-regulate c-MYC at the transcriptional level. Whereas the mRNA levels of Mcl-1 of the Bcl-2 family are significantly upregulated only in TMD-8 cells, and TMD-8 belongs to a more sensitive cell in DHL and thus may be an increased reactivity.

The applicant states that the invention is illustrated by the above examples to a medicament for treating double-hit lymphoma and the application thereof, but the invention is not limited to the above examples, i.e. it does not mean that the invention must be implemented by means of the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.