CN117338786A - Combined medicine for treating acute T lymphocyte leukemia and combined central nervous system leukemia and application thereof - Google Patents

Abstract

The invention provides a combined medicament for treating acute T lymphocyte leukemia and central nervous system leukemia and application thereof, wherein the combined medicament for treating T-ALL comprises dasatinib and dasafil. The invention creatively combines the drug dasatinib and the drug dasatinib as the drug for treating the T-ALL, plays a remarkable synergistic effect, has the effect of remarkably inhibiting the proliferation of T-ALL cells compared with a single drug, prolongs the survival time of a T-ALL mouse model, and relieves the progress of T-ALL combined central nervous system leukemia. The invention provides a new strategy and thought for treating acute T lymphocyte leukemia and acute T lymphocyte leukemia combined central nervous system leukemia, and has very remarkable significance.

Description

Combined medicine for treating acute T lymphocyte leukemia and combined central nervous system leukemia and application thereof

Technical Field

The invention belongs to the technical field of biological medicine. More particularly relates to a combined drug for treating acute T lymphocyte leukemia and application thereof.

Background

Acute lymphoblastic leukemia (Acute lymphoblastic leukemia, ALL) is a lymphocytic hematological tumor. Acute T-cell lymphoblastic leukemia (T cells acute lymphoblastic leukemia, T-ALL) is a subtype of ALL derived from a T cell line, accounting for 10-15% of pediatric ALL patients and 20-25% of adult ALL patients. The clinical manifestations of T-ALL tend to be more aggressive than other types of ALL and are prone to complications of Central Nervous System Leukemia (CNSL), and combination chemotherapy is a first-line treatment for T-ALL, but its efficacy is not ideal. About 20% of children and 50% of adult T-ALL patients fail to achieve sustained complete remission and die from disease progression. To improve efficacy and prognosis, more two-line treatment protocols, including hematopoietic stem cell transplantation (Hematopoietic stem cell transplantation, HSCT) and CAR-T (Chimeric antigen receptor T cells), have been applied in clinical trials and clinical practice. However, these cell therapy methods are costly, complex to manage throughout, and have no medical accessibility in some developing areas. Thus, the highly invasive and limited clinical treatment of T-ALL, poor prognosis, remains a clinical pain point, and there remains a need to study effective and relatively economical T-ALL therapies, especially in relapsed or refractory T-ALL.

Dasatinib was an FDA approved second generation BCR-ABL tyrosine kinase inhibitor (Tyrosine kinase inhibitor, TKI), originally developed for the treatment of chronic myeloid leukemia (Chronicmyeloid leukemia, CML), and is currently also used for the treatment of philadelphia chromosome positive acute lymphoblastic leukemia (ph+all). Given the targeted inhibition of activated ABL1 proteins by dasatinib, dasatinib was also used to treat T-ALL patients with ABL1 gene amplification and carrying NUP214-ABL1 fusion genes. However, BCR/ABL1 positive TALL is rare, unlike CML, which must be BCR/ABL1 positive. Subsequently, researchers began exploring the use of dasatinib to treat T-ALL patients that did not carry ABL1 gene abnormalities. Studies have shown that dasatinib and protein degradation targeting chimeras (PROTACs) utilizing dasatinib as a ligand have targeted killing effects on T-ALL in computer simulation (in silico), in vitro experiments (in vitro) and in vivo experiments (in vivo). After in vitro cell experiments prove that dasatinib can effectively kill T-ALL cells from patients, the T-ALL patients with partial refractory or recurrent disease adopt dasatinib as a rescue treatment means, and the remission is obtained. This unexpected effect is mainly due to the fact that dasatinib can inhibit LCK, which is a key regulator of T Cell Receptor (TCR) signaling pathway and T cell expansion, one of the most vulnerable targets of T-ALL.

Although some studies have shown that dasatinib has been shown to be effective in some of the TALL patients, nearly half of patient-derived T-ALL cells still exhibit resistance to dasatinib in vitro experiments. Studies have shown that in the relevant experiments with cell lines, while LCK gene expression was significantly inhibited by dasatinib, the activity and proliferation of T-ALL cells was not affected. In addition, whether there is a correlation between the sensitivity of samples derived from T-ALL patients to dasatinib and LCK expression, the results in different studies are not consistent. All of these previous studies indicate that in addition to TCR-LCK signaling, there may be other important kinases or pathways that regulate tumor cell responses to dasatinib.

BCR-ABL fusion group positivity is rare in T-ALL and central nervous system white blood incidence is higher compared to CML, so both pathogenesis and therapeutic administration are different in focus. Dasatinib is one of the standard drugs for treating CML, and the effectiveness and drug resistance mechanism of the dasatinib applied to T-ALL have not been elucidated. Therefore, there is a need to further study the pharmacological action targets of dasatinib and the molecular mechanisms affecting its action to find possible drug combination targets, improving therapeutic response and patient prognosis.

Disclosure of Invention

The invention aims to overcome the defects and the shortcomings of the traditional dasatinib in the aspect of treating acute T-cell lymphoblastic leukemia, and provides a novel scheme for treating acute T-cell lymphoblastic leukemia.

The first object of the invention is to provide the application of dasatinib in preparing a synergist for preventing, improving or treating acute T lymphocyte leukemia.

The second object of the invention is to provide the application of dasatinib and dasatinib in preparing medicaments for preventing, improving or treating acute T lymphocyte leukemia.

A further object of the present invention is to provide a combination for the treatment of acute T-lymphocyte leukemia, characterized by comprising dasatinib and dasafil, and pharmaceutically acceptable pharmaceutical excipients.

The above object of the present invention is achieved by the following technical scheme:

the invention creatively combines the drug dasatinib and the drug dasatinib as the drug for treating the acute T-lymphocyte leukemia, wherein the dasatinib acts on T-ALL cells through an LCK (LCK-mediated apoptosis) channel, but other mechanisms influence the effect of the dasatinib to be exerted, so that the effect of treating the T-ALL is limited; wherein dasatinib has synergistic killing effect with dasatinib by inhibiting MAPK pathway on T-ALL cells, and both dasatinib and dasafinib can pass through blood brain barrier. The study of the invention shows that dasatinib combined with dasatinib has the effect of remarkably inhibiting T-ALL cell proliferation compared with single dasatinib or dasatinib, remarkably prolonging the survival time of a T-ALL mouse model compared with single dasatinib or dasatinib, and relieving the progress of T-ALL combined central nervous system leukemia.

In a first aspect, the invention provides the use of dasatinib for the preparation of a potentiator for the prevention, amelioration or treatment of acute T-lymphocyte leukemia.

In a second aspect, the invention provides the use of dasatinib and dasatinib in combination for the manufacture of a medicament for the prevention, amelioration or treatment of acute T-lymphocyte leukaemia.

In a third aspect, the invention provides the use of dasatinib and dasatinib in combination for the preparation of an acute T-lymphocyte leukemia cell proliferation inhibitor or apoptosis inducer.

In a fourth aspect, the invention provides the use of dasatinib and dasatinib in combination for the preparation of an acute T-lymphocyte leukemia cell clone formation inhibitor.

In a fifth aspect, the invention provides the use of dasatinib and dasatinib in combination for the preparation of an acute T-lymphocyte leukemia combined central nervous system leukemia inhibitor.

Specifically, the mass ratio of dasatinib to dasatinib in the application of the combination of dasatinib and dasatinib is 1: (1-100).

Preferably, the mass ratio of dasatinib to dasatinib in the application of the combination of dasatinib and dasatinib is 1: (3-60).

More preferably, the mass ratio of dasatinib to dasafil is 1: (3-15).

More preferably, the mass ratio of dasatinib to dasafil is 1: (3-11).

In a sixth aspect, the invention provides a combination for treating acute T-lymphocyte leukemia, which is characterized by comprising dasatinib and dasafil, and pharmaceutically acceptable pharmaceutical excipients.

The combined medicine can be a single compound preparation or a combination of two independent preparations, and when the combined medicine is a combination of two independent preparations, the administration mode can be simultaneous administration of the two independent preparations, or cross administration or sequential administration. Wherein the preparation is any pharmaceutically acceptable dosage form, such as tablet, powder, suspension, granule, capsule, solution, enema, emulsion, etc.

The combined medicine can be singly administered or can be matched with pharmaceutical excipients to be prepared into proper dosage forms for administration, and the pharmaceutical excipients comprise any one or a combination of at least two of carriers, diluents, excipients, fillers, adhesives, wetting agents, disintegrating agents, emulsifying agents, cosolvent, solubilizers, osmotic pressure regulators, surfactants, coating materials, coloring agents, pH regulators, antioxidants, bacteriostats or buffers. Wherein the carrier comprises liposome, micelle, dendritic macromolecule and the like.

Preferably, the pharmaceutically acceptable carrier comprises a liposome, micelle, dendrimer, microsphere or microcapsule.

The invention has the following beneficial effects:

the invention creatively combines dasatinib and dasatinib as a medicine for treating T-ALL, wherein dasatinib acts on T-ALL cells through an LCK pathway to induce apoptosis of the T-ALL cells, but has limited effect for treating the T-ALL due to other mechanisms. The invention discovers that dasatinib can trans-activate MAPK channel through mechanism research, and if the MAPK channel is inhibited, the effect of dasatinib on T-ALL cell proliferation inhibition can be enhanced. The study of the invention shows that the application of dasatinib combined with dasatinib has the effect of remarkably inhibiting T-ALL cell proliferation compared with single dasatinib or dasatinib, remarkably prolonging the survival time of a T-ALL mouse model, and both dasatinib and dasatinib can pass through a blood brain barrier, thereby having the effect of relieving the progress of T-ALL combined central nervous system leukemia. The invention provides a new strategy and thought for treating acute T lymphocyte leukemia and acute T lymphocyte leukemia combined central nervous system leukemia, and has very remarkable significance.

Drawings

FIG. 1 is a graph showing the results of inhibition of proliferation of HAP1 cell lines by different combinations of drugs (dasatinib and different MAPK pathway inhibitor combinations) in example 1.

FIG. 2 is a graph showing the results of inhibition of proliferation of HAP1 cell lines by a combination of dasatinib and different concentrations of dasatinib in example 1.

FIG. 3 is a graph showing the effect of the combination of drugs at different concentrations on the proliferation inhibition of T-ALL cell lines in example 2.

FIG. 4 is a graph showing the results of inhibition of primary T-ALL cell proliferation by combinations of different concentrations of the drugs of example 3.

Fig. 5 is a graph showing the statistical results of the body weight of each group of NSG mice in example 4.

FIG. 6 is a graph showing survival of NSG mice in each group of example 4.

FIG. 7 is a graph showing the results of HE staining of pathological sections of the spinal cord and subarachnoid space of the control group in example 4.

FIG. 8 is a graph showing HE staining results of spinal cord and subarachnoid space pathological sections in example 4 in combination with drugs.

Detailed Description

The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

The drugs Dasatinib (Dasatinib) and dasafinib (dasafenib) referred to in the following examples are provided by Shanghai blue wood chemical company.

SPF-class NSG mice were supplied from the university of Zhongshan affiliated first hospital animal center, 6-8 week old female mice, and all the operations on the mice were performed in a sterile laminar flow chamber.

Clinical case specimens (2 cases of bone marrow specimens of T-ALL patients) were derived from pediatric secondary departments of the first hospital affiliated to the university of Zhongshan.

T-ALL cell lines (including Jurkat, CCRF-CEM) were derived from the professor Chen Yueqin of the university of Zhongshan student's Ministry of life.

Example 1 drug screening combination drug combinations

The test operation method comprises the following steps: HAP1 cells transferred to 4-5 passages and in good condition were cultured at 1.5X10 ⁴ The seed/hole 96-well plate is put into a cell culture box for culture. After 24h plates, medium was discarded, 100nM dasatinib and 1uM different MAPK pathway inhibitors were added to 96-well plates, respectively, as Lapatinib (Lapatinib), sorafenib (Sorafenib), regorafenib (Regorafenib), lonafanib (Lonafarnib), sotoracib (AMG-510), dasafinib (Dabrafenib), vitamin Mo Feini (Vemurafenib), semmetinib (Selumetinib), cobicitinib (Cobimetanib), trametinib (Trametinib), asiatic acid (Asiatic acid) were set, and a DMSO control group was set without dasatinib (i.e. DMSO was used instead of dasatinib), 48h post-culture broth was used to examine proliferation of different groups of HAP1 cells, as shown in FIG. 1.

In addition, HAP1 cells transferred to 4-5 passages and well-conditioned were cultured at a ratio of 1.5X10 ⁴ The seed/hole 96-well plate is put into a cell culture box for culture. After 24h plating, medium was discarded, 100nM dasatinib and dasatinib at different concentrations (concentration gradients 0. Mu.M, 0.125. Mu.M, 0.25. Mu.M, 0.5. Mu.M, 1. Mu.M) were added to 96 well plates, and a control group without dasatinib was set, and after 48h the medium was discarded, and proliferation of HAP1 cells of different groups was detected using CCK8 kit. The results are shown in FIG. 2.

As shown in fig. 1, the combination of dasatinib and dasatinib has a remarkable effect of inhibiting the proliferation of HAP1 cell lines, and the effect of inhibiting the proliferation of dasatinib is more remarkable than that of dasatinib alone or dasatinib alone. This experiment demonstrates that not all MAPK pathway inhibitors act synergistically with dasatinib and that none of the RTK, RAS, P, MEK1/2 protein inhibitors act significantly to inhibit the proliferation of HAP1 cell lines when used in combination with dasatinib. Even inhibitors against RAF proteins as well, vemurafenib (Vemurafenib) cannot be synergistic with dasafinib, and there may be specific mechanisms for the co-synergy of dasatinib in combination with dasafinib. As is clear from FIG. 2, the inhibition effect of Darafenib alone on HAP1 cell lines was weak, and when used together with 100nM Dasatinib, the inhibition effect of Darafenib on cell proliferation was continuously enhanced with increasing concentration, and the inhibition effect of Darafenib on HAP1 cell lines was more than 50% at concentrations of 0.5. Mu.M and 1. Mu.M. This demonstrates that the combination of dasatinib and dasafil has a significant concentration dependence on the proliferation inhibition effect of HAP1 cell lines.

Example 2 inhibition of proliferation of T-ALL cell lines by combination of drugs

The test operation method comprises the following steps: T-ALL cell lines (including Jurkat, CCRF-CEM) transferred to 4-5 passages in good condition at 1.5X10 ⁴ The seed/hole 96-well plate is put into a cell culture box for culture. After 24h plating, the medium was discarded, 100nM dasatinib and a mixed group of dasatinib of different concentrations (concentration gradient of 0. Mu.M, 0.125. Mu.M, 0.25. Mu.M, 0.5. Mu.M, 1. Mu.M) were added to the 96-well plate, and a control group without dasatinib was set, and after 48h the medium was discarded, and proliferation of T-ALL cells of different groups was detected using CCK8 kit.

The results of the inhibition of T-ALL cell line proliferation by the combination of different concentrations are shown in FIG. 3, wherein FIG. 3A shows the results of Jurkat cell line action for 48h, and FIG. 3B shows the results of CCRF-CEM cell line action for 48 h. As shown in FIG. 3, single Darafenib has a certain proliferation inhibition effect on CCRF-CEM cell lines, but has little proliferation inhibition effect on Jurkat cell lines. The combined use of the dasatinib and the dasatinib has the effect of obviously inhibiting the proliferation of the T-ALL cell strain, the effect of inhibiting the proliferation of the T-ALL cell strain is better and more obvious than that of single dasatinib or dasatinib, the combined use of the dasatinib and the dasatinib has obvious concentration dependence on the proliferation inhibition effect of the T-ALL cell strain, and the combined use effect of the dasatinib and 100nM dasatinib at the concentration of 1 mu M is better.

Example 3 evaluation of the Effect of combination drugs on T-ALL cells from the Primary cell level

The test operation method comprises the following steps: collecting 2 high-risk bone marrow specimens of T-ALL patients, and showeringThe single nuclear cell is extracted from the separating liquid of the baryte. According to 1.5X10 ⁴ The seed/hole 96-well plate is put into a cell culture box for culture. After 24h of plating, the medium was discarded, 100nM dasatinib and a mixed group of dasatinib with different concentrations (concentration gradient of 0. Mu.M, 1. Mu.M, 5. Mu.M) were added to the 96-well plate, and a control group without dasatinib was set, and after 48h of the broth, proliferation of the different groups of primary T-ALL cells was detected using CCK8 kit.

The results of the inhibition of primary T-ALL cell proliferation by different concentrations of the drug combination are shown in FIG. 4. As can be seen from FIG. 4, 100nM dasatinib alone does not necessarily inhibit primary T-ALL cell lines. The combined application of the dasatinib and the dasatinib has the effect of obviously inhibiting the proliferation of the primary T-ALL cell strain, the effect of inhibiting the proliferation of the dasatinib is better and more obvious than that of single dasatinib or dasatinib, and the combined application of the dasatinib and the dasatinib has obvious concentration dependence on the proliferation inhibition effect of the primary T-ALL cell strain, and the better the proliferation inhibition effect on the cell strain is along with the increase of the concentration of the dasatinib.

Example 4 evaluation of the effects of combination drug combinations on T-ALL cells from animal level

The test operation method comprises the following steps:

(1) Construction of NSG mouse tumor-bearing model by T-ALL cell line Jurkat

Jurkat cells were resuspended in 100. Mu.L PBS (10 per 100. Mu.L) ⁶ Individual cells), by tail vein injection into NSG mice, and 4 weeks later, in vivo drug experiments were started;

(2) A blank group (the drug is replaced by an equal volume of solvent), a dasatinib group (10 mg/kg/d), a dasatinib group (30 mg/kg/d), a dasatinib and dasatinib combined group (10 mg/kg/d and 30 mg/kg/d) are respectively arranged, 8 dasatinib and dasatinib are respectively administered daily (the administration mode is oral), the administration is continued until a humane end point, the weight of the mice is monitored daily, and a survival curve is drawn.

From the statistical results of the body weight changes of the groups in fig. 5, the body weight of the combined drug group is reduced more slowly than that of the control group and the single drug treatment group, which shows that the combined use of dasatinib and dasafil has smaller influence on the body weight of animals and lower toxicity to organisms.

From the survival graph results of fig. 6, it can be seen that dasatinib and dasatinib combined mice have about 80% survival rate, dasatinib mice have about 40% survival rate, and dasatinib mice have about 30% survival rate, as seen from the survival rate of the mice at 40 days. And in terms of the total death time of mice, the total death of the mice in the combined group of dasatinib and dasatinib is 45 days, and the survival time is obviously longer than that of a blank control group and a single administration group. In conclusion, the median survival time of the combined drug group is obviously prolonged compared with that of the control group and the single drug treatment group.

From the HE staining results of the spinal cord and subarachnoid space pathological sections of the control group in FIG. 7 and the combined drug group in FIG. 8, the combined drug significantly reduces leukemia cell infiltration in the subarachnoid space and relieves T-ALL-complicated central nervous system leukemia.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (10)

1. The application of dasatinib in preparing a synergist for preventing, improving or treating acute T lymphocyte leukemia.

2. The use of dasatinib and dasatinib in the manufacture of a medicament for the prevention, amelioration or treatment of acute T-lymphocyte leukaemia.

3. The application of dasatinib and dasatinib in preparing acute T lymphocyte leukemia cell proliferation inhibitor or apoptosis inducer.

4. The use of dasatinib and dasatinib in the preparation of an acute T-lymphocyte leukemia cell clone formation inhibitor.

5. The application of dasatinib and dasatinib in preparing medicaments for preventing, improving or treating acute T lymphocyte leukemia combined central nervous system leukemia.

6. The use according to any one of claims 1-5, wherein the mass ratio of dasatinib to dasatinib is 1: (1-100).

7. The combined medicine for treating acute T lymphocyte leukemia is characterized by comprising dasatinib and dasafil and pharmaceutically acceptable pharmaceutic adjuvant.

8. The combination of claim 7, wherein the pharmaceutical formulation is a pharmaceutically acceptable dosage form.

9. The combination of claim 7, wherein the pharmaceutical adjuvant comprises any one or a combination of at least two of a carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, co-solvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH regulator, antioxidant, bacteriostat, or buffer.

10. The combination according to any one of claims 7-9, wherein dasatinib and dasatinib are formulated separately as two separate formulations for reconstitution; or dasatinib and dasatinib are prepared into a single compound preparation.