阅读说明：本技术 Jak抑制剂和p53-mdm2抑制剂联合用于制备治疗t-all药物的用途 (Use of a JAK inhibitor in combination with a P53-MDM2 inhibitor for the preparation of a medicament for the treatment of T-ALL ) 是由 汪晓敏 侯帅兵 袁卫平 袁胜男 初雅婧 于 2021-08-17 设计创作，主要内容包括：本发明公开了一种JAK抑制剂和P53-MDM2抑制剂联合用于制备治疗T-ALL药物的用途。所述JAK抑制剂选用Tofacitinib。所述P53-MDM2抑制剂选用Idasanutlin。本发明将两药联合用于治疗PHF6～(△)和JAK3～(M511I)共突变的T-ALL,克服了现有的治疗药物出现的预后差、耐药及副作用的问题,有效改善单药耐药,较单用一种药物,更能有效缓解疾病,延长生存期和改善预后。(The invention discloses an application of a JAK inhibitor and a P53-MDM2 inhibitor in combination in preparing a medicine for treating T-ALL. Tofacitinib is selected as the JAK inhibitor. The P53-MDM2 inhibitor is Idasanutlin. The invention combines two medicines for treating PHF6 △ And JAK3 M511I The co-mutant T-ALL overcomes the problems of poor prognosis, drug resistance and side effects of the existing treatment drugs, effectively improves single drug resistance, and can more effectively relieve diseases, prolong life cycle and improve prognosis compared with single drug.)

1. JA (JA-JA-JA-Combination of a K inhibitor and a P53-MDM2 inhibitor for the preparation of a medicament for the treatment of PHF6^△And JAK3^M511IUse of a co-mutated T-ALL medicament.

2. Use according to claim 1, characterized in that: tofacitinib is selected as the JAK inhibitor.

3. Use according to claim 1, characterized in that: the P53-MDM2 inhibitor is Idasanutlin.

4. Use according to claim 1, characterized in that: the mass ratio of the JAK inhibitor to the P53-MDM2 inhibitor is (3.5-4.5): (2.5-3.5).

5. A pharmaceutical composition characterized by: including JAK inhibitors and P53-MDM2 inhibitors.

6. The pharmaceutical composition of claim 5, wherein: tofacitinib is selected as the JAK inhibitor.

7. The pharmaceutical composition of claim 5, wherein: the P53-MDM2 inhibitor is Idasanutlin.

8. The pharmaceutical composition of claim 5, wherein: the mass ratio of the JAK inhibitor to the P53-MDM2 inhibitor is 4: 3.

9. Use of a pharmaceutical composition according to any one of claims 5 to 8 in the preparation of a medicament for the treatment of PHF6^△And JAK3^M511IUse of a co-mutated T-ALL medicament.

Technical Field

The invention relates to the field of drug therapy, in particular to application of a JAK inhibitor and a P53-MDM2 inhibitor in preparation of a drug for treating T-ALL.

Background

T-cell acute lymphoblastic leukemia (T-ALL) is the most commonIs one of the hematological malignancies. T-ALL patients have a 5-year survival rate of less than 50% and recurrent adult T-ALL deaths of up to 90%. To date, the low cure rate of T-ALL has not been improved and relapse after reaching complete remission remains a major challenge for T-ALL therapy, and researchers have been working on developing new therapeutic targets and new effective drugs. Recent studies have shown that more than 25% of patients with T-ALL carry JAK-STAT mutations, with the JAK3 mutation being most common in patients with T-ALL. The M511I point mutation was the most common JAK3 mutation, with a mutation rate of 34.7% among all JAK3 mutations. The JAK3 mutation is often accompanied by a number of genetic changes in T-ALL, such as a mutation in the epigenetic regulator PHF 6. Studies have found that the PHF6 mutation is frequently co-present with the JAK3 mutation in T-ALL patients, and that the average survival time of patients with the JAK/STAT mutation and the PHF6 mutation is significantly shorter than that of patients with the JAK/STAT mutation alone. Although various drugs have been applied to the treatment of T-ALL, the prognosis of patients is still poor. Thus, for PHF6^△And JAK3^M511IIn co-mutated patients, a novel and effective treatment scheme is a key problem for prolonging the survival time of patients and improving the prognosis of patients.

JAK kinases are a family of intracellular non-receptor tyrosine kinases of which there are 4 members: JAK-1, JAK-2, JAK-3 and TYK-2. It plays an important role in cytokine receptor signaling pathways through interactions with Signal Transducers and Activators of Transcription (STATs). Therefore, JAK kinase is a very important drug target, and a JAK inhibitor Tofacitinib (Tofacitinib) developed aiming at the target can effectively inhibit the activities of JAK1 and JAK3, but has obvious side effects, such as infection, anemia, cytopenia and the like. The P53 signal path is at Phf6 KO + JAK3^M511IThe Idasanatlin can selectively inhibit P53 and MDM2 from mutually binding, selectively binds to a P53 site on the surface of MDM2, isolates P53 from MDM2, and leads to activation of an apoptosis program after P53 is stabilized, thereby killing cancer cells. However, MDM2 inhibitors do not always induce apoptosis in P53 wild-type cancer cells, probably due to failure of the downstream signaling pathway, and cells that were initially sensitive to MDM2 inhibitors also produce over timeGenerating drug resistance.

Disclosure of Invention

The invention provides an application of a JAK inhibitor and a P53-MDM2 inhibitor in preparation of a T-ALL treatment drug in order to solve the problem of poor treatment effect of the existing T-ALL treatment drug.

The invention is realized by the following technical scheme.

Combined application of JAK inhibitor and P53-MDM2 inhibitor in preparation of PHF6^△And JAK3^M511IUse of a co-mutated T-ALL medicament.

Further, Tofacitinib is selected as the JAK inhibitor.

Further, the P53-MDM2 inhibitor is Idasanutlin.

Further, the mass ratio of the JAK inhibitor to the P53-MDM2 inhibitor is (3.5-4.5): (2.5-3.5).

A pharmaceutical composition comprising a JAK inhibitor and a P53-MDM2 inhibitor.

Further, Tofacitinib is selected as the JAK inhibitor.

Further, the P53-MDM2 inhibitor is Idasanutlin.

Further, the mass ratio of the JAK inhibitor to the P53-MDM2 inhibitor is 4: 3.

A method for preparing PHF6^△And JAK3^M511IUse of a co-mutated T-ALL medicament.

The present application has the following advantageous effects.

This application is made by the PHF6^△And JAK3^M511IIn a co-mutation mouse model, Tofacitinib and Idasanutlin are jointly applied for treatment, the synergistic effect of the Tofacitinib and the Idasanutlin overcomes the drug resistance and toxic and side effect of single drug application, relieves the diseases of the mouse, prolongs the life cycle of the mouse, improves the prognosis, achieves good treatment effect, and also is used for clinically and jointly applying Tofacitinib and Idasanutlin to treat PHF6^△And JAK3^M511IMutant T-ALL patients provide the theoretical basis.

Drawings

FIG. 1 is a PHF6 of the present invention^△And JAK3^M511IConstructing a pattern diagram of a co-mutation T-ALL mouse model;

FIG. 2 shows a PHF6 carried by the present invention^△And JAK3^M511ISurvival monitoring profile of co-mutated T-ALL mice treated with four treatment regimens;

FIG. 3 shows a PHF6 carried by the present invention^△And JAK3^M511IRoutine peripheral blood detection of co-mutated T-ALL mice treated with four treatment regimens;

FIG. 4 shows a PHF6 carried by the present invention^△And JAK3^M511IA graph of the degree of infiltration of leukemic cells in each organ of co-mutated T-ALL mice treated by four treatment regimens;

FIG. 5 PHF6 carried by the present invention^△And JAK3^M511IWeight plots of organs of co-mutated T-ALL mice after treatment with four treatment regimens;

FIG. 6 shows a PHF6 carried by the present invention^△And JAK3^M511IAfter the co-mutant T-ALL mice are treated by four treatment schemes, detecting a leukemia cell infiltration degree chart by HE staining;

FIG. 7 shows a PHF6 carried by the present invention^△And JAK3^M511ITUNEL staining detects leukemic apoptosis patterns in co-mutant T-ALL mice treated with four treatment regimens;

FIG. 8 shows a PHF6 carried by the present invention^△And JAK3^M511IThe proliferation pattern of leukemia cells was examined by Ki67 staining of co-mutated T-ALL mice treated with four treatment regimens.

Detailed Description

Tofacitinib of the present application is available from MedChemexpress (MCE) under the designation HY-40354A.

Idasanatlin of the present application is available from MedChemExpress (MCE) under the designation HY-15676.

EXAMPLE 1 construction of PHF6^△And JAK3^M511ICo-mutant T-ALL mice

Two LoxP sequences were inserted from exon 4 to the two ends of exon 5 of Phf6 using homologous recombination technology, and mice were mated with Vav1-Cre transgenic mice expressing Cre recombinase, resulting in Phf6 mice conditional knockouts in the hematopoietic system.

Wild type Phf6 or Phf6 knockout male mice were sacrificed and bone marrow cells were harvested from the femur and tibia. Lin-cells were enriched and cultured in medium containing 10% Fetal Bovine Serum (FBS), 50ng/ml recombinant mouse stem cell factor, 10ng/ml recombinant mouse interleukin-3, and 50ng/ml recombinant mouse thrombopoietin. Mixing MSCV-JAK3^M511IIRES-GFP retroviral vectors were transfected into 293T cells and the virus was harvested after 48 and 72 hours. Converting JAK3^M511IGFP virus transfected Lin-cells. Intravenous injection of GFP in Male mice⁺Cells, establishment of Vav1-Cre Phf6^fl/y+JAK3^M511IT-ALL mouse model of (PHF 6)^△And JAK3^M511ICo-mutant T-ALL mice) (see figure 1 for construction mode).

EXAMPLE 2 method of administration

PHF6^△And JAK3^M511ICo-mutant T-ALL mice were treated with four regimens of oral gavage of placebo (DMSO), Tofacitinib alone, Idasanutlin alone, or a combination of Tofacitinib and Idasanutlin

Administration dose: tofacitinib (40 mg/kg per day), Idasanutlin (30 mg/kg per day), DMSO (concentration 0.01%, 150ul per day)

The administration route is as follows: administration by oral gavage

The administration time is as follows: receive drug treatment daily until death.

Example 3 Life cycle testing

PHF6^△And JAK3^M511IAfter the development of co-mutant T-ALL mice, which received the drug daily until death, the survival of the mice on their four dosing regimens was calculated (figure 2 is the survival of the mice on the different dosing regimens).

The experimental results show that the survival of mice treated with Tofacitinib and Idasanutlin in combination is significantly longer than that of mice treated with placebo (DMSO), Tofacitinib alone and Idasanutlin alone.

Example 4 routine peripheral blood testing

Adding 480ul pre-dilution (purchased from mindray mairi biology, main components of dodecyl trimethyl ammonium chloride and buffer) into 1.5ml EP tube, adding 20ul mouse eye blood into EP tube, mixing, and detecting with blood conventional instrument (figure 3 is peripheral blood conventional detection result)

The experimental results show that the number of hemoglobin and platelets in peripheral blood of the mice treated by combining Tofacitinib and Idasanutlin is obviously higher than that of the mice treated by the other three groups, and the number of leukemia cells is obviously lower than that of the mice treated by the other three groups.

Example 5 flow assay of leukemia cell proportion

80ul of PBE Buffer (PBS + 2% FBS +2nM EDTA) was added to the flow tube, and 20ul of mouse eye blood was taken and mixed with the PBE Buffer. Adding 1ml of erythrocyte lysate into each tube, standing at room temperature for 10min, centrifuging at 4 ℃ for 1500rpm for 5min, discarding the supernatant, resuspending with 100ul of PBS, and detecting the proportion of GFP and leukemia cells, namely the proportion of leukemia cells by a flow analyzer.

The tibia, femur and ilium of the mouse were separated, bone marrow cavities were repeatedly washed from both ends of the bone using a 1ml syringe in 3ml of PBE to obtain bone marrow cells, the whole bone marrow cells were collected by filtration using a 100-mesh nylon membrane, centrifuged at 4 degrees at 1500rpm for 5min, the supernatant was discarded, resuspended in 100ul of PBS, and GFP was detected by a flow analyzer⁺Leukemia cell ratio.

The spleen, liver and thymus of the mice were removed, ground in 3ml of PBE, respectively, the cells were collected by filtration through a 100-mesh nylon membrane, centrifuged at 4 ℃ at 1500rpm for 5min, the supernatant was discarded, resuspended in 100ul of PBS, and the ratio of each GFP + leukemia cell was determined by a flow analyzer. (FIG. 4 shows the flow-type assay of the ratio of leukemia cells in peripheral blood, bone marrow, liver, spleen, and thymus)

Experimental results show that the proportion of leukemia cells in peripheral blood, bone marrow, liver, spleen and thymus of mice treated by combining Tofacitinib and Idasanutlin is obviously lower than that of the mice treated by the other three groups.

Example 6 measurement of weight of each organ

The liver, thymus and spleen of mice of the four administration schedules were taken out and weighed, respectively, as an index for determining the severity of the disease (fig. 5 shows the weight of each organ).

The experimental results show that the weight of spleen, liver and thymus of the mice treated by combining Tofacitinib and Idasanutlin is obviously reduced compared with the weight of the mice treated by the other three groups.

Example 7HE staining to detect the extent of infiltration of leukemia cells

Mice of four dosing schedules were sliced with bone marrow, spleen, liver, thymus, and brain paraffin, deparaffinized, and then HE-stained to examine the extent of infiltration of leukemia cells in these organ tissues (fig. 6 shows the extent of infiltration of leukemia cells by HE-staining).

The experimental result shows that compared with the other three groups of treated mice, the combined treatment of Tofacitinib and Idasanutlin obviously reduces the leukemia cell infiltration degree of the spleen, the liver, the brain, the thymus and the bone marrow of the mice.

Example 8TUNEL staining for leukemia apoptosis

Paraffin sections of bone marrow from mice of four dosing regimens were deparaffinized and subjected to TUNEL staining to detect apoptosis of leukemic cells in bone marrow (figure 7 shows TUNEL staining to detect leukemic apoptosis).

Experimental results show that compared with the other three groups of treated mice, the mice treated by combining Tofacitinib and Idasanutlin have obviously increased apoptotic cells in bone marrow.

Example 9Ki67 staining for leukemia cell proliferation

Paraffin sections of spleen, liver, brain and thymus of mice of four dosing schedules were deparaffinized, and then subjected to Ki67 staining to examine the proliferation potency of leukemia cells in these organ tissues (Ki 67 staining in fig. 8 is used to examine the proliferation potency of leukemia cells).

Experimental results show that compared with the other three groups of mice treated by Tofacitinib and Idasanutlin, the mice treated by Tofacitinib and Idasanutlin have obviously reduced proliferation capacity of leukemia cells in liver, spleen, brain and thymus.

The embodiments of the present invention are preferred embodiments of the present invention, and the scope of the present invention is not limited by these embodiments, so: all equivalent changes made according to the structure, shape and principle of the invention are covered by the protection scope of the invention.