CN113633639A

CN113633639A - Application of Defatinib in medicine for treating thrombocytopenia

Info

Publication number: CN113633639A
Application number: CN202111122148.9A
Authority: CN
Inventors: 李燕; 吴建明; 王雨情; 李芸霞
Original assignee: Southwest Medical University
Current assignee: Southwest Medical University
Priority date: 2021-09-24
Filing date: 2021-09-24
Publication date: 2021-11-12

Abstract

The invention relates to application of Defatinib in a medicine for treating thrombocytopenia, and the Defatinib which is an existing medicine compound is applied to treating thrombocytopenia, so that a safe and efficient medicine for preventing and treating thrombocytopenia can be provided, and the health level of a patient is improved. Compared with the traditional treatment method, the application of the Defatinib disclosed by the invention is safe and effective as proved by animal experiments, and the life health of the thrombocytopenia patient can be effectively guaranteed.

Description

Application of Defatinib in medicine for treating thrombocytopenia

Technical Field

The invention relates to an application of a pharmaceutical compound, in particular to an application of Defatinib in a medicine for treating thrombocytopenia, belongs to a new application of the pharmaceutical compound, and belongs to the field of medicines.

Background

The prior art situation, the defects or shortcomings of the prior art:

thrombocytopenia refers to a problem caused by a decrease in the proportion of platelets in blood, and may be caused by many reasons, such as leukemia, aplastic anemia, the use of anti-tumor drugs, etc., and spontaneous thrombocytopenia due to immune causes. There are various options for the treatment of thrombocytopenia depending on the etiology, of which the drastic decrease in platelet count caused by chemotherapeutic drugs during antitumor therapy is one of the most dangerous cases and it is often difficult to find a good way to overcome or avoid due to the need for chemotherapeutic drugs. In addition, chemotherapy-induced thrombocytopenia is often acute, and particularly chemotherapy-combined radiotherapy treatment-induced thrombocytopenia is faster in onset, the number of platelets is more rapidly reduced, the bleeding risk is increased, and the time for platelets to fall to the minimum value due to a combined treatment scheme is faster than other cases, so the bleeding risk is higher, and the risk of improper treatment is higher.

For thrombocytopenia symptoms caused by chemotherapy, the current treatment protocols are mainly:

first, platelet growth factor including interleukin-11 and Thrombopoietin (TPO) is infused. It is generally recommended that the administration of platelet growth factor be initiated after chemotherapy, with subcutaneous injections once a day for 7-10 consecutive days. However, the platelet growth factor is not abundant in source, expensive in price, and has a time lag in drug action. Improper dosage control can result in sustained platelet elevation, leading to thrombosis, especially in the elderly.

Second, platelet infusion, which rapidly ameliorates the problem of thrombocytopenia in patients by directly infusing platelets to the patient. Each patient is required to consume large quantities of platelets, which are only available for donation by volunteers, resulting in patient treatment limited by the total amount of donation available to the donor station on a daily basis.

Therefore, there is no stable and reliable method for treating thrombocytopenia, and a drug compound that can effectively treat thrombocytopenia or prevent thrombocytopenia is needed.

Disclosure of Invention

The invention aims to: aiming at the problems of insufficient treatment method or poor effect of thrombocytopenia in the prior art, provides a new application of a pharmaceutical compound. In particular to application of the Defatinib in the preparation of medicines for treating thrombocytopenia, belonging to novel application of the existing medicinal compounds.

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

application of Defatinib in preparation of medicines for treating thrombocytopenia is provided.

By applying the Defatinib as the active ingredient of the medicine for treating thrombocytopenia, a brand-new medicine active ingredient for preventing thrombocytopenia can be provided, good news is brought to more patients, and the defect that the platelet infusion treatment scheme is limited by the total amount of platelets obtained by blood donation is avoided.

Defectinib (Defectinib) is a novel FAK inhibitor, and inhibits FAK phosphorylation at Tyr397 site. The molecular formula is C₂₀H₂₁F₃N₈O₃S, the structural formula is as follows:

as a preferred embodiment of the present invention, the use of delfacitinib in a medicament for the treatment of thrombocytopenia caused by chemotherapy. Namely, the drug is a drug for treating thrombocytopenia caused by chemotherapy.

As a preferred embodiment of the invention, the application of the Defatinib in the medicine for treating thrombocytopenia caused by radiotherapy is provided. Namely, the drug is a drug for treating thrombocytopenia caused by radiation therapy.

As a preferred embodiment of the invention, the use of Defatinib in a medicament for the treatment of thrombocytopenia due to tumor diseases. Namely, the drug is a drug for treating thrombocytopenia caused by tumor diseases.

As a preferred embodiment of the invention, the use of Defatinib in the preparation of a medicament for treating aplastic anemia, hyperfunction of the spleen, myelodysplastic syndrome, leukemia or thrombocytopenia due to systemic lupus erythematosus is provided. Namely, the drug is a drug for treating aplastic anemia, hyperfunction of spleen, myelodysplastic syndrome, leukemia or thrombocytopenia caused by systemic lupus erythematosus.

As a preferred embodiment of the present invention, the use of delfacitinib in a medicament for the treatment of thrombocytopenia. That is, the drug is a drug for treating insufficient thrombopoiesis.

As a preferred embodiment of the invention, the use of Defatinib in a medicament for treating thrombocytopenia caused by bone marrow megakaryocyte developmental maturation disorder. Namely, the drug is a drug for treating thrombocytopenia caused by a developmental maturation disorder of bone marrow megakaryocytes.

As a preferred embodiment of the invention, the application of the Defatinib in the medicine for treating the drug immune thrombocytopenia is provided.

As a preferable scheme of the invention, the application of the Defatinib in the medicine for treating thrombocytopenic purpura is provided.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

1. the existing drug compound of Defatinib is applied to treating thrombocytopenia, so that a safe and efficient drug for preventing and treating thrombocytopenia can be provided, and the health level of patients is improved.

2. Compared with the traditional treatment method, the preparation method disclosed by the invention uses the Defatinib as a marketed drug, and animal experiments prove that the preparation method is safe and effective and can effectively ensure the life health of a patient.

Description of the drawings:

FIG. 1 Effect of Defatinib on cell viability of HEL, Meg-01, K562 cells.

FIG. 2 cytotoxicity of Defatinib on HEL, Meg-01, K562 cells.

FIG. 3 shows the cell growth curves of Defatinib intervention HEL, Meg-01 and K562.

FIG. 4 Effect of Defatinib on the differentiation of HEL, Meg-01 and K562 cells into polyploids.

FIG. 5Giemsa staining to observe the effect of Defatinib on HEL, Meg-01 and K562 cell morphology.

FIG. 6 Coprinus cinereus peptide staining to observe the effect of Definity on HEL, Meg-01 and K562 cell morphology.

FIG. 7 effect of different concentrations of Defatinib on expression of CD41/CD42b in HEL, Meg-01 and K562 cells.

FIG. 8 effect of different concentrations of Defatinib on DNA ploidy of HEL, Meg-01 and K562 cells.

FIG. 9 Effect of Defatinib on mouse platelet count.

Figure 10 effect of dfatinib on mouse organ index.

FIG. 11 Effect of Defect on the proportion of megakaryocyte progenitors.

FIG. 12 Effect of Defacitinib on the proportion of CD41+/CD61+ positive cells in bone marrow.

FIG. 13 Effect of Defacitinib on the number of megakaryocytes in mouse bone marrow.

FIG. 14 Effect of Defacitinib on the number of activated platelets in bone marrow.

Detailed Description

The present invention will be described in detail below.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the following embodiments further describe the present invention in detail. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[ examples ] A method for producing a compound

1 drugs and agents

TABLE 1 Primary drugs and reagents

2 Experimental cells and animals

HEL, Meg-01 and K562 cells, purchased from the Wuhan cell Bank of China Center for Type Culture Collection (CCTCC).

Kunming breed mice, SPF grade, half male and female, 4 weeks old, 18-22g in weight, purchased from Liaoning Biotechnology Ltd, license number: SYXK (Liao) 2020 and 0001. Animals are raised in an animal experiment center of southwest medical university, and are divided into a male cage and a female cage, the temperature is 22-25 ℃, the humidity is 40-60%, and free diet and drinking water are provided.

3 Main Instrument

TABLE 2 Main Experimental instruments

4 Experimental methods

4.1 Defatinib in vitro megakaryocyte differentiation promotion experiment

4.1.1 cell culture

(1) Cell resuscitation

Taking out HEL, Meg-01 and K562 cells from liquid nitrogen tank, thawing in 37 deg.C water bath, centrifuging at 1000r/min for 5min, discarding supernatant, adding 5mL 1640, mixing, transferring to 25cm²And (3) shaking the culture bottle uniformly to enable the cell suspension to be spread at the bottom of the culture bottle, and putting the culture bottle into an incubator with the temperature of 37 ℃, the relative saturation humidity of 95% and the 5% CO2 for culture.

(2) Cell exchange liquid

Taking out the cells from the incubator, observing the state and density of the cells under a microscope, transferring the cell suspension into a 15mL centrifuge tube by using a sterile pipette when the culture solution turns yellow, adding PBS to 10mL, centrifuging at 700r/min for 3min, discarding the supernatant, repeatedly washing for 2 times, adding 5mL 1640, finishing cultivation and resuspension, transferring to a culture flask, shaking uniformly, and replacing every other day.

(3) Cell passage

Observing the state and the density of the cells under a microscope, when the cell density reaches 80-90%, transferring the cells in the culture bottles to a 15mL centrifuge tube, adding PBS to wash the cells for 2 times, centrifuging for 3min at 700r/min, discarding the supernatant, completing the culture and heavy suspension by 1640, separating the cells into two culture bottles, and putting the two culture bottles into an incubator at 37 ℃ for culture.

4.1.2 Effect of Defatinib on HEL, Meg-01, K562 cell viability

Collecting HEL, Meg-01, K562 cells of logarithmic growth phase cultured in 4.1.1, moistening with PBS, adding culture medium to adjust cell density to 2 × 10⁴one/mL, at 180 μ L cell suspension per well, was seeded into 96-well plates. The stock solution of German furtinib was diluted to 0.0625. mu.M, 0.125. mu.M, 0.25. mu.M, 0.5. mu.M, 1. mu.M, 1.5. mu.M, 3. mu.M, 6. mu.M. 20 mu L of Defatinib with different concentrations is added into the drug groups, 20 mu L of Defatinib is added into the control group (Coultrol), the culture is finished, 200 mu L of Defatinib is added into the blank group, the plate is stirred gently, each group has 3 multiple holes, and the culture is carried out in an incubator at 37 ℃. After 24h, 20. mu.L of CCK8 was added to each well, mixed by gentle shaking, and incubated in an incubator for 1 h. And detecting the OD value of the sample by using a multifunctional microplate reader at the wavelength of 450nm, and repeating the experiment for three times. Cell viability was calculated using the following formula:

cell viability (%) - (drug OD value-blank OD value)/(control OD value-blank OD value) × 100%

The effect of Defatinib on the viability of HEL, Meg-01 and K562 cells shows that: the intervention of des fantinib on HEL, Meg-01 and K562 cells for 24h had no effect on cell viability, and 3 and 6 μ M reduced the viability of the three cells, with the difference having statistical significance (p <0.05 and p <0.0001), as shown in FIG. 1.

4.1.3 Defatinib cytotoxicity assay for HEL, Meg-01, K562 cells

HEL, Meg-01, K562 cells were washed with PBS, and the cell density was adjusted to 2X 10 by adding medium⁴one/mL, at 180 μ L cell suspension per well, was seeded into 96-well plates. Respectively setting a drug group, a control group (Coutrol), a maximum enzyme activity group (Coutrol max) and a blank group, respectively adding 20 mu L of 0.5 mu M, 1 mu M and 1.5 mu M Defatinib to the drug group, respectively adding 20 mu L of the control group and the maximum enzyme activity group to the blank group, respectively completing culture, adding 200 mu L of the blank group to the blank group, completing culture, 3 multiple wells in each group, gently shaking the well plate, and continuously culturing for 6 days in an incubator at 37 ℃. Adding 20 mu L of LDH solution into the maximum enzyme activity group at 1, 2, 3, 4, 5 and 6 days, repeatedly blowing and beating until the cells are completely broken, incubating in an incubator at 37 ℃ for 1h, centrifuging at 1000r/min for 3min, and adding 120 mu L of supernatant into a new 96-well plate. The drug group, control group and blank group were the sameThe time points were centrifuged as before and the supernatant was added to a new 96-well plate. And preparing a proper amount of detection solution (lactic acid solution: 1 XINT solution: enzyme solution: 1:1:1) in a dark place, adding 20 mu L of detection solution into each hole, mixing uniformly, reacting at room temperature for 30min, detecting the OD value of the detection solution at 490nm by using a multifunctional microplate reader, and repeating the experiment for three times. The cytotoxicity calculation formula is as follows:

determining the OD value of the drug group, namely the measured OD value of the drug group-the OD value of the blank group;

the maximum enzyme activity group OD value is the measured maximum enzyme activity group OD-blank group OD value;

control OD value-measured control OD-blank OD value.

(S) (%) cytotoxicity (drug OD-control OD)/(maximum enzyme active OD-control OD) × 100%

The cytotoxicity test results of the delta-tinib show that the cells of the control group and the drug group gradually release LDH along with the prolongation of the intervention time, the LDH release rate between the two groups is not obviously different, and the LDH release rate is obviously lower than that of the maximum enzyme release group (p <0.05, p < 0.001). The results show that the three concentrations of Defatinib are non-toxic to the three cells, as shown in FIG. 2.

4.1.4 Effect of Defatinib on proliferation of HEL, Meg-01, K562 cells

Taking HEL, Meg-01 and K562 cells, adjusting cell density to 2 × 10⁴one/mL, at 500 μ L cell suspension per well, in 12-well plates. The drug component is added with 500 μ L of 0.5 μ M, 1 μ M, 1.5 μ M Defatinib, the control component is added with the same amount of complete culture medium, and the mixture is continuously cultured in an incubator at 37 ℃ for 6 d. The cells were counted daily and cell growth curves were plotted.

The experimental results of the influence of Defatinib on the proliferation of HEL, Meg-01 and K562 cells show that the proliferation of three cells can be inhibited to different degrees after 0.5, 1 and 1.5 mu M Defatinib intervenes in HEL, Meg-01 and K562 cells for 6d, and the growth curves are shown in figure 3.

4.1.5 Effect of Defacitinib on megakaryocyte morphology

(1) Observation of the Effect of Devatinib on megakaryocyte morphology under a mirror

HEL, Meg-01, K562 cells cultured and intervened by the method in 4.1.4 were observed in the wells for morphological changes of cell differentiation using an inverted phase contrast microscope at 3, 5, 6 and photographed.

As shown in FIG. 4, the observation of the effect of Defatinib on megakaryocyte morphology under the microscope revealed that the cell volume increased by the intervention of 0.5, 1, 1.5. mu.M Defatinib on HEL, Meg-01, K562 cells for 3d, and the cell number increased by 6d and was concentration-dependent. The results indicate that Defatinib promotes megakaryocyte differentiation, as shown in FIG. 4.

(2) Giemsa staining for observation of effects of Defatinib on megakaryocyte morphology

HEL, Meg-01 and K562 cells cultured and intervened for 6d by the method in 4.1.4 are collected, transferred to a 1.5mL EP tube, added with 500. mu.L of 0.075g/L KCl solution for swelling, mixed evenly, centrifuged for 5min at 1000r/min, 800. mu.L of supernatant is discarded, then 400. mu.L of stationary liquid (methanol: glacial acetic acid ═ 3: 1) is added for resuspension, centrifuged, the supernatant is discarded, then 600. mu.L of stationary liquid is added for resuspension, and the mixture is stood for 5min at room temperature, centrifuged for 5min, and 500. mu.L of supernatant is discarded. Then, 30. mu.L of the cell suspension was dropped on a clean slide glass and air-dried, and 200. mu.L of the prepared Giemsa working solution (Giemsa staining solution: Giemsa dilution: 1: 9) was slowly dropped on the slide glass to uniformly cover the stained area. After 8min, the staining solution was gently flushed with pure water from one end of the slide to the top down, and the slide was observed under a microscope and photographed.

The results are shown in fig. 5, and the effect of dfatinib on megakaryocyte morphology is observed by Giemsa staining, and it is found that when 0.5, 1, 1.5 μ M of dfatinib intervenes in HEL, Meg-01, K562 cells 6d, all three cells show multinucleate phenomenon, and a small amount of cells show 16N or more fold, compared with the control group, as shown in fig. 5.

(3) Coprinus cinereus cyclopeptide staining observation of influence of Defatinib on megakaryocyte morphology

Collecting HEL, Meg-01 and K562 cells cultured and intervened for 6d by the method in 4.1.4, centrifuging for 5min at 1000r/min, discarding the supernatant, rinsing with PBS, adding 1mL of 4% paraformaldehyde for fixing for 10min, and centrifuging for 3min at 700r/min by using a flaker to ensure that the cells are uniformly attached to a glass slide. And then PBS is added for washing for three times, 200 mu.L of 1 XTriton X-100 is added for permeabilization for 5min after natural air drying, and PBS is used for washing for three times. Naturally drying, dripping 200 μ L of 200nM TRITC-labeled phalloidin solution (containing 1% BSA) in dark, incubating for 30min, rinsing with PBS for three times, drying in the air, adding 100 μ L of DAPI for dyeing for 30s, recovering dye solution, drying in the air, sealing with DAPI-resistant quencher, and taking pictures under an inverted fluorescence microscope.

Microscopic observation of a phalloidin staining experiment shows that compared with a control group, after 0.5, 1 and 1.5 mu M Defatinib intervenes in HEL, Meg-01 and K562 cells for 6d, all three cells show multinucleate phenomena, as shown in figure 6.

4.1.6 flow cytometry detection of the Effect of Defacitinib on megakaryocyte surface antigen expression

Collecting HEL, Meg-01, K562 cells cultured and intervened for 6d in the method of 4.1.4, centrifuging at 1000r/min for 5min, discarding supernatant, adding 1mL precooled (4 ℃) PBS for rinsing, collecting 1 × 10⁵mu.L of FITC-CD41 and 5. mu.L of LPE-CD42b antibody were added to each cell, mixed well, and incubated on ice for 30min in the absence of light. The cells were transferred to a flow loading tube in the dark, 400. mu.L of pre-cooled PBS was added, and CD41/CD42b expression was detected by flow cytometry.

The experimental results show that compared with the control group, three cells CD41 are obtained after 0.5, 1 and 1.5 mu M of Defatinib intervenes HEL, Meg-01 and K562 cells for 6d⁺/CD42b⁺The expression rate was significantly increased, and the difference was statistically significant, as shown in fig. 7.

4.1.7 flow cytometry to examine the Effect of Defatinib on megakaryocyte DNA ploidy

HEL, Meg-01 and K562 cells cultured and intervened for 6d by the method in 4.1.4 are collected, centrifuged for 5min at 1000r/min, the supernatant is discarded, rinsed by PBS, fixed by adding 500 mu L of 70% ethanol, and kept in a refrigerator at 4 ℃ overnight. Centrifuging again, rinsing, discarding the supernatant, adding 100 μ L RNase A solution, mixing, incubating at 37 deg.C for 30min, placing on ice, adding 400 μ L PI staining solution in dark place, incubating at 4 deg.C for 30min, transferring to a flow-type sample loading tube, and detecting the DNA content of cells by using a flow cytometer.

Flow cytometry detection shows that after 0.5, 1 and 1.5 mu M Defatinib intervenes in HEL, Meg-01 and K562 cells for 6d, the proportion of diploid cells is reduced, the proportion of tetraploid and octaploid cells is increased, and the difference has statistical significance, as shown in FIG. 8.

4.2 Defatinib in vivo thrombopoietic assay

4.2.1 thrombocytopenia model establishment and administration

After one week of adaptive feeding, the mice were randomly divided into a control group, a model group, a TPO group, and low, medium and high dose groups of Defacitinib, each group containing 12 mice, each half of which is male and female. Except for the control group, 4Gy of X-ray was given for radiation molding. TPO group, low, medium and high dose groups of Defatinib were administered by intraperitoneal injection at doses of 3000U/kg, 2.5mg/kg, 5mg/kg and 10mg/kg, once daily for two weeks. The control group and the model group were given an equal amount of physiological saline.

4.2.2 detection index

(1) Peripheral platelet levels

Orbital bleeds were performed at 0, 4, 7, 10, 12, 14d post-dose, and groups of mice were tested for blood and platelet number changes were plotted using Graphpad prism 8.0.

The effect of Defatinib on the number of mouse platelets was found to be: the number of platelets in the mice in the 7d model group was lowest compared to the control group; the platelet recovery rate was significantly increased in the different concentrations of the dfatinib group and the positive group compared to the model group, as shown in fig. 9.

The test of peripheral blood platelet level and the index analysis of mouse organs were performed simultaneously with the above test of in vivo thrombopoiesis in Defatinib.

(2) Index of visceral organs

At the end of the experiment, the heart, spleen, liver, kidney, lung and thymus of the mice were taken and the organ index was calculated. The organ index formula is as follows:

organ index is organ weight/mouse weight x 100%

The result of the organ index analysis of the mouse shows that compared with the control group, the heart, liver, spleen, lung and kidney indexes of the mouse in the model group have no obvious change, and the thymus index is obviously reduced; compared with the model group, the thymus index of mice in different concentrations of the Defatinib group and the positive group is obviously increased (p is less than 0.001), and the index of other organs is not obviously different (p is more than 0.05), as shown in FIG. 10.

(3) Proportion of megakaryocyte progenitors in bone marrow

After the medium-dose Defatinib is administrated in an abdominal cavity for 12 days, a femur of a mouse is taken, bone marrow cells are flushed out, the femur is filtered by a nylon net, the femur is centrifuged for 5min at 1000r/min, 1mL of PBS is added for heavy suspension, 200 muL of 10 Xerythrocyte lysate is added to the 200 muL of the suspension for lysis for 15min until the cell suspension is red-free, the suspension is centrifuged for 5min at 1000r/min, the supernatant is discarded, and the PBS is used for rinsing. Adjusting the cell density to 1 × 10⁶And (2) adding 100 mu L of cell suspension into a new EP tube, adding 0.3 mu L of PE-CD117(c-Kit) and 0.5 mu L of FITC-CD41 antibody in a dark place, mixing uniformly, and incubating on ice for 25 min. Transfer to flow-up tubes and detect CD117/CD41 expression using flow cytometry.

Through the above experimental analysis, it was found that the effect of dfatinib on the proportion of megakaryocyte progenitor cells in mouse bone marrow is mainly shown in that the number of megakaryocyte progenitor cells in the model group is reduced (p <0.05) compared with the control group, and the number of megakaryocyte progenitor cells in the medium-dose drug group and the positive group is increased compared with the model group, and the difference has statistical significance (p <0.01), as shown in fig. 11.

(4) Megakaryocyte count in bone marrow

Collecting 100 mu L of cell suspension processed by the method in 4.2.2(3), adding a new EP tube, adding 0.5 mu L of FITC-CD41 and 0.5 mu L of PE-CD61 antibody in dark, mixing uniformly, and incubating on ice for 25 min. Transfer to flow loading tube and detect CD41/CD61 expression using flow cytometry.

The results of the experiments on the influence of Defatinib on the number of megakaryocytes in the bone marrow of mice are shown below.

(ii) Drifatinib on CD41 in mouse bone marrow cells⁺/CD61⁺Positive cell proportion influence

Model group versus control group CD41⁺/CD61⁺Positive cell proportion decrease (p)<0.05), medium dose Defatinib group and positive group vs model group, CD41⁺/CD61⁺The proportion of positive cells increases, and the difference has statistical significance (p)<0.01) as shown in fig. 12.

② observing the number of megakaryocytes in the bone marrow by adopting HE staining. After 12 days of administration, the femur of a mouse is taken, decalcified and embedded in paraffin, sliced, dewaxed by dimethylbenzene, dehydrated by 100%, 95%, 80% and 75% ethanol in sequence, stained by hematoxylin after washing by distilled water for 5min, washed by pure water to be purple blue, kept still in hydrochloric alcohol for 30s, washed by pure water, stained by eosin staining solution for 2min, washed by pure water, dehydrated by the ethanol with the concentration gradient, and transparent tissue of the dimethylbenzene, finally, sealed by neutral resin, and observed and photographed under an inverted microscope.

Through the experiments that the ② German Fatinib influences the number of megakaryocytes in the bone marrow of a mouse, the number of the megakaryocytes in a model group is obviously less than that in a control group; the megakaryocyte numbers of the medium-dose drug group and the positive group are obviously higher than those of the model group. The results indicate that dfatinib promoted megakaryocyte production in bone marrow of thrombocytopenic mice, as shown in fig. 13.

(5) Proportion of activated platelets in bone marrow

Collect 100. mu.L of cell suspension processed by the method in 4.2.2(3), add a new EP tube, add 0.5. mu.L of APC-CD62P antibody in dark, mix well, incubate for 25min on ice. Transfer to flow loading tube and detect CD62P expression using flow cytometry.

Through the research experiment of the influence of the Defatinib on the number of activated platelets in the bone marrow of the mice, the model group is discovered to be relative to the control group CD62P⁺Platelet rate reduction, medium dose Defatinib and positive vs model group, CD62P⁺The platelet fraction increased as shown in fig. 14.

The descriptions of each patent, patent application, and publication cited in this application are incorporated herein by reference in their entirety. Citation of any reference shall not be construed as an admission that such reference is available as "prior art" to the present application.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. Application of Defatinib in preparation of medicines for treating thrombocytopenia is provided.

2. Use according to claim 1, characterized in that the use of Defatinib in a medicament for the treatment of thrombocytopenia due to chemotherapy.

3. Use according to claim 1, wherein the use of Defatinib is in a medicament for the treatment of thrombocytopenia due to radiotherapy.

4. The use according to claim 1, wherein the use of dorfatinib is in a medicament for the treatment of thrombocytopenia due to tumor diseases.

5. Use according to claim 1, wherein the use of delfacitinib is in a medicament for the treatment of aplastic anemia, hyperfunction of the spleen, myelodysplastic syndrome, leukemia or thrombocytopenia due to systemic lupus erythematosus.

6. Use according to claim 1, characterized in that the use of dorfatinib in a medicament for the treatment of thrombocytopenia.

7. The use according to claim 1, wherein the use of dorfatinib is in a medicament for the treatment of thrombocytopenia due to myelomegakaryocytic developmental maturation disorder.

8. The use according to claim 1, wherein the use of Defatinib in the treatment of drug-induced immune thrombocytopenia is provided.

9. The use according to claim 1, wherein the use of Defatinib in the treatment of thrombocytopenic purpura is provided.