CN114344467A

CN114344467A - Application of BET protein inhibitor in preparation of chronic graft-versus-host disease drug

Info

Publication number: CN114344467A
Application number: CN202111584709.7A
Authority: CN
Inventors: 王荧; 程巧; 吴德沛; 徐杨; 刘吟; 张文丽; 雷蕾
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2021-12-22
Filing date: 2021-12-22
Publication date: 2022-04-15

Abstract

The invention belongs to the field of biomedicine, and particularly relates to application of a BET protein inhibitor in preparation of a chronic graft-versus-host disease medicine and a mouse model establishing method. The I-BET151 has the function of inhibiting chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation; can inhibit proliferation of donor T cell and T cell function, and inhibit Th1 cell reaction; inhibiting proliferation of dendritic cells; inhibit Tfh proliferation, promote thymocyte proliferation, and promote thymus immune reconstitution. The BET protein inhibitor can be used for preparing a medicine for treating chronic graft-versus-host disease, and provides a new idea for treating the chronic graft-versus-host disease.

Description

Application of BET protein inhibitor in preparation of chronic graft-versus-host disease drug

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to an application of a BET protein inhibitor in preparation of a medicament for resisting a host cell in chronic graft.

Background

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is currently an important approach for the treatment of hematological malignancies, certain inherited diseases, and bone marrow failure diseases. However, the occurrence of chronic graft-versus-host disease (cGVHD) is one of the major reasons that currently restrict the long-term survival and quality of life of patients after transplantation. The clinical manifestations of cGVHD are similar to those of autoimmune diseases, and are characterized by autoimmune vascular collagen diseases with multiple damaged organs, such as scleroderma, bronchiolitis obliterans, deposition of target organ alloantibodies, and the like, and the major involved organs include oral cavity, eyes, skin, reproductive tract, liver, lung, kidney, and digestive tract. The incidence rate of cGVHD reaches 30-70%, and the fatality rate reaches 30-50%. Compared with aGVHD, cGVHD has a more complex pathogenesis and a poorer therapeutic effect, and therefore, it is important to find an effective means for preventing and treating cGVHD.

The small molecule inhibitor I-BET151 is an isoxazole compound, can block combination of BET protein and acetylated lysine residues in a targeted manner, participates in chromosome modification, and plays an important role in regulating and controlling cell functions. Preclinical studies prove that the BET protein inhibitor I-BET151 has an obvious inhibition effect on the proliferation of human and mouse MLL fusion positive leukemia cell lines, and in vivo studies prove that the I-BET151 treatment can benefit the survival of two different mouse models with MLL fusion positive. It has been reported that I-BET151 inhibits proliferation of HEL cell lines and juvenile cells of erythroid isolated from polycythemia vera patients by highly down-regulating the LMO2 gene, and that its inhibitory effect is still present in the JAK2 inhibitor-resistant HEL cell line. Liu (Liu En)^[Etc. show that I-BET151 can inhibit ovarian cancer metastasis by inhibiting Stat3 signaling pathway, and that I-BET151 has significant anti-ovarian tumor activity. Studies by Sun et al show that the BET protein inhibitor I-BET151 can demonstrateThe inhibitor has the advantages of obviously inhibiting the functions of dendritic cells and T cells, and obviously reducing the severity of acute GVHD by early administration of short-range I-BET151 treatment in a mouse allo-HSCT model.

Compared with aGVHD, the pathogenesis of the cGVHD is more complex, the treatment effect is poorer, the glucocorticoid treatment response rate is only 50% -60%, the prognosis of refractory cGVHD patients is poor, adverse reactions such as diabetes, infection, osteoporosis and mental state abnormality can be caused by long-term use of the hormone, the life quality of the patients is influenced, and the lives of the patients are threatened by serious patients. And only the application of the BET protein inhibitor I-BET151 in preventing aGVHD in a mouse model.

In recent years, the research of targeting small molecule compounds that block the combination of BET proteins and acetylated lysine residues has led to great results. In 2010, the first BET protein small molecule inhibitor JQl appeared, but the drugability was inferior to that of the novel small molecule inhibitor I-BET synthesized in 2012. I-BET151 can obviously inhibit dendritic cell and T cell functions, and can reduce the severity of acute GVHD in a mouse allo-HSCT model, but the role of the I-BET151 in cGVHD is not reported so far.

Disclosure of Invention

The invention explores the application of a BET protein inhibitor I-BET151 in the preparation of a medicament for preventing and treating graft-versus-host disease through animal experimental research.

The invention provides an application of a BET protein inhibitor in preparation of a medicine for preventing and/or treating chronic graft-versus-host disease.

Preferably, the BET protein inhibitor is a small molecule inhibitor I-BET 151.

Preferably, the medicament is for inhibiting immune cells in a donor mesenteric lymph node.

Preferably, the immune cell is CD3⁺T cell, CD8⁺One or more of a T cell and a dendritic cell.

Preferably, the medicament is for inhibiting T cell function in the spleen.

Preferably, the medicament is for inhibiting the proportion of Tfh cells in the spleen.

Preferably, the medicament is used for reducing thoracic gland injury after transplantation.

The present invention also provides a medicament for preventing and/or treating chronic graft-versus-host disease comprising the BET protein inhibitor of

claim

1 or 2.

Further, the dosage of the BET protein inhibitor is 8-12 mg/kg.

Further, the administration mode of the medicine is one or more of oral administration, intraperitoneal injection, subcutaneous injection, intravenous injection and intramuscular injection.

Compared with the prior art, the technical scheme of the invention has the following advantages:

the I-BET151 has the function of inhibiting chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation; can inhibit proliferation of donor T cell and T cell function, and inhibit Th1 cell reaction; inhibiting proliferation of dendritic cells; inhibit Tfh proliferation, promote thymocyte proliferation, and promote thymus immune reconstitution.

Drawings

FIG. 1 shows the body weight change (A), cGVHD score (B), and diarrhea (C) after transplantation of mice.

FIG. 2 is H & E staining pattern of lung, kidney, intestine and skin tissues of Vehicle group and I-BET151 mice.

FIG. 3 is a graph of the inhibitory effect of I-BET151 on T cells and dendritic cells in mesenteric lymph nodes.

FIG. 4 is a graph of the inhibitory effect of I-BET151 on T cell function.

FIG. 5 is a graph of I-BET151 vs. Tfh cell inhibition in spleen cells.

FIG. 6 is a graph showing thymus tissue and thymocyte counts of Vehicle group and I-BET151 mice.

FIG. 7 is a graph of I-BET151 vs. CD4 in thymocytes⁺T，CD8⁺T cell promotion effect.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Example 1

Receptor mice (BALB/c, H-2)^d) The acidified water containing gentamicin (gentamicin 32 ten thousand units/liter) was drunk from 7 days before transplantation and was maintained until the end of the experiment.

Example 2

The donor mice were sacrificed by cervical dislocation, the body surface was sterilized with 70% ethanol, the mouse spleen was aseptically taken out in a clean bench, placed in a petri dish containing RPMI 1640 medium, gently ground with sterile rag glass slides until most of the cells had been ground out, and then filtered through a 200 mesh nylon mesh to 50ml clean centrifuge tubes to make single cell suspension. Appropriate RPMI 1640 medium was poured into the plate and washed once to avoid cell loss. Centrifuging, rotating at 1300 deg.C for 5min, pouring the waste liquid, adding 5ml erythrocyte lysate (the amount of the lysate is adjusted according to the number of cells), mixing by gentle blowing, lysing at room temperature for 5min, and adding large volume RPMI 1640 culture medium to stop the erythrolysis. Centrifugation was performed as above, the waste solution was poured, spleen cells were washed twice with an appropriate volume of PBS to resuspend the cells, then the cells were resuspended with an appropriate amount of PBS, and the cells were counted and placed on ice for use (cells were infused within 2 hours from the donor mice ex vivo).

Example 3

Aseptically taking the femur, tibia and spine of a donor mouse in a super clean bench, placing the donor mouse in a mortar containing RPMI 1640 medium, gently grinding the donor mouse by using a pestle head until most cells are ground out, filtering the donor mouse by using a nylon net until a 50ml clean centrifuge tube obtains single cell suspension, pouring an appropriate amount of RPMI 1640 medium into the mortar, and rinsing the single cell suspension once to avoid cell loss. Centrifuging, pouring the waste liquid, adding 5ml of the split red liquid (the amount of the split red liquid is adjusted according to the number of cells), lightly blowing, mixing, splitting at room temperature for 7min, and adding a large volume of RPMI 1640 culture medium to stop the split red. Centrifugation was performed as above, the waste solution was poured, bone marrow cells were washed twice with an appropriate volume of PBS to resuspend the cells, then the cells were resuspended with an appropriate amount of PBS, and counted and placed on ice for future use (cells were infused within 2 hours from the donor mice ex vivo).

Example 4

The recipient mice received a lethal dose of total body radiation (X-ray 6.5Gy, 3.25Gy each, two) one day prior to transplantationInterval of 2 hours between sub-irradiations). On the day of transplantation, mice-derived whole splenocytes were injected via tail vein injection at 5X 10⁵ 1X 10 of bone marrow cells⁷One (total volume 200. mu.l).

Example 5

I-BET151(100 mg/piece, from Selleck, USA) was dissolved in 1ml sterile anhydrous ethanol, mixed well, and each 20ul of the above solution was dispensed into an EP tube containing 1.5ml sterile PBS, mixed well and stored in a refrigerator at-20 ℃. The control group was PBS containing an equal amount of absolute ethanol. It is generally believed that relatively significant cGVHD expression was observed approximately 21 days after transplantation, so mice were randomized into two groups on day 21 post-transplantation, the experimental group started to be injected I-BET151 intraperitoneally at a dose of 10mg/kg, and the control group was given equal volumes of solvent. I-BET151 treatment was performed every three days starting on day 21 after transplantation, and the observation endpoints were set to day 30, day 45, and day 60 after transplantation, respectively.

Example 6

The survival condition and clinical manifestations of the mice are observed every day after transplantation, the cGVHD scoring is carried out from the 21 st day after transplantation, the scoring is carried out once every 3 days, the main observation indexes comprise weight, back, hair loss, activity, diarrhea and other conditions, and the specific scoring standard for cGVHD evaluation is shown in table 1.

TABLE 1 cGVHD clinical scoring criteria after mouse allo-HSCT

Note: GVHD is determined when the crusting or desquamation of each part of sole, tail and ear is counted for 0.3 point, and the skin score exceeds 0.6 point or the total score is more than 2 points; GVHD is considered to occur even if symptoms subside, mice die naturally, or death is caused by human factors; asymptomatic death of mice was considered to be GVHD free.

Example 7

On 30 days, 45 days and 60 days after transplantation, the skin, lung, kidney and small intestine of the mouse are collected after the mouse is killed by cervical dislocation (materials are taken to avoid tissue damage caused by human factors), and the tissue is put into 10% pre-prepared formalin for fixation overnight as soon as possible after being isolated. Replacing water in tissue with increasing concentration of alcohol as dehydrating agent, placing tissue in xylene to be transparent to replace alcohol in tissue, embedding with paraffin and cutting into slices with thickness of about 5 μm, removing paraffin in slices with xylene, gradient alcohol hydrating, staining with hematoxylin-eosin, and sealing with gum. Pictures of HE sections were taken using an Olympus upright fluorescence microscope (japan) at 100-fold magnification. The target organ is then assessed by an experienced pathologist under single-blind conditions. The skin cGVHD pathological observation shows that the epidermis is slightly hyperplastic, the subcutaneous fat is reduced, the hair follicle is reduced, inflammatory cells are increased, collagen deposition and the like; lung cGVHD pathological observation of lung interstitial hyperplasia, inflammatory cell infiltration and the like; small intestine cGVHD pathological observation villus structure destruction, basement membrane continuity interruption, intestinal submucosa lymphocyte infiltration and the like; renal cGVHD pathology observes glomerulosclerosis, focal necrosis, etc.

Example 8

Killing mice by adopting a cervical dislocation method, disinfecting the body surface by using 70% alcohol, sequentially taking out thymus, spleen and mesenteric lymph nodes, placing the thymus, the spleen and the mesenteric lymph nodes in a glass plate containing RPMI 1640, gently and fully grinding the thymus, the spleen and the mesenteric lymph nodes by using a rough edge glass sheet until most cells are ground out, and then filtering the ground cells to a 50ml centrifuge tube by using a 200-mesh nylon net to obtain a single cell suspension. Appropriate RPMI 1640 medium was poured into the plate and washed once to avoid cell loss. Centrifuging, transferring at 1300 deg.C for 5min, pouring waste liquid, adding 5ml of schizophyllic solution (adjusted according to cell number) into thymus and spleen, mixing by gentle blowing, lysing at room temperature for 5min (mesenteric lymph node does not have schizophyllic), and stopping with large volume RPMI 1640 medium. And (3) centrifuging the same as above, pouring the waste liquid, washing the cells once by using an appropriate volume of RPMI 1640 culture medium, centrifuging the same as above, pouring the waste liquid, re-suspending the cells by using a certain volume of RPMI 1640 culture medium containing 10% FBS, sucking 10ul of cell suspension, counting the cell suspension by using a full-automatic counter, and placing the rest cells in a refrigerator at 4 ℃ for later use.

Example 9

(1) Cell membrane staining: spreading the prepared single cell suspension of thymus, spleen and mesenteric lymph node into 96-well flat bottom plate with 1/well10⁶Centrifuging the cells, rotating at 1300 ℃ for 5min, and pouring waste liquid. Add FACS buffer 100ul containing CD16/CD32 antibody (1:400 configuration), block FcR for 20min at 4 ℃ with a discharge gun, wash once with FACS buffer, add FACS buffer 100ul containing cell surface marker antibody (set up staining protocol according to different channel combinations), gently blow, mix well, incubate at 4 ℃ for 30min in the dark. Centrifugation was performed as above, the waste solution was poured, washed twice with FACS buffer, resuspended in 200. mu.l of FACS buffer, and then examined by an Essen flow cytometer. And (5) performing negative control tube and single staining tube adjustment fluorescence compensation at the same time, and performing data analysis by using Flowjo software.

(2) Intracellular staining: the prepared spleen cell suspension was plated in a 96-well flat bottom plate at approximately 2X 10 per well⁶Cells were centrifuged, 1300 rpm, 4 ℃ for 5min, the waste solution was decanted, 200ul of stimulating medium containing LPS (50ng/ml) + PMA (50ng/ml) + Ionomycin (500ng/ml) + BFA (10. mu.g/ml) or PMA (50ng/ml) + Ionomycin (1ug/ml) + BFA (10. mu.g/ml) was added to the well plate and mixed, followed by stimulation in a 37 ℃ cell incubator for 4-6 hours. The cells were centrifuged as above, the waste was poured, and 100ul of FACS buffer containing CD16/CD32 antibody (1:400 configuration) was added after washing with FACS buffer, mixed well and placed in a refrigerator at 4 ℃ to block FcR for 20 min. Centrifugation, as above, was performed by pouring the waste solution, washing the solution once with FACS buffer, adding 100ul FACS buffer containing cell surface marker antibody (staining protocol was set according to different channel combinations), resuspending, and incubating at 4 ℃ for 30min in the dark. The waste was centrifuged as above, the waste was poured out, washed twice with FACS buffer, and then fixed to rupture membranes using the Fixation/Permeabilization Kit, 100. mu.l of the Fixation/Permeabilization Solution was added to each well, and the mixture was placed in a refrigerator at 4 ℃ for 30 min. The waste was centrifuged as above, and washed 2 times by adding 200. mu.l of membrane disruption Buffer (Permeabilization Buffer diluted 1 Xwith deionized water before use). Intracellular antibody (prepared by membrane rupture buffer) is added, and the mixture is incubated for 30min at 4 ℃ in the dark. The resulting mixture was centrifuged as above, and the resulting waste liquid was poured and washed with 200. mu.l of a membrane-broken solution for 2 times. 200ul FACS buffer was resuspended and examined on an Essen flow cytometer. And (5) performing negative control tube and single staining tube adjustment fluorescence compensation at the same time, and performing data analysis by using Flowjo software.

(3) Treg cell staining, spreading spleen cell suspension to 96-wellIn the flat bottom plate, each hole is laid with 2X 10⁶Counting cells, centrifuging, transferring at 1300 deg.C for 5min, pouring waste liquid, adding FACS buffer solution containing CD16/CD32 antibody 100ul, blowing gently, mixing, and sealing FcR in refrigerator at 4 deg.C for 20 min. The cells were centrifuged as above, the waste solution was poured off, washed once with FACS buffer, 100ul of FACS buffer containing the cell surface marker antibody was added, resuspended, and incubated at 4 ℃ in the dark for 30 min. Centrifugation was performed as above, the waste was poured, the FACS buffer was washed twice, and then the nuclear membrane was fixed with FoxP3 stabilizing kit. Adding 200 mul of fixing/membrane-breaking working solution (working solution diluted by concentrated solution and diluent according to the volume of 1: 3) into each hole, mixing uniformly, and incubating for 30 minutes at room temperature in a dark place. The mixture was centrifuged at 1300 ℃ for 5min, the waste was decanted and washed twice with 200. mu.l of membrane disruption Buffer (Permeabilization Buffer diluted 1 Xwith deionized water before use). Foxp3 antibody (in membrane rupture buffer, 1:200) was added and incubated for 30min at room temperature in the absence of light. Centrifuging for 5min at 1300 rpm, pouring waste liquid, washing with rupture buffer for 2 times, centrifuging for 5min at 1300 rpm, pouring waste liquid, resuspending with FACS buffer solution 200 μ l, and detecting with an AINSON flow cytometer. And (5) performing negative control tube and single staining tube adjustment fluorescence compensation at the same time, and performing data analysis by using Flowjo software.

Example 10

Analysis and mapping were performed using GraphPad Prism 8 software. The mean comparison of two samples adopts unpaired t test, and the mean comparison of multiple groups of samples adopts variance analysis. Survival curves were plotted using the Kaplan-Meier method and log-rank was used to test for differences between the two groups. Differences of P <0.05 were statistically significant. P <0.05, P <0.01, and P < 0.01.

Effect evaluation 1

The effect of the drug treatment on mouse cGVHD was as follows:

all mice after TBI pretreatment developed radiosclerosis and decreased food intake, lost weight about one week after transplantation, were medicated with I-BET151 or control vehicle 21 days after transplantation, observed for cGVHD expression such as arch back, hair loss, motility, and diarrhea, and recorded for weight change. The control group showed diarrhea symptoms within one week after the administration, while the I-BET151 group showed no diarrhea at day 45 after the observation point transplantation. The I-BET151 group showed lower weight loss and lower cGVHD score than the vehicle group, as shown in FIG. 1. At three observation points, 30 days, 45 days and 60 days after transplantation, lung, kidney, intestinal tract and skin tissues of the mouse are collected for pathological H & E staining. The results indicate that the interstitial congestion and edema of lung in the group I-BET151 have the effects of obviously relieving inflammatory cell infiltration, obviously widening alveolar space, retaining part of lung structure and reducing inflammatory cell infiltration; the glomerulus structure is complete, and the glomerulus is hardened and atrophied slightly; the intestinal tract shows that villi of the small intestine are broken, the structure is complete, and the crypt structure is reserved; dermal fibrosis, fat loss, inflammation and epidermal interfacial changes in the skin were significantly more severe than in the control group, see fig. 2.

Effect evaluation 2

The effect of the medicine on each immune cell in mesenteric lymph node is as follows:

phenotypic changes of various immune cells in mesenteric lymph nodes of mice were examined on day 30 post-transplantation. The results show that I-BET151 can obviously reduce CD3 in mesenteric lymph nodes of mice⁺Proportion of T cells, in which CD8 is expressed⁺The decrease in T cells was more pronounced, and in addition the proportion of dendritic cells in the I-BET151 group was also decreased (P ═ 0.05). The ratio of B cells to macrophages was not significantly different in the two groups, as shown in FIG. 3.

Effect evaluation 3

The effect of the medicine for inhibiting the function of the T cell is as follows:

and (3) detecting the secretion of cytokines such as IFN-gamma, IL-4, IL-17A and the like of the spleen T cells of the mice at the 30 th day after transplantation. It can be seen that I-BET151 can significantly inhibit CD4 in spleen⁺T cells secreted IFN-. gamma. (FIG. 4), whereas there was no significant difference in the secretion of IL-4, IL-17A. Therefore, the I-BET151 has obvious inhibition capacity on the allogeneic T cell response, and the percentage of Th1 cells in the spleen of a mouse is obviously reduced.

Effect evaluation 4

The percentage effect of the drug on inhibition of Tfh cells was as follows:

t cells in the spleen of mice 60 days after transplantation were detectedThe change in cell and B cell subsets, the results showed that the two groups are CD3⁺T、CD4⁺T、Treg、Tfh、PD-1⁺CD4⁺T, Tfr, PD-1+ Treg, B cell, CD24^hiCD38^hiThe difference in Breg ratios was not statistically significant, but it was seen that Tfh in spleen in the I-BET151 group tended to decrease, suggesting that I-BET151 could mitigate cGVHD by decreasing the ratio of Tfh, as shown in fig. 5.

Effect evaluation 5

The effect of the medicine for reducing the damage of the thymus of the mouse after transplantation is as follows:

thymus of mice 60 days after transplantation was extracted and analyzed, and it was found that the absolute count of total thymocytes was significantly higher in the I-BET151 group than in the control group. And CD3 in thymus of group I-BT151⁺The proportion and the quantity of T are obviously increased, CD4⁺T，CD8⁺The absolute number of T cells also increased significantly, as shown in fig. 6 and 7.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims

Use of a BET protein inhibitor for the manufacture of a medicament for the prophylaxis and/or treatment of chronic graft-versus-host disease.
2. The use of claim 1, wherein the BET protein inhibitor is the small molecule inhibitor I-BET 151.
3. The use of claim 1, wherein the medicament is for inhibiting immune cells in a donor mesenteric lymph node.
4. Use according to claim 3, characterized in thatThe immune cell is CD3⁺T cell, CD8⁺One or more of a T cell and a dendritic cell.
5. The use according to claim 1, wherein the medicament is for inhibiting T cell function in the spleen.
6. The use of claim 1, wherein the medicament is for inhibiting the proportion of Tfh cells in the spleen.
7. The use of claim 1, wherein the medicament is for reducing thoracic gland injury after transplantation.
8. A medicament for preventing and/or treating chronic graft-versus-host disease, comprising the BET protein inhibitor of claim 1 or 2.
9. The medicament of claim 8, wherein the BET protein inhibitor is administered in a dose of 8 to 12 mg/kg.
10. The medicament of claim 8, wherein the medicament is administered by one or more of oral administration, intraperitoneal injection, subcutaneous injection, intravenous injection, and intramuscular injection.