CN111265664B - Composition for preventing and treating acute graft-versus-host disease - Google Patents

Composition for preventing and treating acute graft-versus-host disease Download PDF

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CN111265664B
CN111265664B CN201911183636.3A CN201911183636A CN111265664B CN 111265664 B CN111265664 B CN 111265664B CN 201911183636 A CN201911183636 A CN 201911183636A CN 111265664 B CN111265664 B CN 111265664B
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wdr5
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CN111265664A (en
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马洁娴
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Huadong Hospital
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Abstract

The invention provides a composition for preventing and treating acute graft-versus-host disease, which belongs to the field of compositions for treating graft-versus-host disease and comprises the following components in parts by weight: WDR5 inhibitor: 1 part, DOT1L inhibitor: 1 part; WDR5 inhibitors include WDR5-MLL inhibitors, which inhibit histone methyltransferase MLL1 from its catalytic complex WDR5 protein-protein interaction by WDR5 inhibitors, histone H3 lysine 79 methyltransferase DOT1L by DOT1L inhibitors; the two medicines are used together to play a synergistic effect, the action time is short, the side effect is slight, and the curative effect maintenance time is long; the lymphocyte after organ transplantation is reduced to differentiate towards Th1 and Th17 effector cells, and certain differentiation is maintained, so that the infection resistance and graft anti-tumor effect after transplantation are prevented from being influenced.

Description

Composition for preventing and treating acute graft-versus-host disease
Technical Field
The invention belongs to the field of compositions for treating graft-versus-host disease, and particularly relates to a composition for preventing and treating acute graft-versus-host disease and application thereof.
Background
Graft-versus-host disease is due to the fact that T lymphocytes in allogenic donor grafts after transplantation are stimulated by a series of 'cytokine storms' initiated by the recipients, immune response to recipient antigens is greatly enhanced, cytotoxic attack is initiated by targeting recipient target cells, wherein skin, liver and intestinal tracts are main target targets. According to the time when the graft-versus-host disease occurs after transplantation, if the patient has acute graft-versus-host disease within 100 days, the patient has chronic graft-versus-host disease after 100 days; the incidence rate of acute graft-versus-host disease is 30-45%, the acute graft-versus-host disease occurs in the early stage after transplantation, the acute graft-versus-host disease is one of important complications of early death after transplantation, and the incidence rate of chronic graft-versus-host disease is lower than that of acute graft-versus-host disease.
At present, the drugs for treating acute rejection are all immunosuppressants, such as cyclosporin, tacrolimus and the like, the drugs have relatively serious nephrotoxicity, the drugs are required to be used for a long time, the immunosuppressive effect of the drugs disappears after the drugs are stopped, and part of acute rejection cannot be controlled.
Another class of drugs, such as sully (CD25 mab), eliminates a large number of effector T cells that are produced during acute rejection, which, although slightly more effective than traditional immunosuppressive agents, also require repeated administrations. Late infection complications are high due to the massive clearance of effector T cells, and severely affect graft versus tumor effects.
WDR5-MLL inhibitors and DOT1L inhibitors are currently in clinical trials for the treatment of tumors, such as NCT02141828, NCT03701295, (DOT1L inhibitor) on clinicalterials, WDR5-MLL inhibitors published on Nature Chemical Biology volume 11, pages 571-578 (2015) for the treatment of acute leukemia, and WDR5 MLL inhibitors are currently used in the treatment of acute GVHD and have not been tried worldwide.
Disclosure of Invention
In view of the disadvantages of the prior art, it is an object of the present invention to provide a composition for preventing and treating acute graft-versus-host disease.
In order to achieve the above purpose of the present invention, the present invention provides the following technical solutions: a composition for preventing and treating acute graft-versus-host disease comprises the following components in parts by weight;
WDR5 inhibitor: 1-8 parts;
DOT1L inhibitor: 1-8 parts.
Preferably, the composition comprises the following components in parts by weight;
WDR5 inhibitor: 1-5 parts;
DOT1L inhibitor: 1-5 parts.
Preferably, the composition comprises the following components in parts by weight;
WDR5 inhibitor: 3-5 parts;
DOT1L inhibitor: 1 part.
Preferably, the composition comprises the following components in parts by weight;
WDR5 inhibitor: 5 parts of a mixture;
DOT1L inhibitor: 1 part.
Preferably, the WDR5 inhibitor comprises a WDR5-MLL inhibitor.
The invention also aims to provide application of the composition for preventing and treating acute graft-versus-host disease.
In order to achieve the above purpose of the present invention, the present invention provides the following technical solutions: use of a WDR5 inhibitor and a DOT1L inhibitor for the preparation of a composition for the prevention and treatment of acute graft-versus-host disease.
Through the research of the inventor, the DOT1L inhibitor is only injected into mice, so that the acute GVHD cannot be reduced, and the DOT1L inhibitor and the WDR5 MLL inhibitor are only injected in a combined manner, so that the acute GVHD can be reduced, and the effect of treating the acute GVHD is achieved.
In summary, compared with the prior art, the invention has the following beneficial effects:
(1) the composition of the invention is an epigenetic inhibitor and acts on
Figure BDA0002291894370000021
The differentiation of T cells to effector cells is convenient for reducing the differentiation of lymphocytes to Th1 and Th17 effector cells after organ transplantation, but certain differentiation is still maintained, so the anti-infection capability and graft anti-tumor effect after transplantation are not seriously influenced;
(2) the epigenetic inhibitor can be used for inheriting the genetic information to the next generation at the stage of immune cell differentiation and proliferation, so that the composition has the characteristics of short application time, slight side effect, long curative effect maintaining time and the like;
(3) the composition provided by the invention can be used for preventing acute rejection in organ transplantation and treating acute rejection after organ transplantation.
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Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a schematic diagram showing the proportions of Th1, Th17 and Treg cells of a mouse after the composition for preventing and treating acute graft-versus-host disease of example 1 is applied to an in vitro test of a mixed lymphocyte reaction system;
FIG. 2 is a graph showing the survival time of mice after implantation of donor lymphocytes using OICR-9429 or EPZ5676 or a combination of both drugs in a non-myeloablative xenograft model of mice in the compositions for preventing and treating acute graft versus host disease of examples 1-2 and comparative examples 1-4;
fig. 3 is a HE staining of liver and intestinal tract of a mouse injected with two compounds, a sheet preparation diagram, of the composition for preventing and treating acute graft versus host disease of example 1;
fig. 4 is a ratio diagram of Th1, Th17, Treg cells of mouse spleen slurry after injection of the composition in the composition for preventing and treating acute graft versus host disease of example 1;
FIG. 5 is a schematic view showing the screening process of components of the composition for preventing and treating acute graft versus host disease according to examples 1 to 8.
Detailed Description
The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention. The present invention will be described in detail with reference to the following specific examples:
as shown in fig. 5, compounds that are likely to treat acute rejection were screened from a wide variety of compounds: a mixed lymphocyte reaction is constructed by selecting an epidemic compound library of selelck company, wherein the library comprises 464 compounds, and in vitro experiments, lymphocytes of Balb/C mice (inactivated by mitomycin and used as antigen presenting cells) and lymphocytes of C57BL/6 mice are mixed. Adding various compounds, incubating for 2 days, and detecting the ratio of Th1, Th17 and Treg cells in lymphocyte mixture by flow cytometry (PS: the main effector cells of acute rejection are Th1 cells and Th17 cells, and are mainly Th17 cells
Figure BDA0002291894370000031
T cells received HLA differential antigens and differentiated towards effector). Observation of which compounds reduced the number of Th1 and Th17 cells, but also reduced Treg cellsThe number of cells has little effect. Through in vitro experimental screening, WDR5-MLL inhibitor and DOT1L inhibitor become candidate compounds, and the effects of the compounds are further verified in experiments.
FIG. 1 shows the in vitro assay of WDR5-MLL inhibitor (represented by OICR-9429) and DOT1L inhibitor (represented by EPZ5676) applied at different doses to a mixed lymphocyte reaction system in an in vitro assay (in which case, the lymphocytes undergo proliferative differentiation,
Figure BDA0002291894370000032
t cells are differentiated into Th1, Th17 and Treg cells, the proportions of Th1, Th17 and Treg cells are detected after different doses of drug are incubated by using flow cytometry, and the inventor finds that as the doses of WDR5-MLL inhibitor and DOT1L inhibitor are increased,
Figure BDA0002291894370000033
the differentiation of T cells to Th1 and Th17 is gradually reduced;
FIG. 2 is a graph showing that the survival time of a mouse is remarkably prolonged by establishing a mouse non-myeloablative allogeneic transplantation model and then observing the survival time of the mouse after implanting donor lymphocytes and using OICR-9429 or EPZ5676 or using the two drugs in combination, and the survival time of the mouse is remarkably prolonged after the two drugs are used in combination and reach a certain dosage;
FIG. 3 mice injected with both compounds were sacrificed and liver and intestine were HE stained and tabletted, and we found that liver, compared to the non-dosed group, had significantly reduced intestinal acute GVHD.
FIG. 4 shows that when mice injected with two compounds are sacrificed, spleens are ground and flow cytometry is used for detecting the proportions of Th1, Th17 and Treg cells, and we find that compared with the medicine group without application of Th1, Th17 cells are obviously reduced, but certain differentiation is still maintained, so that certain anti-infection capacity and anti-tumor cell activity are still maintained;
detection method and detection index
1. A test method for preventing acute graft-versus-host reaction after organ transplantation.
In organ transplantation, after donor organs are implanted into a recipient, mice of different species are selected as the recipient and the donor respectively for allogeneic bone marrow transplantation. The acute graft-versus-host disease caused by bone marrow transplantation is quite severe in mice of different species because of HLA incompatibility. In addition to acute rejection caused by skin transplantation, which may be more severe than bone marrow transplantation, compared to other organ transplants, acute rejection caused by other organ transplants is all milder than bone marrow transplantation. Therefore, allogeneic bone marrow transplantation in mice was selected as a model to verify the effect of the compounds.
After the hematopoietic stem cells and lymphocytes of a rat are implanted into a receiving rat, the receiving rat is injected with one part of WDR5-MLL inhibitor and one part of DOT1L inhibitor by means of intravenous administration, intraperitoneal administration or oral administration and the like, and the injections are continuously performed for seven days.
2. A detection method for treating acute rejection after organ transplantation.
Similarly, using a mouse model of allogeneic bone marrow transplantation as a subject, after acute rejection of mice (about 7 to 10 days after transplantation) occurred after transplantation of hematopoietic stem cells and lymphocytes into the recipient mice (mice were shown to have inverted hair, arch back, etc.), the recipient mice were injected with a dose of WDR5-MLL inhibitor and a dose of DOT1L inhibitor for seven consecutive days after transplantation of hematopoietic stem cells and lymphocytes from the recipient mice.
3. Detecting the index
Th1 cell, Th17 cell, median survival time, intestinal pathology.
Examples 1 to 8 and comparative examples 1 to 8
Example 1:
a composition for preventing and treating acute graft-versus-host disease comprises the following components in parts by weight;
WDR5 inhibitor: 1 part;
DOT1L inhibitor: 1 part;
WDR5 inhibitors include WDR5-MLL inhibitors.
In organ transplantation, after donor organs are implanted into a recipient, mice of different species are selected as the recipient and the donor respectively for allogeneic bone marrow transplantation. The acute graft-versus-host disease caused by bone marrow transplantation is quite severe in mice of different species because of HLA incompatibility. In addition to acute rejection caused by skin transplantation, which may be more severe than bone marrow transplantation, compared to other organ transplants, acute rejection caused by other organ transplants is all milder than bone marrow transplantation. Therefore, allogeneic bone marrow transplantation in mice was selected as a model to verify the effect of the compounds.
After the hematopoietic stem cells and lymphocytes implanted into the mice enter the mice, the mice are injected with one part of WDR5-MLL inhibitor and one part of DOT1L inhibitor by intravenous administration, intraperitoneal administration or oral administration, wherein the ratio of the addition amount of each part to the weight of the mice is 1.4mg/kg, and the injections are continuously injected for seven days.
The survival state of the mice after the injection of the drug is found to be good, and the median survival time is 6 to 7 times that of the group without the injection of the drug. And the discovery shows that the number of effector cells Th1 cells and Th17 cell drug injection groups which can cause acute graft-versus-host disease in the lymphoid tissues of mice is obviously reduced compared with the drug-free groups, and the number of intestinal pathological drug injection groups in intestinal tissues which are easy to have acute rejection is obviously reduced compared with the drug-free groups.
Example 2:
a composition for preventing and treating acute graft-versus-host disease comprises the following components in parts by weight;
WDR5 inhibitor: 1 part;
DOT1L inhibitor: 1 part;
the WDR5 inhibitor comprises WDR5-MLL inhibitor, and the ratio of the addition amount of each part to the weight of the mouse is 1.4 mg/kg.
The same mouse model using allogeneic bone marrow transplantation as in example 1 was studied, and after the hematopoietic stem cells and lymphocytes of the donor mice had entered the recipient mice, the mice exhibited acute rejection (about 7-10 days after the transplantation), with inverted hair, dorsal arch, etc., and after about one week of transplantation of the hematopoietic stem cells and lymphocytes of the donor mice, the recipient mice were injected with a dose of WDR5-MLL inhibitor and a dose of DOT1L inhibitor for seven consecutive days, and the median survival time of the drug injection group was found to be 3-4 times longer than that of the drug-free group, and the drug injection groups of Th1 and Th17 cells were found to be significantly shorter than that of the drug-free group, and the drug injection group of intestinal pathology was significantly shorter than that of the drug-free group.
Example 3:
a composition for preventing and treating acute graft-versus-host disease comprises the following components in parts by weight;
WDR5 inhibitor: 3 parts of a mixture;
DOT1L inhibitor: 1 part;
the WDR5 inhibitor comprises WDR5-MLL inhibitor, and the ratio of the addition amount of each part to the weight of the mouse is 1.4 mg/kg.
The same mouse model using allogeneic bone marrow transplantation as in example 1 was studied, and after the hematopoietic stem cells and lymphocytes of the donor mice had entered the recipient mice, the mice exhibited acute rejection (about 7-10 days after the transplantation), i.e., inverted hair, dorsal arch, etc., and after about one week of transplantation of the hematopoietic stem cells and lymphocytes of the donor mice, the recipient mice were injected with three portions of WDR5-MLL inhibitor and one portion of DOT1L inhibitor for seven consecutive days, and the median survival time of the drug injection group was found to be 5-6 times longer than that of the drug non-injection group, and the drug injection groups of Th1 and Th17 were found to be significantly shorter than that of the drug non-injection group, and the drug injection group of the intestinal pathology was significantly shorter than that of the drug non-injection group.
Example 4:
a composition for preventing and treating acute graft-versus-host disease comprises the following components in parts by weight;
WDR5 inhibitor: 5 parts of a mixture;
DOT1L inhibitor: 1 part;
the WDR5 inhibitor comprises WDR5-MLL inhibitor, and the ratio of the addition amount of each part to the weight of the mouse is 1.4 mg/kg.
The same mouse model using allogeneic bone marrow transplantation as in example 1 was studied, and after the hematopoietic stem cells and lymphocytes of the donor mice had entered the recipient mice, the mice exhibited acute rejection (about 7-10 days after the transplantation), with inverted hair, dorsal arch, etc., and after about one week of transplantation of the hematopoietic stem cells and lymphocytes of the donor mice, the recipient mice were injected with five WDR5-MLL inhibitors and one DOT1L inhibitor for seven consecutive days, and the median survival time of the drug injection group was found to be 6 times longer than that of the drug non-injection group, and the drug injection groups of Th1 and Th17 cells were found to be significantly shorter than that of the drug non-injection group, and the drug injection group of intestinal pathology was significantly shorter than that of the drug non-injection group.
Example 5:
a composition for preventing and treating acute graft-versus-host disease comprises the following components in parts by weight;
WDR5 inhibitor: 8 parts of a mixture;
DOT1L inhibitor: 1 part;
the WDR5 inhibitor comprises WDR5-MLL inhibitor, and the ratio of the addition amount of each part to the weight of the mouse is 1.4 mg/kg.
The same mouse model using allogeneic bone marrow transplantation as in example 1 was studied, and after the hematopoietic stem cells and lymphocytes of the donor mice had entered the recipient mice, the mice exhibited acute rejection (about 7-10 days after the transplantation), with inverted hair, dorsal arch, etc., and after about one week of transplantation of the hematopoietic stem cells and lymphocytes of the donor mice, the recipient mice were injected with five WDR5-MLL inhibitors and one DOT1L inhibitor for seven consecutive days, and the median survival time of the drug injection group was found to be 3 times longer than that of the drug non-injection group, and the median survival time of the drug injection group was found to be significantly shorter than that of the drug non-injection group, and the drug injection group for Th1 and Th17 cells was found to be significantly shorter than that of the drug non-injection group, and the drug injection group for intestinal pathology was significantly shorter than that of the drug non-injection group.
Example 6:
a composition for preventing and treating acute graft-versus-host disease comprises the following components in parts by weight;
WDR5 inhibitor: 1 part;
DOT1L inhibitor: 3 parts of a mixture;
the WDR5 inhibitor comprises WDR5-MLL inhibitor, and the ratio of the addition amount of each part to the weight of the mouse is 1.4 mg/kg.
The same mouse model using allogeneic bone marrow transplantation as in example 1 was studied, and after the hematopoietic stem cells and lymphocytes of the donor mice had entered the recipient mice, the mice exhibited acute rejection (about 7-10 days after the transplantation), with inverted hair, dorsal arch, etc., and after about one week of transplantation of the hematopoietic stem cells and lymphocytes of the donor mice, the recipient mice were injected with five WDR5-MLL inhibitors and one DOT1L inhibitor for seven consecutive days, and the median survival time of the drug injection group was found to be 4 times longer than that of the drug non-injection group, and the drug injection groups of Th1 and Th17 cells were found to be significantly shorter than that of the drug non-injection group, and the drug injection group of intestinal pathology was significantly shorter than that of the drug non-injection group.
Example 7:
a composition for preventing and treating acute graft-versus-host disease comprises the following components in parts by weight;
WDR5 inhibitor: 1 part;
DOT1L inhibitor: 5 parts of a mixture;
the WDR5 inhibitor comprises WDR5-MLL inhibitor, and the ratio of the addition amount of each part to the weight of the mouse is 1.4 mg/kg.
The same mouse model using allogeneic bone marrow transplantation as in example 1 was studied, and after the hematopoietic stem cells and lymphocytes of the donor mice had entered the recipient mice, the mice exhibited acute rejection (about 7-10 days after the transplantation), with inverted hair, dorsal arch, etc., and after about one week of transplantation of the hematopoietic stem cells and lymphocytes of the donor mice, the recipient mice were injected with five WDR5-MLL inhibitors and one DOT1L inhibitor for seven consecutive days, and the median survival time of the drug injection group was found to be 3 times longer than that of the drug non-injection group, and the median survival time of the drug injection group was found to be significantly shorter than that of the drug non-injection group, and the drug injection group for Th1 and Th17 cells was found to be significantly shorter than that of the drug non-injection group, and the drug injection group for intestinal pathology was significantly shorter than that of the drug non-injection group.
Example 8:
a composition for preventing and treating acute graft-versus-host disease comprises the following components in parts by weight;
WDR5 inhibitor: 1 part;
DOT1L inhibitor: 8 parts of a mixture;
the WDR5 inhibitor comprises WDR5-MLL inhibitor, and the ratio of the addition amount of each part to the weight of the mouse is 1.4 mg/kg.
The same mouse model using allogeneic bone marrow transplantation as in example 1 was studied, and after the hematopoietic stem cells and lymphocytes of the donor mice had entered the recipient mice, the mice exhibited acute rejection (about 7 to 10 days after the transplantation), with inverted hair, dorsal arch, etc., and after about one week of transplantation of the hematopoietic stem cells and lymphocytes of the donor mice, the recipient mice were injected with five WDR5-MLL inhibitors and one DOT1L inhibitor for seven consecutive days, and the median survival time of the drug injection group was found to be 2 to 3 times longer than that of the drug-free group, and the median survival time of the drug injection group was found to be significantly shorter than that of the drug-free group, Th1 and Th17 cell drug injection group, and the drug injection group of the intestinal pathology was significantly shorter than that of the drug-free group.
Comparative example 1
A composition for preventing and treating acute graft versus host disease, which is different from example 1 only in that the composition comprises the following components in parts by weight: 2 parts WDR5 inhibitor, no DOT1L inhibitor added; the rest of the operation was the same as in example 1.
The survival status of mice after drug injection was found: the median survival time is similar to that of the group without the injected medicament. And the Th1 cells which can cause acute graft-versus-host disease in the mouse lymphoid tissue are found to be similar to the drug injection group without drugs in the Th17 cell drug injection group, and the intestinal pathological drug injection group in the intestinal tissue which is easy to have acute rejection is similar to the drug injection group without drugs in the intestinal tissue.
The WDR5-MLL inhibitor is only used by a mouse, so that the survival time of the mouse cannot be prolonged, but the two compounds can be used together to prevent acute graft-versus-host disease and remarkably prolong the survival time of the mouse.
Comparative example 2
A composition for preventing and treating acute graft versus host disease, which is different from example 1 only in that the composition comprises the following components in parts by weight: 2 parts DOT1L inhibitor, no WDR5 inhibitor added; the rest of the operation was the same as in example 1.
The survival status of mice after drug injection was found: the median survival time is similar to that of the group without the injected medicament. And the Th1 cells which can cause acute graft-versus-host disease in the mouse lymphoid tissue are found to be similar to the drug injection group without drugs in the Th17 cell drug injection group, and the intestinal pathological drug injection group in the intestinal tissue which is easy to have acute rejection is similar to the drug injection group without drugs in the intestinal tissue.
The fact that the survival time of the mice cannot be prolonged only by using the DOT1L inhibitor is shown, but the two compounds can prevent acute graft-versus-host disease and remarkably prolong the survival time of the mice when used together.
Comparative example 3
A composition for preventing and treating acute graft versus host disease, which is different from example 2 only in that the composition comprises the following components in parts by weight: 2 parts WDR5 inhibitor, no DOT1L inhibitor added; the rest of the operation was the same as in example 2.
The survival status of mice after drug injection was found: the median survival time is similar to that of the group without the injected medicament. And the Th1 cells which can cause acute graft-versus-host disease in the mouse lymphoid tissue are found to be similar to the drug injection group without drugs in the Th17 cell drug injection group, and the intestinal pathological drug injection group in the intestinal tissue which is easy to have acute rejection is similar to the drug injection group without drugs in the intestinal tissue.
The WDR5-MLL inhibitor is only used by a mouse, so that the survival time of the mouse cannot be prolonged, but the two compounds can be used together for treating acute graft-versus-host disease, and the survival time of the mouse is prolonged obviously.
Comparative example 4
A composition for preventing and treating acute graft versus host disease, which is different from example 2 only in that the composition comprises the following components in parts by weight: 2 parts DOT1L inhibitor, no WDR5 inhibitor added; the rest of the operation was the same as in example 2.
The survival status of mice after drug injection was found: the median survival time is similar to that of the group without the injected medicament. And the Th1 cells which can cause acute graft-versus-host disease in the mouse lymphoid tissue are found to be similar to the drug injection group without drugs in the Th17 cell drug injection group, and the intestinal pathological drug injection group in the intestinal tissue which is easy to have acute rejection is similar to the drug injection group without drugs in the intestinal tissue.
The fact that the survival time of the mice cannot be prolonged only by using the DOT1L inhibitor is shown, but the two compounds can be used together for treating acute graft-versus-host disease, so that the survival time of the mice is remarkably prolonged.
Comparative example 5
A composition for preventing and treating acute graft versus host disease, which is different from example 1 only in that the composition comprises the following components in parts by weight; the same procedure as in example 1 was repeated except for using 10 parts of WDR5 inhibitor and 1 part of DOT1L inhibitor.
The research finds that the survival state of the mouse after the drug injection is slightly improved compared with the previous survival state, the phenomenon is better than the previous improvement, but the weight loss situation is similar to that of the group without the drug injection, and the median survival time is similar to that of the group without the drug injection. And the discovery shows that effector cells Th1 cells and Th17 cell drug injection groups which can cause acute graft-versus-host disease in mouse lymphoid tissues are obviously reduced compared with non-drug groups, and intestinal pathological drug injection groups in intestinal tissues which are easy to have acute rejection are reduced compared with non-drug groups, but bone marrow inhibition of drug-containing mouse groups is serious, and the mice have severe anemia and platelet reduction.
Comparative example 6
A composition for preventing and treating acute graft versus host disease, which is different from example 2 only in that the composition comprises the following components in parts by weight; the same procedure as in example 2 was repeated except for using 10 parts of WDR5 inhibitor and 1 part of DOT1L inhibitor.
The study finds that the mice in the survival state after the drug injection have shrug hair, and the diarrhea is reduced compared with the drug group without the injection, but the weight loss is similar to that of the drug group without the injection, and the median survival time is similar to that of the drug group without the injection. And the discovery shows that effector cells Th1 cells and Th17 cell drug injection groups which can cause acute graft-versus-host disease in mouse lymphoid tissues are obviously reduced compared with non-drug groups, and intestinal pathological drug injection groups in intestinal tissues which are easy to have acute rejection are obviously reduced compared with non-drug groups, but bone marrow inhibition of drug-containing mouse groups is serious, and the mice have severe anemia and platelet reduction.
Comparative example 7
A composition for preventing and treating acute graft versus host disease, which is different from example 1 only in that the composition comprises the following components in parts by weight; the same procedure as in example 1 was repeated except for using 1 part of WDR5 inhibitor and 10 parts of DOT1L inhibitor.
The study finds that the mice in the survival state after the drug injection have shrug hair, and the diarrhea is reduced compared with the drug group without the injection, but the weight loss is similar to that of the drug group without the injection, and the median survival time is similar to that of the drug group without the injection. And the discovery shows that effector cells Th1 cells and Th17 cell drug injection groups which can cause acute graft-versus-host disease in mouse lymphoid tissues are obviously reduced compared with non-drug groups, and intestinal pathological drug injection groups in intestinal tissues which are easy to have acute rejection are obviously reduced compared with non-drug groups, but bone marrow inhibition of drug-containing mouse groups is serious, and the mice have severe anemia and platelet reduction.
Comparative example 8
A composition for preventing and treating acute graft versus host disease, which is different from example 2 only in that the composition comprises the following components in parts by weight; the same procedure as in example 2 was repeated except for using 1 part of WDR5 inhibitor and 10 parts of DOT1L inhibitor.
The study finds that the mice in the survival state after the drug injection have shrug hair, and the diarrhea is reduced compared with the drug group without the injection, but the weight loss is similar to that of the drug group without the injection, and the median survival time is similar to that of the drug group without the injection. And the discovery shows that effector cells Th1 cells and Th17 cell drug injection groups which can cause acute graft-versus-host disease in mouse lymphoid tissues are obviously reduced compared with non-drug groups, and intestinal pathological drug injection groups in intestinal tissues which are easy to have acute rejection are obviously reduced compared with non-drug groups, but bone marrow inhibition of drug-containing mouse groups is serious, and the mice have severe anemia and platelet reduction.
Third, conclusion of experiment
We have found that administration of only WDR5-MLL inhibitor or only DOT1L inhibitor in mice did not result in prolonged survival, neither prophylactic nor therapeutic effect, but that co-administration of both compounds in prophylactic or therapeutic treatment significantly prolonged survival of mice. In vitro experience finds out the effects achieved by different drug dosages and combinations, and finds that the drug combination has a synergistic effect.
The combination of the two compounds can obviously prolong the survival time of mice.
Different groups of mice are dissected on the seventh day after transplantation, spleens are taken for analysis, and the result shows that the spleen of the mice in the drug combination group has acute rejection of effector cells Th1 cells, and Th17 cells are obviously reduced.
FIG. 1 shows the in vitro assay of WDR5-MLL inhibitor (represented by OICR-9429) and DOT1L inhibitor (represented by EPZ5676) applied at different doses to a mixed lymphocyte reaction system in an in vitro assay (in which case, the lymphocytes undergo proliferative differentiation,
Figure BDA0002291894370000112
t cells are differentiated into Th1, Th17 and Treg cells, the proportions of Th1, Th17 and Treg cells are detected after different doses of drug are incubated by using flow cytometry, and the inventor finds that as the doses of WDR5-MLL inhibitor and DOT1L inhibitor are increased,
Figure BDA0002291894370000111
t cells gradually decreased in differentiation to Th1 and Th 17.
FIG. 2 shows that the survival time of mice was significantly prolonged by establishing a non-myeloablative allogeneic transplant model and then observing the survival time of mice after implanting donor lymphocytes with either OICR-9429 or EPZ5676 or a combination of both drugs.
FIG. 3 mice injected with both compounds were sacrificed and liver and intestine were HE stained and tabletted, and we found that liver, compared to the non-dosed group, had significantly reduced intestinal acute GVHD.
FIG. 4 shows that the ratio of Th1, Th17 and Treg cells is measured by flow cytometry after the mice injected with the two compounds are sacrificed and the spleen is ground, and we find that the Th17 cells are obviously reduced but still keep a certain differentiation compared with the Th1 without the medicine composition, so that a certain anti-infection capacity and anti-tumor cell activity are still kept.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The embodiments and features of the embodiments of the present application may be combined with each other arbitrarily without conflict.

Claims (5)

1. A composition for preventing and treating acute graft-versus-host disease is characterized by comprising the following components in parts by weight:
WDR5 inhibitor: 1-8 parts;
DOT1L inhibitor: 1-8 parts;
the WDR5 inhibitor is OICR-9429 and the DOT1L inhibitor is EPZ 5676.
2. The composition for preventing and treating acute graft versus host disease according to claim 1, comprising the following components in parts by weight:
WDR5 inhibitor: 1-5 parts;
DOT1L inhibitor: 1-5 parts.
3. The composition for preventing and treating acute graft versus host disease according to claim 1 or 2, comprising the following components in parts by weight:
WDR5 inhibitor: 3-5 parts;
DOT1L inhibitor: 1 part.
4. The composition for preventing and treating acute graft versus host disease according to claim 1, comprising the following components in parts by weight:
WDR5 inhibitor: 5 parts of a mixture;
DOT1L inhibitor: 1 part.
5. Use of a composition according to any one of claims 1 to 4 for the preparation of a composition for the prevention and treatment of acute graft-versus-host disease.
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