CN113797345A

CN113797345A - Application of glucocorticoid and glycolysis regulator in preparation of acute graft-versus-host disease (AGP) medicine

Info

Publication number: CN113797345A
Application number: CN202111231383.XA
Authority: CN
Inventors: 黄晓军; 孔圆; 温琦
Original assignee: Peking University Peoples Hospital
Current assignee: Peking University Peoples Hospital
Priority date: 2021-10-22
Filing date: 2021-10-22
Publication date: 2021-12-17
Anticipated expiration: 2041-10-22
Also published as: CN113797345B

Abstract

The invention discloses application of glucocorticoid and glycolysis regulator in preparing a medicine for treating acute graft-versus-host disease. The invention protects the application of glucocorticoid and glycolysis regulator in preparing medicaments; the drug is a drug for acute graft versus host disease. The invention also protects the application of the glycolysis regulator in preparing products; the product has the function of treating and/or preventing glucocorticoid drug resistance of patients suffering from acute graft-versus-host disease. The invention discovers that the function of the T cell can be improved by regulating the glucose metabolism level of the T cell, so that the glucocorticoid and the glycolysis regulator can be used for synergistically treating the aGVHD patient after transplantation, and the invention has very important significance for clinical targeted therapy.

Description

Application of glucocorticoid and glycolysis regulator in preparation of acute graft-versus-host disease (AGP) medicine

Technical Field

The invention belongs to the field of biomedicine, and relates to application of glucocorticoid and glycolysis regulator in preparation of a medicine for treating acute graft-versus-host disease, in particular to synergistic treatment of acute graft-versus-host disease by the glucocorticoid and the glycolysis regulator.

Background

Acute graft versus host disease (aGVHD) is an important complication of allogeneic hematopoietic stem cell transplantation. aGVHD is generally recognized as an immune-mediated disease, and in particular, aGVHD is characterized by "cytokine storm" stimulation, enhanced immune response to the recipient's antigen, and the onset of cytotoxic attack with the recipient's skin, liver and gut as the primary target, leading to increased associated mortality and increased patient medical costs.

Glucocorticoids are the first line treatment of aGVHD at present, but hormone resistance still occurs in 40-60% of patients, and the second line treatment is not good enough. Therefore, the intensive elucidation of the pathogenesis of aGVHD and the establishment of novel therapeutic strategies thereof are important clinical scientific issues to be urgently solved.

Disclosure of Invention

The invention aims to provide application of glucocorticoid and glycolytic regulator in preparing a medicament for treating acute graft-versus-host disease, and particularly relates to synergistic treatment of acute graft-versus-host disease by glucocorticoid and glycolytic regulator.

The invention protects the application of glucocorticoid and glycolysis regulator in preparing medicaments; the drug is a drug for acute graft versus host disease. When the medicine is applied, the glucocorticoid and the glycolytic regulator exert curative effect on acute graft-versus-host disease through synergistic action.

Specifically, the glycolytic regulator is 3 PO.

Specifically, the glycolytic regulator is MTX.

In particular, the glucocorticoid is MP.

Specifically, the molar ratio of MP to 3PO can be 1: 2.5-20.

Specifically, the molar ratio of MP to 3PO is 1: 10.

Specifically, the mass ratio of MP to 3PO is 2: 25.

Specifically, the molar ratio of MP to MTX is 1: 0.25-2.

Specifically, the molar ratio of MP to MTX is 1: 0.25.

Specifically, the mass ratio of MP to MTX is 2: 1.

The invention also protects a medicament, the active ingredients of which are glucocorticoid and glycolysis regulator; the drug is a drug for acute graft versus host disease. When the medicine is applied, the glucocorticoid and the glycolytic regulator exert curative effect on acute graft-versus-host disease through synergistic action.

Specifically, the glycolytic regulator is 3 PO.

Specifically, the glycolytic regulator is MTX.

In particular, the glucocorticoid is MP.

Specifically, the molar ratio of MP to 3PO can be 1: 2.5-20.

Specifically, the molar ratio of MP to 3PO is 1: 10.

Specifically, the mass ratio of MP to 3PO is 2: 25.

Specifically, the molar ratio of MP to MTX is 1: 0.25-2.

Specifically, the molar ratio of MP to MTX is 1: 0.25.

Specifically, the mass ratio of MP to MTX is 2: 1.

The invention also protects the application of the glycolytic regulator in preparing medicaments; the medicine has the function of treating and/or preventing glucocorticoid drug resistance of patients suffering from acute graft-versus-host disease. The glycolytic modulators may be used to treat and/or prevent glucocorticoid resistance in patients with acute graft-versus-host disease, such that the glucocorticoid and glycolytic modulators exert therapeutic effects against acute graft-versus-host disease through a synergistic effect.

Specifically, the glycolytic regulator is 3 PO.

Specifically, the glycolytic regulator is MTX.

In particular, the glucocorticoid is MP.

The invention also provides a medicament, the active ingredient of which is a glycolysis regulator; the medicine has the function of treating and/or preventing glucocorticoid drug resistance of patients suffering from acute graft-versus-host disease. The glycolytic modulators may be used to treat and/or prevent glucocorticoid resistance in patients with acute graft-versus-host disease, such that the glucocorticoid and glycolytic modulators exert therapeutic effects against acute graft-versus-host disease through a synergistic effect.

Specifically, the glycolytic regulator is 3 PO.

Specifically, the glycolytic regulator is MTX.

In particular, the glucocorticoid is MP.

The acute graft versus host disease patient may be an acute graft versus host disease patient after hematopoietic stem cell transplantation.

The metabolic reactions in T cells control cell proliferation, differentiation, activation, and apoptosis, and imbalance in cell metabolism and immune disorders are causal to each other. Therefore, a deep understanding of T cell metabolism and its dynamic regulation will provide effective molecular targets and new methods of potential clinical treatment for the prevention and treatment of immune-related diseases. It was found by animal models that glycolysis becomes the major energy source for effector T cells during the development of graft versus host disease. In a transplanted mouse model where major histocompatibility complex is not suitable, donor alloreactive T cells were found to acquire substances required for activation and proliferation by increasing glycolysis and oxidative phosphorylation, while levels of 6-phosphofructose-2-kinase/fructose-2, 6-bisphosphatase 3 (6-phosphofructo-2-kinase/fluctose-2, 6-bisphosphatase 3, PFKFB3), a key enzyme of cellular metabolism, were significantly increased. By blocking PFKFB3 with drugs, inhibiting glycolysis, the generation of alloreactive T cells can be reduced, thereby reducing the severity of acute graft-versus-host disease.

At present, whether the T cell metabolic state of aGVHD patients after transplantation is abnormal or not is still to be studied deeply. The invention discovers that the function of the T cell can be improved by regulating the glucose metabolism level of the T cell, so that the glucocorticoid and the glycolysis regulator can be used for synergistically treating the aGVHD patient after transplantation, and the invention has very important significance for clinical targeted therapy.

Drawings

FIG. 1 is a graph showing the results of example 1.

FIG. 2 is a graph showing the results of example 2.

FIG. 3 is a graph showing the results of example 3.

Fig. 4 shows the survival results of example 4.

Fig. 5 shows the scoring results in example 4.

FIG. 6 shows the results of flow cytometry in example 4 for detection of PFKFB3 and GLUT 1.

FIG. 7 is the flow cytometry detection of CD4 in example 4⁺And CD8⁺Results of cell ratios.

FIG. 8 is the results of HE staining in example 4.

Fig. 9 shows the results of in vivo bioluminescence imaging in example 4.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Unless otherwise stated, the quantitative tests in the following examples were performed in triplicate, and the results were averaged.

Methylprednisolone (MP), white crystalline powder, CAS registry number 82-43-2. Methotrexate (MTX), yellow crystalline powder, CAS registry 59-05-2. 3PO, all 3- (3-pyridinyl) -1- (4-pyridinyl) -2-propen-1-one: sigma, St.Louis, MO, USA, cat # SML1343, chemical formula C₁₃H₁₀N₂And O. MP belongs to the group of glucocorticoids. MTX belongs to the glycolytic regulator. 3PO belongs to a glycolytic regulator.

CD3 magnetic beads: american Tian-Geng (Miltenyi Biotec, Bergisch Gladbach, Germany) with a designation of 130-. MiniMACS with MS sorting columns^TMA separator: american Tian-Geng (Miltenyi Biotec, Bergisch Gladbach, Germany) with a cat number of 130-.

The phenotype of the Th1 cell is CD3⁺And CD8^-And IFN-gamma⁺. The phenotype of the Th2 cell is CD3⁺And CD8^-And IL-4⁺. The phenotype of the Th17 cell is CD3⁺And CD8^-And IL-17A⁺. The Treg cell phenotype is CD3⁺And CD8^-And CD25⁺And Foxp3⁺. Tc1 cell phenotype is CD3⁺And CD8⁺And IFN-gamma⁺. Tc2 cell phenotype is CD3⁺And CD8⁺And IL-4⁺. Antibodies in flow cytometry were as follows: CD8 antibody (BD, cat # 560347), IFN-. gamma.antibody (Biolegend, cat # 502536), IL-4 antibody (BD, cat # 559333), IL-17A antibody (Biolegend, cat # 512304), CD25 antibody (BD cat # 335807), Foxp3 antibody (BD, cat # 560045). DMEM high-glucose medium: gibco, cat # s: 11965092. CD3/CD28 antibody-coupled magnetic beads (CD3/CD28 monoclonal antibody beads): gibco, cat # s: 11131D.

Example 1 prospective clinical cohort studies found abnormally elevated levels of peripheral T cell glycolysis in aGVHD patients

Study subjects: patients with aGVHD (indicated by GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and patients with non-aGVHD (indicated by non-GVHD). Basic characteristics of aGVHD patients and non-aGVHD patients (e.g., gender, age, pre-transplant basal disease, number of chemotherapy treatments, risk assessment, pretreatment regimen, source of transplanted stem cells, total nucleated cell dose, post-transplant detection time, history of cytomegalovirus infection), and the like.

Abnormally elevated levels of peripheral T cell glycolysis were found in aGVHD patients by prospective clinical cohort studies.

The inventor detects the distribution of each subgroup of peripheral T cells of the aGVHD patient, and compared with a non-aGVHD patient, the T cells of the aGVHD patient are differentiated to Th1 and Tc1, the Th17 cells of the aGVHD patient are increased, the ratio of Th17 to Treg cells is unbalanced, and the T cell differentiation state towards the proinflammatory phenotype is presented.

The inventors further assessed whether there was a difference in the level of glycolysis in peripheral T cells in aGVHD patients versus non-aGVHD patients. Detection of peripheral blood CD3 in a subject using flow cytometry⁺PFKFB3 abundance in T cells, PFKFB3 abundance in aGVHD patients increased compared to non-aGVHD patientsHigh (a of fig. 1). PFKFB3 abundance in total peripheral blood protein of subjects was measured using wseternbot, and PFKFB3 abundance was increased in aGVHD patients compared to non-aGVHD patients (fig. 1D). Detecting the T cell glucose metabolism level of the aGVHD patient by utilizing a seahorse cell energy metabolism analysis kit, a lactic acid determination kit and a glucose determination kit; t cells from aGVHD patients showed higher ECAR values (E and F in figure 1), higher glucose consumption rate (I in figure 1), and higher lactate production rate (J in figure 1) compared to non-aGVHD patients. However, there was no significant change in OCR in T cells of aGVHD patients compared to non-aGVHD patients (G and H of figure 1). T cells, especially CD4, from aGVHD patients compared to non-aGVHD patients were detected by flow cytometry⁺The levels of PFKFB3 protein in naive T cells were significantly increased (B of fig. 1). Further, by real-time fluorescent quantitative PCR, it was found that mRNA levels of metabolic enzymes of glycolytic pathway were increased in aGVHD patients compared to non-aGVHD patients, with significant increases in mRNA levels including hexokinase 3(HK3), PFKFB3, and lactate dehydrogenase b (ldhb) (C of fig. 1).

Taken together, these results indicate that PFKFB 3-mediated increases in glycolysis are present in the T cells of aGVHD patients.

Example 2 PFKFB3 modulators inhibit peripheral T cell proliferation and activation in aGVHD patients by inhibiting glycolysis

The aGVHD patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) were confirmed patients in the Beijing university people hospital and had informed consent for the relevant tests. Peripheral blood from aGVHD patients was collected, peripheral blood mononuclear cells were isolated, and CD3 was isolated from peripheral blood mononuclear cells⁺Cells, i.e. peripheral CD3⁺T cells.

Peripheral CD3⁺T cells were divided into two groups, one group was treated with 3PO (this group is designated GVHD +3 PO) and one group was used as a control group (this group is designated GVHD).

PFKFB3 abundance in cells was detected using flow cytometry and the results are shown in a of figure 2. The glucose consumption of the cells was measured using the glucose assay kit, and the results are shown in B of FIG. 2. The amount of lactic acid produced by the cells was measured using a lactic acid assay kit, and the results are shown in C of FIG. 2. Expression of pro-inflammatory cytokines in T cells was detected by flow cytometry to obtain Th1 cell fraction, Tc1 cell fraction, Th17 cell fraction, and the results are shown in D, E and F of fig. 2. T cell inflammatory transcription factor (T-beta and ROR gamma T) abundance in cells was determined by flow cytometry and the results are shown in FIG. 2, G and H. The abundance of GATA3 and Foxp3 in cells was detected by flow cytometry and the results are shown in fig. 2, I and J. The proliferation capacity and T cell apoptosis of T cells in the cells were examined by flow cytometry, and the results are shown in K and L of FIG. 2.

The results show that: 3PO can be used for remarkably reducing the expression of PFKFB3 in T cells of aGVHD patients, promoting the reduction of glucose consumption rate and the remarkable reduction of lactic acid generation rate; 3PO reduces the expression of proinflammatory cytokines in T cells of aGVHD patients, and the proportion of Th1 cells, Tc1 cells and Th17 cells is reduced; 3PO reduces the expression of T cell inflammatory transcription factors of aGVHD patients, and reduces T-beta and ROR gamma T; 3PO has no obvious influence on the expression of transcription factors GATA3 and Foxp 3; 3PO reduced the proliferative capacity of T cells from aGVHD patients without affecting T cell apoptosis.

Taken together, 3PO inhibits activation and proliferation of peripheral T cells in aGVHD patients by inhibiting glycolysis.

Example 3 Co-treatment of aGVHD with glucocorticoids and glycolytic modulators

First, CD3 of aGVHD patients was obtained⁺T cells

The aGVHD patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) are confirmed patients in the people hospital of Beijing university and have known informed consent for relevant experiments.

1. Peripheral blood of a patient is taken and mononuclear cells are isolated.

2. Using CD3 magnetic beads and MiniMACS with MS sorting columns^TMSeparator separation of CD3 from mononuclear cells obtained in step 1⁺The cell is CD3⁺T cells.

Second, fluorescent microscope observation

The test cells were: step one prepared CD3⁺T cells.

1. A96-well plate was taken and previously coated with 1 × recombinant human fibronectin (sigma, cat # ECM001) for 24 hours.

2. After completion of step 1, inoculate 2X 10 wells per well⁵The test cells were cultured in 100-200. mu.l of DMEM high-glucose medium containing CD3/CD28 antibody-coupled magnetic beads and 10% FBS for 24 hours. The ratio of the number of CD3/CD28 antibody coupled magnetic beads to the number of cells is 1: 1.

3. After step 2 is completed, the grouping process is as follows:

a first group: adding MP and 3PO, and culturing for 48 hr; the concentration of MP in the system was set to 1. mu.M, and the concentration of 3PO in the system was set to 2.5. mu.M, 5. mu.M, 10. mu.M or 20. mu.M, respectively (a treatment without addition of 3PO was set as a0 concentration control of 3 PO).

Second group: adding MP and MTX, and then culturing for 48 hours; the concentration of MP in the system was 1. mu.M, and the concentration of MTX in the system was set to 0.25. mu.M, 0.5. mu.M, 1. mu.M or 2. mu.M, respectively (treatment without MTX was set as a 0-concentration control of MTX).

4. After completion of step 3, the supernatant was aspirated, 5. mu.l of DAPI was added to each well and incubated at room temperature for 10 minutes, followed by washing with PBS buffer and counting by fluorescence microscopy using excitation with green and blue light.

The results are shown in FIG. 3 at I.

Third, detection of other indexes

1. Packet processing

(1) Inoculating 96-well plate with 8 × 10 seed per well⁴Step one preparation of CD3⁺T cells were cultured in 100-200. mu.l DMEM high-glucose medium containing CD3/CD28 antibody-coupled magnetic beads and 10% FBS for 24 hours. The ratio of the number of CD3/CD28 antibody coupled magnetic beads to the number of cells is 1: 1.

(2) After the step (1) is completed, the grouping process is as follows:

first group (GVHD +3PO group): adding 3PO, and culturing; the concentration of 3PO in the system is 10 mu M;

second group (GVHD + MP group): adding MP, and culturing; the concentration of MP in the system is 1 μ M;

third group (GVHD + MTX group): adding MTX, and then culturing; the concentration of MTX in the system is 0.25. mu.M;

fourth group (GVHD + MP +3PO group): adding MP and 3PO, and culturing; the concentration of MP in the system is 1 μ M, and the concentration of 3PO in the system is 10 μ M;

fifth panel (GVHD + MP + MTX panel): adding MP and MTX, and culturing; the concentration of MP in the system is 1 μ M, and the concentration of MTX in the system is 0.25 μ M;

sixth group (GVHD group): no treatment was performed.

The incubation time was 48 hours.

2. Flow cytometry detection of PFKFB3 and GLUT1

(1) And (3) taking the cells treated in the step (1) to a flow detection tube.

(2) Add 5. mu.l of CD3 to the flow assay tube⁺Cell surface flow antibody CD3, incubated at room temperature for 15min in the dark.

(3) Adding 2ml PBS buffer solution and mixing evenly, centrifuging at 1500rpm for 5 minutes; then, the supernatant was discarded and 100. mu.l of FIX was added&PERM^TMThe immobilized medium A (invitrogen, cat # GAS004) in the cell permeabilization kit is mixed evenly and incubated for 15min at room temperature in the dark.

(4) Adding 2ml PBS buffer solution and mixing evenly, centrifuging at 1500rpm for 5 minutes; then, the supernatant was discarded and 100. mu.l of FIX was added&PERM^TMThe permeabilization medium B (invitrogen, cat # GAS004) in the cell permeabilization kit was mixed well, 1. mu.l of intracellular flow antibody was added, and incubated for 15min at room temperature in the dark. The intracellular flow antibodies were: intracellular flow antibody PFKFB3(Cell Signaling Technology, Danvers, MA, USA) or intracellular flow antibody GLUT1(Cell Signaling Technology, Danvers, MA, USA).

(5) Adding 2ml PBS buffer solution and mixing evenly, centrifuging at 1500rpm for 5 minutes; then, the supernatant was discarded and 100. mu.l of FIX was added&PERM^TMThe permeabilization medium B in the Cell permeabilization kit was mixed well, and rabbit anti-mouse secondary antibody (Cell Signaling Technology, Danvers, MA, USA) was added and incubated for 15min at room temperature in the dark.

(6) Adding 2ml PBS buffer solution and mixing evenly, centrifuging at 1500rpm for 5 minutes; then, the supernatant was discarded, 200. mu.l of PBS buffer was added to resuspend the cells, and the cells were tested on the machine within 4 hours. The streaming image analysis employed Diva 7.0 software (BD corporation, usa).

The results are shown in FIGS. 3F and G.

3. Detecting glucose consumption

(1) Inoculating 96-well plate with 2 × 10 seed per well⁵Each of the step 1 treated cells was cultured in 100-200. mu.l of DMEM high-glucose medium containing CD3/CD28 antibody-coupled magnetic beads and 10% FBS for 24 hours. The ratio of the number of CD3/CD28 antibody coupled magnetic beads to the number of cells is 1: 1.

(2) After completion of step (1), the mixture was centrifuged at 1500rpm for 5 minutes, and the supernatant was collected.

(3) And (3) taking the supernatant obtained in the step (2), detecting the content of glucose by adopting a glucose determination kit (Nanjing institute of bioengineering, Cat: 361510), and calculating the glucose consumption rate.

The results are shown in D of FIG. 3.

4. Detection of lactic acid production

(3) And (3) taking the supernatant obtained in the step (2), detecting the content of lactic acid by adopting a lactic acid determination kit (Nanjing institute of bioengineering, Cat: A019-2-1), and calculating the generation rate of the lactic acid.

The results are shown in E of FIG. 3.

5. Detecting expression of proinflammatory cytokines in T cells

(1) Inoculating 96-well plate at 2 × 10⁵Adding 100 mu l of 1 ng/mu l PMA aqueous solution, 2 mu l of 1 mu g/mu l ionomycin aqueous solution and 0.7 mu l Golgistop aqueous solution into the cells treated in the step 1, shaking and uniformly mixing, and incubating for 4-6 hours in an incubator at 37 ℃.

(2) After centrifugation at 1500rpm for 5min, the supernatant was discarded and the cells were flicked off, and surface flow antibodies (CD3 antibody, CD8 antibody and CD25 antibody) were added and incubated at room temperature for 15 min.

(3) 2ml of PBS buffer was added, centrifuged at 1500rpm for 5min, and the supernatant was discarded to eject the cells.

(4) Add 1ml of Etobibioscience^TMFoxp 3/transcription factor fixation concentrate and solvent buffer were mixed as 1: 3, mixing the prepared stationary liquid by vortex, and incubating for 30min in a refrigerator at 4 ℃.

(5) 2ml of water buffer and deionized water were added at a rate of 1: 9 proportion of prepared nuclear membrane breaking working solution (invitrogen, eBioscience)^TM Foxp 3/transcription factor staining buffer kit, cat #: 00-5523-00), centrifuging at 2000rpm for 5min, and discarding the supernatant.

(6) Intracellular antibodies (IFN-. gamma., IL-4, IL-17 and Foxp3) were added, vortexed, and incubated at 4 ℃ for 30min in a refrigerator.

(7) Adding 2ml of the nuclear membrane breaking working solution, rotating at 2000rpm for 5min, discarding the supernatant, adding 200 mu l of the nuclear membrane breaking working solution, and bouncing up the cells.

(8) And (5) performing machine detection within 24 h. The streaming image analysis employed Diva 7.0 software.

The results are shown in FIGS. 3A and B.

6. CCK-8 detection of T cell proliferation capacity

(1) Inoculating 100 μ l of 1 × 10 in 96-well plate⁵The cells treated in step one were cultured for 24 hours.

(2) Add 10. mu.l CCK-8 solution to each well and incubate for 2h in an incubator.

(3) Absorbance at 450nm was measured with a microplate reader.

The results are shown in C of FIG. 3.

7. Detection of NF- κ B p65

(1) And (3) taking the cells treated in the step (1) to a flow detection tube.

(4) Adding 2ml PBS buffer solution and mixing evenly, centrifuging at 1500rpm for 5 minutes; then, the supernatant was discarded and 100. mu.l of FIX was added&PERM^TMThe permeabilization medium B (invitrogen, cat # GAS004) in the cell permeabilization kit was mixed well and 1. mu.l of intracellular flow was addedThe antibody of formula NF-. kappa. B p65(Cell signalling Technology, Danvers, MA, USA) was incubated at room temperature for 15min in the absence of light.

The results are shown in FIG. 3H.

Calculation of four, CI value

Test cells: step one prepared CD3⁺T cells.

Two drug combinations were set: MP and 3PO in combination, MP and MTX in combination.

Each medicine composition is set with various proportioning concentrations.

And processing according to the packet processing method in the step two.

Computing CD3 through Com-puSyn software⁺The inhibition rate of IFN gamma synthesis by T cells is 25%, 50%, 75%, 90%, 95% corresponding to CI value. CD3 pair calculated by Com-puSyn software⁺The inhibition rates of T cell proliferation were 25%, 50%, 75%, 90%, and 95% for the CI values.

The results are shown in J of FIG. 3. The mean CI values for MP and 3PO for T cell IFN γ synthesis were 0.364; the mean CI values for MP and MTX for T cell IFN γ synthesis were 0.554. Mean CI values for MP and 3PO for T cell proliferation were 0.475; mean CI values for MP and MTX for T cell proliferation were 0.406.

The combined use of glucocorticoid (MP) and a glycolytic regulator (3PO or MTX) in vitro on peripheral T cells of a patient suffering from aGVHD has been found to synergistically inhibit glycolysis and thus peripheral T cell activation and proliferation in a patient suffering from aGVHD.

Through the dose-response effect, the effect of MP (1 μ M) and MTX (0.5 μ M) for 48h is found to have the best inhibition effect on the T cells of aGvHD patients, but has no obvious effect on the survival of the T cells. The in vitro application of MP and MTX significantly reduced the glucose consumption rate and the lactic acid production rate. MP reduces glycolytic activity of T cells by down-regulating expression of GLUT1 and PFKFB 3. MTX reduces glycolytic activity of T cells by down-regulating expression of GLUT 1. In addition, MP and MTX reduced the proportion of pro-inflammatory cells, including Th1, Tc1 cells, in the T cells of aGVHD patients. And MP and MTX reduced T cell proliferation in aGvHD patients but did not significantly affect T cell apoptosis. These results indicate that MP inhibits T cell activation and proliferation by inhibiting GLUT1 and PFKFB3 stimulated glycolysis, and MTX inhibits T cell activation and proliferation by inhibiting GLUT1 stimulated glycolysis.

The combination of glucocorticoids and glycolytic modulators has a synergistic effect on the improvement of T cell activity in aGvHD patients, probably by reducing glycolytic activity. The combined use of glucocorticoids and glycolytic modulators has a synergistic inhibitory effect on the differentiation of T cells into the pro-inflammatory Th1, Tc1 and T cell proliferation compared to single drug treated cells. In addition, the combined use of a glucocorticoid and a glycolytic modulator down-regulates glycolytic activity in vitro over the glucocorticoid alone.

Notably, the combination regimen provided a strong synergy for T cells, with MP and 3PO having an average CI of 0.364 for T cell IFN γ synthesis. In addition, the mean CI values for MP and MTX for T cell IFN γ synthesis were 0.554. Mean CI values for MP and 3PO for T cell proliferation were 0.475. In addition, the mean CI values for MP and MTX for T cell proliferation were 0.406.

Example 4 Co-treatment of aGVHD with glucocorticoids and glycolytic modulators

The synergistic therapeutic effect of glucocorticoids and glycolytic modulators was verified using the aGVHD mouse model.

NPG mice (nod. cg-Prkdcscid Il2rgtm1Vst/Vst mice): beijing Wittiulihua laboratory animal technology Co.

Luciferase⁺THP-1 leukemia cells (THP-1-luc cells) described in: azacytidine precursors eximental xenogeneic graft-versus-host diseasewithout abrogating graft-versus-leukemia effects，ONCOIMMUNOLOGY，2017,VOL.6,NO.5,e1314425。

1. Suspending human peripheral blood stem cells by using 1 Xhemolysin, suspending and shaking for 5 seconds, then standing for 15 minutes on ice in a dark place, then centrifuging for 5 minutes at 1500rpm, and discarding the supernatant; then suspending with PBS buffer solution, centrifuging at 1500rpm for 5 minutes, and removing the supernatant; then, the suspension was suspended in PBS buffer to obtain a cell suspension. 1 part by volume of 10 Xhemolysin (BD Biosciences) and 9 parts by volume of sterile water for injection were mixed well to obtain 1 Xhemolysin.

2. The NPG mice are fed with antibiotic water one week before cell injection to prevent infection, and are placed in an air laminar flow cabinet for aseptic breeding.

3. One day before cell injection⁶⁰NPG mice were irradiated systemically with Co gamma radiation at 1.5Gy (semi-lethal dose).

4. Each mouse was injected via tail vein with the cell suspension prepared in step 1 (5X 10)⁶One cell/one) and luciferase⁺THP-1 leukemia cell (1X 10)⁶One cell/one). Days were counted beginning after completion of the injection.

5. Mice completing step 4 were randomly divided into six groups (PBS group, MP group, 3PO group, MTX group, MP-associated 3PO group, MP-associated MTX group) of 11 mice each. The PBS group mice were intraperitoneally injected with PBS buffer 1 time per day in a volume of 200. mu.l/mouse. MP group mice were injected intraperitoneally with 1 MP solution per day at a volume of 200. mu.l/mouse, and MP dose of 2 mg/kg body weight/time. 3PO group mice were intraperitoneally injected with 1 time of 3PO solution per day at a volume of 200. mu.l/mouse, and a 3PO dose of 25 mg/kg body weight/time. MTX group mice were intraperitoneally injected with 1 MTX solution per day at a volume of 200. mu.l/mouse and a dose of 1 mg/kg body weight/mouse. MP combination 3PO group mice were injected intraperitoneally with MP-3PO solution 1 time per day in a volume of 200. mu.l/mouse, at a MP dose of 2 mg/kg body weight/mouse, and at a 3PO dose of 25 mg/kg body weight/mouse. The MP-MTX solution is injected into the abdominal cavity of the mice in the MP combined MTX group 1 time per day, the injection volume is 200 mu l/mouse, the MP dose is 2 mg/kg body weight/time, and the MTX dose is 1 mg/kg body weight/time. The administration was continued for 30 days. The PBS group is also called CTL group.

The MP solution is obtained by dissolving MP in sterile water. The 3PO solution is obtained by dissolving 3PO in sterile water. The MTX solution is obtained by dissolving MTX in sterile water. The MP-3PO solution is obtained by dissolving MP and 3PO in sterile water. The MP-MTX solution is obtained by dissolving MP and MTX in sterile water.

Survival was continuously counted and the results are shown in figure 4.

On day 28, acute GVHD scores were performed. The acute GVHD clinical scoring system is based on six parameters: weight loss, posture, activity, fur texture, skin integrity and diarrhea. The results are shown in FIG. 5.

On day 30, mice were sacrificed, livers and spleens were removed, mononuclear cells were collected, and CD4 was detected by flow cytometry⁺Cell proportion, CD8⁺Cell ratio, Th1 cell ratio, Tc1 cell ratio, and T cell proliferation capacity. The results are shown in FIG. 6.

On day 28, peripheral blood was collected from the mice, mononuclear cells were collected, and CD4 was detected by flow cytometry⁺Cell ratio and CD8⁺Cell ratio. The results are shown in FIG. 7.

On day 28, mice were sacrificed and acute GVHD target organ (liver, spleen, skin, intestinal tract, lung) tissues were taken, fixed with 4% paraformaldehyde, paraffin sections were prepared and HE stained. Mice acute GVHD pathology scoring was performed according to the scoring system. The results are shown in FIG. 8.

On day 21, clearance of leukemic cells after NPG mice transplantation was assessed by in vivo bioluminescence imaging. The results are shown in FIG. 9. Finding ways to effectively treat GVHD while maintaining the GVL effect is important to improve the curative effect of hematopoietic stem cell transplantation and reduce transplantation related complications. To further explore whether MP combined with 3PO or MTX affected the GVL effects, the luciferase was back-transfused⁺THP-1 leukemia cells, and the clearance of leukemia cells after NPG mice transplantation was evaluated by in vivo bioluminescence imaging. Compared to MP alone, MP in combination with 3PO or MTX had no significant effect on GVL. These results indicate that the combination of MP with 3PO or MTX in a humanized mouse model synergistically reduces T cell alloreactivity and improves aGvHD by down-regulating glycolytic activityThe GVL effect was not lost.

The combined use of glucocorticoid (MP) and glycolytic modulators (3PO or MTX) reduces mouse aGvHD clinical score and mortality compared to single drug therapy. Pathological evidence suggests that mouse GVHD target organ pathology scores with MP in combination with 3PO or MTX were significantly reduced. The results indicate that a combination of MP and 3PO or a combination of MP and MTX can be used to synergistically treat aGVHD.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Claims

1. The use of a glucocorticoid and a glycolytic modulator in the preparation of a medicament; the drug is a drug for acute graft versus host disease.

2. A medicine contains glucocorticoid and glycolytic regulator as active ingredients; the drug is a drug for acute graft versus host disease.

3. The use according to claim 1 or the medicament according to claim 2: the method is characterized in that: in the medicine, the glucocorticoid and the glycolysis regulator exert the curative effect on acute graft-versus-host disease through synergistic action.

4. The use of a glycolytic regulator in the manufacture of a medicament; the medicine has the function of treating and/or preventing glucocorticoid drug resistance of patients suffering from acute graft-versus-host disease.

5. A medicine contains glycolytic regulator as active ingredient; the medicine has the function of treating and/or preventing glucocorticoid drug resistance of patients suffering from acute graft-versus-host disease.

6. The use according to claim 4 or the medicament according to claim 5: the method is characterized in that: the glucocorticoid and glycolytic regulator exert curative effect on acute graft-versus-host disease through synergistic effect.

7. The use or medicament as claimed in any one of claims 1 to 6, wherein: the glycolytic regulator is 3 PO.

8. The use or medicament as claimed in any one of claims 1 to 6, wherein: the glycolytic regulator is MTX.

9. The use or medicament as claimed in any one of claims 1 to 8, wherein: the glucocorticoid is MP.