CN113797345B - Application of glucocorticoid and glycolytic demodulator in preparation of medicines for treating acute graft-versus-host disease - Google Patents

Application of glucocorticoid and glycolytic demodulator in preparation of medicines for treating acute graft-versus-host disease Download PDF

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CN113797345B
CN113797345B CN202111231383.XA CN202111231383A CN113797345B CN 113797345 B CN113797345 B CN 113797345B CN 202111231383 A CN202111231383 A CN 202111231383A CN 113797345 B CN113797345 B CN 113797345B
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glucocorticoid
glycolytic
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agvhd
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CN113797345A (en
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黄晓军
孔圆
温琦
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Peking University Peoples Hospital
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Abstract

The invention discloses application of glucocorticoid and glycolytic demodulator in preparing medicines for treating acute graft-versus-host disease. The invention protects the application of glucocorticoid and glycolytic demodulator in preparing medicines; the medicament is a medicament for treating acute graft versus host disease. The invention also protects the application of the glycolysis regulator in preparing products; the function of the product is to treat and/or prevent glucocorticoid resistance in patients with acute graft versus host disease. The invention discovers that the T cell function can be improved by regulating and controlling the glucose metabolism level of the T cell, so that the glucocorticoid and the glycolytic demodulator are used for cooperatively treating the transplanted aGVHD patient, and the invention has very important significance for clinical targeted treatment.

Description

Application of glucocorticoid and glycolytic demodulator in preparation of medicines for treating acute graft-versus-host disease
Technical Field
The invention belongs to the field of biological medicine, relates to application of glucocorticoid and a glycolytic demodulation node agent in preparation of medicines for treating acute graft-versus-host disease, and in particular relates to application of glucocorticoid and glycolytic demodulation node agent in preparation of medicines for treating acute graft-versus-host disease in a synergistic manner.
Background
Acute graft versus host disease (aGVHD) is an important complication of allogeneic hematopoietic stem cell transplantation. aGVHD is generally regarded as an immune-mediated disease, and in particular, aGVHD stimulated with a "cytokine storm" to enhance an immune response to a subject antigen, characterized by a subject's skin, liver and gut as the primary targets for initiating cytotoxic attacks, resulting in increased mortality and increased medical costs for the patient.
Glucocorticoids are the first line treatment regimen for current aGVHD, but there are still 40-60% of patients with hormonal resistance and the existing second line treatment is poorly effective. Thus, the in-depth elucidation of aGVHD pathogenesis and the establishment of new therapeutic strategies are important clinical scientific problems that need to be addressed.
Disclosure of Invention
The invention aims to provide application of glucocorticoid and a glycolytic demodulator in preparation of medicines for treating acute graft-versus-host disease, and in particular relates to application of glucocorticoid and glycolytic demodulator in preparation of medicines for treating acute graft-versus-host disease in a synergistic way.
The invention protects the application of glucocorticoid and glycolytic demodulator in preparing medicines; the medicament is a medicament for treating acute graft versus host disease. When the medicine is applied, the glucocorticoid and the glycolysis regulator exert the curative effect on the acute graft-versus-host disease through the synergistic effect.
Specifically, the glycolytic regulator is 3PO.
Specifically, the glycolytic demodulator is MTX.
Specifically, 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 medicine, the active ingredients of which are glucocorticoid and glycolysis demodulator; the medicament is a medicament for treating acute graft versus host disease. When the medicine is applied, the glucocorticoid and the glycolysis regulator exert the curative effect on the acute graft-versus-host disease through the synergistic effect.
Specifically, the glycolytic regulator is 3PO.
Specifically, the glycolytic demodulator is MTX.
Specifically, 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 glycolysis regulator in preparing medicines; the function of the medicament is to treat and/or prevent glucocorticoid resistance in patients with acute graft versus host disease. The glycolytic modulator can be used to treat and/or prevent glucocorticoid resistance in an acute graft-versus-host patient, such that the glucocorticoid and the glycolytic modulator exert a therapeutic effect against the acute graft-versus-host disease through a synergistic effect.
Specifically, the glycolytic regulator is 3PO.
Specifically, the glycolytic demodulator is MTX.
Specifically, the glucocorticoid is MP.
The invention also protects a medicine, the active ingredient of which is a glycolysis-demodulation agent; the function of the medicament is to treat and/or prevent glucocorticoid resistance in patients with acute graft versus host disease. The glycolytic modulator can be used to treat and/or prevent glucocorticoid resistance in an acute graft-versus-host patient, such that the glucocorticoid and the glycolytic modulator exert a therapeutic effect against the acute graft-versus-host disease through a synergistic effect.
Specifically, the glycolytic regulator is 3PO.
Specifically, the glycolytic demodulator is MTX.
Specifically, the glucocorticoid is MP.
The acute graft-versus-host patient may be an acute graft-versus-host patient following hematopoietic stem cell transplantation.
The metabolic response within T cells controls proliferation, differentiation, activation and apoptosis of cells, and the imbalance in cellular metabolism and immune disorders are causal. Therefore, a deep understanding of T cell metabolism and its dynamic regulation will provide an effective molecular target and a new approach to potential clinical treatment for the prevention and treatment of immune-related diseases. Glycolysis was found by animal models to be the primary energy source for effector T cells during the development of graft versus host disease. In a transplanted mouse model with major histocompatibility complex mismatch, donor alloreactive T cells were found to obtain substances required for activation and proliferation by increasing glycolysis and oxidative phosphorylation, while the level of the cell metabolism key enzyme 6-phosphofructose-2-kinase/fructose-2, 6-bisphosphatase 3 (6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3, pfkfb 3) was significantly increased. The medicine can block PFKFB3 and inhibit glycolysis, so that the generation of alloreactive T cells can be reduced, and the severity of acute graft versus host disease can be reduced.
Currently, whether there is an abnormality in the metabolic state of T cells in aGVHD patients after transplantation has yet to be studied intensively. The invention discovers that the T cell function can be improved by regulating and controlling the glucose metabolism level of the T cell, so that the glucocorticoid and the glycolytic demodulator are used for cooperatively treating the transplanted aGVHD patient, and the invention has very important significance for clinical targeted treatment.
Drawings
FIG. 1 is a graph showing the results of example 1.
FIG. 2 is a graph of the results of example 2.
FIG. 3 is a graph showing the results of example 3.
Fig. 4 is a survival rate result in example 4.
Fig. 5 is the scoring result in example 4.
FIG. 6 shows the results of flow cytometry detection of PFKFB3 and GLUT1 in example 4.
FIG. 7 is a flow cytometry analysis of CD4 in example 4 + And CD8 + Results of cell ratios.
FIG. 8 shows the results of HE staining in example 4.
FIG. 9 is the results of in vivo bioluminescence imaging in example 4.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. Unless otherwise indicated, the quantitative tests in the examples below were all performed in triplicate, and the results averaged.
Methylprednisolone (MP), white crystalline powder, CAS registry number 82-43-2. Methotrexate (MTX), yellow crystalline powder, CAS registry number 59-05-2.3PO, all known as 3- (3-pyridinyl) -1- (4-pyridinyl) -2-propen-1-one: sigma, st.Louis, MO, USA, product number SML1343, chemical formula C 13 H 10 N 2 O. MP belongs to the class of glucocorticoids. MTX belongs to the glycolytic demodulator. 3PO belongs to a glycolytic agent.
CD3 magnetic beads: meitian and gentle (MiltenyiBiotec, bergischGladbach, germany), cat No. 130-050-101. MiniMACS with MS sorting column TM A separator: meitian and gentle (MiltenyiBiotec, bergischGladbach, germany), cat No. 130-090-312.
Th1 cell phenotype is CD3 + And CD8 - And IFN-gamma + . Th2 cell phenotype is CD3 + And CD8 - And IL-4 + . Th17 cell phenotype is CD3 + And CD8 - And IL-17A + . 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 cytometric assays were as follows: CD8 antibody (BD, cat# 560347), IFN-gamma antibody (bioleged, cat# 502536), IL-4 antibody (BD, cat# 559333), IL-17A antibody (bioleged, cat# 512304), CD25 antibody (BD cat# 335807), foxp3 antibody (BD, cat# 560045). DMEM high sugar medium: gibco, cat No.: 11965092.CD3/CD28 antibody-conjugated magnetic beads (CD 3/CD28 monoclonal antibody beads): gibco, cat No.: 11131D.
Example 1 prospective clinical cohort studies found abnormally elevated peripheral T cell glycolysis levels in aGVHD patients
Study object: aGVHD patients (indicated by GVHD) and non-aGVHD patients (indicated by non-GVHD) following allogeneic hematopoietic stem cell transplantation (allogeneic hematopoietic stem cell transplantation, allo-HSCT). The aGVHD patient is similar to the basic characteristics of non-aGVHD patients (e.g., sex, age, pre-transplant basal disease, number of chemotherapies, risk assessment, pretreatment regimen, source of transplanted stem cells, total nucleated cell dose, time of detection after transplantation, history of cytomegalovirus infection) and the like.
Abnormal elevation of peripheral T cell glycolysis levels in aGVHD patients was found by prospective clinical cohort studies.
The inventor detects the distribution situation of each subgroup of peripheral T cells of an aGVHD patient, and discovers that the T cells of the aGVHD patient differentiate to Th1 and Tc1, the Th17 cells of the aGVHD patient rise, the proportion of Th17 and Treg cells is unbalanced, and the T cell differentiation imbalance state towards the pro-inflammatory phenotype is presented.
The inventors further evaluated whether there was a differential glycolytic level of 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 was increased compared to non-aGVHD patients (a of fig. 1). PFKFB3 abundance in the peripheral blood total protein of the subject was detected using wsternblot, with PFKFB3 abundance in aGVHD patients increased compared to non-aGVHD patients (D of fig. 1). Detecting the glucose metabolism level of the T cells of the aGVHD patient by using a seahorse cell energy metabolism analysis, a lactic acid determination kit and a glucose determination kit; compared to non-aGVHD patients, aGVHD patient T cells exhibited higher ECAR values (E and F of fig. 1), higher glucose consumption rates (I of fig. 1) and higher lactate production rates (J of fig. 1). However, there was no significant change in OCR of the aGVHD patient T cells compared to the non-aGVHD patient (G and H of fig. 1). Detection by flow cytometry revealed that the T cells of aGVHD patients, in particular CD4, compared to non-aGVHD patients + The PFKFB3 protein levels of naive T cells were significantly elevated (B of fig. 1). Further discovery by real-time fluorescent quantitative PCRThe mRNA levels of metabolic enzymes of the glycolytic pathway were elevated in aGVHD patients compared to non-aGVHD patients, including those of hexokinase 3 (HK 3), PFKFB3, and lactate dehydrogenase B (LDHB) (C of fig. 1).
Taken together, these results indicate that there is an increase in PFKFB3 mediated glycolysis in the T cells of aGVHD patients.
Example 2 inhibition of peripheral T cell proliferation and activation in aGVHD patients by inhibition of glycolysis by PFKFB3 modulators
The aGVHD patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) were diagnosed in the beijing university people hospital and had informed consent to the relevant experiments. Taking peripheral blood of an aGVHD patient, separating peripheral blood mononuclear cells, and then separating CD3 from the peripheral blood mononuclear cells + Cells, i.e. peripheral CD3 + T cells.
Taking peripheral CD3 + T cells were divided into two groups, one group was subjected to 3PO treatment (this group is denoted gvhd+3po) and one group was used as a control group (this group is denoted GVHD).
PFKFB3 abundance in cells was detected using flow cytometry, and the results are shown in fig. 2 a. The glucose consumption of the cells was measured using a glucose assay kit, and the results are shown in FIG. 2B. The lactic acid production amount of the cells was measured by using a lactic acid measuring kit, and the result is shown in FIG. 2C. The expression of pro-inflammatory cytokines in T cells was examined 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. The abundance of T cell inflammatory transcription factors (T-bet and rorγt) in cells was examined using flow cytometry, and the results are shown in G and H of fig. 2. The abundance of GATA3 and Foxp3 in cells was measured using flow cytometry, and the results are shown in fig. 2I and J. The proliferation capacity and T cell apoptosis of T cells in the cells were examined using flow cytometry, and the results are shown in K and L of fig. 2.
The results show that: the application of 3PO can obviously reduce the expression of PFKFB3 in the T cells of the aGVHD patient, promote the reduction of the glucose consumption rate and the obvious reduction of the lactic acid generation rate; 3PO reduces the expression of pro-inflammatory cytokines in T cells of an aGVHD patient, and reduces the proportion of Th1 cells, tc1 cells and Th17 cells; 3PO reduces the expression of inflammatory transcription factors of T cells of the aGVHD patient, and T-bet and RORgamma T are reduced; 3PO has no obvious effect on the expression of transcription factors GATA3 and Foxp 3; 3PO reduced the proliferative capacity of the aGVHD patient's T cells without affecting T cell apoptosis.
Taken together, 3PO inhibits peripheral T cell activation and proliferation in aGVHD patients by inhibiting glycolysis.
Example 3 synergistic treatment of aGVHD with glucocorticoid and a glycolytic agent
1. Obtaining CD3 of aGVHD patients + T cell
The aGVHD patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) were established patients in the civil hospital at the university of beijing and agreed with the known conditions of the related experiments.
1. Peripheral blood of the patient is taken, and mononuclear cells are isolated.
2. MiniMACS using CD3 magnetic beads and with MS sorting column TM Separator separation of CD3 from the mononuclear cells obtained in step 1 + Cells, i.e. CD3 + T cells.
2. Fluorescent microscope observation
The test cells were: CD3 prepared in step one + T cells.
1. 96-well plates were prepared and pre-coated with 1 Xrecombinant human fibronectin (sigma, cat# ECM 001) for 24 hours.
2. After completion of step 1, 2X 10 cells were inoculated per well 5 The test cells were cultured in 100-200. Mu.l of DMEM high-sugar medium containing CD3/CD28 antibody-conjugated 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 packet processing is as follows:
a first group: MP and 3PO are added and then cultured for 48 hours; 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 (treatment without 3PO addition was set as a0 concentration control for 3 PO).
Second group: MP and MTX were added and then incubated for 48 hours; MP concentration in the system was 1. Mu.M, MTX concentration 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 addition was set as a0 concentration control for 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 then observation counting was excited by using a fluorescence microscope with green light and blue light.
The results are shown in FIG. 3I.
3. Detection of other indicators
1. Packet processing
(1) Taking 96-well plates, inoculating 8×10 each 4 CD3 prepared in step one + T cells were cultured for 24 hours with 100-200. Mu.l of DMEM high-sugar medium containing CD3/CD28 antibody-conjugated magnetic beads and 10% FBS. The ratio of the number of CD3/CD28 antibody coupled magnetic beads to the number of cells is 1:1.
(2) After step (1) is completed, the packet processing is as follows:
first group (gvhd+3po group): adding 3PO and then culturing; the concentration of 3PO in the system was 10. Mu.M;
second group (gvhd+mp group): MP is added and then cultured; MP concentration in the system was 1. Mu.M;
third group (gvhd+mtx group): adding MTX and then culturing; the concentration of MTX in the system was 0.25. Mu.M;
fourth group (gvhd+mp+3po group): adding MP and 3PO, and then culturing; MP concentration in the system was 1. Mu.M, 3PO concentration in the system was 10. Mu.M;
fifth group (gvhd+mp+mtx group): MP and MTX are added and then cultured; MP concentration in the system was 1. Mu.M, MTX concentration in the system was 0.25. Mu.M;
sixth group (GVHD group): no treatment was performed.
The incubation time was 48 hours.
2. Flow cytometry detection of PFKFB3 and GLUT1
(1) Taking the cells treated in the step 1 to a flow detection tube.
(2) Add 5. Mu.l CD3 to the flow tube + Cell surface flow antibody CD3, incubated at room temperature for 15min in the dark.
(3) Adding 2ml PBS buffer solution, mixing, and isolating at 1500rpmHeart for 5 min; then, the supernatant was discarded, and 100. Mu.l of FIX was added&PERM TM The fixed medium A (Invitrogen, cat# GAS 004) in the cell permeabilization kit was mixed well and incubated at room temperature for 15min in the absence of light.
(4) 2ml PBS buffer is added and mixed well, and centrifuged at 1500rpm for 5 minutes; then, the supernatant was discarded, and 100. Mu.l of FIX was added&PERM TM The permeabilization medium B (invitrogen, cat# GAS 004) in the cell permeabilization kit is mixed well, 1 μl of intracellular type antibody is added, and incubated at room temperature in the absence of light for 15min. Intracellular antibodies are respectively: intracellular flow antibody PFKFB3 (Cell Signaling Technology, danvers, MA, USA) or intracellular flow antibody GLUT1 (Cell Signaling Technology, danvers, MA, USA).
(5) 2ml PBS buffer is added and mixed well, and centrifuged at 1500rpm for 5 minutes; then, the supernatant was discarded, and 100. Mu.l of FIX was added&PERM TM And uniformly mixing a permeabilization medium B in the cell permeabilization kit, adding a rabbit anti-mouse secondary antibody (Cell Signaling Technology, danvers, MA, USA), and incubating for 15min at room temperature in a dark place.
(6) 2ml PBS buffer is added and mixed well, and centrifuged 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 detected on the machine within 4 hours. Streaming image analysis employed Diva 7.0 software (BD company, usa).
The results are shown in fig. 3F and G.
3. Detecting glucose consumption
(1) Taking 96-well plates, inoculating 2×10 each 5 The cells treated in step 1 were cultured in 100-200. Mu.l of DMEM high-sugar medium containing CD3/CD28 antibody-conjugated 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) Taking the supernatant obtained in the step (2), detecting the glucose content by using a glucose measuring kit (Nanjing institute of biological engineering, cat# 361510), and calculating the glucose consumption rate.
The results are shown in FIG. 3D.
4. Detection of lactic acid production
(1) 96 wells are takenPlates, inoculated 2X 10 per well 5 The cells treated in step 1 were cultured in 100-200. Mu.l of DMEM high-sugar medium containing CD3/CD28 antibody-conjugated 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 lactic acid by using a lactic acid detection kit (Nanjing institute of biological engineering, product number: A019-2-1), and calculating the lactic acid production rate.
The results are shown in FIG. 3E.
5. Detecting expression of pro-inflammatory cytokines in T cells
(1) Taking 96-well plate, inoculating 2×10 each well 5 After the treatment of step 1, 100. Mu.l of 1 ng/. Mu.l of PMA aqueous solution, 2. Mu.l of 1. Mu.g/. Mu.l of ionomycin aqueous solution and 0.7. Mu.l of Golgiston aqueous solution were added, mixed with shaking, and incubated in a incubator at 37℃for 4-6 hours.
(2) Centrifugation at 1500rpm for 5min, the supernatant was discarded, the cells were sprung, surface flow antibodies (CD 3 antibody, CD8 antibody and CD25 antibody) were added, and incubation was performed for 15min at room temperature.
(3) 2ml of PBS buffer was added, centrifuged at 1500rpm for 5min, and the supernatant was discarded to spring up the cells.
(4) 1ml of the solution obtained from eBioscience was added TM Foxp 3/transcription factor immobilization concentrate and the reagent buffer were mixed according to 1:3, mixing the prepared fixing solution by vortex, and incubating for 30min at 4 ℃.
(5) 2ml of water prepared from wash buffer and deionized water was added at a ratio of 1:9 ratio of formulated Nuclear Membrane disruption working fluid (Invitrogen, eBioscience TM Foxp 3/transcription factor staining buffer kit, cat: 00-5523-00), centrifugation at 2000rpm for 5min, and discarding the supernatant.
(6) Intracellular antibodies (IFN-. Gamma., IL-4, IL-17 and Foxp 3) were added, vortexed and incubated at 4℃for 30min.
(7) 2ml of the working solution for breaking nuclear membrane was added thereto, 2000rpm X5 min, and the supernatant was discarded, and 200. Mu.l of the working solution for breaking nuclear membrane was added thereto to bounce the cells.
(8) And (5) detecting the machine in 24 hours. Streaming image analysis uses Diva 7.0 software.
The results are shown in fig. 3 a and B.
6. CCK-8 detection of T cell proliferation
(1) A96-well plate was used, and 100. Mu.l of 1X 10 was inoculated into each well 5 And/ml the cells treated in the first step, and culturing for 24 hours.
(2) Mu.l of CCK-8 solution was added to each well and incubated in an incubator for 2h.
(3) The absorbance at 450nm was measured with a microplate reader.
The results are shown in FIG. 3C.
7. Detection of NF- κ B p65
(1) Taking the cells treated in the step 1 to a flow detection tube.
(2) Add 5. Mu.l CD3 to the flow tube + Cell surface flow antibody CD3, incubated at room temperature for 15min in the dark.
(3) 2ml PBS buffer is added and mixed well, and centrifuged at 1500rpm for 5 minutes; then, the supernatant was discarded, and 100. Mu.l of FIX was added&PERM TM The fixed medium A (Invitrogen, cat# GAS 004) in the cell permeabilization kit was mixed well and incubated at room temperature for 15min in the absence of light.
(4) 2ml PBS buffer is added and mixed well, and centrifuged at 1500rpm for 5 minutes; then, the supernatant was discarded, and 100. Mu.l of FIX was added&PERM TM The permeabilization medium B (invitrogen, cat# GAS 004) in the cell permeabilization kit was mixed well, 1. Mu.l of intracellular type antibody NF- κ B p65 (Cell Signaling Technology, danvers, mass., USA) was added and incubated at room temperature for 15min in the absence of light.
(5) 2ml PBS buffer is added and mixed well, and centrifuged at 1500rpm for 5 minutes; then, the supernatant was discarded, and 100. Mu.l of FIX was added&PERM TM And uniformly mixing a permeabilization medium B in the cell permeabilization kit, adding a rabbit anti-mouse secondary antibody (Cell Signaling Technology, danvers, MA, USA), and incubating for 15min at room temperature in a dark place.
(6) 2ml PBS buffer is added and mixed well, and centrifuged 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 detected on the machine within 4 hours. Streaming image analysis employed Diva 7.0 software (BD company, usa).
The results are shown in FIG. 3H.
4. Calculation of CI values
Test cells: CD3 prepared in step one + T cells.
Two drug combinations were set up: MP and 3PO in combination, MP and MTX in combination.
Each medicine combination is provided with a plurality of proportioning concentrations.
And (3) processing according to the packet processing method in the step two.
Calculation of CD3 by Com-puSyn software + The inhibition rate of the synthesis of IFNgamma by the T cells is 25%, 50%, 75%, 90% and 95% of the corresponding CI value. Calculation of CD3 by Com-puSyn software + The inhibition rate of T cell proliferation was 25%, 50%, 75%, 90%, 95% of the corresponding CI value.
The results are shown in J of FIG. 3. The average CI value for MP and 3PO for T cell IFNγ synthesis was 0.364; the average CI value for MP and MTX for T cell IFNγ synthesis was 0.554.MP and 3PO had an average CI value for T cell proliferation of 0.475; the average CI for MP and MTX for T cell proliferation was 0.406.
In vitro combined application of glucocorticoid (MP) and a glycolytic agent (3 PO or MTX) to peripheral T cells of an aGVHD patient is found to synergistically inhibit glycolysis by the combined application of glucocorticoid and the glycolytic agent, so that activation and proliferation of peripheral T cells of the aGVHD patient are synergistically inhibited.
MP (1. Mu.M) and MTX (0.5 μm) were found to have the best effect of inhibiting T cells in aGvHD patients by a dose-response effect for 48 hours, but had no significant effect on T cell survival. In vitro application of MP and MTX significantly reduced the rate of glucose consumption and lactate production. MP reduces glycolytic activity of T cells by down regulating GLUT1 and PFKFB3 expression. MTX reduces glycolytic activity of T cells by down regulating GLUT1 expression. In addition, MP and MTX reduced the proportion of pro-inflammatory cells, including Th1, tc1 cells, in the aGVHD patient T cells. And MP and MTX reduced proliferation of T cells 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 combined use of a glucocorticoid and a glycolytic modulator has a synergistic effect on the improvement of T cell activity in aGvHD patients, the mechanism of which may be achieved by decreasing glycolytic activity. The combined use of a glucocorticoid and a glycolytic modulator has a synergistic inhibitory effect on T cell differentiation towards pro-inflammatory Th1, tc1 and T cell proliferation compared to single drug treated cells. Furthermore, the combination of a glucocorticoid and a glycolytic modulator has a superior in vitro down-regulation of glycolytic activity than the glucocorticoid alone.
Notably, the combination protocol was very synergistic for T cells with MP and 3PO having an average CI value of 0.364 for T cell ifnγ synthesis. Furthermore, the average CI value for MP and MTX for T cell IFNγ synthesis was 0.554. The average CI for MP and 3PO for T cell proliferation was 0.475. In addition, the average CI value for MP and MTX for T cell proliferation was 0.406.
Example 4 synergistic treatment of aGVHD with glucocorticoid and a glycolytic agent
The aGVHD mouse model was used to verify the co-therapeutic effect of glucocorticoids with glycolytic modulators.
NPG mice (NOD.Cg-Prkdcscid Il2rgtm1Vst/Vst mice): beijing Vitolihua laboratory animal technologies Co.
Luciferase luciferases + THP-1 leukemia cells (THP-1-luccells) are described in the following documents: azacytidine prevents experimental xenogeneic graft-versus-host diseasewithout abrogating graft-versus-leukemia effects, ONCOIMMUNOLOGY,2017,VOL.6,NO.5,e1314425.
1. Suspending human peripheral blood stem cells with 1 Xhemolysin, shaking for 5 seconds, standing for 15 minutes in ice in the dark, centrifuging at 1500rpm for 5 minutes, and discarding the supernatant; then suspending with PBS buffer solution, centrifuging at 1500rpm for 5 minutes, and discarding the supernatant; then, the cells were suspended in PBS buffer to obtain a cell suspension. 1 part by volume of 10 Xhemolysin (BD Biosciences) and 9 parts by volume of sterilized water for injection were mixed to obtain 1 Xhemolysin.
2. The NPG mice begin drinking antibiotic water a week before injecting cells, prevent infection, and are placed into an air laminar flow cabinet for aseptic feeding.
3. The day before cell injection 60 CoγThe NPG mice were irradiated systemically with 1.5Gy (semi-lethal dose).
4. Each mouse was injected with the cell suspension prepared in step 1 via tail vein (5X 10) 6 Individual cells/individual) and luciferase + THP-1 leukemia cells (1X 10) 6 Individual cells/cell). Days started after the injection was completed.
5. The mice completed in step 4 were randomly divided into six groups (PBS group, MP group, 3PO group, MTX group, MP combined 3PO group, MP combined MTX group), 11 each. PBS group mice were intraperitoneally injected with PBS buffer 1 time per day at a volume of 200 μl/mouse. The MP group of mice were intraperitoneally injected with 1 MP solution per day at a volume of 200 μl/dose of MP at 2 mg/kg body weight/dose. Mice in the 3PO group were intraperitoneally injected 1 time per day with 3PO solution at a volume of 200 μl/dose of 3PO at 25 mg/kg body weight/time. MTX group mice were intraperitoneally injected with 1 MTX solution per day at a volume of 200 μl/dose of MTX at 1 mg/kg body weight/dose. MP-3PO solution was injected intraperitoneally 1 time per day in 200 μl/mouse, with MP at a dose of 2 mg/kg body weight/mouse, and 3PO at a dose of 25 mg/kg body weight/mouse. MP in combination with MTX mice were intraperitoneally injected with 1 MP-MTX solution per day at a volume of 200 μl/dose of MP at 2 mg/kg body weight/dose and MTX at 1 mg/kg body weight/dose. The administration was continued for 30 days. The PBS group is also referred to as CTL group.
MP solution is prepared by dissolving MP in sterile water. The 3PO solution was obtained by dissolving 3PO in sterile water. The MTX solution is prepared by dissolving MTX in sterile water. MP-3PO solution is prepared by dissolving MP and 3PO in sterile water. MP-MTX solution is prepared 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, coat texture, skin integrity, and diarrhea. The results are shown in FIG. 5.
On day 30, mice were sacrificed, livers and spleens were taken, mononuclear cells were collected, and CD4 was detected by flow cytometry + Cell proportion, CD8 + Cell proportion, th1 cellsProportion, tc1 cell proportion, and T cell proliferation potency. The results are shown in FIG. 6.
On day 28, mouse peripheral blood was collected and single nuclear cells were collected and examined for CD4 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 fixed with 4% paraformaldehyde, then paraffin sections were prepared and HE stained. Mice were scored for acute GVHD pathology according to a scoring system. The results are shown in FIG. 8.
On day 21, NPG mice were assessed for clearance of leukemic cells following transplantation by in vivo bioluminescence imaging. The results are shown in FIG. 9. Searching a way for effectively treating GVHD and maintaining GVL effect is important to improving the curative effect of hematopoietic stem cell transplantation and reducing the complications related to the transplantation. To further investigate whether MP in combination with 3PO or MTX treatment affects GVL effect, luciferase was reinfused + THP-1 leukemia cells clearance of leukemia cells after NPG mice were assessed by in vivo bioluminescence imaging. The effect of combination treatment of MP with 3PO or MTX on GVL was not apparent compared to MP alone. These results indicate that MP in combination with 3PO or MTX can synergistically reduce T cell alloreactivity by down-regulating glycolytic activity in a humanized mouse model, improving aGvHD without losing GVL effect.
The combined use of glucocorticoid (MP) and a glycolytic agent (3 PO or MTX) reduced mice aGvHD clinical score and mortality compared to single drug treatment. Pathological evidence suggests that the GVHD target organ pathology scores were significantly reduced in mice with MP combined with 3PO or MTX. The results indicate that either combination of MP and 3PO or combination of MP and MTX can synergistically treat aGVHD.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present 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 respect to specific embodiments, it will be appreciated that the invention may 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 application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims (2)

1. Use of a combination of a glucocorticoid and a glycolytic demodulator for the preparation of a medicament for acute graft versus host disease;
the glycolysis demodulator is 3PO;
the glucocorticoid is methylprednisolone;
in the medicine, the molar ratio of the methylprednisolone to the 3PO is 1:10.
2. A medicine contains glucocorticoid and glycolytic demodulator as active ingredients; the medicine is a medicine for treating acute graft versus host disease;
the glycolysis demodulator is 3PO;
the glucocorticoid is methylprednisolone;
in the medicine, the molar ratio of the methylprednisolone to the 3PO is 1:10.
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