CN116966186A - Application of ticagrelor in preparation of medicines for treating immune diseases - Google Patents
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Classifications
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- 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
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- 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
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- 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 application relates to the technical field of biology, and particularly discloses application of ticagrelor and pharmaceutically acceptable salts thereof in preparation of medicines for treating immune diseases caused by excessive activation and activation of T lymphocytes. The ticagrelor can inhibit the activation and proliferation of T lymphocytes, has obvious inhibition effect on T lymphocyte effector molecules IL-17 and IFN-gamma, and can treat immune diseases (such as immune diseases caused by rejection reaction after organ transplantation and multiple sclerosis-cerebrospinal meningitis) caused by the excessive activation and the activation of the T lymphocytes, thereby slowing down the progress of the diseases.
Description
Technical Field
The application relates to the technical field of biology, in particular to application of ticagrelor and pharmaceutically acceptable salts thereof in preparation of medicines for treating immune diseases.
Background
Ticagrelor (ticagrelor) was the first reversible binding P2Y12 receptor antagonist to inhibit platelet activation in patients with coronary heart disease and was approved by the U.S. food and drug administration in 2011. Compared with Clopidogrel (Clopidogrel), a common P2Y12 receptor inhibitor, ticagrelor has better therapeutic effects on preventing vascular events and death in patients with Acute Coronary Syndrome (ACS). At present, ticagrelor is widely used in patients with respect to clopidogrel and other various antiplatelet drugs, and is recommended as a first-line antiplatelet therapeutic drug in clinical guidelines.
It has been reported that ticagrelor contributes to reduction of mortality of inflammatory diseases such as sepsis and infection, as compared to clopidogrel. However, whether ticagrelor has platelet-independent immunomodulatory effects has not been reported.
Disclosure of Invention
The application aims to overcome the defects of the prior art and provide an application of ticagrelor and pharmaceutically acceptable salts thereof in preparing medicines for treating immune diseases.
In order to achieve the above purpose, the technical scheme adopted by the application is as follows:
the application provides application of ticagrelor and pharmaceutically acceptable salts thereof in preparing medicines for treating immune diseases caused by excessive activation and activation of T lymphocytes.
Through a great deal of researches and experiments, the inventor of the application finds that ticagrelor can inhibit immune diseases caused by excessive activation and activation of T lymphocytes, thereby slowing down the progress of the diseases.
As a preferred embodiment of the use according to the application, the immune disorder comprises one of an immune disorder caused by rejection after organ transplantation, multiple sclerosis-cerebrospinal meningitis.
In the field of organ transplantation, immune rejection after transplantation and complications thereof bring great problems to the long-term survival of patients after transplantation, prevention and treatment of immune rejection are key to the long-term survival of transplanted patients, and T lymphocyte mediated rejection, particularly IL-17 and IFN-gamma secretion, are major factors of rejection after transplantation.
In the field of autoimmune diseases, multiple sclerosis is mainly characterized by inflammatory demyelinating lesions of the central nervous system, the specific mechanism is not clear, but the infiltration of multiple lymphocytes, especially Th17 cells, at the spinal cord injury is shown by the local high expression state of IL-17 and IFN-gamma, which aggravates the injury.
Through specific experimental exploration, the application can obviously relieve T lymphocyte mediated immune response by adopting ticagrelor, thereby treating immune diseases and multiple sclerosis-cerebrospinal meningitis caused by rejection reaction after transplantation.
As a preferred embodiment of the use according to the application, the ticagrelor and the pharmaceutically acceptable salts thereof inhibit the T lymphocyte effector molecules IL-17 and IFN- γ.
Ticagrelor can inhibit the activation and proliferation of T lymphocytes, has obvious inhibition effect on T lymphocyte effector molecules IL-17 and IFN-gamma, and can treat immune diseases caused by the excessive activation and the activation of T lymphocytes.
As a preferred embodiment of the use according to the application, the dose of ticagrelor is in the range of 2 μm to 100 μm in vitro cell experiments. Preferably, the dose of the ticagrelor is 20 mu M-100 mu M.
Ticagrelor exhibits a gradient dose-dependent inhibition of purified CD3+ T lymphocyte effector molecules IFN-gamma and IL-17, and such inhibition occurs at an earlier time (12 h). Meanwhile, ticagrelor can inhibit the expression of T lymphocyte activating molecules CD69 and CD25, and has obvious inhibition effect on T lymphocyte division proliferation (CFSE).
As a preferred embodiment of the use according to the application, the ticagrelor achieves alleviation of symptoms of immune disorders by modulating T lymphocyte infiltration.
In an in vitro experiment, after the ticagrelor is administrated to a BN and Lewis rat heart transplantation model, the transplanted heart is significantly reduced in infiltrating CD3+T lymphocytes, CD45+lymphocytes and CD45+CD3+T cells, which indicates that the ticagrelor can prolong the survival time of the implant by regulating T lymphocyte infiltration.
After ticagrelor is administrated to a multiple sclerosis-encephalomyelitis mouse model, the expression of IL-17 and IFN-gamma in spinal cord infiltration CD4+Tfh cells is obviously reduced, the proportion and quantity of infiltration CD45+lymphocytes, CD3+CD4+Tfh lymphocytes and CD3+CD8+CTL lymphocytes in spinal cord are also obviously reduced, which indicates that the ticagrelor can lighten the spinal cord injury degree in a cerebrospinal meningitis mouse model and lighten the clinical symptoms of multiple sclerosis by regulating T lymphocyte infiltration.
As a preferred embodiment of the use according to the application, the administration of ticagrelor was performed in animal experiments at a dose of 10mg/kg/day.
As a preferred embodiment of the use according to the present application, the T lymphocytes comprise at least one of cd3+ T lymphocytes, cd4+ T lymphocytes, cd8+ T lymphocytes, cd45+ lymphocytes, cd3+cd4+ Tfh lymphocytes and cd3+cd8+ CTL lymphocytes.
The application also provides application of ticagrelor and pharmaceutically acceptable salts thereof in preparing medicines for inhibiting T lymphocyte effector molecules IL-17 and IFN-gamma.
Compared with the prior art, the application has the following beneficial effects:
the application provides application of ticagrelor and pharmaceutically acceptable salts thereof in preparing medicines for treating immune diseases caused by excessive activation and activation of T lymphocytes, the ticagrelor can inhibit activation and proliferation of the T lymphocytes, has obvious inhibition effect on T lymphocyte effector molecules IL-17 and IFN-gamma, and can treat the immune diseases caused by excessive activation and activation of the T lymphocytes, thereby slowing down the progress of the diseases.
Drawings
FIG. 1 is a graph showing the results of the gradient dependent inhibition of purified CD3+ T lymphocyte effector molecules IFN-gamma and IL-17 by ticagrelor;
FIG. 2 is a graph showing the results of the inhibition of purified CD3+ T lymphocyte effector molecules IFN-gamma and IL-17 by ticagrelor at various times;
FIG. 3 is a flow chart of ticagrelor inhibiting the purification of CD3+ T lymphocyte effector molecules IFN-gamma and IL-17;
FIG. 4 is a graph showing the results of ticagrelor inhibiting T lymphocyte activating molecules CD69, CD25 and division proliferation (CFSE);
FIG. 5 is a graph of the results of transplanted heart survival time for a ticagrelor dosed BN and Lewis rat heart transplant model;
FIG. 6 is a graph showing the results of infiltration numbers and ratios of CD45+ lymphocytes and CD45+CD3+ T cells following administration of ticagrelor to a BN and Lewis rat heart transplant model;
FIG. 7 is a graph showing the results of infiltration numbers and ratios of CD45, CD45+ T lymphocytes, CD3, CD3+ T lymphocytes after administration of ticagrelor to a BN and Lewis rat heart transplant model;
FIG. 8 is a graph showing the results of infiltration numbers and ratios of CD4, CD3+CD4+T lymphocytes, CD8 and CD3+CD8+T lymphocytes after administration of BN and Lewis in a rat heart transplant model;
FIG. 9 is a graph I of staining results of a model of cardiac transplantation in rats with ticagrelor administered BN and Lewis;
FIG. 10 is a graph II of staining results of a model of cardiac transplantation in rats with ticagrelor administered BN and Lewis;
FIG. 11 is a graph showing the results of disease progression in a multiple sclerosis-encephalomyelitis mouse model administered with ticagrelor;
fig. 12 is a graph of the results of the proportion and number of infiltrating cd3+cd4+tfh cells, cd45+ lymphocytes in spinal cord of ticagrelor-administered group;
FIG. 13 is a graph showing the results of expression of IL-17 and IFN-gamma in infiltrating CD3+CD4+Tfh cells, CD3+CD8+CTL cells, and the ratio and number of CD4+Tfh cells in spinal cord of ticagrelor-dosed groups;
FIG. 14 is a staining chart of lymphocyte infiltration in a mice model of multiple sclerosis-encephalomyelitis with ticagrelor administration;
Detailed Description
For a better description of the objects, technical solutions and advantages of the present application, the present application will be further described with reference to the accompanying drawings and specific embodiments.
In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available.
EXAMPLE 1 inhibition of purified CD3+ T lymphocyte effector molecules IFN-gamma and IL-17 by ticagrelor
In vitro experiments: the application obtains CD3+ T cells in human peripheral blood lymphocytes through purification by the prior art steps, adopts anti-CD3+ anti-CD28 stimulation, adopts a test group to administer different doses of ticagrelor (tic) 2 mu M-100 mu M, and a control group to administer an equivalent amount of physiological saline, and the inventor discovers that the ticagrelor has gradient dependent inhibition effect on purified CD3+ T lymphocyte effector molecules IFN-gamma and IL-17 (figure 1), and the inhibition occurs at an earlier time (12 h) and lasts for 72h (figure 2).
Streaming results also confirm the previous results (fig. 3). Ticagrelor can inhibit the expression of T lymphocyte activating molecules CD69 and CD25, while ticagrelor has a significant inhibitory effect on T lymphocyte division proliferation (CFSE) (fig. 4).
Example 2 Effect of ticagrelor on BN and Lewis rat heart transplantation models
The construction of the BN and Lewis rat heart transplantation model comprises the following steps:
1) Donor acquisition: BN rat heart was used as donor. After the BN rats were anesthetized, the chest and abdomen hairs were shaved, and then transferred to a constant temperature microsurgery console, with the limbs fixed with adhesive tape. Chest and abdomen skin was sterilized, the abdomen was cut off and the inferior vena cava was exposed, and heparinized anticoagulation was performed by injecting 3-4ml heparin sodium solution (500U/kg) from the inferior vena cava. Cutting the chest cavity to fully expose the heart, separating and ligating the superior vena cava from the inferior vena cava, and cutting off the superior vena cava; the ascending aorta and pulmonary artery trunks are separated and sheared as close to the distal end as possible. Then the heart is pressed rapidly, blood in the heart is emptied fully, and heparin sodium physiological saline is injected into the heart through the inferior vena cava, and the heart is washed and perfused fully. The inferior vena cava and the pulmonary vein are pricked along the proximal end of the heart, the heart is separated and taken down, and immediately put into pre-cooled heparin sodium physiological saline for ice bath preservation.
2) Heart transplantation: lewis rats pre-sensitized with skin grafts were used as recipients. After anesthesia, the abdominal hair is shaved, transferred to a constant temperature microsurgery operating table, and the limbs are fixed by rubberized fabric. The abdominal skin is disinfected, an abdominal median incision is taken, the abdominal wall is cut, the intestinal canal is removed, the peritoneum and the perivascular soft tissues are separated, the abdominal aorta and the inferior vena cava are fully exposed, and the abdominal aortic and the inferior vena cava are blocked simultaneously by using vascular blocking forceps. The right incision is made from the median longitudinal line of the driven vein, then the main artery of the receptor and the inferior vena cava are anastomosed with the main artery and the pulmonary artery of the donor heart respectively in an end-side anastomosis mode, and the 8-0 nylon thread is continuously sutured. After the suturing is completed, the blocking forceps are opened, and the perfusion condition, the pulse rhythm, the pulse force, whether the anastomotic stoma is oozed or not and the like of the transplanted heart are observed. Finally, the intestinal tube is moved back to the abdominal cavity, guan Fu, and the abdominal skin is disinfected again. The anaesthetic mask was removed and the recipient mice were placed on a heating pad to keep warm until complete recovery from anaesthesia.
3) Post-operation treatment: after observing for 1 hour after operation, the recipient rats are returned to the feeder cage after normal activities, feeding and the like. The next day, the pulsation of the transplanted heart is observed and palpated, and if the pulsation disappears, the operation failure is defined and the experiment is not included. If no special, the experiment is continued.
4) Experiment and control: the arrangement of the experimental group and the control group corresponds to the skin transplantation group, the experimental group is continuously given with 10mg/kg/day of gavage ticagrelor or 10mg/kg/day of clopidogrel, and the control group is given with an equivalent amount of physiological saline.
5) Observing and obtaining materials: survival of the graft, such as the disappearance of the transplanted heart beat, is observed and recorded daily and is defined as graft deactivation. In the first survival experiment, rats were euthanized by day 30 after heart transplantation. In the second experiment, rats were euthanized by analysis of the material on day 5 post-implantation.
Experimental results: in isogenic transplantation, the transplanted heart can survive for a long time, and the survival time of the transplanted heart of the control group is about 7 days; the survival time of transplanted hearts is prolonged by three times and can reach 21 days at most by administering ticagrelor 10mg/kg/day groups; while the same P2Y12 receptor antagonist clopidogrel (10 mg/kg/day) group was not significantly different from the control group (FIG. 5).
The transplanted heart can detect the infiltrating lymphocytes by the internal flow method, and the infiltrating quantity and proportion of the CD45+ lymphocytes and CD45+CD3+ T cells are obviously reduced compared with the comparison group after the administration of ticagrelor for gastric lavage. The ratio of cd3+ T lymphocytes, cd4+ T lymphocytes and cd8+ T lymphocytes was not significantly altered, while the numbers were significantly reduced after administration of ticagrelor compared to the control and clopidogrel groups (fig. 6-8).
H & E staining showed that the ticagrelor group was less damaged than the other two groups (fig. 9). The CD 3-specific immunohistochemical results were consistent with the flow results, with significantly reduced infiltration of cd3+ T lymphocytes in the transplanted heart after administration of ticagrelor compared to the placebo and clopidogrel groups (fig. 10). It can be seen from this that ticagrelor can prolong graft survival time by modulating T lymphocyte infiltration.
Example 3 Effect of ticagrelor on multiple sclerosis-encephalomyelitis mouse model
The construction of the clinical multiple sclerosis-encephalomyelitis mouse model comprises the following steps:
1) Model construction: the antigen myelin oligodendrocyte glycoprotein (MOG 35-55) polypeptide was diluted to 300ug/50ul in 6 week male C57BL/6 mice with 1:1 adding equivalent complete Freund adjuvant, supplementing the content of tubercle bacillus to 5mg/ml, mixing, fully emulsifying, and selecting four subcutaneous injections at root and back of mouse tail according to 100 μl each. On the day of immunization, 100 μl of PBS containing 200ng pertussis toxin was intravenously injected via the tail of the mice 24h later.
2) Experimental grouping: experimental group intragastric ticagrelor 10mg/kg/day; the control group was given an equivalent amount of physiological saline for gastric lavage.
3) Model evaluation: neurological scoring: the body weight of the mice was measured from the day of immunization induction, and the neurological score was performed by a double-blind method 32 days after immunization, with the following specific scoring criteria: grade 0, no clinical manifestation; grade 1, listlessness, tail weakness; level 2, abnormal walking gait, weakness of the double hind limbs; level 3, paralysis of the double hind limbs; level 4, double hind limb paralysis with one or two sides of forelimb paralysis; grade 5, dying or dying. Recurrence is defined as the clinical score of the recurrence after the recovery of the first onset is higher than before, or the same score lasts for more than 3 days.
4) Tissue sampling and pathological analysis during the peak period of the EAE model, mice are anesthetized by intraperitoneal injection of chloral hydrate, and the brains and spinal cords are taken and fixed in 4% paraformaldehyde, and paraffin embedded sections, HE and fast green staining are observed. Inflammatory cell infiltration exists in brain and spinal cord parenchyma, but lesion distribution is mainly expanded in spinal cord neck and waist, and is represented by infiltration of a large number of lymphocytes and neutrophils, infiltration extends from outside to inside along envelope and small blood vessels, and blood vessel sleeves are formed around the small blood vessels. Fast green staining can be seen in inflammatory infiltrate areas, with variable-sized lamellar demyelination areas in the spinal cord white matter, loose myelin fibers, and massive vacuoles.
Experimental results: this example constructed a clinical multiple sclerosis-encephalomyelitis mouse model, and it can be seen from the three week clinical scores that clinical scores were significantly reduced at the beginning of day 10 after administration of ticagrelor intragastric administration (10 mg/kg/day) and the progression was slower at the middle and later 18-21 days (fig. 11) compared to the control group. H & E and LFB staining showed significant lymphocyte infiltration in the control group and significant spinal cord demyelination, with significant remission in the ticagrelor group (fig. 14).
The difference in lymphocyte infiltration in spinal cord was seen by flow staining, with the proportion and number of infiltrating cd4+ lymphocytes in spinal cord of ticagrelor-administered group significantly lower than control group, wherein the proportion and number of cd3+cd4+tfh cells and cd3+cd8+ CTL cells were also significantly reduced (fig. 12-13). Functionally, detection of IL-17 and IFN-gamma expression in spinal cord infiltrating CD4+ Tfh cells revealed that both were significantly reduced after administration of ticagrelor compared to the control. Therefore, ticagrelor can reduce the spinal cord injury degree in a mouse model of cerebrospinal meningitis and alleviate the clinical symptoms of multiple sclerosis by regulating T lymphocyte infiltration.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present application and not for limiting the scope of the present application, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present application may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present application.
Claims (8)
1. Application of ticagrelor and pharmaceutically acceptable salts thereof in preparing medicines for treating immune diseases caused by excessive activation and activation of T lymphocytes.
2. The use of claim 1, wherein the immune disorder comprises one of an immune disorder caused by rejection after organ transplantation, multiple sclerosis-cerebrospinal meningitis.
3. The use of claim 1, wherein said ticagrelor and pharmaceutically acceptable salts thereof inhibit T lymphocyte effector molecules IL-17 and IFN- γ.
4. The use according to claim 1, wherein the dose of ticagrelor is 2 μm to 100 μm in an in vitro cell assay.
5. The use of claim 1, wherein ticagrelor achieves alleviation of symptoms of an immune disorder by modulating T lymphocyte infiltration.
6. The use according to claim 1, wherein the ticagrelor is administered in an amount of 10mg/kg/day in animal experiments.
7. The use of claim 1, wherein the T lymphocytes comprise at least one of cd3+ T lymphocytes, cd4+ T lymphocytes, cd8+ T lymphocytes, cd45+ lymphocytes, cd3+ cd4+ Tfh lymphocytes, and cd3+ cd8+ CTL lymphocytes.
8. Application of ticagrelor and pharmaceutically acceptable salts thereof in preparing medicines for inhibiting T lymphocyte effector molecules IL-17 and IFN-gamma.
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