CN111494620A - Application of trametinib in preparation of vaccine - Google Patents
Application of trametinib in preparation of vaccine Download PDFInfo
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- CN111494620A CN111494620A CN202010370525.XA CN202010370525A CN111494620A CN 111494620 A CN111494620 A CN 111494620A CN 202010370525 A CN202010370525 A CN 202010370525A CN 111494620 A CN111494620 A CN 111494620A
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- LIRYPHYGHXZJBZ-UHFFFAOYSA-N trametinib Chemical compound CC(=O)NC1=CC=CC(N2C(N(C3CC3)C(=O)C3=C(NC=4C(=CC(I)=CC=4)F)N(C)C(=O)C(C)=C32)=O)=C1 LIRYPHYGHXZJBZ-UHFFFAOYSA-N 0.000 title claims abstract description 48
- 229960004066 trametinib Drugs 0.000 title claims abstract description 44
- 229960005486 vaccine Drugs 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title claims description 10
- 239000012646 vaccine adjuvant Substances 0.000 claims abstract description 10
- 229940124931 vaccine adjuvant Drugs 0.000 claims abstract description 9
- 230000001737 promoting effect Effects 0.000 claims abstract description 7
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 18
- 230000004069 differentiation Effects 0.000 claims description 11
- 241000700605 Viruses Species 0.000 claims description 8
- 230000002401 inhibitory effect Effects 0.000 claims description 7
- 230000024245 cell differentiation Effects 0.000 claims description 6
- 230000004913 activation Effects 0.000 claims description 4
- 229960004854 viral vaccine Drugs 0.000 claims description 4
- 241001678559 COVID-19 virus Species 0.000 claims description 3
- 241000315672 SARS coronavirus Species 0.000 claims description 3
- 210000002540 macrophage Anatomy 0.000 claims description 3
- 210000001616 monocyte Anatomy 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 7
- 206010035737 Pneumonia viral Diseases 0.000 abstract description 5
- 208000009421 viral pneumonia Diseases 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 230000000694 effects Effects 0.000 abstract description 2
- 230000007365 immunoregulation Effects 0.000 abstract description 2
- 238000011081 inoculation Methods 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- 230000009385 viral infection Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 20
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 12
- 239000002953 phosphate buffered saline Substances 0.000 description 10
- 238000000034 method Methods 0.000 description 8
- 230000000770 proinflammatory effect Effects 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 101001011441 Homo sapiens Interferon regulatory factor 4 Proteins 0.000 description 6
- 102100030126 Interferon regulatory factor 4 Human genes 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 6
- 239000011324 bead Substances 0.000 description 6
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- 239000001963 growth medium Substances 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 230000002757 inflammatory effect Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 238000002255 vaccination Methods 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 210000000662 T-lymphocyte subset Anatomy 0.000 description 2
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- 238000006243 chemical reaction Methods 0.000 description 2
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- 230000001681 protective effect Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
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- 206010002091 Anaesthesia Diseases 0.000 description 1
- 206010013183 Dislocation of vertebra Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 1
- 208000034486 Multi-organ failure Diseases 0.000 description 1
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- 206010035664 Pneumonia Diseases 0.000 description 1
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- 229940079593 drug Drugs 0.000 description 1
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- 210000003743 erythrocyte Anatomy 0.000 description 1
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- 210000000987 immune system Anatomy 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
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- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
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- 239000006166 lysate Substances 0.000 description 1
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- 239000002609 medium Substances 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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Abstract
The invention relates to trametinib, which has a chemical formula as follows: c26H23FIN5O4Further relates to the application of trametinib in preparing vaccines; the use of trametinib as a vaccine adjuvant; the beneficial effects are that: as a broad-spectrum immunoregulation medicine, the medicine has the effect of promoting the generation of antibodies for other viral pneumonia, virus infection or seedling inoculation.
Description
Technical Field
The invention belongs to the technical field of medicine, and particularly relates to application of trametinib in preparation of vaccines.
Background
In order to obtain high-quality blocking antibodies, virus immunization and vaccination are carried out, different subtypes of T cells perform different functions, the Tfh subtype is most important in the process, but simultaneously, excessive T cells of other proinflammatory subtypes can secrete a large amount of inflammatory factors to induce excessive inflammatory response of organisms, and complications occur: fever, organ damage, and even multiple organ failure, which is also the most serious complication of vaccine injection (inflammatory factor storm). The technical problems at present are as follows:
1. in the process of vaccination, an effective antibody can not be obtained when the organism reaction is too weak, an adjuvant or strengthened vaccination is needed, and the death rate is high when the vaccine reaction is too strong due to individual difference;
2. at present, only drugs for enhancing or inhibiting the overall functions of T cells and antibodies for single cytokines are clinically used, the proportion of Tfh cannot be changed for overall intervention of the T cells, viral immunity cannot be enhanced and unnecessary inflammatory reactions cannot be inhibited at the same time, the single cytokines play multiple roles, the corresponding antibodies are used for one purpose, unnecessary complications can be caused, and no more suitable monoclonal antibody is used for viral immunity.
Disclosure of Invention
Aiming at the problems, the invention provides the application of trametinib in preparing a medicament for treating pneumonia, and mainly solves the problem that the prior art relates to the application of trametinib in preparing a vaccine.
In order to solve the problems, the invention adopts the following technical scheme:
trametinib, the chemical formula is: c26H23FIN5O4。
Use of trametinib in the preparation of a vaccine.
The use of trametinib as a vaccine adjuvant.
One mode, use of the trametinib as a vaccine adjuvant that promotes the differentiation of Tfh cells.
One mode, the use of said trametinib as a vaccine adjuvant for promoting Tfh cell differentiation and inhibiting T cell differentiation, monocyte macrophage activation.
In one form, the vaccine is a viral vaccine.
In one form, the virus comprises a COVID-19 virus, a SARS virus.
The invention has the beneficial effects that:
1. can enhance virus immunity, inhibit unnecessary inflammatory reaction and promote antibody generation;
2. as a broad-spectrum immunoregulation medicine, the medicine has the effect of promoting the generation of antibodies for other viral pneumonia, virus infection or seedling inoculation.
Drawings
FIG. 1 is a graph of the results of trametinib decreased IRF4 expression;
FIG. 2 is a graph showing the results of inhibition of total T cell (T lymphocyte) proliferation by trametinib;
FIG. 3 is a graph showing the results of trametinib inhibiting the differentiation of the proinflammatory T cell subsets Th1, Th17 and increasing the differentiation of the proinflammatory cell subset Treg;
FIG. 4 is a graph of the results of trametinib promoting Tfh differentiation, increasing the Tfh cell proportion;
figure 5 is a graph of the results of trametinib increasing the level of protective antibodies.
Detailed Description
The following is further described in conjunction with the detailed description:
use of trametinib in the preparation of a vaccine.
The use of trametinib as a vaccine adjuvant.
The trametinib is applied as a vaccine adjuvant for promoting Tfh cell differentiation and/or inhibiting T cell differentiation and inhibiting activation of mononuclear macrophages.
The vaccine is a viral vaccine.
The virus includes COVID-19 virus and SARS virus.
Example 1: trametinib (Trametinib) reduced IRF4 expression.
The method comprises the following steps:
taking one spleen of a mouse in a sterile environment, adding a proper amount of PBS buffer solution in a sterile platform for grinding, filtering an unground part through a 40-micron screen, centrifuging for 3 minutes at 2000 r/min, discarding a supernatant, cracking red blood cells by using 1ml of ACK lysate for 3 minutes, adding the PBS buffer solution for dilution, and centrifuging again to obtain the lymphocytes. After cell counting lymphocytes were seeded in flat bottom 96 well plates with 1640 complete medium at a density of about 0.5 x 106 cells/well, CD28 was added to a final concentration of 1ug/ml, and Trametinib was added at various concentrations. (96-well plates were coated with 4ug/ml CD3 in advance). After 2-3 days of activation, flow-type staining is carried out, live T cells positive for CD4 are circled, and the IRF4 expression amount is detected (left in figure 1) and counted (right in figure 1).
And (4) analyzing results:
as shown in FIG. 1, the IRF4 expression level is reduced with the increase of Trametinib concentration, the interference of IRF4 expression level is significantly different when the concentration is more than 40nM, and Trametinib reduces the IRF4 expression.
Example 2: trametinib inhibits the proliferation of total T cells.
The method comprises the following steps:
t cell culture medium was mixed at 1: adding CTV cell cycle marker staining (invitrogen C34557) in a ratio of 1000, staining for 20 minutes in a dark place, adding complete culture medium, incubating for 5 minutes, centrifuging cells, removing supernatant, re-suspending cells with the complete culture medium, adding 100nM Trametinib or DMSO (equivalent solvent) in two groups respectively, and performing flow detection on cell proliferation after three days.
And (4) analyzing results:
as shown in fig. 2, T cells stimulated by Trametinib proliferated significantly slower, with the passage number of T cell proliferation being much lower than that of the control group.
Example 3: trametinib inhibits the differentiation of proinflammatory T cell subsets Th1 and Th17, and increases the differentiation of proinflammatory cell subset Treg.
T cell sorting:
to obtain purified CD4+ T cells and T cell depleted (CD3 negative) splenocytes, sorting of specific cell subsets was performed using a levitated and whirlpool magnetic bead sorting system. All manipulations were performed aseptically to prevent cell contamination and sorted according to the instructions of the America whirly and relevant kit.
1. Preparing a cell suspension:
after the operating room is disinfected by ultraviolet irradiation, the mouse is killed by using a cervical vertebra dislocation method and is soaked in 75% alcohol for 5 minutes to reduce pollution; rapidly taking out spleen tissues of the mouse after the abdominal median incision is performed, and putting the spleen tissues into a culture dish which is prepared in advance and is filled with a culture medium; the culture dish was transferred to a clean bench, the spleen was placed in a 200 mesh sieve and gently ground using a 1ml syringe piston, and the filtered single cell suspension was centrifuged, split red and counted for future use.
2. Labeling magnetic beads:
sucking the required amount of cell suspension, and washing the cells for 3 times by using ice PBS; adding 90 ul PBS and 10 ul corresponding labeled magnetic beads into each 107 total cells, mixing uniformly, and incubating for 10 minutes at 4 ℃; after 10 minutes, the cell suspension was removed and washed 3 times with 1ml PBS per 107 cells for use.
3. Sorting cells by a sorter:
placing a specific sorter in a matched magnet, wetting the sorter by PBS, and transferring the cell suspension marked by the magnetic beads into the sorter; collecting the effluent cells by using a centrifugal tube below the sorter, wherein the collected cells are cells which are not labeled by magnetic beads; and taking the sorter out of the magnet, adding a proper amount of PBS, and collecting cells flowing out of the lower part of the sorter, wherein the collected cells are cells marked by the magnetic beads.
Induction of Th cells:
sorted T cells were stimulated for 3 days as follows to obtain different subsets of Th cells. During induction, 100nM Trametinib or DMSO, an equivalent amount, was added to each group and induction was monitored after three days.
And (4) analyzing results:
as shown in fig. 3, the Trametinib intervention has an inhibitory effect on the differentiation of the pro-inflammatory cell subsets Th1 and Th17 and has a promoting effect on the differentiation of Treg suppressor T cells. Trametinib can reduce the proportion of T cells secreting proinflammatory factors and reduce inflammatory factor storm and nonspecific cell injury in the virus immune process.
Example 4: trametinib promotes Tfh differentiation and increases the Tfh cell proportion.
The method comprises the following steps:
the experimental animal and viral pneumonia model construction is that BA L B/c mice are randomly divided into a normal group, a model PBS group and a model Trametinib group, each group comprises 36 mice, the mice are under ether mild anesthesia, each mouse of the two model groups is infected with 25 mu L50L D50 virus liquid drops, the normal group is infected with equal amount of sterile Phosphate Buffered Saline (PBS) liquid drops, 1h after infection, the normal group and the model group are subjected to sterile PBS intragastric perfusion, the Trametinib group is subjected to 3 mg/(kg. D) intragastric perfusion, each group is subjected to 1 time/D administration, the administration is continuously carried out until the detection time point, the spleen of the mice is taken, and the Tfh subgroup proportion is detected.
And (4) result sorting:
as shown in figure 4, the Tfh cell ratio in the mouse viral pneumonia model is obviously higher than that of the model-free model, the model is successfully formed, and the mouse is infected with the viral pneumonia and makes a related immune response. The Tfh cell subset in the Trametinib group is much higher than that in the PBS group, which proves that the Trametinib can induce the Tfh differentiation and increase the Tfh ratio.
Example 5: trametinib increased protective antibody levels.
The method comprises the following steps:
plasma total IgG levels were measured from the mice on days 0, 9, and 15 of the model of example 4.
And (4) analyzing results:
as shown in fig. 5, the content of IgG antibody in mice in the Trametinib group is significantly higher than that in the control group at different time points, indicating that the immune system has stronger resistance to virus, faster antibody production speed and higher content.
It will be apparent to those skilled in the art that various modifications may be made to the above embodiments without departing from the general spirit and concept of the invention. All falling within the scope of protection of the present invention. The protection scheme of the invention is subject to the appended claims.
Claims (6)
1. Use of trametinib in the preparation of a vaccine.
2. Use of trametinib according to claim 1 for the preparation of a vaccine, wherein trametinib is used as a vaccine adjuvant.
3. Use of trametinib according to claim 2, in the preparation of a vaccine, wherein the use of trametinib is as a vaccine adjuvant that promotes the differentiation of Tfh cells.
4. Use of trametinib according to claim 3, as a vaccine adjuvant for promoting Tfh cell differentiation and inhibiting T cell differentiation, monocyte macrophage activation, in the preparation of a vaccine.
5. Use of trametinib according to claim 1, for the preparation of a vaccine, wherein said vaccine is a viral vaccine.
6. The use of trametinib according to claim 4, in a viral vaccine adjuvant, wherein the virus comprises COVID-19 virus, SARS virus.
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CN202010370525.XA CN111494620A (en) | 2020-05-05 | 2020-05-05 | Application of trametinib in preparation of vaccine |
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN112957361A (en) * | 2021-03-24 | 2021-06-15 | 中国科学院武汉病毒研究所 | Application of trametinib in preparation of anti-arenavirus antiviral preparation |
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CN102813920A (en) * | 2011-06-10 | 2012-12-12 | 复旦大学 | Vaccine adjuvant |
CN103357009A (en) * | 2012-04-05 | 2013-10-23 | 中国农业大学 | Application of Hemokinin-1 in preparation of vaccine adjuvant |
CN105101999A (en) * | 2012-10-08 | 2015-11-25 | S·普莱施卡 | Mek inhibitors in the treatment of virus diseases |
CN105983095A (en) * | 2015-02-15 | 2016-10-05 | 北京艾棣维欣生物技术有限公司 | Respiratory syncytial virus vaccine, and preparation method and application thereof |
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2020
- 2020-05-05 CN CN202010370525.XA patent/CN111494620A/en active Pending
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112957361A (en) * | 2021-03-24 | 2021-06-15 | 中国科学院武汉病毒研究所 | Application of trametinib in preparation of anti-arenavirus antiviral preparation |
CN112957361B (en) * | 2021-03-24 | 2022-07-29 | 中国科学院武汉病毒研究所 | Application of trametinib in preparation of anti-arenavirus antiviral preparation |
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