CN116617386A

CN116617386A - Application of tripterine or medicinal derivative thereof in preparation of vaccine adjuvant

Info

Publication number: CN116617386A
Application number: CN202310579417.7A
Authority: CN
Inventors: 王志标; 王欣瑀; 李月; 王旭
Original assignee: China National Biotec Research Institute Co ltd
Current assignee: China National Biotec Research Institute Co ltd
Priority date: 2023-01-05
Filing date: 2023-05-22
Publication date: 2023-08-22
Anticipated expiration: 2043-05-22
Also published as: CN116617386B; CN116077639A; WO2024146068A1; CN118286412A

Abstract

The application discloses application of tripterine or a pharmaceutically acceptable derivative thereof in preparation of a vaccine adjuvant. The application discovers that the use of tripterine or a pharmaceutically acceptable derivative thereof as a vaccine adjuvant can enhance the immune response of an organism to an antigen. Compared with metal salt adjuvants such as aluminum adjuvants which are not easy to metabolize in vivo and have the risk of accumulation, the tripterine or the medicinal derivatives thereof are small molecular compounds, are easy to degrade and excrete, have higher safety, and have better enhancing effect on immunogenicity than the aluminum adjuvants.

Description

Application of tripterine or medicinal derivative thereof in preparation of vaccine adjuvant

Cross-reference specification

The present application claims priority from the chinese patent application filed on 1 month 5 2023, entitled "use of tripterine or pharmaceutically acceptable derivatives thereof in the preparation of vaccine adjuvants", filed in chinese patent office under application No. 202310012605.1, the entire contents of which are incorporated herein by reference.

Technical Field

The application relates to the field of biological pharmacy, in particular to application of tripterine or a medicinal derivative thereof in preparation of vaccine adjuvant.

Background

Vaccines play an important role in combating infectious diseases and can be classified into attenuated vaccines, inactivated vaccines, recombinant subunit protein vaccines and nucleic acid vaccines according to different antigen forms. In addition to the antigen, an adjuvant is often added to the vaccine to enhance its immune effect, thereby inducing a sufficiently strong immune response. Adjuvants currently approved for marketing are of the following classes: delivery systems such AS oil-in-water emulsions MF59, AS01, AS03, etc.; aluminum salts and complex adjuvants based on aluminum salts (e.g., AS04, aluminum + CpG, etc.). The immune response of the body to foreign substances (such as antigens) is a complex process, but as with the marketed adjuvants, the research focus in the field of the existing adjuvants is mostly focused on the stimulation of immune cells or specific immune targets (hot targets such as toll-like receptors, STING receptors, etc.) by the additive substances, and generally speaking, the corresponding stimulating agents can be used as dangerous signals by the body to create local inflammatory environments to induce the aggregation of immune cells, enhance the uptake and subsequent presentation of antigens by the body, thereby enhancing the immune response to antigens.

Small molecule compounds alone are not widely used as vaccine adjuvants. The small molecule compound is easy to be degraded and excreted, and the safety is higher, so that the small molecule compound is an ideal target as a vaccine adjuvant. Tripterine is a pentacyclic triterpene compound, which is separated from Tripterygium wilfordii in the last 30 th century by a famous pharmacy family Zhao Chengxia in China, and is listed as five natural compounds most likely to be developed into modern medicines together with artemisinin, triptolide, capsaicin, curcumin and the like by journal of Cell. The existing researches show that the tripterine can play the roles of anti-inflammatory, blood sugar reducing, weight reducing, anticancer and the like in vivo, but no report on the application of the tripterine to vaccine adjuvants exists.

Disclosure of Invention

In one aspect, the application provides a new application of tripterine or a pharmaceutically acceptable derivative thereof, aiming at the lack of application of small molecular compounds as vaccine adjuvants in the prior art.

The technical scheme provided by the application is as follows:

the use of celastrol or a pharmaceutically acceptable derivative thereof in the preparation of a vaccine adjuvant.

The tripterine is also called celastrol, is a natural product with various biological activities, has strong antioxidation, anti-cancer angiogenesis and anti-rheumatoid effect, is mainly derived from root bark of celastrol of Celastraceae, and is one of the effective components of preparations such as tripterygium wilfordii tablet, tripterygium glycosides tablet and the like for treating rheumatoid diseases. The main activity and pharmacological action of tripterine: 1. cytotoxic activity. Has strong nonspecific cytotoxic activity to P388 and a group of human cancer cell lines in vitro. 2. Immunoregulatory effects. Obviously inhibit the formation of hemolytic plaque cells in the spleen cells of the mice. Obviously inhibit the delayed hypersensitivity of mice. 3. Anti-inflammatory effect. At 0.5mg/kg, the formation of cotton ball granuloma in rats is significantly inhibited. Inhibiting the production of PGE2 induced by yeast sugar at 0.1-1.0 μg/mL; at 1.0 μg/mL, phagocytosis of macrophages was inhibited. 4. The anti-peroxidation effect of tripterine is 15 times that of tocopherol, and the IC50 is 7 mu M. Inhibit peroxidation inside and outside mitochondrial membrane, and directly remove free radicals. 5. Tripterine can prolong sleep time of mice caused by sodium pentobarbital. 6. Immunosuppression effect: inhibit proliferation of spleen cells induced by PHA, conA, LPS and the like in mice, and inhibit proliferation of lymphocytes. 7. Inhibiting the fertilization capacity of the in vitro sperms of guinea pigs, and the activity is obviously stronger than that of gossypol acetate. 8. Anti-arthritic effect. Inhibiting the activity of interleukin-1 in and out of mouse abdominal macrophages, inhibiting the production of interleukin-2 by mouse spleen cells, and reducing the release of PGE2 by rabbit synovial cells.

The molecular formula of the tripterine is C ₂₉ H ₃₈ O ₄ The molecular weight is 450.61, and the structural formula is as follows:

in certain embodiments of the application, the pharmaceutically acceptable derivatives described above are in the form of pharmaceutically acceptable salts thereof. For example, acid salts, basic salts. Preferably, in certain embodiments of the present application, the acid salts include, but are not limited to, hydrochloride, sulfate, phosphate, citrate, hydrobromide, acetate, benzoate, benzenesulfonate, tartrate, carbonate, citrate, gluconate, lactate, malate, methanesulfonate, stearate, valerate, or nitrate; such basic salts include, but are not limited to, sodium, calcium, potassium, zinc or meglumine salts.

In the present application, the above-described tripterine or a pharmaceutically acceptable derivative thereof may be used as any suitable type of vaccine adjuvant. For example, including but not limited to, inactivated vaccines, attenuated live vaccines, protein vaccines, bacterial polysaccharide and polysaccharide protein conjugate vaccines, genetically engineered vaccines or genetically reassortant vaccines. Preferably, in certain embodiments of the present application, the vaccine described above is a protein vaccine (a vaccine with protein as antigen, e.g., a recombinant subunit vaccine). More preferably, in one embodiment of the present application, the antigen of the vaccine is hepatitis B surface antigen.

In the present application, the above-described tripterine or a pharmaceutically acceptable derivative thereof may be packaged in any suitable form and administered using any suitable delivery system. Preferably, in one embodiment of the present application, the above-mentioned tripterine or a pharmaceutically acceptable derivative thereof is in the form of nanoparticles. The above nano-drug may be prepared by a suitable method, for example, the method described in chinese patent publication No. CN114886855A, CN114903872 a.

In order to achieve a better immune effect, in certain embodiments of the present application, the above-described tripterine or a pharmaceutically acceptable derivative thereof may form a complex adjuvant with other adjuvants. Preferably, in certain embodiments of the present application, the additional adjuvant is one or more selected from aluminum adjuvant, MG-132, carfilzomib or bortezomib.

In order to further achieve a better immune effect, in certain embodiments of the present application, the above-mentioned compound adjuvant further comprises an immunopotentiator. The immunopotentiator may be, for example, including, but not limited to, cytokines, chemokines PAMPs, TLR-ligands, immunostimulatory sequences, cpG-containing DNA, dsRNA, endocytic-pattern recognition receptor ligands, LPS, quillaja saponaria, tocardanol, and the like. May be contained in the same container as the compound adjuvant or may be contained in separate containers.

In another aspect of the present application there is provided a vaccine adjuvant comprising celastrol or a pharmaceutically acceptable derivative thereof, and a suitable carrier, excipient, stabilizer or diluent therefor. The vaccine can be injection, oral preparation or nasal inhalant.

The beneficial effects of the application are as follows:

the application discovers that the tripterine or the medicinal derivative thereof can be used as a vaccine adjuvant to enhance the immune response of an organism to an antigen. Compared with metal salt adjuvants such as aluminum adjuvants which are not easy to metabolize in vivo and have the risk of accumulation, the tripterine or the medicinal derivatives thereof are small molecular compounds, are easy to degrade and excrete, have higher safety, and have better enhancing effect on immunogenicity than the aluminum adjuvants.

Drawings

FIG. 1 is a graph showing the evaluation results of the adjuvant effect of compounds such as tripterine;

FIG. 2 is a graph showing the particle size measurement of blank liposomes and tripterine liposomes;

FIG. 3 is an electron microscope of celastrol liposome;

FIG. 4 is a graph showing the results of the adjuvant effect of celastrol liposomes;

FIG. 5 is a graph showing the results of antigen-specific cytokines induced after immunization of tripterine and tripterine liposomes as an adjuvant;

FIG. 6 is a graph showing the evaluation results of the effect of tripterine as a virus-splitting seedling adjuvant;

FIG. 7 is a schematic representation of the interaction of tripterine with aluminum and an antigen;

FIG. 8 is a graph showing the results of evaluation of the effect of tripterine as a novel coronavirus vaccine adjuvant.

Detailed Description

The application discloses application of tripterine or a medicinal derivative thereof in preparing vaccine adjuvants, and a person skilled in the art can properly improve process parameters by referring to the content of the tripterine or the medicinal derivative thereof. It is to be particularly pointed out that all similar substitutes and modifications apparent to those skilled in the art are deemed to be included in the application and that the relevant person can make modifications and appropriate alterations and combinations of what is described herein to make and use the technology without departing from the spirit and scope of the application.

In the present application, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising", etc. will be understood to include the stated element or component without excluding other elements or components. The terms "such as," "for example," and the like are intended to refer to exemplary embodiments and are not intended to limit the scope of the present disclosure.

The following is a description of some of the terms appearing in the present application.

The term "pharmaceutically acceptable derivative" includes any pharmaceutically acceptable salt, solvate, hydrate or prodrug of celastrol.

The term "adjuvant" refers to a nonspecific immunopotentiator that, when injected or pre-injected with an antigen, enhances the body's immune response to the antigen or alters the type of immune response.

In order to enable those skilled in the art to better understand the technical solution of the present application, the present application will be further described in detail with reference to specific embodiments.

EXAMPLE 1 evaluation of the Effect of Tripterine as vaccine adjuvant

In this example, hepatitis B surface antigen (HBsAg) was selected as a protein-like model antigen, and aluminum adjuvant gold standard (Alhydrogel) was used as a positive control.

In addition to tripterine, another small molecule compound was selected as a control in this example: bortezomib and carfilzomib, which are clinically used in the treatment of multiple myeloma, are currently tested globally for tumor treatment by a large number of analogues. Immunization samples were prepared as in table 1 for animal immunization.

Table 1 immunization groups and doses

30 Babl/c mice with the age of 6-8 weeks were randomly divided into 5 groups, the samples prepared in the above table were used for immunization twice at 0W and 3W, 5W was used for blood collection, and after serum separation, antibody titer was measured by enzyme-linked immunosorbent assay (ELISA), and the results are shown in FIG. 1.

The results show that both the aluminium adjuvant group and the triptolide group significantly increased antibody titres (p < 0.05) compared to the antigen group alone, while unexpectedly the adjuvant effect induced by the addition of 5 μg tripterygium was significantly stronger than that induced by 25 μg aluminium adjuvant (p < 0.05).

EXAMPLE 2 preparation of Tripterine liposomes and evaluation of adjuvant Effect

1. Preparation and characterization of liposomes

The celastrol has poor solubility in water, so that the celastrol is coated by the corresponding liposome to form nano particles, and lecithin in the prepared liposome: cholesterol: the mass ratio of the tripterine is 10:1:1, using absolute ethyl alcohol solvent, performing rotary evaporation to form a film, adding a citric acid buffer solution for hydration, homogenizing by using a film extrusion method, wherein the content of the homogenized particles of tripterine is 1mg/ml, and simultaneously, measuring the particle size of the homogenized particles and observing by a transmission electron microscope, wherein the results are shown in fig. 2 and 3.

2. Tripterine can induce extremely strong cellular immunity and humoral immunity

Using hepatitis B surface antigen as model antigen, taking 80 Babl/c mice with the age of 6-8 weeks, randomly dividing into 16 groups, performing immunization twice at 0W and 3W according to samples configured in table 2, taking blood at 5W, separating serum, measuring antibody titer by using an enzyme-linked immunosorbent assay (ELISA), and separating spleen cells to detect antigen-specific cytokines.

Table 2 immunization groups and doses

The results show that tripterine can induce very strong humoral immunity (the result is shown in figure 4) and cellular immunity (the result is shown in figure 5) which are quite level with QS-21, and is far stronger than POLY (I: C), and the adjuvant effect is further enhanced after the tripterine is prepared into liposome. The results also show that the tripterine is combined with other adjuvants (such as aluminum adjuvants, QS-21 and the like) and does not show obvious enhancement effect on cellular immunity, and the induction effect of the tripterine on cellular immunity is inhibited due to the addition of the other adjuvants, so that the tripterine has different action mechanisms.

Example 3 evaluation of the Immunity-potentiating effect of Tripterine on influenza vaccine

15 Babl/c mice with the age of 6-8 weeks were randomly divided into 3 groups using H1N1 influenza virus lytic subunit antigen as model antigen, samples were prepared according to Table 3, and were immunized twice at 0W and 3W, and the 5W was used to collect blood, and then fresh chicken blood was used to measure blood inhibitory titers.

The results show that compared with the single antigen group, the tripterine can improve the neutralizing antibody titer by 20 times; the ratio was increased by about 3-fold over the aluminum adjuvant group (results shown in fig. 6).

Table 3 immunization groups and doses

EXAMPLE 4 interaction of Tripterine with aluminum adjuvant and antigen

In the experiment, the tripterine has low solubility in water, is a suspension and is orange, and the suspension is filtered by using a 0.2 mu m PVDF filter membrane, so that the color is clear, and the tripterine solution alone cannot pass through the filter membrane.

When the antigen and the tripterine are mixed, the tripterine is filtered again, and the tripterine and the antigen can pass through a filter membrane together, so that the interaction between the antigen and the tripterine is indicated, and the solubility of the tripterine is increased.

Meanwhile, when the tripterine is mixed with an aluminum adjuvant, the tripterine can be found to be adsorbed by aluminum to be precipitated.

The results show that the tripterine can interact with the antigen and aluminum, and further analysis shows that the tripterine, aluminum adjuvant and antigen are mixed and reacted as shown in figure 7.

Therefore, the coupling of the tripterine and the antigen can also be used as a preferable technical scheme of the application through the complexation of metals.

EXAMPLE 5 evaluation of the Immunity-enhancing Effect of Tripterine as New coronatine vaccine adjuvant

15 Babl/c mice with age of 6-8 weeks were randomly divided into 3 groups using novel coronavirus omacron strain recombination S1+S2 trimers as model antigen, samples were prepared according to Table 4 and immunized twice at 0W, 2W, 3W was collected, and IgG antibody titer was measured, which showed that 5. Mu.g of tripterine induced 10-fold higher antibody titer than 25. Mu.g of aluminum adjuvant mean (results are shown in FIG. 8).

Table 4 immunization groups and doses

The foregoing is merely a preferred embodiment of the present application and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present application, which are intended to be comprehended within the scope of the present application.

Claims

1. The use of celastrol or a pharmaceutically acceptable derivative thereof in the preparation of a vaccine adjuvant.

2. Use according to claim 1, wherein the pharmaceutically acceptable derivative is in the form of a pharmaceutically acceptable salt thereof.

3. Use according to claim 2, wherein the pharmaceutically acceptable derivative is in the form of a pharmaceutically acceptable acid or basic salt thereof;

preferably, the acid salt is a hydrochloride, sulfate, phosphate, citrate, hydrobromide, acetate, benzoate, benzenesulfonate, tartrate, carbonate, citrate, gluconate, lactate, malate, methanesulfonate, stearate, valerate or nitrate; the basic salt is sodium salt, calcium salt, potassium salt, zinc salt or meglumine salt.

4. The use according to claim 1, wherein the vaccine is an inactivated vaccine, an attenuated live vaccine, a protein vaccine, a bacterial polysaccharide and polysaccharide protein conjugate vaccine, a genetically engineered vaccine or a genetically reassortant vaccine.

5. The use according to claim 4, wherein the antigen of the vaccine is hepatitis b surface antigen.

6. The use according to claim 1, wherein the tripterine or a pharmaceutically acceptable derivative thereof is in the form of nanoparticles.

7. The use according to claim 1, wherein the tripterine or a pharmaceutically acceptable derivative thereof forms a complex adjuvant with other adjuvants.

8. The use according to claim 7, wherein the other adjuvant is one or more selected from MG-132, carfilzomib or bortezomib.

9. The use according to claim 7, wherein the compound adjuvant further comprises an immunopotentiator.

10. A vaccine adjuvant comprising tripterine or a pharmaceutically acceptable derivative thereof, and a suitable carrier, excipient, stabilizer or diluent therefor.