WO2024146068A1 - Use of tripterine or pharmaceutically acceptable derivative thereof in preparation of vaccine adjuvant - Google Patents

Abstract

The present invention provides a use of tripterine or a pharmaceutically acceptable derivative thereof in the preparation of a vaccine adjuvant, and a vaccine adjuvant containing the tripterine or the pharmaceutically acceptable derivative thereof. The present invention finds that the immune response of an organism to an antigen can be enhanced by using the tripterine or the pharmaceutically acceptable derivative thereof as the vaccine adjuvant. Compared with metal salt adjuvants such as an aluminum adjuvant that is not easy to metabolize in vivo and has a risk of accumulation, the tripterine or the pharmaceutically acceptable derivative thereof in the present invention is a small molecule compound, is easy to degrade and excrete, is high in safety, and has a better immunogenicity enhancement effect than that of the aluminum adjuvant.

Description

Use of tripterygium wilfordii or its pharmaceutically acceptable derivatives in preparing vaccine adjuvants

Cross-reference instructions

This application claims priority to the Chinese patent application filed with the Chinese Patent Office on January 5, 2023, with application number 202310012605.1, and invention name "Use of tripterygium wilfordii or its pharmaceutically acceptable derivatives in the preparation of vaccine adjuvants" and the Chinese patent application filed with the Chinese Patent Office on May 22, 2023, with application number 202310579417.7, and invention name "Use of tripterygium wilfordii or its pharmaceutically acceptable derivatives in the preparation of vaccine adjuvants", the entire contents of which are incorporated by reference into this application.

Technical Field

The present invention relates to the field of biopharmaceuticals, and in particular to the use of tripterygium wilfordii or its pharmaceutically acceptable derivatives in the preparation of vaccine adjuvants.

Background technique

Vaccines play an important role in combating infectious diseases. According to different antigen forms, vaccines can be divided into attenuated vaccines, inactivated vaccines, recombinant subunit protein vaccines and nucleic acid vaccines. In addition to antigens, adjuvants are often added to vaccines to enhance their immune effects and induce sufficiently strong immune responses. The following types of adjuvants have been approved for marketing: delivery systems such as water-in-oil emulsions MF59, AS01, AS03, etc.; aluminum salts and aluminum salt-based composite adjuvants (such as AS04, aluminum + CpG, etc.). The body's immune response to exogenous substances (such as antigens) is a complex process, but like the adjuvants that have been marketed, the current research hotspots in the field of adjuvants are mostly focused on the stimulation of immune cells or specific immune targets by added substances (popular targets such as toll-like receptors, STING receptors, etc.). Generally speaking, the corresponding stimulants will be regarded as danger signals by the body, creating a local inflammatory environment to induce the aggregation of immune cells, enhance the body's uptake of antigens and subsequent presentation, and thus enhance the immune response to antigens.

Small molecule compounds are not widely used as vaccine adjuvants. Small molecule compounds are easily degraded and excreted, and are safer, making them ideal targets for vaccine adjuvants. Triptergium wilfordii is a pentacyclic triterpene compound that was isolated from Tripterygium wilfordii by Zhao Chengxia, a famous Chinese pharmacist in the 1930s. It was listed by Cell magazine as one of the five natural compounds most likely to be developed into modern drugs, along with artemisinin, triptolide, capsaicin and curcumin. Existing studies have shown that triptergium wilfordii can produce It has anti-inflammatory, hypoglycemic, weight loss, and anti-cancer effects, but there are no reports on its use as a vaccine adjuvant.

Summary of the invention

One aspect of the present invention is to provide a new use of tripterygium wilfordii or its pharmaceutically acceptable derivatives in view of the lack of application of small molecule compounds alone as vaccine adjuvants in the prior art.

The technical solution provided by the present invention is:

Use of tripterygium wilfordii or its pharmaceutically acceptable derivatives in preparing vaccine adjuvants.

Triptolide, also known as celastrol, is a natural product with multiple biological activities. It has strong antioxidant effects, anti-cancer angiogenesis effects, and anti-rheumatoid effects. It is mainly derived from the root bark of the Celastraceae plant Tripterygium wilfordii. It is one of the effective ingredients of preparations such as Tripterygium wilfordii tablets and Tripterygium wilfordii polyglycoside tablets for the treatment of rheumatoid arthritis. The main activities and pharmacological effects of triptolide: 1. Cytotoxic activity. It has strong nonspecific cytotoxic activity against P388 and a group of human cancer cell lines in vitro. 2. Immunomodulatory effect. It significantly inhibits the formation of hemolytic plaque cells in mouse spleen cells. It significantly inhibits delayed hypersensitivity reactions in mice. 3. Anti-inflammatory effect. At 0.5 mg/kg, it significantly inhibits the formation of cotton ball granulomas in rats. At 0.1-1.0 μg/mL, it inhibits the production of PGE2 induced by yeast sugar; at 1.0 μg/mL, it inhibits the phagocytosis of macrophages. 4. The anti-peroxidation effect of tripterygium wilfordii is 15 times that of tocopherol, with an IC50 of 7μM. It inhibits peroxidation inside and outside the mitochondrial membrane and directly removes free radicals. 5. Tripterygium wilfordii can prolong the sleep time of mice induced by sodium pentobarbital. 6. Immunosuppressive effect: It inhibits the proliferation of spleen cells induced by PHA, ConA, LPS, etc. in mice, and inhibits the proliferation of lymphocytes. 7. It inhibits the fertilization ability of guinea pig sperm in vitro, and its activity is significantly stronger than that of acetic acid gossypol. 8. Anti-arthritis effect. It inhibits the activity of interleukin-1 inside and outside mouse peritoneal macrophages, inhibits the production of interleukin-2 by mouse spleen cells, and reduces the release of PGE2 by rabbit synovial cells.

The molecular formula of tripterygium wilfordii 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 present invention, the above-mentioned pharmaceutically acceptable derivatives are in the form of pharmaceutically acceptable salts thereof. For example, acid salts, basic salts. Preferably, in certain embodiments of the present invention, the above-mentioned acid salts include Including but not limited to hydrochloride, sulfate, phosphate, citrate, hydrobromide, acetate, benzoate, benzenesulfonate, tartrate, carbonate, citrate, gluconate, lactate, malate, methanesulfonate, stearate, valerate or nitrate; the above basic salts include but are not limited to sodium salt, calcium salt, potassium salt, zinc salt or meglumine salt.

In the present invention, the above-mentioned tripterygium wilfordii or its pharmaceutically acceptable derivatives can be used as any suitable type of vaccine adjuvant. For example, including but not limited to, inactivated vaccines, live attenuated vaccines, protein vaccines, bacterial polysaccharide and polysaccharide protein conjugate vaccines, genetically engineered vaccines or genetic reassortment vaccines. Preferably, in certain embodiments of the present invention, the above-mentioned vaccine is a protein vaccine (a vaccine with protein as an antigen, for example, a recombinant subunit vaccine). More preferably, in one embodiment of the present invention, the antigen of the above-mentioned vaccine is hepatitis B surface antigen.

In the present invention, the above-mentioned tripterygium wilfordii or its pharmaceutically acceptable derivatives can be packaged into any suitable form and administered using any suitable delivery system. Preferably, in one embodiment of the present invention, the above-mentioned tripterygium wilfordii or its pharmaceutically acceptable derivatives are in the form of nanoparticles. The above-mentioned nanomedicine can be prepared by a suitable method, for example, the method described in the Chinese patents with publication numbers CN114886855A and CN114903872A.

In order to achieve a better immune effect, in certain embodiments of the present invention, the above-mentioned tripterygium wilfordii or its pharmaceutically acceptable derivatives can form a composite adjuvant with other adjuvants. Preferably, in certain embodiments of the present invention, the above-mentioned other adjuvants are 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 invention, the composite adjuvant further comprises an immunopotentiator. The immunopotentiator may be, for example, but not limited to, cytokines, chemokines PAMP, TLR-ligands, immunostimulatory sequences, DNA containing CpG, dsRNA, ligands of receptors that recognize endocytic patterns, LPS, Quillaja saponins, tucaresol, etc. The immunopotentiator may be contained in the same container as the composite adjuvant, or may be contained in different containers.

Another aspect of the present invention is to provide a vaccine adjuvant, wherein the vaccine adjuvant contains tripterine or its pharmaceutically acceptable derivatives, and appropriate carriers, excipients, stabilizers or diluents. The vaccine can be an injection, oral agent or nasal inhalation agent.

The beneficial effects of the present invention are:

The present invention has found that using tripterygium wilfordii or its pharmaceutically acceptable derivatives as vaccine adjuvants can enhance the body's immune response to antigens. Compared with metal salt adjuvants such as aluminum adjuvants that are not easily metabolized in the body and have the risk of accumulation, the tripterygium wilfordii or its pharmaceutically acceptable derivatives of the present invention are small molecule compounds that are easily degraded and excreted, have higher safety, and have a better immunogenicity enhancement effect than aluminum adjuvants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram showing the evaluation results of the adjuvant effect of tripterygium wilfordii and other compounds;

FIG2 is a graph showing the particle size of blank liposomes and tripterygium wilfordii liposomes;

FIG3 is an electron micrograph of tripterygium wilfordii liposomes;

FIG4 is a graph showing the adjuvant effect of tripterygium wilfordii liposomes;

FIG5 is a graph showing the results of antigen-specific cytokines induced by immunization with tripterygium wilfordii and tripterygium wilfordii liposomes as adjuvants;

FIG6 is a graph showing the evaluation results of the effect of tripterygium wilfordii as an adjuvant for virus-split seedlings;

FIG7 is a schematic diagram of the interaction of tripterygium wilfordii with aluminum and antigen;

Figure 8 is a graph showing the evaluation results of the effect of tripterygium wilfordii as an adjuvant for the new coronavirus vaccine.

Detailed ways

The present invention discloses the use of tripterygium wilfordii or its pharmaceutically acceptable derivatives in the preparation of vaccine adjuvants. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention, and relevant personnel can obviously modify or appropriately change and combine the contents described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly indicated, throughout the specification and claims, the term "including" or its variations such as "comprising" or "including" etc. will be understood to include the stated elements or components without excluding other elements or other components. The terms "such as", "for example", etc. are intended to refer to exemplary embodiments and are not intended to limit the scope of the present disclosure.

Some terms used in the present invention are explained below.

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

The term "adjuvant" refers to a non-specific immunopotentiator that, when injected together with an antigen or pre-injected into the body, can enhance the body's immune response to the antigen or change the type of immune response.

In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with specific embodiments.

Example 1 Evaluation of the effect of tripterygium wilfordii as a vaccine adjuvant

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

In addition to tripterygium wilfordii, another small molecule compound was selected as a control in this example: bortezomib and carfilzomib, which are clinically used for the treatment of multiple myeloma. Currently, a large number of similar drugs are tested for tumor treatment worldwide. The immune samples were prepared according to Table 1 for animal immunization.

Table 1 Immunization groups and doses

Thirty Babl/c mice aged 6 to 8 weeks were randomly divided into 5 groups. They were immunized twice at 0 week and 3 weeks using the samples configured in the above table. Blood was collected at 5 weeks, and then the serum was separated and the antibody titer was measured using enzyme-linked immunosorbent assay (ELISA). The results are shown in Figure 1.

The results showed that compared with the antigen group alone, both the aluminum adjuvant group and the tripterygium wilfordii group could significantly increase the antibody titer (p<0.05). Surprisingly, the adjuvant effect induced by the addition of 5μg of tripterygium wilfordii was significantly stronger than that induced by 25μg of aluminum adjuvant (p<0.05).

Example 2 Preparation of tripterygium wilfordii liposomes and evaluation of adjuvant effect

1. Preparation and characterization of liposomes

Tripterygium wilfordii has poor solubility in water. Therefore, we prepared corresponding liposomes to encapsulate tripterygium wilfordii to form nanoparticles. The mass ratio of lecithin: cholesterol: tripterygium wilfordii in the prepared liposomes is 10:1:1. After using anhydrous ethanol solvent, rotary evaporation was performed to form a film, citric acid buffer was added for hydration, and then thin film extrusion was used for homogenization. The content of tripterygium wilfordii in the homogenized particles was 1 mg/ml. At the same time, the particle size of the homogenized particles was measured and transmission electron microscopy was observed. The results are shown in Figures 2 and 3.

2. Tripterygium wilfordii can induce extremely strong cellular immunity and humoral immunity

Using hepatitis B surface antigen as a model antigen, 80 Babl/c mice aged 6 to 8 weeks were randomly divided into 16 groups. They were immunized twice at 0 week and 3 weeks according to the samples configured in Table 2. Blood was collected at 5 weeks, and then the serum was separated and the antibody titer was measured by enzyme-linked immunosorbent assay (ELISA). At the same time, spleen cells were separated and antigen-specific cytokines were detected by Elispot.

Table 2 Immunization groups and doses

The results showed that tripterygium wilfordii could induce strong humoral immunity (results shown in Figure 4) and cellular immunity (results shown in Figure 5) at the same level as QS-21, which was much stronger than POLY (I: C). At the same time, after being prepared into liposomes, the adjuvant effect was further enhanced. The results also showed that tripterygium wilfordii combined with other adjuvants (such as aluminum adjuvant, QS-21, etc.) did not show obvious enhancement effect on cellular immunity. The induction effect of tripterygium wilfordii on cellular immunity was inhibited by the addition of other adjuvants, suggesting that they have different mechanisms of action.

Example 3 Evaluation of the immunopotentiating effect of tripterygium wilfordii on influenza vaccine

Using H1N1 influenza virus lytic subunit antigen as a model antigen, 15 Babl/c mice aged 6 to 8 weeks were randomly divided into 3 groups, and samples were configured according to Table 3. They were immunized twice at 0 week and 3 weeks, blood was collected at 5 weeks, and then fresh chicken blood was used to measure the hemostatic titer.

The results showed that compared with the single antigen group, tripterygium wilfordii could increase the neutralizing antibody titer by 20 times; The results were shown in Figure 6 , which was about 3 times higher than that of the aluminum adjuvant group.

Table 3 Immunization groups and doses

Example 4 Interaction of tripterygium wilfordii with aluminum adjuvant and antigen

In the experiment, we found that the solubility of tripterygium wilfordii in water was low. It was a suspension with an orange color. The suspension was filtered using a 0.2μm PVDF filter membrane and the color was clear, indicating that the tripterygium wilfordii solution alone failed to pass through the filter membrane.

When the antigen was mixed with celastrol and filtered again, it was found that celastrol could pass through the filter membrane together with the antigen, indicating that the antigen interacted with celastrol, thereby increasing the solubility of celastrol.

At the same time, when celastrol was mixed with aluminum adjuvant, it was found that celastrol would be adsorbed by aluminum and precipitate.

The above results indicate that tripterygium wilfordii can interact with antigens and aluminum. Further analysis shows that the mixture of tripterygium wilfordii + aluminum adjuvant + antigen produced a reaction as shown in FIG7 .

Therefore, coupling tripterygium wilfordii and antigens through metal complexation can also be used as a preferred technical solution of the present invention.

Example 5 Evaluation of the immunopotentiating effect of tripterygium wilfordii as an adjuvant for the COVID-19 vaccine

Using the recombinant S1+S2 trimer of the novel coronavirus omicron strain as a model antigen, 15 Babl/c mice aged 6 to 8 weeks were randomly divided into 3 groups. Samples were configured according to Table 4, and two immunizations were performed at 0W and 2W. Blood was collected at 3W, and the IgG antibody titer was then measured. The results showed that 5μg of tripterygium wilfordii could induce an antibody titer 10 times higher than the average of 25μg aluminum adjuvant (the results are shown in Figure 8).

Table 4 Immunization groups and doses

The above description is only a preferred embodiment of the present invention. It should be noted that the common It is clear to the technicians that several improvements and modifications may be made without departing from the principles of the present invention, and these improvements and modifications should also be considered within the protection scope of the present invention.

Claims (15)

1. Use of tripterygium wilfordii or its pharmaceutically acceptable derivatives in preparing vaccine adjuvants.
2. The use according to claim 1, characterized in that the pharmaceutically acceptable derivative is in the form of a pharmaceutically acceptable salt thereof.
3. The use according to claim 2, characterized in that the pharmaceutically acceptable derivative is in the form of a pharmaceutically acceptable acid salt or basic salt thereof.
4. The use according to claim 3, characterized in that the acid salt is 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.
5. The use according to claim 1 is characterized in that the vaccine is an inactivated vaccine, a live attenuated vaccine, a protein vaccine, a bacterial polysaccharide and polysaccharide protein conjugate vaccine, a genetically engineered vaccine or a genetically reassortant vaccine.
6. The use according to claim 5 is characterized in that the antigen of the vaccine is hepatitis B surface antigen.
7. The use according to claim 1, characterized in that the tripterygium wilfordii or its pharmaceutically acceptable derivative is in the form of nanoparticles.
8. The use according to claim 1 is characterized in that the tripterygium wilfordii or its pharmaceutically acceptable derivative forms a composite adjuvant with other adjuvants.
9. The use according to claim 7, characterized in that the other adjuvant is one or more selected from MG-132, carfilzomib or bortezomib.
10. The use according to claim 7 is characterized in that the composite adjuvant also contains an immunopotentiator.
11. A vaccine adjuvant, characterized in that it contains tripterygium wilfordii or its pharmaceutically acceptable derivatives, and appropriate carriers, excipients, stabilizers or diluents.
12. The use according to claim 10, characterized in that the immunopotentiator is a cytokine, a chemokine PAMP, a TLR-ligand, an immunostimulatory sequence, a DNA containing CpG, a dsRNA, a ligand for a receptor that recognizes the endocytic mode, LPS, a Quillaja saponin or a tucaresol.
13. The use according to claim 5 is characterized in that the antigen of the vaccine is an H1N1 influenza virus split subunit antigen.
14. The use according to claim 5, characterized in that the antigen of the vaccine is a novel coronavirus Antigens in viruses.
15. The use according to claim 7 is characterized in that the nanoparticles are formed by encapsulating tripterygium wilfordii in liposomes.