Rescimod galloyl derivative as well as synthesis method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a racimod galloyl derivative as well as a synthesis method and application thereof. The derivative is formed by connecting rasimod and gallic acid through an ester bond, and the structure of the derivative is shown in the following formula I; according to the invention, the racimod is subjected to galloyl derivatization modification, so that the affinity of the racimod to the antigen is improved, and the racimod is simply mixed with the antigen to form the nanoparticles. A new thought and opportunity are provided for development of antigen and adjuvant co-delivery nanoparticles, so that the urgent demand on cancer vaccines clinically is met. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and particularly relates to a resiquimod galloyl derivative and a synthesis method and application thereof. Background Art

[0002] In today's medical field, cancer poses an extremely serious threat to human health, and its morbidity and mortality continue to rise, becoming the focus of global attention. Tumor vaccines are the introduction of tumor antigens into patients in different forms, which activate immune responses to exert anti-tumor effects. As an important breakthrough in cancer immunotherapy, tumor vaccines have shown remarkable potential. Many tumor therapeutic vaccines have been launched abroad, such as Provenge for prostate cancer, Melacine for malignant melanoma, and Oncophage for renal cell carcinoma. Tumor cell lysates (TCLs) can provide a wide variety of tumor antigens, making them a comprehensive source that can induce strong and lasting anti-tumor immune responses. However, traditional vaccines based on TCLs have not achieved satisfactory results in early clinical trials due to low immunogenicity, instability in the body, and inability to be delivered with adjuvants.

[0003] Toll-like receptor 7 / 8 (TLR7 / 8) agonists can activate antigen-presenting cells and enhance antigen-specific T cell responses by activating the innate immune system. Combining tumor cell lysates with Toll-like receptor 7 / 8 agonists has great potential, but the effect of simple mixed administration is limited. Existing delivery strategies based on microparticle platforms often involve harsh synthesis conditions, which may inhibit the biological activity of the coupled molecules and have low antigen / adjuvant loading efficiency. Therefore, how to find and develop alternative strategies with simple design, high biocompatibility and high antigen / adjuvant loading efficiency is a problem and challenge facing the next generation of drug delivery technology.

[0004] Polyphenols are widely present in nature and are secondary metabolites of plants. They have multiple biological activities such as antioxidant, anti-inflammatory, antibacterial, antiviral, anti-tumor, and prevention of cardiovascular and cerebrovascular diseases. They are characterized by containing multiple galloyl groups, which enable them to form strong interactions (including covalent and non-covalent interactions) with various materials including biomacromolecules. This diverse binding ability makes them valuable in many biomedical applications. In addition, natural polyphenols also have potential uses in cancer treatment due to their powerful anti-inflammatory and antioxidant properties and their ability to regulate key molecular targets and signaling pathways involved in cancer progression. However, there are no reports on the preparation of antigen-binding nanoformulations by galloylation of resiquimod. Summary of the invention

[0005] In order to overcome the limitations and shortcomings of existing cancer vaccines, the present invention aims to provide a resiquimod galloyl derivative and a synthesis method and application thereof.

[0006] To achieve the above purpose, the present invention adopts the following technical solution:

[0007] A resiquimod galloyl derivative, wherein the derivative is resiquimod and gallic acid connected by an ester bond, and the structure is shown in the following formula 1;

[0008]

[0009] The gallic acid may also be replaced by phenolic acid compounds including caffeic acid, p-coumaric acid, ferulic acid, sinapinic acid, rosmarinic acid, 4-hydroxyphenylacetic acid, 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid and 3,4-dihydroxyphenylacetic acid.

[0010] A method for synthesizing the resiquimod galloyl derivative, the reaction formula is as follows:

[0011]

[0012] Under the catalysis of 4-dimethylaminopyridine (DMAP) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), 3,4,5-tri(benzyloxy)benzoic acid reacts with resiquimod, and an intermediate compound is separated and purified; the intermediate compound is catalyzed by palladium-carbon hydrogenation to remove the protecting group, and a resiquimod galloyl derivative is obtained after separation and purification.

[0013] A resiquimod galloyl derivative antigen nanocomposite, the nanocomposite comprising the resiquimod galloyl derivative, antigen and PEG; the weight ratio of the resiquimod galloyl derivative to the antigen is (1:2) to (1:10); the weight ratio of the resiquimod galloyl derivative to the PEG is (1:1) to (1:8);

[0014] The antigen is ovalbumin, carcinoembryonic antigen, alpha-fetoprotein, P1AB antigen, gp95 / p97 antigen, breast cancer cell lysate protein, melanoma cell lysate protein, colorectal cancer cell lysate protein, prostate cancer cell lysate protein, liver cancer cell lysate protein, lung cancer cell lysate protein, gastric cancer cell lysate protein, esophageal cancer cell lysate protein, pancreatic cancer cell lysate protein, ovarian cancer cell lysate protein, bladder cancer cell lysate protein or nasopharyngeal cancer cell lysate protein; the PEG molecular weight is 2k-35k.

[0015] A method for preparing the resiquimod galloyl derivative-antigen nanocomposite comprises the following steps: dissolving the resiquimod galloyl derivative in an organic solvent as an organic phase according to the ratio, dissolving the antigen and PEG in deionized water as an aqueous phase, and rapidly stirring and mixing the organic phase and the aqueous phase so that the resiquimod galloyl derivative and the antigen spontaneously form a uniform nanocomposite; the organic solvent is one or a mixed solvent of DMSO, acetone, ethanol, methanol, acetonitrile or tetrahydrofuran.

[0016] The above-mentioned resiquimod galloyl derivative antigen nanocomplex is prepared by stirring and mixing under mild conditions. The preparation process is simple, easy to scale up production, and can retain the biological activity of the antigen; no antigen modification is required, and it is not limited to specific antigens, and is also suitable for delivering TCLs containing multiple antigens of uncertain properties; the synthesis can be completed within one day, avoiding the typical time-consuming and expensive antigen screening process of cancer vaccines based on new antigens; at the same time, the resulting complex can achieve the synchronous release of antigens and adjuvants in dendritic cells (DCs), enhance the cross-presentation of tumor antigens, promote the production of proinflammatory cytokines, activate dendritic cells, and then induce a strong anti-tumor immune response, with good safety and great potential for clinical application.

[0017] A use of the derivative or the nanocomposite in preparing a drug delivery system.

[0018] A use of the derivative or the nanocomposite in preparing anti-tumor drugs.

[0019] The drug is administered by injection, oral administration or topical administration.

[0020] Beneficial effects of the present invention:

[0021] The present invention improves the affinity of resiquimod to antigens by performing galloyl derivatization modification on resiquimod, thereby simply mixing with the antigen to form nanoparticles. There is no need to modify the antigen, and it is not limited to specific antigens, and is also suitable for delivering TCLs containing multiple antigens of uncertain properties. It provides new ideas and opportunities for the development of antigen and adjuvant co-delivery nanoparticles to meet the urgent clinical demand for cancer vaccines. The prepared nanoparticles can achieve the synchronous release of antigens and adjuvants in dendritic cells (DCs), enhance the cross-presentation of tumor antigens, activate dendritic cells, and thus induce a strong anti-tumor immune response. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The high resolution mass spectrum of the resimod galloyl derivative (R848-GA) whose phenolic acid part is 3,4,5-trihydroxybenzoic acid in Example 1 of the present invention and 1 H-NMR spectrum.

[0023] Figure 2 The particle size distribution diagram and appearance morphology diagram of the resiquimod galloyl derivative ovalbumin nanocomplex, the resiquimod galloyl derivative colon cancer cell (MC38) lysate protein nanocomplex, the resiquimod galloyl derivative melanoma cell (B16) lysate protein nanocomplex, and the resiquimod galloyl derivative breast cancer cell (4T1) lysate protein nanocomplex of Example 2 of the present invention.

[0024] Figure 3 This is a stability diagram of the resiquimod galloyl derivative colon cancer cell (MC38) lysate protein nanocomplex, the resiquimod galloyl derivative melanoma cell (B16) lysate protein nanocomplex, and the resiquimod galloyl derivative breast cancer cell (4T1) lysate protein nanocomplex of Example 3 of the present invention.

[0025] Figure 4 This is a graph showing the tumor volume-time growth curve of MC38 tumor-bearing mice in an in vivo efficacy experiment of the resiquimod galloyl derivative colon cancer cell (MC38) lysate protein nanocomplex of Example 4 of the present invention.

[0026] Figure 5 This is a graph showing the changes in body weight over time in MC38 tumor-bearing mice in an in vivo efficacy experiment of the resiquimod galloyl derivative colon cancer cell (MC38) lysate protein nanocomplex of Example 4 of the present invention.

[0027] Figure 6 This is a graph showing changes in blood indicators in MC38 tumor-bearing mice in an in vivo efficacy experiment of the resiquimod galloyl derivative colon cancer cell (MC38) lysate protein nanocomplex of Example 4 of the present invention.

[0028] Figure 7 This is an immune cell expression diagram of MC38 tumor-bearing mice in an in vivo efficacy experiment of the resiquimod galloyl derivative colon cancer cell (MC38) lysate protein nanocomplex of Example 5 of the present invention.

[0029] Figure 8 This is a graph showing changes in serum cytokines in MC38 tumor-bearing mice in an in vivo efficacy experiment of the resiquimod galloyl derivative colon cancer cell (MC38) lysate protein nanocomplex of Example 5 of the present invention. DETAILED DESCRIPTION

[0030] The present invention is further described below by way of examples, but the invention is not limited to the scope of the examples. This example is only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art may make some non-essential improvements and adjustments based on the contents described in the present invention, which all belong to the scope of protection of the present invention.

[0031] The present invention synthesizes a resiquimod galloyl derivative with high protein affinity, and prepares an antigen resiquimod galloyl derivative nanocomplex with simple design and good stability by simply mixing the galloyl derivative with an antigen, thereby achieving co-delivery of the antigen and the adjuvant and inducing a strong anti-tumor immune response.

[0032] Example 1

[0033] Synthesis of Resiquimod Galloyl Derivative (R848-GA)

[0034] Accurately weigh 3,4,5-tri(benzyloxy)benzoic acid (210.1 mg, 0.477 mmol), EDC (74.0 mg, 0.477 mmol) and DMAP (58.3 mg, 0.477 mmol) and add them to a 100 mL eggplant-shaped bottle, dissolve them with 5 mL of dichloromethane, and stir them in an ice bath for 2 hours. Accurately weigh resimod (100 mg, 0.318 mmol), dissolve them with 5 mL of dichloromethane, slowly drip them into the above reaction solution while stirring in an ice bath, transfer to room temperature and continue the reaction for 24 hours, and monitor the reaction progress by thin layer chromatography. After the reaction is completed, the reaction solution is dried, the sample is dissolved in acetonitrile, and separated and purified by preparative liquid phase (the chromatographic column used is YMC-Pack ODS-A, the mobile phase is acetonitrile: water = 85:15, the flow rate is 5 mL / min, and the 20th minute component is collected) to obtain the intermediate product (white powder, yield 70%). The intermediate product (100 mg) was added to a 100 mL eggplant-shaped bottle and dissolved with 15 mL of anhydrous ethanol. Then Pd / C (10 mg, 10% w / w) with a mass fraction of 10% of the intermediate product was added, and the reaction was carried out at room temperature for 8 hours in the presence of H2, and the reaction process was monitored by thin layer chromatography. After the reaction was completed, Pd / C was removed by microporous membrane filtration, and the filtrate was purified by preparative liquid separation (the chromatographic column used was YMC-Pack ODS-A, the mobile phase was acetonitrile: water = 80:20, the flow rate was 5 mL / min, and the 10th minute component was collected) to obtain the target product R848-GA (yellow solid, yield 90%). High-resolution mass spectrometry and 1 The structure of the target compound was confirmed by H-NMR spectroscopy. Figure 1As shown, the results of nuclear magnetic resonance spectrum analysis are as follows: δ10.20 (s, 1H, Ar-OH), 9.19 (s, 2H, Ar-OH), 8.57 (d, J = 8.3 Hz, 1H, H-4), 8.05-7.91 (d, J = 8.3 Hz, 1H, H-1), 7.65 (t, J = 7.6 Hz, 1H, H-3), 7.58 (t, J = 7.7 Hz, 1H, H-2), 7.06 (s, 2H, H-5and6), 4.95 (s, 2H, 8-CH2), 4.77 (s, 2H, 7-CH2), 3.52 (q, J = 6.9 Hz, 2H, -O CH2 CH3),1.22(s,6H,-C(CH3)2),1.13(t,J=7.0Hz,3H,-OCH2 CH3 ).

[0035] Example 2

[0036] Preparation of resiquimod-galloyl derivative-antigen nanocomplex

[0037] Accurately measure 1 mL of different antigen (ovalbumin (OVA), colon cancer cell (MC38) lysate protein, melanoma cell (B16) lysate protein, breast cancer cell (4T1) lysate protein) solution (concentration of 2 mg / mL, solvent is water) and 80 μL of PEG20000 solution (concentration of 20 mg / mL, solvent is water) in a 2 mL vial and mix and stir. Subsequently, 60 μL of R848-GA (concentration of 10 mg / mL, solvent is DMSO) prepared in the above example was added to the vial for assembly and stirred at room temperature for 20 minutes to obtain stable nanoparticles (R848-GA@OVA, R848-GA@MC38-L, R848-GA@B16-L, R848-GA@4T1-L) (see Figure 2 The particle sizes and particle size distributions of the different particles obtained by the above determinations are shown in Table 1.

[0038] Table 1 Particle size and PDI of different resiquimod derivative-antigen nanocomplexes

[0039]

[0040] Depend on Figure 2 It can be seen that resiquimod galloyl derivatives can effectively bind to antigens to form nanocomplexes with uniform particle size.

[0041] Example 3

[0042] Stability experiment of resiquimod galloyl derivative antigen nanocomplex

[0043] 1 mL of R848-GA@MC38-L, R848-GA@B16-L, and R848-GA@4T1-L prepared in the above examples were added to 9 mL of phosphate buffered saline (PBS, pH 7.4) containing 10% fetal bovine serum, placed in a 37°C water bath, and samples were collected at specified times and tested with a Malvern particle size analyzer to evaluate stability.

[0044] The results are as follows Figure 3 As shown in the figure, R848-GA@MC38-L, R848-GA@B16-L, and R848-GA@4T1-L showed excellent physical stability when incubated in phosphate buffered saline (PBS, 10% fetal bovine serum, pH 7.4) at 37°C. This indicates that R848-GA@TCLs can overcome the instability of antigens and prevent their degradation during delivery.

[0045] Example 4

[0046] In vivo efficacy study of resiquimod-galloyl derivative-antigen nanocomplex

[0047] Take 100 μL MC38 cells (2×10 6 ) was injected subcutaneously above the right buttocks of mice to establish an MC38 mouse ectopic tumor animal model. When the tumor volume of the mouse reached 100 mm 3 Afterwards, the mice were randomly divided into 6 groups, with 5 animals in each group, namely PBS group, R848 group, R848+MC38-L group, R848-GA@OVA group, R848-GA+MC38-L group, and R848-GA@MC38-L group. The equivalent dose of resimod in all groups (except PBS group) was 2 mg / kg. The administration method was subcutaneous injection, once every three days, for a total of 3 times. The weight changes and tumor volume of the mice were measured and recorded every two days after administration. After the treatment, the mouse serum was collected to detect the content of blood white blood cells, lymphocytes, monocytes, red blood cells and platelets.

[0048] The results are as follows Figure 4 As shown, the R848-GA@MC38-L group showed significantly enhanced tumor inhibition, with an average tumor size of only 7% of the tumor size in the PBS group. In addition, the effects of free R848 and the combination of R848-GA+MC38-L were significantly inferior to those of R848-GA@MC38-L, highlighting the advantage of co-delivery of antigens and adjuvants for maximizing anti-tumor effects. The body weights of mice in all groups remained essentially unchanged throughout the study ( Figure 5 ), indicating that these treatments have a good in vivo safety profile. No changes were detected in peripheral blood cell subsets (including leukocytes, lymphocytes, monocytes, erythrocytes, and platelets), further confirming the safety of the nanovaccine ( Figure 6 ).

[0049] Example 5

[0050] Study on the anti-tumor immune response induced by resiquimod galloyl derivative antigen nanocomplex

[0051] Take 100 μL MC38 cells (2×10 6 ) was injected subcutaneously above the right buttocks of mice to establish an MC38 mouse ectopic tumor animal model. When the tumor volume of the mouse reached 100 mm 3 Afterwards, the mice were randomly divided into 3 groups, namely PBS group, R848-GA+MC38-L group, and R848-GA@MC38-L group. The equivalent dose of resiquimod in all groups (except PBS group) was 2 mg / kg. The administration method was subcutaneous injection, once every three days, for a total of 3 times. After the experiment, the lymph nodes and tumor tissues of each group of mice were collected to detect the infiltration of immune cells, and the mouse serum was collected to detect the expression levels of TNF-α, IFN-γ and IL-12 by ELISA kit.

[0052] The results are as follows Figure 7 As shown, in lymph nodes and tumors, the percentage of CD80+CD86+ dendritic cells in the R848-GA@MC38-L treated group was significantly higher than that in the R848-GA+MC38-L group, indicating that R848-GA@TCLs induced stronger dendritic cell maturation. In addition, R848-GA@MC38-L treatment significantly increased the percentage of CD8+T cells and anti-tumor M1-like macrophages (F4 / 80+CD11c+) in lymph nodes, and the percentage of CD25+CD8+T cells and NK cells in tumor tissues. Serum cytokine TNF-α, IFN-γ, and IL-12 levels further indicated that the R848-GA@MC38-L group had the strongest ability to induce immune responses ( Figure 8 ).

Claims

1. A resiquimod galloyl derivative, characterized in that: The derivative is a substance in which resiquimod and gallic acid are connected by an ester bond, and the structure is shown in the following formula 1; 2. A method for synthesizing the resiquimod galloyl derivative according to claim 1, characterized in that: The reaction formula is as follows: Under the catalysis of 4-dimethylaminopyridine (DMAP) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), 3,4,5-tri(benzyloxy)benzoic acid reacts with resiquimod, and an intermediate compound is separated and purified; the intermediate compound is catalyzed by palladium-carbon hydrogenation to remove the protecting group, and a resiquimod galloyl derivative is obtained after separation and purification.

3. A resiquimod galloyl derivative antigen nanocomplex, characterized in that: The nanocomposite is the resiquimod galloyl derivative, antigen and PEG as described in claim 1; the weight ratio of the resiquimod galloyl derivative to the antigen is (1:2) to (1:10); the weight ratio of the resiquimod galloyl derivative to PEG is (1:1) to (1:8).

4. The resiquimod galloyl derivative antigen nanocomplex according to claim 3, characterized in that: The antigen is ovalbumin, carcinoembryonic antigen, alpha-fetoprotein, P1AB antigen, gp95 / p97 antigen, breast cancer cell lysate protein, melanoma cell lysate protein, colorectal cancer cell lysate protein, prostate cancer cell lysate protein, liver cancer cell lysate protein, lung cancer cell lysate protein, gastric cancer cell lysate protein, esophageal cancer cell lysate protein, pancreatic cancer cell lysate protein, ovarian cancer cell lysate protein, bladder cancer cell lysate protein or nasopharyngeal cancer cell lysate protein; the PEG molecular weight is 2k-35k.

5. A method for preparing the resiquimod galloyl derivative antigen nanocomplex according to claim 1, characterized in that: According to the ratio described in claim 3, the resiquimod galloyl derivative described in claim 1 is dissolved in an organic solvent as an organic phase, the antigen and PEG are dissolved in deionized water as an aqueous phase, and the organic phase and the aqueous phase are mixed by rapid stirring so that the resiquimod galloyl derivative and the antigen spontaneously form a uniform nanocomposite; the organic solvent is one or more mixed solvents selected from DMSO, acetone, ethanol, methanol, acetonitrile or tetrahydrofuran.

6. Use of the derivative according to claim 1 or the nanocomposite according to claim 3 in preparing a drug delivery system.

7. Use of the derivative according to claim 1 or the nanocomposite according to claim 3 in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that: The drug is administered by injection, oral administration or topical administration.

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