A gsh-responsive capsaicin prodrug and a preparation method thereof

Abstract

The application discloses a GSH-responsive capsaicin prodrug, which is capsaicin dimer nanoparticles formed by esterification reaction of capsaicin and dithiodipropionic acid and self-assembly, and discloses a preparation method of the prodrug. The capsaicin dimer nanoparticles prepared by the application can react with glutathione highly expressed at an inflammation site, and then release capsaicin. The prodrug has certain targeting property, can reduce drug side effects and improve curative effect. In addition, the prodrug is not easy to be metabolically discharged, and is more stable in blood plasma, so that the half-life and bioavailability of the drug can be improved, and the frequency of drug administration can be reduced. The in-vitro cell test result shows that, compared with capsaicin, the application can significantly reduce the content of inflammatory factors in cells, and has better anti-inflammatory effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of drug synthesis, and particularly relates to a GSH-responsive capsaicin prodrug and a preparation method thereof. BACKGROUND

[0002] Arthritis is a disease caused by inflammation of joints or surrounding tissues, including rheumatoid arthritis, rheumatoid arthritis, osteoarthritis and gouty arthritis, and its main symptoms include pain, swelling, swelling and joint function limitation. Arthritis has a long course and is difficult to cure, which has a significant impact on the daily life of patients and has become a major problem in the medical field. At present, surgery or taking non-steroidal anti-inflammatory drugs is mainly used to treat arthritis, the former is expensive and painful for patients, and the latter can increase the burden on the liver and kidney of patients, stimulate the gastrointestinal tract, and produce drug resistance.

[0003] Capsaicin, also known as trans-8-methyl-N-vanillyl-6-nonenamide, is the main active ingredient in chili peppers. Studies have shown that capsaicin binds to capsaicin receptors, causing an increase in intracellular calcium ion concentration, thereby prompting neurons and their fibers to release a variety of neuropeptides, which have significant anti-inflammatory, antioxidant and analgesic effects, and show good therapeutic effect on inflammatory joint diseases such as rheumatoid arthritis and osteoarthritis. However, capsaicin has poor stability, short half-life and strong irritation, which may cause a burning sensation, thereby limiting its application in the biomedical field.

[0004] The stability of the drug can be improved by constructing small molecule prodrugs with self-assembly ability, thereby prolonging the half-life. Studies have shown that a sulfur-containing linker can effectively improve the self-assembly ability of the prodrug, because the sulfur bond has a bond angle close to 90°, which can balance the intermolecular force. Compared with single sulfur bond (-s-), disulfide bond (-ss-) has more sulfur dihedral angle, resulting in stronger self-assembly ability and in vivo stability. In addition, disulfide bond is widely used in the field of biological medicine due to its strong redox property to improve the targeting of drugs. SUMMARY

[0005] Capsaicin has excellent anti-inflammatory and analgesic effects and is suitable for the treatment of arthritis, but due to its poor stability, short half-life, low bioavailability, strong irritation and other defects, its application in the medical field is limited. To solve this problem, the present application connects capsaicin with GSH-responsive disulfide dipropionic acid by esterification, and then self-assembles into nanoparticles. The prepared GSH-responsive capsaicin dimer prodrug has good inflammation site targeting effect, high bioavailability and low irritation, and can be better used for the treatment of arthritis.

[0006] In order to achieve the above-mentioned purpose, the application provides a GSH-responsive capsaicin prodrug, which is a capsaicin dimer nanoparticle formed by esterification of capsaicin and dithiodipropionic acid and self-assembly.

[0007] The application further provides a preparation method of the capsaicin prodrug, which comprises the following steps:

[0008] 1) Steglich esterification of capsaicin and dithiodipropionic acid is carried out by taking EDC as a dehydrating agent and DMAP as a catalyst to generate capsaicin dimers;

[0009] 2) The synthesized capsaicin dimers are dissolved in dimethyl sulfoxide for self-assembly, impurities and organic solvents are removed by dialysis, and then drying is carried out to obtain capsaicin dimer nanoparticles.

[0010] Preferably, the Steglich esterification is carried out at room temperature and in the dark.

[0011] Preferably, the Steglich esterification is carried out in two steps, in the first step, half of the amount of dehydrating agent and catalyst is added, and the reaction is carried out for 1-5 hours, in the second step, the remaining amount of dehydrating agent and catalyst is added, and the reaction is carried out for 20-30 hours.

[0012] Preferably, the reaction solvent of the Steglich esterification is dichloromethane.

[0013] Preferably, the mass ratio of EDC to DMAP is 20:1.

[0014] Preferably, the dialysis time is 20-30 hours.

[0015] Preferably, the membrane pore size of the dialysis is 1 kd.

[0016] The application has the following beneficial effects:

[0017] The capsaicin dimer nanoparticle prepared by the application has GSH responsiveness, can react with glutathione highly expressed at an inflammatory site, and then release capsaicin, so that the prodrug prepared by the application has a certain targeting property, can reduce drug side effects and improve curative effect. In addition, the nanoparticle dimer is not easy to be metabolically excreted and is more stable in blood plasma, so that the half-life and bioavailability of the drug can be improved, and the frequency of drug administration can be reduced. The in vitro cell test results show that, compared with capsaicin, the dimer nanoparticle prepared by the application can significantly reduce the content of inflammatory factors in cells, and has a better anti-inflammatory effect, so that the prodrug is expected to play a better role in the treatment of arthritis diseases such as rheumatoid arthritis, rheumatoid arthritis, osteoarthritis, gouty arthritis, traumatic arthritis, infectious arthritis, etc.

[0018] Compared with traditional drug delivery systems, the dimer prodrug avoids complex carrier systems, greatly improves drug loading capacity and reduces particle size, avoids potential toxicity caused by carrier materials, and has better biological safety. The present application also has the advantages of simple preparation method, high product yield and the like. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 FTIR spectrum of capsaicin dimer nanoparticles.

[0020] Figure 2 Dynamic light scattering DLS particle size analysis chart of capsaicin dimer nanoparticles.

[0021] Figure 3 Cell toxicity test results of capsaicin dimer nanoparticles.

[0022] Figure 4 In vitro anti-inflammatory test results of capsaicin dimer nanoparticles. DETAILED DESCRIPTION

[0023] The present application will be further described in detail below in combination with specific examples.

[0024] Example 1 Preparation of capsaicin dimer nanoparticles (CAP-S-S-CAP)

[0025] First step: synthesis of capsaicin dimer prodrug

[0026] The reaction formula is as follows. Accurately take capsaicin 30 mg (0.098 mmol), dithiodipropionic acid 10 mg (0.047 mmol), EDC 40 mg, DMAP 2 mg, and dissolve them in a reaction kettle containing 5 ml of dichloromethane, stir at room temperature in the dark for 2 h; then add EDC 40 mg and DMAP 2 mg again to the reaction kettle, dissolve thoroughly, and continue to react at room temperature in the dark for 24 h. After the reaction is completed, remove the dichloromethane by rotary evaporation to obtain a white solid product, capsaicin dimer prodrug, with a yield of 81%.

[0027]

[0028] Second step: self-assembly of nanoparticles

[0029] Dissolve the synthesized prodrug in 5 ml of dimethyl sulfoxide (DMSO), add 20 ml of distilled water, stir until completely dissolved, and then put it into a dialysis membrane (1 kd) in distilled water for 24 h to form a nanoparticle dispersion. Finally, freeze-dry the dispersion in a freeze-dryer for 24 h, and collect the product for storage at room temperature.

[0030] FTIR spectrum confirms the formation of capsaicin dimer (Figure 1 1600, 1500 and 1427 cm −1 The peaks around 1600 cm −1 The absorption peaks around 1750 cm −1 The absence of peaks around 1690 cm −1 indicates the C=0 stretch of carboxyl group is not present, further indicating the reaction of carboxyl and hydroxyl group to form ester bond. This indicates the success of the synthesis and complete consumption of the carboxyl group of the thioether linker.

[0031] Example 2

[0032] Accurately weigh capsaicin 30 mg, dithiodipropionic acid 10 mg, EDC 80 mg, DMAP 4 mg into a reaction flask with 5 ml dichloromethane, dissolve thoroughly, react at room temperature for 30 h in the dark. After the reaction is completed, remove the dichloromethane by rotary evaporation to obtain a white solid product with a yield of 36%. Dissolve the synthesized prodrug in 5 ml dimethyl sulfoxide (DMSO), add 20 ml distilled water, stir until completely dissolved, then put it into a dialysis membrane (1 kd), dialyze in distilled water for 24 h to form a nanoparticle dispersion, and finally freeze-dry the dispersion in a freeze-dryer for 24 h. Collect the product and store it at room temperature for use.

[0033] Example 3

[0034] Accurately weigh capsaicin 30 mg, dithiodipropionic acid 10 mg, DCC 40 mg, DMAP 2 mg into a reaction flask with 5 ml dichloromethane, stir at room temperature for 2 h in the dark, then add another DCC 40 mg, DMAP 2 mg into the reaction flask, dissolve thoroughly, continue to react at room temperature for 24 h in the dark. After the reaction is completed, remove the dichloromethane by rotary evaporation to obtain a white solid product. Dissolve the synthesized prodrug in 5 ml dimethyl sulfoxide (DMSO), add 20 ml distilled water, stir until completely dissolved, then put it into a dialysis membrane (1 kd), it is found that DCC and DMSO can mix to form Pfitzner-Moffatt reagent, which is difficult to remove during dialysis.

[0035] Test Example

[0036] 1. Particle size detection

[0037] Dissolve the prodrug prepared in Example 1 in 10 ml distilled water, take 1.5 ml solution, under the condition of 24.9 degrees and scattering angle 90°, use Malvern nanoparticle size Zeta potential analysis device to measure the particle size, Zeta potential and polydispersity index (PDI) of the product. The average particle size is 170.7 nm (PDI = 0.125), the Zeta potential is -25.1, and the PDI is 0.125. Figure 2

[0038] 2. GSH-responsive assay

[0039] The synthesized capsaicin dimer does not contain free phenolic hydroxyl group, so it cannot react with ferric trichloride, but after reacting with glutathione, the disulfide bond is broken, releasing free capsaicin, so the color development experiment with ferric trichloride and phenolic hydroxyl group is used to judge the GSH responsiveness of the dimer. The prepared capsaicin dimer was dissolved in two test tubes containing an appropriate amount of ethanol solution, glutathione was added to one of the test tubes and mixed well, and then the same amount of ferric trichloride was added to both test tubes. It can be seen that the solution without glutathione shows yellow color, and the solution with glutathione shows green color, indicating that ferric trichloride reacts with the free phenolic hydroxyl group of capsaicin. Thus, it can be judged that the prepared capsaicin dimer has GSH responsiveness.

[0040] 3. Cytotoxicity assay

[0041] MTT method was used to evaluate the cytotoxicity of capsaicin dimer. First, HaCaT cells were subcultured and inoculated in sterile culture dishes, and cultured in a 37°C, humidity saturated, 5% CO2 incubator. When the cells grew to 80%-90% confluence, the original culture medium was replaced with culture medium containing different concentrations (100, 200, 300, 400, 500 ug / ml) of capsaicin dimer, and continued to culture for 24 hours. Subsequently, the cells were transferred to a 96-well adhesion plate, 10 μL of MTT reagent (dissolved in DMEM) was added to each well, and continued to culture for 4 hours. Then, 100 μL of DMSO was added to each well, followed by shaking in a 37°C full-temperature shaking incubator for 10 minutes. Finally, the optical density value of each well at 510 nm was determined using an enzyme-labeled instrument, and the results showed that the capsaicin dimer had no cytotoxicity Figure 3 ).

[0042] 4. Anti-inflammatory effect assay

[0043] The anti-inflammatory effect of free capsaicin and capsaicin dimer was detected by ELISA experiment. A 24-well plate was used, 80000 mouse macrophages (RAW264.7 cells) were added to each well, and 40 μL of Tris buffer solution was added to the blank well; 20 μL of LPS-containing Tris buffer and 20 μL of Tris solution were added to the well for inducing cell inflammation by lipopolysaccharide (LPS); 20 μL of LPS-containing Tris buffer and 20 μL of CAP dispersion were added to the well for free capsaicin (CAP); 20 μL of LPS-containing Tris buffer and 20 μL of CAP-S-S-CAP nps were added to the well for capsaicin dimer nanoparticles (CAP-S-S-CAP nps). Each group above occupied 6 wells, and was cultured for 24 h and 48 h, respectively. After the culture, the anti-inflammatory effect was detected by tumor necrosis factor TNF-α (mouse) ELISA kit. TNF-α is a multi-effect systemic inflammatory response mediator and a pro-inflammatory cytokine. The results can be judged by the content of TNF-α. The lower the content of TNF-α, the stronger the anti-inflammatory ability of the drug. It can be seen that the anti-inflammatory effect of capsaicin dimer at 24 h and 48 h is better than that of free capsaicin (see Figure 4 ),which shows that the prepared capsaicin dimer can maintain a longer action time and a better effect.​

Claims

1. A GSH-responsive capsaicin prodrug, which is a capsaicin dimer nanoparticle formed by esterification of capsaicin with dithiodipropionic acid and self-assembly.

2. A method of making the capsaicin prodrug of claim 1, characterized by The method comprises the following steps: 1.A Steglich esterification reaction is performed on capsaicin with dithiodipropionic acid using EDC as a dehydrating agent and DMAP as a catalyst to generate capsaicin dimers; 2.The synthesized capsaicin dimers are dissolved in dimethyl sulfoxide for self-assembly, impurities and organic solvents are removed by dialysis, and then dried to obtain capsaicin dimer nanoparticles.

3. The method for preparing the capsaicin prodrug according to claim 2, wherein: The Steglich esterification reaction is performed at room temperature and in the dark.

4. The method of claim 2, wherein the preparation of the prodrug of capsaicin is characterized by: The Steglich esterification reaction is performed in two steps, in the first step, half of the amount of dehydrating agent and catalyst is added, and the reaction is performed for 1-5 hours, in the second step, the remaining amount of dehydrating agent and catalyst is added, and the reaction is performed for 20-30 hours.

5. The method of claim 2, wherein the preparation of the prodrug of capsaicin is characterized by: The reaction solvent of the Steglich esterification reaction is dichloromethane.

6. The method of claim 2, wherein the preparation of the prodrug of capsaicin is characterized by: The mass ratio of EDC to DMAP is 20:

1.

7. The method of claim 2, wherein the method is carried out in the presence of a base. The dialysis time is 20-30 hours.

8. The method of claim 2, wherein the preparation of the prodrug of capsaicin is characterized by: The membrane pore size of the dialysis is 1 kd.

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