GSH responsive capsaicin prodrug and preparation method thereof

Abstract

The invention discloses a GSH (glutathione) responsive capsaicin prodrug which is capsaicin dimer nano-particles formed by performing esterification reaction on capsaicin and dithiodipropionic acid and performing self-assembly, and further discloses a preparation method of the prodrug. The capsaicin dimer nano-particles prepared by the invention can react with glutathione highly expressed at an inflammation part so as to release capsaicin, and the prodrug has a certain targeting property, so that the side effect of the drug can be reduced, and the curative effect can be improved. In addition, the prodrug is not easy to be metabolized and discharged, and is more stable in plasma, so that the half-life period and bioavailability of the drug can be improved, and the administration frequency is reduced. An in-vitro cell test result shows that compared with capsaicin, the content of inflammatory factors in cells can be remarkably reduced, and a better anti-inflammatory effect is achieved.

Description

Technical Field

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

[0002] Arthritis is a type of disease caused by inflammation of the joints or surrounding tissues, including rheumatoid arthritis, osteoarthritis, gouty arthritis and other types. Its main symptoms include pain, soreness, swelling and limited joint function. Arthritis has a long course and is difficult to cure, which has a significant impact on patients' daily lives and has become a major problem in the medical field. Currently, arthritis is mainly treated by surgery or taking non-steroidal anti-inflammatory drugs. The former is expensive and painful for patients, while the latter will increase the burden on the patient's liver and kidneys, irritate the gastrointestinal tract, and produce drug resistance if taken for a long time.

[0003] Capsaicin, whose scientific name is trans-8-methyl-N-vanillyl-6-nonenamide, is the main active ingredient in chili peppers. Studies have shown that capsaicin binds to capsaicin receptors, increasing the intracellular calcium ion concentration, thereby prompting neurons and their fibers to release a variety of neuropeptides. It has significant anti-inflammatory, antioxidant and analgesic effects, and shows good therapeutic effects on inflammatory joint diseases such as rheumatoid arthritis and osteoarthritis. However, capsaicin has poor stability, a short half-life, and strong irritation, which may cause a burning sensation, resulting in its application in the field of biomedicine being greatly limited.

[0004] The stability of drugs can be improved by constructing small molecule prodrugs with self-assembly ability, thereby extending the half-life. Studies have shown that connecting chains containing sulfur bonds can effectively improve the self-assembly ability of prodrugs. This is because sulfur bonds have a bond angle close to 90°, which can balance the intermolecular forces. Compared with single sulfur bonds (-s-), disulfide bonds (-ss-) have more sulfur-containing dihedral angles, resulting in stronger self-assembly ability and in vivo stability. In addition, disulfide bonds are widely used in the biomedical field to improve the targeting of drugs due to their strong redox properties. Summary of the invention

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

[0006] To achieve the above object, the present invention provides a GSH-responsive capsaicin prodrug, which is a capsaicin dimer nanoparticle formed by esterification reaction of capsaicin and dithiodipropionic acid and self-assembly.

[0007] The present invention further provides a method for preparing the capsaicin prodrug, which comprises the following steps: 1) Using EDC as a dehydrating agent and DMAP as a catalyst, capsaicin and dithiodipropionic acid were subjected to a Steglich esterification reaction to generate capsaicin dimer; 2) The synthesized capsaicin dimer is dissolved in dimethyl sulfoxide for self-assembly, and the impurities and organic solvents are removed by dialysis, and then dried to obtain capsaicin dimer nanoparticles.

[0008] Preferably, the Steglich esterification reaction is carried out at room temperature in the dark.

[0009] Preferably, the Steglich esterification reaction is carried out in two steps. In the first step, half of the dehydrating agent and catalyst are added and the reaction is carried out for 1-5 hours. In the second step, the remaining amount of the dehydrating agent and catalyst are added and the reaction is carried out for 20-30 hours.

[0010] Preferably, the reaction solvent of the Steglich esterification reaction is dichloromethane.

[0011] Preferably, the mass ratio of EDC to DMAP is 20:1.

[0012] Preferably, the dialysis time is 20-30 hours.

[0013] Preferably, the pore size of the dialysis membrane is 1 kd.

[0014] The beneficial effects of the present invention are: The capsaicin dimer nanoparticles prepared by the present invention have GSH responsiveness, can react with glutathione highly expressed in the inflammatory site, and then release capsaicin, so the prodrug prepared by the present invention has certain targeting, can reduce drug side effects and improve the efficacy. In addition, since the nanoparticle dimer is not easily metabolized and discharged, it is also more stable in blood plasma, so the half-life and bioavailability of the drug can be improved, and the number of administrations can be reduced. The results of in vitro cell tests show that compared with capsaicin, the dimer nanoparticles prepared by the present invention can significantly reduce the content of inflammatory factors in cells, and have better anti-inflammatory effects, so 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.

[0015] Compared with the traditional drug delivery system, the dimer prodrug avoids the complex carrier system, greatly increases the drug loading and reduces the particle size, avoids the potential toxicity caused by the carrier material, and has better biosafety. The present invention also has the advantages of simple preparation method and high product yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 FTIR spectrum of capsaicin dimer nanoparticles.

[0017] Figure 2 This is the dynamic light scattering DLS particle size analysis diagram of capsaicin dimer nanoparticles.

[0018] Figure 3 These are the cytotoxicity test results of capsaicin dimer nanoparticles.

[0019] Figure 4 These are the results of in vitro anti-inflammatory tests on capsaicin dimer nanoparticles. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with specific embodiments.

[0021] Example 1 Preparation of Capsaicin Dimer Nanoparticles (CAP-SS-CAP) Step 1: Synthesis of capsaicin dimer prodrug Obtained by esterification reaction of capsaicin and dithiodipropionic acid, the reaction formula is as follows. Accurately measure 30 mg (0.098 mmol) of capsaicin, 10 mg (0.047 mmol) of dithiodipropionic acid, 40 mg of EDC, and 2 mg of DMAP and dissolve them in a reactor containing 5 ml of dichloromethane, stir at room temperature and avoid light for 2 hours; then take another 40 mg of EDC and 2 mg of DMAP and add them to the reactor again to fully dissolve, and continue to react at room temperature and avoid light for 24 hours. After the reaction is completed, rotary evaporation is performed to remove dichloromethane to obtain a white solid product, capsaicin dimer prodrug, with a yield of 81%.

[0022]

[0023] Step 2: Self-assembly of nanoparticles The synthesized prodrug was dissolved in 5 ml of dimethyl sulfoxide (DMSO), and 20 ml of distilled water was added. After stirring until completely dissolved, it was placed in a dialysis membrane (1 kd) and dialyzed in distilled water for 24 hours to form a nanoparticle dispersion. Finally, the dispersion was freeze-dried in a freeze dryer for 24 hours, and the product was collected and stored at room temperature for later use.

[0024] FTIR spectroscopy confirmed the formation of capsaicin dimers ( Figure 1 ). 1600, 1500 and 1427cm −1The peaks on the left and right are caused by the C=C stretching vibration of the benzene ring, at 1750cm −1 The absorption peaks on the left and right are due to the C=O stretching vibrations generated by the ester bond. In addition, there is no 1690cm −1 The peaks on the left and right indicate that the C=O stretching vibration of the carboxyl group does not exist, which further indicates that the carboxyl group and the hydroxyl group react to produce an ester bond. This indicates that the synthesis is successful and the carboxyl group of the thioether linker is completely consumed.

[0025] Example 2 Accurately measure 30 mg of capsaicin, 10 mg of dithiodipropionic acid, 80 mg of EDC, and 4 mg of DMAP and dissolve them in a reactor containing 5 ml of dichloromethane and react for 30 hours at room temperature in the dark. After the reaction, remove dichloromethane by rotary evaporation to obtain a white solid product with a yield of 36%. 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 (1kd). Dialyze in distilled water for 24 hours to form a nanoparticle dispersion. Finally, freeze-dry the dispersion in a freeze dryer for 24 hours, collect the product and store it at room temperature for use.

[0026] Example 3 Accurately measure 30 mg of capsaicin, 10 mg of dithiodipropionic acid, 40 mg of DCC, and 2 mg of DMAP and dissolve them in a reactor containing 5 ml of dichloromethane. Stir for 2 hours at room temperature and away from light. Then take another 40 mg of DCC and 2 mg of DMAP and add them to the reactor again to fully dissolve. Continue to react at room temperature and away from light for 24 hours. After the reaction is completed, remove dichloromethane by rotary evaporation to obtain a white solid product. 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 (1kd). It is found that DCC and DMSO will mix to form Pfitzner-Moffatt reagent, which is difficult to remove during dialysis.

[0027] Test example 1. Particle size detection The prodrug prepared in Example 1 was dissolved in 10 ml of distilled water, and 1.5 ml of the solution was taken and measured at 24.9 degrees and a scattering angle of 90 degrees using a Malvern nanoparticle size Zeta potential analyzer to measure the particle size, Zeta potential, and polydispersity index (PDI) of the product. The average particle size was 170.7 nm ( Figure 2 ), Zeta potential is -25.1, and PDI is 0.125.

[0028] 2. GSH responsiveness assay The synthesized capsaicin dimer does not contain free phenolic hydroxyl groups, so it cannot react with ferric chloride. However, after reacting with glutathione, the disulfide bond breaks and free capsaicin is released. Therefore, the colorimetric experiment of ferric chloride and phenolic hydroxyl groups is used to determine the GSH responsiveness of the dimer. The prepared capsaicin dimer is dissolved in two test tubes containing an appropriate amount of ethanol solution, glutathione is added to one of the test tubes and mixed, and then the same amount of ferric chloride is added to the two test tubes. It can be seen that the solution without glutathione is yellow, and the solution with glutathione is green, indicating that ferric chloride reacts with the phenolic hydroxyl groups of free capsaicin. It can be judged that the prepared capsaicin dimer has GSH responsiveness.

[0029] 3. Cytotoxicity Assay The cytotoxicity of capsaicin dimer was evaluated by MTT method. First, HaCaT cells were passaged and inoculated in sterile culture dishes and cultured in an incubator at 37°C, saturated humidity, and 5% CO2. 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 cultured for 24 hours. Subsequently, the cells were transferred to a 96-well adherent plate, and 10 μL of MTT reagent (dissolved in DMEM) was added to each well. After 4 hours of continuous culture, 100 μL of DMSO was added to each well, followed by shaking in a full-temperature shaking incubator at 37°C for 10 minutes. Finally, the optical density of each well at 510 nm was measured using a microplate reader. The results showed that capsaicin dimer had little cytotoxicity ( Figure 3 ).

[0030] 4. Determination of anti-inflammatory effect ELISA was used to detect the anti-inflammatory effects of free capsaicin and capsaicin dimer. A 24-well plate was used, and 80,000 mouse macrophages (RAW264.7 cells) were added to each well. 40 μL of Tris buffer solution was added to the blank wells; 20 μL of Tris buffer containing LPS and 20 μL of Tris solution were added to the wells where lipopolysaccharide (LPS) induced cell inflammation; 20 μL of Tris buffer containing LPS and 20 μL of CAP dispersion were added to the wells of free capsaicin (CAP); 20 μL of Tris buffer containing LPS and 20 μL of CAP-SS-CAP nps were added to the wells of capsaicin dimer nanoparticles (CAP-SS-CAP nps). Each group occupied 6 wells and was cultured for 24 h and 48 h, respectively. After culture, the anti-inflammatory effect was detected using a tumor necrosis factor TNF-α (mouse) ELISA kit. TNF-α is a multi-effect systemic inflammatory response mediator and a pro-inflammatory cytokine. The anti-inflammatory effect 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 is better than that of free capsaicin at 24 hours and 48 hours ( Figure 4 ), indicating that the prepared capsaicin dimer can maintain a longer action time and better effect.

Claims

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

2. A method for preparing the capsaicin prodrug according to claim 1, characterized in that The following steps are involved: 1) Using EDC as a dehydrating agent and DMAP as a catalyst, capsaicin and dithiodipropionic acid were subjected to a Steglich esterification reaction to generate capsaicin dimer; 2) The synthesized capsaicin dimer is dissolved in dimethyl sulfoxide for self-assembly, and the impurities and organic solvents are removed by dialysis, and then dried to obtain capsaicin dimer nanoparticles.

3. The method for preparing a capsaicin prodrug as claimed in claim 2, wherein: The Steglich esterification reaction is carried out at room temperature and in the dark.

4. The method for preparing a capsaicin prodrug as claimed in claim 2, wherein: The Steglich esterification reaction is carried out in two steps. In the first step, half of the dehydrating agent and catalyst are added and the reaction is carried out for 1-5 hours. In the second step, the remaining amount of the dehydrating agent and catalyst are added and the reaction is carried out for 20-30 hours.

5. The method for preparing the capsaicin prodrug according to claim 2, wherein: The reaction solvent of the Steglich esterification reaction is dichloromethane.

6. The method for preparing the capsaicin prodrug according to claim 2, wherein: The mass ratio of EDC to DMAP is 20:

1.

7. The method for preparing the capsaicin prodrug according to claim 2, wherein: The dialysis time is 20-30h.

8. The method for preparing the capsaicin prodrug according to claim 2, wherein: The pore size of the dialysis membrane is 1 kd.

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