C6-phenylboronic acid-locked tripterine prodrug nanoparticles, preparation and application thereof

Abstract

The application discloses C6 phenyl borate locking tripterine prodrug nanoparticles and preparation and application, relates to the technical field of natural medicine chemistry and targeted drug preparation.The application inhibits the reverse decomposition reaction of a C6 thiol addition product from the thermodynamic level through the "locking" effect of a borate ester bond, the storage stability of the product at room temperature is improved compared with that of a traditional addition product, and the stability requirement of large-scale production and preparation processing can be met;the prodrug molecule has a hydrophobic tripterine mother nucleus and a hydrophilic polyethylene glycol segment, the amphiphilic structure can self-assemble into uniform nanomicelles in an aqueous phase, the solubility in water is improved compared with that of the tripterine mother nucleus, and the preparation forming difficulty is significantly reduced;the application has the pH / glutathione dual-response characteristics through the borate ester bond and the disulfide bond in the connecting arm, and the drug release is triggered under the condition of the weak acid environment of inflammatory skin and high concentration of glutathione, and the release amount of normal skin tissue is extremely small.

Description

Technical Field

[0001] This invention relates to the fields of natural product chemistry and targeted drug delivery formulation technology, specifically to C6 phenylboronic acid ester-locked triptolide prodrug nanoparticles and their preparation and application. Background Technology

[0002] Tripterygium wilfordii extract is an oleanane-type triterpenoid active ingredient extracted from the Celastraceae plant Tripterygium wilfordii. It possesses significant anti-inflammatory, immunosuppressive, and inhibitory effects on excessive keratinocyte proliferation pharmacological activities, showing clear therapeutic potential for inflammatory skin diseases such as psoriasis and atopic dermatitis. However, this ingredient faces three major industrialization bottlenecks:

[0003] Firstly, it has extremely poor water solubility, with a solubility of less than 10 μg / mL in water at room temperature. It is difficult to disperse evenly in conventional topical creams, resulting in low transdermal absorption efficiency and insufficient bioavailability at the lesion site.

[0004] Secondly, it has strong systemic toxicity, and its non-specific distribution can easily lead to exposure of normal tissues. Long-term topical use carries the risk of liver and kidney toxicity caused by transdermal absorption.

[0005] Third, structural modification technology is limited. Existing studies mostly use Michael addition of C6-thiol for modification. Although it can reduce toxicity to a certain extent, the chemical bonds of the addition products are highly reversible, easily decomposed at room temperature, and have poor kinetic stability. It can only be used for basic laboratory research and cannot meet the stability requirements of large-scale production and formulation processing.

[0006] Currently, most publicly available triptolide nanoformations employ physical encapsulation strategies, which suffer from low encapsulation efficiency, severe drug burst release, and lack of lesion microenvironment responsiveness. Structural modifications at the C6 position have not solved the product stability problem, and no triptolide prodrug nanodelivery system has yet been found that combines "structural stability locking, inflammatory microenvironment responsive drug release, and efficient transdermal delivery". Summary of the Invention

[0007] The purpose of this invention is to provide C6 phenylboronic acid ester-locked triptolide prodrug nanoparticles and their preparation and application, so as to solve the technical problems mentioned in the background art.

[0008] To achieve the above objectives, the present invention provides the following technical solution: C6 phenylboronic acid ester locked triptolide prodrug nanoparticles, comprising triptolide;

[0009] Using triptolide as the parent compound, a hydrophilic linker containing a catechol structure is bonded at the C6 position via a thiol-Michael addition bond.

[0010] It then forms a borate ester bond with polyethylene glycol-modified phenylboronic acid to form a locked prodrug molecule.

[0011] Furthermore, the hydrophilic linker includes reduced glutathione, L-cysteine, and reduced lipoic acid.

[0012] Furthermore, the polyethylene glycol-modified phenylboronic acid is methoxy polyethylene glycol-2-formylphenylboronic acid, and the polyethylene glycol segment has a molecular weight of 1000 Da to 5000 Da.

[0013] Preparation of C6 phenylboronic acid ester-locked triptolide prodrug nanoparticles: The preparation of C6 phenylboronic acid ester-locked triptolide prodrug nanoparticles includes the following steps:

[0014] Tripterygium wilfordii was dissolved in an anhydrous alcohol solvent, and a thiol compound and an organic base catalyst were added. The reaction was carried out under nitrogen protection and at room temperature in the dark for 12-24 hours. After purification, a C6-thiol addition intermediate was obtained.

[0015] The intermediate was dissolved in a tetrahydrofuran-water mixed solvent, and polyethylene glycol-modified 2-formylphenylboronic acid was added. The mixture was reacted at room temperature for 4–8 h, and then dialyzed and freeze-dried to obtain the target prodrug.

[0016] Furthermore, the organic base is triethylamine, and the molar ratio of the thiol compound to triptolide is 1.2~1.5:1.

[0017] Further, the prodrug was dissolved in an organic solvent, slowly added dropwise to a phosphate buffer solution and stirred to self-assemble. After removing the organic solvent by ultrafiltration, a nanoparticle dispersion was obtained.

[0018] Application of C6 phenylboronic acid ester-locked triptolide prodrug nanoparticles: C6 phenylboronic acid ester-locked triptolide prodrug nanoparticles are used to prepare topical treatment drugs for inflammatory skin diseases.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] 1. This invention inhibits the reverse decomposition reaction of C6 mercapto addition products from a thermodynamic perspective through the "locking" effect of borate ester bonds. The product has improved room temperature storage stability compared with traditional addition products, which can meet the stability requirements of large-scale production and formulation processing.

[0021] 2. The present invention utilizes a prodrug molecule that combines a hydrophobic triptolide core with a hydrophilic polyethylene glycol segment. The amphiphilic structure can self-assemble in the aqueous phase to form uniform nanomicelles, which improves the solubility in water compared to the parent compound triptolide and significantly reduces the difficulty of formulation.

[0022] 3. This invention possesses pH / glutathione dual-response characteristics through the borate ester bond and the disulfide bond in the linker arm. It triggers drug release in the weakly acidic environment of inflamed skin and under high concentration of glutathione conditions, while the release amount in normal skin tissue is extremely low.

[0023] 4. Both steps of the reaction in this invention are carried out at room temperature in the dark, using a mild aqueous / alcoholic reaction system, eliminating the need for complex high-pressure equipment, improving the overall conversion rate, simplifying the purification process, and possessing the potential for industrial production. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a particle size distribution curve of the prodrug nanoparticles of the present invention;

[0026] Figure 2 These are in vitro drug release curves under different microenvironments according to the present invention;

[0027] Figure 3 This is a bar chart comparing the PASI scores of the psoriasis model mice of this invention. Detailed Implementation

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0029] Example 1:

[0030] C6 phenylboronic acid ester-locked triptolide prodrug nanoparticles, including triptolide;

[0031] Using triptolide as the parent compound, a hydrophilic linker containing a catechol structure is bonded at the C6 position via a thiol-Michael addition bond.

[0032] It then forms a borate ester bond with polyethylene glycol-modified phenylboronic acid to form a locked prodrug molecule.

[0033] The C6 phenylboronic acid ester locks the triptolide prodrug, and its molecular structure is characterized as follows: with triptolide as the parent compound, a hydrophilic linker containing a catechol structure is bonded to the C6 position through a thiol-Michael addition reaction, and then covalently bonded to polyethylene glycol-modified phenylboronic acid through a borate bond to achieve the "locking" of the molecular structure; the hydrophilic linker is any one of reduced glutathione, L-cysteine, and reduced lipoic acid.

[0034] Benzylboronic acid is 2-formylbenzeneboronic acid modified with methoxy polyethylene glycol, and the polyethylene glycol segment has a molecular weight of 1000~5000 Da.

[0035] Example 2:

[0036] The preparation method of C6 phenylboronic acid ester-locked triptolide prodrug used in Example 1 above includes the following steps:

[0037] (1) Preparation of C6-thiol addition intermediate: Tripterygium wilfordii was dissolved in anhydrous alcohol solvent, and thiol compound and organic base catalyst were added. The reaction was carried out under nitrogen protection and at room temperature in the dark for 12-24 h. After the reaction was completed, the solvent was removed under reduced pressure and purified by column chromatography to obtain C6-thiol addition intermediate.

[0038] (2) Boron ester bond locking reaction: The above intermediate was dissolved in a tetrahydrofuran-water mixed solvent, and polyethylene glycol-modified 2-formylphenylboronic acid was added. The mixture was stirred at room temperature for 4-8 hours. The reaction solution was dialyzed to remove small molecule impurities, and then freeze-dried to obtain the phenylboronic ester-locked triptolide prodrug.

[0039] In step (1), the anhydrous alcohol solvent is anhydrous ethanol or anhydrous methanol; the organic base catalyst is triethylamine, and the amount used is 0.5 to 1 times the molar amount of triptolide; the molar ratio of thiol compound to triptolide is 1.2 to 1.5:1.

[0040] In step (2), the volume ratio of tetrahydrofuran to water is 1:1 to 2:1; dialysis is performed using a dialysis bag with a molecular weight cutoff of 3000 to 5000 Da, and the dialysis time is 24 to 36 hours.

[0041] C6 phenylboronic acid ester-locked triptolide prodrug nanoparticles are formed by the self-assembly of the above-mentioned amphiphilic prodrug molecules in an aqueous phase. The nanoparticles have a particle size of 200~250nm, a polydispersity index ≤0.2, and exhibit dual-response drug release characteristics based on pH value and glutathione.

[0042] Preparation method of prodrug nanoparticles: Dissolve triptolide prodrug in an organic solvent and slowly add it dropwise to a phosphate buffer solution under stirring. After the addition is complete, continue stirring for 30 to 60 minutes. Remove the organic solvent by ultrafiltration and centrifugation to obtain the drug-loaded nanoparticle dispersion.

[0043] The organic solvent is dimethyl sulfoxide or anhydrous ethanol; the volume ratio of prodrug solution to buffer solution is 1:10 to 1:15; ultrafiltration is performed using an ultrafiltration tube with a molecular weight cutoff of 100 kDa.

[0044] Example 3:

[0045] Based on the above embodiment one;

[0046] Applications of C6 phenylboronic acid ester-locked triptolide prodrug and prodrug nanoparticles: used to prepare topical treatment drugs for inflammatory skin diseases such as psoriasis and atopic dermatitis, and can be formulated into topical dosage forms such as nanoemulsions, gels, and creams.

[0047] In vitro responsive drug release and transdermal performance validation

[0048] See Figure 1 and Figure 2 The drug release behavior was investigated using the dialysis method: experiments were conducted in two release media: pH 5.5 + 10 mmol / L glutathione (inflammatory microenvironment) and pH 7.4 (physiological environment), with constant temperature oscillation at 37℃. The results showed that the cumulative drug release rate reached 76.3% after 12 hours in the inflammatory microenvironment, while the cumulative drug release rate was only 12.7% after 12 hours in the physiological environment, demonstrating significant microenvironment-responsive drug release characteristics.

[0049] Transdermal experiments were conducted using Franz diffusion cells on isolated mouse skin. After 12 hours, the skin retention of nanoparticles in this embodiment was 21.4 μg / cm², which is 5.1 times that of ordinary triptolide cream, indicating a significant improvement in transdermal efficiency.

[0050] Validation of drug efficacy in a psoriasis-like mouse model

[0051] See Figure 3 A BALB / c mouse psoriasis model was established by inducing imiquimod, and the mice were randomly divided into a blank control group, a matrix group, a triptolide cream group, and a nanoparticle group of the present invention, with 10 mice in each group. The mice were given topical treatment once a day for 7 consecutive days.

[0052] The results showed that on day 7 of administration, the total PASI score of mice with skin erythema, scaling, and thickening in the nanoparticle group of this invention was 1.2 points, which was significantly lower than the 3.5 points in the ordinary cream group; the expression levels of IL-17A and IL-23 inflammatory factors in the skin lesions decreased by 72% and 68%, respectively, and the anti-inflammatory effect was better than that of the commercially available positive control mometasone furoate cream; no obvious pathological damage was observed in the liver and kidney tissues of mice, and the systemic toxicity was significantly reduced.

[0053] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. C6 phenylboronic acid ester-locked triptolide prodrug nanoparticles, characterized in that: Including triptolide; Using triptolide as the parent compound, a hydrophilic linker containing a catechol structure is bonded at the C6 position via a thiol-Michael addition bond. It then forms a borate ester bond with polyethylene glycol-modified phenylboronic acid to form a locked prodrug molecule.

2. The C6 phenylboronic acid ester-locked triptolide prodrug nanoparticles according to claim 1, characterized in that: The hydrophilic linker includes reduced glutathione, L-cysteine, and reduced lipoic acid.

3. The C6 phenylboronic acid ester-locked triptolide prodrug nanoparticles according to claim 1, characterized in that: The polyethylene glycol-modified phenylboronic acid is methoxy polyethylene glycol-2-formylphenylboronic acid, and the polyethylene glycol segment has a molecular weight of 1000 Da to 5000 Da.

4. Preparation of C6 phenylboronic acid ester-locked triptolide prodrug nanoparticles, characterized in that: The preparation of C6 phenylboronic acid ester-locked triptolide prodrug nanoparticles according to any one of claims 1-3 comprises the following steps: Tripterygium wilfordii was dissolved in an anhydrous alcohol solvent, and a thiol compound and an organic base catalyst were added. The reaction was carried out under nitrogen protection and at room temperature in the dark for 12-24 hours. After purification, a C6-thiol addition intermediate was obtained. The intermediate was dissolved in a tetrahydrofuran-water mixed solvent, and polyethylene glycol-modified 2-formylphenylboronic acid was added. The mixture was reacted at room temperature for 4–8 h, and then dialyzed and freeze-dried to obtain the target prodrug.

5. The preparation of C6 phenylboronic acid ester-locked triptolide prodrug nanoparticles according to claim 4, characterized in that: The organic base is triethylamine, and the molar ratio of the thiol compound to triptolide is 1.2~1.5:

1.

6. The preparation of C6 phenylboronic acid ester-locked triptolide prodrug nanoparticles according to claim 4, characterized in that: The prodrug was dissolved in an organic solvent and slowly added dropwise to a phosphate buffer solution. The mixture was stirred and then self-assembled. After removing the organic solvent by ultrafiltration, a nanoparticle dispersion was obtained.

7. The application of C6 phenylboronic acid ester-locked triptolide prodrug nanoparticles, characterized in that: The C6 phenylboronic acid ester-locked triptolide prodrug nanoparticles as described in any one of claims 1-3 are used to prepare topical treatment drugs for inflammatory skin diseases.

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