Chiral ibuprofen prodrug based on mannosamine covalent modification, cell-penetrating peptide self-assembly body and preparation and application of chiral ibuprofen prodrug and cell-penetrating peptide self-assembly body

Abstract

The invention relates to a chiral ibuprofen prodrug based on mannosamine covalent modification, a cell-penetrating peptide self-assembly body and preparation and application of the chiral ibuprofen prodrug. The chiral ibuprofen prodrug is prepared by connecting a chiral drug ibuprofen and azide mannosamine Ac4ManNAz through an amido bond. Compared with the prior art, the preparation method disclosed by the invention is simple, and synthesis can be realized only through simple operation and equipment and instruments. The prepared F6P-Mibu self-assembly is small and uniform in particle size, stable in property and capable of being stored for a long time; meanwhile, the surface of the small-size drug self-assembly body carries positive charges, so that the drug self-assembly body is easy to carry drugs to penetrate through extracellular matrixes outside cartilage cells to achieve the effect of deeply treating OA, and the blank of treating OA by charged small-particle-size organic chiral small-molecule drugs is filled up. The F6P-coated Mibu self-assembled drug has a wide application prospect in the prevention and treatment of OA.

Description

Technical Field

[0001] The invention belongs to the technical field of drug structure modification and new nano drug preparation, and relates to a chiral ibuprofen prodrug based on mannosamine covalent modification, a cell-penetrating peptide self-assembly, and preparation and application thereof. Background Art

[0002] Osteoarthritis (OA) is the most common degenerative joint disease. Its occurrence is often accompanied by joint swelling, chronic pain, and even permanent sports injuries. In worse cases, it may cause deformity and disability (L. Tong, H. Yu, X. Huang, J. Shen, G. Xiao, L. Chen, H. Wang, L. Xing, D. Chen, Bone Research 2022, 10, 1-17.). Due to the aging population and the rising proportion of obese people, the number of people suffering from OA is accelerating worldwide (AR Sun, A. Udduttula, J. Li, Y. Liu, P.-G. Ren, P. Zhang, Journal of Orthopaedic Translation 2021, 26, 3-15.). According to statistics, about 300 million adults worldwide suffer from OA, especially in the elderly and obese population. However, there is currently no drug that can cure OA, which poses a huge challenge to social economy and public health. [3] . Joint diseases are often accompanied by pain, which in turn limits people's activities. Cartilage damage caused by injury, degenerative diseases or other pathological reasons usually leads to the development of osteoarthritis, and ultimately leads to progressive total joint destruction. Although current progress shows that biological agents can slow the progression of deterioration, such drugs cannot promote tissue repair. Due to the lack of blood supply to articular cartilage and its limited regeneration capacity due to its special structure, it is difficult to complete the self-repair process once damaged. What's worse, local joint damage may cause degeneration of the entire joint, seriously affecting the quality of human life. The pathogenesis of OA is still incomplete. It is currently believed to be related to multiple factors such as genetic inheritance, gender, age, weight, mechanical damage, metabolic disorders, etc. (C. Wen, G. Xiao, Journal of Orthopaedic Translation 2022, 32, A1.). Treatment methods mainly include drug therapy, physical therapy, surgical treatment, etc., but there is currently no cure in clinical practice.

[0003] Drug therapy is the basic treatment for OA and can be used to relieve pain and improve joint function. Commonly used drugs include nonsteroidal anti-inflammatory drugs (NSAIDs), acetaminophen, chondroprotectants, etc. Among them, NSAIDs are the most commonly used analgesics, but their long-term use may cause serious adverse reactions in the gastrointestinal and cardiovascular systems. Therefore, the development of new, safe and effective OA drugs is of great clinical significance.

[0004] The ibuprofen drugs on the market are mainly racemates with two mixed configurations. A large number of studies have shown that only the S-configuration ibuprofen has analgesic and anti-inflammatory effects. The use of racemates increases the dosage of the drug and the risk of drug use. Ibuprofen has a wide range of applications, and long-term clinical data suggest that oral use has an increased risk of cardiovascular disease. At the same time, there is a lack of research on the stability of the chiral configuration of ibuprofen.

[0005] Chinese patent CN112891311A discloses a low nephrotoxic ibuprofen nanoparticle and its preparation method, which uses plant-derived polymer raw materials and ibuprofen to be coated by ball milling to reduce the nephrotoxicity in the body. However, this method will produce a high temperature environment through ball milling, which has a great influence on the stability of drug molecules. The high temperature environment can flip the chiral configuration of ibuprofen in the S configuration to the invalid R configuration, reducing the efficacy. Summary of the invention

[0006] The purpose of the present invention is to provide a chiral ibuprofen prodrug based on covalent modification of mannosamine, a membrane-penetrating peptide self-assembly, and its preparation and application, so as to maintain the stability of the drug structure, achieve deep local penetration of the drug, maintain low-dose medication to reduce the systemic toxicity of the drug, etc.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] In a first aspect, the present invention provides a chiral ibuprofen prodrug based on covalent modification of mannosamine, which is prepared by connecting the chiral drug ibuprofen and azidomannosamine Ac4ManNAz through an amide bond.

[0009] In a second aspect, the present invention provides a method for preparing a chiral ibuprofen prodrug based on covalent modification of mannosamine, comprising the following steps:

[0010] (1) Dissolve azidomannosamine Ac4ManNAz in anhydrous methanol and add Molecular sieves, and continue to stir the reaction at room temperature. After the reaction is completed, the reaction solution is filtered and concentrated to obtain the deacetylated product Ac3ManNAz;

[0011] (2) The product Ac3ManNAz is dissolved in tetrahydrofuran, and a HATU coupling agent and a chiral drug ibuprofen are added to react at room temperature. After the reaction is completed, the reaction product solution is extracted, dried, filtered, concentrated, and column chromatographed to obtain the prodrug Man-ibu, which is the target product.

[0012] Furthermore, in step (1), the mass ratio of azidomannosamine Ac4ManNAz to anhydrous methanol is 1:10 to 1:1000;

[0013] Azidomannosamine Ac4ManNAz and The mass ratio of the molecular sieve is 1:2 to 1:100. In addition, preferably, the reaction concentration of the Ac4ManNAz powder in step (1) is not less than 0.5 mg / mL.

[0014] Furthermore, in step (1), the stirring reaction time is 3 to 4 hours.

[0015] Furthermore, in step (2), the molar ratio of the product Ac3ManNAz and the HATU coupling agent is 1:1 to 1:50;

[0016] The mass ratio of the product Ac3ManNAz to the chiral drug ibuprofen is (2-10): 1. In addition, preferably, the concentration of Ac3ManNAz is not less than 0.2 mg / mL.

[0017] Furthermore, the desiccant used in the drying process includes but is not limited to sodium sulfate, solid magnesium sulfate, etc.

[0018] Furthermore, in step (2), the chiral drug ibuprofen is in S configuration, R configuration or racemic form.

[0019] Furthermore, in step (2), the reaction time is 8 to 48 hours.

[0020] In addition, in the actual operation process, since the solubility of (2S)-2-(4-isobutylphenyl)propionic acid (i.e., ibuprofen) used in the present invention is poor, in order to make it dissolve better, methanol, an organic solvent with a greater polarity, is selected as the reaction solvent. The stability of the molecular sieve is further improved by using anhydrous methanol as a reaction solvent to reduce the amount of molecular sieve. In the present invention, the reaction can be kept at room temperature for 3 to 4 hours or the reaction time can be appropriately extended at a lower temperature to effectively control the progress and amount of deacetylation in the reaction. This step is simple in the operation steps of using thin layer chromatography to indicate the reaction end point, and a judgment can be made quickly whether the reaction is completed. However, due to the small number of conjugated groups in (2S)-2-(4-isobutylbenzene) propionic acid, the color development is not clear. After thin layer chromatography, iodine can be used to enhance the color development to more clearly indicate the reaction end point.

[0021] At the same time, in the present invention, after the reaction in step (1), a product Ac3ManNAz with a single acetyl group removed can be obtained with high purity by simple filtration and washing. The obtained sample may also contain products with multiple acetyl groups removed. These products do not need additional purification treatment in step (2). After further extraction and other operations, they are separated by column chromatography. This synthesis method simplifies the operation steps, reduces the number of cumbersome chromatographic separation operations, achieves the purpose of green chemical synthesis, and is conducive to industrial production.

[0022] In a third aspect, the present invention provides a cell-penetrating peptide self-assembly, which is self-assembled by any of the chiral ibuprofen prodrugs described above and the F6G6(rR)3R2 cell-penetrating peptide.

[0023] In a fourth aspect, the present invention provides a method for preparing a cell-penetrating peptide self-assembly. First, the F6G6(rR)3R2 cell-penetrating peptide and a chiral ibuprofen prodrug are dissolved in tetrahydrofuran, mixed and then added dropwise to water. The mixture is stirred at room temperature to form a stable colloidal solution. The colloidal solution is then dialyzed to obtain a F6P@Mibu self-assembly, which is the target product.

[0024] Furthermore, the dialysis process is carried out in an aqueous solution, the number of dialysis is generally not less than 5 times, and the duration of each dialysis is 3 to 6 hours.

[0025] Furthermore, the volume ratio of tetrahydrofuran to water is in the range of 1:5 to 1:50.

[0026] Furthermore, the mass ratio of F6G6(rR)3R2 and prodrug Man-ibu ranges from 3:1 to 20:1.

[0027] Furthermore, the encapsulation efficiency of Man-ibu was detected by UV-visible spectrophotometer and was 48% to 64%.

[0028] In the present invention, the usage amount and treatment process of various chemical reagents are also limited. In this experiment, the loading of Man-ibu is involved, and the polypeptide F6G6(rR)3R2 is used as the encapsulation material. Since the molecular weight of F6G6(rR)3R2 is relatively large, it has poor solubility in general low-polarity organic solvents or water. It is necessary to use an amphiphilic solvent as a solvent and fully mix it with Man-ibu and then drop it into pure water to form a self-assembled colloid. The stability of the formed colloid is related to the stirring speed. If the stirring speed is too high, it may affect the accumulation of surface charges on the polypeptide and cause rapid sedimentation, which has a greater impact on the stability of the colloid. Therefore, medium and low speed stirring is used to increase its stability.

[0029] In a fifth aspect, the present invention also provides the use of a membrane-penetrating peptide self-assembly in the preparation of a drug for inhibiting the progression of osteoarthritis and treating osteoarthritis. The drug carrier solves the problem of toxicity caused by systemic drug distribution of traditional oral NSAIDs, while maintaining the chiral drug configuration to improve the stability of ibuprofen and further enhance the drug's permeability in joints. It is an innovative OA drug design strategy with the advantages of simplicity, high efficiency, safety and targeting, and has broad application prospects in the prevention and treatment of OA.

[0030] Furthermore, the colloidal particles prepared above are small in size, with a diameter of 15 to 20 nm.

[0031] Furthermore, the colloidal solution prepared above can be co-incubated with rat chondrocytes without affecting the viability and proliferation of the cells and without obvious cytotoxicity;

[0032] Furthermore, after the colloidal solution prepared above is co-incubated with rat chondrocytes induced by inflammatory factors, the apoptosis and senescence of chondrocytes can be reduced and cell activity can be restored;

[0033] Furthermore, the colloidal particles prepared above are small in size, and can maintain drug stability after chemical modification. At the same time, they can deeply penetrate cartilage tissue due to the positive charge on their surface, thus achieving deep-level treatment.

[0034] Furthermore, the colloidal solution prepared above significantly improved the structure of knee cartilage after being injected into the knee joint of rats with knee osteoarthritis model.

[0035] Ibuprofen is a widely used NSAIDs with good analgesic and anti-inflammatory effects, but it has a racemization phenomenon, which causes its active isomer S-ibuprofen to be converted into inactive or highly toxic R-ibuprofen in vivo. In order to solve this problem, the present invention connects S-ibuprofen with azidomannosamine through an amide bond to first prepare a prodrug molecule Man-ibu. The prodrug molecule can be hydrolyzed by enzymes in vivo to release S-ibuprofen, thereby avoiding the racemization phenomenon and increasing the stability and selectivity of the drug in vivo.

[0036] At the same time, in order to improve the targeted delivery and penetration ability of drugs in joints, the present invention self-assembles Man-ibu and F6G6 (rR) 3R2 transmembrane peptide (F6P) to prepare a drug self-assembly F6P@Mibu. The self-assembly has a small and uniform particle size (about 15 to 20nm) and carries a positive charge (about +20mV). It can use electrostatic attraction to pass through the extracellular matrix of cartilage cells and enter the cartilage cells through transmembrane transport mediated by transmembrane peptides to release S-ibuprofen. The self-assembly can not only increase the concentration and bioavailability of drugs in joints, but also reduce toxic side effects on other organs and tissues.

[0037] The present invention uses azide mannosamine ibuprofen encapsulated by a transmembrane peptide (F6P@Mibu) as a drug delivery carrier, firstly forms a carbonyl oxygen bond through the dehydration condensation coupling of azide mannosamine and ibuprofen, and then forms small particles with a particle size of about 15 to 20 nm through the self-assembly of the transmembrane peptide F6G6(rR)3R2 with a positive charge. The delivery carrier is small in size and rich in positive charges on the surface, which improves the point charge of ibuprofen while protecting the chemical structure of ibuprofen from flipping the chiral center, increases the penetration depth of the drug in the cartilage layer, and also reduces the amount of the drug, thereby achieving efficient and safe knee joint cartilage drug delivery.

[0038] Compared with the traditional preparation method, firstly, the preparation method disclosed in the present invention is simpler and milder, with low requirements for equipment, and can prepare new nano drug particles with stable structure, uniform morphology and size, and positive charge on the surface without severe ball milling and other steps; secondly, the present invention uses NSAIDs ibuprofen, which has been on the market for many years, as the drug molecules for research, and has a good and rich research basis. The present invention further modifies azidomannosamine (Ac4ManNAz) at the α position on the original S-configuration drug molecule to increase steric hindrance to maintain configuration stability, reduce the amount of effective drugs used, and reduce drug toxicity; finally, the newly synthesized mannosamine-ibuprofen molecule is coated with the transmembrane peptide F6G6 (rR) 3R2 (F6P), further changing the electrical properties of the particle surface, so that it presents a higher positive charge, further increasing the stability of the drug particles, while enhancing the permeability of the drug molecules in cartilage tissue, and achieving the effect of deep treatment.

[0039] In general, the present invention has the following advantages:

[0040] (1) The nanomedicine preparation method is simple, has short operation steps, and has low equipment dependence;

[0041] (2) The prepared nanomedicine has good biosafety;

[0042] (3) The prepared nanomedicine protects the chiral drug configuration and avoids drug configuration reversal;

[0043] (4) The prepared nanomedicine has a small size and can deeply penetrate cartilage to achieve deep treatment of osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 and Figure 2 It is a schematic diagram of the two-step synthesis method of azidomannosamine ibuprofen (Man-ibu);

[0045] Figure 3 is the NMR spectrum of Man-ibu;

[0046] Figure 4 is the morphology characterization diagram of F6P@Mibu particles;

[0047] Figure 5 This is the cell biosafety assessment diagram of F6P@Mibu;

[0048] Figure 6 This is a graph evaluating the effect of F6P@Mibu in treating osteoarthritis in rats. DETAILED DESCRIPTION

[0049] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0050] In the following examples, (2S)-2-(4-isobutylphenyl)propionic acid (S-ibuprofen) was purchased from Tokyo Chemical Industry Co., Ltd. (Cat. No. 100126035); F6G6(rR)3R2 was purchased from Shanghai Qiangyao Biotechnology Co., Ltd.

[0051] Unless otherwise specified or used in conjunction with special processing techniques, the remaining reagents are conventional commercially available reagents or conventional processing techniques in the art.

[0052] Embodiment 1:

[0053] Chemical synthesis of azidomannosamine ibuprofen (Man-ibu), see Figure 1 As shown: The synthesis of Man-ibu mainly includes the hydrolysis of azidomannosamine (Ac4ManNAz) and the esterification reaction of S-ibuprofen. The specific operation steps are as follows: weigh 22 mg of Ac4ManNAz and fully dissolve it in 5 mL of anhydrous methanol, then add 137 mg of Molecular sieves, react at room temperature for 3-4 hours, and monitor the end point with thin layer chromatography. After filtration, washing, and further concentration, 13 mg of the deacetylated product Ac3ManNAz was obtained.

[0054] Add 13 mg HATU peptide coupling reagent, 7.0 mg S-ibuprofen and 7 mL tetrahydrofuran solution to Ac3ManAz, react at room temperature for 24 hours, extract with dichloromethane / water system and concentrate to dryness. The viscous liquid is chromatographed on a silica gel column to obtain 11.4 mg Man-ibu powder. Figure 3 , and its structure was confirmed by nuclear magnetic resonance (NMR) spectrum.

[0055] Embodiment 2:

[0056] The specific steps for preparing F6P@Mibu nanoparticles are as follows: take 5 mg of F6G6(rR)3R2 transmembrane peptide and dissolve 4 mg of Man-ibu solid prepared in Example 1 in 200 μL of tetrahydrofuran solution, mix thoroughly, add the mixed solution dropwise into 10 mL of double distilled water, stir at room temperature for about 1.5 h, and rotate at 200 rpm to form a stable colloid, then dialyze in an aqueous solution with a dialysis bag with a molecular weight of 300 KDa for 48 h to remove residual solvent and unassembled raw materials, replace the dialysate every 4 h or so, and finally obtain a F6P@Mibu colloidal solution.

[0057] Figure 4 This is the TEM image of the prepared F6P@Mibu nanoparticles. Figure 4 (Scale bar is 200 nm) It can be seen that F6P@Mibu is round particles with good dispersibility, uniform particle size, narrow distribution, and a particle size of about 15 to 20 nm.

[0058] Embodiment 3:

[0059] 2 × 10 3 Rat chondrocytes (n=5) were planted at a concentration of 1 μM, 10 μM, and 100 μM of S-ibuprofen (lbuprofen) and F6P@Mibu (prepared in Example 2) in a complete culture medium solution and co-incubated with the chondrocytes in the 96-well plate. The cells cultured in the complete culture medium supplemented with PBS were used as the control group, and the cells were co-incubated for 24 hours. After 24 hours, the culture medium was removed, and the chondrocytes were incubated with a culture medium containing 10% CCK-8 for 2 to 4 hours, and the cell activity was measured on an ELISA instrument (wavelength 496 nm).

[0060] Figure 5 This is the result of the cell activity test. The figure shows that both high and low concentrations of F6P@Mibu and Ibuprofen have good biosafety. Compared with the blank control group, they have no obvious toxicity to rat chondrocytes, and their vitality and proliferation are not affected.

[0061] Embodiment 4:

[0062] To evaluate the therapeutic effect of F6P@Mibu on osteoarthritis model mice.

[0063] SD rats weighing between 260g and 280g were purchased, with 5 rats in each group. The rats were randomly divided into a control group and an experimental group. The rats were anesthetized with Shutai-50 at a ratio of 50mg / kg. After anesthesia, the rats were skinned and disinfected, and 6μL type VII collagenase (10μg / mL) was injected into the knee joint cavity of the hind limb of the rat with a microinjection solution. The treated SD rats were treated 10 to 14 days after surgery. 30μL 500μM Ibuprofen (S-ibuprofen) and F6P@Mibu were injected into the knee joint cavity of the rats, respectively, and PBS was injected into the control group. After 2 weeks, 4 weeks and 6 weeks of observation, the knee joint tissues of the rats were collected in combination with histological evaluation, and histological staining was performed to evaluate the degree of cartilage damage.

[0064] Figure 6 This is a pathological section of the joints of SD rats with osteoarthritis after treatment. The results show that the effect of F6P@Mibu treatment is more significant, indicating that the cartilage joint surface is smooth after treatment, the cartilage layer structure is not damaged by inflammatory factors, and the progression of osteoarthritis is greatly delayed.

[0065] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A chiral ibuprofen prodrug based on covalent modification of mannosamine, characterized in that: It is prepared by connecting the chiral drug ibuprofen and azidomannosamine Ac4ManNAz through an amide bond.

2. The method for preparing a chiral ibuprofen prodrug based on covalent modification of mannosamine according to claim 1, characterized in that: The following steps are involved: (1) Dissolve azidomannosamine Ac4ManNAz in anhydrous methanol and add Molecular sieves, and continue to stir the reaction at room temperature. After the reaction is completed, the reaction solution is filtered and concentrated to obtain the deacetylated product Ac3ManNAz; (2) The product Ac3ManNAz is dissolved in tetrahydrofuran, and a HATU coupling agent and a chiral drug ibuprofen are added to react at room temperature. After the reaction is completed, the reaction product solution is extracted, dried, filtered, concentrated, and column chromatographed to obtain the prodrug Man-ibu, which is the target product.

3. The method for preparing a chiral ibuprofen prodrug based on covalent modification of mannosamine according to claim 2, characterized in that: In step (1), the mass ratio of azidomannosamine Ac4ManNAz to anhydrous methanol is 1:10 to 1:1000; Azidomannosamine Ac4ManNAz and The mass ratio of the molecular sieve is 1:2 to 1:

100.

4. The method for preparing a chiral ibuprofen prodrug based on covalent modification of mannosamine according to claim 2, characterized in that: In step (1), the stirring reaction time is 3 to 4 hours.

5. The method for preparing a chiral ibuprofen prodrug based on covalent modification of mannosamine according to claim 2, characterized in that: In step (2), the molar ratio of the product Ac3ManNAz and the HATU coupling agent is 1:1 to 1:50; The mass ratio of the product Ac3ManNAz to the chiral drug ibuprofen is (2-10):

1.

6. The method for preparing a chiral ibuprofen prodrug based on covalent modification of mannosamine according to claim 2, characterized in that: In step (2), the chiral drug ibuprofen is in S configuration, R configuration or racemic form.

7. The method for preparing a chiral ibuprofen prodrug based on covalent modification of mannosamine according to claim 2, characterized in that: In step (2), the reaction time is 8 to 48 hours.

8. A membrane-penetrating peptide self-assembly, characterized in that: The chiral ibuprofen prodrug is self-assembled by the chiral ibuprofen prodrug as claimed in claim 1 and the F6G6(rR)3R2 membrane-penetrating peptide.

9. The method for preparing a cell-penetrating peptide self-assembly according to claim 8, characterized in that: First, the F6G6(rR)3R2 membrane-penetrating peptide and the chiral ibuprofen prodrug are dissolved in tetrahydrofuran at a mass ratio of 3:1 to 20:1, mixed and added dropwise to water, stirred at room temperature to form a stable colloidal solution, and then the colloidal solution is dialyzed to obtain the F6P@Mibu self-assembly, which is the target product.

10. Use of the cell-penetrating peptide self-assembly according to claim 8 in the preparation of a drug for inhibiting the progression of osteoarthritis and treating osteoarthritis.

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