Metabolic enzyme-based related flavonoid prodrug nanoparticles as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of new dosage forms of pharmaceutical preparations and targeted delivery, and relates to a composite preparation of nanoparticles and inulin gel based on metabolic enzyme related icaritin prodrugs or pharmaceutically acceptable salts thereof, and a preparation method and application thereof. According to the invention, an icaritin carbamate prodrug or a pharmaceutically acceptable salt thereof is prepared into PLGA nanoparticles to improve the solubility of the icaritin carbamate prodrug, and the nanoparticles are embedded into inulin gel to construct an oral colon-targeted drug delivery system. And a new strategy and choice are provided for delivery of insoluble drugs and efficient treatment of inflammatory bowel diseases and complications thereof.

Description

Technical Field

[0001] This invention belongs to the field of novel drug formulations and targeted delivery technology, specifically relating to a nanoparticle inulin gel composite formulation based on metabolic enzyme-related icariin carbamate prodrug with colon-targeting and sustained-release functions, its preparation method, and its application in the treatment of inflammatory bowel disease and secondary liver injury. Background Technology

[0002] Inflammatory bowel disease (IBD), primarily including ulcerative colitis (UC) and Crohn's disease (CD), is a group of chronic, nonspecific inflammatory bowel diseases with unknown etiologies. IBD is a protracted and recurring condition, often accompanied by various extraintestinal complications in addition to intestinal symptoms, among which secondary liver injury is particularly common, severely impacting patients' quality of life and prognosis. Current clinical treatment mainly relies on small-molecule anti-inflammatory drugs and immunosuppressants; however, these drugs have drawbacks such as significant systemic side effects and poor intestinal targeting, making precise administration to the site of intestinal inflammation difficult, and their intervention effect on secondary liver injury is limited. Icaritin, an active flavonoid compound extracted from the traditional Chinese medicine Epimedium, has been shown by numerous studies to possess multiple pharmacological effects, including anti-inflammatory, immunomodulatory, antioxidant, and hepatocyte-protective properties, demonstrating potential therapeutic value for IBD and related liver injury. However, icaritin suffers from poor metabolic stability, low water solubility, and low oral bioavailability, limiting its clinical application. Although the inventors previously prepared it into a carbamate prodrug (patent publication number CN119841798A) to improve metabolic stability and solve the key problem of phase II metabolic inactivation in the colon and liver, the prodrug still has problems such as insufficient solubility and limited in vivo absorption. Moreover, the diarrhea symptoms of IBD patients will further accelerate the emptying of the drug in the colon, resulting in the local drug concentration failing to meet the treatment requirements. To address the issues of insufficient solubility and difficulty in effective drug accumulation at the lesion site in the context of colitis associated with icariin prodrug therapy, the inventors propose a solution: encapsulating the icariin prodrug in biodegradable PLGA nanoparticles to enhance its solubility, and further embedding the nanoparticles in inulin gel (IG). Utilizing the specific degradation of IG by the colonic flora, the drug is protected from destruction by gastrointestinal digestive juices and enzymes in the upper digestive tract, exhibiting good colonic targeting and adhesion. This solves the problem of rapid drug emptying caused by diarrhea in IBD patients, improving drug accumulation and release in the colon. Simultaneously, the absorbed drug can act on the liver, thereby achieving highly effective treatment for colitis and secondary liver damage. Summary of the Invention

[0003] The technical problem solved by this invention is to overcome the defects of poor metabolic stability and insufficient solubility of flavonoids, improve their colon-targeted delivery efficiency and increase accumulation in the colon, and design a composite inulin gel with good biosafety and long-acting drug release.

[0004] The primary objective of this invention is to provide a nanoparticle inulin gel composite formulation of icariin prodrug. This formulation effectively improves the solubility of the icariin prodrug, enabling colon-targeted delivery, prolonging its retention time at the lesion site in the colon, and increasing drug absorption in vivo. Furthermore, it has applications in the treatment of inflammatory bowel disease and secondary liver injury, achieving simultaneous treatment of the intestine and liver, and providing a new strategy for the comprehensive treatment of IBD.

[0005] The present invention achieves the above objectives through the following technical solutions: In a first aspect, the present invention provides flavonoid prodrug nanoparticles based on metabolic enzymes, wherein the flavonoid prodrug nanoparticles are PLGA nanoparticles of icariin carbamate prodrug or a pharmaceutically acceptable salt thereof.

[0006] The icariin carbamate prodrug or its pharmaceutically acceptable salt is a known compound with the following structural formula, and the synthesis method is described in Chinese Patent Publication CN119841798A.

[0007] The PLGA nanoparticles are prepared by emulsifying and solvent evaporation of the above-mentioned icariin carbamate prodrug or its pharmaceutically acceptable salt with PLGA material to increase drug solubility.

[0008] Preferably, the mass concentration of the above-mentioned icariin carbamate prodrug or its pharmaceutically acceptable salt is 2-5 mg / mL; the mass concentration of the PLGA material is 45-60 mg / mL. Accurately weigh the PLGA material and the icariin prodrug or its pharmaceutically acceptable salt, and dissolve them in 1 mL of a mixed solvent of tetrahydrofuran and dichloromethane in a 1:1 volume ratio (organic phase); then slowly add the organic phase to an aqueous phase containing 1% PVA solution, with a volume ratio of organic phase to aqueous phase of 10:1; sonicate at 210 W for 5 min under ice bath conditions. After sonication, remove the organic solvent by rotary evaporation to obtain a homogeneous and transparent nano-formulation.

[0009] In a second aspect, the present invention provides a metabolic enzyme-related flavonoid prodrug nanoparticle-inulin gel system, the gel system comprising PLGA nanoparticles of the above-mentioned icariin carbamate prodrug or a pharmaceutically acceptable salt thereof and inulin.

[0010] Preferably, the mass of the PLGA nanoparticles of the above-mentioned icariin carbamate prodrug or its pharmaceutically acceptable salt is 1.6 mg, and the mass of inulin is 400-600 mg.

[0011] Furthermore, the PLGA nanoparticles of the icariin carbamate prodrug or its pharmaceutically acceptable salt are physically mixed with inulin through a heating and cooling process to form a nanoparticle-inulin gel system.

[0012] Furthermore, inulin was fully dissolved in an appropriate amount of pure water, heated at 70 °C for 5 min, and rapidly cooled to room temperature in ice water. The prepared PLGA nanoparticles of icariin carbamate prodrug or its pharmaceutically acceptable salt were freeze-dried and reconstituted with a small amount of pure water. They were then added to the above inulin solution and rapidly mixed to a final volume of 1 mL. The nanoparticle-inulin gel system was prepared by continuously shaking and cooling to a gel state.

[0013] In a third aspect, the present invention also provides the use of the above-described flavonoid prodrugs based on metabolic enzymes or their PLGA nanoparticles or gel systems in the preparation of delivery carriers for poorly soluble flavonoid compounds.

[0014] Preferably, the poorly soluble flavonoid delivery carrier is used for oral administration.

[0015] In a fourth aspect, the present invention also provides the use of the above-described flavonoid prodrug based on metabolic enzymes or its PLGA nanoparticle or gel system in the preparation of intestinal targeted drug delivery formulations.

[0016] In a fifth aspect, the present invention also provides the use of the above-described flavonoid prodrug based on metabolic enzymes or its PLGA nanoparticle or gel system in the preparation of a medicament for treating inflammatory bowel disease.

[0017] Alternatively, the drug may be used for ulcerative colitis or Crohn's disease.

[0018] In a sixth aspect, the present invention also provides the use of the above-described flavonoid prodrug based on metabolic enzymes or its PLGA nanoparticle or gel system in the preparation of medicaments for treating inflammatory bowel disease and extraintestinal complications.

[0019] Furthermore, the parenteral complications include secondary liver injury.

[0020] The present invention has the following beneficial effects: By preparing icariin into a carbamate prodrug, phase II metabolic reactions in the colon and liver can be avoided, increasing metabolic stability. Preparing the prodrug into PLGA nanoparticles can significantly improve drug solubility. Introducing inulin gel utilizes its properties of not being decomposed by digestive enzymes in the stomach and small intestine and being specifically degraded by intestinal flora in the colon to achieve colon-targeted delivery. At the same time, the bioadhesiveness of inulin gel can prolong the local retention time of the drug in the colon, continuously releasing the drug, and can also address the problem of accelerated drug emptying caused by diarrhea in IBD patients, increasing drug accumulation in the colon and increasing in vivo absorption. The absorbed drug can then directly act on the liver, achieving synergistic treatment of inflammatory bowel disease and its secondary liver damage. Attached Figure Description

[0021] Figure 1 This is a transmission electron microscope image of PLGA nanoparticles, the prodrug of epimedium, in Example 1 of the present invention.

[0022] Figure 2 This image shows the in vitro stability of PLGA nanoparticles, the prodrug of epimedium, in Example 1 of this invention.

[0023] Figure 3 The image shows the appearance and scanning electron microscope (SEM) of the blank inulin gel and the icariin prodrug nanoparticle inulin gel system in Example 2 of this invention.

[0024] Figure 4 These are rheological images of the blank inulin gel and the icariin prodrug nanoparticle inulin gel system in Example 2 of this invention.

[0025] Figure 5 This is a gastrointestinal stability diagram of the blank inulin gel and the icariin prodrug nanoparticle inulin gel system in Example 3 of the present invention.

[0026] Figure 6 The figure shows the tissue distribution of the inulin composite gel in Example 4 of the present invention, and the figure also shows the fluorescence imaging of the distribution of each group of preparations in the gastrointestinal tract.

[0027] Figure 7 This is a semi-quantitative fluorescence diagram of the tissue distribution of the inulin composite gel in Example 4 of the present invention.

[0028] Figure 8 Images showing the stability of the prodrug and its nanoparticles in colon tissue homogenate and plasma in Example 5 of this invention.

[0029] Figure 9 This image shows the phase II metabolic stability of the prodrug and its nanoparticles in colon tissue microsomes in Example 6 of the present invention.

[0030] Figure 10 This is a blood drug concentration-time curve from Example 7 of the present invention.

[0031] Figure 11 This is an experimental diagram illustrating the pharmacodynamic effects of the compound inulin gel in Example 8 of this invention on colitis and secondary liver injury in mice. A: Images of the colon of mice in different treatment groups; B: Experimental groups; C: Weight changes of mice in different treatment groups during the experiment; D: DAI scores of mice in different treatment groups; E: Quantitative diagrams of colon length in different groups of mice.

[0032] Figure 12 The values ​​represent the serum levels of colitis-related factors in mice during the efficacy experiment of the compound inulin gel in Example 8 of this invention on colitis and secondary liver injury. Specifically, A represents the serum level of interleukin-1-β in mice; B represents the serum level of tumor necrosis factor-α in mice; C represents the serum level of interleukin-6 in mice; and D represents the serum level of myeloperoxidase in mice.

[0033] Figure 13 These are liver indicators of mice in different treatment groups during the efficacy experiment of the composite gel in Example 8 of this invention on colitis and secondary liver injury in mice. Wherein A: liver coefficient of mice in different treatment groups; B: level of alanine aminotransferase (ALT) in mouse serum; C: level of aspartate aminotransferase (AST) in mouse serum; D: level of lipopolysaccharide (LPS) in mouse serum. Detailed Implementation

[0034] The following specific examples further illustrate the above-mentioned content of the present invention in detail, but do not imply that the embodiments limit the present invention.

[0035] Example 1: Preparation of PLGA nanoparticles containing icariin prodrug 55 mg of PLGA and 4 mg of icariin prodrug 3N-Me were accurately weighed and dissolved in 1 mL of a 1:1 mixture of tetrahydrofuran and dichloromethane (organic phase). The organic phase was then slowly added to an aqueous phase containing 1% PVA solution, with a volume ratio of organic to aqueous phase of 10:1. The mixture was sonicated at 210 W for 5 min under ice bath conditions. After sonication, the organic solvent was removed by rotary evaporation to obtain uniform and transparent nano-formulation 3N-Me NPs.

[0036] The results are shown in Table 1. The particle size of the nanoparticles is 162.1 nm, and the encapsulation efficiency is about 90%. Figure 1 Transmission electron microscopy results show that 3N-Me NPs have a spherical particle structure. Figure 2 The results showed that the particle size of 3N-Me NPs remained almost unchanged after being placed at 4℃ for one week, indicating that 3N-Me NPs have good stability.

[0037] Table 1: Particle size and encapsulation efficiency of PLGA nanoparticles containing icariin prodrug Example 2: Preparation of an inulin-based nanoparticle gel system for icariin prodrug The prepared 3N-Me NPs were freeze-dried and reconstituted in a small amount of pure water. 500 mg of inulin was fully dissolved in an appropriate amount of pure water, heated at 70 °C for 5 min, and rapidly cooled to room temperature in ice water. The 3N-Me NPs prepared above were added to the inulin solution and rapidly mixed to a final volume of 1 mL, wherein the concentration of 3N-Me NPs was 1.6 mg / mL. IG@3N-Me NPs were then prepared by continuously shaking and cooling to a gel state.

[0038] like Figure 3 As shown, compared to the blank inulin gel, the drug-loaded inulin gel exhibits a pale yellow color, and both possess typical porous hydrogel structures and excellent injectability; furthermore, the IG@3N-Me NPs remained at the bottom of the vial after inversion, demonstrating good adhesion of the formulation. Figure 4 As shown, the storage modulus G' of both the blank inulin gel and the drug-loaded inulin gel exceeded the loss modulus G'', indicating that moderate drug incorporation does not affect the hydrogel properties of inulin.

[0039] Example 3: Gastrointestinal stability experiment of the icariin prodrug nanoparticle inulin gel system A mouse colitis model was established by dissolving sodium dextran sulfate (DSS) at a concentration of 4% in water and allowing C57BL / 6 mice to freely drink the 4% DSS solution for 6 days. Mice were fasted for 12 hours before being orally administered 0.4 mL of IG@3N-Me NPs. Gastric, small intestinal, and colonic contents were collected at 0.5 h, 4 h, and 6 h, respectively. The gastrointestinal contents were freeze-dried, and the morphology of the formulation was observed using scanning electron microscopy.

[0040] like Figure 5 As shown, in the stomach and small intestine, the intestinal contents exhibited a similar morphology to the blank inulin, indicating that the inulin gel was not degraded and remained stable in the upper gastrointestinal formulation. Electron microscopy of the colonic contents revealed some exposed spherical nanoparticles from the inulin gel. These gastrointestinal stability results demonstrate that the inulin gel can protect 3N-Me NPs from the acidic environment of the stomach and gradually degrades in the colon, thus playing a colon-targeting role.

[0041] Example 4: Tissue distribution assay of icariin prodrug nanoparticle inulin gel system Mice with a colitis model were randomly divided into two groups of nine each, and fasted for 12 hours before administration. Mice were administered 3N-MeNPs@Dio and IG@3N-MeNPs@Dio by gavage, respectively, at a dose of 27.71 mg / kg / day (calculated as icariin). Three mice from each group were sacrificed by cervical dislocation at 4, 8, and 12 hours. The gastrointestinal tract, heart, liver, spleen, lungs, and kidneys were collected and placed in a small animal in vivo imaging system to observe the distribution of prodrug nanoparticles in the isolated tissues.

[0042] like Figure 6 The gastrointestinal distribution results showed that at 4 h, the fluorescence signals of 3N-Me NPs@Dio and IG@3N-Me NPs@Dio were mainly distributed in the small intestine. At 8 h and 12 h, compared with the control group, IG@3N-Me NPs@Dio still showed strong fluorescence signal intensity in the cecum and colon. Figure 7 As shown, IG@3N-Me NPs@Dio still exhibited a strong fluorescence signal at 12 hours. These results indicate that inulin gel coating can prolong drug retention time in the colon and increase drug accumulation there.

[0043] Example 5: Biomedia stability test of prodrug nanoparticles Preparation of colonic homogenate: For the colitis model mice, fasting was allowed for 12 hours prior to the experiment, but free access to water was permitted. Blood was collected from the mice, and they were euthanized. Colonic tissue was quickly extracted, washed with ice-cold PBS (4°C), and adipose tissue was removed. Excess water was wiped off with filter paper, and the tissue was weighed. The homogenate was then carefully ground with ice-cold PBS (4°C) at a 1:4 (w / v) ratio using high-speed shear homogenization (ice bath). After centrifugation at 12000×g for 20 min (4°C), the supernatant was collected to obtain a 20% colonic tissue homogenate, which was stored at -80°C for later use.

[0044] Plasma preparation: Mice with colitis model were fasted for 12 hours prior to the experiment, but allowed free access to water. Fresh whole blood was collected from the mice and plasma was pumped at 13000 r·min. -1 Centrifuge for 5 minutes, collect the upper layer of blank plasma, and store at -80℃ for later use.

[0045] Accurately measure 2.0 mL of colon tissue homogenate and plasma, preheat in a 37℃ water bath shaker for 5 min, and add 3N-Me or 3N-Me NPs to bring the final drug concentration to 10 μM. Using the classic isothermal method, 150 μL samples of colon tissue homogenate and plasma were taken at different time points: 0, 2, 4, 8, 12 h and 0, 5, 15, 30, 45, 60, 120 min. 150 μL of glacial acetonitrile (containing 0.3% formic acid) was added, and the mixture was immediately vortexed at 13000 r·min. -1Centrifuge for 10 min, accurately measure 50 μL of the supernatant and inject it for analysis, record the peak area, and calculate the concentration of the remaining prodrug in the medium at different time points using the standard curve method.

[0046] Chromatographic conditions: Chromatographic column: Silversil column (4.6 × 250 mm, 5 μm) Mobile phase composition: Acetonitrile: Water (containing 0.1% phosphoric acid) Flow rate: 1.0 mL / min Detection wavelength: 270 nm Column temperature: 30℃ Injection volume: 50 μL gradient: like Figure 8 As shown, after 12 hours of incubation in colon tissue homogenate, approximately 23% of the prodrug nanoparticles were hydrolyzed into the parent drug, and after 2 hours of incubation in plasma, approximately 88% were hydrolyzed into the parent drug, suggesting that plasma may be the main site of 3N-Me activation.

[0047] Example 6: Phase II metabolic stability test of prodrug nanoparticles in colon tissue microsomes Preparation of homogenate: Weigh 85.58 g (0.25 mol) sucrose, 1.21 g (10 mmol) tris(hydroxymethyl)aminomethane (Tris) and 0.34 g (1 mmol) disodium ethylenediaminetetraacetate (EDTA-2Na) and place them in the same beaker. Add an appropriate amount of purified water and sonicate to dissolve. Add purified water to a final volume of about 1000 mL. Adjust the pH of the solution to 7.4 with hydrochloric acid and store at 4°C for later use.

[0048] Preparation of resuspension: Weigh 0.34 g (1 mmol) EDTA-2Na, 12.11 g (0.1 mol) Tris and 15.4 mg (0.1 mmol) dithiothreitol into the same beaker, add an appropriate amount of purified water and sonicate to dissolve, add purified water to about 800 mL, add hydrochloric acid to adjust the pH of the solution to 7.4, add 200 mL of glycerol, mix well and store in a refrigerator at 4℃ for later use.

[0049] Preparation of 0.1 mol / L Tris-HCl buffer: Weigh 6.06 g (50 mmol) of Tris into a beaker, add an appropriate amount of purified water and sonicate to dissolve, add purified water to about 500 mL, add an appropriate amount of hydrochloric acid to adjust the pH of the solution to 7.4, and store in a refrigerator at 4℃ for later use.

[0050] Preparation of colon tissue microsomes: For the colitis model mice, fasting was allowed for 12 hours prior to the experiment, but free access to water was permitted. Blood was collected from the mice, and they were euthanized. Colon tissue was quickly extracted, washed with ice-cold PBS (4℃), and adipose tissue was removed. Excess water was wiped off with filter paper, and the tissue was chopped, weighed, and added to the homogenate at a 1:4 (w / v) ratio. The homogenate was carefully homogenized using a tissue homogenizer under ice bath conditions to prepare a 20% colon tissue homogenate. The homogenate was centrifuged at 12000×g (4℃) for 20 min. The supernatant was transferred to an ultracentrifuge tube and centrifuged at 100000×g (4℃) for 60 min. The resulting precipitate was the colon tissue microsomes. The colon microsomes were resuspended in a resuspending solution at a specific ratio, and the protein content was determined using a BCA kit. The colon microsome suspension was diluted to an appropriate concentration with the resuspending solution, aliquoted, and stored at -80℃ for later use.

[0051] Triton, MgCl2, colonic microsomes, and the test drug were prepared to the following suitable concentrations using 0.1 mol / L Tris-HCl buffer: final concentration of triton 0.01%, final concentration of MgCl2 10 mmol / L, final concentration of colonic microsomal protein 50 μg / mL, and final concentration of icariin, 3N-Me, or 3N-Me NPs 10 μM. The incubation buffer was adjusted to 900 μL using the above 0.1 mol / L Tris-HCl buffer and incubated in a 37°C water bath with a shaker for 5 min. Then, 100 µL of trisodium 5′-uridine diphosphate glucuronide solution (final concentration 1 mmol / L) was added to initiate the glucuronidation reaction. The final total volume of the incubation system was 1 mL, and it was incubated in a 37°C water bath with a shaker for 60 min. At different time points, 150 μL samples were taken from the incubation system and added to 150 μL of glacial acetonitrile (containing 0.3% formic acid), vortexed at 13000 r·min. -1 Centrifuge for 10 min, accurately measure 50 μL of supernatant for analysis, record the peak area, and calculate the concentration of residual icariin or prodrug in the buffer medium at different time points using the standard curve method.

[0052] like Figure 9 As shown, with prolonged incubation time, icariin was rapidly metabolized, while 3N-Me and 3N-Me NPs were metabolized slowly. After 60 min of incubation, 11.09% of icariin, 57.91% of 3N-Me, and 65.06% of 3N-Me NPs remained. This demonstrates that the prodrug and its nanoparticles significantly improved the phase II metabolic stability of icariin in colonic tissue, and that the nanoparticles may have a certain protective effect on the prodrug.

[0053] Example 7: Pharmacokinetic Study of Oral Icariin Prodrug Nanoparticle Inulin Gel System Healthy male SD rats were randomly divided into four groups (n=4): ICT group, 3N-Me group, 3N-Me NPs group, and IG@3N-Me NPs group. Rats were administered a single oral dose of 27.71 mg / kg (based on icariin). The formulation for the ICT group consisted of 50% PEG400, 40% Tween 80, and 10% water. Blood samples were collected from rats at 0.083, 0.17, 0.33, 0.5, 0.75, 1, 2, 4, 8, 12, and 24 h post-administration and centrifuged in heparin-coated anticoagulant tubes at 13000 r·min⁻¹. -1 Centrifuge for 5 min, separate 100 μL of plasma and place it in another EP tube (pre-added with 0.5 μL of glacial acetic acid), vortex and immediately store at -80℃ for later use.

[0054] Accurately measure 50 μL of rat plasma sample (containing 0.5% glacial acetic acid) into an EP tube, add 5 μL of acetonitrile (containing 0.3% formic acid) (equivalent to the volume of the working standard solution), 5 μL of IS (daidzein) solution, and 50 μL of purified water. Vortex for 1 min, add 1 mL of extraction reagent methyl tert-butyl ether, vortex for 3 min, centrifuge at 13000 r / min for 10 min, collect the supernatant, and dry under a nitrogen stream. Redissolve the residue in 50 μL of acetonitrile (containing 0.3% formic acid), vortex, and centrifuge at 13000 r·min. -1 Centrifuge for 10 min, obtain the supernatant and inject it for analysis. Determine the concentration of epimedium and prodrug in plasma samples of SD rats at various time points. Process the blood drug concentration data using DAS2.0 pharmacokinetic software and calculate various pharmacokinetic parameters.

[0055] Chromatographic conditions: Column: ACE Excel 2 C 18 -Amide column (50 mm × 2.1 mm, 2 μm) Mobile phase composition: Acetonitrile: Water (containing 0.3% formic acid) Flow rate: 0.2 mL / min Column temperature: 40℃ Injection volume: 10 μL gradient: Mass spectrometry conditions: Ion source: Electrospray ionization source with positive ion detection scanning (ESI) +Scanning mode: Multiple reaction monitoring (MRM); Capillary voltage: 3.5 kV; Cone voltage: 40 V; Ion source temperature: 120℃; Interface temperature: 300℃; DL temperature: 280℃; Heating block temperature: 400℃; Desolventizing temperature: 450℃; Desolventizing gas flow rate: 700 L / h; Nebulizer flow rate: 3.0 L / min; Cone gas flow rate: 50 L / h; Heating gas flow rate: 10.0 L / min; Drying gas flow rate: 10.0 L / min; Collision energy (icariin): 25 V, Collision energy (3N-Me): 27 V, Collision energy (DAN): 28 V. Quantitative ion (icariin) m / z 369.2 → 313.2, (3N-Me) m / z 426.2 → 338.8, (IS) m / z 255.1 → 181.1.

[0056] like Figure 10 The plasma concentration-time curves and Tables 2 and 3 show the plasma concentrations of icariin in rats after oral administration of icariin raw material, 3N-Me, 3N-Me NPs, and IG@3N-Me NPs, respectively. C max The concentrations were 5.025±2.05, 11.588±1.966, 12.478±3.717, and 22.758±6.766 nmol / L, respectively. AUC 0-t The values ​​were 15.099±7.228, 60.298±33.274, 90.828±23.021, and 299.357±72.926, respectively. In SD rats, using icariin as a control, the relative bioavailability of icariin in the 3N-Me group, 3N-Me NPs group, and IG@3N-Me NPs group were 399.35%, 601.55%, and 1982.63%, respectively; the relative bioavailability of the prodrug and the total icariin were 907.22%, 1191.89%, and 3291.58%, respectively. These results indicate that IG@3N-Me NPs significantly improved the bioavailability of icariin in rats, suggesting its potential for direct treatment of extraintestinal complications associated with inflammatory bowel disease.

[0057] Table 2: Pharmacokinetic parameters of icariin in each group Table 3: Pharmacokinetic parameters of icariin and its prodrugs in each group Example 8: Pharmacodynamic study of an oral icariin prodrug nanoparticle inulin gel system Healthy C57BL / 6 mice were randomly divided into 7 groups (n=5): normal control group, model control group, 5-ASA group (150 mg / kg / d), IG group (10 g / kg / d), ICT group, 3N-Me NPs group, and IG@3N-Me NPs group (27.71 mg / kg / d, calculated as icariin). The ICT group was given a formulation of 50% PEG400, 40% Tween 80, and 10% water. Except for the normal control group, all mice had free access to water, while the others had free access to 4% DSS solution for 7 days to establish a mouse model of colitis and secondary liver injury. During the modeling period, the mice had free access to food, and the DSS solution was changed daily to prevent deterioration. The drug-treated groups were administered the drug by gavage daily, while the normal control group and the model group were given the same volume of physiological saline simultaneously. The mice were weighed daily during the experiment, and their living conditions and fecal characteristics were observed. On the 7th day, colon and liver tissues were collected from the mice.

[0058] Effects on colitis-related markers in mice: like Figure 11 As shown in Figure C, the overall weight change of the normal control group mice showed an upward trend. The weight of the remaining mice given the modeling agent fluctuated slightly for the first three days without a significant trend. From the fourth day onwards, the weight of the modeling mice began to decrease, with the control group experiencing the most severe weight loss. The drug-treated groups showed varying degrees of improvement in weight loss. The improvement was most significant with IG@3N-Me NPs.

[0059] like Figure 11 As shown in Figure D, the DAI scores of mice indicate that IG@3N-Me NPs showed a significant improvement compared to the DSS model group; the disease index of mice in the other groups, 5-ASA, IG, ICT and 3N-Me NPs, were relatively similar and also showed slight improvement compared to the DSS model group.

[0060] like Figure 11 A and Figure 11 As shown in Figure E, observation of the colon's appearance and measurement of the length from the lower end of the ileocecal junction to the anus, along with photographic results of the mouse intestinal segments, revealed that compared to the normal group, the model group mice had significantly shorter colons, no formed feces visible in the intestinal lumen, a thinner and shorter lumen with reduced elasticity, and dark red watery bloody stools. After drug administration, each treatment group alleviated colon shortening and bloody stools to varying degrees. The IG@3N-Me NPs group showed a significant difference, with mouse intestinal segments approaching the length of normal mice and formed feces visible in the intestinal lumen.

[0061] like Figure 12 A- Figure 12As shown in Figure C, IG@3N-Me NPs reduced the concentration of pro-inflammatory cytokines in the serum of colitis mice. While the other treatment groups also alleviated the expression of inflammatory factors to varying degrees, the differences compared to IG@3N-Me NPs were significant. Figure 12 As shown in Figure D, compared with the normal group, the MPO activity in the serum of the model control group mice was significantly increased, while the drug-treated groups reduced the MPO activity in the serum of IBD mice to varying degrees; compared with other drug-treated groups, IG@3N-Me NPs significantly reduced the MPO level in the serum of mice.

[0062] Effects on indicators related to secondary liver injury in mice: like Figure 13 As shown in Figure A, the liver coefficient of the model group mice was significantly increased compared with that of the control group. The drug-treated groups could reduce the increase in liver coefficient caused by DSS to varying degrees, with IG@3N-Me NPs showing the most significant reduction, approaching that of the normal group. Figure 13 As shown in B and C, the serum ALT and AST levels in the model group mice were significantly increased, and they were significantly reduced after treatment with IG@3N-Me NPs, showing significant differences from other treatment groups.

[0063] like Figure 13 As shown in Figure D, LPS is an endotoxin derived from Gram-negative bacteria. When the intestinal barrier function is impaired, large amounts of LPS translocate into the liver via the portal vein, directly damaging hepatocytes and triggering inflammatory responses and tissue damage. The results showed that serum LPS levels in the model control group were significantly higher than in the blank control group. Oral administration of IG@3N-Me NPs significantly reduced the increase in serum LPS induced by DSS-induced colitis. This suggests that IG@3N-Me NPs may indirectly alleviate the degree of related liver damage through highly effective treatment of colitis.

[0064] The results show that the oral icariin prodrug nanoparticle inulin gel system has good colon-targeted drug release performance, achieving precise drug release. The system exhibits good biocompatibility, effectively prolongs drug retention time in the colon, significantly improves symptoms of inflammatory bowel disease, and has a significant alleviating effect on secondary liver injury. It demonstrates comprehensive therapeutic potential in a DSS-induced colitis and related liver injury model.

Claims

1. A flavonoid prodrug nanoparticle based on metabolic enzymes, characterized in that: The flavonoid prodrug is an icariin carbamate prodrug or a pharmaceutically acceptable salt thereof, which is used to inhibit the phase II metabolic response of icariin in the colon and liver. The nanoparticles are PLGA nanoparticles of the icariin carbamate prodrug or a pharmaceutically acceptable salt thereof. The structural formula of the icariin carbamate prodrug or a pharmaceutically acceptable salt is as follows: 。 2. The flavonoid prodrug nanoparticles according to claim 1, characterized in that: The nanoparticles are prepared by emulsification solvent evaporation method using icariin carbamate prodrug or its pharmaceutically acceptable salt and PLGA material to increase drug solubility.

3. An inulin gel delivery system, characterized in that: The delivery system comprises a flavonoid prodrug or a pharmaceutically acceptable salt thereof, or its nanoparticles, prepared by physically mixing inulin with heating and cooling to form a colon-targeting gel delivery system.

4. The use of the flavonoid prodrug nanoparticles according to any one of claims 1 to 2 or the gel delivery system according to claim 3 in the preparation of delivery carriers for poorly soluble flavonoid compounds.

5. The application according to claim 4, characterized in that: The poorly soluble flavonoid delivery carrier is used for oral administration.

6. The use of the flavonoid prodrug nanoparticles of any one of claims 1 to 2 or the gel delivery system of claim 3 in the preparation of a medicament for treating intestinal diseases.

7. The application according to claim 6, characterized in that: The drug is an intestinal-targeted drug delivery formulation used to treat inflammatory bowel disease and its extraintestinal complications.

8. The application according to claim 7, characterized in that: The inflammatory bowel disease is ulcerative colitis or Crohn's disease, and the extraintestinal complications include secondary liver injury.

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