BE1031925B1 - AN ORAL, LIVER-TARGETED NANOEMULSION FOR PIRFENIDONE LOADING, AS WELL AS ITS MANUFACTURING METHOD AND USE - Google Patents

Abstract

The present invention provides an oral, liver-targeted nanoemulsion for loading with pirfenidone, as well as a method for its preparation and use. The nanoemulsion is essentially formed by protein-polysaccharide-folic acid conjugates made from zein peptide, dextran, and folic acid, loaded with pirfenidone. Furthermore, the invention provides for the use of the nanoemulsion in the manufacture of drugs for the prevention or treatment of liver fibrosis, in particular liver fibrosis caused by TACE surgery. The nanoemulsion according to the invention exhibits good stability, high drug loading, and a simple manufacturing process. The surface-modified folic acid can promote intestinal epithelial penetration of the drug-loaded nanoemulsion, increase the accumulation of the drug-loaded nanoemulsion in the liver, and ultimately target activated hepatic stellate cells to exert an antifibrotic effect, so that pirfenidone can exhibit a higher antifibrotic effect at a lower oral dose. The protein-polysaccharide-folic acid nanoemulsion according to the invention can serve as an oral delivery platform for the antifibrotic drug pirfenidone.

Description

'BE2024/5892

AN ORAL, LIVER-TARGETED NANOEMULSION FOR LOADING WITH

Pirfenidone, as well as its manufacturing process and use

TECHNICAL AREA

The present invention belongs to the technical field of oral, liver-targeted

Formulations and in particular concerns an oral, liver-targeted nanoemulsion for

Loading with the antifibrotic drug pirfenidone and a method for its

Production. Furthermore, it concerns the use of the pirfenidone nanoemulsion for

Prevention or treatment of liver fibrosis diseases caused by various factors.

STATE OF THE ART

Liver fibrosis is a pathophysiological process primarily caused by excessive liver function.

Deposition of extracellular matrix (especially collagen) in the liver is characterized by this condition, leading to destruction of liver structure and function. When the factors that cause this condition are present, the liver becomes more susceptible to the deposition of extracellular matrix (especially collagen) in the liver.

Causes of liver damage that cannot be eliminated in the long term can lead to...

Disruption of liver structure, nodular regeneration of liver cells and the formation of

Pseudolobular structure, i.e., liver cirrhosis. Liver fibrosis is histologically reversible and can be reversed by early, active treatment. Numerous experimental studies have shown that the activation of hepatic stellate cells (HSCs) is the cytological basis for the development of liver fibrosis. Activated HSCs can be...

Proliferation and secretion of extracellular matrix (ECM) may be involved in the formation of liver fibrosis and the remodeling of the intrahepatic structure, and in the origin and development of

Liver fibrosis can be promoted. Currently, however, there are no drugs approved for the clinical treatment of liver fibrosis. Pirfenidone (PFD) is the first new broad-spectrum antifibrotic drug to be used clinically. Its unique mechanism of anti-inflammatory action,

Antifibrotic and antioxidant stress effects not only delay the progression of chronic fibrosis

stage of fibrosis, but can also be the acute inflammation caused by idiopathic

Pulmonary fibrosis (IPF) is caused, to effectively alleviate the symptoms. The Food and Drug Administration of

The United States and the European Medicines Agency have approved PFD for clinical trials.

Treatment of patients with IPF is approved.

PFD is primarily administered orally in clinical practice, which offers a significant advantage.

This relates to improving patient compliance. However, after oral absorption, PFD is widely distributed throughout the tissues and can be detected in tissues such as the liver, kidneys, heart, and lungs. Clinical studies have shown that oral administration of PFD can cause systemic side effects, such as pronounced gastrointestinal symptoms.

Skin reactions to sunlight or ultraviolet radiation, as well as weight loss, are common side effects. Currently, PFD-induced side effects are primarily managed by dose reduction or discontinuation.

* BE2024/5892 of the treatment controlled. Therefore, the question is how the targeted distribution of PFD in

Liver tissue and uptake into target cells can be increased to achieve lower levels of absorption.

Dosage to achieve a better therapeutic effect and the occurrence of toxic effects

Avoiding side effects is a pressing problem in the clinical application of

The question of how to treat liver fibrosis (PFD) needs to be addressed. In recent years, the combined strategy of oral administration and targeted therapy has become increasingly important.

It attracts attention. Oral targeting is a non-invasive delivery strategy that uses nanotransport systems to deliver a systemic or distant substance via the oral route.

Targeting has been achieved. It can identify target tissues or cells more accurately and efficiently, while offering the advantages of convenient administration and good compliance.

Oral targeting must overcome the gastrointestinal barrier and prevent rejection by the

To bypass the immune system, in order to increase the likelihood that the drug delivery system will enter the systemic circulation and ultimately fulfill its intended targeting function.

Nanoemulsions as promising oral drug delivery systems have already been used for

Nanoemulsions are used to treat various diseases. Due to their easy absorption, they can significantly improve the bioavailability and efficacy of medications. Many dietary proteins are nutrient-rich, non-toxic, inexpensive, and have good properties.

Emulsifying capabilities, so that they can be used as emulsifiers for nanoemulsions.

Zein is a natural plant protein derived from corn protein powder and is a major ingredient.

It contains a number of hydrophobic residues and polar groups. It is a typical amphiphilic polymer.

However, proteins often carry a certain charge and form a thin interface in the

Protein emulsion, so protein emulsions are strongly influenced by environmental factors in the gastrointestinal tract (pH value, ionic strength and enzymatic hydrolysis, etc.).

Furthermore, oral nano-drug delivery systems must not only...

not only survive gastrointestinal digestion, but are also able to sem, biological

Breaking down membrane barriers also requires further research.

CONTENT OF THE PRESENT INVENTION

Objective of the invention: In order to remedy the shortcomings of the prior art, the present invention presents

The invention comprises an oral, liver-targeted nanoemulsion for loading with pirfenidone, as well as a

Methods for their manufacture and application are available.

Technical solution: In order to achieve the above-mentioned objective of the invention, the present invention is realized by the following technical solutions:

An oral, liver-targeted nanoemulsion for loading with pirfenidone, wherein the

The nanoemulsion is essentially formed by protein-polysaccharide-folic acid conjugates made from zein peptide, dextran and folic acid, and loaded with pirfenidone.

In a specific embodiment, pirfenidone comprises pirfenidone or a derivative thereof.

In a specific embodiment, the zein peptide is mainly characterized by the following

* BE2024/5892

Process manufactured:

First, zein is dissolved in an aqueous ethanol solution; alkali is used for a

Alkali hydrolysis reaction is carried out; after completion of the reaction, ethanol and water are removed from the solution, the pH is adjusted to acidic, the solution is allowed to stand, the supernatant is discarded, the precipitate is washed, then the precipitate is dissolved, and finally the solution is freeze-dried to obtain the zein peptide; preferably the concentration of the aqueous ethanol solution is 65-75%, the alkali is obtained from

Sodium hydroxide is selected, the mass ratio of zein to sodium hydroxide is 1:(1-3); the alkali hydrolysis reaction is carried out under stirring, the reaction temperature is 35-40 °C, the reaction time is 30-40 h.

The present invention also provides a method for preparing the oral, liver-targeted nanoemulsion for loading with pirfenidone, comprising the following steps: (1) First, dextran and folic acid are removed to prepare dextran-folic acid conjugates, which are then reacted with zein peptide to

(1) to synthesize protein-polysaccharide-folic acid polymers; (2) The protein-polysaccharide-folic acid polymers are extracted and treated with deionized

Water is added to form a solution as the aqueous phase; pirfenidone is dissolved in oil and serves as

Oil phase; (3) The oil phase and the water phase are mixed to produce the nanoemulsion.

In a specific embodiment, the molar ratio of dextran to in step (1) is

Folic acid 1:(0.2-0.6), preferably 1.04:0.4; the reaction for the preparation of the

Dextran-folic acid conjugates are prepared in an organic solvent in the presence of EDCI and DMAP under stirring; the reaction temperature is 35-40 °C.

The reaction time is 20-28 h, preferably at 37 °C for 24 h; after completion of the reaction, dialyzing and freeze-drying are carried out to obtain the dextran-folic acid conjugates; preferably the organic solvent is selected from dimethyl sulfoxide.

In a specific embodiment, in step (1) the mass ratio of zine peptide to

Dextran-folic acid conjugates 1:(4-8), preferably 1:6; the synthesis of

Protein-polysaccharide-folic acid polymers are produced via the Maillard reaction and include the following steps:

Zein peptide and dextran-folic acid conjugates are mixed and dissolved, freeze-dried, and the freeze-dried powder is placed in a sealed chamber filled with saturated KBr and reacted at 55–65 °C and 75–85% relative humidity for 20–28 h. Preferably, the reaction is carried out at 60 °C and 79% relative humidity for 24 h.

In a specific embodiment, in step (2) the oil is selected from one or more of medium-chain triglycerides, soybean oil, corn oil, peanut oil and castor oil. Preferably

* BE2024/5892 become medium-chain triglycerides.

In a specific embodiment, in step (3) the oil phase and the water phase are mixed, whereby the mass ratio of protein-polysaccharide-folic acid polymers in the water phase is adjusted to

Pirfenidone in the ol phase, relative to the mass of zein in the

Protein-polysaccharide-folic acid polymers, 1:(1-4), preferably 1:2.5.

As a specific embodiment, step (3) includes the method for producing the

Nanoemulsion: The following: Mixing the oil phase and the water phase and then

Ultrasonic treatment to obtain a primary emulsion; high-pressure homogenization of the

Primary emulsion to obtain the nanoemulsion; preferably, the ultrasonic treatment is carried out at 60-70 W in an ice water bath for 2.5-3.5 min (3 s pulse, 3 s pause), more preferably at 65 W in an ice water bath for 3 mm (3 s pulse, 3 s pause); the

High-pressure homogenization is performed 2-4 times at 5000-7000 psi, preferably 3 times at 6000 psi.

Finally, the present invention presents the use of oral, liver-targeted

Nanoemulsion for loading with pirfenidone in the manufacture of a drug for

Prevention or treatment of liver fibrosis is available.

Furthermore, liver fibrosis is caused by TACE surgery.

The present invention initially obtains a

Dextran-folic acid (DEX-FA) conjugate and synthesized using a green and safe process

Maillard reaction with zein peptide (ZP) zein peptide-dextran-folic acid (ZD-FA) polymers. Oral

Liver targeting differs from conventional bloodstream targeting because it requires not only overcoming the gastrointestinal barrier but also effectively targeting the liver.

Disease focus requires. Therefore, the present invention uses zein peptide as the basis for the nanoemulsion, which not only exhibits good biocompatibility but can also be readily combined with other ligands to compensate for the shortcomings of conventional proteins. For example, the use of the more hydrophilic dextran (DEX) in the aqueous phase can form an extended spatial network structure that creates steric hindrance and the

The disadvantage of protem emulsions is compensated for, as they are susceptible to environmental influences 1m

Gastrointestinal tract (pH value, ionic strength and enzymatic hydrolysis, etc.) are involved in the

To improve the stability of the delivery system, the production of nanoemulsions by grafting zein peptide (ZP) with DEX to synthesize a conjugate can be used to...

to survive gastrointestinal digestion and increase the stability of the system in the gastrointestinal tract. Furthermore, the expression of the folic acid receptor α (FRo) during the

Activation process of HSC: significantly upregulated. The present invention utilizes FA for

Ligand modification, which can not only target the FA receptor on intestinal epithelial cells and improve the intestinal wall permeability of the nanoemulsion, but also the targeted

The effect of the nanoemulsion on activated HSCs is enhanced to increase the therapeutic effect of the

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to have a better effect of the medication on liver fibrosis.

Secondly, the present invention uses a

Microjet high-pressure homogenization process for the production of a PFD-loaded

Nanoemulsion (PFD@ZD-FA) for oral administration for the treatment of CCl4-induced

Liver fibrosis or TACE-induced liver fibrosis. The results show that the FA-modified

Protein-polysaccharide nanoemulsion (PFD) effectively encapsulates protein-polysaccharide nanoemulsions, thus facilitating transport through the

The nanoemulsion can improve the function of small intestinal epithelial cells and increase the oral bioavailability of PFD. Furthermore, it prolongs the residence time of PFD in the gastrointestinal tract and, following TACE, selectively accumulates in the liver, particularly targeting activated HSCs, resulting in a higher antifibrotic effect of PFD at lower doses. Therefore, the PFD@ZD-FA nanoemulsion developed in this study has great potential for the targeted oral treatment of liver fibrosis.

Advantageous effects: The PFD nanoemulsion produced according to the present invention exhibits good stability, high drug loading, and a simple manufacturing process. The surface-modified FA can promote intestinal epithelial penetration of the drug-loaded nanoemulsion and the accumulation of the drug-loaded nanoemulsion.

Nanoemulsion increase in the liver and ultimately target HSCs to have an antifibrotic effect

to exert an effect, so that PFD has a higher antifibrotic effect at a lower oral dose.

The @ZD-FA nanoemulsion developed within this project can serve as an oral delivery platform for the antifibrotic drug PFD.

BRIEF DESCRIPTION OF THE DRAWING

Figures 1A-1B show the production and characterization of the inventive

Protein-polysaccharide-folic acid conjugates.

Figure 2A-2C shows the results of the formulation characterization of PFD@ZD-FA.

Figure 3A-3C shows the results of the stability study of PFD@ZD-FA.

Figure 4A-4J shows the results of the in vitro permeability study of PFD@ZD-FA.

Figure 5A-5B shows the results of the pharmacokinetic study of PFD@ZD-FA.

Figure 6A-6C shows the results of the liver targeting study after oral administration of

PFD@ZD-FA.

Figure 7A-7B shows the results of the HSCs targeting study in the liver after oral administration

Administration of PFD@ZD-FA.

Figure 8 shows the results of the study on the antifibrotic effect of PFD@ZD-FA against

CCL-induced liver fibrosis: Measurement of liver tissue stiffness using

Ultrasound shear wave plastography.

Figure 9 shows the results of the study on the antifibrotic effect of PFD@ZD-FA against

CCL-induced liver fibrosis: Histopathological staining of liver tissue.

BE2024/5892

Figure 10 shows the results of the study on the antifibrotic effect of PFD@ZD-FA against TACE-induced liver fibrosis: measurement of liver tissue stiffness (peritumoral)

liver tissue) using ultrasound shear wave elastography.

Figure 11 shows the results of the study on the antifibrotic effect of PFD@ZD-FA against TACE-induced liver fibrosis: Histopathological staining of liver tissue.

DETAILED DESCRIPTION

The present invention can be better understood with reference to the following examples.

However, the content described in the examples serves only to illustrate the

The invention and shall and will be described in detail in the patent claims.

Do not limit the scope of the invention.

Example 1: Production and characterization of protein-polysaccharide-folic acid polymers

Experimental method: Folic acid (FA, 0.4586 g, 1.04 mmol) was dissolved in 20 ml

Dimethyl sulfoxide (DMSO) was dissolved, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 0.199 g, 1.04 mmol), 4-dimethylaminopyridine (DMAP, 0.127 g, 1.04 mmol), and dextran (DEX, 10 kDa, 4 g, 0.4 mmol) were added successively. The reaction was then carried out for 24 h at 37 °C with magnetic stirring, and the product was then dialyzed for 24 h against phosphate buffer (PBS, 0.01 M, pH 7.4) and water (MWCO, 14 kDa). Finally, the

Polysaccharide-folic acid conjugates (DEX-FA) are obtained by freeze-drying. The structure of

DEX-FA was characterized using ¹H-NMR.

The protein-polysaccharide-folic acid polymers (ZD-FA) were prepared via the Maillard reaction. First, Zem (5 g) was dissolved in 70% solution at a concentration of 10 mg/ml.

Ethanol solution was dissolved, and solid NaOH was added until a final concentration of 0.5 mol/L was reached. The solution was then heated for 36 hours at 37 °C in a water bath with stirring.

The solution was subjected to an alkaline hydrolysis reaction. After completion of the reaction, ethanol and water were removed from the solution by rotary evaporation, and then the pH of the resulting solution was determined.

A solution containing 5 mol/l HCl was adjusted to pH 3.1. During pH adjustment, the solution became cloudy, and the amount of precipitate gradually increased. After allowing the solution to stand overnight, the supernatant was discarded. The zein peptide precipitate at the bottom was washed three times with deionized water. Then, a specific amount of deionized water was added to the precipitate, and 2 mol of NaOH were added until a pH of 9.0 was reached to completely dissolve the precipitate. Finally, the solution was freeze-dried to obtain the zein peptide (ZP). ZP and DEX-FA were recombined in a

Dissolved in a mass ratio of 1:6 in deionized water, and the pH was set to 1 mol/l.

NaOH was adjusted to 8.8. After freeze-drying the reaction solution, the freeze-dried powder was placed in a sealed container filled with saturated KBr and reacted for 24 h at 60 °C and 79% relative humidity to obtain ZD-FA.

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Simultaneously, the protein-polysaccharide conjugate without folic acid modification (ZD) was synthesized in the same manner. To confirm the formation of ZD and ZD-FA, the following were performed:

FTIR spectra of FA, DEX, ZP, ZD and ZD-FA in KBr pellets with a

Recorded using an infrared spectrometer.

Experimental results: The ¹H NMR spectrum of DEX-FA showed the characteristic peaks of DEX and FA (Figure 1A). The characteristic signal of DEX (proton, a) was observed at 4.68 ppm. Simultaneously, the characteristic

Resonance signals of FA at 8.64 ppm (pteride proton, β), 7.58 ppm and 6.64 ppm (γ- and β-aromatic)

protons) the presence of FA residues. The formation of ZD and ZD-FA was further verified by FT-IR. The FT-IR spectra of FA, DEX, ZP, ZD, and ZD-FA are shown in Figure 1B. ZP showed characteristic peaks at 3445 cm″, 1656 cm″, and 1556 cm″, which the

Vibrations were assigned to the hydroxyl, amide I, and amide II groups. At

At wavenumbers of 1656 cm⁻¹ and 1556 cm⁻¹, the absorption intensity of ZD and ZD-FA was lower than that of ZP. This is due to the fact that the carbonyl group of DEX is associated with the

The amino group in ZP has undergone a Maillard reaction, leading to a consumption of the

The bands belonging to amino groups and carbonyl groups are involved. Furthermore, the

The absorption intensity of ZD at wavenumbers of 1017-1155 em is significantly higher than that of

ZP, which suggests that the newly formed C-N covalent bond in the conjugate is strong

exhibits stretching vibration. Remarkably, ZD-FA and ZD showed similar

FT-IR spectra. This could be due to the overlap of the Amide I and Amide II peaks of ZP at 1656 and 1556 cm⁻¹ with the characteristic peaks of FA at 1628 cm⁻¹, 1601 cm⁻¹ and 1510 cm⁻¹!

Example 2: Production and characterization of PFD@ZD-FA

Experimental method: ZD-FA was diluted to 10 mg/ml (corresponding to the

ZP concentration) solution dissolved in deionized water, and the pH was adjusted to 1 mol/l.

HCI was adjusted to 4.5 to obtain the aqueous phase. PFD was dissolved in medium-chain triglycerides to a concentration of 100 mg/ml to obtain the oil phase. Then the

The oil phase and the water phase are mixed in an oil-to-water volume ratio of 1:4.

The solution was shaken and then treated with ultrasound for 3 mm at 65 W in an ice water bath (3 s pulse, 3 s pause) to obtain a primary emulsion. Subsequently, the

Emulsion homogenized three times at 6000 psi using a microjet high-pressure homogenizer to

to obtain PFD@ZD-FA. PFD@Z and PFD@ZD were produced in the same way.

The particle size (PS), the polydispersity index (PDI) and the zeta potential of the

PFD-loaded nanoemulsions were measured using a Zetasizer Nano ZS.

Drug encapsulation rate (LE) and drug loading (LC) of PFD were determined using

Determined by high-performance liquid chromatography (HPLC) with a UV detector.

PFD was detected using a mobile phase of acetonitrile/water (65:35, v/v) in a

, BE2024/5892

Wavelength of à = 316 nm. The LE (%) and LC (%) of PFD were determined according to the following

Formulas calculated:

LE (Gow.-%) = ee een Se x 100 (1)

LC (wt%) = a Wists 100 2)

Experimental results: As shown in Figure 2A, the particle size of the particles with ZP as

Emulsifier-produced active ingredient-loaded nanoemulsion PFD@Z approximately 117.77 nm with a

PDI of 0.3. After the introduction of DEX, the particle size of the nanoemulsion decreased significantly, and the particle size of PFD@ZD was only 28.48 nm. In comparison, the particle size of PFD@ZD-FA was slightly larger at 44.79 nm. The PDI of PFD@ZD and

PFD@ZD-FA was lower than that of PFD@Z. The results show that the introduction of

DEX enhances the emulsifying ability of ZP and forms smaller droplets, and that the

The introduction of FA had little effect on the emulsifying ability. All nanoemulsions were negatively charged (Figure 2B); the introduction of DEX had almost no effect on the

Potential, while the introduction of FA further reduced the potential. The PFD-laden

All nanoemulsions showed good PFD loading, with a LE of over 80%, and the

LE of PFD@ZD and PFD@ZD-FA was slightly higher than that of PFD@Z (Figure 2C).

Example 3: Stability study of PFD@ZD-FA

Experimental method: The stability of the nanoemulsions at 4 °C was investigated by monitoring the changes in PS and PDI of PFD@Z, PFD@ZD, and PFD@ZD-FA over 28 days. Furthermore, the gastrointestinal stability of the

PFD nanoemulsions were investigated in vitro. In short, PFD@Z, PFD@ZD, and

PFD@ZD-FA is mixed with simulated gastric juice (SGF) for the first 2 hours and then in simulated

Intestinal fluid (SIF) was transferred, and then the mixtures were placed in a shaker (100 rpm/min, 37 ± 0.5 °C). Finally, the changes in the PS of the nanoemulsions were measured at predetermined time points using a Zetasizer Nano ZS. To evaluate the release of PFD, PFD@Z, PFD@ZD, and PFD@ZD-FA were each placed in dialysis tubing (MWCO, 3.5 kDa) and dispersed in 20 ml of SGF during the first 2 h, then transferred to SIF. In a shaking incubator (100 rpm/min, 37 ± 0.5 °C), 0.5 ml of medium was withdrawn at predetermined time points and replaced with the same amount of fresh medium. The released

The amount of PFD was analyzed using HPLC.

Experimental results: The stability of the nanoemulsions at 4 °C was investigated by monitoring the changes in PS and PDI over 28 days (Figure 3A).

Compared to PFD@ZD and PFD@ZD-FA, PFD@Z showed a faster increase in horsepower.

Furthermore, PFD@ZD and PFD@ZD-FA showed excellent stability in SGF and SIF (Figure 3B). PFD@Z was rapidly released in SGF and SIF, with the cumulative release amount reaching 43.39% within 12 h, while the cumulative release amount of PFD@ZD

) BE2024/5892 and PFD@ZD-FA was significantly delayed and reduced (Figure 3C). The results show that the polysaccharide-modified nanoemulsions exhibit good stability in SGF and SIF.

Example 4: In-vitro permeability study of PFD@ZD-FA

Experimental Method: The permeability of the nanoemulsions through the mucus layer was measured using a Transwell system. In short, a 10 mg/ml mucin solution was prepared by dissolving porcine gastric mucin in PBS, and 0.2 ml of mucin was added to the upper chamber of the Transwell (12 inserts/24-well plate, pore size 3 µm), while the lower chamber was filled with 0.8 ml of PBS. Then, 0.2 ml of free PFD, PFD@Z,

PFD@ZD and PFD@ZD-FA (1 mg/ml PFD) were added to the upper chamber. After incubation in a 37 °C water bath, 0.5 ml of solution was taken from the lower chamber for analysis at 0.5, 1, 2, and 4 h. The concentration of PFD in the lower chamber was determined by HPLC, and the ratio of PFD that had penetrated the mucus layer was calculated for each group.

Caco-2 cells were used as an in vitro model for the gastrointestinal epithelium. In short, Caco-2 cells were introduced into the upper chamber of the gastrointestinal tract at a density of 5 x 10 cells/well.

Transwells (pore size 0.4 µm) were sown and incubated for 21 days in MEM medium with 20% v/v fetal

Cattle serum (FBS) was cultured. The transepithelial electrical resistance (TEER) of the

The Caco-2 cell monolayer was measured with a voltmeter. When the TEER value reached 500 Q-cm², subsequent experiments were performed. For sample preparation, free PFD, PFD@Z, PFD@ZD, and PFD@ZD-FA (1 mg/ml PFD) were diluted with PBS.

Then the medium was removed and the cells were heated for 30 minutes at 37 °C with pre-warmed water.

PBS was equilibrated. The PBS in the upper chamber was then replaced with 0.2 ml of sample, and the PBS in the lower chamber with 0.8 ml of fresh PBS. After 2 h of incubation, 0.5 ml was added.

Solution was taken from the lower chamber and the PFD content was quantitatively analyzed by HPLC. The apparent permeability coefficient (Papp) of PFD was calculated as follows:

Pap = eu 6) where Q is the cumulative amount of PFD (ng) transported into the lower chamber,

A is the area of the Caco-2 cell monolayer (cm²), Co is the initial PFD concentration in the upper chamber (ng/ml), and / is the duration of the permeability experiment (s). To determine the cellular

To investigate uptake, cells were collected, lysed, and extracted with acetonitrile. Cellular uptake of PFD was then determined by HPLC. Furthermore, various inhibitors were added during the permeability experiment to investigate the transcellular mechanism of PFD@ZD-FA. In short, the medium was removed, the cells were incubated with pre-warmed PBS for 30 minutes, and then the

Caco-2 monolayer washed and treated for 30 min with 37 °C PBS containing colchicine (4 pg/ml),

Cells containing chlorpromazine (10 µg/ml), genistein (100 µg/ml), and FA (10 µg/ml) were incubated, and then treated for 2 h with PFD@ZD-FA (0.2 ml, 10 mg/ml PFD) to remove the Pappus from

PFD was calculated. Finally, the TEER values were measured to determine the influence of the

To monitor the integrity of the Caco-2 monolayer using PFD formulations. First, the initial TEER value of the Caco-2 cell monolayer was measured. Then, the medium in the upper chamber was replaced with 0.2 ml of PFD formulation (1 mg/ml PFD). After 2 h, the

PFD formulation was removed from the upper chamber and replaced with PBS, and the TEER value of the Caco-2 cell monolayer was remeasured after the different treatments.

For imaging, Nile Red (NR)-labeled nanoemulsions were prepared. Rats with

Patients with CCL-induced liver fibrosis were randomly assigned to 3 groups (n = 3) and received oral administration of free NR/PFD, NR/PFD@ZD and NR/PFD@ZD-FA (2 mg/kg NR, 100 mg/kg NR/PFD@ZD-FA).

PFD). The rats were killed 0.5, 2, 6, and 12 hours after administration. The duodenum, jejunum, and ileum were removed, opened, and carefully rinsed with PBS.

Tissues were embedded in OCT, frozen and sectioned, and then stained with DAPI.

Finally, the fluorescence images of the small bowel sections were examined under a confocal microscope.

Observed and recorded using a laser scanning microscope (CLSM).

Experimental results: In vitro models of the mucus layer and the

Caco-2 cell monolayer established to form the intestinal mucus barrier or the intestinal

to simulate epithelial cells and their tight junctions (Figure 4A). At time points of 0.5, 1, 2, and 4 h, the amount of PFD that had penetrated the mucus layer was measured to...

To determine the efficiency of mucus penetration. The PFD penetration of all PFD nanoemulsions increased over time. At time 1 h, the penetration of PFD at PFD@ZD and

PFD@ZD-FA was significantly higher than with free PFD and PFD@Z, and at time 4 h it showed

PFD@ZD-FA exhibits the highest penetration (Figure 4B). It is suggested that the DEX modification improves the stability and transmucosal permeability of the PFD nanoemulsions, and that

PFD@ZD-FA exhibits the best permeability through the mucus layer. Furthermore,

Papp is used as the main indicator for assessing permeability to determine the transport of

PFD nanoemulsions were evaluated through the Caco-2 cell monolayer (Figure 4C). Compared to free PFD, the nanoemulsions increased the permeability of PFD through the intestinal

Epithelial cells significantly, especially in the PFD@ZD-FA group. Both the transcellular

Both the transcellular pathway and paracellular transport could contribute to PFD permeability. The role of the transcellular pathway in PFD permeability was demonstrated by the cellular uptake of

PFD formulations were evaluated by Caco-2 cells (Figure 4D). The nanoemulsions increased the cellular uptake of PFD, particularly PFD@ZD-FA, possibly due to the promotion of folic acid receptor-mediated endocytosis. To further investigate the mechanism of cellular uptake of PFD@ZD-FA, various

Absorption inhibitors were added to assess their effect on the uptake of PFD@ZD-FA by

Caco-2 cells were examined (Figure 4E). The results showed that colchicine, chlorpromazine

U BE2024/5892 and genistein significantly reduced the Papp values of PFD, suggesting that

PFD@ZD-FA is primarily taken up via macropinocytosis-, clathrin-, and caveolin-mediated pathways. Importantly, the competitive inhibition of endocytosis by

PFD@ZD-FA through free FA the role of folic acid receptor-mediated uptake in

Caco-2 cells were confirmed. Furthermore, the TEER, which assesses the integrity of the

Monolayer membrane reflects, used to demonstrate the influence of nanoemulsions on the

To assess tight junctions between cells, which are closely related to paracellular transport (Figure 4F). The addition of PFD-loaded nanoemulsions to the apical side of the cell monolayer resulted in an immediate reduction of TEER, which gradually recovered after removal of the formulation. This suggests that the nanoemulsions inhibit the tight junctions.

They can temporarily and reversibly open junctions between cells, confirming that paracellular transport contributes to the transcellular transport of PFD nanoemulsions.

The rats were euthanized 0.5, 2, 6, and 12 h after administration, and the small intestine was dissected. Segments of the duodenum, jejunum, and ileum were sectioned, and the fluorescence intensity at various sites was observed and quantified using CLSM (Figure 4G-J). The overall fluorescence intensity of the nanoemulsions was higher than that of free NR/PFD, and the fluorescence intensity of NR/PFD@ZD-FA was higher in the small intestinal segments than that of NR/PFD@ZD. Two h after oral administration, the fluorescence intensity of free NR/PFD in the duodenum was very weak, while the

Nanoemulsions retained the fluorescence intensity of NR in the duodenum or jejunum to varying degrees, particularly in the NR/PFD@ZD-FA group. This could be due to the nanoemulsions exhibiting strong resistance to enzymatic degradation and the slow elimination of the drug from the stomach and...

Intestinal juice is released, leading to early retention of the drug in the small intestine.

Furthermore, the high expression of the folic acid receptor on the apical

Brush border membrane of the duodenum and proximal jejunum is one of the reasons for the increased NR distribution in the villi of the duodenum and jejunum in the

The NR/PFD@ZD-FA group. In summary, NR/PFD@ZD-FA can effectively promote the transepithelial transport of NR in the duodenum and jejunum, which contributes to improved drug bioavailability.

Example 5: Pharmacokinetic study of PFD@ZD-FA

Experimental method: Rats with CCl-induced liver fibrosis were randomly assigned to 4

Groups (n = 6) were divided and received an oral dose of free PFD, PFD@ZD and

PFD@ZD-FA (100 mg/kg PFD). Simultaneously, the pharmacokinetics of free PFD at high concentrations were studied.

The dose (150 mg/kg PFD) was evaluated. At the specified time points, the rats were measured at each

The group was anesthetized, and 0.5 ml of blood was drawn from the tail vein. The blood was centrifuged for 5 minutes at 3000 x g to obtain the plasma. 100 µl of plasma was mixed with 400 µl of

Acetonitrile was mixed. The mixture was centrifuged for 10 minutes at 4 °C and 10,000 x g.

The supernatant was removed and analyzed by HPLC. Finally, the most important pharmacokinetic parameters were analyzed using the pharmacokinetic software WinNonlin 8.3.

Experimental results: To determine the pharmacokinetic behavior of PFD in rats with

To investigate liver fibrosis, free PFD was administered orally at doses of 100 mg/kg or 150 mg/kg, while PFD@ZD and PFD@ZD-FA were administered at doses of 100 mg/kg PFD to obtain plasma concentration-time curves (Figure 5A), and key pharmacokinetic parameters were calculated (Figure 5B). Compared to 100 mg/kg free

The high-dose free PFD group (150 mg/kg) prolonged the mean

PFD@ZD prolonged the duration of MRI by 1.38-fold and increased AUCo by 1.61-fold. However, there was no significant difference in apparent plasma clearance (CL/F) between the two groups. Compared to the free PFD (100 mg/kg) group, PFD@ZD and

PFD@ZD-FA increased the MRI of PFD by a factor of 1.85 and 1.88, respectively. Regarding AUCow, PFD@ZD and PFD@ZD-FA increased AUCO-0o by a factor of 2.18 and 2.80, respectively.

At the same time, a reduction in CL/F was observed. The CL/F of the PFD@ZD- and

In the PFD@ZD-FA groups, the concentration of PFD was 46.2% and 36.5%, respectively, in the free PFD (100 mg/kg) group. Therefore, the nanoemulsions effectively prolonged the blood circulation time of PFD and delayed its onset.

Kidney and liver clearance. Interestingly, there was hardly any difference in MRI between the PFD@ZD and PFD@ZD-FA groups. Compared to the PFD@ZD group, Cmax and AUC increased by 1.12-fold and 1.25-fold, respectively, in the PFD@ZD-FA group.

This suggests that PFD@ZD-FA may improve the oral absorption of PFD, possibly due to the promotion of PFD@ZD-FA transport in the intestine by the

is due to folic acid receptor-mediated endocytosis.

Example 6: Liver targeting study of PFD@ZD-FA after oral administration

Experimental method: First, Lipiodol-labeled nanoemulsions were prepared. Rats with CCl4-induced liver fibrosis (CCI, mixed with olive oil in a 1:3 ratio and then administered intraperitoneally at a dose of 0.1 ml/100 g body weight over 8 weeks) were randomly assigned to 3 groups (n = 5) and, after anesthesia, treated with 1%

Isoflurane was administered orally with Lipiodol/PFD, Lipiodol/PFD@ZD, and Lipiodol/PFD@ZD-FA (2 ml/kg Lipiodol, 100 mg/kg PFD). Abdominal CT scans were performed at 0, 0.08, 0.17, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours. The CT scan parameters were as follows:

Tube voltage 120 kV, current 120 mA, layer thickness 3.5 mm, layer spacing 3.5 mm

Pitch 0.8, field of view 22 cm. The distribution of the nanoemulsions in the gastrointestinal tract was observed using localization images, and the CT values of the liver were determined using the

Cross-sectional images were measured. The region of interest (ROT) was measured in the right lobe of the liver using a

A size of approximately 2 mm was selected, avoiding artifacts, and the mean was calculated after several measurements.

Experimental results: The Lipiodol/PFD solution as well as Lipiodol/PFD@ZD and

Lipiodol/PFD@ZD-FA was administered orally to rats with liver fibrosis, and various

Localization images and liver cross-sections were taken at various time points.

Localization images (Figure 6A) showed that 0.08 h after oral administration of 5 Lipiodol/PFD alone, a high-density Lipiodol shadow appeared in the 1 m stomach, while part of the

Lipiodol was emptied from the stomach into the small intestine. The residence time of Lipiodol in the

Small intestine was about 4 hours old; 6 hours later it was essentially completely gone from the small intestine into the

It passed into the large intestine and was excreted after 24 hours. The residence time of

Lipiodol/PFD@ZD and Lipiodol/PFD@ZD-FA in the small intestine segment lasted for over 6 hours. 6 hours later, some of the formulation was emptied from the stomach into the small intestine.

At time 24 h, part of the drug-loaded nanoemulsion still remained in the large intestine of the

Rats. The CT images of the liver cross-sections (Figure 6B) and the quantitative results (Figure 6C) showed that the CT values of the liver increased over time in all formulation groups.

Liver CT values in the Lipiodol/PFD@ZD and Lipiodol/PFD@ZD-FA groups were significantly higher at various time points than in the Lipiodol/PFD group. At time point 24 h, the liver CT value in the Lipiodol/PFD@ZD-FA group was 95.62, which is 1.39 times and 1.22 times the value in the Lipiodol/PFD group and the Lipiodol/PFD@ZD group, respectively. The results above indicate that Lipiodol/PFD@ZD-FA reduces the loaded

The drug can be continuously released into the intestine, remain in the small intestine for a longer period of time, and reach the liver after effective absorption by the intestinal epithelial cells, which helps PFD to exert its therapeutic effect more effectively.

Example 7: HSCs targeting study in the liver after oral administration of PFD@ZD-FA

Experimental method: A Fin liver fibrosis model was used by 8-week intraperitoneal

Injection of CCl4 (CCl4 mixed with olive oil in a 1:3 ratio, dose 0.1 ml/100 g body weight) was established in rats. After completion of the modeling, the rats with liver fibrosis were randomly assigned to 3 groups (n = 3) and received an oral dose of free

NR/PFD, NR/PFD@ZD and NR/PFD@ZD-FA (2 mg/kg NR, 100 mg/kg PFD). 6 h after oral administration

The livers were removed and fixed in 4% paraformaldehyde for 24 hours.

Tissues were dehydrated, embedded, and sectioned. Prior to staining, the sections were deparaffinized and rehydrated in water. Subsequently, the sections were blocked for 1 hour with 10% goat serum blocking solution and incubated overnight at 4°C with α-SMA antibodies, followed by 1 hour of incubation with goat anti-mouse IgG.

H&L secondary antibodies were applied at room temperature. After DAPI staining, the sections were mounted with an antifade reagent. Finally, all sections were observed using CLSM.

Experimental results: Rats with liver fibrosis received an oral dose of free

NR/PFD, NR/PFD@ZD and NR/PFD@ZD-FA. 6 hours later the rats were killed, and that

Liver tissue was extracted. The distribution of α-SMA-positive activated HSCs in the

Liver tissue was examined using immunofluorescence staining. Furthermore, it was determined whether the NR-labeled nanoemulsions could be taken up by the activated HSCs in the liver tissue. The fluorescence signal of free NR in the liver tissue HSCs was very weak, indicating that free NR cannot reach the HSCs in the liver after oral administration. However, both NR-labeled nanoemulsions increased the

Fluorescence signal in the HSCs of the liver (Figure 7A). In particular, the fluorescence intensity of NR/PFD@ZD-FA in the liver was 5.73 times and 1.8 times higher, respectively, than that of free NR/PFD and NR/PFD@ZD (Figure 7B).

Example 8: Study on the antifibrotic effect of PFD@ZD-FA against CClı-induced

Liver fibrosis

Experimental method: A Fin liver fibrosis model was used by 8-week intraperitoneal

Injection of CCl4 (twice weekly, CCl was mixed with olive oil in a ratio of 1:3, which

The dose was 0.1 mcg/100 g body weight) established in rats. The control group (normal) received intraperitoneal injections of olive oil. After completion of the modeling, the rats were treated with

CCl4-induced liver fibrosis randomly divided into 6 groups (n = 6): model group (PBS), free

PFD (150 mg/kg), free PFD (100 mg/kg), PFD@ZD (100 mg/kg) and PFD@ZD-FA (100 mg/kg).

The rats were then given the different substances once a day for 4 weeks.

PFD formulations were administered orally. The normal and model groups received an oral dose of physiological saline solution. After treatment, the stiffness of the

Liver tissue of rats was measured using ultrasound shear wave elastography while they were alive. Subsequently, the rats were euthanized, and the liver tissue was examined histologically, including H&E staining, Sirius Red staining, Masson's trichrome staining, and immunohistochemical detection of α-SMA and collagen III to assess the degree of liver fibrosis.

Experimental results: To determine the influence of the different PFD formulations on the

To investigate the progression of liver fibrosis, rats were examined after CCl₂-induced

Liver fibrosis with high-dose (150 mg/kg) or low-dose (100 mg/kg) PFD,

PFD@ZD solution (100 mg/kg) and PFD@ZD-FA (100 mg/kg) were administered orally once daily for 4 weeks. Following treatment, liver tissue stiffness was initially assessed using...

Ultrasound shear wave elastography was used to measure (Figure 8). The results showed that the liver in the model group exhibited increased stiffness compared to the normal group. After the

Treatment with the different PFD formulations reduced liver stiffness in a dose-dependent manner with both high- and low-dose free PFD, suggesting that free PFD has an antifibrotic effect. At a dose of 100 mg/kg, the different PFD emulsions reduced liver stiffness to varying degrees, particularly in the PFD@ZD-FA group, where liver stiffness was only 67.55% of that in the model group.

Rats were killed, liver tissue was extracted and examined histologically. As in

As shown in Figure 9, the liver surface in the normal group was smooth, while the liver capsule in the model group was thickened and grayish-white. After treatment with the various

With PFD formulations, the thickness of the liver capsule was reduced to varying degrees, and the grayish-white color of the surface decreased, suggesting that the different PFD formulations may attenuate liver fibrosis. Histological staining of the

Liver sections stained with Sirius Red, Masson's Trichrome, and immunohistochemical stains further confirmed the above findings. Initially, the H&E staining showed that the hepatocytes in the normal group were regularly arranged and spread radially from the central vein.

In the model group, the cells were enlarged, the cytoplasm was stained paler, there were slight signs of liver edema, and fibrosis was visible near the portal tracts. Sirius Red and Masson's trichrome staining further confirmed that in the

In the model group, strong collagen fiber formation occurred. Collagen III and o-SMA staining confirmed that the different PFD formulations inhibited the deposition of collagen HI or the

They were able to reduce o-SMA expression to varying degrees, particularly in the

PFD@ZD-FA-Group.

Example 9: Study on the antifibrotic effect of PFD@ZD-FA against TACE-induced

Liver fibrosis

Experimental method: First, a VX2 liver cancer transplantation model was used in

Rabbits were established by percutaneous ultrasound-guided injection. Subsequently, the

Rabbits were randomly assigned to 6 groups (n = 6): model group (model), TACE + PBS, TACE + free PFD (135 mg/kg), TACE + free PFD (90 mg/kg), TACE + PFD@ZD (90 mg/kg), and TACE + PFD@ZD-FA (90 mg/kg). In the first week after modeling, oral administration of the PFD was initiated.

Administration of the various PFD formulations began. In the second week after the

Modeling was performed using TACE treatment, and after the operation, oral medication was administered.

Administration of the different PFD formulations continued for 2 weeks. Subsequently, the stiffness of the rabbits' liver tissue within 2 cm of the edge of the

VX2 tumors were measured using ultrasound shear wave elastography. The rabbits were euthanized, and liver tissue samples from this area were taken and inoculated in 4% solution for 24 hours.

Paraformaldehyde was used to fix the tissues. They were then dehydrated, embedded, and cut.

Prior to staining, the sections were deparaffinized and rehydrated in water. The sections were stained with H&E, Sirius Red, and Masson's Trichrome, and α-SMA and collagen HI were detected immunohistochemically to assess the degree of liver fibrosis after TACE in the rabbits.

Experimental results: Measurement of liver tissue stiffness using

Ultrasound shear wave elastography revealed that tissue stiffness after the

TACE treatment was increased. Treatment with the different PFD formulations significantly reduced liver tissue stiffness, particularly in the PFD@ZD-FA group, where the reduction in liver tissue stiffness was most pronounced (Figure 10). The H&E staining of the

Liver tissue showed that the hepatocytes were slightly enlarged after TACE treatment and that

The cytoplasm was paler. Sirius Red and Masson's trichrome staining confirmed that the

Staining of the fibrous tissue in the liver increased after TACE treatment, while the

Treatment with the different PFD formulations improved the degree of liver fibrosis to varying degrees. The PFD@ZD-FA group showed the most significant reduction in the degree of liver fibrosis. This was further confirmed by immunohistochemical staining, which showed that collagen III deposition and α-SMA expression in the liver were reduced after treatment with PFD@ZD-FA (Figure 11).

Claims (10)

Claim 1. An oral, liver-targeted nanoemulsion for loading with pirfenidone, characterized in that the nanoemulsion is essentially formed by protein-polysaccharide-folic acid conjugates made from zein peptide, dextran and folic acid and loaded with pirfenidone. 2. The oral, liver-targeted nanoemulsion for loading with pirfenidone according to claim 1, characterized in that the zein peptide is mainly produced by the following process: First, zein is dissolved in an aqueous ethanol solution, alkali is added for an alkaline hydrolysis reaction, after completion of the reaction, ethanol and water are removed from the solution, the pH is adjusted to acidic, the solution is allowed to stand, the supernatant is discarded, the precipitate is washed, then the precipitate is dissolved, and finally the solution is freeze-dried to obtain the zein peptide; preferably the concentration of the aqueous ethanol solution is 65-75%, the alkali is selected from sodium hydroxide, the mass ratio of zein to sodium hydroxide is 1:(1-3); the alkaline hydrolysis reaction is carried out with stirring, the reaction temperature is 35-40 °C, and the reaction time is 30-40 h. 3. A method for producing the oral, liver-targeted nanoemulsion for loading with pirfenidone according to claim 1 or 2, characterized in that it comprises the following steps: (1) First, dextran and folic acid are removed to produce dextran-folic acid conjugates, which are then reacted with zein peptide to synthesize protein-polysaccharide-folic acid polymers; (2) The protein-polysaccharide-folic acid polymers are removed and dissolved in deionized water to form an aqueous phase; pirfenidone is dissolved in oil and serves as the oil phase; (3) The oil phase and the aqueous phase are mixed to produce the nanoemulsion. 4. A process for the preparation of the oral, liver-targeted nanoemulsion for loading with pirfenidone according to claim 3, characterized in that in step (1) the molar ratio of dextran to folic acid is 1:(0.2-0.6), the reaction to prepare the dextran-folic acid conjugates is carried out in an organic solvent in the presence of EDCI and DMAP under stirring, the temperature of the reaction is 35-40 °C, the reaction time is 20-28 h; after completion of the reaction, dialyzing and freeze-drying are carried out to obtain the dextran-folic acid conjugates; preferably the organic solvent is selected from dimethyl sulfoxide. 5. Method for producing the oral, liver-targeted nanoemulsion for loading with © BE2024/5892 Pirfenidone according to claim 3, characterized in that in step (1) the mass ratio of zein peptide to dextran-folic acid conjugates is 1:(4-8); the synthesis of the protein-polysaccharide-folic acid polymers is carried out by Maillard reaction and comprises the following steps: zein peptide and dextran-folic acid conjugates are mixed and dissolved, freeze-dried, the freeze-dried powder is placed in a sealed chamber filled with saturated KBr and reacted at 55-65 °C and 75-85% relative humidity for 20-28 h. 6. Method for producing the oral, liver-targeted nanoemulsion for loading with pirfenidone according to claim 3, characterized in that in step (2) the oil is selected from one or more of medium-chain triglycerides, soybean oil, corn oil, peanut oil and castor oil. 7. Method for producing the oral, liver-targeted nanoemulsion for loading with pirfenidone according to claim 3, characterized in that in step (3) the oil phase and the aqueous phase are mixed, wherein the mass ratio of protein-polysaccharide-folic acid polymers in the aqueous phase to pirfenidone in the oil phase, based on the mass of zein in the protein-polysaccharide-folic acid polymers, is 1:(1-4). 8. A method for producing the oral, liver-targeted nanoemulsion for loading with pirfenidone according to claim 3, characterized in that in step (3) the method for producing the nanoemulsion comprises: mixing the oil phase and the water phase and subsequent ultrasonic treatment to obtain a primary emulsion; high-pressure homogenization of the primary emulsion to obtain the nanoemulsion; preferably the ultrasonic treatment is performed at 60-70 W in an ice water bath for 2.5-3.5 min (3 s pulse, 3 s pause); the high-pressure homogenization is performed 2-4 times at 5000-7000 psi. 9. Use of the oral, liver-targeted nanoemulsion for loading with pirfenidone according to claim 1 or 2 in the manufacture of a medicament for the prevention or treatment of liver fibrosis. 10. Use according to claim 9, characterized in that the liver fibrosis is caused by a TACE operation.