Oral nanoparticles of physiologically active substances and methods for producing the same

Abstract

The present invention relates to oral nanoparticles for orally administering physiologically active substances that are mainly administered as injections due to problems of low bioavailability caused by solubility, digestive degradation, and intestinal permeability, a method for preparing the same, and uses thereof.The present invention also relates to oral nanoparticles that have the effect of maintaining or protecting the balance of the intestinal microbiome by minimizing the exposure of the physiologically active substance to the intestinal microbiome, a preparation thereof, and uses thereof.

Description

[Technical Field]

[0001] The present invention relates to oral nanoparticles and a method for producing the same, which improve the bioavailability of physiologically active substances that have low bioavailability due to problems with solubility, digestive tract degradation, and intestinal permeability. Furthermore, the present invention relates to oral nanoparticles and a method for producing the same, which have the effect of maintaining or protecting the balance of the intestinal microbiome by minimizing exposure of the physiologically active substance to the intestinal microbiome. [Background technology]

[0002] Drugs, nutritional supplements, and dietary supplements containing bioactive compounds for human health are most commonly prescribed as oral tablets. Injectable formulations typically cost more than ten times as much to produce as tablets, resulting in significantly higher selling prices. Furthermore, unlike convenient oral medications, injections require a direct hospital visit, incurring additional time and expense for the patient. Injectable formulations also inevitably cause pain during administration. Thus, oral formulations offer significant advantages over injections, making it most common practice to first formulate bioactive compounds requiring absorption into the body into tablet form.

[0003] However, many of the physiologically active substances in pharmaceuticals, nutritional supplements, and dietary supplements have very low oral bioavailability, making it extremely difficult to formulate them as oral drugs, and only injectable formulations have been developed. Drugs with low oral bioavailability have characteristics such as low solubility, degradation by digestive enzymes, and low permeability from the intestines to the bloodstream. As a result, when administered orally, the amount absorbed into the bloodstream is significantly less than the amount administered, compared to when injected directly into the bloodstream. Therefore, drugs with low bioavailability are either developed only as injectable formulations or as oral formulations with low bioavailability.

[0004] Another problem with drugs with low bioavailability is that only a portion of the ingested amount is absorbed, while the majority remains in the digestive tract, leading to intestinal health problems such as indigestion. Furthermore, exposure of the gut microbiota, which are normally present in the digestive tract, to drugs in the tract significantly impacts the survival rate of each species, disrupting the balance of the gut microbiome and causing various health problems.

[0005] The gut microbiome significantly impacts overall human health, including various diseases related to digestive activity such as diabetes, dementia, cancer, hypertension, and hyperlipidemia. Due to these characteristics, maintaining a healthy gut microbiome is crucial for overall health. Chemicals ingested by humans are absorbed into the body through a process of uniform mixing with the contents of the intestines. Therefore, ingested chemicals can have a very serious impact on the survival and community of gut microorganisms, disrupting the balance of the gut microbiome. In particular, taking antibiotics with microbial-killing effects as oral tablets can kill specific species of gut microorganisms, disrupting the balance of the gut microbiome and causing not only gastrointestinal diseases such as diarrhea and abdominal pain, but also various health problems related to the gut microbiome. Therefore, there is a pressing need to develop technologies that increase the bioavailability of physiologically active substances so that they do not affect the gut microbiome.

[0006] For the reasons mentioned above, development of oral formulations with excellent bioavailability is underway, and the following technologies have been developed.

[0007] U.S. Patent No. 7060708 describes an invention that involves covalently conjugating amino acids to a substance to be absorbed into the body, so that when the amino acids are absorbed in the digestive tract, the conjugated substance is also absorbed into the body. However, this invention has the drawback that when chemical changes occur due to covalent bonding, the inherent properties of the substance change, leading to problems such as decreased activity, deformation, and side effects.

[0008] U.S. Patents No. 7144877, No. 7598235, and No. 7678782, among others, all present novel compound structures in which drug-conjugate-bile acid systems are chemically linked to each other by covalent bonds. This invention has the problem that the drug is broken down by digestive enzymes in the gastrointestinal tract, and because chemical changes occur due to covalent bonding, the intrinsic properties of the substance change, leading to problems of decreased activity, deformation, and side effects.

[0009] U.S. Patent No. 7736679 describes a method for increasing the absorption rate of curcumin and improving its bioavailability by preparing a mixture of curcumin and turmeric oil. However, this invention is limited to curcumin and turmeric oil, and the degree of improvement in bioavailability is minimal.

[0010] U.S. Published Patent 2019-0247313 provides nanoparticles made by electrically stimulating a central drug to become positively charged, and then mixing it with a negatively charged polymer material to which bile acids are covalently bonded. This invention has limitations in drug delivery to the human body because the amount of encapsulable drug is too small, and because the negatively charged polymer-bile acid covalent compound is a chemically novel substance, it may cause unexpected side effects.

[0011] U.S. Published Patent No. 2020-0009067 provides a drug solution prepared by dissolving a hydrophobic drug in a mixed solution consisting of three components: lipid, surfactant, and emulsifying solvent. When this drug solution is taken orally, it may mix with digestive fluids in the body, potentially increasing absorption and thus increasing bioavailability. However, it is limited to hydrophobic drugs only, and the improvement in bioavailability is only slight.

[0012] As mentioned above, technologies to improve bioavailability employ methods that covalently bond substances to improve solubility, intestinal permeability, and absorption rates. However, during chemical bonding (covalent bonding), a new chemical entity is created, altering the structure and chemical properties of the original substance. This can lead to the loss of the drug's inherent properties or the occurrence of unexpected toxicity and immunological side effects. In other words, conventional technologies have not only failed to improve the bioavailability of oral drugs, but have also failed to address the intestinal microbiome issues associated with oral administration.

[0013] Replacing injectable drugs with oral tablets requires fundamentally resolving the problems of existing oral administration methods. Specifically, there is a pressing need for technology that completely eliminates the denaturation of orally administered substances and dramatically increases their bioavailability after absorption. Furthermore, technology that minimizes exposure of orally administered substances to gut microbiota, thereby maintaining the balance of the gut microbiome, is crucial. The development of such technologies is urgently needed for the health of humankind. [Overview of the Initiative] [Problems that the invention aims to solve]

[0014] The inventors have made diligent efforts to solve the problems of low bioavailability of physiologically active substances and disruption of the balance of the gut microbiome. Therefore, the object of the present invention is to provide oral nanoparticles with improved bioavailability of physiologically active substances and a method for producing the same. Another object of the present invention is to provide oral nanoparticles and a method for producing the same that have the effect of maintaining and protecting the balance of the gut microbiome by minimizing the exposure of orally administered substances to gut microorganisms. [Means for solving the problem]

[0015] To achieve the above objective, the present invention provides oral nanoparticles and a method for producing the same, which dramatically improve the bioavailability of physiologically active substances and have the effect of maintaining and protecting the balance of the intestinal microbiome.

[0016] The inventors focused on the fact that bile acids are amphiphilic, meaning they are composed of a hydrophobic portion and a hydrophilic portion. Therefore, the inventors hypothesized that if nanoparticles were created with the hydrophilic portion of bile acid on the outside and the hydrophobic portion on the inside surrounding the substance, the substance encapsulated inside the nanoparticles would not be broken down by digestive fluids in the digestive tract and would not be exposed to intestinal microorganisms, thus maintaining the balance of the intestinal microbiome, even when administered orally and reaching the digestive tract.

[0017] Furthermore, the inventors focused on the fact that bile acids excreted into the digestive tract have the property of being reabsorbed into the blood by more than 90% via the enterohepatic circulation through the active uptake / transport system. Bile acids are a type of emulsifier that allows lipid components to mix well with digestive enzymes. Therefore, the inventors hypothesized that if a nanoformulation could be created using only bile acids as the physiologically active substance without any denaturation, it would be reabsorbed into the blood from the digestive tract along with the bile acids.

[0018] Therefore, after numerous experiments, the inventors succeeded in formulating oral nanoparticles encapsulated with bile acid, and experiments with the manufactured oral nanoparticles confirmed that the majority of the bile acid-encapsulated substance was absorbed into the body.

[0019] To achieve the above object, the nanoparticles of the present invention consist of (1) a physiologically active substance to be absorbed in the body and (2) a non-covalent bond of a bile acid surrounding and encapsulating the substance. The nanoparticles of the present invention can: 1) epochally improve the bioavailability of the encapsulated substance; 2) minimize the exposure of the encapsulated substance to digestive juices, thereby preventing denaturation or degradation in the digestive organs; 3) increase intestinal permeability by allowing the encapsulated substance to be reabsorbed into the blood together with bile acids; 4) maintain and protect the balance of the intestinal microbiota by minimizing the exposure of the encapsulated substance to intestinal microorganisms due to the increased bioavailability of the encapsulated substance.

[0020] Particularly, different from existing technologies, the substances constituting the nanoparticles of the present invention are bonded only by non-covalent bonds without an integral covalent bond with bile acids. Therefore, 1) the efficacy of the chemical substance is maintained as it is without any chemical denaturation; 2) since no new chemical entity is generated, it is characterized by having no unexpected new side effects.

[0021] Therefore, the present invention provides orally administered nanoparticles with innovatively improved bioavailability and a method for producing the same.

Advantages of the Invention

[0022] The orally administered nanoparticles according to the present invention have the following effects: 1) an effect of epochally improving the bioavailability of the encapsulated substance; 2) an effect of preventing denaturation or degradation in the digestive organs by minimizing the exposure of the encapsulated substance to digestive juices; 3) an effect of increasing intestinal permeability by allowing the encapsulated substance to be reabsorbed into the blood together with bile acids; 4) an effect of maintaining and protecting the balance of the intestinal microbiota by minimizing the exposure of the encapsulated substance to intestinal microorganisms.

Brief Description of the Drawings

[0023] [Figure 1]This is the result of making an aqueous solution (expressed concentration mg / ml) with the niclosamide nanoparticles produced according to Example 1 of the present invention and then comparing and confirming it with the original non-nanoparticulated niclosamide substance (A: Particle size analysis of the produced niclosamide nanoparticles, B: Niclosamide solution in which precipitates are confirmed, C: Niclosamide nanoparticle solution without precipitates, D: Low magnification electron microscope image of niclosamide nanoparticles, E: High magnification electron microscope image of niclosamide nanoparticles). [Figure 2] This is the result of confirming the structural stability of the niclosamide nanoparticles produced according to Example 1 of the present invention during long-term storage or at various pH values (A: Niclosamide nanoparticle solution stored for a long time, B: Particle size analysis result of the niclosamide nanoparticle solution stored for a long time, C: Niclosamide nanoparticle solution at various pH values, D: Particle size analysis result in the niclosamide nanoparticle solution at various pH values). [Figure 3] This is the electron microscope image of the paclitaxel nanoparticles produced according to Example 1 of the present invention and the analysis result of a particle size analyzer (A: Low magnification electron microscope image of paclitaxel nanoparticles, B: High magnification electron microscope image of paclitaxel nanoparticles, C: Analysis result of paclitaxel nanoparticles produced with an average particle size of 189 nm). [Figure 4] This is the electron microscope image of the 5-fluorouracil nanoparticles and metformin nanoparticles produced according to Example 2 of the present invention and the analysis result of a particle size analyzer (A: Electron microscope image of 5-fluorouracil nanoparticles, B: Electron microscope image of metformin nanoparticles, C: Analysis result of 5-fluorouracil nanoparticles produced with an average particle size of 119 nm, D: Analysis result of metformin nanoparticles produced with an average particle size of 108 nm). [Figure 5]These are electron microscope images and particle size analyzer analysis results of azithromycin nanoparticles and ciprofloxacin nanoparticles produced according to Example 3 of the present invention (A: electron microscope image of azithromycin nanoparticles, B: electron microscope image of ciprofloxacin nanoparticles, C: nanoparticle analysis results of azithromycin nanoparticles produced with an average particle size of 172 nm, D: analysis results of ciprofloxacin nanoparticles produced with an average particle size of 142 nm). [Figure 6] In Experimental Example 1 of the present invention, these are electron microscope images of remdesivir nanoparticles produced by Example 1 of the present invention and glutathione nanoparticles produced by Example 2 of the present invention (A: remdesivir nanoparticles, B: glutathione nanoparticles). [Figure 7] In Experimental Example 2 of the present invention, the intestinal microbiome of mice orally administered azithromycin nanoparticles was metagenomically sequenced, and then the intestinal microbiome diversity was analyzed using the Shannon alpha-diversity analysis program (Group 1: Antibiotic-free control group, Group 2: Azithromycin nanoparticle administration group, Group 3: Azithromycin administration group). [Figure 8] In Experimental Example 3 of the present invention, the antiviral therapeutic efficacy was confirmed after orally administering niclosamide or niclosamide nanoparticles to an SH101 hamster animal model infected with coronavirus (A: design of the antiviral efficacy evaluation experiment and a certain schematic diagram, B: results of body temperature measurement, C: results of body weight measurement, D: results of survival rate (% survival) measurement). [Figure 9] In Experimental Example 3 of the present invention, the antiviral therapeutic efficacy was confirmed after orally administering niclosamide or niclosamide nanoparticles to a hACE transgenic mouse animal model infected with coronavirus (A: design of the antiviral efficacy evaluation experiment and a certain schematic diagram, B: results of body temperature measurement, C: results of body weight measurement, D: results of survival rate (% survival) measurement). [Figure 10]In Experimental Example 3 of the present invention, the following are the results of observing lung tissue obtained after orally administering niclosamide or niclosamide nanoparticles to SH101 hamsters and hACE transgenic mouse animal models infected with coronavirus (arrows indicate sites of inflammation due to infection) and quantitatively analyzing the coronavirus levels in the lung tissue (A: H&E stained image of lung tissue from SH101 hamsters, B: H&E stained image of lung tissue from hACE transgenic mice, C: SARS-CoV-2 virus RNA copies per gram of lung tissue from SH101 hamsters, D: SARS-CoV-2 virus RNA copies per gram of lung tissue from hACE transgenic mice). [Modes for carrying out the invention]

[0024] Oral medications, which are effective against various diseases, are far more convenient and economical to administer than injectable medications. Therefore, various efforts have been made to improve the low oral bioavailability of these drugs. However, the technologies invented to date only offer minimal improvements in bioavailability, making it difficult to formulate oral medications as replacements for injectables. Furthermore, the drugs are inevitably broken down by digestive enzymes, and the chemical binding methods used alter the drug's structure, physical properties, and chemical characteristics—a serious problem. Consequently, these technologies have not been successful or widely adopted, and many drugs continue to be used as injectables despite their various drawbacks, or as oral medications with extremely low bioavailability. Therefore, replacing injectables with oral medications requires the invention of a revolutionary concept. This invention can solve all the above problems with oral medications, making it possible to replace all injectables with edible tablets. If this invention becomes widespread, it is expected to bring about a medical revolution for humanity.

[0025] Unlike existing inventions that link physiologically active chemical substances to bile acids by chemical bonds such as covalent bonds, the present inventors focused on the fact that if the process of creating stable nanoparticles by surrounding a desired substance with bile acids can be carried out non-covalently without using covalent bonds, then there will be no structural changes between the bile acids located on the surface of these nanoparticles and the chemical substance located in the center, and as a result, the chemical properties will be maintained, allowing for extremely efficient absorption into the blood via the enterohepatic circulation, which is the absorption pathway for bile acids, and thus the present invention was completed.

[0026] Conventional oral nanoparticle manufacturing processes have always required covalent bonding (conjugation) processes that cause chemical changes in the reactants. This not only makes the nanoparticle manufacturing process extremely complex, but also leads to structural deformation of the reactants in covalently bonded products, causing them to lose their inherent properties. This makes it difficult to maintain the original function of the drug and simultaneously increases the possibility of unexpected side effects from the new material. Furthermore, because the nano-dosage form must be capable of chemically reacting with the drug, the types of drugs that can be encapsulated in nanoparticles are limited.

[0027] The oral nanoparticles of the present invention are linked only by non-covalent bonds between exactly two components: (1) a substance to be absorbed into the body and (2) bile acids that act as drug delivery bodies surrounding it. This has the following effects: 1) dramatically improves the bioavailability of the encapsulated substance; 2) minimizes exposure of the encapsulated substance to digestive fluids, thereby preventing denaturation or degradation in the digestive tract; 3) increases intestinal permeability as the encapsulated substance is reabsorbed into the bloodstream along with bile acids; and 4) maintains and protects the balance of the intestinal microbiome by minimizing exposure of the encapsulated substance to intestinal microorganisms. Due to these excellent advantages, the nanoparticles of the present invention are a revolutionary invention that can convert all drugs currently used as injectable formulations into oral formulations.

[0028] One embodiment of the present invention provides orally administered nanoparticles of a substance with low bioavailability, a formulation thereof, and a method for using the same.

[0029] Accordingly, the present invention relates to an oral nanoparticle characterized in that, in one embodiment, it is composed of a bioactive substance located in the center and bile acids surrounding the bioactive substance, wherein the bioactive substance and the bile acids are non-covalently bonded.

[0030] The aforementioned physiologically active substances include all substances that have drug, nutritional component, or functional use in pharmaceuticals, nutritional supplements, and dietary supplements, and are intended for absorption into the body. Specific examples include 5-fluorouracil, remdesivir, azithromycin, paclitaxel, doxorubicin, oxaliplatin, and phenylephrine hydrochloride. Hydrochloride, glutathione, acetazolamide, amphotericin, aprepitant, azathioprine, chlorothiazide, chlorthalidone, ciprofloxacin, colistin, cyclosporine AA) Digoxin, docetaxel, furosemide, etravirine, famotidine, griseofulvin, hydrochlorothiazide, mebendazol, methotrexate, neomycin, niclosamide, nystatin, ritonavir, albendazole, artemther, chlorpromazine, efavirenz, glibenclamide, ivermectin, lopinavir, mefloxacin Mefloquine, retinol, spironolactone, sulfadiazine, sulfasalazine, triclabendazole, acyclovir, amoxicillin, bidisomide, biperiden, captopril, cefazolin, chloroquine, cimetidine, cloxacillin, didanosine, ephedrine, erythromycin, famotidine, fluconazole, folinic acidacid), furosemide, ganciclovir, lisinopril, methotrexate, metformin, nifurtimox, nadolol, nystatin, pravastatin, penicillin, ranitidine, reserpine, tetracycline, balsa Examples include, but are not limited to, valsartan, vancomycin, doxycycline, chlorpheniramine, clomiphene, clomipramine, dexamethasone, ethinylestradiol, metoclopramide, morphine, and quinine.

[0031] The aforementioned bile acids are amphiphilic and have the characteristic of being actively absorbed in the intestines, and preferably include bile acids or their derivatives, specifically the bile acids being cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, ursodeoxycholic acid, tauroursodeoxycholic acid, hyodeoxycholic acid, 7-oxolithocholic acid, iodine cholic acid, iodine cholic acid, taurolithocholic acid, glycoursodeoxycholic acid, taurocholic acid Examples include, but are not limited to, acids and glycocholic acid.

[0032] Furthermore, the "oral nanoparticles of physiologically active substances" of the present invention are characterized in that the physiologically active substance and the bile acid are bonded to each other only by non-covalent bonds, without any covalent bonds, and there is absolutely no chemical modification, so that the properties of the chemical substance are maintained as they are.

[0033] In another embodiment, the present invention relates to an oral nanoparticle composition comprising the oral nanoparticles.

[0034] The oral nanoparticle composition of the present invention may contain pharmaceutically acceptable carriers commonly used in formulation, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

[0035] In addition to these components, the oral nanoparticle composition of the present invention may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0036] The appropriate dosage of the oral nanoparticle composition of the present invention can be formulated in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, medical condition, diet, administration time, administration route, excretion rate, and response sensitivity. On the other hand, a preferred dosage of the oral nanoparticle composition of the present invention is 0.0001 to 1000 μg per day.

[0037] The oral nanoparticle composition of the present invention can be manufactured in unit dose form by formulation using pharmaceutically acceptable carriers and / or excipients by a method readily available to a person with ordinary skill in the art to which the invention pertains, or in multi-volume containers. In this case, the dosage form may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablet or capsule, and may further contain a dispersant or stabilizer.

[0038] The oral nanoparticle composition of the present invention has the effect of preventing denaturation or degradation before absorption in the digestive tract by minimizing the exposure of the substance encapsulated in the nanoparticles to digestive enzymes.

[0039] Furthermore, the oral nanoparticle composition of the present invention has the effect of maintaining and protecting the balance of the intestinal microbiome by minimizing the exposure of substances encapsulated in nanoparticles to intestinal microorganisms.

[0040] In another embodiment, the present invention relates to a method for producing oral nanoparticles, comprising the steps of (a) dissolving a physiologically active substance in a solvent, (b) dissolving bile acid in a solvent, (c) mixing the physiologically active substance solution and the bile acid solution, and (d) freeze-drying the mixed solution of the physiologically active substance and bile acid.

[0041] Examples of solvents for dissolving the aforementioned physiologically active substance include, but are not limited to, water, DMSO, ethanol, methanol, and acetone.

[0042] Examples of solvents for dissolving the bile acids include, but are not limited to, ethanol, water, and methanol.

[0043] To produce oral nanoparticles characterized by being composed of a physiologically active substance located at the center of the present invention and bile acids surrounding the physiologically active substance, and wherein the physiologically active substance and the bile acids are non-covalently bonded, it is necessary to mix a physiologically active substance solution and a bile acid solution. This mixing can be achieved by mixing the physiologically active substance solution and the bile acid solution together and then stirring or ultrasonically treating them.

[0044] The method for producing oral nanoparticles of the present invention may further include, in order to further promote the formation of oral nanoparticles, a step of mixing a physiologically active substance solution and a bile acid solution, and then subjecting the mixture of the physiologically active substance and bile acid to low-temperature treatment or salt treatment, or subjecting it to low-temperature treatment after salt treatment.

[0045] The low-temperature treatment can be carried out by stirring a mixture of the physiologically active substance and bile acid while lowering the temperature from room temperature to a range of -20 to +20°C at a rate of 1°C / min or less, and the salt treatment can be carried out by stirring a mixture of the physiologically active substance and bile acid while adding Na + Mg 2+ Li + Ca 2+ Fe 2+ A salt selected from the group consisting of such salts can be added in a range of 0.1 to 20 M.

[0046] If the rate or temperature during the low-temperature processing falls outside the above range, the effect of promoting the formation of oral nanoparticles may be minimal.

[0047] Furthermore, if the concentration of the salt treated during the salt treatment falls outside the above range, the effect of promoting the formation of oral nanoparticles may be minimal. [Examples]

[0048] The present invention will be described in more detail below with reference to examples. These examples are for illustrative purposes only and it will be obvious to those who are ordinarily skilled in the art that the scope of the present invention is not to be construed as being limited to these examples.

[0049] Example 1. Production of oral nanoparticles using a mixed reaction Niclosamide is known for its excellent and diverse pharmacological effects, including antibacterial, antiparasitic, anticancer, antiviral, and neuroprotective properties. However, due to issues with absorption and water solubility, it is only approved by the FDA as a tablet for intestinal parasites where absorption into the body is not necessary. Despite its excellent antibacterial and antiviral effects, niclosamide has not been developed as a treatment for COVID-19, tuberculosis, or as an antiviral or anticancer agent. This is because niclosamide, being a hydrophobic chemical, dissolves only in limited hydrophobic organic solvents, making it difficult to develop as an injectable drug. Furthermore, it is not well absorbed into the body when administered orally, resulting in a bioavailability of less than 10% for tablets. Similarly, paclitaxel, a representative cytotoxic anticancer drug, also has a bioavailability of only 10%, and is therefore only prescribed as an injectable drug. If paclitaxel were produced as oral nanoparticles, it could be prescribed as an oral medication instead of an injectable drug, which would reduce drug side effects and improve convenience of use.

[0050] Therefore, the present inventors, in developing an oral formulation with improved bioavailability, synthesized niclosamide and paclitaxel, which are expected to offer dose-increasing effects, reduced side effects, and ease of use, into nanoparticles using the following method.

[0051] Niclosamide powder was dissolved in DMSO solvent at a concentration of 6 mg / mL, and cholic acid powder was dissolved in water at a concentration of 6 mg / mL. 1 mL of the niclosamide solution and 3 mL of the cholic acid solution were mixed, and both solutions were thoroughly mixed in a water-filled ultrasonic generator for 5 minutes. This solution was then freeze-dried to produce a powder. The produced niclosamide nanoparticles were completely water-soluble nanoparticles, unlike pure niclosamide, soluble in water without precipitate, and had an average particle size of 120–150 nm, as confirmed by electron microscopy (Figure 1).

[0052] Furthermore, we confirmed that the particle size remained stable even when the manufactured niclosamide nanoparticles were stored for a long period of time, more than 6 weeks, or left at pH 6.8 and pH 7.4 for more than 12 hours (Figure 2).

[0053] Paclitaxel powder was dissolved in DMSO solvent at a concentration of 10 mg / mL, and cholic acid powder was dissolved in ethanol at a concentration of 6 mg / mL. After mixing 1 mL of this paclitaxel solution with 3 mL of the cholic acid solution, the container holding this mixture of paclitaxel and cholic acid was placed in an ultrasonic generator filled with water and thoroughly mixed using ultrasound for 5 minutes. The solution after this mixing process was freeze-dried to obtain a powder.

[0054] The manufactured paclitaxel nanoparticles were completely water-soluble, unlike pure paclitaxel, readily soluble in water, and had an average particle size of 150–250 nm, as confirmed by electron microscopy and particle size analyzer (Figure 3).

[0055] Each of the manufactured nanoparticles was orally administered to rats at a dose of 20 mg / kg (based on the test substance), and blood samples were collected from the rats at different time intervals. A control group was administered the same amount orally to rats using the pure bioactive substance used in nanoparticle production, and the same experiment was performed. 0.1 mL of blood collected from the rats at different time intervals was mixed with 0.9 mL of acetone, and this mixture was thoroughly mixed using a vortex mixer. The mixed solution was centrifuged at 15,000 rpm to remove precipitates, and only the resulting solution was analyzed by LC-MS / MS. The bioavailability is shown in Table 1.

[0056] [Table 1]

[0057] This experiment demonstrates the process of manufacturing niclosamide nanoparticles, but also shows the low bioavailability of hydrophobic substances in the BCS classification class IV group (acetazolamide, amphotericin, aprepitant), azathioprine, chlorothiazide, chlorthalidone, ciprofloxacin, colistin, and cyclosporine A. A) Digoxin, docetaxel, furosemide, etravirine, famotidine, furosemide, griseofulvin, hydrochlorothiazide, mebendazole, methotrexate, neomycin, nystatin, paclitaxel, ritonavir, etc.), or class Hydrophobic substances other than those in Group IV that require improved bioavailability (such as albendazole, artemther, chlorpromazine, efavirenz, glibenclamide, ivermectin, lopinavir, mefloquine, retinol, spironolactone, sulfadiazine, sulfasalazine, and tricalbendazole) can also be produced as nanoparticles of the present invention using a similar method. In these cases, it has been confirmed that oral nanoparticles can be produced even when ethanol, methanol, acetone, etc. are used as the solvent instead of DMSO, or when other bile acids that constitute bile acids are used instead of cholic acid.

[0058] Example 2. Production of oral nanoparticles using salt treatment 5-fluorouracil is a representative cytotoxic anticancer drug and one of the most widely used anticancer drugs in cancer treatment, but due to its low oral bioavailability, it is currently only used as an injectable drug. Metformin is a representative treatment for type 2 diabetes and is mainly prescribed in tablet form for convenience, but its bioavailability is only about 30%.

[0059] Therefore, when developing oral formulations with improved bioavailability, the inventors experimented with 5-fluorouracil and metformin, which are expected to offer dose-increasing effects, reduced side effects, and ease of use, using the technology of the present invention under various conditions, and as a result, they synthesized them into nanoparticles using the following method.

[0060] 5-fluorouracil powder was dissolved in water at a concentration of 6 mg / mL, and cholic acid powder was dissolved in ethanol at a concentration of 6 mg / mL. 1 mL of this 5-fluorouracil solution and 3 mL of the cholic acid solution were mixed together, and 10 mg of NaCl was added to this solution. The two solutions were then thoroughly mixed for 5 minutes using an ultrasonic generator containing ethanol. Next, while maintaining ultrasonic generation, dry ice was added to the ultrasonic generator to freeze the 5-fluorouracil solution, and then freeze-dried to produce a powder. The produced 5-fluorouracil nanoparticles were confirmed to be 90-120 nm in size (Figure 4).

[0061] Metformin powder was dissolved in water at a concentration of 5 mg / mL, and cholic acid powder was dissolved in ethanol at a concentration of 5 mg / mL. 1 mL of this metformin solution and 3 mL of the cholic acid solution were mixed, and 60 mg of NaCl was added to this solution. The two solutions were then thoroughly mixed for 5 minutes using an ultrasonic generator containing ethanol. Next, while maintaining ultrasonic generation, dry ice was added to the ultrasonic generator to freeze the metformin solution, and then freeze-dried to produce a powder. The produced metformin nanoparticles were confirmed to be 90-120 nm in size (Figure 4).

[0062] Each of the manufactured nanoparticles was orally administered to rats at a dose of 20 mg / kg (based on the bioactive substance), and blood samples were collected from the rats at different time intervals. As a control group, the same experiment was performed on rats orally administered the same amount of pure bioactive substance used in nanoparticle production. 0.1 mL of blood collected from the rats at different time intervals was mixed with 0.9 mL of acetone, and this mixture was thoroughly mixed using a vortex mixer. The mixed solution was centrifuged at 15,000 rpm to remove precipitates, and only the resulting solution was analyzed by LC-MS / MS. The bioavailability is shown in Table 2.

[0063] [Table 2]

[0064] This experiment demonstrates the process of producing nanoparticles of 5-fluorouracil and metformin, but also shows the low permeability of hydrophilic substances in the BCS classification class III group (acyclovir, amoxicillin, bidisomide, biperiden, captopril, cefazolin, chloroquine, cimetidine, cloxacillin, didanosine, ephedrine, erythromycin, famotidine, fluconazole, folinic acid). (e.g., acid), furosemide, ganciclovir, lisinopril, methotrexate, metformin, nifurtimox, nadolol, nystatin, pravastatin, penicillin, ranitidine, reserpine, tetracycline, valsartan, vancomycin, etc.) or class Hydrophilic substances other than III that require improved absorption in the body (such as chlorpheniramine, clomiphene, clomipramine, dexamethasone, ethinylestradiol, metoclopramide, morphine, and quinine) can also be manufactured into oral nanoparticles of the present invention using a similar method.In these cases, we confirmed that oral nanoparticles can be produced even if ethanol, methanol, acetone, or other solvents are used instead of water as needed, or if other bile acids that make up bile acids are used instead of cholic acid.

[0065] Example 3. Production of oral nanoparticles using low-temperature processing Azithromycin is a representative broad-spectrum antibiotic and one of the most widely used antibiotics for treating bacterial infections. However, due to its low oral bioavailability, it is not absorbed by the human body and remains in large quantities in the intestines. This residual azithromycin kills intestinal microorganisms and disrupts the gut microbiota, causing many serious side effects in people.

[0066] Ciprofloxacin is also a representative broad-spectrum antibiotic, but its low bioavailability causes the same problems as azithromycin. Therefore, it is necessary to reduce drug side effects by improving bioavailability and reducing the amount remaining in the intestines.

[0067] Therefore, the present inventors conducted experiments using the technology of the present invention under various conditions with azithromycin and ciprofloxacin, which are expected to have dose-increasing effects, reduced side effects, and ease of use when developed as oral formulations with improved bioavailability. As a result, they synthesized them into nanoparticles using the following method.

[0068] Azithromycin powder was dissolved in ethanol at a concentration of 6 mg / mL, and cholic acid powder was dissolved in ethanol at a concentration of 6 mg / mL. Next, 1 mL of the azithromycin solution and 3 mL of the cholic acid solution were mixed together, and 1 mg of NaCl was added to this solution. Then, both solutions were thoroughly mixed for 5 minutes using an ultrasonic generator containing ethanol. Next, the temperature of the azithromycin and cholic acid mixture was lowered from room temperature to -4°C at a rate of 0.001°C / min while stirring using a temperature control device. Finally, this mixture was frozen and then freeze-dried to obtain a powder. The produced azithromycin nanoparticles were confirmed to be 130-220 nm in size (Figure 5).

[0069] Ciprofloxacin powder was dissolved in water at a concentration of 5 mg / mL, and cholic acid powder was dissolved in ethanol at a concentration of 5 mg / mL. 1 mL of this ciprofloxacin solution and 3 mL of the cholic acid solution were mixed together, and 21 mg of FeCl was added to this solution. The two solutions were then thoroughly mixed for 5 minutes using an ultrasonic generator containing ethanol. Next, the temperature of the ciprofloxacin and cholic acid mixture was lowered from room temperature to -2°C at a rate of 0.001°C / min while stirring using a temperature control device. Finally, this mixture was frozen and then freeze-dried to obtain a powder. The produced ciprofloxacin nanoparticles were confirmed to be 100-200 nm in size (Figure 5).

[0070] Each of the manufactured nanoparticles was orally administered to rats at a dose of 20 mg / kg (based on the bioactive substance), and blood samples were collected from the rats at different time intervals. As a control group, the same experiment was performed on rats orally administered the same amount of pure bioactive substance used in nanoparticle production. 0.1 mL of blood collected from the rats at different time intervals was mixed with 0.9 mL of acetone, and this mixture was thoroughly mixed using a vortex mixer. The mixed solution was centrifuged at 15,000 rpm to remove precipitates, and only the resulting solution was analyzed by LC-MS / MS. The bioavailability is shown in Table 3.

[0071] [Table 3]

[0072] This experiment demonstrates the process for producing nanoparticles of azithromycin and ciprofloxacin, but also shows the low permeability of substances in the BCS classification class III group (chloroquine, cimetidine, cloxacillin, didanosine, ephedrine, erythromycin, famotidine, fluconazole, folinic acid). (e.g., acid), furosemide, ganciclovir, lisinopril, methotrexate, metformin, nifurtimox, nadolol, nystatin, pravastatin, penicillin, ranitidine, reserpine, tetracycline, valsartan, vancomycin, doxycycline, etc.) or class Hydrophilic substances other than III that require improved absorption in the body (such as chlorpheniramine, clomiphene, clomipramine, dexamethasone, ethinylestradiol, metoclopramide, morphine, and quinine) can also be produced as oral nanoparticles of the present invention by a similar method. In these cases, it has been confirmed that oral nanoparticles can be produced even if ethanol, methanol, acetone, etc. are used as solvents instead of water as needed, or if other bile acids that constitute bile acids are used instead of cholic acid.

[0073] Experimental Example 1. Evaluation of the effect of the nanoparticle formation of the present invention on the decomposition of encapsulated materials. Remdesivir is a type of nucleoside analog and was the first coronavirus treatment approved due to its excellent antiviral effect. Although remdesivir is the most effective treatment for COVID-19, it is currently only available as an injectable drug. This is because, upon ingestion, remdesivir is broken down almost immediately by nucleases in digestive fluids, resulting in a significantly lower bioavailability of the oral formulation compared to the injectable form, which is absorbed into the bloodstream.

[0074] Glutathione, derived from a peptide formed by the peptide bond of three amino acids, possesses powerful antioxidant properties as well as skin-whitening effects. However, when glutathione is ingested, it is immediately broken down by proteolytic enzymes in digestive fluids, and only a very small amount is absorbed, resulting in very low bioavailability of glutathione. Therefore, it is practically difficult to expect the desired skin-whitening effect from oral administration of glutathione, and it is more commonly used as an injection than orally. Due to several drawbacks of injections, research to improve the bioavailability of glutathione has been actively pursued, but the problem of breakdown by digestive enzymes has not yet been solved, and there are no successful examples of developing oral glutathione formulations.

[0075] Therefore, in order to develop an oral formulation with improved bioavailability, the present inventors conducted experiments under various conditions using the technology of the present invention. As a result, they produced orally administered remdesivir nanoparticles using the technology of Example 1 (Figure 6), and produced orally administered glutathione nanoparticles using the technology of Example 2 (Figure 6).

[0076] The manufactured remdesivir nanoparticles and glutathione nanoparticles were orally administered to rats at a dose of 20 mg / kg (drug standard), respectively, and blood samples were collected from the rats at different time intervals. For the control group, the same experiment was performed by orally administering the same amount of the pure bioactive substance (remdesivir or glutathione) used in the nanoparticle manufacturing process to rats. 0.1 mL of blood collected from the rats at different time intervals was mixed with 0.9 mL of acetone, and this mixture was thoroughly mixed using a vortex mixer. The mixed solution was centrifuged at 15,000 rpm to remove precipitates, and only the resulting solution was analyzed by LC-MS / MS. The bioavailability is shown in Table 4.

[0077] [Table 4]

[0078] As can be seen from Table 4, the present invention is an innovative technology that allows physiologically active substances, which were previously only usable as injectable drugs due to the problem of digestion during oral administration, to be formulated as oral drugs, thereby protecting the physiologically active substances from digestive enzymes in the body and significantly improving their bioavailability.

[0079] This experiment illustrates the process for producing nanoparticles of remdesivir and glutathione, but other biochemical substances that are broken down by digestive enzymes in the digestive tract upon oral administration can also be produced as oral nanoparticles of the present invention using a similar method. It was also confirmed that oral nanoparticles can be produced by using ethanol, methanol, acetone, etc., instead of DMSO as the solvent, or by using other bile acids that constitute bile acids instead of cholic acid, if necessary.

[0080] Experimental Example 2. Evaluation of the effect of the nanoparticle formulation of the present invention on the balance of the gut microbiome. Amoxicillin, azithromycin, clindamycin, ceftriaxone, erythromycin, fluconazole, penicillin, and tetracycline are commonly used antibiotics in hospitals. Because they have excellent solubility and permeability, and therefore high bioavailability, they are generally used orally instead of by injection. However, antibiotics that remain in the intestinal lumen after oral ingestion, before being absorbed into the bloodstream, kill surrounding intestinal microorganisms, disrupting the balance of the gut microbiome and causing various digestive disorders such as diarrhea and abdominal pain.

[0081] Therefore, in order to analyze the effect of nanoparticle production of antibiotics on the balance of the intestinal microbiome, the inventors produced oral azithromycin nanoparticles using the technique of Example 3. The produced nanoparticles were orally administered to rats at a dose of 20 mg / kg, and blood was collected from the rats at different time intervals. At the same time, a comparative experiment was conducted by orally administering the same amount of azithromycin to a control group of rats. 0.1 mL of blood collected from the rats at different time intervals was mixed with 0.9 mL of acetone, and this mixed solution was thoroughly mixed using a vortex mixer. The mixed solution was centrifuged at 15,000 rpm to remove the precipitate, and only the obtained solution was analyzed by LC-MS / MS. The bioavailability is shown in Table 5.

[0082] [Table 5]

[0083] Azithromycin nanoparticles produced by the method of Example 3 were orally administered to each rat at a dose of 5 mg / kg (drug reference) daily for 5 days, and the gut microbiome was then analyzed by metagenomic sequencing. A control group was administered the same amount of the original bioactive substance (azithromycin) orally to the animals, and the same experiment was performed together. The results showed that, compared to the azithromycin-administered group, the azithromycin nanoparticle-administered group did not experience any disruption to the gut microbiome balance because the antibiotic azithromycin was not exposed to the gut microbiome (Figure 7).

[0084] Experimental Example 3: Evaluation of the effect of nanoparticle formation according to the present invention on the efficacy of drugs. The inventors confirmed the structural stability and improved bioavailability of niclosamide nanoparticles produced by the method of Example 1, and then confirmed the therapeutic effect of oral niclosamide nanoparticles against viral infection in in vivo animal experiments. All experiments were conducted in BSL Level 3. For this purpose, SH101 hamsters, a coronavirus animal model, were first prepared, and then the lethal dose (TCID) was administered. 50 Participants were infected with the SARS-CoV-2 virus. One hour after viral infection, niclosamide nanoparticles were administered orally daily at a dose of 5 or 10 mg / kg (drug standard). A control group was administered orally either 10 mg / kg of the original bioactive substance niclosamide or PBS (phosphorate-buffered saline) for comparison. Subsequently, infection symptoms (weight loss, decreased body temperature after fever, death, etc.) were observed during a 7-day day-post-infection period (dPI) (Figure 8).

[0085] The same experiment was conducted using hACE2 transgenic mice, another animal model of coronavirus, and infection symptoms (weight loss, decreased body temperature after fever, death, etc.) were observed during the 7-day dpi (day-post-infection) period (Figure 9).

[0086] Lung tissue samples taken at 3 dpi and 7 dpi were stained with hematoxyline and eosin (H&E) and then observed under a microscope (Figure 10). Furthermore, the amount of SARS-CoV-2 virus in the lung tissue was quantitatively analyzed by performing PCR against the SARS-CoV-2 virus envelope (E) gene (forward primers: 5'-GCCTCTTCTCGTTCCTCATCAC-3', reverse primer: 5'-AGCAGCATCACCGCCATTG-3') at 50°C for 2 minutes, then at 95°C for 2 minutes, followed by 40 cycles of [95°C for 15 seconds, 60°C for 30 seconds], and finally RT-qPCR at 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 45 seconds (Figure 10).

[0087] As described above, experimental results using SH101 hamsters and hACE2 transgenic mice, which are animal models of coronavirus, showed that in the niclosamide nanoparticle administration group, the bioavailability of the antiviral agent niclosamide was improved by delivering it to infected tissue, and by killing the SARS-CoV-2 virus (Figure 10C, D), it was confirmed that, unlike the PBS administration group and the niclosamide administration group, it had a significant therapeutic effect against SARS-CoV-2 infection. [Industrial applicability]

[0088] The nanoparticles of this invention represent a revolutionary invention that can transform all drugs currently used as injectable formulations into oral formulations. This specification includes the disclosures in the following appendices 1 to 10. <Note 1> An oral nanoparticle characterized by being composed of a bioactive substance located in the center and bile acids surrounding the bioactive substance, wherein the bioactive substance and bile acids are non-covalently bonded. <Note 2> The oral nanoparticles according to Appendix 1, characterized in that the bioactive substance is present in an amount of 10-90% by weight and the bile acid is present in an amount of 10-90% by weight. <Note 3> The aforementioned physiologically active substances include 5-fluorouracil, remdesivir, azithromycin, paclitaxel, doxorubicin, oxaliplatin, phenylephrine hydrochloride, glutathione, acetazolamide, amphotericin, aprepitant, azathioprine, chlorothiazide, chlorthalidone, ciprofloxacin, colistin, and cyclosporine A.A) Digoxin, docetaxel, furosemide, etravirine, famotidine, griseofulvin, hydrochlorothiazide, mebendazol, methotrexate, neomycin, niclosamide, nystatin, ritonavir, albendazole, artemther, chlorpromazine, efavirenz, glibenclamide, ivermectin, lopinavir, mefloxacin Mefloquine, retinol, spironolactone, sulfadiazine, sulfasalazine, triclabendazole, acyclovir, amoxicillin, bidisomide, biperiden, captopril, cefazolin, chloroquine, cimetidine, cloxacillin, didanosine, ephedrine, erythromycin, famotidine, fluconazole, folinic acidAcid), furosemide, ganciclovir, lisinopril, methotrexate, metformin, nifurtimox, nadolol, nystatin, pravastatin, penicillin, ranitidine, reserpine, tetracycline, valsartan The oral nanoparticles described in Appendix 1 are characterized by being at least one selected from the group consisting of tan, vancomycin, doxycycline, chlorpheniramine, clomiphene, clomipramine, dexamethasone, ethinylestradiol, metoclopramide, morphine, and quinine. <Note 4> The oral nanoparticles described in Appendix 1, characterized in that the bile acid is at least one selected from the group consisting of cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, ursodeoxycholic acid, tauroursodeoxycholic acid, hyodeoxycholic acid, 7-oxolithocholic acid, iododeoxycholic acid, iodine cholic acid, taurolithocholic acid, glycoursodeoxycholic acid, taurocholic acid, and glycocholic acid. <Note 5> (a) The step of dissolving the physiologically active substance in a solvent, (b) The step of dissolving bile acids in a solvent, (c) A step of mixing a physiologically active substance solution with a bile acid solution, (d) A method for producing oral nanoparticles, comprising the step of freeze-drying a mixed solution of a mixed physiologically active substance and a bile acid. <Note 6> A method for producing oral nanoparticles as described in Appendix 5, characterized in that the physiologically active substance solution and the bile acid solution are mixed in a volume percentage ratio of 10-90:90-10. <Note 7> A method for producing oral nanoparticles as described in Appendix 5, characterized by further comprising the step of mixing a physiologically active substance solution with a bile acid solution, and then subjecting the mixture of the physiologically active substance and bile acid to low-temperature treatment or salt treatment. <Note 8> The method for producing oral nanoparticles as described in Appendix 7, characterized in that the low-temperature treatment is carried out while stirring a mixture of a physiologically active substance and bile acid, and lowering the temperature from room temperature to a temperature in the range of -20 to +20°C at a rate of 1°C / min or less. <Note 9> The aforementioned salt treatment involves stirring a mixture of physiologically active substances and bile acids, and then adding Na + Mg 2+ Li + Ca 2+ , and Fe 2+ A method for producing oral nanoparticles as described in Appendix 7, characterized by adding a salt selected from the group to a range of 0.1 to 20 M. <Note 10> An oral nanoparticle composition comprising oral nanoparticles described in any one of the appendices 1 to 4.

Claims

1. It consists of a bioactive substance located in the center and bile acids surrounding the bioactive substance, and the bioactive substance and bile acids are non-covalently bonded. The aforementioned physiologically active substance is selected from the group consisting of niclosamide, paclitaxel, and remdesivir. The aforementioned bile acid is cholic acid. Oral nanoparticles characterized in that the physiologically active substance is present in an amount of 10 to 90% by weight, and the bile acid is present in an amount of 10 to 90% by weight.

2. It consists of a bioactive substance located in the center and bile acids surrounding the bioactive substance, and the bioactive substance and bile acids are non-covalently bonded, Na ＋ and Fe ２＋ It further contains a salt containing ions selected from a larger group, The aforementioned physiologically active substance is selected from the group consisting of 5-fluorouracil, metformin, azithromycin, ciprofloxacin, and glutathione. The aforementioned bile acid is cholic acid. Oral nanoparticles characterized in that the physiologically active substance is present in an amount of 10 to 90% by weight, and the bile acid is present in an amount of 10 to 90% by weight.

3. A method for producing orally administered nanoparticles, (a) The step of dissolving the physiologically active substance in a solvent, (b) The step of dissolving bile acids in a solvent, (c) The step of mixing the physiologically active substance solution and the bile acid solution, (d) The step of freeze-drying the mixed solution of the mixed physiologically active substance and bile acid, The aforementioned physiologically active substance is selected from the group consisting of niclosamide, paclitaxel, and remdesivir. The aforementioned bile acid is cholic acid. The physiologically active substance solution and the bile acid solution are mixed such that the physiologically active substance in the oral nanoparticles is 10 to 90% by weight and the bile acid is 10 to 90% by weight. A method for producing orally administered nanoparticles.

4. A method for producing orally administered nanoparticles, (a) The step of dissolving the physiologically active substance in a solvent, (b) The step of dissolving bile acids in a solvent, (c) The step of mixing the physiologically active substance solution and the bile acid solution, (d) Add Na to the mixture of the physiologically active substance solution and the bile acid solution. ＋ and Fe ２＋ A salt treatment step in which a salt formed by bonding with an ion selected from a group is added to a range of 0.002 to 0.26 M, (e) The step of freeze-drying the mixed solution of the mixed physiologically active substance and bile acid, The aforementioned physiologically active substance is selected from the group consisting of 5-fluorouracil, metformin, azithromycin, ciprofloxacin, and glutathione. The aforementioned bile acid is cholic acid. The physiologically active substance solution and the bile acid solution are mixed such that the physiologically active substance in the oral nanoparticles is 10 to 90% by weight and the bile acid is 10 to 90% by weight. A method for producing orally administered nanoparticles.

5. A pharmaceutical composition for antibacterial, antiparasitic, anticancer, antiviral, antidiabetic, antioxidant, antibiotic, or neuroprotective purposes, comprising oral nanoparticles as described in claim 1 or 2.

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