CN113975233B - Preparation method of enteric stable ursodeoxycholic acid nanosuspension - Google Patents
Preparation method of enteric stable ursodeoxycholic acid nanosuspension Download PDFInfo
- Publication number
- CN113975233B CN113975233B CN202111230180.9A CN202111230180A CN113975233B CN 113975233 B CN113975233 B CN 113975233B CN 202111230180 A CN202111230180 A CN 202111230180A CN 113975233 B CN113975233 B CN 113975233B
- Authority
- CN
- China
- Prior art keywords
- ursodeoxycholic acid
- nanosuspension
- poloxamer
- particle size
- suspension
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
- A61K47/38—Cellulose; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
- A61K9/145—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
- A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Inorganic Chemistry (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Dispersion Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention belongs to the field of medicines, and particularly relates to ursodeoxycholic acid nanosuspension as well as a preparation method and application thereof. Specifically, the ursodeoxycholic acid nanosuspension is prepared by a medium grinding process, the appearance and physical stability of the ursodeoxycholic acid nanosuspension are improved by selecting specific auxiliary materials, and the intestinal stability of the preparation is improved. The nano suspension is used as an intermediate product to be further prepared into oral suspension or dry suspension, can be successfully applied to digestive tract diseases caused by cholestasis, and fully exerts the pharmacological action of ursodeoxycholic acid.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a preparation method and application of ursodeoxycholic acid nanosuspension.
Background
Ursodeoxycholic acid, namely 3 alpha, 7 beta-dihydroxy-5 beta-cholestane-24-acid, is endogenous bile acid of human bodies. Ursodeoxycholic acid water has low solubility, belongs to class II Biopharmaceutical Classification System (BCS) medicine, and can be used for treating cholesterol calculus, hyperlipidemia, bile secretion disorder disease, primary biliary cirrhosis, chronic hepatitis, bile reflux gastritis and preventing liver transplantation acute rejection. After oral administration, ursodeoxycholic acid absorption occurs in the entire small intestine (approximately 80%) and in parts of the colon (approximately 20%). Once absorbed, ursodeoxycholic acid is combined with glycine or taurine in the liver to become conjugated bile acid and stored in the gallbladder, which contracts after a meal and is excreted into the duodenum. Approximately 95% of the bound bile acids are reabsorbed into the portal vein by active transport or passive diffusion at the terminal ileum and then efficiently taken up by the liver from the portal vein blood, and newly synthesized and reabsorbed bile acids are again secreted into the biliary tract, and this transport of bile acids between the intestine and the liver is called enterohepatic circulation of bile acids. In addition, the bound bile acids can also be broken down by intestinal enzymes in the ileum into free ursodeoxycholic acid, which is then absorbed by the intestine and taken up by the liver.
As a medicine for enterohepatic circulation, ursodeoxycholic acid only slightly enters the systemic blood circulation, so the systemic blood concentration is very low. In addition, the therapeutic effect of ursodeoxycholic acid is related to the concentration of ursodeoxycholic acid in bile, and no direct correlation is established with the systemic circulating blood concentration, so that the clinical significance of monitoring the blood concentration is yet to be researched.
Nanocrystal technology is suitable for the formulation and delivery of class II and class IV Biopharmaceutical Classification System (BCS) drugs (low solubility drugs). The production method of drug nanocrystals is typically media milling or high pressure homogenization, reducing the drug particle size from coarse particles to nanoparticles; in addition, the drug in a molecularly dispersed state is caused to construct drug nanocrystals by agglomeration by non-solvent precipitation or different kinds of liquid atomization techniques. To improve efficiency, several techniques are often combined, such as media milling in combination with high pressure homogenization, non-solvent precipitation in combination with spray drying, and the like.
The intestinal tract is a very complex environment, and similar to other oral nano preparations (lipid nanoparticles, polymer micelles, microemulsions, liposomes and the like), the nanosuspension (or nanocrystal) entering the intestinal tract also faces the complex environment and spans various absorption barriers, so that the nanosuspension (or nanocrystal) can be effectively transported to enter the small intestine to be absorbed. Various electrolytes are contained in intestinal fluid, which negatively affects the surface charge of nanoparticles, resulting in reduced stability of nanoparticles, and aggregation may eventually occur in intestinal fluid (see Shahbazi et al, current Drug Metabolism,2013, 14 (1), 28-56).
One of the key factors determining the quality of the nanosuspension is a stabilizer, including the type and dosage of the stabilizer and the strength of interaction between the stabilizer and an active drug, which becomes a key element of the stability of the nanosuspension in the intestinal tract, and the complex intestinal environment may cause the stabilizer molecules on the surface of the nanosuspension to fall off or be replaced by other molecules, which causes the aggregation of the active drug nanoparticles and influences the subsequent intestinal absorption efficacy. Due to the fact thatThus, the stability of nanosuspensions in the intestinal tract is one of the key evaluation criteria (see 1.Wang et al, journal of Colloid and Interface Science,2017, 507 (1), 119-130, 2.Jain et al, AAPS PharmSciTech,2018, 19, 3152-3164
Et al, polymers,2019, 11 (10), 1632).
Some reports on ursodeoxycholic acid nanosuspensions exist in the prior art, but the technologies reported in the documents have poor control effect on particle size, have unknown gastrointestinal stability, and cannot prove that the oral administration is effective only by the reported data, and in addition, the reports also use a surfactant with certain toxic action, which are specifically as follows:
document 1 (Li et al, pharmaceutical Development and Technology,2014, 19 (6), 662-670) reports two preparation processes, a simple high pressure homogenization process and a non-solvent precipitation combined high pressure homogenization process, for the preparation of ursodeoxycholic acid (UDCA) Nanosuspensions (NS). Poloxamer 188 (P188) is selected as a single stabilizer, the weight ratio of P188 to the drug is considered to significantly influence the particle size of UDCA-NS, the particle size is in negative correlation with the proportion of P188 to the drug, the ideal particle size can be obtained when the dosage of P188 to the drug is 10-20 times, and the dosage of P188 respectively reaches 10%, 20%, 40% and 80% (w/v) relative to 1% (w/v) of UDCA in an experiment, namely the dosage of P188 is at least 10 times of that of UDCA. In reference 1, the preparation of UDCA-NS is followed by the addition of hypromellose HPMC (0%, 50%, 100% or 200% relative to the weight of UDCA), followed by freezing of UDCA-NS under liquid nitrogen conditions. It can be seen that hypromellose is used as a lyoprotectant in document 1 to prepare a lyophilized formulation of a nanosuspension. The particle size can be effectively reduced only by singly using P188 with the dosage of more than 10 times that of UDCA, which is very low in efficiency, and the dosage of the stabilizer can be reduced on the premise of ensuring safety and effectiveness, so that the method is the direction of efforts of professionals.
Literature 2 (Ma et al, pharmaceutical Development and Technology,2016, 21 (2), 180-188) reports the effect of the freeze-drying process on the redispersibility of ursodeoxycholic acid Nanocrystals (NC). Wherein, the stabilizer comprises poloxamer (P188, P407), polyvidone K30 (PVP K30), polyoxyethylene hydrogenated castor oil (RH 40), hydroxypropyl methylcellulose (HPMC, methocel E15), polysorbate 80, sodium carboxymethyl starch (CMS-Na), vitamin E polyethylene glycol succinate (TPGS), sodium Dodecyl Sulfate (SDS), polyethylene glycol 4000 (PEG 4000), trehalose, microcrystalline cellulose, sodium carboxymethylcellulose and the like. Several lyoprotectants are also used, including sucrose, glucose, trehalose, lactose, mannitol, sorbitol, and polyethylene glycol 4000. This document emphasizes that the PVP K30/sucrose system is more protective, better protecting the UDCA-NC crystal growth performance than other systems, and suggests that the synergy of the polymeric stabilizer with the lyoprotectant affects the redispersibility of NC. The document mainly focuses on how to reduce the particle size of ursodeoxycholic acid, and overcomes the limitation of the traditional particle size reduction technology, but the particle size is not the only index of oral absorption, only the particle size is reduced, and professionals do not consider that the oral absorption can be improved.
Document 3 (yupeng, "redispersion of nanocrystals of Chinese medicinal ingredients brain-targeted drug delivery system," Shanghai medicine university, med. Pharmacology (doctor) thesis, 2015) discloses the use of surface active stabilizers (poloxamer 188, polysorbate 80, polyoxyethylene (hydrogenated) castor oil, etc.) and polymer stabilizers (hypromellose, povidone K30, etc.) for the preparation of ursodeoxycholic acid nanocrystals, respectively. Unfortunately, a single stabilizer does not work well. The surface active stabilizer with the concentration of 10-50% (w/w relative to the mass of the medicine) and the average success rate of redispersion and reconstruction of the ursodeoxycholic acid nanocrystal are not more than 15 percent; the average success rate of redispersion and reconstruction of the nano-crystal is not more than 25% under the same concentration of the polymer stabilizer.
Chinese patent CN101606906A discloses a nano suspension of ursodeoxycholic acid, wherein SDS and PVP K30 are used together as a stabilizer, and the weight ratio of the SDS to the PVP K30 is preferably 1: 1-1: 6. Among them, SDS is an anionic surfactant, which has the functions of solubilization, emulsification, moistening and disintegration, etc. and can enhance the absorption of some medicines by mucosa. However, SDS has irritation to skin, eyes, mucous membranes, upper respiratory tract and stomach, especially the mucous membranes of digestive tracts of children are delicate and more vulnerable to SDS, and there is a certain potential safety hazard when SDS is used as an auxiliary agent in oral drugs.
Therefore, the prescription and the preparation process of the ursodeoxycholic acid nanosuspension are designed, so that the ursodeoxycholic acid nanosuspension has good safety and intestinal stability, meets the requirement of oral administration of the ursodeoxycholic acid nanosuspension, effectively and safely plays the pharmacological action of the ursodeoxycholic acid, and is a technical problem which is urgently needed to be solved in the field.
Disclosure of Invention
In order to overcome the limitations and defects of the prior art, the inventors of the present application conducted a great deal of research, and recognized that the disadvantage of using a single stabilizer in the preparation process of ursodeoxycholic acid nanosuspension exists, and found that using a specific composite stabilizer in the preparation process of ursodeoxycholic acid nanosuspension can significantly reduce the amount of the stabilizer, so that the ursodeoxycholic acid nanosuspension has good intestinal stability and safety, and lays a foundation for providing a ursodeoxycholic acid nanosuspension which can be directly orally administered clinically.
The invention aims to improve the prescription composition of the ursodeoxycholic acid nanosuspension, enhance the drug effect of the ursodeoxycholic acid after oral administration, ensure the stable existence of the ursodeoxycholic acid in the form of nanoparticles on the basis of ensuring the external indexes (including particle size, charge and crystal form) of the basic nanosuspension, indicate better intestinal transmembrane transport efficacy, enhance the anti-cholestasis activity and further highlight the advantages of the invention compared with the prior art. The compound stabilizer is selected, and the suspension can be directly orally taken, or be re-dissolved and orally taken after freeze drying or spray drying, so that the excellent physicochemical property of the suspension system can be ensured, and the application range of the preparation is widened; in addition, stabilizers with potential safety hazards, such as sodium dodecyl sulfate, polysorbate and the like, are eliminated, a surfactant poloxamer with good safety is selected to form a composite stabilizer with hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone or polyvinyl alcohol respectively, the synergistic effect is achieved, and compared with other composite stabilizers, the composite stabilizer has a more excellent technical effect.
The ursodeoxycholic acid nanosuspension has a simple preparation process, is easy to industrialize and convenient for children to take, so that the compliance of children with the medicine can be improved. Ursodeoxycholic acid belongs to indissolvable drugs, and the preparation can improve the solubility of the drugs and improve the curative effect and the medication safety of the drugs.
Stabilizers are essential to avoid aggregation and sedimentation of the active drug nanoparticles during preparation and storage of the nanocrystal suspension. The invention optimizes and screens the type and dosage proportion of the stabilizer aiming at the ursodeoxycholic acid nano suspension, optimizes and screens a large number of different types of surfactants and polymers, and finally finds that the composite stabilizer formed by combining poloxamer, hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone and polyvinyl alcohol in a specific proportion can play the synergistic effect of the poloxamer and the hydroxypropyl methylcellulose, the hydroxypropyl cellulose, the povidone and the polyvinyl alcohol, thereby maximally stabilizing the ursodeoxycholic acid nano suspension.
Wherein the dosage ratio of the ursodeoxycholic acid, the poloxamer and the hydroxypropyl methylcellulose is 5: 1-5: 4: 2; the dosage ratio of the ursodeoxycholic acid, the poloxamer and the hydroxypropyl cellulose is 5: 1-5: 2: 3; the dosage ratio of the ursodeoxycholic acid, the poloxamer and the polyvidone K30 is 5: 1: 0.25-5: 3: 1; the dosage ratio of the ursodeoxycholic acid, the poloxamer and the polyvinyl alcohol is 5: 1: 0.5-5: 4: 1.5.
The ursodeoxycholic acid raw material used in the present invention has an average particle diameter (D90) of 72 μm or less.
The poloxamer used in the invention contains 70-84% of oxyethylene, has average molecular weight of 7680-14600, preferably poloxamer 188 and poloxamer 407, and more preferably poloxamer 188.
The viscosity of the hypromellose used in the invention is 2.4-7.2 mPas, the determination conditions are that the temperature is 20 ℃, the concentration is 2 percent w/v, and the solvent is water; selected from hypromellose E3, hypromellose E5, and hypromellose E6.
The polyvinyl alcohol used in the present invention is preferably polyvinyl alcohol 0588.
The average grain diameter of the suspension formed by the ursodeoxycholic acid nanocrystal in water is about 60-800 nm; preferably 80 to 600nm; more preferably 100 to 500nm; most preferably 120 to 400nm.
The ursodeoxycholic acid nanosuspension can exist in the form of suspension or dry suspension, the dry suspension can contain pharmaceutically acceptable drying protective agents, and the types of the drying protective agents comprise mannitol, sorbitol, trehalose, sucrose, glucose, dextrin, microcrystalline cellulose or lactose.
The preparation method of the ursodeoxycholic acid nanosuspension is a medium grinding method. The specific process comprises the following steps: weighing the composite stabilizer, adding water to completely dissolve the composite stabilizer, transferring the composite stabilizer into a grinding tank of a ball mill, adding the ursodeoxycholic acid raw material and zirconium oxide grinding beads with the diameter of 0.1mm, tightly covering the cover of the grinding tank, starting the ball mill, setting the rotating speed to be 600-1200 rpm, starting the ball mill, pouring out grinding liquid after grinding for 4-8 hours, and filtering to remove the zirconium oxide grinding beads to obtain the ursodeoxycholic acid nano suspension.
The preparation method of the ursodeoxycholic acid nano dry suspension is a drying method, and comprises a spray drying method or a freeze drying method.
Wherein, the spray drying process comprises the following steps: taking the suspension, adding a drying protective agent, uniformly mixing, placing in a spray drying instrument, setting the spray drying conditions as follows, wherein the inlet temperature is as follows: 120-140 ℃, outlet temperature: 50-60 ℃, atomization pressure: 7-24 kPa and feed rate: 2.0-5 mL/min, and then the ursodeoxycholic acid nano dry suspension (nano spray-dried powder) is obtained.
The freeze drying process comprises the following steps: and (3) uniformly mixing the suspension with a drying protective agent, placing the mixture in a freeze dryer, pre-freezing for 4 hours at the temperature of minus 40 ℃, then setting the temperature of minus 20 ℃, starting a vacuum pump, and freeze-drying for 48 hours to obtain the ursodeoxycholic acid nano dry suspension, namely nano freeze-dried powder.
The ursodeoxycholic acid nanosuspension can be added with a sweetening agent and an aromatic agent to improve the compliance of children taking.
The flavoring agent used in the present invention is selected from banana, cherry, grape, grapefruit, mixed berry, orange vanilla, chocolate, mint, raspberry, strawberry flavor; the sweetener is selected from acesulfame potassium, ammonium glycyrrhizinate, stevioside, aspartame, glucose, maltitol, xylitol, saccharin sodium, sorbitol, mannitol, sucralose, sucrose, and fructose.
The ursodeoxycholic acid nanosuspension can be used for treating cholestatic diseases. Cholestatic disorders selected from, but not limited to, childhood/infant cholestasis, pregnant woman cholestasis, gall bladder cholesterol stones, primary sclerosing cholangitis, primary biliary cholangitis, autoimmune hepatitis, ulcerative colitis, biliary tract locking, fatty liver and overlap syndrome; preferably children/infants cholestasis, gall bladder cholesterol stones, primary sclerosing cholangitis, primary biliary cholangitis; more preferably, cholestasis in children/infants, cholecystolithiasis, primary biliary cholangitis.
The meanings of the english abbreviations mentioned in the present application are as follows: alanine aminotransferase ALT, alkaline phosphatase ALP, gamma-glutamyl transpeptidase GGT, immunoglobulin IgM, total bilirubin TBIL, hydroxyproline HyP, autotaxin ATX, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer Soluplus, polyoxyethylene hydrogenated castor oil (RH 40), vitamin E polyethylene glycol succinate (TPGS), and the D90 particle diameter is the equivalent diameter (average particle diameter) of the largest particle when the cumulative distribution in the particle size distribution curve is 90%.
Drawings
FIG. 1 is a graph showing the particle size distribution of ursodeoxycholic acid nanosuspension (example 44)
FIG. 2 is a transmission electron micrograph of ursodeoxycholic acid nanosuspension (example 44)
FIG. 3 is the powder X-ray diffraction pattern of ursodeoxycholic acid nanosuspension (examples 44, 47-49)
FIG. 4 is a graph showing the change in mean particle size of ursodeoxycholic acid nanosuspensions (examples 56 to 67) incubated with simulated intestinal fluid for different periods of time
FIG. 5 is a graph showing the change in mean particle size of ursodeoxycholic acid nanosuspensions (examples 68 to 77) incubated with simulated intestinal fluid for different periods of time
FIG. 6 is a graph showing the change in mean particle size of ursodeoxycholic acid nanosuspensions (examples 78 to 87) incubated with simulated intestinal fluid for different periods of time
FIG. 7 is a graph showing the change in mean particle size of ursodeoxycholic acid nanosuspensions (examples 88-97) incubated with simulated intestinal fluid for different periods of time
FIG. 8 is a graph showing the change in mean particle size of ursodeoxycholic acid nanosuspensions (examples 98 to 104) incubated with simulated intestinal fluid for different periods of time
FIG. 9 is a graph showing the effect of ursodeoxycholic acid nanosuspensions (examples 58, 72, 80, 92 and examples 105, 106) on MDCK cell survival
FIG. 10A shows the ALT measurement results of ursodeoxycholic acid suspensions (examples 107 to 112)
FIG. 10B shows ALP measurement results of ursodeoxycholic acid suspensions (examples 107 to 112)
FIG. 10C shows the GGT measurement results of ursodeoxycholic acid suspensions (examples 107 to 112)
FIG. 10D shows the IgM measurement results of ursodeoxycholic acid suspensions (examples 107 to 112)
FIG. 10E shows the results of TBIL measurement of ursodeoxycholic acid suspensions (examples 107 to 112)
FIG. 10F shows the results of HyP measurement of ursodeoxycholic acid suspensions (examples 107 to 112)
FIG. 10G shows the results of ATX measurement of ursodeoxycholic acid suspensions (examples 107 to 112)
FIG. 11 is a diagram showing the appearance of the liver and gallbladder of a suspension of ursodeoxycholic acid (example 109)
FIG. 12 is a photograph of pathological section of liver tissue of ursodeoxycholic acid nanosuspension (example 109)
Detailed Description
The present invention is described in further detail below with reference to examples, but the present invention is not limited thereto.
Examples 1 to 4
Weighing 4 parts of poloxamer 1880.1g and hydroxypropyl cellulose 0.1g respectively, placing the weighed materials into ball milling tanks respectively, adding 10mL of deionized water to dissolve a stabilizing agent, then adding 0.5g of ursodeoxycholic acid raw materials with different particle sizes (D90) and 20g of zirconia milling beads (with the diameter of 0.1 mm), covering the ball milling tanks, opening the ball mills, setting the rotating speed to 900rpm, grinding the materials for 1.5h, closing the ball mills, pouring out grinding fluid, removing the zirconia milling beads, and respectively measuring the particle size of the grinding fluid by adopting a dynamic light scattering instrument.
| Numbering | Ursodeoxycholic acid raw material D90 particle size (mum) | Particle size of suspension after grinding (nm) |
| Example 1 | 156 | 943 |
| Example 2 | 116 | 881 |
| Example 3 | 72 | 487 |
| Example 4 | 45 | 334 |
The results are shown in the table above, the particle size of ursodeoxycholic acid raw material is reduced, and the particle size of the prepared nano suspension is smaller under the same process conditions, which indicates that the grinding efficiency can be improved by reducing the particle size of the raw material drug. The D90 particle size of ursodeoxycholic acid used in the invention is less than 72 microns.
Examples 5 to 31
Weighing the stabilizer according to the formula shown in the table, placing the stabilizer in a ball milling tank, adding 10mL of deionized water to dissolve or uniformly disperse the stabilizer, adding 20g of 0.1mm zirconium oxide grinding beads and ursodeoxycholic acid, sealing the ball milling tank with a cover, starting the ball milling tank, setting the rotating speed to be 900rpm, grinding for 1.5h, pouring out the grinding fluid, and filtering out the zirconium oxide grinding beads to obtain the ursodeoxycholic acid nano suspension.
The particle size, polydispersity and Zeta potential of the nanosuspension were measured by dynamic light scattering and the results were as follows:
as can be seen from the above table, the concentration of ursodeoxycholic acid in the suspension is within the range of 0.5% -7.5%, the dosage ratio of the ursodeoxycholic acid to the stabilizer is within the range of 1: 6-5: 1, the particle size of the prepared nano suspension is 178-655 nm, and the polydispersity is 0.17-0.54. In addition, zeta potential results indicate that all nanocrystals exhibit a negative charge, which is beneficial to the physical stability of the formulation.
Examples 32 to 41
Weighing two stabilizers according to the formula shown in the following table, placing the two stabilizers in a ball milling tank, adding 10mL of deionized water, uniformly stirring, weighing ursodeoxycholic acid, adding the ursodeoxycholic acid, uniformly stirring, adding 20g of 0.1mm zirconium oxide grinding beads, covering and sealing, setting the rotating speed to be 900rpm, starting a ball mill, grinding for 1.5h, pouring out grinding liquid, and filtering out the zirconium oxide grinding beads to obtain the ursodeoxycholic acid nano suspension.
| Example 32 | Ursodeoxycholic acid 0.5g | Polysorbate 80.1g | Hydroxypropyl methylcellulose E5.1 g |
| Example 33 | Ursodeoxycholic acid 0.5g | Poloxamer 407 0.2g | Hydroxypropyl methylcellulose E6.1 g |
| Example 34 | Ursodeoxycholic acid 0.5g | Polysorbate 80.2g | Hydroxypropyl methylcellulose E6.1 g |
| Example 35 | Ursodeoxycholic acid 0.5g | TPGS 0.1g | Polyvinyl alcohol 0588.1g |
| Example 36 | Ursodeoxycholic acid 0.5g | Poloxamer 188.2g | Hydroxypropyl methylcellulose E3.1 g |
| Example 37 | Ursodeoxycholic acid 0.5g | Poloxamer 188.2g | Polyvinyl alcohol 0588.1g |
| Example 38 | Ursodeoxycholic acid 0.5g | Poloxamer 407 0.2g | Hydroxypropyl methylcellulose E3.1 g |
| Example 39 | Ursodeoxycholic acid 0.5g | Poloxamer 407 0.2g | Polyvinyl alcohol 0588.1g |
| Example 40 | Ursodeoxycholic acid 0.5g | Soluplus 0.2g | Povidone K30.1 g |
| EXAMPLE 41 | Ursodeoxycholic acid 0.5g | Polysorbate 80.1g | Povidone K30.1 g |
The particle size, polydispersity and Zeta potential of the nanosuspension were measured by dynamic light scattering and the results were as follows:
| particle size (nm) | Polydispersity index | Zeta potential (mV) | |
| Example 32 | 436.5 | 0.234 | -22.2 |
| Example 33 | 287.5 | 0.159 | -16.1 |
| Example 34 | 275.9 | 0.212 | -18.8 |
| Example 35 | 415.6 | 0.273 | -22.8 |
| Example 36 | 298.5 | 0.221 | -24.2 |
| Example 37 | 358.0 | 0.264 | -19.9 |
| Example 38 | 581.0 | 0.370 | -15.6 |
| Example 39 | 270.3 | 0.152 | -14.4 |
| Example 40 | 272.2 | 0.226 | -25.4 |
| EXAMPLE 41 | 368.1 | 0.310 | -15.3 |
The results show that the particle size of the ursodeoxycholic acid nano suspension prepared by different stabilizer types and dosages is 270-581 nm, the polydispersity is 0.15-0.37, and the prepared nano suspension has narrow particle size distribution and shows negative charges.
Examples 42 to 43
Weighing ursodeoxycholic acid and a stabilizer according to the formula in the table, placing the weighed ursodeoxycholic acid and the stabilizer in a ball mill tank, adding 20g of 0.1mm zirconium oxide grinding beads and 10mL of deionized water, sealing the ball mill tank with a cover, starting the ball mill, setting the rotating speed to 900rpm, grinding the mixture for 1.5h, taking out a small amount of grinding suspension for measuring the particle size, and continuously grinding the rest for 2.5h. After removing the zirconia beads by filtration, the particle size and polydispersity of the nanosuspension were determined by dynamic light scattering, the results of which are shown in the table below.
Research results show that the grinding time is increased from 1.5h to 4h, the particle size and the polydispersity of the prepared nano suspension are obviously reduced, and the particle size distribution is obviously improved.
Examples 44 to 52
The preparation of examples 44 to 52 is as follows:
weighing the stabilizer according to the formula shown in the table, adding water to completely dissolve the stabilizer, transferring the stabilizer into a ball mill grinding tank, adding ursodeoxycholic acid and zirconium oxide grinding beads with the diameter of 0.1mm, covering the grinding tank, setting the rotating speed at 600-1200 rpm, and grinding for 4-8 h to obtain the nano suspension.
The nanosuspensions of examples 47, 48 and 52 were taken, the corresponding drying protector was added according to the above formula, mixed well, spray dried using a Buchi B-290 spray dryer, the inlet temperature was set: 120-140 ℃, outlet temperature: 50-60 ℃, atomization pressure: 7-24 kPa and feed rate: 2.0-5 mL/min to obtain the nanometer spray-dried powder.
Taking the nano suspension of the embodiments 44-46 and 49-51, adding the corresponding drying protective agent according to the formula in the table, pre-freezing the mixture in a Labconco freeze dryer at minus 40 ℃ for 4h, then starting a vacuum pump, and freeze-drying the mixture at minus 20 ℃ for 48h to obtain the nano freeze-dried powder.
Taking the dry suspension powder, adding a proper amount of water, uniformly stirring and filtering, and then measuring the particle size, the polydispersity index and the Zeta potential by using a dynamic light scattering method, wherein the results are shown in the following table:
| particle size (nm) | Polydispersity index | Zeta potential (mV) | |
| Example 44 | 246.5 | 0.207 | -20.4 |
| Example 45 | 264.1 | 0.121 | -22.2 |
| Example 46 | 259.4 | 0.149 | -25.9 |
| Example 47 | 196.7 | 0.204 | -28.5 |
| Example 48 | 299.0 | 0.256 | -27.5 |
| Example 49 | 230.8 | 0.119 | -18.5 |
| Example 50 | 200.0 | 0.136 | -18.3 |
| Example 51 | 207.6 | 0.135 | -17.4 |
| Example 52 | 224.7 | 0.187 | -23.1 |
The results show that the particle sizes of examples 44-52 are all in the nanometer range, the polydispersity is less than 0.3, and the particle size distribution is uniform (the particle size distribution diagram of example 44 is shown in fig. 1), which indicates that the ursodeoxycholic acid nano dry suspension can be successfully prepared by adopting the freeze drying or spray drying process.
The dry suspension powder of example 44 was diluted with distilled water, mixed well, pipetted in appropriate amount onto a copper mesh, stained with uranyl acetate, air dried, and observed on a JEM1200EX electron microscope. The results (as shown in fig. 2) show that ursodeoxycholic acid nanocrystals are elliptical in shape.
And (3) taking appropriate amount of the nano powder in the examples 44, 47-49 and ursodeoxycholic acid raw material powder, respectively placing the powders in grooves of a sample rack matched with an X-ray powder diffractometer, compacting and flattening the sample, and carrying out X-ray powder diffraction measurement. The results (as shown in fig. 3) show that the crystal forms of the nanosuspensions of examples 44 and 47 to 49 are not obviously changed compared with the ursodeoxycholic acid raw material; the crystallinity is in the range of 60-90%, which shows that the ursodeoxycholic acid in all the examples is a nano crystal, and the crystal form is consistent with the ursodeoxycholic acid raw material.
It is fully explained above that the ursodeoxycholic acid prepared by the above examples has good stability when spray drying or freeze drying is used.
Examples 53 to 55
Nanosuspensions were prepared separately according to the following table, using one or two stabilizers each, and using dynamic light scattering techniques to determine particle size, as specified in the following table:
the result shows that the nanosuspension prepared from the composite stabilizer has more ideal particle size under the same preparation process condition, which indicates that the particle size of the nanosuspension can be effectively reduced by using the surfactant and the polymer in a composite way.
Examples 56 to 67
Suspensions of nanoparticles were prepared separately according to the following table, containing different amounts and different specifications of the stabilizer.
The particle size was measured by dynamic light scattering technique and the content was measured by HPLC, with the results as follows:
| sample name | Average particle diameter (nm) | Content (%) |
| Example 56 | 235 | 99 |
| Example 57 | 261 | 97 |
| Example 58 | 287 | 97 |
| Example 59 | 222 | 98 |
| Example 60 | 241 | 94 |
| Example 61 | 274 | 98 |
| Example 62 | 278 | 90 |
| Example 63 | 366 | 97 |
| Example 64 | 501 | 99 |
| Example 65 | 494 | 99 |
| Example 66 | 277 | 99 |
| Example 67 | 535 | 98 |
The results show that in the nano suspension composed of poloxamer 188 and hypromellose composite stabilizer, the concentration of ursodeoxycholic acid is fixed at 50mg/mL, the concentration of poloxamer 188 is within the range of 5-50 mg/mL, and the concentration of hypromellose is within the range of 5-30 mg/mL, so that the nano suspension is successfully obtained, the average particle size is within the range of 222-535 nm, and the content is more than 90%.
Examples 68 to 77
The particle size was measured by dynamic light scattering technique and the content was measured by HPLC, with the results as follows:
| sample name | Average particle diameter (nm) | Content (%) |
| Example 68 | 286 | 95 |
| Example 69 | 313 | 98 |
| Example 70 | 255 | 98 |
| Example 71 | 310 | 97 |
| Example 72 | 378 | 98 |
| Example 73 | 388 | 99 |
| Example 74 | 264 | 94 |
| Example 75 | 284 | 96 |
| Example 76 | 415 | 98 |
| Example 77 | 277 | 92 |
The results show that in the nanosuspension composed of poloxamer 188 and hydroxypropylcellulose composite stabilizer, the concentration of ursodeoxycholic acid is fixed at 50mg/mL, the concentration of poloxamer 188 is within the range of 5-30 mg/mL, and the concentration of hydroxypropylcellulose is within the range of 5-40 mg/mL, so that the nanosuspension is successfully prepared, the average particle size is within the range of 255-415 nm, and the content is more than 90%.
Examples 78 to 87
The particle size was measured by dynamic light scattering technique and the content was measured by HPLC, with the results as follows:
| sample name | Average particle diameter (nm) | Content (%) |
| Example 78 | 248 | 98 |
| Example 79 | 354 | 98 |
| Example 80 | 311 | 96 |
| Example 81 | 265 | 97 |
| Example 82 | 278 | 98 |
| Example 83 | 298 | 95 |
| Example 84 | 430 | 98 |
| Example 85 | 258 | 90 |
| Example 86 | 344 | 97 |
| Example 87 | 387 | 96 |
The results show that in the nanosuspension composed of the poloxamer 188 and the povidone K30 composite stabilizer, the ursodeoxycholic acid concentration is fixed at 50mg/mL, the poloxamer 188 concentration is within the range of 5-40 mg/mL, and the povidone K30 concentration is within the range of 1-20 mg/mL, so that the nanosuspension is successfully prepared, the average particle size is within the range of 248-430 nm, and the content is more than 90%.
Examples 88 to 97
The particle size was measured by dynamic light scattering technique and the content was measured by HPLC, with the results as follows:
| sample name | Average particle diameter (nm) | Content (%) |
| Example 88 | 341 | 98 |
| Example 89 | 372 | 99 |
| Example 90 | 298 | 98 |
| Example 91 | 265 | 100 |
| Example 92 | 374 | 96 |
| Example 93 | 316 | 96 |
| Example 94 | 424 | 98 |
| Example 95 | 382 | 97 |
| Example 96 | 309 | 93 |
| Example 97 | 333 | 97 |
The results show that in the nanosuspension composed of the poloxamer 188 and the polyvinyl alcohol 0588 composite stabilizer, the ursodeoxycholic acid concentration is fixed at 50mg/mL, the poloxamer 188 concentration is within the range of 5-50 mg/mL, and the polyvinyl alcohol 0588 concentration is within the range of 2.5-20 mg/mL, so that the nanosuspension is successfully obtained, the average particle size is within the range of 265-424 nm, and the content is more than 90%.
Nanosuspensions were prepared according to reference 1, with the following formulation and procedure:
the following nanosuspensions were prepared according to reference 2, with the following formulation and procedure as follows:
study of intestinal stability
Reference (Jingyihong et al, colloids and Surfaces B: biointerfaces,2016, 145, 319-327) designed comparative experimental methods for intestinal stability of ursodeoxycholic acid nanosuspensions. The method comprises the following specific steps:
the nanosuspensions prepared in examples 98 to 104 were taken, redispersed in water, and, together with examples 56 to 97, incubated with simulated intestinal fluid at 37 ℃ for 8 hours with shaking, samples were taken at 2,4 and 8 hours, and the average particle size was determined using a dynamic light scattering instrument, the results being shown in FIGS. 4 to 8.
As shown in fig. 4, in the nanosuspension composed of poloxamer 188 and hypromellose composite stabilizer, the particle diameters of examples 56-61 and example 66 gradually increased with the increase of the incubation time, and the average particle diameter of the nanoparticles increased to 700nm or below at 8 h; in comparison, in examples 62 to 65, the average particle size increased to 800nm in 4 hours and exceeded 1000nm in 8 hours, indicating that the combined usage of poloxamer and hypromellose was too high or too low, and the stability of the nanoparticles in intestinal juice was reduced; example 67 also shows a significant increase in average particle size, indicating that the nanosuspensions prepared using hypromellose of greater viscosity are also unstable in intestinal fluid.
By combining the results, the dosage ratio of the ursodeoxycholic acid, the poloxamer 188 and the hydroxypropyl methylcellulose is within the range of 5: 1-5: 4: 2, and the prepared nano suspension has more excellent stability in intestinal juice. In addition, the nano-suspension prepared by using hypromellose (E3, E5 and E6 respectively) with different specifications also shows excellent intestinal stability. Each of these hypromelloses was dissolved in water to prepare a solution having a concentration of 2% w/v and a viscosity of 2.4 to 7.2 mPas (20 ℃ C.).
As shown in FIG. 5, in the nanosuspension composed of poloxamer 188 and hydroxypropylcellulose composite stabilizer, the average particle size of examples 68-73 slowly increased with the increase of the incubation time, the average particle size of 8h was close to 700nm, the average particle size of examples 74-77 significantly increased with the increase of the incubation time, and the average particle size of 8h was over 1000nm. By combining the results, the dosage ratio of the ursodeoxycholic acid, the poloxamer 188 and the hydroxypropyl cellulose is within the range of 5: 1-5: 2: 3, and the prepared nano suspension has more excellent stability in intestinal juice.
As shown in fig. 6, in the nanosuspension composed of poloxamer 188 and povidone K30 composite stabilizer, the average particle size of examples 78 to 83 slowly increased with the increase of the incubation time, the average particle size of 8h was 590 to 650nm, the average particle size of examples 84 to 87 significantly increased with the increase of the incubation time, and the average particle size of 8h was more than 750nm. By combining the results, the dosage ratio of the ursodeoxycholic acid, the poloxamer 188 and the povidone K30 is within the range of 5: 1: 0.25-5: 3: 1, and the prepared nano suspension has more excellent stability in intestinal juice.
As shown in FIG. 7, in the nanosuspension composed of poloxamer 188 and polyvinyl alcohol 0588 composite stabilizer, the average particle size of examples 88-93 slowly increased with the increase of the incubation time, the average particle size of 8h was 570-720 nm, the average particle size of examples 94-97 significantly increased with the increase of the incubation time, and the average particle size of 8h exceeded 920-1040 nm. Combining the results, the dosage ratio of ursodeoxycholic acid, poloxamer 188 and polyvinyl alcohol 0588 is in the range of 5: 1: 0.5-5: 4: 1.5, and the prepared nano suspension has more excellent stability in intestinal juice.
According to documents 1 and 2, the nanosuspensions are prepared by respectively adopting a high-pressure homogenization method (example 98) and a non-solvent precipitation combined high-pressure homogenization method (examples 99-104), and after incubation with simulated intestinal juice, the particle size is remarkably increased along with time extension (as shown in figure 8), which indicates that the nanosuspensions are rapidly aggregated in intestinal tracts to form micron-sized precipitates, and the effect of oral absorption is not ideal.
The cytotoxic effects of nanoparticles on organs and normal cells are a serious limiting factor that hinders their clinical application (see Madni et al, AAPS PharmSciTech,2021, 22, 3). The effect of the examples and control formulations on intestinal epithelial cell viability was determined according to conventional cytotoxic assay methods, as follows:
nanosuspensions (examples 105, 106) were prepared as in CN101606906A, with the following formulation:
examples 58, 72, 80, 92, 105, 106 were each quantitatively diluted in PBS to a range of concentrations and incubated with MDCK cells (as model intestinal epithelial cells) for 24h, respectively, and cell viability was determined by the SRB method, and the results are shown in fig. 9. The results show that the MDCK cell viability is significantly reduced in examples 105 and 106 under the condition of high ursodeoxycholic acid concentration (> 5 mg/mL), which significantly interferes with the transport function of intestinal tract cells, thereby affecting oral absorption, and in addition, adverse effects may be caused on the safety of intestinal tracts; in contrast, the suspension of the embodiment of the application has no obvious cytotoxicity on MDCK cells and good safety.
Examples 107 to 112
Examples 107 to 111 the preparation method was as follows:
weighing the stabilizer according to the formula shown in the table, adding water to completely dissolve the stabilizer, transferring the stabilizer into a ball mill grinding tank, adding the ursodeoxycholic acid raw material and zirconia grinding beads with the diameter of 0.1mm, covering the grinding tank, starting the ball mill, setting the rotating speed to be 600-1200 rpm, and grinding for 4-8 hours to obtain the nano suspension.
The nano-suspension of the embodiment 107 and 108 is taken and put into a freeze dryer to be pre-frozen for 4h at minus 40 ℃, and then vacuum freeze-dried for 48h, thus obtaining the nano freeze-dried powder.
The nanosuspensions of examples 109, 110 and 111 were taken, the drying protectant was added according to the above formula, mixed well, spray dried using a spray dryer, the inlet temperature was set: 120-140 ℃, outlet temperature: 50-60 ℃, atomization pressure: 7-24 kPa and feed rate: 2.0-5 mL/min to obtain the nanometer spray-dried powder.
And (3) weighing the aromatic and the sweetening agent according to the formula in the table, and uniformly mixing to obtain the ursodeoxycholic acid nanosuspension.
The nanopowder (examples 107 to 111) was mixed with an appropriate amount of water, stirred and filtered, and the filtrate was collected and measured for particle size, polydispersity and Zeta potential by dynamic light scattering method, the results are shown in the following table:
| particle size (nm) | Polydispersity index | Zeta potential (mV) | |
| Example 107 | 219.4 | 0.113 | -16.1 |
| Example 108 | 228.0 | 0.137 | -18.4 |
| Example 109 | 223.9 | 0.127 | -23.5 |
| Example 110 | 220.4 | 0.144 | -20.4 |
| Example 111 | 274.1 | 0.186 | -29.3 |
The results show that the particle size of the suspension prepared by diluting the water in the examples 107 to 111 is in the nano-scale range, and the result of the polydispersity coefficient shows that the particle size distribution is narrow, thereby meeting the requirements of subsequent experiments.
Example 112 was prepared as follows: weighing the raw and auxiliary materials according to the formula, adding water, stirring uniformly, standing to fully dissolve the auxiliary materials, and shearing the suspension by using a high-speed shearing machine to obtain the suspension. The particle size of the suspension is measured by a laser particle sizer, and as a result, the average particle size (D50) of the suspension is 16 microns, which meets the requirement of oral suspension on the particle size.
Male ICR mice were selected and acclimatized for one week and divided into a blank group, a model group and a test group, each group consisting of 6 mice. At the beginning of the experiment, each test group of mice was continuously fed with a feed containing 0.1% of 3, 5-diethoxycarbonyl-1, 4-dihydro-2, 4, 6-collidine (DDC) for 24 days, and a blank group (fed with a feed containing no DDC only) was set. On the 10 th day, weighing the weight of the mice, and administering ursodeoxycholic acid nanosuspension to the mice of each test group in an oral gavage mode according to the dosage of 137mg/kg (examples 107-111, each example is diluted with water quantitatively); the model group (oral gavage 0.25mL water) and the general suspension group (example 112) were set simultaneously. On the 25 th day, each group of mice was sacrificed, the eyeballs were picked and blood was taken, the blood was collected at room temperature for 1 to 1.5 hours and centrifuged to obtain serum, and a number of biochemical indicators of serum, including glutamic-pyruvic transaminase ALT, alkaline phosphatase ALP, γ -glutamyl transpeptidase GGT, immunoglobulin IgM, total bilirubin TBIL, hydroxyproline HyP, and autotaxin ATX, were measured respectively according to the instructions of the kit (see fig. 10A-10G).
Compared with the model group, the ursodeoxycholic acid nanosuspension remarkably reduces the expression quantity of serum ALT, ALP and GGT, and shows that the liver function of a model mouse is improved; reducing the expression level of serum IgM, indicating that the inflammation of the liver tissue is reduced; the expression level of serum TBIL is reduced, which shows that the nanosuspension can reduce bile toxicity; the expression level of serum HyP is reduced, which indicates that the progress of liver and gall fibrosis is delayed; reduce the expression level of serum ATX, and indicate that the pruritus of the model mouse is relieved. In addition, all nanosuspension groups had greater efficacy compared to the common suspension group (example 112), which was in accordance with the commercial formula, and the comparison showed that each nanosuspension example was superior to the simulated commercial formula.
The intact liver was dissected out, and the morphology of fresh liver and gallbladder was observed and photographed, as shown in FIG. 11, and the liver of the mouse (example 109) of the test group was soft and bright red, and the liver lobe and gallbladder were not enlarged. The results show that the tested group has no obvious adverse effect on the liver.
Cutting off a liver, fixing in 4% neutral formaldehyde solution for 48h, cutting off appropriate tissue with a blade, placing in an embedding box, washing with running water, dehydrating with ethanol, embedding, preparing wax block, slicing, staining with hematoxylin-eosin (HE), sealing, etc., and observing the prepared pathological section under a biological microscope. As shown in fig. 12, the liver tissue structure was intact and normal, no degenerated and necrotic tissue was observed, bile duct did not show cholestasis, inflammatory cell infiltration and connective tissue proliferation were not observed in the region of the sink. The results show that ursodeoxycholic acid nanosuspension can reduce inflammation and injury.
The experimental results show that after the formula of the ursodeoxycholic acid nanosuspension is improved, the stability, safety and pharmacodynamic effect of the preparation are obviously improved. The prior art does not recognize that the above-described technical effects can be achieved by incorporating a specific combination of complex stabilizers in ursodeoxycholic acid nanosuspensions, nor does it suggest or suggest any way for one skilled in the art to achieve such improvements, which the present application is unexpected with respect to the technical effects achieved by the prior art.
Claims (10)
1. A ursodeoxycholic acid nanosuspension is characterized by comprising ursodeoxycholic acid and a composite stabilizer, wherein the composite stabilizer is a combination formed by poloxamer and any one of hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone K30 or polyvinyl alcohol 0588; wherein the dosage ratio of ursodeoxycholic acid, poloxamer and hydroxypropyl methylcellulose is 5: 1-5: 4: 2; the dosage ratio of ursodeoxycholic acid, poloxamer and hydroxypropyl cellulose is 5: 1-5: 2: 3; the dosage ratio of ursodeoxycholic acid, poloxamer and polyvidone K30 is 5: 1: 0.25-5: 3: 1; the dosage ratio of ursodeoxycholic acid, poloxamer and polyvinyl alcohol 0588 is 5: 1: 0.5-5: 4: 1.5; wherein the poloxamer is selected from poloxamer 188 or poloxamer 407; the hypromellose is selected from hypromellose E3, E5 or E6; particle diameter D of ursodeoxycholic acid raw material powder 90 Below 72 μm.
2. The ursodeoxycholic acid nanosuspension according to claim 1, wherein the poloxamer is poloxamer 188, at a concentration of 5-50 mg/mL.
3. The ursodeoxycholic acid nanosuspension according to claim 1, wherein the nanosuspension forms a suspension in water having an average particle size of 60 to 800nm.
4. The ursodeoxycholic acid nanosuspension according to claim 3, wherein the nanosuspension forms a suspension in water having an average particle size of 80 to 600nm.
5. The ursodeoxycholic acid nanosuspension according to claim 4, wherein the nanosuspension forms a suspension in water having an average particle size of 100 to 500nm.
6. The ursodeoxycholic acid nanosuspension according to claim 5, wherein the nanosuspension forms a suspension in water having an average particle size of 120 to 400nm.
7. A ursodeoxycholic acid nanosuspension, which is characterized in that the ursodeoxycholic acid nanosuspension is prepared from the ursodeoxycholic acid nanosuspension of any one of claims 1 to 6 by a drying method; the drying method is selected from spray drying method or freeze drying method.
8. A process for preparing the ursodeoxycholic acid nanosuspension according to any one of claims 1-6, comprising the steps of: weighing the composite stabilizer, adding water to completely dissolve the composite stabilizer, transferring the composite stabilizer into a grinding tank of a ball mill, adding the ursodeoxycholic acid raw material and zirconium oxide grinding beads, setting the rotating speed to be 600-1200 rpm, starting the ball mill, grinding for 4-8 hours, pouring out grinding liquid, and filtering to remove the zirconium oxide grinding beads to obtain the ursodeoxycholic acid nano suspension.
9. According to claim 8The ursodeoxycholic acid nanosuspension is prepared by using ursodeoxycholic acid as active ingredient, wherein the structural formula is 3 alpha, 7 beta-dihydroxy-5 beta-cholestane-24-acid, and the particle size D of ursodeoxycholic acid raw material powder 90 Below 72 μm.
10. Use of the ursodeoxycholic acid nanosuspension according to any one of claims 1-6 in the manufacture of a medicament for the treatment of cholestatic disorders including childhood/infant cholestasis, gall bladder cholesterol stones, primary biliary cholangitis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111230180.9A CN113975233B (en) | 2021-10-22 | 2021-10-22 | Preparation method of enteric stable ursodeoxycholic acid nanosuspension |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111230180.9A CN113975233B (en) | 2021-10-22 | 2021-10-22 | Preparation method of enteric stable ursodeoxycholic acid nanosuspension |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113975233A CN113975233A (en) | 2022-01-28 |
| CN113975233B true CN113975233B (en) | 2023-04-18 |
Family
ID=79740103
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111230180.9A Active CN113975233B (en) | 2021-10-22 | 2021-10-22 | Preparation method of enteric stable ursodeoxycholic acid nanosuspension |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113975233B (en) |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008013416A1 (en) * | 2006-07-27 | 2008-01-31 | Amorepacific Corporation | Process for preparing powder comprising nanoparticles of sparingly soluble drug |
| CN101606906B (en) * | 2009-07-23 | 2012-05-30 | 中国人民解放军第三○二医院 | Ursodeoxycholic acid nano suspension and preparation method thereof |
| FR2981572B1 (en) * | 2011-10-21 | 2018-01-19 | Inopharm Limited | PHARMACEUTICAL COMPOSITIONS OF URSODESOXYCHOLIC ACID |
| CN107496928B (en) * | 2017-09-11 | 2020-07-14 | 石家庄学院 | Ursodeoxycholic acid dry suspension and preparation method thereof |
-
2021
- 2021-10-22 CN CN202111230180.9A patent/CN113975233B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN113975233A (en) | 2022-01-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Mohammad et al. | Drug nanocrystals: Fabrication methods and promising therapeutic applications | |
| US6592903B2 (en) | Nanoparticulate dispersions comprising a synergistic combination of a polymeric surface stabilizer and dioctyl sodium sulfosuccinate | |
| US5622938A (en) | Sugar base surfactant for nanocrystals | |
| CN101636150B (en) | Method for producing pulverized organic compound particle | |
| EP0812187B1 (en) | Solid nanoparticulate pharmaceutical composition | |
| EP1423096B1 (en) | A process for preparing crystalline drug particles by means of precipitation | |
| JP2005527536A (en) | Preparation process and use of microparticles or nanoparticles by constructive refinement. | |
| KR101455446B1 (en) | Composite organic compound powder for medical use, method for producing same and suspension of same | |
| CA2353809A1 (en) | Nanoparticulate core-shell systems and use thereof in pharmaceutical and cosmetic preparations | |
| JP2009518300A (en) | Mometasone composition and methods for making and using the same | |
| JP2010506886A (en) | Chemical micelle nanoparticles | |
| JP7386967B2 (en) | Icaritin nanomicelle preparation, its preparation method and its application | |
| EP1494649B1 (en) | Nanoparticulate megestrol formulations | |
| Liu | Nanocrystal formulation for poorly soluble drugs | |
| EP1658052B1 (en) | Particle size reduction of bioactive compounds | |
| CN108403646B (en) | Albendazole nano micropowder and preparation method thereof | |
| WO2019214715A1 (en) | Meloxicam composition, preparation and preparation method and use thereof | |
| CN110604747B (en) | Propolis nanoparticle, preparation method and application thereof, and preparation method of freeze-dried powder of propolis nanoparticle | |
| CN109568264B (en) | Andrographolide nanosuspension | |
| CN113975233B (en) | Preparation method of enteric stable ursodeoxycholic acid nanosuspension | |
| CN106420607B (en) | A kind of sirolimus nano suspension and preparation method thereof | |
| CN113069415A (en) | Insoluble drug nanosuspension and preparation method thereof | |
| CN103070836A (en) | Ciclesonide nanometer freeze-dried powder and preparation method thereof | |
| CN109568265A (en) | A kind of andrographolide nano suspension | |
| Godge et al. | Nanosuspension Technology for Delivery of Poorly Soluble Drugs and Its Applications: A Review |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |