KR102653853B1 - Nanosuspension for Drug Delivery and Use of the Same - Google Patents

Abstract

The present invention relates to a nanosuspension for drug delivery and a pharmaceutical composition for the treatment or prevention of diseases containing nanosuspension as an active ingredient. The nanosuspension for drug delivery according to the present invention safely delivers drugs to the inner ear through a route within the eardrum. and has the advantage of being able to communicate effectively.
In addition, the nanosuspension for drug delivery according to the present invention effectively maintains the drug delivered to the inner ear and has excellent effects in terms of safety and efficacy, so it can be applied clinically.

Description

Nanosuspension for drug delivery and use thereof {Nanosuspension for Drug Delivery and Use of the Same}

The present invention relates to a nanosuspension for drug delivery and a pharmaceutical composition for the treatment or prevention of diseases containing the nanosuspension as an active ingredient.

Hearing loss can be divided into chronic hearing loss, congenital hearing loss, and acute hearing loss depending on the time of onset. Of these, only acute hearing loss that occurs within one month is a disease that can be recovered with drug treatment. Diseases such as ototoxic hearing loss, sudden hearing loss, and acoustic trauma fall into this category. , the standard treatment method to date is the administration of systemic oral steroids. However, as a result of systemic steroid administration to healthy hearing loss patients, one or more systemic side effects occurred in 33.0%, and serious side effects such as hip fracture, toxic hepatitis, and death occurred in approximately 1%.

Recently, in addition to systemic high-dose steroid administration, intratympanic steroid injection has been used to treat acute hearing loss, including sudden hearing loss. Steroids are injected into the tympanic cavity through the eardrum, and the injected steroid is delivered through the round window located between the tympanic cavity and the inner ear. It is expected to spread, but there are two obstacles while the drug injected into the tympanic cavity enters the inner ear. First, the drug does not stay in the middle ear space for a long enough time and easily escapes into the nasopharynx through the eustachian tube, leaving insufficient time for absorption into the inner ear. Second, there is a limitation in that it cannot efficiently pass over the anatomical structure called the round window made of mucous membrane, which is the passage into the inner ear. According to a recent report, it has been reported that hydrophobic drugs are absorbed much better than hydrophilic drugs in mucous membranes such as the cervix.

Meanwhile, in addition to steroids, which are existing treatments for acute hearing loss disease, experimental results have been reported showing that various antioxidants, including alpha-lipoic acid, are effective. Most steroids and antioxidants are hydrophobic drugs, so they do not dissolve in water, so they are used in clinical practice. There is a problem that it is difficult to develop an injectable drug for intratympanic injection. Dexamethasone sodium phosphate (Dex-SP), which is currently used as a standard intratympanic injection for acute hearing loss, is made by chemically combining phosphate with the originally hydrophobic dexamethasone to make it hydrophilic. Dex is stable as an injection, but has been changed to hydrophilic. -SP had a limitation in that it could not easily pass through the mucous membrane of the round window.

Therefore, in order to develop hydrophobic drugs such as steroids or antioxidants as injectables for intratympanic injection, a dilemma arises in which the drug must be dissolved in water while maintaining its hydrophobicity. To solve this problem, the present inventors developed a nanosuspension (nano-size). Among the nanosuspension production technologies, focusing on the technology of split hydrophobic drug particles that do not dissolve in a liquid but are irregularly dispersed as fine particles (or a mixture thereof), a nanosuspension was produced by combining dexamethasone with a technology called fat and supercritical fluid (NUFS). Thus, an injectable agent for intratympanic injection was created, and the newly developed agent was discovered to have much better absorption through the eardrum and better drug delivery to the inner ear than the existing one, and the present invention was completed. In addition, a comparative evaluation was conducted between the newly created injection and Dex-SP, which is currently in clinical use, in terms of drug delivery ability to inner ear tissue and efficacy of the delivered drug.

Republic of Korea Patent Publication No. 10-2018-0065811 (2018.06.18)

The purpose of the present invention is to provide a nanosuspension for inner ear-specific drug delivery that is water-dispersed in a solid dispersion containing steroid drugs, lipids, biocompatible polymers, and surfactants, including dexamethasone, by removing the lipids with a supercritical fluid. It is done.

Another object of the present invention is to treat diseases containing as an active ingredient a drug delivery nanosuspension prepared by treating a supercritical fluid with a solid dispersion containing steroid drugs, lipids, biocompatible polymers and surfactants, including dexamethasone. To provide a pharmaceutical composition for treatment or prevention.

Other objects and technical features of the present invention are presented in more detail by the following description, claims, and drawings.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention relates to a nanosuspension for drug delivery in which a solid dispersion containing a steroid drug, lipid, biocompatible polymer, and surfactant, including dexamethasone, is dispersed in water by removing the lipid using a supercritical fluid.

Nanosuspension in the present invention can be easily made by preparing a solid dispersion containing steroid drugs including dexamethasone, lipids, biocompatible polymers, and surfactants, and removing lipids from this solid dispersion using a supercritical fluid. As a nanosuspension for dispersing drug delivery, the present invention provides a nanopowder manufactured from the nanosuspension.

“Nanosuspension” in the present invention refers to particles of various materials having nanoscale diameters. In one embodiment of the present invention, “nanosuspension” can be formed by using the property of inducing dispersion of steroid-based drug particles containing dexamethasone divided into nano sizes when manufacturing nanosuspension in a dispersion medium. Specifically, the nanosuspension is made by dispersing and mixing steroid drug particles, lipids, biocompatible polymers, and surfactants containing nano-sized dexamethasone, and then performing continuous milling on the resulting product to ensure that the composition is uniform. A well-mixed dispersion-type mixture can be obtained, and the obtained mixture can be formed by treating the supercritical fluid to remove the used lipid.

In one embodiment of the present invention, the “nanosuspension” may be composed of a steroid drug containing dexamethasone to be delivered. Additionally, the nanosuspension does not contain any other carriers or vehicles. Therefore, nanosuspension has a high drug loading value and can deliver the drug to target cells very efficiently, achieving high therapeutic concentration and maximizing pharmacological effect.

In one embodiment of the present invention, the nanosuspension can deliver a steroid drug, including dexamethasone, to hair cells or the organ of Corti by penetrating the round window membrane.

In one embodiment of the present invention, the nanosuspension may have a size of 1nm to 1㎛, 10nm to 900nm, 10nm to 800nm, 100nm to 900nm or 100nm to 500nm, preferably 250 to 350nm, but is not limited thereto. no.

“Lipid” in the present invention is used as a lubricant in the nanosuspension manufacturing process and includes Myristyl alcohol, Cetyl alcohol, Stearyl alcohol, and Lauryl Alcohol. Fatty alcohols such as stearic acid, palmitic acid, myristic acid, lauric acid and their methyl, ethyl, propyl and butyl alcohols Fatty acid monoglycerides such as esters, stearyl monoglyceride, palmityl monoglyceride, myristyl monoglyceride, lauryl monoglyceride, distearyl glyceride, dipalmityl glyceride, dimyristyl glyceride, etc. Fatty acid diglycerides such as glyceride, dilaurylglyceride, etc. tetradecane, pentadecane, hexadecane, heptadecane, octadecane, nonadecane, icosane, heneicosane, doco It may be selected from hydrocarbons such as acid (docosane), tricosane, tetracosane, etc., and combinations thereof, but is not limited thereto. In addition, it is a material that acts as a lubricant in the milling process according to the method of the present invention and can be removed by supercritical fluid, but is not limited thereto.

“Biocompatible polymer” in the present invention is a biocompatible polymer with a glass transition temperature (Tg) of 80°C or higher, preferably 90°C or higher, such as methylcellulose (MC), MC with a Tg of about 184 to 197°C. ), hydroxyethylcellulose (HEC), such as HEC with a Tg of about 127°C), hydroxypropylcellulose (HPC), such as HPC with a Tg of about 105°C), hydroxypropyl methylcellulose ( hydroxypropyl methylcellulose (HPMC), e.g., HPMC with a Tg of about 180° C., hydroxypropyl methylcellulose acetate succinate (HPMC-AS), e.g., HPMC-AS with a Tg of about 117° C., hyde hydroxypropyl methylcellulose phthalate (HPMC-P), e.g., HPMC-P with a Tg of about 145° C., carboxymethyl cellulose (CMC), e.g., CMC with a Tg of about 135° C., cellulose Cellulose-based biocompatible polymers with a Tg of 80°C or higher, such as acetate (cellulose acetate (CA), for example, CA with a Tg of about 180°C) and dextran (e.g., dextran with a Tg of about 200°C), Polyvinyl pyrrolidone (PVP) with a Tg of 80°C or higher, such as PVP k17, k30, k90 with a Tg of about 138 to 156°C, polyvinyl alcohol with a Tg of 80°C or higher, and Tg It may be one or more types selected from the Eudragit series of biocompatible polymers having a temperature of 80°C or higher, but is not limited thereto.

“Surfactant” in the present invention includes cetyl pyridinium chloride, phospholipids, fatty acid, benzalkonium chloride, calcium stearate, and glycerin fatty acid ester. (glycerin esters of fatty acid), fatty alcohol, cetomacrogol, polyoxyethylene alkyl ethers, sorbitan esters, polyoxyethylene castor oil derivatives oil derivatives, polyoxyethylene sorbitan fatty acid esters, dodecyl trimethyl ammonium bromide, polyoxyethylene stearate, sodium lauryl sulfate ), sucrose fatty acid ester, polyethylene glycol (PEG; polyethylene glycol)-cholesterol, polyethylene glycol (PEG; polyethylene glycol)-40 stearate, PEG-vitamin E (PEG-vitamin E) There may be one or more types selected, but it is not limited thereto.

Meanwhile, the solid dispersion may contain additional ingredients. The additional ingredients include known biocompatible polymers with a glass transition temperature of less than 80°C, surfactants, and anti-agglomerates that are known to be usable for nanosuspension of active materials, or even if new, can be used for nanosuspension of active materials. Anything can be applied to the present invention. Examples of such optional additional ingredients include gelatin, casein, gum acacia, tragacanth, polyethylene glycols, poloxamers, glass transition temperature. Biocompatible polymers with a glass transition temperature of less than 80℃, such as eudragit ®, lysozyme, and albumin; Examples include anti-aggregation agents such as sugars.

In the present invention, “saccharides” include monosaccharide compounds, disaccharide compounds, polysaccharide compounds, and sugar alcohols, and especially include glucose, lactose, mannitol, sucrose, xylitol, chitosan, starch fiber, etc., alone or in mixtures. It can be used in the form of . These optional additional ingredients may be used alone or in combination of two or more, but are not limited thereto.

In the present invention, “supercritical fluid” is a gas that is non-reactive and inert, such as carbon dioxide or nitrogen, and can become a supercritical fluid under a specific temperature and specific pressure, that is, supercritical temperature and supercritical pressure. Or it means liquid. Preferably, the supercritical fluid may be carbon dioxide, but is not limited thereto.

In the present invention, “supercritical pressure” means a specific pressure at which supercritical fluid gas can become a supercritical fluid under a pressure higher than that pressure.

“Drug” in the present invention may be a steroid-based drug including dexamethasone.

In the present invention, “drug delivery” refers to delivering a drug to a specific area in the body. In order to minimize side effects and maximize efficacy and effectiveness, the necessary amount of drug is selectively delivered to the treatment area without exposing healthy tissues to the drug. At the same time, it means efficient delivery so that excellent therapeutic effects can be achieved with only a small amount of drug. The purpose of drug delivery in the present invention is local delivery that penetrates the round window and delivers the drug to hair cells, organ of Corti, or spiral ganglion cells, which are the main areas of hearing loss.

In one embodiment of the present invention, “permeation” refers to the ability of the nanosuspension to penetrate the round window and effectively deliver drugs to the inner ear. Specifically, the permeation means that the drug is placed in the middle ear space through the eardrum and the drug permeates the cochlear window from the middle ear space to the inner ear.

Another aspect of the present invention relates to a pharmaceutical composition for the treatment or prevention of diseases containing the nanosuspension as an active ingredient.

“Disease” in the present invention refers to a condition in which the physical and mental functions of an organism are abnormal. Preferably, in one embodiment of the present invention, the disease may be an inner ear disease.

In the present invention, “inner ear disease” refers to a disease that can be caused by various disorders, diseases, or trauma, and is one of the diseases classified as incurable diseases because it is difficult to deliver drugs or genes to the inner ear. The inner ear diseases include tinnitus, vertigo such as paroxysmal positional vertigo (BPPV), hearing loss, sensorineural hearing loss, tinnitus, chronic otalgia, perilymph fistula, secondary endolymphatic hydrops, labyrinthitis and vestibular neuritis, acoustic neuroma, ototoxicity, autologous Including, but not limited to, immune-mediated inner ear disease (AIED) and Meniere's disease.

In one embodiment of the present invention, the inner ear disease refers to sensorineural hearing loss, and more specifically, hair cells, spiral ganglion neurons (SGN) in the cochlea of the inner ear, It refers to hearing loss caused by damage to the organ of Corti.

“Prevention” in the present invention refers to any action that suppresses or delays inner ear disease by administering nanosuspension.

“Treatment” in the present invention means reversing, alleviating, inhibiting the progression of, or preventing a disease or disease, or one or more symptoms of the disease or disease.

In one embodiment of the present invention, the pharmaceutical composition is injected into the eardrum, that is, into the tympanum. The injected drug is located within the eardrum and is delivered to the inner ear through the round window.

The present invention relates to a nanosuspension for drug delivery. The nanosuspension for drug delivery according to the present invention has the advantage of safely and effectively delivering a drug to the inner ear through an intratympanic route.

In addition, the nanosuspension for drug delivery according to the present invention effectively maintains the drug delivered to the inner ear and has excellent effects in terms of stability and efficacy, so it has the advantage of being clinically applicable and highly usable.

Figure 1 shows the dispersion results of dexamethasone nanosuspension and micronized dexamethasone powder in water over time.
Figure 2 shows the dissolution profile of dexamethasone nanosuspension compared to micronized dexamethasone powder.
Figure 3 shows the cytotoxicity results of three types of dexamethasone nanosuspension (NUFS) and micronized dexamethasone (micro-dex) on HEI-OC1 cells.
Figure 4 (A) shows a comparison of dexamethasone concentrations in perilymph, and (B) shows a comparison of dexamethasone concentrations for NUFS B and Dex-SP in perilymph.
Figure 5 (A) shows the amount of dexamethasone per 25 μg of protein in the cochlear homogenate quantified by LC/MS, and (B) shows the results of dexamethasone immunochemical staining in the cochlea collected 6 hours after drug administration. will be.
Figure 6 (A) shows the expression level of P-GR or GR according to Dex-SP or NUFS B treatment in HEI-OC1 cell line, and (B) shows the expression level of P-GR or GR according to Dex-SP or NUFS B treatment in cochlear homogenate. It shows GR or the expression level of GR.
Figure 7 (A) shows the results of in vivo safety evaluation after intratympanic injection of NUFS B, and (B) shows the results of histological evaluation.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Experimental method

1. Preparation of dexamethasone nanosuspension of the present invention

The nanosuspension in the present invention was provided with materials from Biosynetics Co., Ltd., and the production of dexamethasone nanosuspension of the present invention was synthesized based on the method described in Korean Patent Publication No. 10-2018-0118519 of Biosynetics Co., Ltd. You can.

First, polyvinylpyrrolidone (PVP), poloxamer, polyethylene glycol (PEG)-40 stearate, and saccharide were mixed in deionized water at a ratio determined by the experimental design. dissolved in. After mixing the above polymer solution with fatty alcohol as an active ingredient, the mixture was milled with a roller press and then dried under reduced pressure at room temperature. Afterwards, the resulting solid dispersion was placed in a pressure reactor (Bio-Synectics, Inc., BS-SF-1, Korea) and fatty alcohol was removed from the solid dispersion using a continuous flow of liquid carbon dioxide to prepare nanopowder. Then, this nanopowder was dispersed in water to prepare the nanosuspension of the present invention.

The composition of the nanosuspension formulation according to the present invention is summarized in Table 1. The nanosuspension according to the present invention used PVP and poloxamer as the basic composition, and the effect of lactose and dextrose was tested (NUFS A, B). In addition, we sought to compare the effect of adding polymer ratio and PEG-40 stearate (NUFS B, C).

Formulation Ratio (weight ratio) Dexamethasone PVP K17 Poloxamer 188 PEG-40 stearate Lactose Dextrose NUFS A One 0.2 0.2 0.5 NUFS B One 0.2 0.2 One NUFS C One 0.3 0.3 0.3 One

2. Characterization of dexamethasone nanocrystals

2.1 Particle size measurement

The hydrodynamic particle size of various nanosuspension formulations was measured using dynamic light scattering (DLS) (ELSX-1000, Otsuka Electronics Co., Ltd, Osaka, Japan).

After being dispersed by sonication, dexamethasone nanosuspension was collected at time points of 0, 1, 2, 6, and 8 hours and particle size was measured by DLS. The nanosuspension was visually analyzed using a 120 kV biotransmission electron microscope (JEM-1400 plus, JEOL, Tokyo, Japan) according to the method used in a previous study (J.Y. Yoon et al., International journal of nanomedicine 11, 2016). observed.

2.2. In vitro dissolution studies

In vitro dissolution tests of the formulations were performed using a USP Dissolution Apparatus 2 (Vision Elite 8, Hanson, CA, USA) at 75 rpm. For each measurement, the powder (100 mg of dexamethasone) was placed in 900 mL deionized water at 37 ± 0.5 °C. At time points after 3, 7, 15, 30, 60, and 120 minutes, 3 mL samples were collected and replaced with an equal volume of fresh lysis medium. Aliquots were filtered through a 0.2 μm membrane filter.

Drug content was analyzed using an Alliance series high-performance liquid chromatography instrument (HPLC) with ultraviolet (UV) detection at 249 nm (Waters Corporation, Milford, MA, USA). An amount of 20 μl per sample was injected into an Epic C18 column (particle size 5 μm, 4.6x250 mm) at a rate of 0.8 mL/min at 25°C. The mobile phase consisted of 0.01 M KH2PO4 buffer and acetonitrile (5:4% v/v).

3. In vitro safety and drug efficacy evaluation of dexamethasone nanosuspension in HEI-OC1 cells

3.1. HEI-OC1 cell culture

The organ of Corti cell line HEI-OC1 was evaluated using immortalized mice. HEI-OC1 cells were grown and subcultured in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal bovine serum, 50 U/mL recombinant mouse interferon-γ, and 10 ng/mL ampicillin, and then cultured for 10 days at 33°C. Cultured in a % humidity environment.

3.2. CCK (Cell Counting Kit) analysis

The safety of three types of dexamethasone nanocrystal nanosuspensions in HEI-OC1 cells was evaluated using the CCK-8 (Dojindo Molecular Technologies, Rockville, MD) assay. Cells were incubated with various concentrations of dexamethasone nanosuspension (0.1-100 μg/ml) for 24 hours. Cell viability was determined using the CCK-8 assay according to the manufacturer's instructions. Optical density was measured at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).

3.3. western blotting

First, HEI-OC1 cells were treated with dexamethasone sodium phosphate (Dex-SP0 and NUFS-B of the present invention for 1 hour and 6 hours. Afterwards, the cells were incubated with RIPA buffer containing protease and phosphatase inhibitors (50mM Tris; 150mM NaCl; 1mM EDTA; 1% sodium deoxycholate (DOC); 1% Triton

Cell lysates were centrifuged at 13,200 rpm for 15 min at 4°C, and the supernatant was used for Western blotting analysis. For each sample, equal amounts of protein were electrophoresed and transferred to a nitrocellulose membrane. Gastric membranes were incubated with blocking solution (Translab, Daejeon, Republic of Korea) for 1 h and then incubated with glucocorticoid receptor (Cell Signaling, Danvers, Massachusetts, US) and phospho-glucocorticoid receptor (Cell Signaling, Danvers, Massachusetts, were incubated overnight with primary antibodies against US).

Protein expression was detected using the ChemiDoc XRS imaging system (Bio-Rad, Hercules, California, United States).

4. In vivo evaluation of inner ear drug delivery efficiency and safety of dexamethasone nanosuspension

Three types of dexamethasone nanosuspension or a control drug were injected into the middle ear cavity of the animals, and the concentration of dexamethasone in the perilymph was monitored for up to 24 hours. After comparing the drug concentration in the perilymph between three types of nanosuspension, one of them, dexamethasone nanosuspension, was selected to further investigate its safety and efficacy in vivo.

4.1. Middle ear administration of drugs

Eight-week-old male BALB/c mice (Orient Bio, Seoul, Republic of Korea) weighing 20-23 g were used for in vivo experiments. Mice were anesthetized using a mixture of 30 mg/kg zoletil (Virbac, Carros, France) and 10 mg/kg Lumpun (Bayer, Leverkusen, Germany) and placed in a supine position on a heat-controlled heating pad. A midline incision was made and the left bulla was exposed. A hole was made in the blister using fine forceps, and the drug solution was injected into the blister using an insulin syringe. Meanwhile, for clinical practice, the use of gel foam to maintain drugs in the middle ear was excluded.

Afterwards, 1.0 mg/kg Rimadyl (Pfizer, Walton Oaks, UK) was injected to relieve pain. Vitril (Orion, Hamburg, Germany) 10 mg/kg was injected intraperitoneally once daily as a preventive measure against middle ear infection.

4.2. Perilymph sampling and dexamethasone concentration measurement

Perilymph was sampled from the lateral semicircular canal (SCC) using the same anesthesia regimen as above. During perilymph collection, an incision was made behind the animal's left ear to prevent contamination by perilymph, and the drug was injected into the middle ear cavity. Afterwards, the animal's pinna was retracted anteriorly and the temporal bone muscle was bluntly incised to expose the lateral and posterior lateral semicircular canals. A small hole was created in the middle part of the lateral semicircular canal with a 26G needle. After observing perilymph leakage through the hole, approximately 3 μL of perilymph was sampled with a calibrated capillary.

Each perilymph sample was transferred to an Eppendorf tube and centrifuged at 13,200 rpm for 5 minutes. Then, 2 μL of supernatant was collected, diluted 25-fold in 50% methanol to a volume of 50 μL, and analyzed by liquid chromatography-mass spectrometry (LC/MS; 1290 Infinity II/Qtrap 6500; Sciex, Washington, DC. , USA) was performed on diluted perilymph samples.

4.3. Cochlear homogenate preparation and dexamethasone concentration determination

To determine the concentration of dexamethasone absorbed into cochlear tissue, the concentration of dexamethasone in cochlear homogenate was analyzed by LC/MS. The cochlea was collected from the animal, immersed in PBS (phosphate buffered saline), and then washed to remove drug residues from the outside of the cochlea. Additionally, the round window membrane was opened and the inside of the cochlea was washed with PBS to remove perilymph.

Afterwards, the cochlea was homogenized using Tissue Lyser II (Qiagen, Hilden, Germany) with RIPA buffer. The lysate was centrifuged at 13,200 rpm for 15 min at 4°C, and the supernatant was collected. To compare the concentration of dexamethasone in the cochlear tissue of each animal, the protein concentration of the supernatant was adjusted to the same value as in Western blotting.

30 μL of the supernatant containing about 25 μg of protein was diluted and mixed with 30 μL of methanol to make a 60 μL sample, and the concentration of dexamethasone in the sample was examined by LC/MS. The results were expressed as the amount of dexamethasone per 25 μg of protein in cochlear tissue.

4.4. Western blotting

To compare the efficacy of NUFS and Dex-SP in the cochlea, the amount of phosphorylated glucocorticoid receptors (P-GR) in cochlear tissue was compared. Western blotting was performed on cochlear homogenates using the same method used for the in vitro evaluation above.

4.5. Histological evaluation

Two weeks after the drug was injected into the middle ear cavity, the animals' hearing was evaluated using auditory brainstem responses (ABR). The cochlea and bulla were collected, and histological damage was observed under a light microscope.

The cochlea and alveoli were fixed in 4% paraformaldehyde (Merck, Darmstadt, Germany) and decalcified with 0.3M 5% ethylenediaminetetraacetic acid (EDTA). Cochlear sections on slides were stained with hematoxylin (YD Diagnostics Corp., Gyeonggi-do, Republic of Korea) and eosin (BBC Biochemical, McKinney, TX, United States). Images were examined using an optical microscope (Nikon Eclipse E400, Tokyo, Japan). Immunofluorescence staining for dexamethasone was performed to investigate the uptake of dexamethasone in cochlear tissue.

Anti-dexamethasone antibody (Abcam, UK) was used as the primary antibody, and tissues were stained using Vectastain Elite ABC HRP Kit and Vector NovaRED Peroxidase Substrate (Vector Laboratories, Burlingame, CA). Images were captured using an optical microscope (Nikon Eclipse E400, Tokyo, Japan).

4.6. In vivo ABR testing

ABR was recorded and evaluated 2 weeks after surgery. Mice were anesthetized with a mixture of 10 mg/kg Lumpun (Bayer, Leverkusen, Germany) and 30 mg/kg Zoletil (Virbac, Carros, France). The active electrode was placed approximately at the vertex of the skull, the reference electrode was inserted under the auricle of the left ear, and the ground electrode was inserted under the opposite ear. ABR thresholds were measured in response to clicks and frequencies from 4 to 32 kHz, and only the operated ear was tested. ABR was recorded using TDT System-3 (Tucker Davies Technologies, Gainesville, FL, USA) hardware and software.

Computer-generated tone pips were used for stimulation. Click and tone burst stimuli (4, 8, 16 and 32 kHz) with a duration of 4 ms and a rise-fall time of 1 ms were used. The sound intensity was changed at 5 dB intervals for click stimuli near the threshold and at 10 dB intervals for tone burst stimuli. ABR was analyzed using a custom program (BioSig RP, ver. 4.4.1). Threshold differences between mouse groups were compared statistically.

5. Statistical analysis

All data were expressed as the mean ± error of measurements performed three times. Statistical significance was determined for differences between groups through one-way analysis of variance (ANOVA) for ABR, Kruskal-Wallis test for comparison of dexamethasone concentrations, and Student's t-test for all other results. For all analyses, P<0.05 indicates statistical significance.

Experiment result

1. Evaluation of the properties of dexamethasone

The dispersion of dexamethasone nanosuspension and micronized dexamethasone powder in water over time was evaluated and is shown in Table 2.

Formulation Time (h) Particle size (nm) NUFS A 0 382.0 One 452.3 2 451.1 4 457.2 6 477.5 8 504.9 NUFS B 0 239.3 One 264.0 2 258.6 4 258.4 6 258.9 8 259.1 NUFS C 0 287.3 One 356.6 2 351.1 4 336.9 6 336.8 8 336.9

Saccharides contribute to the formation of nanosuspensions in the API mixture during the milling process, and the effect of dextrose appears to be greater on the particles of dexamethasone nanosuspensions than lactose (NUFS A, B).

Dextrose appears to be more beneficial to particle stability in suspension than lactose, but the addition of polymers and surfactants does not seem to have a significant effect on particle size formation (NUFS B, C). Increasing the polymer and surfactant concentration appears to have a negative impact on the stability of the nanosuspension.

The particle size of NUFS B and C did not change until 8 hours after the nanosuspension was dispersed in deionized water. However, the particle size of NUFS A with a high solubilizer ratio increased after dispersion, and after 8 hours of dispersion, the particle size of NUFS A increased by more than 100 nm.

The dissolution status of dexamethasone nanosuspension and micronized dexamethasone powder was confirmed through Figures 1 and 2. Dexamethasone nanosuspension was dispersed in the medium within a short time and reached the highest dissolution rate within 10 minutes after the start of dissolution. The dissolution rate of the nanosuspension was proportional to particle size and consistently exceeded 70%. NUFS C showed a lower dissolution rate than the other two formulations despite containing more polymer and surfactant. In conclusion, particle size appears to play an important role in dissolution rate.

On the other hand, micronized dexamethasone had poor dispersibility in the medium and reached a maximum dissolution rate of about 50% 2 hours after the start of dissolution.

2. In vitro safety evaluation

HEI-OC1 cells were treated with NUFS and micro-dex (0-100 μg/mL) for 24 hours, and cell viability was detected by CCK-8 assay. Since NUFS and micro-dex treatment for 24 h at concentrations up to 100 μg/ml had no significant effect on cell viability, neither micronized dexamethasone powder nor all three types of nanosuspension were used in HEI-OC 1 at concentrations up to 100 μg/mL. So far, it has not caused any toxic effects. Assuming that the drug was absorbed into the cochlea, all four preparations were evaluated to have no particular toxicity (Figure 3).

3. Comparison of dexamethasone inner ear delivery efficiency and drug efficacy

Three types of dexamethasone nanosuspension, micronized dexamethasone powder, and Dex-SP 5 mg/mL solution were injected into the middle ear, and then the dexamethasone concentration in the perilymph was compared over time (Figure 4A). The mean concentrations of dexamethasone measured after 1 and 6 hours were significantly different between groups, as calculated by the Kruskal-Wallis test. The perilymph dexamethasone concentrations of the three types of dexamethasone nanosuspensions were similar to those of Dex-SP measured after 1 hour, but were higher than those of Dex-SP measured after 6 hours. However, there was no significant difference in concentration between the three types of nanosuspension in the results measured after 1 hour or 6 hours.

Because the results of the three types of nanosuspension were similar, NUFS B, which had the smallest particle size, was selected and compared with Dex-SP to specifically analyze the drug delivery efficiency and efficacy of dexamethasone nanosuspension.

First, to determine whether the NUFS B nanosuspension produces a higher drug concentration in the perilymph than Dex-SP 6 hours after injection, the two drugs were injected into the middle ear at various concentrations and the drug concentrations in the perilymph were compared (Figure 4B). When comparing the two groups by Student's t-test, the NUFS B group showed significantly higher concentrations in the perilymph at all three dose concentrations.

Next, because the amount of drug absorbed into the tissue is important for the effect of the drug, the amount of dexamethasone absorbed into the cochlear tissue was compared between the two groups (Figure 5). Six hours after drug administration of 10 mg/mL NUFS B nanosuspension or Dex-SP to the middle ear, the amount of dexamethasone per 25 μg of protein in cochlear homogenates was examined by LC/MS. The result of NUFS B (36.3±17.8 ng) was confirmed to be approximately 26 times that of Dex-SP (1.4±1.8 ng). 24 hours after drug administration, the levels decreased to 1.6±1.1 ng in the NUFS B group and 0.0±0.0 ng in the Dex-SP group. Immunochemical staining for dexamethasone in the cochlea collected 6 hours after drug administration confirmed that the NUFS B group absorbed more dexamethasone than the Dex-SP group.

Next, to evaluate the efficacy of NUFS B, the P-GR of Dex-SP and NUFS B were compared in in vitro and in vivo experiments (Figure 6). As a result of Western blotting, it was confirmed that the level of P-GR was increased in the NUFS B treatment group 1 hour and 6 hours after treatment with 50 μg/mL of Dex-SP or NUFS B in vitro. Through an in vivo experiment, changes in P-GR levels in the cochlea were investigated after treatment with 5 mg/mL and 10 mg/mL concentrations of NUFS B and Dex-SP in the middle ear cavity for 24 hours. As a result of Western blotting, it was confirmed that the level of P-GR was increased in the NUFS B group compared to the Dex-SP group.

4. In vivo safety evaluation

Since Dex-SP is generally injected into the middle ear space at 5 mg/mL in clinical practice, the concentration of NUFS B in the middle and inner ear was set to 20 mg/mL, which is 4 times the normal concentration of Dex-SP, to evaluate toxicity. . The animals' hearing was evaluated by ABR 2 weeks after drug injection into the middle ear, and it was confirmed that the NUFS B group had no hearing loss up to a concentration of 20 mg/mL compared to the normal group (FIG. 7).

Histological evaluation was performed after the hearing test, and it was confirmed that there was no inflammatory reaction in the eardrum or middle ear mucosa in all animals treated with NUFS B, and that no damage was observed in the inner ear tissue of these animals.

Claims (15)

dexamethasone;
Fatty alcohol containing at least one selected from the group consisting of Myristyl alcohol, Cetyl alcohol, Stearyl alcohol, and Lauryl Alcohol;
polyvinyl pyrrolidone (PVP);
polysaccharide;
Surfactants including poloxamer or polyethylene glycol (PEG)-40 stearate; and
A nanosuspension for inner ear-specific drug delivery that is dispersed in water by removing the fatty alcohol in a solid dispersion containing dextrose by supercritical fluid. delete delete delete delete delete The nanosuspension for drug delivery according to claim 1, wherein the supercritical fluid is carbon dioxide or nitrogen. The nanosuspension for drug delivery according to claim 1, wherein the drug is dispersed within the nanosuspension. delete delete The nanosuspension for drug delivery according to claim 1, wherein the nanosuspension has a size of 100 nm to 500 nm. The nanosuspension for drug delivery according to claim 1, wherein the nanosuspension penetrates the round window membrane and delivers the drug to hair cells or the organ of Corti. A pharmaceutical composition for the treatment or prevention of inner ear diseases comprising the nanosuspension according to any one of claims 1, 7, 8, 11, and 12 as an active ingredient,
The inner ear diseases include tinnitus, paroxysmal positional vertigo (BPPV), hearing loss, sensorineural hearing loss, tinnitus, chronic otalgia, perilymph fistula, secondary endolymphatic hydrops, labyrinthitis and vestibular neuritis, acoustic neuroma, ototoxicity, and autoimmune inner ear disease. A pharmaceutical composition comprising at least one selected from the group consisting of (AIED) and Meniere's disease. delete The pharmaceutical composition according to claim 13, wherein the pharmaceutical composition is injected into the eardrum.