Controlled release formulation for treating hearing loss and method for preparing the same

Abstract

The present invention relates to a controlled-release formulation for treating hearing loss and a method for preparing the same, and more particularly, to a controlled-release formulation for treating hearing loss prepared by dispersing a steroidal anti-inflammatory agent encapsulated in a low-molecular-weight hyaluronic acid in an aqueous solution of a high-molecular-weight hyaluronic acid and a method for preparing the same.

Description

Technical Field

[0001] This application claims priority to Korean Patent Application No. 10-2020-0012749, filed on February 3, 2020, which is hereby incorporated by reference in its entirety into this application.

[0002] The present invention relates to a controlled-release formulation for treating hearing loss and a method for preparing the same, and more particularly, to a controlled-release formulation for treating hearing loss prepared by dispersing a steroidal anti-inflammatory agent encapsulated in a low-molecular-weight hyaluronic acid in an aqueous solution of a high-molecular-weight hyaluronic acid and a method for preparing the same. Background Art

[0003] Hearing loss refers to a state of hearing loss due to impaired hearing or hearing abnormality caused by various reasons. The ear is composed of the outer ear (from the auricle to the eardrum), the middle ear (from the eardrum to the entrance of the cochlea) and the inner ear (inside the cochlea), and any problem occurring in any of these parts will lead to hearing loss. Hearing loss is divided into hearing loss due to outer ear and middle ear disorders (conductive hearing loss) and hearing loss due to inner ear and auditory nervous system disorders (sensoreneural hearing loss). However, hearing loss due to outer ear and middle ear disorders will also be restored when the disorders are treated, but hearing loss due to inner ear and auditory nervous system disorders will not be restored in many cases, even if the disorders are treated, causing great inconvenience to the patient.

[0004] To date, the most standard approach for treating hearing loss is systemic oral steroid administration. However, as a result of systemic steroid administration to healthy hearing loss patients, 33.0% of patients reportedly experienced one or more systemic side effects, and approximately 1% of patients experienced severe side effects, such as hip fracture, toxic hepatitis, and death (Min KH and Suh MW, Kor J Audiol 2012).

[0005] Recently, in addition to systemic high-dose steroid administration, intratympanic steroid injection has been used to treat sudden hearing loss. Steroids are injected into the tympanic cavity through the tympanic membrane, and it is expected that the injected steroid will diffuse through the cochlear window located between the tympanic cavity and the inner ear. However, since the drug injected into the tympanic cavity is released through the ear canal within tens of minutes, the actual delivery time of the drug to the inner ear is shorter than 24 hours, which does not achieve a sufficient therapeutic effect.

[0006] Meanwhile, in Korean Patent Registration No. 10-1877894, a drug controlled-release formulation for treating hearing loss prepared by dispersing a steroidal anti-inflammatory agent encapsulated in biocompatible polymer-derived microspheres in a hyaluronic acid hydrogel is disclosed. The drug controlled-release formulation for treating hearing loss plays a role in extending the release period of the steroidal anti-inflammatory agent, but the use of two types of chemically modified hyaluronic acid derivatives is inconvenient and indispensable for hydrogel production, and there is a problem in that inflammation in the tympanic membrane is caused by a cross-linking agent necessarily involved in hydrogel production.

[0007] Therefore, there is a need for a formulation for treating hearing loss that reduces the side effects caused by systemic steroid administration and delivers the drug in the tympanic cavity for a sufficient time to treat the hearing loss.

[0008] public

[0009] Technical issues

[0010] The present inventors conducted many studies to develop a formulation for treating hearing loss that can reduce the side effects caused by systemic steroid administration and deliver the drug in the tympanic cavity for a sufficient time to treat hearing loss, and as a result found that a composite formulation prepared by dispersing a steroidal anti-inflammatory agent encapsulated in a low molecular weight hyaluronic acid in an aqueous solution of a high molecular weight hyaluronic acid not only significantly increased the drug release period in the tympanic cavity but also did not show a side effect of inflammatory response, and then completed the present invention.

[0011] Therefore, an object of the present invention is to provide a controlled-release drug formulation in which a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or hyaluronate is dispersed in an aqueous solution of hyaluronic acid or hyaluronate having a molecular weight greater than 5,000,000 g / mol.

[0012] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating hearing loss, comprising a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or hyaluronate as an active ingredient, wherein the microspheres are dispersed in an aqueous solution of hyaluronic acid or hyaluronate having a molecular weight greater than 5,000,000 g / mol.

[0013] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating hearing loss, which consists of a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or hyaluronate, wherein the microspheres are dispersed in an aqueous solution of hyaluronic acid or hyaluronate having a molecular weight greater than 5,000,000 g / mol.

[0014] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating hearing loss, which consists essentially of a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or hyaluronate, wherein the microspheres are dispersed in an aqueous solution of hyaluronic acid or hyaluronate having a molecular weight greater than 5,000,000 g / mol.

[0015] Another object of the present invention is to provide a method for preparing a controlled-release drug formulation, which comprises (a) encapsulating a steroidal anti-inflammatory agent in microspheres of hyaluronic acid or hyaluronate; (b) preparing an aqueous solution of hyaluronic acid or hyaluronate having a molecular weight greater than 5,000,000 g / mol; and (c) dispersing the microspheres in the aqueous solution.

[0016] Another object of the present invention is to provide a use of a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or hyaluronate in the preparation of a formulation for treating hearing loss, wherein the microspheres are dispersed in an aqueous solution of hyaluronic acid or hyaluronate having a molecular weight greater than 5,000,000 g / mol.

[0017] Another object of the present invention is to provide a method for treating hearing loss by administering to a subject in need thereof an effective amount of a composition comprising a steroidal anti-inflammatory agent as an active ingredient encapsulated in microspheres of hyaluronic acid or hyaluronate, wherein the microspheres are dispersed in an aqueous solution of hyaluronic acid or hyaluronate having a molecular weight greater than 5,000,000 g / mol.

[0018] Technical Solution

[0019] To achieve the object of the present invention, the present invention provides a controlled-release drug formulation, wherein a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or hyaluronate is dispersed in an aqueous solution of hyaluronic acid or hyaluronate having a molecular weight greater than 5,000,000 g / mol.

[0020] In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating hearing loss, comprising a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or hyaluronate as an active ingredient, wherein the microspheres are dispersed in an aqueous solution of hyaluronic acid or hyaluronate having a molecular weight greater than 5,000,000 g / mol.

[0021] In addition, the present invention provides a pharmaceutical composition for preventing or treating hearing loss, which consists of a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or hyaluronate, wherein the microspheres are dispersed in an aqueous solution of hyaluronic acid or hyaluronate having a molecular weight greater than 5,000,000 g / mol.

[0022] In addition, the present invention provides a pharmaceutical composition for preventing or treating hearing loss, which is essentially composed of a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or hyaluronate, wherein the microspheres are dispersed in an aqueous solution of hyaluronic acid or hyaluronate having a molecular weight greater than 5,000,000 g / mol.

[0023] To achieve another object of the present invention, the present invention provides a method for preparing a controlled-release drug formulation, which comprises (a) encapsulating a steroidal anti-inflammatory agent in microspheres of hyaluronic acid or hyaluronate; (b) preparing an aqueous solution of hyaluronic acid or hyaluronate having a molecular weight greater than 5,000,000 g / mol; and (c) dispersing the microspheres in the aqueous solution.

[0024] In order to achieve another object of the present invention, the present invention provides the use of a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or hyaluronate in preparing a formulation for treating hearing loss, wherein the microspheres are dispersed in an aqueous solution of hyaluronic acid or hyaluronate having a molecular weight greater than 5,000,000 g / mol.

[0025] In order to achieve another object of the present invention, the present invention provides a method for treating hearing loss by administering an effective amount of a composition to a subject in need thereof, the composition comprising a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or hyaluronate as an active ingredient, wherein the microspheres are dispersed in an aqueous solution of hyaluronic acid or hyaluronate having a molecular weight greater than 5,000,000 g / mol.

[0026] Hereinafter, the present invention will be described in detail.

[0027] The present invention provides a controlled-release drug formulation, wherein a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or hyaluronate is dispersed in an aqueous solution of hyaluronic acid or hyaluronate having a molecular weight greater than 5,000,000 g / mol.

[0028] In the present invention, hyaluronic acid is a linear polymeric polysaccharide in which β-DN-acetylglucosamine and β-D-glucuronic acid are alternately combined. Known hyaluronic acid is not only distributed in connective tissues such as subcutaneous tissue and cartilage tissue of mammals, but also present in vitreous body of eyes or umbilical cord, and also present in the capsule of streptococcus or bacillus. Natural hyaluronic acid with excellent biocompatibility does not have cross-species specificity and tissue or organ specificity, and enhances the moisturizing power of skin, maintains skin elasticity, and reduces damage to the lower layer of skin when skin is damaged, and is used as a lubricant to promote movement between collagen cells (the main component of skin).

[0029] On the other hand, since natural hyaluronic acid is rapidly decomposed by hyaluronidase when injected into the body, hyaluronic acid derivatives that are cross-linked by various methods or structurally modified using chemicals are also prepared and used in order to control the decomposition rate. However, in the present invention, hyaluronic acid can be characterized as including only natural hyaluronic acid or its salt that is not chemically modified.

[0030] In the present invention, the hyaluronate may be sodium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate, tetrabutylammonium hyaluronate or a combination thereof, but is not limited thereto.

[0031] In the present invention, "microspheres" are spherical fine particles with a diameter of less than several micrometers, and refer to particles used as drug carriers due to excellent biocompatibility and biodegradability. In the present invention, low molecular weight hyaluronic acid having a molecular weight of 100,000 g / mol or less, preferably hyaluronic acid having a molecular weight of 50,000 g / mol or less, more preferably hyaluronic acid having a molecular weight of 2,000 g / mol or less, and most preferably hyaluronic acid having a molecular weight of 10,000 g / mol or less can be used to prepare microspheres.

[0032] In the present invention, "steroidal anti-inflammatory agent" refers to an agent with a steroid nucleus and anti-inflammatory effect, for example, a corticosteroid, such as dexamethasone, prednisolone, methylprednisolone, betamethasone, hydrocortisone or a combination thereof. Preferably, the steroidal anti-inflammatory agent may be dexamethasone.

[0033] In the present invention, microspheres can be prepared by known methods such as a phase separation method, a spray drying method, a solvent evaporation drying method, and a low-temperature solvent extraction method.

[0034] According to an embodiment of the present invention, microspheres are prepared by adding a steroidal anti-inflammatory agent to an aqueous solution of low molecular weight hyaluronic acid and then adding an electrolyte aqueous solution to the solution to induce complex formation between the hyaluronic acid and the electrolyte.

[0035] The drug controlled release formulation of the present invention can be characterized by the above-mentioned hyaluronic acid microspheres dispersed in an aqueous solution of ultra-high molecular weight hyaluronic acid or hyaluronate with a molecular weight greater than 5,000,000 g / mol.

[0036] The ultra-high molecular weight hyaluronic acid or its salt is not chemically modified, for example, natural ultra-high molecular weight hyaluronic acid that is not modified with chemical functional groups, such as hyaluronic acid diaminobutane, hyaluronic acid succinimide, and the like.

[0037] The molecular weight of the ultra-high molecular weight hyaluronic acid may be greater than 5,000,000 g / mol, preferably greater than 5,100,000 g / mol, more preferably greater than 5,200,000 g / mol, even more preferably greater than 5,300,000 g / mol, and most preferably greater than 5,400,000 g / mol.

[0038] There is no particular restriction on the upper molecular weight limit of ultra-high molecular weight hyaluronic acid, and it is obvious to those skilled in the art that any ultra-high molecular weight hyaluronic acid with a molecular weight greater than 5,000,000 g / mol, as a high molecular weight hyaluronic acid naturally present in nature, is applied to the present invention to show the same effect. The upper molecular weight limit of ultra-high molecular weight hyaluronic acid may preferably be 10,000,000 g / mol, more preferably 9,000,000 g / mol, more preferably 8,000,000 g / mol, and most preferably 7,500,000 g / mol.

[0039] According to one embodiment of the present invention, ultra-high molecular weight hyaluronic acid can be produced by Streptococcus UBC-U46 (registration number KCTC13556BP) strain. The hyaluronic acid produced by microorganisms can be prepared with reference to Korean Patent No. 10-2018-0130275, which is cited in the present invention.

[0040] In the present invention, the concentration of the hyaluronic acid or hyaluronate aqueous solution can be appropriately adjusted by those skilled in the art in consideration of the release time of the steroidal anti-inflammatory agent to be controlled-released, but may be 1.0 to 10.0% (w / v), preferably 1.5 to 8% (w / v), more preferably 2.0 to 7% (w / v), still more preferably 2.5 to 6% (w / v), most preferably 3.0 to 5% (w / v) of the hyaluronic acid aqueous solution.

[0041] The release rate and release period of the steroidal anti-inflammatory agent in the controlled release formulation of the present invention can be controlled by the concentration of the ultra-high molecular weight hyaluronic acid aqueous solution and the molecular weight of the ultra-high molecular weight hyaluronic acid, and can be adjusted by those skilled in the art according to the target site to be treated, the severity of the disease to be treated, etc.

[0042] The aqueous solution of ultra-high molecular weight hyaluronic acid does not contain any materials other than ultra-high molecular weight hyaluronic acid and water, such as a cross-linking agent, a curing agent, a chemical modifier, a fatty acid, a cationic polymer that forms a complex with hyaluronic acid, and the like.

[0043] The cross-linking agent is a compound that reacts with hyaluronic acid to form a three-dimensional network structure, and may be at least one compound selected from the following: a multivalent epoxy compound such as polyglycidyl ether, divinyl sulfone, formaldehyde, phosphorus oxychloride, a combination of a carbodiimide compound and an amino acid ester, and a combination of a carbodiimide compound and a dihydrazide compound.

[0044] The chemical modifier is a compound that reacts with a carboxyl group, a hydroxyl group or an acetylamino group of hyaluronic acid to form a covalent bond, and may be at least one compound selected from the group consisting of a combination of acetic anhydride and concentrated sulfuric acid, a combination of trifluoroacetic anhydride and an organic acid, and an alkyl iodide compound.

[0045] The cationic polymer that forms a complex with hyaluronic acid is a polymer that forms a complex through an ionic bond between a carboxyl group of hyaluronic acid and an amino group or an imino group of a polymer compound, and may be at least one cationic polymer selected from the group consisting of chitosan, polylysine, polyvinylpyridine, polyethyleneimine, and polydimethylaminoethylmethacrylate.

[0046] In the case of preparing hyaluronic acid hydrogel using chemical crosslinking agents and / or curing agents, these chemicals remain inside the hyaluronic acid hydrogel or in the external carrier, thereby increasing the possibility of inducing harmful reactions such as inflammatory reactions. However, the drug controlled release formulation of the present invention can exhibit physiological activity very safely after being injected into the body because no chemicals are added to the aqueous solution of ultra-high molecular weight hyaluronic acid.

[0047] In one embodiment of the present invention, the aqueous solution of ultra-high molecular weight hyaluronic acid can be characterized as native hyaluronic acid, wherein cross-linking is not induced by any means such as radiation, heat, and the like.

[0048] According to an embodiment of the present invention, the drug controlled release formulation of the present invention using an aqueous solution of ultra-high molecular weight hyaluronic acid as a dispersion does not induce any inflammatory response after being applied to the tympanic cavity. However, in the case where the formulation uses a hydrogel prepared by treating a hyaluronic acid derivative with a cross-linking agent as a dispersion, it was confirmed that the formulation induces a severe inflammatory response after being applied to the tympanic cavity.

[0049] In the present invention, in the controlled-release drug formulation, the concentration of the microspheres of hyaluronic acid or hyaluronate dispersed in the aqueous solution of ultra-high molecular weight hyaluronic acid or hyaluronate can be appropriately adjusted by those skilled in the art according to the release period, release concentration, etc. of the steroidal anti-inflammatory agent contained in the microspheres. For example, the microspheres can be dispersed at a concentration of 0.1 to 5% (w / v), preferably 0.5 to 4% (w / v), more preferably 0.5 to 3% (w / v), even more preferably 0.5 to 2.5% (w / v), and most preferably 1 to 2% (w / v).

[0050] In one embodiment of the invention, formulation can be a controlled release formulation, so that the target site releases the predetermined time period of active drug. Formulation can release steroidal anti-inflammatory agent up to 20 days or longer, 1 month or longer, 40 days or longer, 2 months or longer, 4 months or longer, 6 months or longer, 6 months to 12 months or 12 months or longer. In one embodiment, the release period can be adjusted according to the molecular weight of ultra-high molecular weight hyaluronic acid or the concentration of its aqueous solution. Target site refers to the specific organ of object, and is not limited to eyes, nose, ear, heart, ventricle, atrium, intestinal tract, blood vessel or joint. In one embodiment, target site can be ear, and ear can include middle ear, inner ear, cochlea, vestibular organ, eustachian tube or tympanic cavity.

[0051] The present invention provides a pharmaceutical composition for preventing or treating hearing loss, comprising a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or hyaluronate as an active ingredient, wherein the microspheres are dispersed in an aqueous solution of hyaluronic acid or hyaluronate having a molecular weight greater than 5,000,000 g / mol.

[0052] In the present invention, hearing loss includes ototoxic hearing loss caused by presbycusis, anticancer drugs, antipyretics, analgesics, antibiotics, etc., noise-induced hearing loss caused by industrial noise, gunshots, explosion noise, etc., traumatic hearing loss such as Meniere's disease, hearing loss due to inner ear diseases such as sudden hearing loss, perilymph laceration, labyrinthine concussion and temporal bone fracture, and other hearing loss caused according to its cause.

[0053] In one embodiment of the invention, the hearing loss may be selected from noise-induced hearing loss, ototoxic hearing loss, hearing loss due to inner ear disease and traumatic hearing loss.

[0054] The pharmaceutical composition of the present invention may be a composition for administration in the middle ear, the inner ear, the cochlea, the vestibular organ, the Eustachian tube or the tympanic cavity, preferably a composition for administration in the tympanic cavity.

[0055] The pharmaceutical composition according to the present invention can be formulated in a suitable form together with a pharmaceutically acceptable carrier, and may further include an excipient or diluent. Carriers include all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microparticles.

[0056] Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. In addition, various drug delivery materials for oral administration may be included. In addition, carriers for parenteral administration may include water, suitable oils, saline, aqueous glucose, ethylene glycol, etc., and also include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methylparaben or propylparaben and chlorobutanol. In addition to the above-mentioned ingredients, the pharmaceutical composition of the present invention may also include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, etc. Other pharmaceutically acceptable carriers and formulations may refer to those known in the art.

[0057] The composition of the present invention can be administered to mammals including humans by any method. For example, the composition can be administered by a parenteral method. The parenteral administration method is not limited thereto, but can be intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, rectal, intraarticular or intratympanic administration, most preferably intratympanic administration.

[0058] Intratympanic injections are injections of medication into the middle and / or inner ear behind the eardrum.

[0059] In one embodiment of the invention, the pharmaceutical composition of the invention may be administered to the middle ear by transtympanic injection. In another embodiment, the pharmaceutical composition of the invention may be administered directly to the inner ear via a non-tympanic route into the inner ear.

[0060] In one embodiment of the invention, the pharmaceutical composition can be administered by a syringe and needle that passes through the tympanic membrane and directly approaches the round window membrane or oval window of the inner ear. The needle of the syringe is preferably used with a needle as small as possible to prevent perforation of the tympanic membrane, and can be, for example, 22 gauge to 24 gauge.

[0061] The pharmaceutical composition of the present invention can be formulated into a formulation for oral administration or parenteral administration according to the administration route as described above.

[0062] In the case of a formulation for oral administration, the composition of the present invention can be formulated into a powder, granules, tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, etc. using methods known in the art. For example, oral formulations can be obtained by mixing the active ingredient with a solid excipient, crushing the mixture, adding a suitable adjuvant, and then processing the mixture into a granular mixture to obtain a tablet or a sugar-coated tablet. Examples of suitable excipients may include: sugars, including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol; starches, including corn starch, wheat starch, rice starch, and potato starch; celluloses, including methylcellulose, sodium carboxymethylcellulose, and hydroxypropyl methylcellulose; fillers, such as gelatin, polyvinyl pyrrolidone, etc. In addition, in some cases, cross-linked polyvinyl pyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant. In addition, the pharmaceutical composition may also include an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, and a preservative.

[0063] The formulations for parenteral administration can be formulated into the form of injections, creams, lotions, external ointments, oils, moisturizers, gels, aerosols and nasal inhalants by methods known in the art. These formulations are described in all the commonly known prescriptions in pharmaceutical chemistry.

[0064] The total effective dose of the composition of the present invention can be administered to the patient in a single dose, or can be administered for a long time in multiple doses according to a fractionated treatment regimen. In the pharmaceutical composition of the present invention, the content of the active ingredient can vary according to the severity of the disease. Preferably, the preferred total dose of the pharmaceutical composition of the present invention can be about 0.01g to 10,000mg per 1kg patient body weight per day, most preferably 0.1g to 500mg. However, in addition to the route of administration and the number of treatments of the pharmaceutical composition, the dosage of the pharmaceutical composition is determined by considering various factors, including the patient's age, weight, health status and gender, the severity of the disease, diet and excretion rate. Taking this aspect into account, those skilled in the art can determine the appropriate effective dose of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited to formulations, routes of administration and methods of administration, as long as the effect of the present invention is shown.

[0065] In one embodiment of the invention, the pharmaceutical composition can be administered before or after the onset of hearing loss or during the onset of hearing loss. The dosage can vary according to the method of administration, the duration of treatment, the condition of the patient to be treated and the severity of the hearing loss, and is determined by the attending physician. The duration of treatment can range from about 1 hour to several days, weeks or months, and can be longer if necessary.

[0066] As used herein, the term "prevention" refers to a therapy that protects against the onset of a disease or condition so that clinical symptoms of the disease do not develop. Thus, "prevention" refers to administering a therapy (e.g., administering a therapeutic agent) to a subject before symptoms of the disease can be detected in the subject (e.g., administering a therapeutic material to a subject in the absence of a detectable infectious agent (e.g., a virus) in the subject). A subject can be an individual at risk of developing a disease or condition, such as an individual with one or more risk factors known to be associated with the development or onset of a disease or condition.

[0067] In the present invention, the term "treatment" refers to a method for obtaining a beneficial or desired clinical result. For the purposes of the present invention, a beneficial or desired clinical result includes a non-limiting alleviation of symptoms, a reduction in the extent of the disease, a stabilization of the disease condition (i.e., no worsening), a delay in the progression of the disease or a reduced rate, an improvement in the disease condition or a temporary alleviation and reduction (which may be partial or complete), and whether it is detectable or undetectable. "Treatment" refers to all therapeutic treatments and preventive or defensive measures. Treatment includes treatment required for conditions that have already occurred as well as conditions to be prevented. "Relief" of a disease refers to a reduction in the extent of the disease condition and / or undesirable clinical symptoms or a delay or prolongation of the time course of progression compared to no treatment.

[0068] Meanwhile, it is obvious to those skilled in the art that the pharmaceutical composition of the present invention may further include known materials for treating hearing loss.

[0069] The present invention provides a method for preparing a controlled-release drug formulation, which comprises (a) encapsulating a steroidal anti-inflammatory agent in microspheres of hyaluronic acid or hyaluronate; (b) preparing an aqueous solution of hyaluronic acid or hyaluronate having a molecular weight greater than 5,000,000 g / mol; and (c) dispersing the microspheres in the aqueous solution.

[0070] The present invention also provides the use of a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or hyaluronate in the preparation of a formulation for treating hearing loss, wherein the microspheres are dispersed in an aqueous solution of hyaluronic acid or hyaluronate having a molecular weight greater than 5,000,000 g / mol.

[0071] The present invention also provides a method for treating hearing loss by administering to a subject in need thereof an effective amount of a composition comprising a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or hyaluronate as an active ingredient, wherein the microspheres are dispersed in an aqueous solution of hyaluronic acid or hyaluronate having a molecular weight greater than 5,000,000 g / mol.

[0072] The "effective dose" of the present invention refers to an amount that, when administered to a subject, shows an effect of improving, treating, detecting and diagnosing hearing loss or inhibiting or reducing hearing loss. The "subject" may be an animal, preferably a mammal, particularly an animal including a human being, and may also be a cell, tissue and organ derived from an animal. The individual may be a patient in need of the effect.

[0073] "Treatment" of the present invention comprehensively refers to improving hearing loss or symptoms caused by hearing loss, and may include treating or substantially preventing a disease or improving its symptoms, and includes alleviating, treating or preventing symptoms or most symptoms caused by a disease, but is not limited thereto.

[0074] As used herein, the term "comprising" is used in the same sense as "including" or "characterized by", and does not exclude additional ingredients or method steps not specifically mentioned in the composition or method according to the present invention. Unless otherwise described, the term "consisting of" means excluding additional elements, steps or ingredients, etc. The term "consisting essentially of" means that within the scope of the composition or method, in addition to the described materials or steps, materials or steps that do not substantially affect its basic properties are included.

[0075] Beneficial effects

[0076] According to the present invention, since the pharmaceutical controlled-release formulation and pharmaceutical composition for treating hearing loss containing an aqueous solution of ultra-high molecular weight hyaluronic acid as a drug carrier can show therapeutic effects for weeks to months at a single dose, the disease can be effectively treated without repeated procedures and without causing systemic side effects. In addition, since a chemical cross-linking agent for cross-linking hyaluronic acid is not used in the pharmaceutical controlled-release formulation of the present invention, the risk of side effects such as inflammatory reactions is very low. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 is a diagram showing a test schedule for evaluating hearing improvement testing in an animal model of hearing loss induction.

[0078] Figure 2 The retention period of drug / vehicle in the tympanic cavity of the animals was assessed by CT scanning.

[0079] Figure 3 is a diagram observing the process of healing a tympanic membrane perforation that occurs during injection of a drug / vehicle into the tympanic membrane over time.

[0080] Figure 4 It is a graph observing whether inflammation occurs in the tympanic membrane of animals over time after administration of the drug / carrier according to the present invention (SUREDEN).

[0081] Figure 5is the result of evaluating the hearing improvement effect in an animal model of hearing loss (SU02+D: drug / carrier according to the present invention).

[0082] Figure 6 is a schematic diagram showing an experimental method of injecting a prior art drug / carrier into the tympanic membrane of an animal model of hearing loss.

[0083] Figure 7 The results are to evaluate the extent of inflammation after injection of prior art drugs / vectors into the tympanic membrane of an animal model of hearing loss.

[0084] Figure 8 The graph is a graph observing contrast effect over time using 9.4T MRI after a vehicle obtained by mixing a contrast agent with an aqueous solution of hyaluronic acid having a weight average molecular weight of 7,000,000 g / mol (SURDEN01+Magnevist) and a vehicle obtained by mixing a contrast agent with an aqueous solution of hyaluronic acid having a molecular weight of 1,000,000 to 3,000,000 g / mol (low-viscosity vehicle+Magnevist) were administered to the tympanic membrane of rats.

[0085] Fig. 9 It is shown in Figure 8 Figure quantification of the amount of contrast agent at each location in the rat tympanic membrane over time during the course of the experiment.

[0086] Methods for use in the present invention

[0087] Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0088] Example 1: Preparation of dexamethasone microcapsules using ultra-low molecular weight hyaluronic acid

[0089] An aqueous solution containing 20% ​​of ultra-low molecular weight hyaluronic acid having a molecular weight of less than 10,000 Da was prepared in ultrapure water, and 2.4% of dexamethasone disodium phosphate was completely dissolved therein.

[0090] The prepared hyaluronic acid-dexamethasone aqueous solution was mixed with 10% CaCl2 (containing Ca 2+ An aqueous solution of hyaluronic acid (ionic compound) was mixed at a ratio of 1:1 (v / v) to induce an ionic complex (precipitation) between hyaluronic acid and CaCl2. Thereafter, the precipitate was centrifuged at 12,000 rpm for 1 minute to remove the supernatant. In addition, in order to remove free ions that do not participate in the ionic complex, the washing and centrifugation process was performed three or more times using an excess of ultrapure water.

[0091] The ion-complexed drug mixture was lyophilized to prepare dexamethasone encapsulated in ultra-low molecular weight hyaluronic acid.

[0092] Example 2: Preparation of Encapsulated Dexamethasone / Hyaluronic Acid Hydrogel Complex (SURDEN; Sustained Release Drug Encapsulation)

[0093] After preparing ultra-high molecular weight hyaluronic acid having adhesiveness, in vivo stability in the tympanic cavity, and excellent biocompatibility as a carrier for encapsulated dexamethasone, microencapsulated dexamethasone / hyaluronic acid complexes were prepared using ultra-high molecular weight hyaluronic acid.

[0094] Ultra-high molecular weight hyaluronic acid was prepared using Streptococcus UBC-U46 strain (accession number: KCTC13556BP) according to the production method specified in a previous patent (KR1020180130275).

[0095] The weight average molecular weight of the ultra-high molecular weight hyaluronic acid prepared according to the method is 7,000,000 g / mol.

[0096] Ultrahigh molecular weight hyaluronic acid was dissolved in ultrapure water to prepare an aqueous solution of ultrahigh molecular weight hyaluronic acid with a concentration of 3%. The dexamethasone powder encapsulated in ultralow molecular weight hyaluronic acid prepared in Example 1 was added to the ultrahigh molecular weight hyaluronic acid aqueous solution to a concentration of 1.5%, and uniformly mixed using a homogenizer to prepare a microencapsulated dexamethasone / hyaluronic acid hydrogel complex (SURDEN), in which the dexamethasone encapsulated in the ultralow molecular weight hyaluronic acid was uniformly mixed.

[0097] Example 3: Evaluation of drug carrier retention period and drug release performance

[0098] In order to evaluate the performance of the composite (SURDEN) prepared in Example 2 as a drug carrier, a hearing loss animal model was established and the following experiments were performed.

[0099] (1) Preparation of animal model of hearing loss

[0100] To exclude the effects of hormones, male SD rats were used for the experiments. To induce ototoxic hearing loss, a combination drug of cisplatin (2 mg / kg), gentamicin (120 mg / kg), and furosemide (90 mg / kg) was injected into the caudal jugular vein once a day for 2 days on day 4 after IT inoculation.

[0101] (2) Experimental design

[0102] Dexamethasone (D) was administered to animals by two different methods using the intratympanic (IT) drug delivery method, which delivers the drug through the tympanic membrane.

[0103] The first method is a method of treatment used in existing hospitals, and a method of delivering 12 mg / ml of D into saline. The second method is a method of delivering 12 mg / ml of D into SURDEN prepared in Example 2 above. As a control, a hearing loss control group without treatment was prepared. Each sample prepared as described above was administered at 0.04 ml / animal (per ear). Figure 1 The specific experimental schedule is shown.

[0104] (3) Hearing measurement

[0105] Hearing was measured every 1 to 14 days before and for approximately 2 months after induction of hearing loss. Auditory brainstem response (ABR) testing was performed at a total of four frequencies (click, 8 kHz, 16 kHz, and 32 kHz) to evaluate the characteristics of each frequency.

[0106] (4) Evaluation of drug carrier retention period

[0107] exist Figure 1 The retention period of the drug / vehicle in the animal tympanic cavity confirmed by the CT scanning time indicated in the experimental plan of was 1.1±0.3 days in the case of saline+D, and 41.1±27.0 days in the case of SURDEN. That is, it was confirmed that when SURDEN was used, the retention period of the drug / vehicle could be about 40 times longer than that of saline ( Figure 2 ).

[0108] (5) Safety assessment of drug carriers

[0109] As a result of regularly taking tympanic membrane endoscopy, the tympanic membrane perforation was completely blocked after 21.6±12.6 days in the saline+D group. In the case of the SERDEN+D group, the tympanic membrane perforation was completely blocked after 16.7±11.9 days. That is, when SURDEN is used, since the time period for the healing of the tympanic membrane perforation is quite short or at least not long, it is confirmed that the treatment can be performed without side effects ( Figure 3 ).

[0110] In addition, as a result of tympanic membrane endoscopy, the subjects did not develop otitis media due to SURSEN administration. Otitis media occurs when the drug / carrier is not biocompatible ( Figure 4 ), and in the case of the drug / carrier complex (SURDEN) of the present invention, since there was no inflammatory side effect, it was determined that the drug / carrier complex (SURDEN) could be successfully used in humans.

[0111] Example 4: Hearing Improvement Assessment

[0112] In the case of SURDEN, hearing began to improve from the first day (46.7dB) based on 32kHz-induced hearing loss, compared with saline+D (70.0dB) or the control group (62.9dB), and this trend was well maintained until 4 days (46.7dB), 8 days (52.5dB), 12 days (47.5dB), 21 days (46.7dB), and 30 days (50.0dB). Compared with the existing standard treatment method of saline+D, the treatment effect of SERDEN was 23.3dB to 26.7dB better. Generally, considering that the clinically significant hearing difference is 15dB, a treatment effect of 23.3dB to 26.7dB better is a clinically significant difference.

[0113] This difference was observed even at the mid-range frequency of 16kHz. Compared with the existing standard treatment, saline + D, SERDEN's treatment effect was 16.8dB to 23.3dB better. Considering that the clinically significant hearing difference is 15dB, a treatment effect of 16.8dB to 23.3dB better at 16kHz is a clinically significant difference.

[0114] Even at the lowest frequency of 8kHz, the therapeutic effect was optimal in SURDEN. Compared with the existing standard treatment method of saline + D, the therapeutic effect through SERDEN was 1.7dB to 10.8dB better ( Figure 5 ).

[0115] The comprehensive results confirmed that SURDEN's hearing loss treatment effect is significantly better than the existing standard treatment method of saline + D.

[0116] Example 5: Confirmation of inflammatory response

[0117] The present inventors compared whether the method according to the present invention induces an inflammatory response in the inner ear with the method disclosed in the existing registered patent (10-1877894).

[0118] First, after noise-induced hearing loss in normal-hearing rats, the rats were divided into Figure 6 The three groups shown in the figure ((1) dexamethasone + saline (D + saline), (2) dexamethasone + IGF-1 + saline (D&G + saline), and (3) LGA microcapsules loaded with dexamethasone + IGF-1 + HA (cD&G + HA)) were then injected into the tympanic membrane, and hearing tests, tympanoscopy, and CT scans were performed for 45 days.

[0119] In addition, the SURDEN administration group according to the present invention was experimented in the same manner as described above.

[0120] The results are as follows Figure 7 shown.

[0121] like Figure 8 As shown, when the ratio of each group relative to the subject in which an inflammatory response was induced in the middle ear tissue after drug injection through the tympanic membrane was calculated, the inflammation ratio in the dexamethasone-loaded PLGA microcapsule + IGF-1 + HA group was 42.3%, which was twice as high as that of the other groups.

[0122] On the other hand, no inflammatory reaction was observed in the SURDEN-administered group (not shown in the results).

[0123] Example 6: Comparison of controlled release ability and hearing loss treatment effect based on hyaluronic acid molecular weight

[0124] In order to comparatively evaluate the controlled release effect according to the molecular weight of the ultra-high molecular weight hyaluronic acid used in Example 2, the controlled release ability of a carrier using hyaluronic acid having a relatively lower molecular weight (1,000,000 to 3,000,000 g / mol) than the ultra-high molecular weight (weight average molecular weight 7,000,000 g / mol) used to prepare SURDEN in Example 2 was comparatively evaluated.

[0125] Specifically, the contrast agent was added to an aqueous hyaluronic acid solution with a weight average molecular weight of 7,000,000 g / mol (SURDEN) or an aqueous hyaluronic acid solution with a molecular weight of 1,000,000 to 3,000,000 g / mol (low viscosity carrier), and each of these samples was injected into the tympanic membrane of rats, and then photographed and quantitatively analyzed using 9.4T MRI.

[0126] The results are as follows Fig. 9 shown.

[0127] like Fig. 9 As shown, it was confirmed that the contrast intensity increased in the vestibular system and the tympanic system, and that the SURDEN-administered group showed a stronger synergistic effect than the low-viscosity vehicle group at all time points and in all regions.

[0128] In addition, as a result of quantification, it was confirmed that the signal intensity of the SURDEN-administered group in the lower, middle, and upper layers of the cochlea was higher than that of the low-viscosity vehicle group.

[0129] In conclusion, in the case of SURDEN, it was confirmed that the drug release ability is excellent in inner ear drug delivery by maintaining the drug at a higher concentration for a longer period of time and in a larger amount compared with a carrier using relatively low molecular weight hyaluronic acid.

[0130] Industrial Applicability

[0131] According to the present invention, since the drug controlled-release formulation and the pharmaceutical composition for treating hearing loss containing an aqueous solution of ultra-high molecular weight hyaluronic acid as a drug carrier can show a therapeutic effect for weeks to months with a single dose, the disease can be effectively treated without repeated procedures and without causing systemic side effects. In addition, since a chemical cross-linking agent for cross-linking hyaluronic acid is not used in the drug controlled-release formulation of the present invention, the risk of side effects such as inflammatory reactions is very low, and thus the industrial applicability is very high.

Claims

1. A drug controlled release formulation, wherein a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or hyaluronate having a molecular weight of 100,000 g / mol or less is dispersed in an aqueous solution of hyaluronic acid or hyaluronate having a molecular weight of 7,000,000 g / mol or more, wherein the steroidal anti-inflammatory agent is dexamethasone, prednisolone, methylprednisolone, betamethasone, hydrocortisone or a combination thereof, and wherein the drug controlled release formulation does not contain a chemical cross-linking agent.

2. The pharmaceutical controlled-release formulation of claim 1, wherein the hyaluronic acid having a molecular weight of 7,000,000 g / mol or more is produced by Streptococcus sp. UBC-U46 (Accession No. KCTC13556BP) strain.

3. The controlled-release drug formulation of claim 1, wherein the controlled-release drug formulation releases the steroidal anti-inflammatory agent at a target site for 20 days or longer.

4. The pharmaceutical controlled-release formulation of claim 1, wherein the aqueous solution of hyaluronic acid or hyaluronate is a 1 to 10% (w / v) aqueous solution.

5. The pharmaceutical controlled-release formulation of claim 1, wherein the formulation is a formulation for administration into the middle ear, the inner ear, the cochlea, the vestibular organ, the Eustachian tube, or the tympanic cavity.

6. A pharmaceutical composition for preventing or treating hearing loss, comprising as an active ingredient a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or hyaluronate having a molecular weight of 100,000 g / mol or less, wherein the microspheres are dispersed in an aqueous solution of hyaluronic acid or hyaluronate having a molecular weight of 7,000,000 g / mol or more, wherein the steroidal anti-inflammatory agent is dexamethasone, prednisolone, methylprednisolone, betamethasone, hydrocortisone or a combination thereof, and wherein the drug controlled-release formulation does not contain a chemical cross-linking agent.

7. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition is injected into the middle ear, the inner ear, the cochlea, the vestibular organ, the Eustachian tube or the tympanic cavity.

8. The pharmaceutical composition of claim 6, wherein the hearing loss is selected from the group consisting of noise-induced hearing loss, ototoxic hearing loss, hearing loss due to inner ear disease, and traumatic hearing loss.

9. A method for preparing a controlled-release pharmaceutical formulation according to any one of claims 1 to 5, comprising the following steps: (a) encapsulating a steroidal anti-inflammatory agent in microspheres of hyaluronic acid or hyaluronate having a molecular weight of 100,000 g / mol or less, wherein the steroidal anti-inflammatory agent is dexamethasone, prednisolone, methylprednisolone, betamethasone, hydrocortisone or a combination thereof; (b) preparing an aqueous solution of hyaluronic acid or a hyaluronate salt having a molecular weight of 7,000,000 g / mol or more; as well as (c) dispersing the microspheres in the aqueous solution; The drug controlled-release formulation does not contain a chemical cross-linking agent.

10. Use of the pharmaceutical controlled-release formulation of claim 1 in the preparation of a medicament for treating hearing loss.

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