Controlled release formulation for treating hearing loss and method for producing same

Abstract

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

Description

[Technical field]

[0001] This application claims priority to Korean Patent Application No. 10-2020-0012749, filed on February 3, 2020, the entire specification of which is incorporated herein by reference.

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

[0003] Hearing loss is a condition in which hearing is impaired or lost due to various causes. The ear is composed of the outer ear (from the earlobe to the eardrum), the middle ear (from the eardrum to the entrance of the cochlea), and the inner ear (inside the cochlea), and hearing loss occurs when a problem occurs in any one of these areas. Hearing loss is classified as hearing loss caused by disorders of the outer and middle ear (total hearing loss) and hearing loss caused by disorders of the inner ear and auditory nervous system (sensory neurological hearing loss). Hearing loss caused by disorders of the outer and middle ear can be recovered if the disorders are treated, but hearing loss caused by disorders of the inner ear and auditory nervous system often cannot be recovered even if the disorders are treated, which causes great inconvenience to patients.

[0004] The most common treatment for hearing loss to date is systemic oral steroid administration. However, it has been reported that 33.0% of healthy patients with hearing loss experienced one or more systemic side effects after systemic steroid administration, and approximately 1% experienced serious 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 is used to treat sudden hearing loss. Steroids are injected into the tympanic cavity through the tympanic membrane, and it is expected that the injected steroids will diffuse through the porcelain window located between the tympanic cavity and the inner ear. However, the drug injected into the tympanic cavity is discharged through the Eustachian tube within a few minutes, so the time for the drug to reach the inner ear is shorter than 24 hours, and sufficient therapeutic effects cannot be achieved.

[0006] Meanwhile, Korean Patent No. 10-1877894 discloses a drug release controlled hearing loss therapeutic agent prepared by dispersing a steroidal anti-inflammatory agent encapsulated in microspheres derived from a biocompatible polymer in a hyaluronic acid hydrogel. The drug release controlled hearing loss therapeutic agent has the effect of extending the release period of the steroidal anti-inflammatory agent, but it is troublesome to use two types of chemically modified hyaluronic acid derivatives, and has a problem of inflammation in the eardrum caused by the crosslinking agent that is required for the preparation of the hydrogel.

[0007] Therefore, there is a need for a drug for the treatment of hearing loss that can reduce the side effects of systemic steroid administration and deliver the drug intratympanically for a sufficient period of time to treat hearing loss. Summary of the Invention [Problem to be solved by the invention]

[0008] The inventors have conducted extensive research to develop a drug for the treatment of hearing loss that reduces the side effects of systemic steroid administration and allows the drug to be delivered into the tympanic cavity for a sufficient period of time to treat hearing loss. As a result, they have found that a composite preparation made by dispersing a steroid anti-inflammatory agent encapsulated in low molecular weight hyaluronic acid in an aqueous solution of high molecular weight hyaluronic acid significantly extends the drug release period in the tympanic cavity. They found that this not only increases the amount of erythrocyte secretion but also does not cause side effects such as inflammatory reactions, which led to the completion of the present invention.

[0009] It is therefore an object of the present invention to provide a controlled release drug formulation in which a steroidal anti-inflammatory drug encapsulated in microspheres of hyaluronic acid or a hyaluronate is dispersed in an aqueous solution of hyaluronic acid or a hyaluronate having a molecular weight of more than 5,000,000 g / mol.

[0010] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of hearing loss, comprising as an active ingredient a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or a hyaluronate, said microspheres being dispersed in an aqueous solution of hyaluronic acid or a hyaluronate having a molecular weight exceeding 5,000,000 g / mol. It is another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of hearing loss, comprising a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or a hyaluronate, said microspheres being dispersed in an aqueous solution of hyaluronic acid or a hyaluronate having a molecular weight exceeding 5,000,000 g / mol. 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 a hyaluronate, said microspheres being dispersed in an aqueous solution of hyaluronic acid or a hyaluronate having a molecular weight exceeding 5,000,000 g / mol.

[0011] Another object of the present invention is to provide a method for preparing said controlled release drug formulation, comprising the steps of: (a) encapsulating a steroidal anti-inflammatory drug in microspheres of hyaluronic acid or a hyaluronate; (b) preparing an aqueous solution of hyaluronic acid or a hyaluronate having a molecular weight of more than 5,000,000 g / mol; and (c) dispersing said microspheres in said aqueous solution.

[0012] Another object of the present invention is to provide the use of a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or a hyaluronate for the manufacture of a preparation for the treatment of hearing loss, said microspheres being dispersed in an aqueous solution of hyaluronic acid or a hyaluronate having a molecular weight of more than 5,000,000 g / mol.

[0013] Another object of the present invention is to provide a method for treating hearing loss by administering to an individual in need thereof an effective amount of a composition comprising as an active ingredient a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or a hyaluronate, said microspheres being dispersed in an aqueous solution of hyaluronic acid or a hyaluronate having a molecular weight exceeding 5,000,000 g / mol. [Means for solving the problem]

[0014] In order to achieve the above-mentioned object of the present invention, the present invention provides a controlled release drug formulation in which a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or a hyaluronate is dispersed in an aqueous solution of hyaluronic acid or a hyaluronate having a molecular weight of more than 5,000,000 g / mol.

[0015] In order to achieve another object of the present invention, the present invention provides 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 a hyaluronate, the microspheres being dispersed in an aqueous solution of hyaluronic acid or a hyaluronate having a molecular weight of more than 5,000,000 g / mol. The present invention also comprises a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or a hyaluronate, said microspheres having a molecular weight of 5,000,000 g / mol. The present invention provides a pharmaceutical composition for preventing or treating hearing loss, comprising more than 100% hyaluronic acid or hyaluronate dispersed in an aqueous solution. The present invention also provides 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 a hyaluronate, said microspheres being dispersed in an aqueous solution of hyaluronic acid or a hyaluronate having a molecular weight exceeding 5,000,000 g / mol.

[0016] In order to achieve another object of the present invention, the present invention provides a method for preparing the controlled release drug formulation, comprising the steps of: (a) encapsulating a steroidal anti-inflammatory drug in microspheres of hyaluronic acid or a hyaluronate; (b) preparing an aqueous solution of hyaluronic acid or a hyaluronate having a molecular weight of more than 5,000,000 g / mol; and (c) dispersing the microspheres in the aqueous solution.

[0017] 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 a hyaluronate for the manufacture of a preparation for treating hearing loss, the microspheres being dispersed in an aqueous solution of hyaluronic acid or a hyaluronate having a molecular weight of more than 5,000,000 g / mol.

[0018] In order to achieve another object of the present invention, the present invention provides a method for treating hearing loss by administering to an individual in need thereof an effective amount of a composition comprising as an active ingredient a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or a hyaluronate, wherein the microspheres are dispersed in an aqueous solution of hyaluronic acid or a hyaluronate having a molecular weight exceeding 5,000,000 g / mol.

[0019] The present invention will be described in detail below.

[0020] The present invention provides a controlled release drug formulation in which a steroidal anti-inflammatory drug encapsulated in microspheres of hyaluronic acid or a hyaluronate is dispersed in an aqueous solution of hyaluronic acid or a hyaluronate having a molecular weight of more than 5,000,000 g / mol.

[0021] In the present invention, the hyaluronic acid is a linear polymeric polysaccharide in which β-DN-acetylglucosamine and β-D-glucuronic acid are alternately linked. Hyaluronic acid is known to be distributed in connective tissues such as subcutaneous tissue and cartilage tissue of mammals, as well as in the vitreous body of the eye, umbilical cord, and narrow membrane of streptococci and bacilli. Natural hyaluronic acid has excellent biocompatibility, does not have interspecies specificity, tissue specificity, or long-term specificity, and promotes the moisturizing power of the skin, maintains the elasticity of the skin, reduces damage to the lower layer of the skin when the skin is damaged, and also plays a role like a lubricant to facilitate the movement of collagen, the main component of the skin, between cells.

[0022] On the other hand, natural hyaluronic acid is decomposed quickly by hyaluronic acid decomposition enzymes when injected into the body, so in order to adjust the decomposition rate, hyaluronic acid derivatives may be prepared and used by crosslinking in various ways or modifying the structure using chemicals, but the hyaluronic acid in the present invention may be characterized as containing only natural hyaluronic acid or its salt that has not been chemically modified.

[0023] In the present invention, the hyaluronate may be, but is not limited to, sodium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate, tetrabutylammonium hyaluronate, or combinations thereof.

[0024] In the present invention, the "microsphere" refers to a spherical microparticle with a diameter of less than several micrometers, which has excellent biocompatibility and biodegradability and is used as a drug delivery agent.The microsphere in the present invention may be made with low molecular weight hyaluronic acid having a molecular weight of 100,000g / mol or less, preferably with hyaluronic acid having a molecular weight of 50,000g / mol or less, more preferably with hyaluronic acid having a molecular weight of 20,000g / mol or less, and most preferably with hyaluronic acid having a molecular weight of 10,000g / mol or less.

[0025] In the present invention, the "steroidal anti-inflammatory agent" refers to a preparation having a steroid nucleus and playing an anti-inflammatory role, and may include, for example, corticosteroids such as dexamethasone, prednisolone, methylprednisolone, betamethasone, hydrocortisone, or a combination thereof. Preferably, the steroidal anti-inflammatory agent may be dexamethasone.

[0026] In the present invention, the microspheres can be prepared by known methods such as phase separation, spray drying, solvent evaporation drying, and low-temperature solvent extraction.

[0027] According to one embodiment of the present invention, the microspheres were prepared by adding a steroidal anti-inflammatory agent to an aqueous solution of low molecular weight hyaluronic acid, and then adding an aqueous electrolyte solution to the solution to induce the formation of a complex between the hyaluronic acid and the electrolyte.

[0028] The controlled release drug preparation of the present invention may be characterized in that the hyaluronic acid microspheres are dispersed in an aqueous solution of ultra-high molecular weight hyaluronic acid or hyaluronate having a molecular weight exceeding 5,000,000 g / mol.

[0029] The ultra-high molecular weight hyaluronic acid or its salt is a natural ultra-high molecular weight hyaluronic acid that has not been chemically modified, for example, modified with a chemical functional group such as diaminobutane hyaluronate, succinimide hyaluronate, etc.

[0030] The ultra-high molecular weight hyaluronic acid may be hyaluronic acid having a molecular weight of more than 5,000,000 g / mol, preferably more than 5,100,000 g / mol, more preferably more than 5,200,000 g / mol, even more preferably more than 5,300,000 g / mol, and most preferably more than 5,400,000 g / mol. The upper limit of the molecular weight of the ultra-high molecular weight hyaluronic acid is not particularly limited, and as the high molecular weight hyaluronic acid that exists in nature in the natural form, as long as its molecular weight is more than 5,000,000g / mol, it can be obviously understood by ordinary skilled artisans that it can be applied to the present invention and achieve the same effect.Preferably, the upper limit of the molecular weight of the ultra-high molecular weight hyaluronic acid can be 10,000,000g / mol, more preferably, can be 9,000,000g / mol, even more preferably, can be 8,000,000g / mol, and most preferably, can be 7,500,000g / mol.

[0031] According to one embodiment of the present invention, the ultra-high molecular weight hyaluronic acid may be produced by Streptococcus sp. UBC-U46 (Accession No. KCTC13556BP) strain. The hyaluronic acid produced by the microorganism may be produced with reference to Korean Patent No. 10-2018-0130275, the disclosure of which is incorporated herein by reference.

[0032] In the present invention, the concentration of the hyaluronic acid or hyaluronate aqueous solution can be appropriately adjusted by a person skilled in the art, taking into consideration the release time of the steroidal anti-inflammatory agent to be controlled. However, the hyaluronic acid solution may be 1.0 to 10.0% (w / v), preferably 1.5 to 8% (w / v), more preferably 2.0 to 7% (w / v), even more preferably 2.5 to 6% (w / v), and most preferably 3.0 to 5% (w / v) aqueous hyaluronic acid solution.

[0033] The release rate and release period of the steroidal anti-inflammatory agent in the controlled release formulation of the present invention can be adjusted 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 a skilled artisan according to the target site to be treated, the severity of the disease to be treated, etc.

[0034] The ultra-high molecular weight hyaluronic acid aqueous solution is characterized by the fact that it does not contain any substances such as crosslinkers, hardeners, chemical denaturants, fatty acids, or cationic polymers that form complexes with hyaluronic acid, other than ultra-high molecular weight hyaluronic acid and water.

[0035] The crosslinking agent is a compound that reacts with hyaluronic acid to form a three-dimensional network structure, and may be one or more compounds selected from the group consisting of polyfunctional epoxy compounds 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. The chemical denaturant is a compound that reacts with a carboxy group, a hydroxy group, or an acetamide group of hyaluronic acid to form a covalent bond, and may be one or more compounds selected from the group consisting of a mixture of acetic anhydride and concentrated sulfuric acid, a mixture of trifluoroacetic anhydride and an organic acid, and an alkyl iodine compound. The cationic polymer that forms a complex with hyaluronic acid is a polymer that forms a complex via an ionic bond between a carboxy group of hyaluronic acid and an amino group or imino group of a polymer compound, and may be one or more cationic polymers selected from the group consisting of chitosan, polylysine, polyvinylpyridine, polyethyleneimine, and polydimethylaminoethyl methacrylate.

[0036] When chemical crosslinkers and / or hardeners are used to manufacture hyaluronic acid hydrogel, these chemicals may remain in the carrier inside or outside the hyaluronic acid hydrogel, which may increase the possibility of inducing adverse reactions such as inflammatory reactions. However, the controlled release drug preparation of the present invention does not contain any chemicals in the ultra-high molecular weight hyaluronic acid aqueous solution, so it can be very safe to show physiological activity after being injected into the body. In one embodiment of the present invention, the ultra-high molecular weight hyaluronic acid aqueous solution may be characterized as being natural hyaluronic acid in which cross-linking is not induced by any means such as radiation or heat.

[0037] According to one embodiment of the present invention, the controlled release drug formulation of the present invention using an aqueous solution of ultra-high molecular weight hyaluronic acid as a dispersion medium did not induce any inflammatory reaction after intratympanic administration, whereas the formulation using a hydrogel prepared by treating a hyaluronic acid derivative with a crosslinker as a dispersion medium caused a severe inflammatory reaction after intratympanic administration.

[0038] In the present invention, the concentration of the microspheres of hyaluronic acid or hyaluronate in the controlled release drug preparation dispersed in the aqueous solution of ultra-high molecular weight hyaluronic acid or hyaluronate can be appropriately adjusted by a skilled artisan depending on the release period or release concentration of the steroidal anti-inflammatory agent contained in the microspheres, and may be, for example, dispersed at a concentration of 0.1-5% (w / v), preferably 0.5-4% (w / v), more preferably 0.5-3% (w / v), more preferably 0.5-2.5% (w / v), and most preferably 1-2% (w / v).

[0039] In one embodiment of the present invention, the dosage form may be a controlled release formulation that allows the active drug to be released at the target site for a predetermined period of time. The dosage form may release the steroidal anti-inflammatory agent for a long period of time, such as 20 days or more, 1 month or more, 40 days or more, 2 months or more, 4 months or more, 6 months or more, 6 months to 12 months, or 12 months or more. In one embodiment, the release period may be adjusted according to the molecular weight of the ultra-high molecular weight hyaluronic acid or the concentration of its aqueous solution. The target site refers to a specific organ of an individual, and is not limited to the eyes, nose, ears, heart, ventricles, atria, intestinal tract, blood vessels, or joints. In one embodiment, the target site may be the ear, and the ear may include the middle ear, inner ear, cochlea, vestibular organ, Eustachian tube, or tympanic cavity.

[0040] The present invention also provides 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 a hyaluronate, the microspheres being dispersed in an aqueous solution of hyaluronic acid or a hyaluronate having a molecular weight exceeding 5,000,000 g / mol.

[0041] In the present invention, the hearing loss may be, depending on the cause, presbycusis, ototoxic hearing loss caused by anticancer drugs, antipyretics, analgesics, antibiotics, etc., noise-induced hearing loss caused by industrial noise, gun noise, explosion noise, etc., hearing loss caused by inner ear diseases such as Meniere's disease and sudden deafness, and traumatic hearing loss such as perilymph fistula, labyrinthine concussion, and temporal bone fracture.

[0042] In one embodiment of the present invention, the hearing loss may be selected from the group consisting of noise-induced hearing loss, ototoxic hearing loss, hearing loss due to inner ear disease, and traumatic hearing loss.

[0043] The pharmaceutical composition of the present invention may be a composition for administration into the middle ear, inner ear, cochlea, vestibular apparatus, Eustachian tube or intratympanic, preferably for intratympanic administration.

[0044] The pharmaceutical compositions according to the present invention may be formulated in a suitable form with a pharma- ceutically acceptable carrier and may further contain an excipient or diluent, including any type of solvent, dispersion medium, oil-in-water or water-in-oil emulsion, aqueous composition, liposome, microbeads and microsomes.

[0045] The pharma- ceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. The carrier for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. In addition, various drug delivery substances used for oral administration may be included. The carrier for parenteral administration may also include water, suitable oil, physiological saline, aqueous glucose and glycol, etc., and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. In addition to the above-mentioned components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, etc. Other pharma- ceutically acceptable carriers and formulations may be referred to those known in the art.

[0046] The composition of the present invention can be administered to mammals, including humans, by any method. For example, it can be administered parenterally. The parenteral administration method can be, but is not limited to, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, percutaneous, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, rectal, intraarticular or intratympanic administration, and most preferably intratympanic administration. Intratympanic injection of drugs is a procedure in which drugs are injected behind the eardrum into the middle and / or inner ear. It is. In one embodiment of the invention, the pharmaceutical compositions of the invention may be administered to the middle ear by transtympanic injection. In yet another embodiment, the pharmaceutical compositions of the invention may be administered directly to the inner ear by a non-transtympanic approach to the inner ear. In one embodiment of the present invention, the pharmaceutical composition may be administered using a syringe and needle that can penetrate the tympanic membrane and directly approach the round window membrane or oval window of the inner ear. The needle of the syringe is as small as possible to prevent perforation of the tympanic membrane, and may be, for example, 22 gauge to 24 gauge.

[0047] The pharmaceutical composition of the present invention can be formulated into a preparation for oral administration or parenteral administration depending on the administration route as described above. For oral administration, the composition of the present invention can be formulated into powder, granule, tablet, pill, sugar-coated tablet, capsule, liquid, gel, syrup, slurry, suspension, etc., by methods known in the art. For example, oral preparations can be obtained by blending the active ingredient with a solid excipient, then grinding it, adding suitable auxiliary agents, and processing it with a granular mixture to obtain tablets or sugar-coated tablets. Examples of suitable excipients 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 cellulose, methylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose, and fillers such as gelatin and polyvinylpyrrolidone. In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant in some cases. Furthermore, the pharmaceutical composition of the present invention may further contain an anti-coagulant, a lubricant, a wetting agent, a flavoring, an emulsifier, a preservative, and the like. For parenteral administration, the preparations may be formulated in the form of injections, creams, lotions, topical ointments, oils, moisturizers, gels, aerosols and nasal inhalants by methods known in the art, and these dosage forms are described in formularies commonly known in all pharmaceutical chemistry.

[0048] The total effective amount of the composition of the present invention may be administered to a patient in a single dose or may be administered by a divided treatment method in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may vary in the content of the active ingredient depending on the severity of the disease. Preferably, the total dose of the pharmaceutical composition of the present invention may be about 0.01 μg to 10,000 mg per kg of patient's body weight per day, most preferably 0.1 μg to 500 mg. However, the dosage of the pharmaceutical composition is determined by taking into consideration various factors such as the formulation method, administration route and number of treatments, as well as the patient's age, body weight, health condition, sex, severity of the disease, diet and excretion rate, so that a person with conventional knowledge in the field can determine an appropriate effective dosage of the composition of the present invention by taking these points into consideration. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route and administration method as long as it shows the effect of the present invention.

[0049] In one embodiment of the present 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 depending on the method of administration, the duration of treatment, the condition of the patient being treated, and the severity of the hearing loss, and is determined by the attending physician. The duration of treatment can range from about an hour to several days, weeks, or months, and can proceed for a long period of time as needed.

[0050] The term "prevention" as used herein refers to a therapy that protects against the onset of a disease or disorder, such that clinical symptoms of the disease do not develop. Thus, "prevention" refers to administering a therapy to a subject (e.g., administering a therapeutic agent) before symptoms of the disease become detectable in the subject. ) (e.g., administering a therapeutic agent to a subject in the absence of a detectable infectious agent (e.g., a virus) in the subject). The subject may be an individual at risk for developing a disease or disorder, e.g., an individual having one or more risk factors known to be associated with the development or onset of a disease or disorder.

[0051] The term "treatment" in the present invention refers to an approach to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delay or reduction in the rate of disease progression, improvement or palliation and relief of the disease state (which may be partial or total), detectable or undetectable. "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. The treatment includes not only disorders that are prevented, but also those that are necessary for disorders that have already occurred. "Palliating" a disease means reducing the extent and / or undesirable clinical signs of the disease state, or slowing or prolonging the time course of progression, as compared to the absence of treatment.

[0052] Meanwhile, it will be obvious to those skilled in the art that the pharmaceutical composition of the present invention may further contain known substances used in the treatment of hearing loss.

[0053] The present invention also provides a method for preparing the controlled release drug formulation, comprising the steps of: (a) encapsulating a steroidal anti-inflammatory drug within microspheres of hyaluronic acid or a hyaluronate; (b) preparing an aqueous solution of hyaluronic acid or a hyaluronate having a molecular weight of more than 5,000,000 g / mol; and (c) dispersing the microspheres in the aqueous solution.

[0054] The present invention also provides the use of a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or a hyaluronate for the manufacture of a preparation for treating hearing loss, the microspheres being dispersed in an aqueous solution of hyaluronic acid or a hyaluronate having a molecular weight of more than 5,000,000 g / mol.

[0055] The present invention also provides a method for treating hearing loss by administering to an individual in need thereof an effective amount of a composition comprising as an active ingredient a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or a hyaluronate, wherein the microspheres are dispersed in an aqueous solution of hyaluronic acid or a hyaluronate having a molecular weight of greater than 5,000,000 g / mol.

[0056] The "effective amount" of the present invention refers to an amount that, when administered to an individual, shows an effect of improving, treating, detecting, diagnosing, or suppressing or reducing hearing loss, and the "individual" may be an animal, preferably a mammal, particularly an animal including a human, or a cell, tissue, organ, etc. derived from an animal. The individual may be a patient in need of the effect. The term "treatment" as used herein refers generally to improving hearing loss or symptoms associated with hearing loss, which may include curing, substantially preventing, or improving the condition of the disease, including, but not limited to, alleviating, curing, or preventing one or most symptoms of the disease.

[0057] As used herein, the term "comprising" is used interchangeably with "including" or "characterized by" and does not exclude additional components, method steps, etc., not specifically mentioned in a composition or method according to the invention. Also, the term "consisting of" means excluding additional elements, steps, ingredients, etc., not otherwise stated. The term "essentially consisting of" means "essentially consisting of" or "consisting ... "Sting of" means that within the scope of a composition or method, in addition to the recited materials or steps, materials or steps, etc. that do not substantially affect the essential properties of the composition or method may be included. Effect of the Invention

[0058] The controlled release drug preparation and pharmaceutical composition for hearing loss treatment containing the ultra-high molecular weight hyaluronic acid aqueous solution according to the present invention as a drug carrier can exert a therapeutic effect for several weeks to several months with a single administration, so that it does not induce systemic side effects and enables effective treatment of diseases without repeated treatment. In addition, the controlled release drug preparation of the present invention does not use a chemical crosslinking agent for crosslinking hyaluronic acid, so the risk of side effects such as inflammatory reactions is extremely low. [Brief description of the drawings]

[0059] [Figure 1] FIG. 1 shows the test schedule for evaluating hearing improvement tests in an induced hearing loss animal model. [Diagram 2] Figure 2 shows the results of CT imaging evaluation of the duration of intratympanic drug / transmitter retention in animals. [Diagram 3] FIG. 3 shows the time course of recovery from a tympanic membrane perforation that occurred after injection of a drug / transmitter into the tympanic membrane. [Figure 4] FIG. 4 is a graph showing the time course of observation of whether inflammation occurs in the tympanic membrane of an animal after administration of the drug / delivery vehicle (SURDEN) according to the present invention. [Diagram 5] FIG. 5 shows the results of evaluating the effect of improving hearing in an animal model of hearing loss (SU02+D: drug / transmitter according to the present invention). [Figure 6] FIG. 6 is a schematic diagram showing an experimental method for injecting a drug / transmitter into the tympanic membrane of an animal model of hearing loss, as described in the prior art. [Figure 7] FIG. 7 shows the results of evaluating the degree of inflammation after injecting the drug / transmitter described in the prior art into the tympanic membrane of an animal model of hearing loss. [Figure 8]Figure 8 shows the contrast effect over time observed using 9.4T MRI after a vehicle (SURDEN01 + Magnevist) made by mixing a contrast agent with an aqueous solution of hyaluronic acid with a weight-average molecular weight of 7,000,000 g / mol, and a vehicle (low viscosity vehicle + magvenist) made by mixing a contrast agent with an aqueous solution of hyaluronic acid with a molecular weight of 1,000,000 to 3,000,000 g / mol were administered into the eardrum of a rat. [Figure 9] FIG. 9 is a graph showing the amount of contrast at each site in the rat eardrum quantified over time during the experimental process of FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] The present invention will now be described in more detail with reference to the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

[0061] Example 1: Preparation of dexamethasone microcapsules using ultra-low molecular weight hyaluronic acid An aqueous solution containing 20% ​​ultra-low molecular weight hyaluronic acid with a molecular weight of less than 10,000 Da was prepared using ultrapure water, and 2.4% dexamethasone disodium phosphate was completely dissolved in it. The hyaluronic acid-dexamethasone aqueous solution and CaCl2 (Ca 2+ The hyaluronic acid was mixed with an aqueous solution of hyaluronic acid and CaCl2 (ionic compound containing hyaluronic acid) at a ratio of 1:1 (v / v) to induce an ionic complex (precipitate) between hyaluronic acid and CaCl2. The precipitate was then centrifuged at 12,000 rpm for 1 minute to remove the supernatant. In addition, the washing and centrifugation process was performed more than three times using excess ultrapure water to remove ions not involved in the ionic complex (free ions). The ion-complexed drug mixture was encapsulated in ultra-low molecular weight hyaluronic acid by freeze-drying. Monomerized dexamethasone was produced.

[0062] Example 2: Preparation of encapsulated dexamethasone / hyaluronic acid hydrogel complex (SURDEN; Sustained Release Drug Encapsulation) As a carrier for the encapsulated dexamethasone, we prepared ultra-high molecular weight hyaluronic acid, which has adhesive properties, is stable in the tympanic cavity, and has excellent biocompatibility, and then used it to prepare a microcapsule dexamethasone / hyaluronic acid complex. Ultra-high molecular weight hyaluronic acid was produced using Streptococcus sp. UBC-U46 strain (accession number: KCTC13556BP) according to the production method specified in the prior patent (KR1020180130275). The weight average molecular weight of the ultra-high molecular weight hyaluronic acid produced by the above method was 7,000,000 g / mol.

[0063] The ultra-high molecular weight hyaluronic acid was dissolved in ultra-pure water to prepare an ultra-high molecular weight hyaluronic acid aqueous solution with a concentration of 3%. The dexamethasone powder encapsulated in ultra-low molecular weight hyaluronic acid prepared in Example 1 was added to the ultra-high molecular weight hyaluronic acid aqueous solution to a concentration of 1.5%, and the mixture was uniformly mixed using a homogenizer to prepare a microcapsule dexamethasone / hyaluronic acid hydrogel complex (SURDEN) in which dexamethasone encapsulated in ultra-low molecular weight hyaluronic acid was uniformly mixed.

[0064] Example 3: Evaluation of drug delivery system duration and drug release performance In order to evaluate the performance of the complex (SURDEN) prepared in Example 2 as a drug delivery agent, the following experiment was carried out using an animal model of hearing loss.

[0065] (1) Creation of an animal model of hearing loss To exclude hormonal effects, male SD rats were used in the experiment. Four days after IT prophylaxis to induce ototoxic hearing loss, a combination of cisplatin (2 mg / kg), gentamicin (120 mg / kg), and furosemide (90 mg / kg) was injected into the caudal jugular vein once daily for 2 days.

[0066] (2) Experimental design Dexamethasone (D) was administered to animals in two different ways using the intratympanic (IT) method, which delivers the drug through the tympanic membrane.

[0067] The first method is a method of delivering D at 12 mg / ml to saline, which is a treatment method used in existing hospitals. The second method is a method of delivering D at 12 mg / ml to SURDEN prepared in Example 2. As a control, a hearing loss control group that did not receive any treatment was prepared. Each sample prepared as described above was administered to each animal (each ear) at 0.04 ml. The specific experimental schedule is shown in Figure 1.

[0068] (3) Hearing test Hearing was measured before and after induction of hearing loss at 1-14 day intervals for approximately two months. To evaluate frequency characteristics, auditory brainstem response (ABR) tests were performed at four frequencies: click, 8 kHz, 16 kHz, and 32 kHz.

[0069] (4) Evaluation of drug carrier maintenance period The duration of drug / transmitter retention in the animal's intratympanic cavity confirmed at the time of CT scan shown in the experimental schedule in Figure 1 was 1.1 ± 0.3 days for Saline + D and 41.1 ± 27.0 days for SURDEN. In other words, it was confirmed that the duration of drug / transmitter retention could be extended by approximately 40 times longer when using SURDEN compared to Sarin (Figure 2).

[0070] (5) Safety evaluation of drug delivery systems Periodic tympanoscope imaging revealed that the tympanic membrane perforation had completely healed after 21.6±12.6 days in the Sarin+D group. In the SURDEN+D group, the tympanic membrane perforation had completely healed after 16.7±11.9 days. In other words, the use of SURDEN did not shorten or at least prolong the healing period for the tympanic membrane perforation, confirming that treatment is possible without side effects (Figure 3).

[0071] Furthermore, tympanic endoscopy revealed that no individual developed middle ear inflammation as a result of administration of SURDEN. Middle ear inflammation occurs when a drug / carrier is not biocompatible (Figure 4). In the case of the drug / carrier complex (SURDEN) of the present invention, no such inflammatory side effects occurred, and it was determined that it can be successfully used in humans.

[0072] Example 4: Hearing improvement evaluation In the case of SURDEN, hearing began to improve from the first day (46.7dB) after hearing loss was induced, compared to the Sarin+D (70.0dB) or control group (62.9dB), based on 32kHz, and this trend was well maintained on the fourth day (46.7dB), eighth day (52.5dB), 12th day (47.5dB), 21st day (46.7dB), and 30th day (50.0dB). Compared to Sarin+D, the conventional standard treatment method, the therapeutic effect of SERDEN was about 23.3dB to 26.7dB better. In general, a clinically significant hearing difference of 15 Considering that the difference is based on the level of hearing loss, a treatment effect of 23.3 dB - 26.7 dB greater was a clinically significant difference. A similar difference was observed at the mid-frequency of 16 kHz. Compared to the conventional standard treatment, Sarin+D, the therapeutic effect of SERDEN was 16.8 dB-23.3 dB better. Considering that a clinically significant hearing difference is 15 dB, the therapeutic effect of 16.8 dB-23.3 dB better at 16 kHz was a clinically significant difference. Even at the lowest frequency of 8 kHz, SURDEN had the best therapeutic effect. Compared to the conventional standard treatment method, Sarin+D, the therapeutic effect of SERDEN was 1.7 dB-10.8 dB better (Figure 5).

[0073] Taking all these results into consideration, it was confirmed that SURDEN's effectiveness in treating hearing loss is significantly superior to that of the existing standard treatment method, Sarin+D.

[0074] Example 5: Confirmation of the presence or absence of an inflammatory reaction The present inventors compared the method disclosed in the prior patent (10-1877894) with the method according to the present invention to determine whether they induce an inflammatory response in the inner ear.

[0075] First, hearing loss was induced by noise exposure in rats with normal hearing, and then, according to the method disclosed in the prior patent, the rats were divided into three groups shown in Figure 6 ((1) dexamethasone + sarin (D+sarin), (2) dexamethasone + IGF-1 + sarin (D&G+sarin), (3) dexamethasone-loaded PLGA microcapsules + IGF-1 + HA (cD&G+HA)), and each drug was injected into the tympanic membrane. Hearing tests, tympanic endoscopy, and CT scans were performed until the 45th day. The group administered with SURDEN according to the present invention was also subjected to the experiment in the same manner as described above.

[0076] The results are shown in FIG. As shown in Figure 8, when the ratio of individuals who developed an inflammatory reaction in the middle ear tissue after injecting a drug through the tympanic membrane was calculated for each group, the incidence of inflammation in the dexamethasone-loaded PLGA microcapsules + IGF-1 + HA group was 42.3%, which was about twice as high as the other groups. On the other hand, no inflammatory reaction was observed in the SURDEN-administered group (results not shown).

[0077] Example 6: Comparison of controlled release ability and hearing loss treatment effect depending on molecular weight of hyaluronic acid In order to compare and evaluate the controlled release effect depending on the molecular weight of the ultra-high molecular weight hyaluronic acid used in Example 2, the controlled release ability of a carrier using a relatively low molecular weight hyaluronic acid (1,000,000 to 3,000,000 g / mol) was compared to the ultra-high molecular weight (weight average molecular weight of 7,000,000 g / mol) used to prepare SURDEN in Example 2.

[0078] Specifically, a contrast agent was added to a hyaluronic acid aqueous solution having a weight-average molecular weight of 7,000,000 g / mol (SURDEN) or a hyaluronic acid aqueous solution having a molecular weight of 1,000,000 to 3,000,000 g / mol (Low viscosity vehicle), and each of these samples was injected into the eardrum of a rat with normal hearing. After imaging using a 9.4T MRI, quantitative analysis was performed.

[0079] The results are shown in FIG. As shown in Figure 9, it was confirmed that the contrast intensity increased in the vestibular and tympanic systems, and that the SURDEN administration group showed a stronger synergistic effect than the low viscosity veh group at all time points and in all regions. Furthermore, as shown in FIG. 10, the results of quantifying this confirmed that the signal intensity in the lower, middle, and upper layers of the cochlea in the SURDEN administration group was higher than that in the low viscosity vehicle group. In conclusion, SURDEN was found to deliver higher concentrations of drugs for longer periods of time and in larger amounts than vehicles using relatively low molecular weight hyaluronic acid, demonstrating superior drug release capabilities in drug delivery to the inner ear. [Industrial Applicability]

[0080] The drug controlled release formulation and pharmaceutical composition for hearing loss treatment containing the ultra-high molecular weight hyaluronic acid aqueous solution according to the present invention as a drug carrier can exert a therapeutic effect for several weeks to several months with one administration, so that it does not induce systemic side effects and enables effective treatment of diseases without repeated treatment.In addition, the drug controlled release formulation of the present invention does not use a chemical crosslinking agent for crosslinking hyaluronic acid, so the risk of side effects such as inflammatory reactions is very low, and it has high industrial applicability.

Claims

1. A steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or a hyaluronate is dispersed in an aqueous solution of hyaluronic acid having a molecular weight of more than 5,000,000 g / mol or a hyaluronate having a molecular weight of more than 5,000,000 g / mol; A controlled release drug formulation, wherein the hyaluronic acid does not have cross-linking.

2. 2. The controlled release drug formulation according to claim 1, wherein the hyaluronic acid forming the microspheres has a molecular weight of 100,000 g / mol or less.

3. 2. The controlled release drug formulation of claim 1, wherein the steroidal anti-inflammatory agent is dexamethasone, prednisolone, methylprednisolone, betamethasone, hydrocortisone, or a combination thereof.

4. 2. The controlled release drug formulation according to claim 1, wherein the controlled release drug formulation releases the steroidal anti-inflammatory agent at a target site for 20 days or more.

5. 2. The controlled release drug formulation according to claim 1, wherein the aqueous solution of hyaluronic acid or hyaluronate is a 1 to 10% (w / v) aqueous solution.

6. 2. The controlled release drug formulation according to claim 1, characterized in that the formulation is for administration into the middle ear, inner ear, cochlea, vestibular apparatus, Eustachian tube or intratympanic cavity.

7. The present invention relates to a method for treating inflammation comprising the steps of: (a) administering to a patient a therapeutically effective amount of a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or a hyaluronate salt, the microspheres being encapsulated in water of hyaluronic acid having a molecular weight of more than 5,000,000 g / mol or hyaluronate having a molecular weight of more than 5,000,000 g / mol; Dispersed in a solution, A pharmaceutical composition for preventing or treating hearing loss, wherein the hyaluronic acid does not have cross-links.

8. 8. The pharmaceutical composition according to claim 7, characterized in that the pharmaceutical composition is injected into the middle ear, inner ear, cochlea, vestibular apparatus, Eustachian tube or tympanic cavity.

9. 8. The pharmaceutical composition according to claim 7, wherein the hearing loss is selected from the group consisting of noise-induced hearing loss, ototoxic hearing loss, hearing loss due to an inner ear disease and traumatic hearing loss.

10. (a) encapsulating a steroidal anti-inflammatory agent within microspheres of hyaluronic acid or a hyaluronate; (b) preparing an aqueous solution of hyaluronic acid having a molecular weight of more than 5,000,000 g / mol or a hyaluronate having a molecular weight of more than 5,000,000 g / mol; and (c) dispersing the microspheres in the aqueous solution; The method for producing a controlled release drug formulation according to any one of claims 1 to 6, wherein the hyaluronic acid is not cross-linked.

11. 1. Use of a steroidal anti-inflammatory agent encapsulated in microspheres of hyaluronic acid or a hyaluronate for the manufacture of a preparation for the treatment of hearing loss, said microspheres being dispersed in an aqueous solution of hyaluronic acid having a molecular weight of more than 5,000,000 g / mol or a hyaluronate having a molecular weight of more than 5,000,000 g / mol; The hyaluronic acid does not have cross-links, and the use of a steroidal anti-inflammatory agent.

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