Patent Application
20250135009
This application claims priority based on Korean Patent Application No. 10-2021-0131088, filed on Oct. 1, 2021.
The present invention relates to an intratympanic controlled drug release formulation comprising N-acylated glycol chitosan.
When a drug for treating inner ear disease is administered systemically, drug delivery to the inner ear is not efficient due to the blood-labyrinthine barrier. In addition, systemic drug administration at high doses to achieve and maintain drug concentrations in the inner ear is accompanied by toxicity and side effects. Thus, local administration is efficient for drug delivery to the inner ear.
Intratympanic administration refers to the administration of a drug by injection into a space called the middle ear cavity or tympanic cavity, and is a widely used method for local administration to the inner ear. Intratympanic administration delivers drugs to the inner car more efficiently and has fewer side effects than systemic administration.
Drugs administered intratympanically are delivered by diffusion to the inner ear through the round window membrane (RWM). Thus, in order to efficiently deliver a drug to the inner ear, the drug must be in contact with the round window membrane at a high concentration for a long time. However, since a drug located in the middle ear cavity by intratympanic administration is easily discharged to the outside through the Eustachian tube, it is difficult to contact the round window membrane at a high concentration for a long time. Therefore, prolonging drug retention in the middle ear cavity is considered an approach to enhance drug delivery to the inner ear.
Thermogel, which exhibits thermo-reversible sol-gel transition properties, can be injected in a sol state and forms a gel in the body, and thus it is suitable for intratympanic drug delivery. Thermogel in a sol state may be mixed with a drug and loaded with the drug. When thermogel mixed with a drug is injected, it is solidified into a gel in response to body temperature. Poloxamers, representative thermogels, have been used for drug delivery to the inner ear. However, poloxamers require a high concentration (more than 20 wt %) for effective thermogelation, and are not sufficiently biodegradable and physically stable, and side effects thereof have been reported.
Glycol chitosan (GC), a water-soluble derivative of chitosan, has promising properties as a biomaterial, including high biocompatibility, biodegradability, and water solubility at neutral pH (Korean Patent Application Publication No. 10-2012-0020386 (Mar. 8, 2012). However, it is not known whether intratympanic administration of a mixture of N-acyl glycol chitosan and a drug enables efficient drug delivery to the inner ear and is accompanied by side effects.
According to one embodiment, there is provided a pharmaceutical composition for intratympanic administration for treating inner ear disease comprising N-acylated glycol chitosan.
According to another embodiment, there is provided a formulation for intratympanic administration comprising N-acylated glycol chitosan.
One aspect provides a pharmaceutical composition for treating inner ear disease by administration through intratympanic injection, comprising a polymer having a unit represented by Formula 1 below, and a drug for treating inner ear disease, wherein the polymer undergoes a sol-gel phase transition depending on temperature:
In Formula 1 above, R1 is H or an acyl group having 1 to 10, 1 to 9, 1 to 8, 1 to 7, or 1 to 6 carbon atoms, and n is 10 to 10,000, 10 to 9,500, 10 to 9,000, 10 to 8,500, 10 to 8,000, 10 to 7,500, 10 to 7,000, 10 to 6,500, 10 to 6,000, 10 to 5,500, 10 to 5,000, 10 to 4,500, 10 to 4,000, 10 to 3,500, 10 to 3,000, 10 to 2,500, 10 to 2,000, 10 to 1,500, 10 to 1,000, 10 to 900, 10 to 800, 10 to 700, 10 to 600, or 10 to 500.
The acyl group may be (—(C═O)-alkyl group), and the alkyl group in the acyl group may have 1 to 10, 1 to 9, 1 to 8, 1 to 7, or 1 to 6 carbon atoms, and may be a straight or branched chain group.
According to one embodiment, the polymer is in the state of sol that can be administered by injection at room temperature, and it may be a thermogel that undergoes a phase transition to a gel in response to body temperature when administered intratympanically. The polymer is a drug carrier that can be loaded with a drug, and when the polymer and the drug are mixed together and administered intratympanically, the mixture undergoes a phase transition to a drug-loaded gel. The gelled pharmaceutical composition may have an increased residence time in the middle ear cavity, and thus may increase the contact time between the drug and the round window membrane and continuously deliver the drug to the inner ear. According to one example, it was confirmed that, when the pharmaceutical composition was administered intratympanically, side effects such as inflammatory responses and loss of auditory hair cells did not occur, indicating that the pharmaceutical composition is highly safe. Such safety was not previously known.
According to one embodiment, the polymer contains a hydrophilic glycol chitosan backbone and a hydrophobic acyl group (e.g., hexanoyl group), and thus it can be loaded with both hydrophilic and hydrophobic drugs.
According to one embodiment, R1 may be H, an acetyl group, or a hexanoyl group.
According to one embodiment, the polymer may have a degree of polymerization (DP) of 150 to 400, 150 to 350, 150 to 300, 150 to 250, 200 to 400, 200 to 350, 200 to 300, or 200 to 250.
According to one embodiment, the temperature at which the sol-gel phase transition occurs may be 30 to 34° C., 30 to 33° C., 30 to 32° C., 31 to 34° C., 31 to 33° C., 31 to 32° C., 32 to 34° C., or 32 to 33° C.
According to one embodiment, the polymer may be a polymer comprising 8 to 10% of N-acetylated glycol chitosan units, 30 to 40% of N-hexanoylated glycol chitosan units, and the balance of glycol chitosan units.
According to one embodiment, the polymer may be a compound having a unit represented by Formula 2 below:
In Formula 2 above, R2 may be H or an acetyl group (—CO—CH3), R3 may be a hexanoyl group (—CO—CH2CH2CH2CH2CH3), and y may be 10 to 10,000, 10 to 9,500, 10 to 9,000, 10 to 8,500, 10 to 8,000, 10 to 7,500, 10 to 7,000, 10 to 6,500, 10 to 6,000, 10 to 5,500, 10 to 5,000, 10 to 4,500, 10 to 4,000, 10 to 3,500, 10 to 3,000, 10 to 2,500, 10 to 2,000, 10 to 1,500, 10 to 1,000, 10 to 900, 10 to 800, 10 to 700, 10 to 600, or 10 to 500.
According to one embodiment, the drug for treating inner car disease may be a corticosteroid-based drug.
The corticosteroid-based drug may be, for example, clobetasol, halometasone, dexamethasone, diflorasone, fluocinonide, halobetasol, amcinonide, halcinonide, hydrocortisone, fluticasone, mometasone, fluocinolone, desonide, prednisone, methylprednisolone, prednisolone, a hydrate or solvate thereof, or a combination thereof. According to one example, it may be dexamethasone, dexamethasone phosphate, or a pharmaceutically acceptable salt thereof.
The drug for treating inner ear disease may be dispersed in the polymer.
According to one embodiment, the drug for treating inner ear disease may be the drug itself, a form in which the drug is dispersed in microspheres, or a combination thereof. The composition may be in a form wherein microspheres having the drug dispersed therein and the polymer are mixed and dispersed, or a form wherein microspheres having the drug dispersed therein, the drug itself, and the polymer are mixed and dispersed. The microspheres are fine particles composed of a biocompatible and biodegradable polymer, and are well known in the field of drug delivery technology. The material forming the microsphere is not particularly limited as long as it is a polymer having excellent biocompatibility and biodegradability. For example, the biocompatible polymer may be PLGA, PEG, PLA, PGA, PHA, a copolymer thereof, or a combination thereof. The microspheres may be composed of a biodegradable polymer having a molecular weight of 5,000 to 200,000. The diameter of the microsphere may be 10 to 100 μm.
The pharmaceutical composition may comprise the polymer in an amount of 0.5 to 4 wt %, 0.6 to 4 wt %, 0.7 to 4 wt %, 0.8 to 4 wt %, 0.9 to 4 wt %, or 1 to 4 wt %.
The pharmaceutical composition may remain in the tympanic cavity for at least 30 minutes, at least 90 minutes, at least 3 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, or at least 12 days.
According to one embodiment, the pharmaceutical composition may release a hydrophilic drug in an immediate release manner and release a hydrophobic drug in a sustained release manner, in the tympanic cavity.
The immediate release refers to the property of releasing the drug immediately after administration so that the initial effect of the drug appears quickly, and the sustained release refers to the property of releasing the drug slowly and continuously so that the therapeutically effective amount or effective concentration of the drug may be maintained for a long period of time. The therapeutically effective amount refers to an amount effective for ameliorating or reducing hearing loss in the subject being treated, and may vary depending on the severity of the subject, the drug administered, and the method of administration.
The mixing ratio of the polymer to the drug may be, based on parts by weight, 4:0.5 to 4:4, 4:1 to 4:4, 4:1.5 to 4:4, 4:2 to 4:4, 4:2.5 to 4:4, 4:3 to 4:4, 4:3.5 to 4:4, 4:0.5 to 4:3.5, 4:1 to 4:3.5, 4:1.5 to 4:3.5, 4:2 to 4:3.5, 4:2.5 to 4:3.5, 4:3 to 4:3.5, 4:0.5 to 4:3, 4:1 to 4:3, 4:1.5 to 4:3, 4:2 to 4:3, 4:2.5 to 4:3, 4:0.5 to 4:2.5, 4:1 to 4:2.5, 4:1.5 to 4:2.5, 4:2 to 4:2.5, 4:0.5 to 4:2, 4:1 to 4:2, 4:1.5 to 4:2, 4:0.5 to 4:1.5, 4:1 to 4:1.5, or 4:0.5 to 4:1.
The content of the drug may be 0.5 to 4 wt %, 1 to 4 wt %, 1.5 to 4 wt %, 2 to 4 wt %, 2.5 to 4 wt %, 3 to 4 wt %, 3.5 to 4 wt %, 0.5 to 3.5 wt %, 1 to 3.5 wt %, 1.5 to 3.5 wt %, 2 to 3.5 wt %, 2.5 to 3.5 wt %, 3 to 3.5 wt %, 0.5 to 3 wt %, 1 to 3 wt %, 1.5 to 3 wt %, 2 to 3 wt %, 2.5 to 3 wt %, 0.5 to 2.5 wt %, 1 to 2.5 wt %, 1.5 to 2.5 wt %, 2 to 2.5 wt %, 0.5 to 2 wt %, 1 to 2 wt %, 1.5 to 2 wt %, 0.5 to 1.5 wt %, 1 to 1.5 wt %, or 0.5 to 1 wt %.
The pharmaceutical composition may release the drug continuously for at least 30 minutes, at least 90 minutes, at least 3 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, or at least 12 days.
According to one embodiment, the pharmaceutical composition may release the drug in an immediate or sustained release manner depending on the concentration of the drug contained therein. Specifically, the pharmaceutical composition may release the drug in a sustained release manner when the concentration of the drug contained therein is 1.5 to 4 wt %, 1.6 to 4 wt %, 1.7 to 4 wt %, 1,8 to 4 wt %, 1.9 to 4 wt %, or 2 to 4 wt %, and may release the drug in an immediate release manner when the concentration of the drug is lower than the lower limit of the above range.
Referring to the results of an in vitro drug release experiment in one example, a pharmaceutical composition containing 0.5 wt % or 1 wt % of the drug released the drug in vitro in an immediate release manner, and a pharmaceutical composition containing 2 wt % or 4 wt % of the drug released the drug in vitro in a sustained release manner for up to 14 days (see
The inner ear disease may be, for example, Meniere's disease; sensorineural hearing loss; ototoxic hearing loss caused by drugs such as antipyretics, anticancer drugs, and antibiotics; noise-induced hearing loss; age-related heating loss; tinnitus; vestibular neuritis; auditory nerve tumor; osteosclerosis; traumatic hearing loss caused by perilymph fistula, labyrinth concussion, and temporal bone fracture; or autoimmune inner car disease. The autoimmune inner ear disease may be caused by ankylosing spondylitis, systemic lupus erythematosus (SLE), Sjögren's syndrome, Cogan's disease, ulcerative colitis, Wegener's granulomatosis, rheumatoid arthritis, scleroderma, or Behcet's disease.
The intratympanic administration may also be referred to as administration into the tympanum, and is a method of injecting the composition into the middle ear or inner ear behind the tympanum. The intratympanic administration may be administering the composition to the middle ear so that the composition can come into contact with the round window membrane. The intratympanic administration may be performed using a syringe, a pump, a microinjection device, a sponge material, or the like. The dosage of the composition may vary depending on the patient's condition and body weight, the severity of the disease, the form of the drug, and the route and duration of administration, and may be appropriately selected by a person skilled in the art.
The pharmaceutical composition may further comprise a Na/K ATPase modulator, chemotherapeutic agent, collagen, gamma-globulin, interferon, antibacterial agent, antibiotic, local acting anesthetic, platelet activating factor antagonist, ear protector, nitric oxide synthase inhibitor, vertigo inhibitor, vasopressin antagonist, antiviral agent, antiemetic, anti-TNF agent, vasopressin receptor modulator, methotrexate, cyclophosphamide, immunosuppressant, macrolide, latanoprost, TNF converting enzyme inhibitor, IKK inhibitor, glutamate receptor modulator, anti-apoptotic agent, neuroprotectant, thalidomide, c-jun inhibitor compound, hyaluronidase, antioxidant, IL-1 beta modulator, ERR-beta antagonist, IGF-modulator, Toll-like receptor, KCNQ channel modulator, neurotrophin modulator, ATOH modulator, or a combination thereof.
The pharmaceutical composition may further comprise carriers, excipients, and diluents, which are commonly used in the manufacture of medicaments. The pharmaceutical composition may be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as external preparations, suppositories, and sterile injectable solutions. Carriers, excipients, and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, without being particularly limited thereto.
Another aspect provides an intratympanic controlled drug release formulation comprising a polymer having a unit represented by Formula 1 below, and a drug for treating inner ear disease dispersed in the polymer, wherein the formulation undergoes a sol-gel phase transition after intratympanic injection thereof:
In Formula 1 above, R1 is H or an acyl group having 1 to 10, 1 to 9, 1 to 8, 1 to 7, or 1 to 6 carbon atoms, and n is 10 to 10,000, 10 to 9,500, 10 to 9,000, 10 to 8,500, 10 to 8,000, 10 to 7,500, 10 to 7,000, 10 to 6,500, 10 to 6,000, 10 to 5,500, 10 to 5,000, 10 to 4,500, 10 to 4,000, 10 to 3,500, 10 to 3,000, 10 to 2,500, 10 to 2,000, 10 to 1,500, 10 to 1,000, 10 to 900, 10 to 800, 10 to 700, 10 to 600, or 10 to 500.
The acyl group may be (—(C═O)-alkyl group), and the alkyl group in the acyl group may have 1 to 10, 1 to 9, 1 to 8, 1 to 7, or 1 to 6 carbon atoms, and may be a straight or branched chain group.
The controlled drug release refers to controlling the release rate and delivery rate of the drug in the tympanic cavity.
According to one embodiment, R1 may be H, an acetyl group, or a hexanoyl group.
According to one embodiment, the polymer may have a degree of polymerization (DP) of 150 to 400, 150 to 350, 150 to 300, 150 to 250, 200 to 400, 200 to 350, 200 to 300, or 200 to 250.
According to one embodiment, the temperature at which the sol-gel phase transition occurs may be 30 to 34° C., 30 to 33° C., 30 to 32° C., 31 to 34° C., 31 to 33° C., 31 to 32° C., 32 to 34° C., or 32 to 33° C.
According to one embodiment, the polymer may be a polymer comprising 8 to 10% of N-acetylated glycol chitosan units, 30 to 40% of N-hexanoylated glycol chitosan units, and the balance of glycol chitosan units.
According to one embodiment, the polymer may be a compound having a unit represented by Formula 2 below:
In Formula 2 above, R2 may be H or an acetyl group (—CO—CH3), R3 may be a hexanoyl group (—CO—CH2CH2CH2CH2CH3), and y may be 10 to 10,000, 10 to 9,500, 10 to 9,000, 10 to 8,500, 10 to 8,000, 10 to 7,500, 10 to 7,000, 10 to 6,500, 10 to 6,000, 10 to 5,500, 10 to 5,000, 10 to 4,500, 10 to 4,000, 10 to 3,500, 10 to 3,000, 10 to 2,500, 10 to 2,000, 10 to 1,500, 10 to 1,000, 10 to 900, 10 to 800, 10 to 700, 10 to 600, or 10 to 500.
According to one embodiment, the drug for treating inner ear disease may be a corticosteroid-based drug.
The preparation may be an injectable formulation.
Other contents regarding the formulation can be understood by referring to the contents regarding the pharmaceutical composition.
A pharmaceutical composition for treating inner ear diseases according to one embodiment, when administered intratympanically, can effectively deliver the drug to the inner ear without side effects.
The controlled drug release formulation according to one embodiment, when administered intratympanically, can deliver the drug in a sustained or immediate release manner without side effects.
Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these examples are intended to illustrate one or more embodiments and the scope of the present invention is not limited to these examples.
Glycol chitosan (GC, degree of polymerization (DP)≥200, degree of acetylation=9.34±2.5%, as measured by 1H-NMR) was purchased from Wako (Japan). Hexanoic acid anhydride (97%) was purchased from Sigma-Aldrich (USA). Acetone and methanol were supplied by Samchun Chemical (Korea). Dialysis membranes (MWCO=12 to 14 kDa) were purchased from Spectrum Laboratories (USA). Deuterium oxide (D2O) and Dulbecco's phosphate buffered saline (PBS) were purchased from Sigma-Aldrich (USA). Dexamethasone (DEX, micronized) and DEX phosphate disodium salt (DSP) were purchased from Farmabios (Italy) and Steraloids (USA), respectively.
HGC was synthesized via the N-hexanoylation reaction of glycol chitosan (GC). GC (3 g) and hexanoic acid anhydride (1.106 mL) were dissolved in 700 mL of a mixed solvent of water and methanol (50:50) and magnetically stirred at room temperature for 24 hours. The reaction solution was poured into an excess amount of cold acetone, and the polymerized product, HGC, was precipitated. The product was dialyzed against distilled water using a dialysis membrane (molecular weight cut-off: 12-14 kDa) for 2 days to purify HGC, followed by lyophilization.
The chemical composition of HGC was analyzed by 1H-NMR spectroscopy at 600 MHz using an AVANCE III 600 spectrometer (Bruker, Germany). The HGC polymer sample was dissolved in D2O at 0.5 wt %. The D2O peak at 4.85 ppm was set as the reference peak. The chemical composition of HGC was characterized by ATR-FTIR using Nicolet iS 5 (Thermo Scientific, USA). The ATR-FTIR spectra of GC and HGC were recorded in a circumstance with 32 scans at a resolution of 4 cm-1 in the frequency range of 4,000 to 750 cm-1.
Changes in water solubility of dexamethasone (DEX) with changes in HGC concentration were analyzed. An excessive amount of DEX was added to 3 mL of an aqueous solution of HGC (PBS, HGC concentration=0 to 4%, w/v). The samples were then stirred for 30 minutes and incubated in a shaking water bath (100 rpm, 37° C.) for 24 hours. All the samples were filtered through a syringe filter (0.8 μm) to remove precipitates and then analyzed using a UV-visible spectrophotometer (V-730, JASCO, Korea). The solubility value of DEX was determined using a standard curve of various DEX concentrations obtained at 242 nm. The test was repeated three times.
Drug-loaded HGC thermogels were prepared by simply physically mixing an aqueous solution of HGC (PBS, 4 wt %) with DSP (hydrophilic DEX) or DEX (hydrophobic DEX). First, 40 mg of HGC was dissolved in 1 mL of PBS (pH 7.4) and stored in a refrigerator at 4° C. DSP or DEX (5 or 10 mg) was added to the HGC solution and mixed by vortexing to obtain DSP-loaded HGC (HGC-DSP) and DEX-loaded HGC (HGC-DEX) samples having a drug concentration of 0.5 or 1.0 wt %.
The sol-gel transition behaviors of the HGC thermogel formulations were observed using the tilting tube method at a heating rate of 1° C./min. The sol-gel transition temperature was determined as the temperature at which the formulation exhibited a non-flowing gel state within 1 minute after tilting the vial. The experiment was performed in triplicate.
Rheological analysis of the HGC solution and the drug-loaded HGC solution was performed using a MARS-40 rheometer (Thermo Scientific, Germany). Aqueous solutions of GC, HGC, HGC-DSP and HGC-DEX samples were placed between parallel plates (60 mm diameter and 1 mm gap). Measurements were performed at a frequency of 1 Hz and a constant stress of 10 Pa. The temperature was increased from 10° C. to 45° C. at a heating rate of 0.05° C./s.
The in vitro drug release profiles of the thermogel formulations were examined in PBS (pH 7.4) using a dialysis membrane. The prepared thermogel formulations (HGC-DSP and HGC-DEX) having a drug concentration of 0.5 or 1 wt % were placed in dialysis membrane bags (MWCO=12 to 14 kDa, width: 10 mm). Then, the dialysis bags were immersed in 50 mL of PBS and incubated in a shaking water bath (SI-600R, Jeio Tech, Seoul, Korea) at 37° C. and 100 rpm or less.
After incubation, 10 ml of the release medium was taken out and the same amount of fresh medium was added again. The collected samples were analyzed by UV-Vis measurement. The DEX release profile was determined by measuring the UV absorbance of DEX at 242 nm. The experiment was performed in triplicate and data are expressed as mean±SD.
All animal experiments were approved by the Chungnam National University Committee of Animal Experiments (202006A-CNU-085). Ninety eight male albino guinea pigs, each weighing 200 to 250 g, were used in this study. To analyze the residual stability of the HGC thermogel after injection into the middle ear, twenty animals were used to perform micro-computed tomography (CT) and magnetic resonance imaging (MRI) at various injection time points (1, 3, 7, 14, and 21 days) and to perform histopathological analysis. Seventy two animals were used for perilymph sampling at various time points (30 minutes, 90 minutes, 3 hours, 1 day, 3 days, and 7 days) after middle ear injection of the HGC thermogel. Other six animals were used as normal controls for CT and T2-weighed MRI (N=4) and used for histopathological studies after intratympanic injection of saline on day 21 (N=2).
Before surgery, animals were anesthetized by intramuscular injection of alfaxan (15 mg/mL, Careside) and Rumpun (23 mg/mL, Careside). In addition, 0.5 ml of 1% lidocaine was injected subcutaneously into the postauricular area for local anesthesia. The anesthetized animals were placed in a prone position on a thermoregulated heated pad. After incising the retro auricular, the temporal bone was exposed and the round window membrane was visualized with a surgical microscope (Carl Zeiss OPMI f 170 Surgical Tilting Head connected to a Hitachi KP-D50 color digital microscope camera). Each of the HGC-DSP and HGC-DEX thermogel formulations (sample volume=100 μL, drug concentration=5 mg/mL (corresponding to 0.5 wt %)) was administered to the tympanic membrane using a 26 gauge needle. After injection, the needle was carefully removed, dental cement (Durelon™ Carboxylate Luting Cement, 3M) was applied to the area, and the skin incision was sutured. No animals were sacrificed in this series of experiments.
Image analysis was performed at various time points after injection (days 1, 3, 7, 14, and 21) using computed tomography (CT) and T2-weighted magnetic resonance imaging (MRI). CT images were acquired on a Quantum GX2 micro-CT Imaging System (PerkinElmer, Waltham, MA, US). MR images were obtained using 4.7 T BioSpec, 47/40 USR (Bruker Biospin, Germany).
To investigate whether the HGC thermogel administered intratympanically has a negative effect on the middle ear, cochlea was obtained from the animals 21 days after intratympanic injection of saline or the HGC thermogel, and inflammation of the middle ear mucosa tissue was assessed. Cochlear samples were placed in 4% paraformaldehyde in PBS for 2 hours, decalcified in EDTA for 3 weeks, embedded in paraffin, serially sectioned at 4 μm thickness, and stained with hemotoxylin and eosin. The stained tissue sections were examined and representative fields were photographed using an optical microscope (Olympus BX51).
To assess the uptake of DEX in the cochlear tissue, animals were sacrificed 30 or 90 minutes after surgery. The tissues were rinsed in PBS for 30 min and incubated for 1 hour in a solution containing 10% normal goat serum (Vector Laboratories, Inc.) and 0.3% Triton X-100 (Sigma-Aldrich Co.) to block nonspecific antibody binding.
The tissues were then stained with rabbit anti-DEX primary antibody (Abcam, Cambridge, MA) at a concentration of 1:200 in blocking solution overnight at 4° C. After rinsing in PBS for 1 hour, the tissues were incubated with the corresponding Alexa Fluor 594 goat anti-rabbit secondary antibody (Molecular Probes, Eugene, OR) at a 1:200 dilution. After incubation at room temperature for 2 hours, the tissues were rinsed in PBS for 30 minutes and stained with 1:200 diluted Hoestch 33342 (Invitrogen) for 5 minutes. After incubation at room temperature, the tissues were rinsed in PBS for 30 minutes and mounted on glass slides using Crystal Mount (Biomeda). The uptake of the drug in the cochlear tissue was observed using a fluorescence microscope (BX53F2, Olympus, Tokyo, Japan).
To evaluate the survival of cochlear hair cells, the animals were sacrificed 21 days after intratympanic injection of the HGC thermogels. Tissues were fixed in 4% paraformaldehyde in PBS for 30 min at 4° C. The cochlear bony walls and lateral wall tissues were first removed and the remaining cochlear tissues were prepared for immunostaining. The tissues were rinsed in PBS for 30 minutes and incubated for 1 hour in a PBS solution containing 10% normal goat serum and 0.3% Triton X-100 to block nonspecific antibody binding. After blocking, the tissues were stained with 1:200 diluted monoclonal anti-myosin VIIa primary antibody (Proteus BioSciences, Inc.) overnight at 4° C. The tissues were rinsed in PBS for 60 minutes. After rinsing, the tissues were stained by incubation with 1:200 diluted Alexa Fluor 594 goat anti-mouse secondary antibody and Alexa Fluor 488 Phalloidin antibody for 2 hours at room temperature. The tissues were rinsed in PBS for 15 minutes. After rinsing, the tissues were mounted on glass slides using Crystal Mount. The tissues were observed using a fluorescence microscope (BX53F2, Olympus, Tokyo, Japan).
To assess the concentration of DEX in cochlear perilymph, perilymph sampling was performed at various time points (30 minutes, 90 minutes, 3 hours, 1 day, 3 days, and 7 days) after intratympanic injection of the DEX-loaded HGC thermogel. Under anesthesia, the temporal bone was removed, and the tympanic bulla was washed with 10 mL of saline. Small apical cochleostomy was performed using a sharp pick, and perilymph was collected in hand-held graduated glass capillary tubes (IntraMAPK micropipettes) marked at every 4 μl volume. Cochlear fluid and DEX standard samples were diluted 1:11 with artificial perilymph, and each mixture was diluted again 1:3 with 50% MeOH. All samples were analyzed using an operator system (QTRAP 6500) with a UHPLC/Tandem mass spectrometer (QTRAP 6500 Low Mass BL210251506) to measure the DEX concentration in the perilymph by LC-MS/MS. Each sample was injected onto a C18 column (Atlantis dC18 column) with solvent A, 0.1% formic acid/DW, and solvent B, MeOH. The flow rate was 0.3 ml/min. Data scans were performed with multiple reaction monitoring (MRM).
Adjustment of image contrast, superimposition of images, and colorization of monochrome fluorescence images were performed using Adobe Photoshop (version 7.0). Data graphing and all statistical analyses were performed using GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). Two-way ANOVA was used to measure the DEX concentration. All experiments were repeated multiple times. The differences between groups were considered significant at p<0.05 in each case.
According to the Examples below, it has been proven that N-acyl glycol chitosan-based thermogels have better properties for injection into the inner ear than block copolymer-based thermogels such as PEG-PPG and PEG-PLGA in terms of physical stability, biocompatibility, biodegradability, thermogelation, and other biofunctions.
Among N-acyl glycol chitosans, N-hexanoyl glycol chitosan (HGC) exhibits sol-gel transition in response to body temperature, and the physicochemical properties and thermogelation properties thereof can be modified by changing the degree of N-hexanoylation (DH). Thus, N-hexanoyl glycol chitosan (HGC) may be considered as a suitable drug delivery platform for intratympanic administration by injection.
N-hexanoyl glycol chitosan (HGC) was synthesized by N-hexanoylation of glycol chitosan (GC) and evaluated as an injectable formulation for inner ear delivery (see
The results of synthesis of HGC were characterized by H-NMR and ATR-FTIR measurements.
The HGC polymer is composed of a hydrophilic GC backbone and a hydrophobic hexanoyl group and is amphiphilic in nature, and thus it is expected that the HGC polymer can be loaded with both hydrophilic and hydrophobic drugs.
The solubilization effect of HGC on hydrophobic dexamethasone (DEX) was evaluated below.
Table 1 below shows the chemical compositions of drug-loaded thermogels.
aThe sol-gel transition temperature determined by tube tilting method
bThe sol-gel transition temperature determined by rheological measurement
As listed in Table 1 above, drug-loaded thermogels were prepared by simply mixing an aqueous solution of HGC (4 wt %) and DSP or DEX (0.5 wt % and 1.0 wt %).
DSP is a hydrophilic form of DEX and has high solubility in aqueous solvents. DSP was well mixed with HGC, thus preparing transparent HGC-DSP mixtures (see
On the other hand, because DEX has low solubility, the HGC-DEX mixtures were not transparent. However, referring to FE-SEM images of the surfaces and cross-sections of lyophilized samples of gelled HGC-DEX, it was observed that micronized DEX particles were uniformly dispersed in the thermogel matrix.
All the HGC-DEX thermogels were shown to stably maintain drug suspensions without aggregation or precipitation. Therefore, HGC has amphiphilic properties and effectively disperses a hydrophobic drug therein, and thus it may be used as an injectable formulation of a hydrophobic drug.
The thermo-sensitive sol-gel phase transition behaviors of HGC, HGC-DSP, and HGC-DEX were analyzed by the tube tilting method and rheological analysis.
The sol-gel transition of HGC-DSP and HGC-DEX was observed to determine whether the presence of hydrophilic DSP or hydrophobic DEX could affect the thermogelation behavior of HGC. Both HGC-DSP and HGC-DEX showed a slightly lower gelation temperature than HGC, and the gelation temperature decreased as the concentration of the drug increased. This may be because the mixing of DSP (sodium phosphate salt form of DEX) or hydrophobic DEX promoted hydrophobic interactions between hexanoyl groups.
The viscoelastic properties, elastic modulus (G′) and loss modulus (G″) of GC, HGC, HGC-DSP and HGC-DEX were observed through rheological experiments in the temperature range of 10 to 45° C. The crossover temperature between G′ and G″ was defined as the gelation point to determine the sol-gel phase transition.
Referring to
In vitro drug release tests were performed on hydrophilic DSP, hydrophobic DEX, DSP-loaded HGC (HGC-DSP), and DEX-loaded HGC (HGC-DEX) in PBS at 37° C.
Referring to
Meanwhile, the HGC-DEX formulation showed various drug release profiles in the range of 4 to 14 days depending on the drug concentration (0.5, 1.0, 2.0, and 4.0 wt %). The release rates of HGC-DEX 0.5 and HGC-DEX 1.0 were faster than that of the free DEX form under the same concentration conditions due to the solubilization effect of the HGC thermogel, which can accelerate the release behavior. HGC-DEX 2 and HGC-DEX 4, which have higher drug concentrations, showed slower release behavior than HGC-DEX 0.5 and HGC-DEX 1.0 having lower drug concentrations, and this may be because when the drug is loaded at a high concentration, more time is needed for the drug to be solubilized and released. Referring to the above results, the HGC thermogel formulation can be optimized to release the hydrophobic drug at an appropriate rate and dose as needed.
The HGC thermogel can improve drug uptake in the inner ear by improving the residual stability of DEX in the middle ear cavity. This is because thermogelation can prevent early leakage of the drug through the Eustachian tube and prolong the residence time of the drug.
In an in vivo experiment, the residual stability of the HGC thermogel in the middle ear cavity was first evaluated by CT and T2-weighted MRI Referring to
Whether intratympanic administration of HGC had an adverse effect was examined by histopathological analysis and observation of the survival of auditory hair cells.
Histological evaluation of the middle ear and inner ear was performed 21 days after intratympanic administration of HGC. Inflammation of the mucosa in the middle ear was detected using hematoxylin and eosin (H&E) staining. Referring to the H&E staining results in
Referring to
The above data suggest that the HGC thermogel has no adverse effects on the middle or inner ear. In fact, no inflammatory response in the middle ear mucosa of the guinea pig and no loss of auditory hair cells in the inner ear of the guinea pig were observed. The day after intratympanic injection of the HGC thermogel, all the guinea pigs did not exhibit pathological vestibular behavior such as rolling and moved normally. Therefore, the HGC thermogel is expected to have no side effects such as acute inflammation, progression of middle ear fibrosis, or hearing changes even when injected intratympanically, and may be a safe means for local drug delivery to the inner ear.
The effect of the HGC thermogel formulation was evaluated by administering HGC-DEX intratympanically and observing changes in the DEX concentration in intracochlear perilymph and DEX distribution in cochlear tissue. Dexamethasone was chosen as a model drug because it has anti-inflammatory and hair cell protective effects and is one of the most commonly used drugs for treating inner ear diseases.
30 minutes, 90 minutes, 3 hours, 1 day, 3 days, or 7 days after intratympanic injection of DSP alone, HGC-DSP (0.5 wt % of DSP), or HGC-DEX (0.5 wt % of DEX), perilymph was collected from the cochlea and the DEX concentration was measured using liquid chromatography/tandem mass spectrometry (LC-MS/MS).
Referring to
Referring to the results of analyzing the intensity of immunohistochemical staining as shown in
Referring to the above in vitro drug release test, the drug release rate of the HGC-DEX thermogel was dependent on the drug concentration, and the higher the concentration of the loaded drug, the slower the release rate was. Therefore, if an HGC thermogel formulation having a higher drug concentration is used, the effective drug concentration will be maintained for a longer period of time. Thus, the HGC thermogel formulation can be considered an effective and useful means for local drug delivery to the inner ear.
Referring to the above experimental results, the HGC-based thermogel was found to be an intratympanically injectable drug delivery platform that can be loaded with both a hydrophobic drug (e.g., dexamethasone) and a hydrophilic drug (e.g., dexamethasone phosphate disodium salt (DSP)). The HGC-based thermogel could be loaded with the drug by a simple method of physically mixing the thermogel in a sol state with the drug, and in particular, the hydrophobic drug (DEX) was effectively dispersed and solubilized therein. Referring to the animal experiment results, it was confirmed that the HGC thermogel remained in the middle ear and inner ear cavity for up to 21 days without significant cytotoxicity or inflammation, suggesting that it has excellent residual safety and is suitable for maintaining the drug concentration in the inner ear at a high level for a long time. In addition, the HGC thermogel formulation exhibited diverse release kinetics depending on the drug type and concentration. Referring to the in vitro drug release test, HGC thermogel could release the drug in an immediate release manner when loaded with 0.5 wt % or 1.0 wt % of the drug, and could release the drug continuously for 2 weeks when loaded with 2 wt % or 4 wt % of the drug. In addition, the HGC thermogel loaded with 0.5 wt % of the drug released the drug for 1 day as a result of the intratympanic administration test.
Since it was confirmed in the in vitro drug release test that the HGC thermogel released the drug in an immediate or sustained release manner depending on the concentration of the drug, it is expected that the HGC thermogel can release the drug sustainedly in the tympanic cavity depending on the concentration of the drug. Therefore, the HGC thermogel was found to have great potential as an effective and safe platform for drug delivery to the inner ear.
Drug-loaded microspheres made of a polymer having excellent biocompatibility and biodegradability can be used for sustained release of drugs. However, the microspheres have a disadvantage in that, since they can easily move to other areas even when injected intratympanically, it is difficult for the microspheres to deliver the drug continuously while remaining in the tympanic cavity.
To overcome this disadvantage, HGC thermogels containing dexamethasone-loaded microspheres dispersed therein were fabricated and their in vitro drug release rates were measured.
Four types of microspheres (GB-5313-023, GB-5313-061, GB-5313-062, and GB-5313-075) with differences in terms of formulation method, drug content, and particle size were prepared (hereinafter referred to as 023, 061, 062, and 075). Their compositions are shown in Tables 2 and 3 below.
Here, the particle size (D50) means the average diameter of 50% of the particles.
The S/O/W denotes Solid-in-Oil-in-Water Emulsion, and each S/O/W was prepared by adding the solid drug(S) encapsulated with 061 or 062 to the polymer/oil phase (O) and adding water thereto to form an emulsion. The O/W was prepared by adding the non-encapsulated solid drug to the polymer/oil phase (O) and adding water thereto to form an emulsion.
The 061 is a formulation that is expected to release the drug faster due to its higher drug content than the 023. The 062 is a formulation that is expected to release the drug more slowly because it contains the 503H polymer having a higher molecular weight than the 023. The 075 is a formulation prepared under the same conditions as the 023, except that the particle size is smaller. The fabricated microspheres are shown in
An in vitro drug release test was conducted as follows.
023 microspheres loaded with dexamethasone were mixed with 4 wt % of HGC hydrogel. An in vitro drug release test was conducted in a PBS (pH 7.4, 50 ml) environment in the same manner as in Example 4. Sampling was performed at days 0, 0.5, 1, 2, 4, 7, 14, 21, 28, and 30. The dialysis membrane had a thickness of 10 mm and a MWCO of 12 to 14 kDa. The in vitro drug release test conditions for 023 microspheres, etc. are shown in Table 4 below.
Dexamethasone-loaded 061 or 062 microspheres were mixed with 4 wt % of HGC hydrogel, and an in vitro drug release test therefor was conducted in the same manner as the test for the 023. The in vitro drug release test conditions for 061 microspheres, etc. are shown in Table 5 below.
Referring to
Referring to
Referring to the above experimental results, the release of DEX loaded in HGC can be promoted due to the solubilization effect of HGC, and DEX can be released sustainedly by being loaded in microspheres made of a biocompatible polymer, and when both DEX and DEX-loaded microspheres are mixed with HGC, high release can occur initially and long-term sustained release can also occur. In addition, since HGC, when injected intratympanically, gels in response to body temperature, thereby inhibiting the drug from moving elsewhere, it is possible to achieve desired drug release characteristics by mixing HGC with one appropriately selected from a drug, a drug loaded in microspheres, or a combination thereof.
The physical properties of drug-loaded microspheres and the HGC thermogel containing the same were analyzed. Dexamethasone was used as the drug, and PLGA was used as the microspheres.
LA (lactide) and GA (glycolide) were dissolved in dichloromethane, and then dexamethasone was suspended in the solution, thus preparing a dispersed phase. The dispersed phase was injected into an aqueous solution of PVA (0.5% w/w) as a continuous phase using a membrane emulsification system (LDC-1, Micropore Technologies), thus preparing an emulsion. The organic solvent was removed from the emulsion to obtain solidified microspheres, which were then washed with distilled water to obtain drug-loaded microspheres (M-DEX).
First, the loading content and encapsulation efficiency of the drug in the microspheres were analyzed. A solution obtained by dissolving 10 mg of dexamethasone in 1 mL of DMSO, followed by dilution with 50% CAN, was used as a standard solution, and a solution obtained by dissolving 10 mg of the microspheres in 1 mL of DMSO, followed by 1,250-fold dilution with 50% CAN, was as a sample solution. Using these solutions, measurements were repeated three times. The content and encapsulation efficiency of dexamethasone were determined by measuring the absorbance of dexamethasone at 254 nm by HPLC (CM5000, Hitachi) using 50% CAN as a mobile phase. The content of the drug was calculated as a percentage of the amount of dexamethasone contained in the microspheres relative to the amount of the microspheres, and the content of the drug was shown to be about 33.3%. The encapsulation efficiency of the drug was calculated as a percentage of the amount of dexamethasone contained in the microspheres relative to the initial amount of dexamethasone, and the encapsulation efficiency was shown to be about 83.3%.
Additionally, according to the same method as the above Example, a HGC thermogel (HGC), a dexamethasone-loaded HGC thermogel (HGC/DEX), a formulation (HGC/M-DEX) comprising the HGC thermogel and dexamethasone-loaded microspheres, and a formulation (HGC/DEX/M-DEX) comprising the dexamethasone-loaded HGC thermogel and dexamethasone-loaded microspheres were prepared as shown in Table 6, and the morphological characteristics and physical properties thereof were investigated.
In Table 6 above, Tgel* is the transition temperature determined by the tube tilting method, and Tgel** is the transition temperature determined by rheological measurement. In the case of HGC without the drug, a sol-gel phase transition was observed at about 28° C., and in the case of the drug-loaded formulations, a sol-gel phase transition was observed at 24 to 26° C. This indicates that gelation occurred at a lower temperature because hydrophobic interactions were enhanced due to the introduction of the hydrophobic drug or the microspheres.
An aqueous solution formulation of DSP was used as a water-soluble drug control, and it was shown that the control completely released the drug quickly within a few hours. On the other hand, dexamethasone (DEX) was used as a hydrophobic drug control, and dexamethasone exhibited sustained release compared to DSP, but it was believed to have a solubility limit due to its poor solubility rather than controlled release. HGC/DEX exhibited more effective release behavior than the hydrophobic drug control (DEX) by virtue of the solubilization effect of HGC. The HGC/M-DEX and HGC/DEX/M-DEX formulations exhibited sustained release for about a month at rates similar to that of HGC/DEX. Since the formulation comprising g the microspheres can exhibit stable and controllable release behavior, sustained drug release of this formulation is easier to control than that of the HGC/DEX formulation, which is dependent on the drug concentration.
Referring to the above-described experimental results, the release of DEX loaded in HGC can be promoted due to the solubilization effect of HGC, and the formulation containing the microspheres and the formulation comprising a mixture of the thermogel and the microspheres have high drug encapsulation efficiency due to the introduction of the microspheres, drug release therefrom is easily controlled, and it is possible to control the drug release rate therefrom from several weeks to several months by controlling the biodegradability thereof.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2021-0131088 | Oct 2021 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2022/013938 | 9/19/2022 | WO |
