Preparation and kit preparation

Abstract

This preparation for delaying or preventing gingivitis and bone resorption associated with periodontal disease includes particles of a nano-sized or micro-sized biodegradable polymer containing a macrolide-based antimicrobial agent.

Description

Formulations and kit formulations

[0001] The present invention relates to formulations and kit formulations.

[0002] Periodontal disease is a disease in which inflammation of the gums caused by oral bacteria spreads to the tissues that support the teeth. Periodontal disease destroys bone and eventually leads to tooth loss. Patients with severe periodontal disease often lose their teeth due to alveolar bone resorption. In the drug treatment of periodontal disease, macrolide antibiotics such as clarithromycin, azithromycin, and erythromycin are used to eliminate the bacteria that cause periodontal disease.

[0003] Patients with dental infections who take bisphosphonates may develop osteonecrosis of the jaw (BRONJ), a condition characterized by impaired wound healing following oral surgery such as tooth extraction, periodontal surgery, or endodontic treatment.

[0004] It has been reported that developmental endothelial locus-1 (DEL-1) is important for bone resorption and destruction (see Non-Patent Document 1). When DEL-1 is reduced by inflammation, bone resorption is observed, but it has been shown that macrolide antibiotics re-induce the reduced DEL-1 and suppress bone resorption (see Non-Patent Document 2). DEL-1 is important not only for suppressing bone resorption but also for bone regeneration (see Non-Patent Document 3). It has been found that the expression level of DEL-1 decreases with age, which is thought to be one of the factors that makes inflammation and bone resorption more likely. Furthermore, DEL-1 has been reported to induce α-smooth muscle actin (αSMA), which is important for mucosal regeneration (see Non-Patent Document 4).

[0005] Tomoki Maekawa et al., Antagonistic effects of IL-17 and D-resolvins on endothelial Del-1 expression through a GSK-3β-C / EBPβ pathway, Nature Communications, 2015, 6, 8272Tomoki Maekawa et al., Erythromycin inhibits neutrophilic inflammation and mucosal disease by upregulating DEL-1, JCI Insight., 2020, 5(15), e136706Da-Yo Yuh et al., The secreted protein DEL-1 activates a β3 integrin-FAK-ERK1 / 2-RUNX2 pathway and promotes osteogenic differentiation and bone regeneration, J. Biol. Chem., 2020, 295(21), 7261-7273Sirisereephap K et al., A novel macrolide-Del-1 axis to regenerate bone in old age, iScience, 2024, 27(2), 108798

[0006] Macrolide antibiotics are administered orally in the pharmacological treatment of periodontal disease. To minimize the side effects of macrolide antibiotics, it is preferable to increase their concentration only at the site of action, the periodontal disease site. However, oral systemic administration does not allow for a limited increase in the concentration of macrolide antibiotics at the site of periodontal disease. Furthermore, considering the emergence of drug-resistant bacteria, it is desirable to avoid long-term administration of macrolide antibiotics. On the other hand, local administration of macrolide antibiotics to the site of periodontal disease is difficult because frequent administration is required to maintain the concentration.

[0007] At present, there are no established methods for preventing or treating BRONJ. When using macrolide antibacterial drugs to treat oral diseases such as gingivitis associated with periodontal disease and BRONJ, it is important to limit the frequency and dosage of administration.

[0008] The present invention has been made in view of the above circumstances, and aims to provide a formulation and a formulation kit that can provide high efficacy of a macrolide antibacterial drug with fewer administrations and at lower doses.

[0009] A formulation for delaying or preventing gingivitis and bone resorption associated with periodontal disease, a formulation for delaying or treating bone resorption and bone destruction associated with bisphosphonate-associated osteonecrosis of the jaw, or a formulation for delaying or treating stomatitis according to a first aspect of the present invention comprises nano- or micro-sized particles of a biodegradable polymer containing a macrolide antibacterial agent.

[0010] The macrolide antibacterial agent may be clarithromycin, azithromycin, or erythromycin.

[0011] The biodegradable polymer may be polylactic acid, polylactic acid-glycolic acid, or a mixture thereof.

[0012] The content of the macrolide antibacterial agent in the particles may be 5 to 30% by weight.

[0013] The 50% particle size of the particles may be 0.1 to 100 μm.

[0014] The formulation according to the first aspect of the present invention may further comprise an excipient.

[0015] The excipient may be a sugar or an amino acid.

[0016] The preparation according to the first aspect of the present invention may be dispersed in a dispersion medium, a gel base or an ointment base and administered intragingivally or into a periodontal pocket.

[0017] The preparation according to the first aspect of the present invention may be injected into the gums or placed on the bone surface, and may release the macrolide antibacterial agent for a period of 1 day to 4 weeks.

[0018] A kit preparation according to a second aspect of the present invention comprises a dispersion medium, a gel base or an ointment base, and the preparation according to the first aspect of the present invention, wherein the preparation is dispersed in the dispersion medium, the gel base or the ointment base and administered into the gums, tissues after tooth extraction, an area affected by stomatitis or a periodontal pocket.

[0019] According to the present invention, a macrolide antibacterial drug can be administered at a lower dose and with fewer administrations to obtain a high efficacy.

[0020] 1 is a diagram showing a scanning electron microscope (SEM) image of microparticles according to Example 2. FIG. 1 is a diagram showing the change over time in the residual rate of erythromycin in the formulation in Test Example 1. FIG. 2 is a diagram showing the change over time in the residual rate of erythromycin in Test Example 2. FIG. 3 is a diagram showing the appearance of teeth and alveolar bone in Test Example 3. FIG. 4 is a diagram showing bone levels in Test Example 3. FIG. 5 is a diagram showing osteoblasts stained with Alizarin Red S in a wild-type mouse in Test Example 3. FIG. 6 is a diagram showing the proportion of bone nodule areas in Test Example 3. FIG. 7 is a diagram showing cells after tartrate-resistant acid phosphatase (TRAP) staining in Test Example 3. FIG. 8 is a diagram showing the proportion of TRAP-positive cells in Test Example 3. FIG. 9 is an observation image of a slice preparation fluorescently immunostained with an anti-DEL-1 monoclonal antibody in Test Example 3. FIG. 10 is a diagram showing the expression levels of DEL-1 compared by mean fluorescence intensity in Test Example 3. FIG. 11 is a diagram showing an observation image of a slice preparation in the alveolar bone of an experimental periodontitis model mouse subjected to TRAP staining in Test Example 3. FIG. 12 is a diagram showing the proportion of TRAP-positive areas in Test Example 3. 4 is a diagram showing an observation image of a slice specimen from the alveolar bone of an experimental periodontitis model mouse subjected to alkaline phosphatase (ALP) staining in Test Example 3. FIG. 5 is a diagram showing the proportion of ALP-positive areas in Test Example 3. FIG. 6 is a diagram showing the relative expression level of the Oscar gene in Test Example 3. FIG. 7 is a diagram showing the relative expression level of the Ocstamp gene in Test Example 3. FIG. 8 is a diagram showing cells after ALP staining in Test Example 3. FIG. 9 is a diagram showing the proportion of ALP-positive cells in Test Example 3. FIG. 10 is a diagram showing the appearance of the gingiva or the surface of the alveolar bone after tooth extraction in Test Example 4. FIG. 11 is a diagram showing an image obtained by analyzing the alveolar bone by micro-computed tomography (μCT) in Test Example 4. FIG. 12 is a diagram showing an observation image of a slice specimen from the alveolar bone subjected to hematoxylin-eosin staining (HE staining) in Test Example 4. FIG. 13 is a diagram showing trabecular width in Test Example 4. FIG. 14 is a diagram showing trabecular spaces in Test Example 4. FIG. 15 is a diagram showing an observation image of a slice specimen from the alveolar bone subjected to Masson's trichrome staining in Test Example 4. Fig. 1 shows an observation image of a 10 μm frozen section of alveolar bone subjected to fluorescent immunostaining with an anti-DEL-1 monoclonal antibody in Test Example 4. Fig. 2 shows the expression intensity of DEL-1 in Test Example 4. Fig. 3 shows the appearance of a site where stomatitis has occurred in Test Example 5.FIG. 1 is a diagram showing an observation image of a slice preparation of tongue tissue stained with HE in Test Example 5. FIG. 2 is a diagram showing the number of neutrophils per unit area in Test Example 5. FIG. 3 is a diagram showing a histoimmunological staining image of a section of tongue tissue fluorescently immunostained with an anti-DEL-1 monoclonal antibody in Test Example 5. FIG. 4 is a diagram showing the expression level of DEL-1 based on fluorescence intensity in Test Example 5. FIG. 5 is a diagram showing an observation image of a slice preparation of tongue tissue stained with Masson's Trichrome in Test Example 5. FIG. 6 is a diagram showing the amount of collagen in tongue tissue in Test Example 5.

[0021] Hereinafter, embodiments of the present invention will be described with reference to the drawings. In each drawing, the same or equivalent parts are designated by the same reference numerals. It should be noted that the present invention is not limited to the following embodiments and drawings. It should be noted that in the following embodiments, the expressions "have," "include," or "contain" also include the meaning of "consisting of" or "consisting of."

[0022] The use of the formulation according to this embodiment is to delay or prevent gingivitis and bone resorption associated with periodontal disease, or to delay or treat bone resorption and bone destruction associated with BRONJ. The formulation may be a medicament for delaying gingivitis associated with periodontal disease, a medicament for preventing gingivitis associated with periodontal disease, a medicament for delaying bone resorption associated with periodontal disease, or a medicament for preventing bone resorption associated with periodontal disease. The formulation may be a medicament for delaying bone resorption associated with BRONJ, a medicament for treating bone resorption associated with BRONJ, a medicament for delaying bone destruction associated with BRONJ, or a medicament for treating bone destruction associated with BRONJ.

[0023] The formulation comprises nano- or micro-sized particles of a biodegradable polymer containing a macrolide antibacterial drug, such as clarithromycin, azithromycin, erythromycin, josamycin, spiramycin, etc. Preferably, the macrolide antibacterial drug is clarithromycin, azithromycin, or erythromycin.

[0024] The biodegradable polymer may be any polymer that has biodegradable properties. The biodegradable polymer is a polymer that is low in irritation and toxicity to the living body and is degraded and metabolized after administration. After administration to the living body, the biodegradable polymer remains in the living body, hydrates and swells, and gradually releases the macrolide antibacterial agent as hydrolysis progresses.

[0025] The biodegradable polymer can be produced from a biodegradable polyester. The biodegradable polyester is a polyester synthesized by further copolymerizing a polymer of one or more monomers or dimers selected from, for example, D,L-lactide, D-lactide, L-lactide, D,L-lactic acid, D-lactic acid, L-lactic acid, glycolic acid, glycolide, ε-caprolactone, ε-hydrohexanoic acid, γ-butyrolactone, γ-hydroxybutyric acid, δ-valerolactone, δ-hydroxyvaleric acid, hydroxybutyric acid, malic acid, etc. The biodegradable polymer may be obtained by mixing the above-mentioned monomers or dimers in an appropriate ratio and polymerizing the mixture.

[0026] Preferably, the biodegradable polymer is polylactic acid (PLA), polyglycolic acid, lactic acid-glycolic acid copolymer (PLGA), or lactic acid-aspartic acid copolymer. The molecular weight of PLA is, for example, 10,000 to 20,000. PLGA is, for example, a copolymer of lactic acid and glycolic acid with a polymerization ratio of 1:99 (lactic acid:glycolic acid) to 99:1. The molecular weight of PLGA is, for example, 10,000 to 60,000. PLGA can be synthesized from any monomer or dimer by a common method such as dehydration condensation or ring-opening polymerization. Preferably, the biodegradable polymer is PLA, PLGA, or a mixture thereof. The biodegradable polymer may be a polyethylene glycol (PEG)-modified PLGA or polyethylene glycol / chitosan-modified PLGA (PEG / CS-PLGA).

[0027] The formulation of this embodiment may further contain an excipient to improve redispersibility during use and stability during storage. Any pharmaceutically acceptable excipient can be used. For example, the excipient is a sugar or an amino acid. More specifically, examples of the excipient include lactose, mannitol, trehalose, inositol, erythritol, sucrose, pullulan, sorbitol, starches, dextrin, dextran, sodium alginate, crystalline cellulose, methylcellulose, carmellose sodium, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), natural polymers, synthetic polymers, glycine, leucine, isoleucine, arginine, histidine, etc. Preferably, the excipient is mannitol.

[0028] The particle diameter (particle size) of the particles is 10 to 1000 nm, preferably 50 to 950 nm, and more preferably 100 to 900 nm if they are nano-sized. The particle diameter is 5 to 100 μm, 5 to 50 μm, and preferably 10 to 40 μm if they are micro-sized. Preferably, the particle size range is 5 to 40 μm, which allows the use of a thin injection needle of about 23 to 26 G that does not require local anesthesia, particularly when injecting into the gums, and ensures sustained release.

[0029] The particle size of particles can be measured by sieving, sedimentation, microscopy, light scattering, laser diffraction / scattering, electrical resistance testing, observation with a transmission electron microscope, observation with a scanning electron microscope, etc. The particle size of particles can be expressed as a Stokes equivalent diameter, a circle equivalent diameter, a sphere equivalent diameter, etc. depending on the measurement method. Furthermore, the particle size of particles shown here may be expressed as an average particle size, particularly a volume average particle size or a mass average particle size, calculated as an average from the number distribution based on measurements such as laser diffraction / scattering, using multiple particles as the measurement subject.

[0030] For example, the particle diameter of the particles may be an average particle diameter calculated from a volume distribution based on measurement by a laser diffraction / scattering method. Specifically, when a cumulative curve is calculated assuming the total volume of a particle group to be 100%, the volume average particle diameter (50% diameter; D) is the particle diameter at the point where the cumulative curve is 50%. ５０ ) may be used as the particle diameter.５０ can be determined using a commercially available particle size distribution analyzer. When the particles are nano-sized, the particle size distribution analyzer may be a Microtrac particle size distribution and particle shape analyzer Nanotrac Wave (manufactured by Microtrac BEL). When the particles are micro-sized, the particle size distribution analyzer may be a Sync Analyzer (manufactured by Microtrac BEL). ５０ is preferably 0.1 to 100 μm, more preferably 0.5 to 30 μm.

[0031] The span value of the particles is preferably 3.5 or less. ９０ -D １０ ) / D ５０ where D ９０ is the particle diameter at the point where the cumulative curve reaches 90%. １０ is the 10% diameter, which is the particle diameter at the point where the cumulative curve reaches 10%. ９０ and D １０ The span value of the particles is more preferably 3.0 or less.

[0032] Next, a method for producing the formulation according to this embodiment will be described. Any known production method, such as a phase separation method or an emulsion-in-water method, can be used to produce the particles contained in the formulation. In the emulsion-in-water method, two types of solvents are used: a good solvent in which the biodegradable polymer and the macrolide antibacterial drug dissolve, and a poor solvent in which the biodegradable polymer does not dissolve. Alternatively, an organic solvent in which the biodegradable polymer dissolves and which is miscible with the poor solvent is used as the good solvent. The types of the good solvent and the poor solvent are not particularly limited.

[0033] Examples of good solvents include poorly water-soluble organic solvents such as dichloromethane, chloroform, halogenated alkanes, ethyl acetate, diethyl ether, hexane, and cyclohexane. Dichloromethane and chloroform are preferred because they are easily removed from the formulation. Examples of poor solvents include water.

[0034] A dispersant may be used in the production of particles. In principle, the dispersant must be removed. Examples of dispersants include surfactants, polyethylene glycol, PVA, PVP, hydroxymethylcellulose, hydroxypropylcellulose, glycerin, fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, polyglycerin fatty acid esters, polysorbates, etc. The dispersant may have both hydrophilic and hydrophobic functional groups and function as a protective colloid or surfactant. The dispersant may also be added to water as a poor solvent. Depending on the poor solvent, a dispersant may not be necessary.

[0035] In the water emulsion method, a macrolide antibacterial agent and a biodegradable polymer are first dissolved in a good solvent. When the mixture containing the macrolide antibacterial agent and the biodegradable polymer is added dropwise to a stirred poor solvent, the good solvent in the mixture rapidly diffuses and migrates into the poor solvent. As a result, the good solvent emulsifies in the poor solvent, forming emulsion droplets of the good solvent. At this time, a small amount of an organic solvent such as methanol or ethanol, which dissolves in both solvents, may be added to increase the solubility of the macrolide antibacterial agent.

[0036] The good solvent diffuses from within the emulsion droplets into the poor solvent and lightly solidifies. Further gentle stirring under normal pressure causes the good solvent to continuously diffuse and evaporate, thereby reducing the solubility of the biodegradable polymer and macrolide antibacterial within the emulsion droplets. Ultimately, biodegradable polymer particles containing the macrolide antibacterial are produced. The biodegradable polymer particles are then re-dispersed in an aqueous solution containing an excipient such as mannitol, if necessary, and freeze-dried or otherwise treated to obtain redispersible biodegradable polymer particles containing the macrolide antibacterial. The content of the macrolide antibacterial in the biodegradable polymer particles is, for example, 5 to 30% by weight.

[0037] Alternatively, for hydrophilic macrolide antibacterial agents that are insoluble in organic solvents, particles may be produced by a W / O / W emulsion method, in which a W / O emulsion is prepared with an aqueous solution of the macrolide antibacterial agent as the internal phase, and then the W / O emulsion is poured into a stirred aqueous phase to produce a W / O / W emulsion.

[0038] The formulation according to this embodiment may contain, as necessary, other pharmacologically acceptable ingredients in addition to the particles, such as carriers, lubricants, binders, disintegrants, solvents, solubilizers, suspending agents, isotonicity agents, buffers, preservatives, antioxidants, colorants, and sweeteners.

[0039] The formulations of this embodiment are administered to humans and non-human animals, preferably mammals such as dogs, cats, cows, pigs, horses, sheep, and deer.

[0040] The route of administration of the formulation according to this embodiment is not particularly limited, but preferably, the formulation is dispersed in a dispersion medium, gel base, or ointment base and administered intragingivally or into a periodontal pocket. When the formulation is an injection, it is preferable to disperse the formulation in a dispersion medium. Examples of dispersion mediums include distilled water for injection in which a surfactant such as Tween 80, a thickener such as sodium carboxymethylcellulose, or an isotonic agent such as D-mannitol has been dissolved. The amount of injection to be administered is preferably an amount physically injectable into the gingiva. The injection is dispersed in a dispersion medium immediately before use and administered to the affected site, more specifically, the gingiva or periodontal pocket exhibiting gingivitis associated with periodontal disease, or the vicinity thereof. Particularly in the case of delaying or treating bone resorption and bone destruction associated with BRONJ, the injection may be dispersed in a dispersion medium immediately before use and administered to the tissue or the vicinity thereof after tooth extraction.

[0041] When the formulation is an oral ointment, it is preferable to disperse the formulation in a gel base or ointment base. The gel base may be an aqueous gel base or an oily gel base. Examples of ointment bases include petrolatum, gelling hydrocarbons, gelling glycerin fatty acid esters, hydrogenated lanolin, and concentrated glycerin. Oral ointments may contain excipients or dispersants as needed. Examples of excipients include lactose, mannitol, trehalose, inositol, erythritol, sucrose, sorbitol, starches, dextrin, dextran, sodium alginate, crystalline cellulose, methylcellulose, and carmellose sodium. Examples of dispersants include polyethylene glycol, PVA, PVP, hydroxymethylcellulose, hydroxypropylcellulose, glycerin, fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, polyglycerin fatty acid esters, and polysorbates. The amount of oral ointment administered is preferably an amount that can be applied to the gums. The oral ointment is applied to the affected area, more specifically, to the gums or their vicinity where gingivitis occurs due to periodontal disease, or injected into periodontal pockets. The oral ointment may also be applied to or its vicinity after tooth extraction, particularly in the retardation or treatment of bone resorption and bone destruction associated with BRONJ.

[0042] The dosage of the formulation according to this embodiment is determined appropriately depending on the gender, age, weight, symptoms, etc. of the recipient. The formulation is administered so that the macrolide antibacterial agent is in an effective amount. An effective amount is the amount of macrolide antibacterial agent required to achieve the desired result, i.e., the amount required to delay, inhibit, prevent, or cure the condition being treated or treated. When the formulation is an injectable solution, the amount of the macrolide antibacterial agent is, for example, 100 to 20,000 μg, preferably 200 to 15,000 μg, and more preferably 400 to 10,000 μg. When the formulation is an oral ointment, the amount of the macrolide antibacterial agent is, for example, 100 to 20,000 μg, preferably 200 to 15,000 μg, and more preferably 400 to 10,000 μg.

[0043] Preferably, the preparation is administered not daily but once every few days, preferably once every one or two weeks. In this case, the dosage of the preparation is appropriately determined taking into consideration the administration interval, the release rate of the macrolide antibacterial agent, localization, and changes in the concentration in the preparation over time. Preferably, the dosage of the preparation is set so as to maintain a constant or higher concentration of the macrolide antibacterial agent in the gingiva or localized diseased area until the next administration.

[0044] Regardless of the formulation, the preparation may be injected into the gums or placed on the bone surface, whereby the preparation will release the macrolide antibacterial agent for 1 day to 4 weeks or 1 day to 2 weeks.

[0045] As shown in the examples below, the formulation of this embodiment has sustained release properties that suppress the initial burst after administration and gradually release the macrolide antibacterial drug, allowing the formulation to achieve high efficacy with fewer administrations and at lower doses.

[0046] In another embodiment, a kit preparation is provided. The kit preparation comprises a dispersion medium, a gel base, or an ointment base, and the above-described preparation. In this kit preparation, the preparation is dispersed in the dispersion medium, gel base, or ointment base and administered intragingivally or into a periodontal pocket. This kit preparation allows the preparation to be dispersed and administered immediately before use, minimizing deterioration in quality due to decomposition of the preparation during storage. In particular, in delaying or treating bone resorption and bone destruction associated with BRONJ, the above-described preparation in this kit preparation may be dispersed in a dispersion medium, gel base, or ointment base and administered to tissues after tooth extraction.

[0047] In another aspect of the present embodiment, there is provided use of nano- or micro-sized biodegradable polymer particles containing a macrolide antibacterial agent for the manufacture of a medicament for delaying gingivitis associated with periodontal disease, a medicament for preventing gingivitis associated with periodontal disease, a medicament for delaying bone resorption associated with periodontal disease, or a medicament for preventing bone resorption associated with periodontal disease. In another aspect of the present embodiment, there is provided a method for delaying or preventing gingivitis and bone resorption associated with periodontal disease, comprising the step of administering nano- or micro-sized biodegradable polymer particles containing a macrolide antibacterial agent to a subject. In another aspect of the present embodiment, there is provided nano- or micro-sized biodegradable polymer particles containing a macrolide antibacterial agent for use in delaying or preventing gingivitis and bone resorption associated with periodontal disease.

[0048] Another aspect of the present embodiment provides use of nano- or micro-sized biodegradable polymer particles containing a macrolide antibacterial agent for the manufacture of a medicament for delaying bone resorption associated with BRONJ, a medicament for treating bone resorption associated with BRONJ, a medicament for delaying bone destruction associated with BRONJ, or a medicament for treating bone destruction associated with BRONJ. Also, another aspect of the present embodiment provides a method for delaying or treating bone resorption and bone destruction associated with BRONJ, comprising the step of administering nano- or micro-sized biodegradable polymer particles containing a macrolide antibacterial agent to a subject. Also, another aspect of the present embodiment provides nano- or micro-sized biodegradable polymer particles containing a macrolide antibacterial agent for use in delaying or treating bone resorption and bone destruction associated with BRONJ.

[0049] Furthermore, as shown in the examples below, the preparation according to this embodiment is also effective in treating stomatitis. Stomatitis is a general term for inflammation occurring in the mucous membranes of the oral cavity, such as the lips, the inside of the cheeks, the surface of the tongue, and the gums. Stomatitis is caused by a weakened immune system due to stress or malnutrition, physical irritation such as biting the inside of the mouth, viral infection, etc. The preparation according to this embodiment may be used to delay or treat stomatitis. The preparation may be a medicine for delaying stomatitis, a medicine for treating stomatitis, a medicine for delaying stomatitis, or a medicine for treating stomatitis.

[0050] In delaying or treating stomatitis, the injection preparation may be dispersed in a dispersion medium immediately before use and administered to the affected area or its vicinity. Furthermore, in delaying or treating stomatitis, the oral ointment may be applied to the affected area or its vicinity. When using the kit preparation in delaying or treating stomatitis, the preparation may be dispersed in a dispersion medium, gel base, or ointment base and administered to the affected area.

[0051] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0052] Example 1 [Preparation of Nanoparticles] Nanoparticles containing erythromycin and a polymer were prepared by the above-mentioned emulsion-in-water method (O / W method) as follows: In the following, PLGA is represented as PLGA(x / y)-z, where x / y represents the polymerization ratio of lactic acid / glycolic acid, and z represents the weight-average molecular weight of the polymer.

[0053] Erythromycin 50.1 mg (preparation concentration 20.0%) and PLGA (75 / 25)-20000 199.9 mg were dissolved in CHCl ３ The resulting oil phase solution was dissolved in 10 mL of water. To optimize the particle shape, the oil phase temperature was adjusted to 20°C in advance, and the mixture was injected into 90 mL of 0.1% PVA aqueous solution while stirring (room temperature, 24,000 rpm) using a Hiscotron (Microtec Nition Co., Ltd.). After injection, the mixture was emulsified by stirring under the same conditions for 3 minutes. The organic solvent was then distilled off while gently stirring the emulsion phase (room temperature, 1000 rpm, 3 hours) to obtain nanoparticles. The resulting nanoparticles were recovered by centrifugation (4°C, 22,540 G, 15 minutes). The free erythromycin was washed from the recovered nanoparticles with 30 mL of MilliQ water and then recovered by centrifugation (4°C, 22,540 G, 15 minutes). The nanoparticles were washed twice in the same manner, and the washed nanoparticles were dispersed in 30 mL of MilliQ water and solidified by lyophilization to obtain a nanoparticle formulation (Formulation 1).

[0054] [Measurement of Particle Size Distribution] The particle size distribution of Formulation 1 was measured in a wet state using a microtrac particle size distribution and particle shape analyzer Nanotrac Wave (manufactured by Microtrac BEL).

[0055] [Measurement of drug content] 3.0 mL of acetonitrile was added to 10 mg of Formulation 1, and the mixture was stirred for 30 seconds using a vortex mixer to dissolve the drug, followed by ultrasonic treatment for 10 minutes to prepare a sample solution. The sample solution was analyzed by high performance liquid chromatography (HPLC) under the following measurement conditions (n=3), and the erythromycin content in the sample solution was determined from the calibration curve of the standard solution.

[0056] HPLC measurement conditions: Instrument: LC-2000plus series (JASCO Corporation) Column: InertSustain C18, 4.6 x 250 mm, ID: 5 μm Column temperature: 35°C Mobile phase: 25 mmol phosphate buffer (pH 7.0):acetonitrile = 2:3 Flow rate: 0.75 mL / min Sample solution injection amount: 20 μL Detection: UV 215 nm Measurement time: 20 min

[0057] The physical properties and erythromycin content of Preparation 1 are shown in Table 1. Nanoparticles containing erythromycin were obtained in high yield.

[0058]

[0059] Example 2 [Preparation of Microparticles] Microparticles containing erythromycin and a polymer were prepared by the above-mentioned emulsion-in-water method (O / W method) as follows. The preparation conditions for the microparticles (Formulations 2 to 5) are shown in Table 2. In the following, the m in the abbreviation for PLA, PLA-m, represents the weight-average molecular weight of the polymer.

[0060]

[0061] Erythromycin and polymer were ２ Cl ２The oil phase solution was obtained by dissolving the erythromycin in water. To optimize the particle shape, the temperature of the oil phase was adjusted to 20°C in advance, and the mixture was poured into 1.0 L of 0.1% PVA aqueous solution while stirring (room temperature, 6,000 rpm) using a Clearmix (M Technique Co., Ltd.). After the pouring, the mixture was stirred under the same conditions for 3 minutes to emulsify. The organic solvent was then distilled off with gentle stirring (room temperature, 1,000 rpm, 3 hours) to obtain microparticles. This suspension was sieved through a 75 μm mesh sieve, and the passed microparticles were recovered by centrifugation (4°C, 350 G, 5 minutes). The recovered microparticles were washed with 100 mL of MilliQ water to remove free erythromycin, and then centrifuged (4°C, 350 G, 5 minutes) to recover them. The microparticles were washed twice in the same manner, dispersed in 100 mL of MilliQ water, and solidified by freeze-drying to obtain microparticle formulations (formulations 2 to 5).

[0062] [Particle size distribution measurement and SEM observation] The particle size distribution of Formulations 2 to 5 was measured using a Microtrac particle size distribution and particle shape analyzer, Sync Analyzer (Microtrac BEL). Furthermore, the microparticles were observed using a SEM TM4000Plus II Miniscope (HITACHI).

[0063] [Measurement of drug content] 3.0 mL of acetonitrile was added to 10 mg of each of Formulations 2, 3, 4, and 5, and the mixture was dissolved by stirring for 30 seconds using a vortex mixer, followed by ultrasonic treatment for 10 minutes to prepare a sample solution. The sample solution was analyzed by HPLC in the same manner as in Example 1 above, and the erythromycin content in the sample solution was determined.

[0064] SEM images of the microparticles of Formulations 2 to 5 are shown in Figure 1. The physical properties of the microparticles and the erythromycin content are shown in Table 3. Erythromycin was able to be encapsulated at a high encapsulation rate in all samples, yielding spherical microparticles that could be easily injected.

[0065]

[0066] Example 3 [Preparation of Ointment Formulations] Preparation 5 prepared in Example 2 above, macrogol ointment, glycerin, and hydroxypropyl methylcellulose (HPMC) in the amounts shown in Table 4 were kneaded using an ointment spatula and an ointment plate to obtain Ointment Formulations A and B. The erythromycin contents in Ointment Formulations A and B were 2.1% and 5.3%, respectively.

[0067]

[0068] [Measurement of Drug Content] 1.0 mL of water was added to 10 mg of Ointment Formulation A or Ointment Formulation B, and the mixture was stirred for 30 seconds using a vortex mixer. The mixture was then centrifuged at room temperature (room temperature, 10,000 rpm, 5 minutes) to separate the supernatant and precipitate. 1.0 mL of acetonitrile was added to the precipitate, which was dissolved by stirring using a vortex mixer for 30 seconds and then ultrasonicated for 10 minutes to obtain a sample solution. The sample solution was analyzed by HPLC in the same manner as in Example 1 above, and the erythromycin content in the sample solution was determined.

[0069] The erythromycin contents of Ointment Preparation A and Ointment Preparation B are shown in Table 5. It was confirmed that the erythromycin contents of Ointment Preparation A and Ointment Preparation B were equivalent to the charged content.

[0070]

[0071] Test Example 1: Evaluation of in vitro release properties The resulting formulations 2 to 5 were added to 1 / 15 mol / L phosphate buffer (pH 7.0) containing 0.2% SDS to a concentration of 0.3 mg / mL, and dispersed by ultrasonic treatment. While stirring with a stirrer, 1 mL aliquots were dispensed into microtubes (manufactured by Bioramo) and stored at 37°C. Formulations 2 and 3 were sampled at 0 hours, 1 day, 2 days, 3 days, 4 days, and 7 days, and formulations 4 and 5 were sampled at 0 hours, 1 day, 2 days, 6 days, 7 days, and 14 days, and these were used as samples for measuring the amount of erythromycin remaining in the particles.

[0072] The amount of erythromycin remaining in the microparticles of the measurement sample was measured by the following method. The measurement sample was centrifuged (20°C, 12,000 rpm, 5 minutes) to separate the supernatant and precipitate. 1.0 mL of acetonitrile was added to the precipitate after centrifugation, and the mixture was stirred in a vortex mixer for 30 seconds to dissolve the precipitate, followed by ultrasonic treatment for 10 minutes to obtain a sample solution. The sample solution was analyzed by HPLC in the same manner as in Example 1 above, and the erythromycin content in the sample solution was determined.

[0073] The change in the percentage of erythromycin remaining in the formulation over time is shown in Figure 2. The microparticles of Formulation 5 released erythromycin gradually over a period of one week, with an initial burst of less than 10% released over one day.

[0074] Test Example 2: Evaluation of in vivo release properties. Formulations 2 and 5 were each suspended in a dispersion medium (containing 5% mannitol, 0.5% carboxymethylcellulose sodium salt (CMC-Na), and 0.1% polysorbate 80) and injected subcutaneously into the occipital region of 6-week-old male SD rats (Charles River Laboratories) under isoflurane inhalation anesthesia. The dose was 10 mg erythromycin / 0.2 mL / rat. After subcutaneous administration, the punctured skin hole was immediately sealed with Araldite. After administration, the administration site was incised 2, 3, and 4 days after administration for Formulation 2, and 1, 3, 1 week, and 2 weeks after administration for Formulation 5. The microparticles were collected along with the collagen coating, and used as samples for measuring the amount of erythromycin remaining inside. Microparticles were collected under isoflurane inhalation anesthesia after the rats were bled to death.

[0075] The amount of erythromycin remaining in the microparticles was measured by the following method. The measurement sample was placed in a 50 mL screw-capped glass centrifuge tube, 15 mL of PBS (pH = 7.4) was added, and the mixture was homogenized twice (10,000 rpm, 2 minutes) using a Hiscotron NS-56S (Microtec Nichion Co., Ltd.). To extract erythromycin, CH ２ Cl ２ After adding 5.0 mL of the solution, the mixture was stirred for 30 seconds using a vortex mixer, ultrasonicated for 10 minutes, centrifuged (room temperature, 1,100 G, 10 minutes), and then centrifuged.２ Cl ２ The extracted CH was collected (a total of two extraction operations). ２ Cl ２ 1.0 mL of acetonitrile was added to the solution, and the mixture was stirred for 30 seconds using a vortex mixer to prepare a measurement sample. The sample solution was analyzed by HPLC in the same manner as in Example 1 above, and the erythromycin content in the sample solution was determined.

[0076] The change in the residual rate of erythromycin over time is shown in Figure 3. Formulations 2 and 5 showed release over 3 days and 1 week that corresponded to the physical properties of the polymer used. These results were also similar to the in vitro release profile. In particular, Formulation 5 was able to suppress the initial burst of erythromycin release over 1 day immediately after administration to approximately 20%, demonstrating the desired continuous, satisfactory sustained release over 1 week.

[0077] Test Example 3: Bone Resorption Inhibitory Effect Experimental periodontitis model mice, in which the teeth are ligated with silk thread, exhibit inflammation and bone resorption approximately 10 days after ligation, making them a useful model for studying inflammatory bone destruction. The experimental periodontitis model was created using 8-week-old wild-type mice (N=8, male C57BL / 6N mice, CLEA Japan Inc.), and erythromycin or Formulations 1, 2, 3, or 5 (5 μL, 1 mg / mL) were inoculated directly into the gums. The erythromycin doses for Formulations 1, 2, 3, and 5 were 0.50 μg, 0.80 μg, 0.93 μg, and 0.61 μg, respectively. After 9 days, bone levels were measured.

[0078] Figure 4 shows the appearance of the teeth and alveolar bone. Figure 5 shows the bone level. Formulations 1, 2, 3, and 5 all showed bone resorption inhibitory effects. In particular, formulation 1 showed a significant bone resorption inhibitory effect.

[0079] Osteoblasts were isolated from the skulls of 3-day-old wild-type mice (N=6, male C57BL / 6N mice, CLEA Japan) by treating them with 0.1% type 1 collagenase and 0.2% dispase at 37°C for 20 minutes, and then cultured in α-MEM and bone differentiation medium (containing 50 μg / mL ascorbic acid and 10 mM β-glycerol 2-phosphate). Wild-type mouse osteoblasts (1 × 10 ４Ethanol (final concentration 0.02%), erythromycin (10 μg / mL), and formulations 1, 2, 3, and 5 (10 μg / mL, 1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL, respectively, were added to the medium during culture of 1000 cells / mL. The medium was replaced with fresh medium of the same composition every 7 days, and after 21 days, the cells were stained with Alizarin Red S and bone nodules were measured. The concentrations (μg / mL) of administered erythromycin were shown in Table 6.

[0080]

[0081] As shown in Figure 6, bone nodule formation was observed in the erythromycin-treated (ERM) group of wild-type mouse osteoblasts and in the groups treated with Formulations 1, 2, 3, and 5. The percentage of bone nodule area relative to the observed area in the osteoblasts was calculated from microscopic images using image processing software (NIH Image). As shown in Figure 7, a significant increase in the percentage of bone nodule area was observed in Formulation 5, which exhibited a 6-day sustained release, at a drug concentration of approximately 1 / 10 or less.

[0082] Osteoclast precursor cells were isolated from the femoral bone marrow of 8-week-old wild-type mice (N=6, male C57BL / 6N mice, CLEA Japan) and cultured in α-MEM and macrophage colony-stimulating factor (M-CSF 30 μg / mL). ４ To the medium in which 1000 osteoclasts (1000 cells / mL) were cultured, ethanol (final concentration: 0.02%), erythromycin (10 μg / mL), and formulations 1, 2, 3, and 5 (10 μg / mL, 1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL, respectively) were added along with 60 μg / mL recombinant RANKL (manufactured by R&D). After 9 days, the cells were stained with a TRAP staining kit (manufactured by Sigma), and the number of osteoclasts was counted.

[0083] As shown in Figure 8, inhibition of osteoclast formation (TRAP-positive cells) was observed in the groups treated with formulations 1, 2, 3, and 5. Erythromycin did not have a strong osteoclast-inhibitory effect. As shown in Figure 9, formulations 1, 2, 3, and 5 exhibited an osteoclast-inhibitory effect.

[0084] The experimental periodontitis model was prepared as described above, and erythromycin or formulations 1, 2, 3, or 5 (5 μL, 1 mg / mL) was inoculated directly into the gums. The erythromycin doses for formulations 1, 2, 3, and 5 were 0.50 μg, 0.80 μg, 0.93 μg, and 0.61 μg, respectively. After 9 days, the jawbones of the mice were excised, fixed, and sliced. Fluorescent immunostaining was performed using an anti-DEL-1 monoclonal antibody (Abcam) fluorescently labeled with Alexa Fluor™ 488 (Thermo Fisher Scientific).

[0085] An image (magnification: 200x) of the slice preparation observed under a fluorescence microscope (Nikon, ECLIPSE Ni-E) is shown in Figure 10. As shown in Figure 11, it was revealed that the expression level of DEL-1 was increased by the administration of erythromycin and Preparations 1, 2, 3, and 5. In particular, Preparations 3 and 5 showed strong DEL-1 induction ability.

[0086] The experimental periodontitis model was prepared as described above, and erythromycin or formulations 1, 2, 3, or 5 (5 μL, 1 mg / mL) was inoculated directly into the gums. The erythromycin doses for formulations 1, 2, 3, and 5 were 0.50 μg, 0.80 μg, 0.93 μg, and 0.61 μg, respectively. After 9 days, the mouse jawbones were excised, fixed, and sliced. TRAP-positive osteoclasts in the mouse alveolar bones were then stained using a TRAP staining kit (Sigma). The percentage of the TRAP-positive area relative to the observed area was calculated from the microscopic images using image processing software (NIH Image).

[0087] The stained slice specimens are shown in Figure 12. As shown in Figure 13, TRAP staining revealed that osteoclasts (TRAP-positive cells) in the mouse alveolar bone were suppressed by the administration of erythromycin and Preparations 1, 2, 3, or 5.

[0088] The same slice specimen stained for ALP is shown in Figure 14. Cells with osteogenic activity show an ALP-positive reaction, and when the ratio of the ALP-positive area to the observed area was calculated from microscopic images using image processing software (NIH Image), a significant increase in the ALP-positive area was observed in the groups administered Formulation 1, 2, 3, or 5, as shown in Figure 15. This suggests that Formulations 1, 2, 3, and 5 contribute to bone regeneration.

[0089] Gingival samples from mice treated with Formulation 5, which has particularly significant bone resorption inhibitory and bone regenerative effects, and from gingival samples from the gingivitis group were analyzed. RNA was extracted using an RNeasy kit (Qiagen) and comprehensive gene expression analysis (mRNA-seq) was performed using DNBSEQ-T7 (MGI). Results showed that, among genes with a log2-fold change of more than 1 in the Formulation 5 group compared to the gingivitis group, 56 genes showed increased expression and 355 genes showed decreased expression, which were statistically significant (p<0.05). The relative expression levels of the Oscar and Ocstamp genes, which are osteoclast activators, are shown in Figures 16 and 17, respectively. Administration of Formulation 5 clearly suppressed the expression levels of the Oscar and Ocstamp genes to approximately 1 / 10, demonstrating the potent osteoclast inhibitory effect of Formulation 5 at the gene level.

[0090] The periodontal ligament, a tissue that supports the teeth, is essential for alveolar bone regeneration, and mesenchymal stem cells contained in the periodontal ligament are important for bone regeneration. Human periodontal ligament cell line (Lonza) (1 × 10 ４ Cells / mL) were cultured in a human mesenchymal stem cell medium kit and osteogenic differentiation medium (containing 50 μg / mL ascorbic acid and 10 mM β-glycerol diphosphate), to which ethanol (final concentration 0.02%), erythromycin (1 μg / mL), and formulations 1, 2, 3, and 5 (1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL, respectively) were added. The medium was replaced with fresh medium of the same composition every 7 days, and after 21 days, the cells were stained with ALP (Sigma) to evaluate ALP activity, an indicator of bone regeneration, and the percentage of ALP-positive cells was calculated.

[0091] The cells after ALP staining are shown in Figure 18. As shown in Figure 19, an increase in ALP-positive cells was observed in the groups administered with Formulation 1, 2, 3, or 5. In particular, a significant increase in ALP-positive cells was observed with Formulation 5, which exhibits a one-week sustained release of erythromycin, suggesting that the sustained release of erythromycin contributes more strongly to the regeneration of alveolar bone.

[0092] Test Example 4: Prevention and Treatment of BRONJ Development The experimental mouse BRONJ model mimics human BRONJ by administering a bisphosphonate used in the treatment of human osteoporosis to mice, ligating the teeth with silk thread to create a severe periodontitis model, and then extracting the teeth. The dose of zoledronic acid as a bisphosphonate was adjusted according to the body weight of the mice.

[0093] Zoledronic acid was intraperitoneally administered twice weekly (20 μL, 250 μg / kg) to 7-week-old wild-type mice (N=8, female C57BL / 6N mice, CLEA Japan) for 8 weeks. Two weeks after zoledronic acid administration, the experimental periodontitis model was prepared, and four weeks later, teeth were extracted. Physiological saline (5 μL), Formulation 5 (5 μL, 1 mg / mL), erythromycin (5 μL, 1 mg / mL), or a blank formulation (5 μL, 1 mg / mL) prepared identically to Formulation 5 except that it did not contain erythromycin were directly inoculated into the gingiva after tooth extraction. The dose of erythromycin in Formulation 5 was 0.61 μg. After a two-week healing period, BRONJ was evaluated.

[0094] The appearance of the gingiva and the surface of the alveolar bone after tooth extraction are shown in Figure 20. The site of tooth extraction on the gingiva is indicated by a > symbol. Rapid healing of the gingiva after tooth extraction was observed in the group administered with Formulation 5. Only in the group administered with Formulation 5 was the alveolar bone flattened, and it was confirmed that the tooth extraction socket had closed. Figure 21 shows an image of the alveolar bone after tooth extraction analyzed by μCT. Closure of the tooth extraction socket was observed only in the group administered with Formulation 5.

[0095] Figure 22 shows an image of an HE-stained slice specimen of alveolar bone after tooth extraction. Figures 23 and 24 show the measured values ​​of trabecular thickness and trabecular spacing, respectively. In the group administered with Formulation 5, normal bone regeneration was observed, with large trabecular thickness and narrow trabecular spacing.

[0096] The observed image of slice specimens from alveolar bone stained with Masson's trichrome is shown in Figure 25. A strong production of bone matrix collagen was observed in the group administered with Preparation 5.

[0097] An image of a 10 μm frozen section of alveolar bone subjected to fluorescent immunostaining with an anti-DEL-1 monoclonal antibody is shown in Figure 26. Based on the DEL-1 expression intensity shown in Figure 27, strong DEL-1 expression was observed in the group administered with Formulation 5.

[0098] These results demonstrate that Formulation 5 suppresses inflammation and promotes normal healing by inducing DEL-1 in the tooth extraction socket. Treatment with Formulation 5 may be a potential treatment for patients taking bisphosphonates who are at risk of osteonecrosis of the jaw following tooth extraction. Furthermore, Formulation 5 makes it possible to avoid unnecessary tooth extractions before bisphosphonate administration.

[0099] Test Example 5: Treatment of Stomatitis Filter paper soaked in 50% acetic acid was placed in contact with the left side of the tongue of 7-week-old wild-type mice (N=8, female C57BL / 6N mice, CLEA Japan, Inc.) for 60 seconds. 24 hours later, a yellow stomatitis was induced on the tongue surface, creating an experimental mouse stomatitis model. Physiological saline (5 μL), Formulation 5 (5 μL, 1 mg / mL), erythromycin (5 μL, 1 mg / mL), or a blank formulation (5 μL, 1 mg / mL) prepared similarly to Formulation 5 but without erythromycin was injected directly into the stomatitis site. Evaluation of stomatitis was performed 3 days after injection.

[0100] The appearance of the tongue of a stomatitis model mouse is shown in Figure 28. A favorable healing effect on stomatitis and mucosal coverage were observed only in the group administered with Formulation 5. HE-stained slice specimens of tongue tissue are shown in Figure 29. Coverage of the ulcerated area by epithelium was observed on the left side of the tongue in the group administered with Formulation 5. When the number of neutrophils per unit area in the tongue tissue was quantified, a decrease in the number of neutrophils was observed in the group administered with Formulation 5, as shown in Figure 30.

[0101] The immunohistochemical staining image of a section (10 μm) of tongue tissue fluorescently immunostained with anti-DEL-1 monoclonal antibody is shown in Figure 31. Strong DEL-1 expression was observed only in the group administered with Formulation 5.

[0102] Proteins were extracted from the collected tongues with stomatitis, and the expression level of DEL-1 was analyzed by Western blotting. The results are shown in Figure 32. As with the immunohistochemical staining image shown in Figure 31, strong DEL-1 expression was observed in the group administered with Formulation 5.

[0103] Masson's trichrome stained slice specimens of tongue tissue and the amount of collagen are shown in Figure 33 and Figure 34. Strong collagen production was observed in the group administered with formulation 5.

[0104] These results demonstrate that Formulation 5 inhibits excessive neutrophil migration and suppresses inflammation by strongly inducing DEL-1 at the site of stomatitis. Furthermore, Formulation 5 contributes to the healing of stomatitis by promoting collagen production.

[0105] The above-described embodiments are intended to explain the present invention and are not intended to limit the scope of the present invention. That is, the scope of the present invention is defined by the claims, not by the embodiments. Various modifications made within the scope of the claims and within the scope of the meaning of the invention equivalent thereto are considered to be within the scope of the present invention.

[0106] This application is based on Japanese Patent Application No. 2024-89728, filed on June 3, 2024. The entire specification, claims, and drawings of Japanese Patent Application No. 2024-89728 are incorporated herein by reference.

[0107] The present invention is useful for retarding or preventing disease in the oral cavity.

Claims

1. A preparation for delaying or preventing gingivitis and bone resorption associated with periodontal disease, a preparation for delaying or treating bone resorption and bone destruction associated with bisphosphonate-associated osteonecrosis of the jaw, or a preparation for delaying or treating stomatitis, comprising nano- or micro-sized particles of a biodegradable polymer containing a macrolide antibacterial agent.

2. The formulation according to claim 1, wherein the macrolide antibacterial drug is clarithromycin, azithromycin, or erythromycin.

3. The formulation according to claim 1 or 2, wherein the biodegradable polymer is polylactic acid, polylactic acid-glycolic acid, or a mixture thereof.

4. The formulation according to claim 1 or 2, wherein the content of the macrolide antibacterial agent in the particles is 5 to 30% by weight.

5. The preparation according to claim 1 or 2, wherein the 50% particle size of the particles is 0.1 to 100 μm.

6. The formulation according to claim 1 or 2, further comprising an excipient.

7. The formulation according to claim 6, wherein the excipient is a sugar or an amino acid.

8. The preparation according to claim 1 or 2, which is dispersed in a dispersion medium, a gel base or an ointment base and administered into the gums or periodontal pockets.

9. The formulation according to claim 1 or 2, which is injected into the gums or placed on the bone surface and releases the macrolide antibacterial agent for 1 day to 4 weeks.

10. A kit preparation comprising a dispersion medium, a gel base or an ointment base, and the preparation according to claim 1 or 2, wherein the preparation is dispersed in the dispersion medium, the gel base or the ointment base and administered into the gums, tissues after tooth extraction, areas affected by stomatitis or periodontal pockets.

Images