JP7186976B2 - Periodontal disease drug - Google Patents

Description

The present invention contains 5-aminolevulinic acid (hereinafter sometimes referred to as "ALA") or a derivative thereof or a salt thereof, and ALA-photodynamic therapy (hereinafter referred to as "ALA") is irradiated with light having a wavelength of 360 nm to 700 nm. -PDT".) It relates to a therapeutic agent for periodontal disease used.

In recent years, photodynamic therapy (hereinafter referred to as PDT) has been developed, which is a method of treating a target site by administering a compound that reacts to light and irradiating it with light. PDT is attracting attention as a new cancer treatment method considering quality of life (QOL) because the treatment is simple, the bioinvasiveness is low, and organs can be preserved. .

ALA, which is one of the drugs used in PDT, is known as an intermediate in the pigment biosynthetic pathway that exists widely in animals, plants, and fungi, and is usually biosynthesized from succinyl-CoA and glycine by ALA synthetase. . ALA itself has no photosensitivity, but it is metabolically activated to protoporphyrin IX (hereinafter also referred to as "PpIX") by a series of enzymes in the heme biosynthetic pathway in cells, and is directly directed to tumor tissues and new blood vessels. It is known that cancer cells are degenerated and necrotic by reactive oxygen species typified by singlet oxygen and/or hydroxyl radicals, superoxide, etc. generated by the excitation of light when PpIX is accumulated and laser light is irradiated to the site where PpIX is accumulated. It is

In 1986, Professor Kennedy of Queen's University in Canada reported that skin cancer can be treated by applying ALA and irradiating it with light (see, for example, Non-Patent Document 1). For example, when ALA is administered into the body, PpIX, which is induced by ALA, accumulates in the cancer and emits fluorescence when exposed to light. A tumor diagnostic agent and the like have been proposed (see, for example, Patent Documents 1 and 2).

On the other hand, in the field of periodontal disease treatment, as a broadly defined phototherapy, treatment using various light energy including high-power laser has been performed for about 20 years. Antimicrobial photodynamic therapy (a-PDT), which uses a photochemical reaction in combination with a dye, is attracting attention as a new means.

Enterococcus faecalis (E. faecalis), which is one of the causative bacteria of periodontal disease, was cultured in a sterilized single-rooted tooth using a culture solution AC Broth (manufactured by Aldrich) for 4 weeks, and then added with a 100 μmol methylene blue solution. After washing, it was irradiated with light of 660 nm. It is known that Faecalis does not die (see Patent Document 3).

In addition, a semiconductor laser with a higher energy wavelength of 405 nm is used to treat periodontal disease-causing bacteria such as Porphynomonas gingivalis (P. gingivalis), Prevotella intermedia (P. intermedia), E. When P. faecalis was irradiated, P. faecalis increased in proportion to the irradiation time and the irradiation light intensity. gingivalis and P. Intermedia reduced the number of bacteria, but E. It is known that the number of bacteria of Faecalis did not decrease (Non-Patent Document 2).

Japanese Patent No. 2731032 JP 2006-124372 A Japanese Patent Publication No. 2017-534412

J.C Kennedy, R.H Pottier and DC Pross, Photodynamic therapy with endogeneous protoprophyrin IX: basic principles and present clinical experience, J. Photochem., Photobiol. B: Biol., 6 (1990) 143-148 International Journal of Photoenergy, Volume 2014, Article ID 387215, 7 pages

Although PDT is a safe and simple treatment method that is non-invasive, causes almost no side effects and causes little physical pain to the patient, among the periodontal disease-causing bacteria, there are bacteria that do not show efficacy with conventional methods. I was faced with the problem of
An object of the present invention is to provide a therapeutic drug, a treatment method, etc. that enable PDT against periodontal disease-causing bacteria for which conventional PDT is ineffective.

The inventors have found that E. We presume that the reason why E. faecalis does not die even when exposed to light is that PpIX cannot be biosynthesized in the cells. Faecalis was cultured and exposed to light, but the bacteria did not die. Therefore, as a result of examining various culture conditions, it was found that E. coli was cultured under anaerobic conditions in the presence of ALA and then irradiated with light. The inventors have found that it is possible to kill Faecalis, and have completed the present invention.

（式中、Ｒ^１は、水素原子又はアシル基を表し、Ｒ^２は、水素原子、アルキル基、シクロアルキル基、アリール基又はアラルキル基を表す。）で表される化合物又は薬学的に許容されるそれらの塩（以下「ＡＬＡ類」ということがある。）を含み、歯周病患部に式（I）で表される化合物又は薬学的に許容されるそれらの塩を投与し、その後３６０ｎｍ～７００ｎｍの波長の光を照射する５－アミノレブリン酸－光線力学的療法のための歯周病の治療薬。
（２）歯周病における歯周病原因菌が、Ｅ．フェカリスである（１）に記載の治療薬。
（３）３６０ｎｍ～７００ｎｍの波長の光が、青色発光ダイオードである（１）に記載の治療薬。
また本発明の他の実施態様は以下のとおりである。
（４）上記式（I）で表される化合物又は薬学的に許容されるそれらの塩を歯周病患部に投与し、その後３６０ｎｍ～７００ｎｍの波長の光を照射する歯周病の治療方法。
（５）歯周病患部に投与し、その後３６０ｎｍ～７００ｎｍの波長の光を照射する歯周病の治療に用いるための上記式（I）で表される化合物又は薬学的に許容されるそれらの塩。
（６）歯周病患部に投与し、その後３６０ｎｍ～７００ｎｍの波長の光を照射する歯周病の治療薬の製造に用いるための上記式（I）で表される化合物又は薬学的に許容されるそれらの塩の利用。That is, the present invention is as follows specified by the following matters.
(1) Formula (I) below

(wherein R ¹ represents a hydrogen atom or an acyl group, and R ² represents a hydrogen atom, an alkyl group, a cycloalkyl group, an aryl group or an aralkyl group) or a pharmaceutically acceptable containing those salts (hereinafter sometimes referred to as "ALAs"), administering the compound represented by formula (I) or a pharmaceutically acceptable salt thereof to the affected area of periodontal disease, and then 360 nm 5-Aminolevulinic acid emitting light with a wavelength of ˜700 nm—periodontal disease treatment for photodynamic therapy.
(2) Periodontal disease-causing bacteria in periodontal disease are E. The therapeutic agent according to (1), which is faecalis.
(3) The therapeutic agent according to (1), wherein the light with a wavelength of 360 nm to 700 nm is a blue light emitting diode.
Other embodiments of the present invention are as follows.
(4) A method for treating periodontal disease comprising administering the compound represented by the above formula (I) or a pharmaceutically acceptable salt thereof to a periodontal disease-affected area and then irradiating light with a wavelength of 360 nm to 700 nm. .
(5) A compound represented by the above formula (I) or a pharmaceutically acceptable compound for use in the treatment of periodontal disease, which is administered to a periodontal disease-affected area and then irradiated with light having a wavelength of 360 nm to 700 nm. salt.
(6) A compound represented by the above formula (I) or a pharmaceutically acceptable compound for use in the production of a therapeutic agent for periodontal disease which is administered to a periodontal disease-affected area and then irradiated with light having a wavelength of 360 nm to 700 nm Utilization of those salts that are made.

Treatment of periodontal disease using the therapeutic agent for periodontal disease of the present invention is non-invasive, causes almost no side effects and physical pain to the patient, is safe and simple, and is not effective by conventional methods. Periodontal disease caused by periodontal disease-causing bacteria can also be treated.

FIG. Faecalis was cultured under anaerobic conditions with the addition of ALA to various concentrations, and then irradiated with light to measure the number of cells in the culture solution obtained by the dilution plate method. show. FIG. Faecalis was cultured under aerobic conditions by adding ALA and ALA + EDTA to various concentrations, and then irradiated with light to measure the number of cells in the culture solution. Show the measurement results.

The therapeutic agent for periodontal disease of the present invention is not particularly limited as long as it is a therapeutic agent for ALA-PDT that contains ALA and is used in ALA-PDT that irradiates light with a wavelength of 360 nm to 700 nm. 5-aminolevulinic acid-photodynamic diagnosis (ALA-PDD) in which PpIX accumulation sites are detected by irradiating excitation light with a wavelength of 360 nm to 420 nm prior to ALA-PDT by irradiating light with a wavelength of 360 nm to 700 nm. can also be performed, but therapeutic agents that do not require such ALA-PDD can be particularly preferably exemplified. Further, the therapeutic system to which the therapeutic agent for periodontal disease of the present invention is applied may be a system equipped with an ALA-PDT device, and may be a system equipped with an ALA administration device or ALA-PDD. .

In the compound represented by formula (I), ^R1 represents a hydrogen atom or an acyl group, and ^R2 represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group or an aralkyl group.

The acyl group for R ¹ includes linear or branched carbon atoms such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, octanoyl, and benzylcarbonyl groups. Examples include alkanoyl groups of 1 to 8 carbon atoms and aroyl groups of 7 to 14 carbon atoms such as benzoyl, 1-naphthoyl and 2-naphthoyl groups. In the present invention, acyl groups include alkoxycarbonyl groups such as methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl and isopropoxycarbonyl groups for convenience.

Alkyl groups for R ² include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl and heptyl groups. , an octyl group, and other linear or branched alkyl groups having 1 to 8 carbon atoms.

Cycloalkyl groups for R ² include saturated or partially unsaturated bonds such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclododecyl, and 1-cyclohexenyl groups. and a cycloalkyl group having 3 to 8 carbon atoms.

Examples of the aryl group for R ² include aryl groups having 6 to 14 carbon atoms such as phenyl group, naphthyl group, anthryl group and phenanthryl group.

As the aralkyl group for R ² , the aryl moiety can be exemplified by the same examples as the above aryl group, and the alkyl moiety can be exemplified by the same examples as the alkyl group above. Specifically, benzyl group, phenethyl group, phenylpropyl group and phenylbutyl group. , a benzhydryl group, a trityl group, a naphthylmethyl group, a naphthylethyl group, and other aralkyl groups having 7 to 20 carbon atoms.

The above R ¹ and R ² may optionally have a substituent within a chemically acceptable range, and examples of such substituents include halogen atoms, alkyl groups, haloalkyl groups, alkoxy groups , a nitro group, an aryl group, and the like.

As compounds of formula (I), they preferably act in vivo as any ALA derivative of ALA capable of forming PpIX, for example ALA prodrugs or intermediates capable of forming ALA in vivo. Included are ALA prodrugs that are converted (eg, enzymatically) to porphyrins without forming ALA as a body, and among these, ALA esters are preferred.

ALA esters such as ALA methyl ester, ALA ethyl ester, ALA n-propyl ester, ALA n-butyl ester, ALA n-pentyl ester, ALA n-hexyl ester, ALA n-octyl ester, and ALA 2-methoxyethyl ester , ALA 2-methyl-n-pentyl ester, ALA 4-methyl-n-pentyl ester, ALA 1-ethyl-n-butyl ester, ALA 3,3-dimethyl-n-butyl ester, ALA benzyl ester, ALA 4- isopropylbenzyl ester, ALA 4-methylbenzyl ester, ALA 2-methylbenzyl ester, ALA 3-methylbenzyl ester, ALA 4-(t-butyl)benzyl ester, ALA 4-(trifluoromethyl)benzyl ester, ALA 4- methoxybenzyl ester, ALA 3,4-(dichloro)benzyl ester, ALA 4-chlorobenzyl ester, ALA 4-fluorobenzyl ester, ALA 2-fluorobenzyl ester, ALA 3-fluorobenzyl ester, ALA 2,3,4, 5,6-pentafluorobenzyl ester, ALA 3-nitrobenzyl ester, ALA 4-nitrobenzyl ester, ALA 2-phenylethyl ester, ALA 4-phenylbutyl ester, ALA 3-pyridyl-methyl ester, ALA 4-phenylbenzyl esters, N-[(1-acetyloxy)ethoxycarbonyl]ALA benzyl ester, N-formyl-ALA methyl ester, N-acetyl-ALA ethyl ester, N-propionyl-ALA methyl ester, N-butyryl-ALA methyl ester, N-formyl-ALA ethyl ester, N-acetyl-ALA ethyl ester, N-propionyl-ALA ethyl ester, N-butyryl-ALA ethyl ester.

The compound of formula (I) can be administered as various salts to increase solubility, depending on the mode of administration. Salts of the compound represented by formula (I) include, for example, pharmacologically acceptable acid addition salts, metal salts, ammonium salts, organic amine addition salts and the like. Acid addition salts include inorganic salts such as hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, formate, acetate, propionate, and toluenesulfonic acid. salt, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, malate, etc. Organic acid addition salts and the like can be exemplified. Examples of metal salts include alkali metal salts such as lithium salt, sodium salt and potassium salt, alkaline earth metal salts such as magnesium and calcium salts, and metal salts such as aluminum and zinc. Examples of ammonium salts include alkylammonium salts such as ammonium salts and tetramethylammonium salts. Examples of organic amine salts include salts such as triethylamine salts, piperidine salts, morpholine salts, and toluidine salts. These salts can also be used as solutions at the time of use.

Preferred examples of ALA derivatives include ALA, various esters such as ALA methyl ester, ALA ethyl ester, ALA propyl ester, ALA butyl ester, and ALA pentyl ester, and hydrochlorides, phosphates, and sulfates thereof. Hydrochloride and ALA phosphate are particularly preferred.

ALA derivatives can be produced by any of the known methods of chemical synthesis, microbial production, and enzymatic production. In addition, the above ALA derivatives may form hydrates or solvates, and either of them can be used alone or two or more of them can be used in appropriate combination.

When the above ALA derivative is prepared as an aqueous solution, care must be taken not to make the aqueous solution alkaline in order to prevent decomposition of the ALA derivative. If it becomes alkaline, decomposition can be prevented by removing oxygen.

In addition to the above-mentioned ALAs, if necessary, the following photosensitizers can be mixed and used, or can be administered simultaneously.
Hematoporphyrin derivatives (HpD); Hematoporphyrins such as Photofrin® (Quadra Logic Technologies, Inc., Vancouver, Canada), Hematoporphyrin IX (HpIX); Photosan III (Seehoff Laboratories, Inc.; Seehof, Wesselbrenerkof, Germany); tetra(m-hydroxyphenyl)chlorin (m-THPC), bacteriochlorin (Scotia Pharmaceuticals, Surrey, UK), mono-L-aspartylchlorin e6 (NPe6) (Japan) Petrochemical Company, California, USA), chlorin e6 (Porphyrin Products, Inc.), benzoporphyrin (Quadralogic Technologies, Vancouver, Canada) (e.g., benzoporphyrin derivative monoacid ring A, BPD-MA), purpurine (PDT). chlorins such as (e.g. tin-ethyl etiopurpurin, SnET2); phthalocyanines (e.g. zinc-(Quadralogic Technologies, Vancouver, Canada); some aluminum - or silicon phthalocyanines, which may be sulfonated, especially sulfonated phthalocyanines such as aluminum phthalocyanine disulfonate (AlPcS _2a ), aluminum phthalocyanine tetrasulfonate (AlPcS ₄ ); porphycenes; hypocrellin; protoporphyrin IX (PpIX); hematoporphyrin di-ester; uroporphyrin; coproporphyrin; deuteroporphyrin; Shire).
These can be used individually by 1 type or in mixture of 2 or more types.

In addition, ALAs can be administered with other active compounds that can increase the photosensitizing effect and thus promote PDT. Such compounds include, for example, chelating agents, more specifically aminopolycarboxylic acids, in the literature on metal detoxification or in the literature on chelation of paramagnetic metal ions in contrast agents used in magnetic resonance imaging. described chelating agents, more specifically, ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA), 1,2-diaminocyclohexane-N,N,N',N '-tetraacetic acid (CDTA), diethylenetriamine-N,N,N',N'',N''-pentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7, 10-tetraacetic acid (DOTA), desferrioxamine or known derivatives/analogs thereof, which can be used singly or in admixture of two or more.
If a chelating agent is present, the chelating agent is typically used at a concentration of 0.05-20% (w/v), for example at a concentration of 0.1-10% (w/v).

In the present invention, administration of ALAs to periodontal disease-affected areas means local administration to periodontal disease-affected areas or their vicinity, and local application is particularly preferred. Local administration into periodontal pockets can be accomplished by using techniques known in the art, such as syringes, catheters, or other suitable drug delivery systems.

Dosage forms of the therapeutic agent of the present invention include, for example, gels, creams, ointments, sprays, lotions, aerosols, liquids for external use, and the like.

In the therapeutic agents of the present invention, the concentration of ALAs included varies depending on a variety of factors, including the chemical nature of the compound, chemical composition, method of administration and degree of disease to be treated, but 20% (w /v), more preferably 0.05-16% (w/v), particularly preferably 0.5-14% (w/v), for example 1.5-12.0% (w/v) or the range of 2 to 10% (w/v) can be preferably exemplified.

In the method for treating periodontal disease of the present invention, when performing PDT for treating periodontal disease by administering a compound that reacts to light to periodontal disease-causing bacteria and irradiating with light, the compound itself is photosensitized. Inactive ALAs were administered to the affected area of periodontal disease, and PpIX induced through the pigment biosynthetic pathway accumulated in the cells of periodontal disease-causing bacteria, and accumulated in the cells of periodontal disease-causing bacteria. By irradiating PpIX with light to excite it, the surrounding oxygen molecules are photoexcited, and as a result, active oxygen species typified by singlet oxygen and/or hydroxyl radicals, superoxide, etc. generated by the excitation of the light are generated. , is a treatment method for periodontal disease utilizing the fact that it has a cell-killing effect due to its strong oxidizing power.

The therapeutic agent of the present invention is administered to the affected area of periodontal disease and its surroundings, especially in the periodontal pocket, and a specific time elapses before the area to be treated is exposed to light to obtain the desired photosensitizing effect. preferably. Excess therapeutic agent is preferably removed prior to exposure.

The time between administration and exposure to light depends on the type of ALA, the condition to be treated and the mode of administration. The time is, for example, about 3 to 6 hours, preferably 0 to 90 minutes, 5 to 90 minutes, 30 to 90 minutes, particularly preferably 10 to 50 minutes.

After the therapeutic agent of the present invention is administered to a periodontal disease-affected area such as a periodontal pocket, the periodontal disease-affected area such as the periodontal pocket must be placed under anaerobic conditions. In the present invention, subjecting the periodontal disease-affected area to anaerobic conditions means subjecting the periodontal disease-affected area to anaerobic conditions when the periodontal disease-affected area is not under anaerobic conditions. , when the periodontal disease-affected area is already under anaerobic conditions, the state is maintained, even when the periodontal disease-affected area is already under anaerobic conditions, the present invention means to revert to or under anaerobic conditions, if lost under anaerobic conditions when the therapeutic agent is administered. In order to place a periodontal disease-affected area such as a periodontal pocket under anaerobic conditions, for example, a method of covering the periodontal disease-affected area with a material capable of blocking oxygen can be considered. In addition, the area affected by periodontal disease is originally a portion near the root of the tooth, and the environment in which periodontal disease-causing bacteria propagate is anaerobic. It is also contemplated to use physical or chemical means to allow therapeutic agent penetration.

After administration of the therapeutic agent of the present invention, photoactivation can be performed using light sources known in the art, such as light emitting diodes such as blue LEDs, red LEDs, semiconductors such as blue semiconductor lasers, red semiconductor lasers, and the like. Examples include lasers, discharge lamps with a strong blue or red emission spectrum, and the like. Blue LEDs and red LEDs are preferable examples because they are advantageous in terms of compactness, cost and portability. can. The wavelength of light used for irradiation can be selected to obtain a more efficient photosensitizing effect and includes light in the range of 360-700 nm. Also, the energy density is preferably in the range of 10 to 200 J/cm ² , more preferably in the range of 10 to 100 J/cm ² , particularly preferably in the range of 20 to 60 J/cm ² .

The power density of ^{the light} source to be used is not particularly limited as long as the effect of the present invention is achieved. ² , a range of 10 to 35 mW/cm ² is more preferred, and a range of 15 to 35 mW/cm ² is particularly preferred.

Irradiation light may be continuous light or pulsed light, but pulsed light is more preferable because the use of pulsed light can reduce damage to the normal skin surface.

The light irradiation time is desirably 5 to 30 minutes, preferably 15 minutes, depending on the energy density and power density. Irradiation may be performed only once, or light irradiation may be used in which the light irradiation amount is divided into several portions, for example, with an irradiation interval of 1 to 10 minutes.

As the excitation light irradiation device, a fine optical fiber for a light source can be mentioned, and as the light source of the excitation light irradiated in the ALA-PDT step that excites the accumulated PpIX, the breeding site of minute periodontal disease-causing bacteria A semiconductor laser or LED light source having a high irradiance and a narrow irradiation area for enabling accurate automatic identification is preferable, and the excitation light is guided from one end to the outside. It is preferable to have an excitation light guide section for emitting light to the excitation light guide section, and a specific example of the excitation light guide section is a thin optical fiber.

As described above, ALA-PDD can also be performed in the therapeutic methods using the therapeutic agents of the present invention. ALA-PDD emits red fluorescence when PpIX accumulated in periodontal disease-causing bacteria cells is irradiated with violet light before the ALA-PDT of the present invention. This is a determination method for specifying an attachment site. Examples of the ALA-PDD device used in the ALA-PDD step include a PpIX excitation light irradiation device, a red fluorescence detection device specific to PpIX in an excited state, or a device in which these are integrated. The light emitted from the excitation light irradiation device for PpIX is preferably light having a wavelength at which PpIX-specific red fluorescence can be observed by exciting PpIX. and the wavelength is at least within the range of 360 nm to 420 nm. Examples thereof include 360 to 420 nm and 403 to 410 nm.

As the light source for irradiating the excitation light in the ALA-PDD step, a known light source can be used. Violet LEDs, particularly flashlight-type violet LEDs and violet semiconductor diodes, can be suitably exemplified, which are advantageous in terms of cost and portability as well as a compact device. In addition, for detecting PpIX accumulation sites and determining the breeding range of periodontal pathogens to be irradiated, red fluorescence, specifically fluorescence with a wavelength of 610 nm to 750 nm, preferably 625 to 638 nm. Examples of the red fluorescence detection device include a naked eye detection device and a CCD camera detection device.

The ALA-PDD device in which the excitation light irradiation device and the red fluorescence detection device are integrated can include a light source and a small-diameter optical fiber for measurement. As a light source, a semiconductor with a high irradiance and a narrow irradiation area to enable accurate automatic identification so that PpIX can be detected even in the microscopic breeding sites of periodontal disease-causing bacteria A laser light source is preferable, and it is preferable to have an excitation light guide section that guides excitation light and emits it from one end to the outside. Specific examples of the excitation light guide section include a thin optical fiber. A semiconductor mixed crystal such as InGaN can be used as an element used for the light source, and violet light can be oscillated by changing the compounding ratio of InGaN. Specifically, a compact laser diode having a diameter of about 5.6 mm can be preferably exemplified. A device the size of a desktop PC can be exemplified, with a port for four laser outputs from a laser diode and a port for spectral measurement coupled with a built-in high-sensitivity spectroscope. In addition, in the light receiving step of receiving the fluorescence emitted by PpIX excited by the excitation light, a small diameter optical fiber for measurement is used, and the small diameter optical fiber for measurement is integrated with the small diameter optical fiber for light source, and the received fluorescence is guided to the detector to determine the PpIX accumulation site.

EXAMPLES The present invention will be specifically described below with reference to examples, but the technical scope of the present invention is not limited by these examples.

E. Faecalis JCM5803 was cultured in brain heart infusion medium (BHI) (Difco Laboratories, Detroit, Mich, USA) at 37° C. for 8 hours and adjusted to 0.8×10 ⁹ CFU/ml. Then, after standing for 6 hours under anaerobic conditions (80% N ₂ , 10% CO ₂ , 10% H ₂ ) at 37° C., it was adjusted to 1.0×10 ⁹ CFU/ml and used as a cell sample. . ALA was dissolved in 1.2 ml of PBS (nacalai tesque, Tokyo), and 0.3 ml of 10N NaOH solution was added to prepare an ALA solution of pH 5.0. The above-prepared E.P. Faecalis cells and the ALA solution prepared above were added, and the final concentrations of ALA were adjusted to 0% (w/v), 0.05% (w/v), 0.5% (w/v), 5% (w/v), v), adjusted to 10% (w/v). After standing in the dark under anaerobic conditions at 37° C., after 2 hours, a blue LED (wavelength 397±13 nm, output 35 mW, irradiation diameter 15.5 mm, average power density 19 mW/cm ² , G-Light Prima-II Plus, GC, Tokyo) was irradiated for 1 minute on the optical stage. Based on the dilution plate method, the culture solution obtained by light irradiation was diluted with BHI to OD=0.1, and each diluted culture solution was cultured on an agar medium for 1 day. The results are shown in FIG. A group in which the final concentration of ALA was adjusted to 0% (w/v) was also provided in which light irradiation by the blue LED was not performed.
In addition, the colonies on each agar medium in FIG. , 5% (w/v), 10% (w/v), 0% (w/v) + colonies formed from non-irradiated culture medium (1 to 6 in FIG. 1).

As is clear from FIG. 1, when the final concentrations of ALA were 5% (w/v) and 10% (w/v), E. The viable count of faecalis was remarkably reduced, suggesting that the therapeutic agent of the present invention can be used to treat periodontal disease without invading the human body.

[Reference example 1]
E. Faecalis JCM5803 was cultured in brain heart infusion medium (BHI) (Difco Laboratories, Detroit, Mich, USA) at 37° C. for 8 hours and adjusted to 0.8×10 ⁹ CFU/ml. ALA was dissolved in 1.2 ml of PBS (nacalai tesque, Tokyo), and 0.3 ml of 10N NaOH solution was added to adjust the ALA solution to pH 5.0. An ALA-EDTA solution was prepared by adding EDTA to 0.05% (w/v) of the ALA solution prepared above. The above-prepared E.P. Faecalis cells and the ALA solution or ALA+EDTA solution prepared above were added to adjust the final concentrations of ALA to 0% (w/v), 0.05% (w/v) and 0.5% (w/v), respectively. . After 2 hours and 4 hours, the blue LED (wavelength 397 ± 13 nm, output 35 mW, irradiation diameter 15.5 mm, average power density 19 mW / cm ² , G-Light Prima -II Plus, GC, Tokyo) was irradiated for 1 minute on the optical stage. Based on the dilution plate method, the culture solution obtained by light irradiation was diluted 1-fold, 10-fold, 100-fold and 1000-fold using BHI, and each diluted culture solution was cultured on an agar medium for 1 day. The results are shown in FIG.
From the left in FIG. 2, the results of 1-fold, 10-fold, 100-fold and 1000-fold dilutions of the culture solution are shown. Colonies formed from culture solutions with ALA concentrations of 0% (w/v), 0.05% (w/v), and 0.5% (w/v) (1 to 3 in FIG. 2) ), the ALA concentration in the culture medium to which the ALA + EDTA solution was added was 0% (w/v), 0.05% (w/v), and 0.5% (w/v). Colonies (4-6 in FIG. 2) are represented.

As is clear from FIG. 2, when cultured under aerobic conditions and irradiated with light, E. Even when ALA was added, the viable cell count of faecalis did not change from that of the blank, and the effect of adding ALA was not observed.

INDUSTRIAL APPLICABILITY The therapeutic agent and therapeutic method of the present invention are effective against periodontal disease-causing bacteria, which conventional methods are ineffective against, and therefore are useful in the field of treating periodontal disease.

Claims (3)

Formula (I) below
[Chemical 1]

(wherein R ¹ represents a hydrogen atom or an acyl group, and R ² represents a hydrogen atom, an alkyl group, a cycloalkyl group, an aryl group or an aralkyl group) or a pharmaceutically acceptable 5-aminolevulinic acid comprising a salt thereof, wherein the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is administered to the area affected by periodontal disease, and then irradiated with light having a wavelength of 397 ± 13 nm - Treatment of periodontal disease for photodynamic therapy (ALA-PDT). The therapeutic agent according to claim 1, wherein the periodontal disease-causing bacterium in periodontal disease is Enterococcus faecalis (E.faecalis). 397±13 2. The therapeutic drug according to claim 1, wherein the light with a wavelength of nm is a blue light emitting diode.

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