WO2016204060A1 - Enhancer of photodynamic effect in ala-pdt or ala-pdd - Google Patents

Abstract

[Problem] To provide a pharmaceutical for enhancing the photodynamic response in ALA-PDT or ALA-PDD. [Solution] Provided is a pharmaceutical for enhancing the photodynamic response in photodynamic therapy or photodynamic diagnosis, the pharmaceutical characterized in containing ALAs and being used in combination with nobiletin or a composition containing nobiletin.

Description

Photodynamic effect enhancer in ALA-PDT or ALA-PDD

The present invention relates to a photodynamic effect enhancer for ALA-PDT or ALA-PDD.

One of the cancer treatment options different from anticancer drug treatment and radiation therapy is photodynamic therapy (PDT). Cancer treatment by PDT is a treatment of cancer by administering a photosensitizer (photosensitizer) having the property of accumulating in cancer cells to the target and irradiating the cancer tissue with light of a specific wavelength. Since it is a treatment method to be performed and is a treatment method that is minimally invasive and hardly leaves a treatment mark, it has attracted attention in recent years.

In recent years, photodynamic diagnosis (hereinafter abbreviated as PDD) has attracted attention as a method for diagnosing diseased tissues such as cancer, warts and acne. PDD is a diagnostic method in which a photosensitizer (photosensitizer) having the property of accumulating in a target tissue is administered to a target, and a target location is identified by irradiating light of a specific wavelength. Compared to diagnostic methods, there is an advantage that the burden on the patient is small because it is less invasive and has fewer side effects.

As described above, PDT and PDD use a compound (photosensitizer) that reacts with light and has a property of accumulating in a tissue to be treated or detected. Various photosensitizers that are suitably used in PDT and PDD have been studied. Recently, 5-aminolevulinic acid compounds (in this specification, “ALA”, which are porphyrin precursors and biological substances). "ALA-PDT" or "ALA-PDD", which is administered to a subject and combined with PDT or PDD, has attracted attention (for example, Patent Document 1).

Although ALAs themselves are not light sensitive, ALAs are metabolically activated in cells by a series of enzymes in the heme biosynthetic pathway to become porphyrins (mainly protoporphyrin IX (PpIX)). Porphyrins have the property of being taken up and accumulated in tumor cells, and PpIX is a photosensitizer having peaks at 410 nm, 510 nm, 545 nm, 580 nm, 630 nm, etc., and is therefore used for PDT or PDD be able to. Since ALA is metabolized in vivo and excreted within 48 hours, it has a feature that it hardly affects the photosensitivity of the whole body.

Japanese Patent Laid-Open No. 11-12197

In ALA-PDT or ALA-PDD, treatment or detection using a photodynamic reaction is possible by administering ALAs to a subject and accumulating PpIX in the tissue to be treated or detected. However, when performing ALA-PDT or ALA-PDD in a clinical setting, the present inventors do not accumulate PpIX sufficiently in the target tissue depending on the constitution or physical condition of the target, and the photodynamic response I realized the existence of the problem that the effect might not be obtained satisfactorily.
In the conventional methods, ALAs are dissolved in physiological saline or the like and orally administered to the subject. However, ALAs have bitterness and the subject is vomited after administration. Was big. Therefore, the present inventors can reduce the bitterness of ALAs by using in combination with ALAs, and further enhance the effects of ALA-PDT or ALA-PDD (that is, the above two problems are simultaneously solved). We have intensively studied foods and beverages that have effects that can be solved.

The present inventors surprisingly found that the combined use of ALAs and citrus extracts enhances the photodynamic response in PDT or PDD compared to the case of using ALAs alone. I found it. Furthermore, when the present inventors screened the component contained in a citrus extract in order to search the factor which causes the above-mentioned effect, the nobiletin contained in a citrus extract enhances the accumulation of PpIX in a tissue, thereby causing PDT or It has been found that the photodynamic response in PDD is enhanced.

（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、直鎖若しくは分岐状アルキル基、シクロアルキル基、アリール基又はアラルキル基を表す）又はその塩、を含み、前記医薬は、ノビレチン又はノビレチンを含む組成物と併用されることを特徴とする。 That is, the present invention is, in one embodiment, a medicament for enhancing a photodynamic response in photodynamic therapy or photodynamic diagnosis, wherein the medicament is a compound represented by the following formula (I):

(Wherein R ¹ represents a hydrogen atom or an acyl group, and R ² represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group) or a salt thereof, The medicine is used in combination with nobiletin or a composition containing nobiletin.

（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、直鎖若しくは分岐状アルキル基、シクロアルキル基、アリール基又はアラルキル基を表す）又はその塩と、（Ｂ）ノビレチン又はノビレチンを含む組成物とを含むことを特徴とする、医薬に関する。 Another embodiment of the present invention is a medicament for enhancing photodynamic response in photodynamic therapy or photodynamic diagnosis, wherein (A) a compound represented by the following formula (I):

(Wherein R ¹ represents a hydrogen atom or an acyl group, R ² represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group or an aralkyl group) or a salt thereof; And a composition containing nobiletin.

（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、直鎖若しくは分岐状アルキル基、シクロアルキル基、アリール基又はアラルキル基を表す）又はその塩と、（Ｂ）ノビレチン又はノビレチンを含む組成物、との組み合わせ医薬に関する。 In addition, another embodiment of the present invention is a combination drug administered simultaneously or simultaneously for enhancing photodynamic response in photodynamic therapy or photodynamic diagnosis, comprising: (A) Compound represented by formula (I):

(Wherein R ¹ represents a hydrogen atom or an acyl group, R ² represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group or an aralkyl group) or a salt thereof; ) Nobiletin or a composition comprising nobiletin, and a combination medicament.

In one embodiment of the present invention, in the above medicament, R ¹ is selected from the group consisting of a hydrogen atom, an alkanoyl group having 1 to 8 carbon atoms, and an aroyl group having 7 to 14 carbon atoms, ² is a hydrogen atom, a linear or branched alkyl group having 1 to 8 carbon atoms, a cycloalkyl group having 3 to 8 carbon atoms, an aryl group having 6 to 14 carbon atoms, and an aralkyl group having 7 to 15 carbon atoms. It is selected from the group consisting of

In one embodiment of the present invention, R ¹ and R ² in the above medicament are hydrogen atoms.

Moreover, in one embodiment of the present invention, in the above medicine, the composition containing nobiletin is a composition containing a citrus extract.

Further, in one embodiment of the present invention, in the above medicament, the citrus extract is a citrus fruit selected from the group consisting of Citrus genus, Kumquat genus, Karatachi genus, Krimenia genus, Eremocitrus genus and Microcitrus genus. It is an extract.

Moreover, in one embodiment of the present invention, in the above combination medicine, (A) and (B) are prepared as a kit.

Note that an invention in which one or more features of the present invention described above are arbitrarily combined is also included in the scope of the present invention.

FIG. 1 shows a chromatogram of a 1 mg / mL citrus extract solution. FIG. 2 shows a graph in which the accumulation of PpIX was measured when citrus extract and ALA were administered to cancer cells (n = 3). FIG. 3 shows a graph in which the accumulation of PpIX was measured when nobiletin and ALA were administered to cancer cells (n = 3). FIG. 4 shows a graph in which the effect of PDT (cell survival rate) is measured when nobiletin and ALA are administered to cancer cells (n = 3). FIG. 5 shows the experimental method for evaluating the ALA-PDT enhancing effect of nobiletin in tumor-transplanted chicken eggs. FIG. 6 shows the results of an experiment for evaluating the ALA-PDT enhancing effect of nobiletin in tumor-transplanted chicken eggs.

In this specification, ALA refers to ALA or a derivative thereof or a salt thereof.

In this specification, ALA means 5-aminolevulinic acid. ALA is also called δ-aminolevulinic acid and is one of amino acids.

Examples of ALA derivatives include compounds represented by the following formula (I). In the formula (I), R ¹ represents a hydrogen atom or an acyl group, and R ² represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group. In formula (I), ALA corresponds to the case where R ¹ and R ² are hydrogen atoms.

ALAs may act as active ingredients in the state of ALA of the formula (I) or derivatives thereof in vivo, and can also be administered as prodrugs (precursors) that are degraded by enzymes in the body.

Examples of the acyl group in R ¹ of the formula (I) include linear or branched C 1-8 carbon atoms such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, octanoyl, benzylcarbonyl group and the like. Examples thereof include alkanoyl groups and aroyl groups having 7 to 14 carbon atoms such as benzoyl, 1-naphthoyl and 2-naphthoyl groups.

Examples of the alkyl group in R ² of the formula (I) include a straight chain such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl group, etc. Examples include branched alkyl groups having 1 to 8 carbon atoms.

As the cycloalkyl group in R ² of formula (I), there is a saturated or partially unsaturated bond such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclododecyl, 1-cyclohexenyl group and the like. Examples thereof may include a cycloalkyl group having 3 to 8 carbon atoms.

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

As the aralkyl group in R ² of formula (I), the aryl moiety can be exemplified as the above aryl group, and the alkyl moiety can be exemplified as the above alkyl group. Specifically, benzyl, phenethyl, phenylpropyl, phenyl Examples thereof include aralkyl groups having 7 to 15 carbon atoms such as butyl, benzhydryl, trityl, naphthylmethyl, and naphthylethyl groups.

Preferable ALA derivatives include compounds in which R ¹ is formyl, acetyl, propionyl, butyryl group or the like. Further, preferable ALA derivatives include compounds in which R ² is a methyl, ethyl, propyl, butyl, pentyl group or the like. As preferred ALA derivatives, the combination of R ¹ and R ² is (formyl and methyl), (acetyl and methyl), (propionyl and methyl), (butyryl and methyl), (formyl and ethyl), (acetyl) And ethyl), (propionyl and ethyl), and (butyryl and ethyl).

Among the ALAs, examples of the salt of ALA or a derivative thereof include pharmacologically acceptable acid addition salts, metal salts, ammonium salts, and organic amine addition salts. Examples of acid addition salts include hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, and other inorganic acid salts, formate, acetate, propionate, toluenesulfonic acid Salt, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, malate, etc. Organic acid addition salts 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 salt, and metal salts such as aluminum and zinc. Examples of ammonium salts include ammonium salts and alkylammonium salts such as tetramethylammonium salts. Examples of the organic amine salt include salts such as triethylamine salt, piperidine salt, morpholine salt, and toluidine salt. These salts can also be used as a solution at the time of use.

Of the above ALAs, the most desirable ones are ALA and various esters such as ALA methyl ester, ALA ethyl ester, ALA propyl ester, ALA butyl ester, ALA pentyl ester, and their hydrochlorides and phosphoric acids. Salt, sulfate. In particular, ALA hydrochloride and ALA phosphate can be exemplified as particularly suitable.

The ALAs can be produced by known methods such as chemical synthesis, production by microorganisms, production by enzymes, and the like. The ALAs may form hydrates or solvates, and ALAs may be used alone or in combination of two or more.

When preparing the ALAs as an aqueous solution, care must be taken so that the aqueous solution does not become alkaline in order to prevent decomposition of the ALAs. When it becomes alkaline, decomposition can be prevented by removing oxygen.

In this specification, ALA-PDT means photodynamic therapy (PDT) using ALAs, and most typically means PDT using ALA. ALA-PDD means photodynamic diagnosis (PDD) using ALAs, and most typically means PDD using ALA.

The ALA-PDT is a method in which a photosensitizer (photosensitizer) having a property of accumulating in a target tissue is administered to a subject, and when performing PDT for treating a target site by irradiating light of a specific wavelength, ALAs that do not themselves have a photosensitizing effect are administered to the subject. In the subject's body, porphyrins (mainly PpIX) derived from ALAs via the pigment biosynthesis pathway are accumulated at the target site, and the accumulated PpIX is excited to photoexcite surrounding oxygen molecules. The resulting singlet oxygen has a cell killing effect due to its strong oxidizing power. For ALA-PDT, it is preferable to use, for example, a light beam having a peak at 400 nm to 420 nm or a light beam having a peak at 600 nm to 650 nm.

On the other hand, the ALA-PDD itself is administered with a photosensitizer (photosensitizer) having a property of accumulating in a target tissue, and when PDD is performed to treat a target site by irradiating light. Administers ALAs having no photosensitizing action to the subject. In the subject's body, the presence or absence of the target tissue is detected by accumulating porphyrins (mainly PpIX) derived from ALA via the pigment biosynthetic pathway at the target site and exciting the accumulated PpIX to detect fluorescence. Detect or diagnose For ALA-PDD, for example, it is preferable to use light having a peak at 400 nm to 420 nm.

Nobiletin is a flavonoid abundantly contained in citrus peels and is a compound represented by the following formula (II).

The composition containing nobiletin used in the present invention may be a liquid (for example, prepared as a beverage) or a solid (for example, a powder).

The citrus extract used in the present invention may be any citrus extract as long as it contains nobiletin. For example, the citrus genus, kumquat genus, Karatachi genus, krimenia genus, elemositrus genus and micro genus A citrus extract selected from the group consisting of Citrus is preferable.

The most preferred citrus used in the present invention is Citrus citrus, and examples of Citrus citrus include Mexican lime, Tahiti lime, Bergamot, Biroro, and Citron. (Citron), Eureka lemon (Sweet lemon), Rumie, Lumie, Hirado Buntan, Shatenyu, Marsh grapefruit (Marsh grapepek), Marsh grapepek ), Natsudaidai, Sambokan, Sour Orange Sour orange, Valencia orange, Morita navel, Iyo, Hyuganatsu, Shunkokan, Yuzu, Sud king, Sudach (King), Satsuma (Satsuma), Yatsushiro, Keraji, Oto, Ponkan, Dancy tangerine, Dancy tangerine, Clementinei, Jimikan ), Shikaikan, Tachibana, Kobenimika ), Distichum (Kishu), Sankitsu (Sunki), Shikuwasa (Shiikuwasha), Koji (Koji) Shikikitsu (Shikikitsu), and the like.

The present invention relates to a combination of ALAs and nobiletin or a composition containing nobiletin, but the mode of the combination is not particularly limited, and those skilled in the art can use ALAs in various ways depending on the embodiment of PDT or PDD. , Nobiletin or a composition comprising nobiletin can be combined.

In the present invention, ALAs and nobiletin or a composition containing nobiletin may be administered to a subject simultaneously or at different times. In the present invention, ALAs and nobiletin or a composition containing nobiletin may be prepared as a combination drug containing each and administered to a subject. Further, in the present invention, ALAs and nobiletin or a composition containing nobiletin may be prepared as separate kits, and each may be administered to a subject simultaneously or differently at the time of use.

In the present invention, ALAs and nobiletin or a composition containing nobiletin are administered at the same time. For example, ALA is contained in a liquid containing nobiletin (for example, a citrus juice or a liquid in which purified nobiletin is dissolved). The composition may be administered to the subject after dissolving the varieties, and the composition containing nobiletin (eg, citrus extract powder, purified nobiletin) is dissolved in the liquid containing the ALAs and administered to the subject. Well, it may be a mode in which a liquid containing nobiletin and a liquid containing ALAs are mixed and administered to a subject, and in a mode in which a combination comprising ALAs and nobiletin or a composition containing nobiletin is administered to the subject There may be.

In the present invention, the ALA and the composition containing nobiletin or nobiletin are administered at different times. For example, the ALA and the composition containing nobiletin or nobiletin are administered at different times. Alternatively, for example, ALAs and nobiletin or a composition containing nobiletin may be administered from different administration routes.

The administration route of ALAs and the administration route of nobiletin or a composition containing nobiletin in the present invention are not limited, and may be systemic administration or local administration. Examples of the administration route include oral administration including sublingual administration, inhalation administration, direct administration to a target tissue or organ by catheter, intravenous administration including infusion, transdermal administration by a patch, suppository, or trans Examples include parenteral administration such as administration by forced enteral nutrition using a nasogastric tube, naso-intestinal tract, gastrostomy tube, or gastrointestinal fistula tube. Moreover, as described above, ALAs and nobiletin or a composition containing nobiletin may be administered from different routes.

The dosage form of the composition containing ALAs, nobiletin or nobiletin used in the present invention, or a combination of these may be appropriately determined according to the administration route, and is not limited. , Drops, tablets, capsules, fine granules, powders, solutions, syrups and the like, liquids, patches, suppositories and the like.

It goes without saying that the pharmaceutical agent according to the present invention can be added with other optional components such as other medicinal ingredients, nutrients, and carriers as necessary. As an optional component, for example, crystalline cellulose, gelatin, lactose, starch, magnesium stearate, talc, vegetable and animal fats, fats and oils, gums, polyalkylene glycols and the like, pharmaceutically acceptable ordinary carriers and binders Various formulation ingredients such as stabilizers, solvents, dispersion media, extenders, excipients, diluents, pH buffers, disintegrants, solubilizers, solubilizers, isotonic agents, etc. it can.

In the present invention, the dose of ALAs to the subject can be appropriately determined by those skilled in the art according to the embodiment of PDT or PDD. Although not limited, 1 mg to 1,000 mg, preferably 5 mg to 100 mg, more preferably 10 mg to 30 mg, and further preferably 15 mg to 25 mg can be administered per 1 kg of the body weight of the subject in terms of ALA.

Examples of the frequency of administration of ALAs include one to several times daily administration or continuous administration by infusion.

The administration period of ALAs is, for example, a method known by pharmacologists and clinicians in the technical field based on various clinical indicators in view of symptoms or conditions to be prevented or treated. Can be determined.

In the present invention, nobiletin or a composition containing nobiletin is administered for the purpose of enhancing the accumulation of PpIX, which is purified by metabolism of ALAs, in the target tissue, and is therefore administered together with the administration of ALAs. Although it is preferred, it is not necessary to administer completely at the same time.

The dose of nobiletin or a composition containing nobiletin in the present invention can be appropriately determined by those skilled in the art according to the embodiment of PDT or PDD. Although not limited, 0.01 mg to 100 mg, preferably 0.05 mg to 20 mg, more preferably 0.1 mg to 10 mg can be administered per 1 kg body weight of the subject in terms of nobiletin.

The ALA-PDT or ALA-PDD using the present invention can be suitably used for, for example, treatment or diagnosis of tumors, treatment or diagnosis of infectious diseases. When the present invention is used for treating or diagnosing a tumor, the type of tumor is not particularly limited. Examples of PDT subjects include sputum, cervical cancer, skin cancer, thyroid cancer, and malignant brain tumor. Superficial cancers and subcutaneous cancers, especially cancers of several millimeters subcutaneously can be preferably exemplified, and sentinel lymph nodes can be suitably exemplified as PDD targets. In addition, it becomes possible to perform pre-extraction lymph node metastasis diagnosis by PDD. When the present invention is used for treating or diagnosing an infection, for example, the infection may be a bacterial infection, a fungal infection, a viral infection or a parasitic infection, preferably for a bacterial infection More preferably, it can be used for S. aureus infection or Pseudomonas aeruginosa infection.

The terms used in this specification are used to describe specific embodiments except for those specifically defined, and are not intended to limit the invention.

In addition, the term “comprising” as used herein is intended to mean that there is a matter (member, step, element, number, etc.) described, unless the context clearly requires a different understanding. It does not exclude the presence of other items (members, steps, elements, numbers, etc.).

Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms used herein should be interpreted as having a meaning consistent with the meaning in this specification and the related technical field, unless otherwise defined, idealized, or overly formal. It should not be interpreted in a general sense.

In the following, the present invention will be described in more detail with reference to examples. However, the invention can be embodied in various ways and should not be construed as limited to the embodiments set forth herein.

Example 1: Determination of nobiletin content in citrus fruits (citrus extract powder)

The present inventors have intensively studied foods and beverages that can reduce the bitterness of ALAs when used in combination with ALAs, and further have an effect of enhancing the effects of ALA-PDT or ALA-PDD.

In the above examination process, the present inventors administered the solvent for dissolving ALA to the patient instead of the citrus extract (orange juice) from physiological saline, and it became easy to drink, not only reducing the burden on the patient, Surprisingly, it has been found that the sensitivity of ALA-PDD and ALA-PDT is improved compared to when ALA is administered alone (data not shown).

Based on the above findings, the present inventors first screened for substances contained in citrus extracts in order to search for factors that improve the sensitivity of ALA-PDD and ALA-PDT when used in combination with ALA.

In this example, citrus extract powder (“Unshu mandarin orange extract-P”, manufactured by Oriza Oil Co., Ltd.) was used as a citrus component. 1 g of citrus extract powder was weighed, 5 mL of dimethyl sulfoxide (DMSO, manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was completely dissolved by stirring and ultrasonic treatment. Then, the volume up to 10 mL with DMSO was 100 mg / mL citrus extract solution, and further diluted 100 times with methanol (Wako Pure Chemical Industries, HPLC grade) was used as the 1 mg / mL citrus extract solution used in this example. This was injected into the HPLC at 5 μL.

HPLC was Agilent 1290 series manufactured by Agilent, and the detector was 1290 DAD of the same company, and absorption at a wavelength of 280 nm was measured. The mobile phase is A for acetonitrile, B for milliQ water, and the flow rate is 1 mL / min. 0 to 10 minutes is held at 22% for solution A, 10 to 35 minutes for linear gradient, 61% for solution A, 35 to 40 minutes. Was a linear gradient with liquid A being 100%, 40 to 40.1 minutes being linear gradient, liquid A being 22%, and 40.1 to 55 minutes being held with liquid A being 22%. The column was Symmetry C18 (3.9 mm × 150 mm, 5 μm, manufactured by weights), and the column temperature was 25 ° C.

The chromatogram of the 1 mg / mL citrus extract solution is shown in FIG. As shown in FIG. 1, six peaks were obtained from the 1 mg / mL citrus extract solution. Of the six peaks, “peak 4” having a retention time of 24.1 minutes was found to be nobiletin. Moreover, the nobiletin content in a 1 mg / mL citrus extract solution was 3.8 μM (1.52 μg / mL), and it was found that 1.52 mg of nobiletin was contained per 1 g of citrus extract powder. Note that a biochemical reagent manufactured by Wako Pure Chemical Industries, Ltd. was used as a nobiletin standard for quantification. The solvent was methanol, a 2 mM nobiletin standard solution was prepared, and the nobiletin content in the 1 mg / mL citrus extract solution was determined by the standard addition method.

These results suggested that nobiletin contained in the citrus extract may be a factor that improved the sensitivity of cancer diagnosis in ALA-PDD when used in combination with ALA.

Example 2: Enhancement effect of citrus components on PpIX accumulation in cancer cells

Experimental Method As a cancer cell, TMK-1, which is a human gastric cancer cell line, was used. The medium used was RPMI-1640 (Wako) supplemented with 10% inactivated fetal bovine serum (FBS) (hereinafter referred to as RPMI medium). Citrus extract powder (Unshu mandarin orange extract, manufactured by Oriza Oil Chemical Co., Ltd.) was used as a citrus component.

To 1 g of citrus extract powder, 5 mL of dimethyl sulfoxide (DMSO, manufactured by Wako) was added, mixed, and then dissolved by ultrasonic treatment to obtain a 200 mg / mL citrus extract solution. A 200 mg / mL citrus extract solution was serially diluted with an equal amount of DMSO to prepare 7 dilution series. 10 μl of these citrus extract solutions was added to RPMI medium or 1 ml of RPMI medium containing 1 mM aminolevulinic acid (ALA) hydrochloride to prepare a citrus extract-containing medium (citrus extract final concentration 0-1 mg / mL). Fumitremgin C (FTC, manufactured by Funakoshi Co., Ltd., product code BVT-0189-C250) was dissolved in DMSO to prepare a 1 mM FTC solution, diluted with an RPMI medium containing 1 mM ALA hydrochloride, and a 1 mM ALA hydrochloride + 10 μM FTC medium was prepared. This was used as a positive control (PC). In addition, FTC is an inhibitor of ABCG2 (transporter for excretion of PpIX out of cells), and can be used as a positive control in examining the effect of PpIX on the extracellular excretion (enhancement effect of PpIX accumulation).

Cancer cells were seeded on a 96-well plate at a density of 2 × 10 ⁴ cells / well and cultured at 37 ° C. overnight. After removing the culture supernatant from the 96-well plate of cancer cells cultured overnight, 100 μL of the above-mentioned citrus extract-containing medium was added to each well and cultured at 37 ° C. for 4.5 hours. Subsequently, the culture supernatant was removed, and the cells were washed with phosphate buffered saline (PBS, manufactured by Wako), and then solubilized by adding 100 μL of 1% sodium dodecyl sulfate (SDS, manufactured by Wako) aqueous solution per well. Intracellular fractionation. Protoporphyrin IX preparation (PpIX, product code P562-9, manufactured by Funakoshi) was dissolved in DMSO to obtain a 1 mM PpIX stock solution. A 1 mM PpIX stock solution was diluted with a 1% SDS solution to a final concentration of 0-500 nM to obtain an intracellular PpIX standard solution. 100 μL of each of these PpIX standard solutions was added to each unused well of the intracellular fraction plate. Using a microplate reader (manufactured by Tecan Co., Ltd.), the fluorescence intensity of each well of these plates was measured under conditions of an excitation wavelength of 405 nm and a measurement wavelength of 635 nm. After the measurement, a calibration curve was created from the fluorescence intensity of the PpIX standard solution, and the PpIX amount of each well was calculated from the calibration curve. The results are shown in FIG.

Results As shown in FIG. 2, when combined with citrus extract, intracellular PpIX increased depending on the amount added, and ALA hydrochloride + 1 mg / mL citrus extract (equivalent to 3.8 μM nobiletin), as shown in FIG. ) Increased by a maximum of 2 times. From this result, it was shown that the accumulation of intracellular PpIX was enhanced by the combined use of ALA and a citrus component compared to the case of using ALA alone. Moreover, since the citrus extract contains nobiletin, it was suggested that nobiletin in the citrus extract may be involved in the PpIX accumulation enhancing effect.

Example 3: Enhancement effect of nobiletin on PpIX accumulation in cancer cells

Method TMK-1, which is a human gastric cancer cell line, was used as a cancer cell. Nobiletin (manufactured by Wako) was dissolved in DMSO to give a 1 mM nobiletin solution. A 1 mM nobiletin solution was diluted with an equal amount of DMSO and a 2-fold dilution series was prepared to obtain a nobiletin solution. These nobiletin solutions were diluted 100-fold with RPMI medium or RPMI medium containing 2.3 mM ALA hydrochloride to prepare a 0-10 μM nobiletin-containing medium. FTC was dissolved in DMSO to prepare a 1 mM FTC solution and then diluted with an RPMI medium containing 2.3 mM ALA hydrochloride to prepare 2.3 mM ALA hydrochloride + 10 μM FTC medium, which was used as a positive control (PC). .

Cancer cells were seeded on a 96-well plate at a density of 2 × 10 ⁴ cells / well and cultured at 37 ° C. overnight. After removing the culture supernatant from the 96-well plate of cancer cells cultured overnight, 100 μL of the above-mentioned 0-10 μM nobiletin-containing medium and 10 μM FTC-containing medium were added to each well and cultured at 37 ° C. for 4 hours. Subsequently, the culture supernatant was removed, and the cells were washed with PBS and solubilized by adding 100 μL of 1% SDS aqueous solution per well to obtain an intracellular fraction. PpIX standard was dissolved in DMSO to make a 1 mM PpIX stock solution. A 1 mM PpIX stock solution was diluted with 1% SDS to a final concentration of 0-500 nM to obtain an intracellular PpIX standard solution. 100 μL of each of these PpIX standard solutions was added to each unused well of the intracellular fraction plate. Using a microplate reader (manufactured by Tecan Co., Ltd.), the fluorescence intensity of each well of these plates was measured under the conditions of an excitation wavelength of 405 nm and a measurement wavelength of 635 nm. After the measurement, a calibration curve was created from the fluorescence intensity of the PpIX standard solution, and the PpIX amount of each well was calculated from the calibration curve. The results are shown in FIG.

Results As is apparent from FIG. 3, intracellular PpIX increased in a concentration-dependent manner when nobiletin was used in combination with the addition of ALA hydrochloride alone, and increased up to twice as much by adding 10 μM nobiletin. From this result, it was shown that the accumulation of intracellular PpIX was enhanced by the combined use of ALA and nobiletin compared to the case of using ALA alone.

Example 4: Enhancement effect of nobiletin on photodynamic therapy (PDT) of cancer cells

Method TMK-1, which is a human gastric cancer cell line, was used as a cancer cell. The cancer cells were cultivated overnight at 37 ° C. after seeding 5 × 10 ³ cells / well in a 96-well plate. After culturing, 1/6 amount of RPMI medium containing 13.8 mM ALA hydrochloride and RPMI medium containing 60 μM nobiletin was added and cultured for 4 hours in the presence of 2.3 mM ALA hydrochloride and 10 μM nobiletin. After culturing for 4 hours, the culture supernatant was removed, the cells were washed with PBS, 100 μL of RPMI medium was added to each well, and irradiation was performed at a wavelength of 631 nm for 5 minutes using a PDT irradiation apparatus to perform PDT.

The cell killing effect was measured using Cell-Counting Kit-8 (manufactured by Dojindo Laboratories) as follows. RPMI medium and Cell counting kit-8 solution were mixed at a ratio of 10: 1 to prepare a WST-8 reaction solution. The medium was removed from the plate after 24 hours of ALA-PDT treatment, washed with 100 μL of PBS, 110 μL of WST-8 reaction solution was dispensed into each well, and further cultured at 37 ° C. in a CO 2 incubator. Using a microplate reader (manufactured by Tecan), the absorbance at 450 nm of the WST-8 formazan dye was measured over time until the absorbance reached about 1. The cell viability of each treatment group was calculated from the relative value with respect to the absorbance of the untreated drug sample. The results are shown in FIG.

Results As is apparent from FIG. 4, the cell viability of the PDT-treated group was 63% in the 2.3 mM ALA hydrochloride alone treatment group, whereas in the 2.3 mM ALA hydrochloride + 10 μM nobiletin combination treatment group, the cell viability was 18%. % Significantly decreased. On the other hand, the PDT untreated group showed a cell survival rate of 95% or more in any of the treatment groups. From this result, it was shown that the cell killing effect of ALA-PDT was enhanced by the combined use of ALA and nobiletin compared with the case of using ALA alone.

Example 5: ALA-PDT enhancing effect of nobiletin on tumor transplanted chicken egg model

Experimental Method EMT-6, a mouse mammary tumor, was used as a cancer cell. The culture medium used was Wayout's MB 752/1 Medium, Liquid (manufactured by Life Technologies) with 10% inactivated fetal bovine serum (FBS) added (hereinafter referred to as Wayout's medium). As nobiletin, nobiletin powder (Funakoshi Co., Ltd.) was used.

FIG. 5 shows an outline of the test schedule using the tumor transplanted chicken egg model and the conditions of the treatment group.
After sterilizing eggshells of white sperm eggs (Goto chicken egg farm) with 70% ethanol cotton, the eggs were placed vertically in a 37 ° C. incubator (the air chamber part was up) and incubated to Day 11. During the culture, the eggs were turned in a timely manner.

A light was applied from the air chamber of Day 11's growing egg, and a thick blood vessel existing in the chorioallantoic membrane under the eggshell and a frame of about 2 × 2 cm around it were marked. After the eggshell was disinfected with 70% ethanol cotton, a deep cut was made at one position of the frame with a grinder, and then a shallow cut was made with the grinder along the frame. After removing the eggshell from the deeply cut site with tweezers, the chorioallantoic membrane and eggshell membrane were peeled off with tweezers to create an artificial air chamber. After attaching Opsite (made by Smith & Nephew) so as to cover the air chamber part, only the Opsite in the opening was removed with scissors.

After placing the Teflon ring at the branching site of the blood vessel, 20 μL of EMT-6 cell suspension (1.25 × 10 ⁷ Cells / mL) was added into the Teflon ring, and 2.5 × 10 ⁵ Cells / egg. The cells were transplanted into chicken eggs. After the opening was covered with Tegaderm (manufactured by 3M), the eggs were returned to the incubator and cultured for 2 days. Two days after transplantation of EMT-6 cell suspension, the Teflon ring was removed and returned to the incubator again.

1 mL of dimethyl sulfoxide (DMSO, manufactured by Wako) was added to 81.21 mg of nobiletin powder, and after mixing, dissolved by heating at 37 ° C. to obtain an 81 mg / mL nobiletin solution. 850 μL of physiological saline and 150 μL of 8.4% sodium bicarbonate were added to 10 mg of ALA hydrochloride to obtain a 10 mg / mL ALA hydrochloride solution. 10 μL of an 81 mg / mL nobiletin solution and 1000 μL of a 10 mg / mL ALA hydrochloride solution were mixed to obtain a 10 mg / mL ALA hydrochloride + 810 μg / mL nobiletin solution. 10 mL of DMSO was added to 1 mL of physiological saline to obtain a Control solution. A Control solution, a 10 mg / mL ALA hydrochloride solution, a 10 mg / mL ALA hydrochloride + 810 μg / mL nobiletin solution was collected with a syringe, and intravenously injected at 100 μL / egg into the blood vessel of a Day 15 tumor-implanted chicken egg. The chicken eggs were returned to the incubator and cultured for 4 hours, and then irradiated with light at 100 mW / cm ² for 250 seconds, and cultured again. Three days after light irradiation (Day 18), the tumor was removed and the weight of the tumor was measured with a microelectronic balance.

Results As shown in FIG. 6, tumor weight decreased when nobiletin was used in combination as compared with ALA hydrochloride alone treatment. From these results, it was shown that the combined use of ALA and nobiletin enhances the antitumor effect of ALA-PDT compared to the case of using ALA alone.

Claims (8)

A medicament for enhancing photodynamic response in photodynamic therapy or photodynamic diagnosis,
The medicament is a compound represented by the following formula (I):

(In the formula, R ¹ represents a hydrogen atom or an acyl group, and R ² represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group).
Or its salt,
Including
The medicament is used in combination with nobiletin or a composition containing nobiletin,
Medicine. A medicament for enhancing photodynamic response in photodynamic therapy or photodynamic diagnosis,
(A) Compound represented by the following formula (I):

(In the formula, R ¹ represents a hydrogen atom or an acyl group, and R ² represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group).
Or its salt,
(B) Nobiletin or a composition containing nobiletin,
Medicine. A combination drug administered simultaneously or simultaneously to enhance photodynamic response in photodynamic therapy or photodynamic diagnosis,
(A) Compound represented by the following formula (I):

(In the formula, R ¹ represents a hydrogen atom or an acyl group, and R ² represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group).
Or its salt,
(B) Combination medicine with nobiletin or a composition containing nobiletin. A medicament according to claims 1-3,
R ¹ is selected from the group consisting of a hydrogen atom, an alkanoyl group having 1 to 8 carbon atoms, and an aroyl group having 7 to 14 carbon atoms,
R ² is a hydrogen atom, a linear or branched alkyl group having 1 to 8 carbon atoms, a cycloalkyl group having 3 to 8 carbon atoms, an aryl group having 6 to 14 carbon atoms, and an aralkyl having 7 to 15 carbon atoms. Selected from the group consisting of groups,
Medicine. The medicine according to claim 4, wherein
R ¹ and R ² are hydrogen atoms,
Medicine. The medicament according to any one of claims 1 to 5,
The nobiletin-containing composition is a composition containing a citrus extract,
Medicine. The medicine according to claim 6, wherein
The citrus extract is an extract of citrus selected from the group consisting of Citrus, Kumquat, Karatachi, Krimenia, Eremocitrus and Microcitrus,
Medicine. A combination medicine according to claim 3,
The (A) and the (B) are prepared as a kit,
Combination medicine.

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