JP5583945B2 - CYP1B1 inhibitor - Google Patents

Description

  The present invention relates to a drug that inhibits the expression of cytochrome P450-1B1 (CYP1B1).

  Cytochrome P450 (hereinafter abbreviated as CYP) plays an important role in the metabolism of exogenous foreign substances such as endogenous compounds and drugs and toxins in cell control and cell transmission mechanisms including arachidonic acid, steroid hormones and fatty acids. Yes. Three types of CYP, CYP1, CYP2, and CYP3, have been reported so far. For example, in the CYP2A family belonging to CYP2, it is reported that CYP2A6 is a major nicotine metabolizing enzyme in human liver. (Patent Document 1) and CYP2A6 has been reported to activate carcinogenic substances. And a soybean saponin extract fraction and a licorice extract are reported as a component which inhibits this CYP2A6 activity (patent document 2).

In addition, CYP3, which is another type of CYP, is a drug-metabolizing enzyme expressed in the human liver and small intestine, and is included in grapefruit juice, a component that inhibits the activity of CYP3 for the purpose of efficient drug administration to the living body. A compound having a psoralen skeleton (Patent Document 3), a processed product of star fruit (Patent Document 4), a strawberry-derived polyphenol (Patent Document 5), a polyunsaturated fatty acid (Patent Document 6), and the like have been reported.

  On the other hand, regarding cytochrome P450 belonging to the family of CYP1, CYP1A1 expressed in tissues other than the liver, CYP1A2 expressed in the liver, and CYP1B1 expressed widely in tumor cells have been reported, and in particular, CYP It has been studied to improve the effectiveness of an anticancer agent by inhibiting CYP1B1 related to inactivation or metabolism of an anticancer agent which is one of metabolic substrates, and α-naphthoflavone, akakatine, diosmethine, hesperetin as inhibitory components , Homeriodictyol, 2-ethynylpyrene and the like have been reported (Patent Document 7). Furthermore, in recent years, it has been confirmed that expression of CYP1A1 is induced by UVB irradiation in mouse keratinocytes (HaCaT cells) other than tumor cells (Non-patent Document 1), and in human epidermal skin fibroblasts. It has been reported that tobacco smoke extract induces CYP1B1 expression and MMP-1 expression, and 3-methoxy-4-nitroflavone and α-naphthoflavanone inhibit the expression induction (Non-patent Document 2). Moreover, it has been reported that the expression of CYP1A1 and CYP1B1 is induced by dioxins in mouse and human melanocytes (Non-patent Document 3).

The CYP to be particularly noted here is CYP1B1 belonging to the family of CYP1 which is expressed in a wide range of specific tissues other than the liver including small intestine, kidney and lung, and also skin tissue, and is also a major marker of tumor cells. Effective administration of drugs and anticancer agents to normal tissues in the body (eg liver and small intestine) and tumor cells, or effective inhibitors such as enzyme inhibitors and activators to the skin in the cosmetics field In considering application, it is important how to suppress the expression of CYP1B1 in the tissue to be administered when a drug or an active ingredient is used. However, the types of active ingredients that inhibit the expression of CYP1 that have been reported to date are rare, and specifically, akakatin, diosmethine, hesperetin, homeriodictyol, 2-ethynylpyrene, and 3-methoxy-4- Nitroflavone and α-naphthoflavanone are mentioned, but these effects have strong inhibitory activity as a positive control when testing inhibition of CYP1 expression, but on the other hand have strong toxicity to living organisms However, when taking it as a drug, there is always concern about its safety.

International Publication No. WO 98/03171 JP 2006-169186 A JP-A-10-204091 JP 2006-22071 A JP 2006-111597 A JP 2006-273798 A Special table 2003-522143

Ellen Fritsche et al., Proceedings of the National Academy of Sciences of the United States of America, 2007, 104 (21), 8851-8856 Y Ono et al., Journal of Investigative Dermatology, 2008, 28, Abstract248 I Kurtmann et al., Journal of Investigative Dermatology, 2008, 28, Abstract 1315

  Therefore, an object of the present invention is to develop a drug that is safer to the living body than conventional CYP1 expression-inhibiting components and that sufficiently inhibits the expression of cytochrome P450-1B1 (CYP1B1).

  The present inventors have found that loquat extract has an excellent CYP1B1 expression inhibitory action. And from this knowledge, this invention provides the chemical | medical agent which inhibits the expression of CYP1B1 which uses a loquat extract as an active ingredient.

  The drug of the present invention has an excellent CYP1B1 expression inhibitory action. By using this drug, it suppresses the metabolism and inactivation of living tissues such as the liver, small intestine, and skin, or other drugs administered to tumor cells, and the bioavailability of these drugs in tissues or cells ( Bioavailability) can be improved. Moreover, the loquat extract used as an active ingredient of this invention has been used widely as what is conventionally mix | blended with a pharmaceutical, foodstuffs, cosmetics, etc., and those safety | security with respect to a biological body is very high.

The loquat extract used as an active ingredient in the medicament of the present invention is obtained by extracting leaves of Eriobotrya japonica Thunb. Lindl. As it is or after pulverization, with a solvent.

As an extraction solvent for obtaining the loquat extract used in the present invention, a mixed solvent of water and 1,3-butylene glycol is used.

  Regarding the extraction method, the temperature of the solvent, the weight ratio of the solvent to the raw material, or the extraction time can be arbitrarily set for the portion of the raw material used and the solvent used. Although it can set arbitrarily as the temperature of a solvent in the range of -4 degreeC to 100 degreeC, 10-40 degreeC vicinity is preferable from the stability point of the component contained in a raw material. Moreover, the weight ratio of the solvent with respect to the raw material can be arbitrarily set within a range of, for example, the raw material: solvent of 4: 1 to 1: 100, and a weight ratio of 1: 1 to 1:20 is particularly preferable.

  The loquat extract used in the present invention also includes those subjected to solvent purification and further subjected to appropriate purification operations within the scope of the extract. Purification operations include decomposition by adding acid (hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, organic acid, etc.) or alkali (sodium hydroxide, calcium hydroxide, ammonia, etc.), fermentation or metabolic conversion by microorganisms, ion exchange resin or activated carbon. , Component adsorption by diatomaceous earth, etc., fractionation using chromatography with various separation modes (ion exchange, hydrophilic adsorption, hydrophobic adsorption, size exclusion, ligand exchange, affinity, etc.), filter paper and membrane filter , Filtration using an ultrafiltration membrane, etc., pressurization or decompression, heating or cooling, drying, pH adjustment, deodorization, decolorization, long-time storage, etc. Can be done.

  In producing the drug of the present invention, the shape of the loquat extract to be used may be any shape such as liquid, solid, powder, paste, etc., and the optimum shape for carrying out the present invention is appropriately selected. Select

  The drug of the present invention is a drug whose content component is composed only of loquat extract, and other components that are acceptable for constituting a drug formulation (for example, carrier, excipient, disintegrant, buffer) , Emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological saline, organic solvents, etc.) are also included in the scope of the drugs. The dosage form of the drug is not particularly limited as long as it can be administered orally or parenterally. Capsule, powder, granule, solid, liquid, gel, foam, emulsion, cream, ointment Form, sheet form, mousse form, powder dispersion form, multilayer form, aerosol form and the like. Content of the said loquat extract in the chemical | medical agent of this invention is the range of 0.01-100 mass%, Preferably it is the range of 1-50 mass%.

  The drug of the present invention is preferably an external preparation, which is one of dosage forms for parenteral administration. The drug of the present invention itself can be used as an external preparation, and is also used as an additive component in the production of cosmetics and quasi drugs. Specific forms for external use include lotion (including whitening and anti-aging), emulsion (including whitening and anti-aging), cream (including whitening and anti-aging), lotion (whitening). And cosmetics such as soaps, body shampoos, facial cleansers, lipsticks and foundations. Further, pharmaceuticals such as ointments, patches, lotions, poultices, liniments, and quasi-drugs may be mentioned. Content of the chemical | medical agent of this invention in cosmetics and quasi-drugs is the range of 0.001-50 mass%.

  EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to these contents.

(Production Example 1) Manufacture of loquat extract After adding 100 mL of 50% 1,3-butylene glycol aqueous solution to 10 g of ground loquat leaves, soaked and extracted at room temperature for 10 days, and then removed by suction filtration. As a result, loquat extract was obtained.

(Test Example 1) Measurement of CYP1B1 expression inhibitory activity
1. Preparation of CYP1B1 Expression Inducing Component (Tobacco Extract) Main smoke for 5 cigarettes was sucked with an aspirator and bubbled in 100 mL of hexane. Next, the hexane extract was concentrated to dryness and then dissolved in 5 mL of DMSO and used for the following measurements.
2. Measurement of CYP1B1 Expression Inhibitory Activity Skin fibroblasts collected from healthy non-smokers were seeded in 24-well plates and cultured to 10% FBS-containing DMEM medium (Sigma) until subconfluent. The cells were then starved of serum, 10 mL of the loquat extract of Production Example 1 was added to 3 mL of the medium, and cultured for 2 hours. The negative control was subjected to the same treatment using 50% 1,3-butylene glycol aqueous solution. Next, 20.1 μL of the above tobacco extract was added to these media, and the cells were collected after 24 hours of culture. Total RNA was extracted using Fast Pure RNA kit (manufactured by Takara Bio Inc.), and then the expression level of CYP1B1 was analyzed by RT-PCR. As an internal standard, glycerol-3-phosphate dehydrogenase (hereinafter referred to as G3PDH) was examined. After calculating the ratio with respect to the internal standard, the ratio with the negative control was calculated to obtain CYP1B1 expression activity. The CYP1B1 and G3PDH primers used for the analysis are shown below.
GA3PDH sense ； CTGGGCTACACTGAGCACCA
G3APDH antisense ； CAGCGTCAAAGGTGGAGGGA
CYP1B1 sense ； CGCAACTTCAGCAACTTCTAGG
CYP1B1 antisense ； CCGCAGAGAGGATAAAGG

(Result of Test Example 1)
As a result of testing the CYP1B1 expression activity of the loquat extract of Production Example 1, the loquat extract showed 70% CYP1B1 expression activity in the tobacco extract-induced stimulation compared to the negative control. Therefore, it was found that loquat extract used as an active ingredient in the present invention has an excellent CYP1B1 expression inhibitory action.

(Production Example 2) Production of CYP1B1 expression inhibitor (oral administration)
1. 10g loquat extract from Production Example 1
2. Anhydrous caffeine 0.5g
3. Sucrose 4.0g
4). Sorbitol 7.0g
5. Citric acid 10.0g
6). Sodium benzoate 0.05g
7). Mixed fruit flavor 0.1g
After the components 1 to 7 were dissolved in purified water, the pH was adjusted to 4.0, and purified water was added to make up a total volume of 100 mL. This solution was filtered with filter paper, and the filtrate was sterilized by heating at 80 ° C. for 25 minutes using a sterilizer to obtain a CYP1B1 expression inhibitor for oral administration.

(Production Example 3) Production of CYP1B1 expression inhibitor (transdermal administration agent)
1. Squalane 10%
2. Behenyl alcohol 1.0%
3. Cetearyl alcohol 1.5%
4). Dimethicone 2.4%
5. Glyceryl stearate 5.0%
6). Butylparaben 0.1%
7). Loquat extract from Production Example 1 10.0%
8). Glycerin 5.0%
9. Sodium stearoyl glutamate 0.2%
10. PEG-60 hydrogenated castor oil 1.0%
11. Xanthan gum 0.2%
12 Methylparaben 0.2%
13. Purified water The above components 1 to 6 were heated and mixed, then added to an aqueous solution mixed with 7 to 13 and emulsified to obtain a CYP1B1 expression inhibitor for transdermal administration.

Claims (1)

A drug that inhibits the expression of cytochrome P450-1B1 (CYP1B1), comprising an extract of loquat leaves using a 1,3-butylene glycol aqueous solution as an active ingredient.