JP4813041B2 - Drug-metabolizing enzyme inhibitor derived from strawberry and method for producing the same - Google Patents

Description

  The present invention relates to a strawberry-derived drug metabolizing enzyme inhibitor, a novel polyphenol exhibiting drug metabolizing enzyme inhibitory activity, and a method for producing the same.

  Currently, more than 50% of pharmaceuticals used in clinical practice are said to be metabolized by cytochrome P-450 3A4 (hereinafter referred to as “CYP3A4”), which is a drug-metabolizing enzyme, when metabolized. And it is said that many of foods and herbal medicines and herbs that humans frequently take daily have CYP3A4 inhibitory action. However, many of them have not been identified. Ingestion of foods that have CYP3A4 inhibitory action while taking the drug inhibits the metabolism of the drug, and as a result, the blood concentration of the drug may increase, resulting in dangerous symptoms and unexpected side effects. There is sex. Therefore, in order to prevent dangerous symptoms caused by drugs, it is considered to be very important to examine what kind of CYP3A4 inhibitor is contained in foods, herbal medicines and herbs that are taken daily by humans.

On the other hand, taking a CYP3A4 inhibitor at the same time as the drug is expected to reduce the intake of expensive drugs and lead to a reduction in patient medical costs. Since drug efficacy can be maintained for a long time even when the dose of the drug is low, the safety range (LD ₅₀ / ED ₅₀ ) is narrow and the application range of drugs that are difficult to use is expanded, and special formulation technology is used. It is also possible to achieve the same purpose as that of the long-lasting preparation without the use of the same.

  As a CYP3A4 inhibitory substance, Patent Document 1 discloses a monoclonal antibody against human CYP3A4.

On the other hand, for strawberry extract, Patent Document 2 discloses the use as an anti-inflammatory agent and Patent Document 3 discloses the use as an anti-allergic agent, but it is known that strawberry-derived substances exhibit CYP3A4 inhibitory activity. It is not done.
Japanese translation of PCT publication No. 2003-506084 JP 2003-261454 A JP 2003-261455 A

  An object of the present invention is to provide a highly safe drug-metabolizing enzyme inhibitor.

  In order to solve the above-mentioned problems, the present inventors have searched for a CYP3A4 inhibitor from a food that has been confirmed to be safe by daily ingestion by humans. As a result, the strawberry extract was found to have CYP3A4 inhibitory activity, and a polyphenol having CYP3A4 inhibitory activity was successfully isolated from the strawberry extract to determine the structure, thereby completing the present invention.

That is, the present invention includes the following inventions.
(1) A drug metabolizing enzyme inhibitor containing, as an active ingredient, a substance derived from strawberries and exhibiting cytochrome P-450 3A4 inhibitory activity.
(2) The drug-metabolizing enzyme inhibitor according to (1), wherein the substance derived from strawberries and exhibiting cytochrome P-450 3A4 inhibitory activity is polyphenol.
(3) The drug-metabolizing enzyme inhibitor according to (2), wherein the polyphenol is a flavonoid derivative.
(4) The polyphenol is represented by the following formula (1) or (2):

で示されるフラボノイド誘導体及び次式（３）：

And a flavonoid derivative represented by the following formula (3):

で示される化合物から選ばれる少なくとも１種である前記（２）に記載の薬物代謝酵素阻害剤。
（５）イチゴの抽出物又はその処理物を有効成分として含有する薬物代謝酵素阻害剤。
（６）薬物活性調節剤として用いられる前記（１）〜（５）のいずれかに記載の薬物代謝酵素阻害剤。
（７）前記（６）に記載の薬物代謝酵素阻害剤を含有する医薬組成物。
（８）次式（２）：

The drug metabolizing enzyme inhibitor according to the above (2), which is at least one selected from the compounds represented by
(5) A drug-metabolizing enzyme inhibitor containing a strawberry extract or a processed product thereof as an active ingredient.
(6) The drug metabolizing enzyme inhibitor according to any one of (1) to (5), which is used as a drug activity regulator.
(7) A pharmaceutical composition comprising the drug metabolizing enzyme inhibitor according to (6).
(8) Formula (2):

で示される化合物。
（９）次式（３）：

A compound represented by
(9) The following formula (3):

で示される化合物。
（１０）イチゴを抽出し、得られた抽出物を精製することを特徴とする、次式（１）もしくは（２）：

A compound represented by
(10) Extracting strawberries and purifying the obtained extract, the following formula (1) or (2):

又は次式（３）：

Or the following formula (3):

で示されるポリフェノールの製造法。
（１１）イチゴがトチオトメである前記（１０）に記載の製造法。

The manufacturing method of the polyphenol shown by this.
(11) The production method according to the above (10), wherein the strawberry is Tochiotome.

  ADVANTAGE OF THE INVENTION According to this invention, the drug-metabolizing enzyme inhibitor which can reduce the intake of a drug by ingesting simultaneously with a drug can be provided.

  In the present invention, strawberry is used as an extract or a processed product thereof. The type of strawberry used as an extraction raw material is not particularly limited, but preferably Dutch strawberry (scientific name: Fragaria ananassa, English name: garden strawberry) is exemplified. Examples include kousei, Fukuha, Nyomine, Kurume 49, Tochinomine, preferably Tochiome. Examples of the strawberry portion include fruits, leaves, stems, roots, and preferably fruits.

  In the present invention, the substance derived from strawberries and exhibiting CYP3A4 inhibitory activity is obtained from strawberries and is not particularly limited as long as it inhibits the activity of CYP3A4. For example, a strawberry extract or this Is subjected to at least one of various treatments such as separation, purification and isolation, and retains CYP3A4 inhibitory activity.

  The extraction solvent is generally an organic solvent, preferably a water-miscible solvent such as methanol, ethanol, acetone, propanol. The obtained extract is preferably concentrated, and then the obtained aqueous solution is partitioned with a low-polar organic solvent such as ethyl acetate, chloroform, dichloromethane, ether, etc. Obtainable.

  Purification can be performed by appropriately combining silica gel column chromatography, high performance liquid chromatography and the like.

  By purifying as described above, the compound represented by the formula (1), (2) or (3) having CYP3A4 inhibitory activity can be obtained. Among these, all the compounds represented by the formula (2) or (3) are novel compounds.

  The extract, processed product and compound obtained as described above all have CYP3A4 inhibitory activity, and the intake of the drug can be reduced by ingesting it simultaneously with the drug.

  The drug-metabolizing enzyme inhibitor and pharmaceutical composition of the present invention can be formulated by combining a strawberry extract or a processed product thereof, or the aforementioned compound with a known food carrier or pharmaceutical carrier. The dosage form is not particularly limited and is appropriately selected as necessary. In general, tablets, capsules, granules, fine granules, powders, solutions, syrups, suspensions, emulsions, elixirs, etc. Used as an oral agent. In addition, the drug-metabolizing enzyme inhibitor and pharmaceutical composition of the present invention are used as parenteral agents such as injections, drops, suppositories, inhalants, transdermal absorption agents, transmucosal absorption agents, patches, ointments and the like. May be. Moreover, the drug-metabolizing enzyme inhibitor of the present invention can be added to foods, chewing gums, beverages and the like to produce so-called foods for specified health use, thereby reducing the content of medicinal ingredients and maintaining the medicinal effects.

  The drug-metabolizing enzyme inhibitor and the pharmaceutical composition of the present invention may contain a drug to be reduced in intake in the same preparation, or may be administered simultaneously with a separate preparation containing the drug. Good. Further, the administration time of a separate preparation containing a drug may be shifted within a range in which the effects of the drug metabolizing enzyme inhibitor and the pharmaceutical composition of the present invention are obtained.

  The dosage of the drug-metabolizing enzyme inhibitor and the pharmaceutical composition of the present invention varies depending on the age, body weight, degree of disease, and administration route of the patient, but in the oral administration, the above formula (1), (2) or (3 ) Is usually 10 to 50 mg per day, and the administration frequency is usually 1 to 3 times per day for oral administration.

  Oral preparations are produced according to a conventional method using excipients such as starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, and inorganic salts.

  In this type of preparation, a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a corrigent, a colorant, a fragrance and the like can be appropriately used in addition to the above-mentioned excipients.

  Specific examples of the binder include crystalline cellulose, crystalline cellulose / carmellose sodium, methylcellulose, hydroxypropylcellulose, low-substituted hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, carmellose sodium , Ethylcellulose, carboxymethylethylcellulose, hydroxyethylcellulose, wheat starch, rice starch, corn starch, potato starch, dextrin, pregelatinized starch, partially pregelatinized starch, hydroxypropyl starch, pullulan, polyvinylpyrrolidone, aminoalkyl methacrylate copolymer E, aminoalkyl METAKU Rate copolymer RS, methacrylic acid copolymer L, methacrylic acid copolymer, polyvinyl acetal diethylamino acetate, polyvinyl alcohol, gum arabic, gum arabic powder, agar, gelatin, white shellac, tragacanth, purified sucrose, macrogol.

  Specific examples of disintegrants include crystalline cellulose, methylcellulose, low-substituted hydroxypropylcellulose, carmellose, carmellose calcium, carmellose sodium, croscarmellose sodium, wheat starch, rice starch, corn starch, potato starch, and partially pregelatinized. Starch, hydroxypropyl starch, sodium carboxymethyl starch, tragacanth can be mentioned.

  Specific examples of surfactants include soybean lecithin, sucrose fatty acid ester, polyoxyl stearate, polyoxyethylene hydrogenated castor oil, polyoxyethylene polyoxypropylene glycol, sorbitan sesquioleate, sorbitan trioleate, sorbitan monostearate Sorbitan monopalmitate, sorbitan monolaurate, polysorbate, glyceryl monostearate, sodium lauryl sulfate, lauromacrogol.

  Specific examples of lubricants include wheat starch, rice starch, corn starch, stearic acid, calcium stearate, magnesium stearate, hydrous silicon dioxide, light anhydrous silicic acid, synthetic aluminum silicate, dry aluminum hydroxide gel, talc, Examples thereof include magnesium aluminate metasilicate, calcium hydrogen phosphate, anhydrous calcium hydrogen phosphate, sucrose fatty acid ester, waxes, hydrogenated vegetable oil, and polyethylene glycol.

  Specific examples of the fluidity promoter include hydrous silicon dioxide, light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, and magnesium silicate.

  Moreover, the drug-metabolizing enzyme inhibitor and pharmaceutical composition of the present invention may contain a flavoring agent and a coloring agent when administered as a liquid, syrup, suspension, emulsion, or elixir.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to this Example.

(Example 1)
1. Isolation operation The fruit (2.0 kg) of thiothiome was extracted with methanol (3 L) and concentrated. The extract was partitioned between ethyl acetate and water. The ethyl acetate soluble part (18.4 g) was further separated with hexane and 90% methanol-water, and the 90% methanol-water fraction was concentrated. The obtained 90% methanol-water fraction (3.35 g) was fractionated by reverse phase column chromatography (40% methanol-water, 60% methanol-water, 80% methanol-water, methanol), and then 60% methanol. -The fraction eluted with water (0.13 g) was further purified by high performance liquid chromatography (50% methanol-water) using a reverse phase column. Then, 7.5 mg of compound (1), 1.0 mg of compound (2) and 2.6 mg of compound (3) were isolated.

Compound (1): [α] ²⁶ _D -62 ° (c 0.28, MeOH), FABMS (positive) m / z 595 [M + H] ⁺ ; ¹ H NMR (CD ₃ OD): δ 3.31 (1H, m ), 3.44 (1H, m), 3.45 (2H, m), 4.18 (1H, dd, J = 11.6, 6.6 Hz), 4.29 (1H, dd, J = 11.6, 2.2 Hz), 5.22 (1H, d, J = 7.1 Hz), 6.06 (1H, d, J = 16.0 Hz), 6.11 (1H, br s), 6.28 (1H, br s), 6.79 (2x1H's, d, J = 8.8 Hz), 6.80 (2x1H's, d, J = 8.8 Hz), 7.30 (2x1H's, d, J = 8.8 Hz), 7.39 (1H, d, J = 16.0 Hz), 7.98 (2x1H's, d, J = 8.8 Hz); ¹³ C NMR (CD ₃ OD): δ 65.1 (t), 72.5 (d), 76.5 (d), 76.6 (d), 78.8 (d), 95.8 (d), 101.1 (d), 104.9 (d), 106.2 (s), 115.6 (d), 116.8 (dx 2), 117.6 (dx 2), 123.6 (s), 127.9 (s), 132.0 (dx 2), 133.0 (dx 2), 136.0 (s), 147.4 (d), 159.3 ( s), 160.1 (s), 162.0 (s), 162.3 (s), 163.7 (s), 167.6 (s), 169.6 (s), 180.1 (s).

Compound (2): [α] ²⁶ _D −57 ° (c 0.045, MeOH), FABMS (positive) m / z 595 [M + H] ⁺ ; ¹ H NMR (CD ₃ OD): δ 3.27 (1H, t , J = 9.9 Hz), 3.39 (1H, m), 3.41 (1H, dd, J = 9.9, 7.1), 3.44 (1H, m), 4.15 (1H, dd, J = 12.5, 6.0 Hz), 4.19 ( 1H, dd, J = 12.5, 2.2 Hz), 5.14 (1H, d, J = 7.1 Hz), 5.51 (1H, d, J = 13.2 Hz), 6.14 (1H, d, J = 2.2 Hz), 6.26 ( 1H, d, J = 2.2 Hz), 6.67 (2x1H's, d, J = 8.8 Hz), 6.71 (1H, d, J = 13.2 Hz), 6.80 (2x1H's, d, J = 8.8 Hz), 7.50 (2x1H's, d, J = 8.8 Hz), 7.95 (2x1H's, d, J = 8.8 Hz); ¹³ C NMR (CD ₃ OD): δ 64.9 (t), 72.4 (d), 76.4 (d), 76.5 (d), 78.9 (d), 96.3 (d), 101.7 (d), 105.1 (d), 105.1 (s), 116.5 (dx 2), 116.8 (dx 2), 117.0 (d), 123.6 (s), 128.4 (s ), 133.0 (dx 2), 134.5 (dx 2), 135.9 (s), 146.2 (d), 159.5 (s), 159.9 (s), 160.9 (s), 162.3 (s), 163.7 (s), 166.3 (s), 168.6 (s), 179.9 (s).

Compound (3): [α] ²⁶ _D −55 ° (c 0.14, MeOH); HRFABMS (positive) m / z 373.1508 [M + H] ⁺ (calcd for C ₁₇ H ₂₅ O ₉ , Δ +1.0 mmu); UV (MeOH) λ _max (log ε) 285.5 (4.2), 225.0 nm (4.0); IR (film) ν _max 3400, 1630 cm ^-1 ; ¹ H NMR (CD ₃ OD): δ 0.92 (1H, d, J = 6.8 Hz), 0.96 (1H, d, J = 6.8 Hz), 2.24 (1H, heptet, J = 6.8 Hz), 2.87 (1H, dd, J = 15.6, 6.8 Hz), 3.16 (1H, dd, J = 15.6, 6.8 Hz), 3.39 (1H, t, J = 9.0 Hz), 3.45 (1H, m), 3.46 (1H, t, J = 9.0 Hz), 3.53 (1H, dd, J = 9.0, 7.7 Hz), 3.71 (1H, dd, J = 12.2, 5.6 Hz), 3.91 (1H, dd, J = 12.2, 2.1 Hz), 5.01 (1H, d, J = 7.7), 5.93 (1H, d, J = 2.1), 6.15 (1H, d, J = 2.1 Hz); ¹³ C NMR (CD ₃ OD): δ 23.7 (q), 27.1 (d), 24.2 (q), 54.9 (t), 63.3 (t), 72.0 (d), 75.7 (d), 79.2 (d), 79.4 (d), 96.4 (d), 99.3 (d), 102.7 (d), 107.7 (s), 162.9 (s), 167.3 (s), 168.4 (s), 207.9 (s).

2. Structure Determination As a result of structure determination of the compound (1) by NMR spectrum and FAB mass spectrum, the known compound kaempferol-3-β-D- (6-Op-coumarylglucopyranoside) (Phytochemisty, 1978, 17, 787) -791). Since the NMR data of this compound has not been reported, this assignment is the first.

  The spectral data of compound (2) was similar to that of compound (1), but it was revealed that the double bond was cis.

  The structure of the compound (3) was determined to be a novel substance 4,6-dihydroxy-2-O- (β-D-glucopyranosyl) isovalerophenone by NMR spectrum and FAB mass spectrum.

3. CYP3A4 inhibition test The sample was used as a DMSO solution and added to 192 μL of 100 mM phosphate buffer (pH 7.4). In addition to the substrate nifedipine (50 μM), the buffer includes glucose-6-phosphate (5 mM), β-NADP ⁺ (0.5 mM), MgCl ₂ (0.5 mM), glucose-6-phosphate. Dehydrogenase (4.3 μg / mL) is included. The reaction solution was preincubated at 37 ° C. for 5 minutes, CYP3A4 was added, and the mixture was reacted at 37 ° C. for 1 hour. 100 μL of methanol was added to terminate the reaction, and 6-methoxycarbonyl-5-methyl-7- (2-nitrophenyl) -4,7-dihydrofuro [3,4-b] pyridine-1- was used as an internal standard substance. (3H) -one in DMSO was added. The mixture was extracted with 1 mL of ether and the ether layer was dried. The residue was analyzed by high performance liquid chromatography using a reverse phase column, and the inhibition rate was calculated.

When the methanol extract of Tochiotome was separated with ethyl acetate and water, the inhibition rates at a concentration of 0.01 w / v% were 79% and 48%, respectively. The CYP3A4 inhibitory activity of each compound was as follows: IC ₅₀ value: Compound (1) 0.7 μM, Compound (2) 0.7 μM, Compound (3) 120 μM.

As flavonoids that are said to have strong CYP3A4 inhibitory activity, there are the following compounds, but compounds (1) and (2) showed about 15 to 100 times stronger inhibitory activity.
Kaempferol: IC _{50 50} μM
Quercetin: IC ₅₀ 10 μM
Naringenin: IC _{50 50} μM
Naringin: IC ₅₀ > 1000 μM

Claims (9)

The following formula (1) or (2):

And a flavonoid derivative represented by the following formula (3):

A drug metabolizing enzyme inhibitor comprising as an active ingredient at least one selected from the compounds represented by:   The drug metabolizing enzyme inhibitor according to claim 1, which is used as a drug activity regulator.   A pharmaceutical composition comprising the drug-metabolizing enzyme inhibitor according to claim 2. Extract an extract obtained by extracting a strawberry with a water-miscible solvent selected from methanol, ethanol, acetone and propanol, and separate the organic layer with a low-polar organic solvent selected from ethyl acetate, chloroform, dichloromethane and ether and water. A CYP3A4 inhibitor containing, as an active ingredient, a fraction obtained by treatment including   The CYP3A4 inhibitor according to claim 4, wherein the strawberry is Tochiome.   The CYP3A4 inhibitor according to claim 4 or 5, which is used as a drug activity regulator.   A pharmaceutical composition comprising the CYP3A4 inhibitor according to claim 6. Extracting strawberries and purifying the resulting extract, the following formula (1) or (2):

Or the following formula (3):

The manufacturing method of the polyphenol shown by this.   The method according to claim 8, wherein the strawberry is Tochiome.