JP2001302511A - Medicine exhaustion suppressant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a medicine exhaustion suppressant agent which comprises flavone derivative or its pharmeceutically acceptable salt as an active ingredient. SOLUTION: The objective medicine exhaustion suppressant agent contains flavone derivative represented by formula (I) (wherein R1, R2, R3, R4, R5 and R6 are identical or different from each other and represent each H, hydroxy or a lower alkoxy group, R7a, R7b, R7c and R7d are identical or different from each other and represent each a lower alkoxy group) or its pharmaceutically acceptable salt as an active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a drug excretion inhibitor containing a flavone derivative, an agent for improving and / or promoting gastrointestinal absorption of a drug, or an agent for overcoming multidrug resistance.

[0002]

2. Description of the Related Art Conventionally, it is known that P-glycoprotein (hereinafter, referred to as P-gp) is closely involved in the efflux transport system of a drug in the digestive tract. On the other hand, it has been reported that many of the drugs known as P-gp substrates or inhibitors of the action of P-gp are also substrates for the metabolic enzyme CYP3A4 [Molecular Calcinogenesis (Mol Carcinogenesis), 13: 129-134 (1995)
Year)]. When a drug that is a P-gp substrate or an inhibitor of the action of P-gp and also a CYP3A4 substrate is used in combination with other drugs, the interaction is complicated by the inhibitory effects of both P-gp and CYP3A4 proteins. Problem.

[0003] On the other hand, immunosuppressants such as cyclosporin and tacrolimus, and HIV protease inhibitors such as saquinavir and ritonavir have low bioavailability (bioavailability, hereinafter referred to as BA). . It is believed that one of the reasons for the low BA of these drugs is due to the excretion of the drug by P-gp in the gastrointestinal tract. Therefore, a specific inhibitor of the action of P-gp, which has no effect on CYP3A4 metabolism, specifically suppresses drug excretion by P-gp in the gastrointestinal tract,
It is considered to be useful as a gastrointestinal absorption improving and / or promoting agent for a drug showing low BA.

[0004] Conventionally, P-gp is contained in grapefruit juice.
It is known that there is a component having the action of CYP3A4 and the activity of CYP3A4 [Biol. Pharm.
l.), 21, 1062-1066 (1998)], and that vinblastine is known to be a substrate of P-gp [Annual Review of Biochemistry (Annu. Re.
v. Biochem.), 62, 385-427 (1993)].

[0005] Further, as one of the causes of cancer cells expressing multidrug resistance, a mechanism is known in which a drug is excreted from cancer cells by P-gp expressed in the cancer cells [Annual Review of Japan].・ Biochemistry (Annu. Rev. Bioche)
m.), 58, pp. 137-171 (1989)]. Therefore, it is considered that a specific inhibitor of the action of P-gp is also useful as an agent for overcoming multidrug resistance of cancer cells.

It is known that the flavonoid quercetin (formula (II)) inhibits both P-gp and CYP3A4 [Cancer Chemother. Pharmacol., Vol. 36, 448-450
P. (1995)]. Quercetin is also known to be useful as an agent for overcoming multidrug resistance (JP-A-5-70348).
issue).

[0007]

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[0008]

SUMMARY OF THE INVENTION An object of the present invention is to provide a drug efflux inhibitor containing a flavone derivative as an active ingredient,
It is an object of the present invention to provide a gastrointestinal absorption improving and / or promoting agent for a drug containing a flavone derivative as an active ingredient, and a multidrug resistance overcoming agent containing a flavone derivative as an active ingredient.

[0009]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, it has been found that certain flavone derivatives suppress drug excretion with little effect on CYP3A4 metabolism. The present invention was found to have an action (inhibiting the action of P-gp to thereby suppress the excretion of a drug from cells), and completed the present invention. By the flavone derivative used in the present invention, it is possible to suppress the cells in the gastrointestinal tract from excreting the drug and to improve and / or promote the absorption of the drug in the gastrointestinal tract. In addition, due to the flavone derivative used in the present invention, it is present in cancer cells.
The action of P-gp is inhibited, and the multidrug resistance of cancer cells is overcome.

That is, the present invention provides the following (1) to (1)
Regarding 7). (1) Formula (I)

[0011]

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Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are the same or different and represent hydrogen, hydroxy or lower alkoxy, and R ^7a , R ^7b , R ^7c and R ^7d Are the same or different and represent lower alkoxy) or a pharmacologically acceptable salt thereof as an active ingredient. (2) The drug elimination inhibitor according to (1), wherein R ⁴ is lower alkoxy. (3) R ¹ and R ³ are lower alkoxy, and R ² and R ⁵
And the drug elimination inhibitor according to item (1) or (2), wherein R ⁶ is hydrogen. (4) The drug elimination inhibitor according to the above (1) or (2), wherein R ¹ , R ² , R ³ , R ⁵ and R ⁶ are hydrogen. (5) The drug elimination inhibitor according to any of (1) to (4), wherein the lower alkoxy is methoxy. (6) An agent for improving and / or promoting gastrointestinal absorption of a drug, comprising the flavone derivative or the pharmacologically acceptable salt thereof according to item (1) as an active ingredient. (7) The agent for improving and / or promoting gastrointestinal absorption of a drug according to (6), wherein R ⁴ is lower alkoxy. (8) R ¹ and R ³ are lower alkoxy, and R ² and R ⁵
And the agent for improving and / or promoting gastrointestinal absorption of the drug according to the above (6) or (7), wherein R ⁶ is hydrogen. (9) The agent for improving and / or promoting gastrointestinal absorption of a drug according to the above (6) or (7), wherein R ¹ , R ² , R ³ , R ⁵ and R ⁶ are hydrogen. (10) Items (6) to (6) wherein the lower alkoxy is methoxy
An agent for improving and / or promoting gastrointestinal absorption of a drug according to any one of the above items (9). (11) An agent for overcoming multidrug resistance comprising, as an active ingredient, the flavone derivative or the pharmacologically acceptable salt thereof described in (1). (12) The agent for overcoming multidrug resistance according to (11), wherein R ⁴ is lower alkoxy. (13) R ¹ and R ³ are lower alkoxy, R ² ,
Item (11) or (1) wherein R ⁵ and R ⁶ are hydrogen.
2) The agent for overcoming multidrug resistance according to the above item. (14) The agent for overcoming multidrug resistance according to the above (11) or (12), wherein R ¹ , R ² , R ³ , R ⁵ and R ⁶ are hydrogen. (15) The agent for overcoming multidrug resistance according to any one of (11) to (14), wherein the lower alkoxy is methoxy. (16) The agent for overcoming multidrug resistance according to any one of (11) to (15), wherein the subject in which multidrug resistance is expressed is an antitumor agent. (17) It is cancer cells that express multidrug resistance
The agent for overcoming multidrug resistance according to any one of Items 1) to (15).

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION A drug efflux inhibitor is intended to suppress the excretion of a drug administered from a cell or tissue when the drug is administered to the cell or tissue. A thing.

For example, P-gp is closely involved in the excretion of a drug from epithelial cells in the small intestinal epithelial cells to the gastrointestinal tract lumen, and P-gp substrates or inhibitors of the action of P-gp include:
It is possible to inhibit the excretion of the drug from the epithelial cells into the digestive tract lumen. Therefore, when the cell into which the drug is taken up is a small intestinal epithelial cell or the like, the substrate of P-gp or the inhibitor of the action of P-gp can be an agent for improving and / or promoting gastrointestinal absorption of the drug. In addition, P-gp is involved in the expression of multidrug resistance in target cells such as cancer cells (eg, the mechanism by which P-gp expressed in cancer cells causes the drug to be extruded out of cancer cells is known). Are involved. Therefore, when the cell into which the drug is taken up is a target cell such as a cancer cell, an inhibitor of the action of P-gp can inhibit the excretion of the drug from the target cell such as a cancer cell, Inhibitors of the action of -gp can be multidrug resistance overcomers.

Hereinafter, the compound represented by the formula (I) is referred to as compound (I). The same applies to compounds represented by other formula numbers. In the present invention, the lower alkyl moiety of the lower alkoxy has 1 to 8 carbon atoms, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl and the like. No.

Some of the compounds (I) are present in orange juice, and these are purification methods commonly used in organic synthetic chemistry, for example, filtration, extraction, washing, drying, concentration, recrystallization, various types of chromatography. Can be isolated and purified. Compound (I) can also be newly synthesized by using a synthetic organic chemistry technique such as the production method described below.

Compound (I) can be prepared by a method described in the literature [Chemical Pharmaceutical Bretane (Chem. Pharm.
Bull.), 43, 2054 (1995) and Bulletin of Chemical Society Japan (Bull. Chem.
Soc. Jpn.), 69, 1033 (1996), etc.] or a modification thereof. <Production Method 1> Compound (Ia) in which R ^{2 to} R ⁶ are the same or different and represent hydrogen or lower alkoxy and R ¹ represents hydrogen in compound (I), and R ² in compound (I)
-R ⁶ are the same or different and represent hydrogen or lower alkoxy and R ¹ represents hydroxy in compound (Ib) and compound (I) wherein R ² -R ⁶ are the same or different and represent hydrogen or lower alkoxy and R ¹ is lower Compound (Ic) representing alkoxy can be produced by the following steps 1 to 4.

[0018]

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(Wherein R ^1a represents lower alkoxy,
R ^{2a to} R ^6a are the same or different and each represents hydrogen or lower alkoxy, and R ^7a , R ^7b , R ^7c and R ^7d have the same meanings as described above, wherein R ^1a and R ^{2a to} R ^6a are as defined above. Lower alkoxy is as defined above.

(Step 1) Compound (IV) is converted to compound (I)
I) is dissolved in a solvent such as pyridine, 1 to 5 equivalents of the compound (IIIa) is added, and the mixture is heated and stirred at 50 to 80 ° C. for 1 to 5 hours, and then 1 to 50 equivalents of a base such as potassium hydroxide is added. In addition, it can be obtained by further stirring at 50 to 80 ° C for 1 to 5 hours.

(Step 2) Compound (Ia) is converted to compound (I
V) can be obtained by dissolving V) in a solvent such as acetic acid and stirring at 20 to 100 ° C for 10 minutes to 5 hours in the presence of a catalytic amount of sulfuric acid or the like.

(Step 3) Compound (Ib) is converted to compound (I
a) is dissolved in an inert solvent such as dichloromethane,
Add 0 equivalent of dimethyldioxirane, -20 ~ 30 ° C
For 1 to 50 hours.

(Step 4) Compound (Ic) is converted to compound (I
b) is dissolved in a solvent such as acetone, and in the presence of 1 to 50 equivalents of a base such as potassium carbonate, 1 to 10 equivalents of an alkylating agent such as di-lower alkylsulfuric acid or halogenated lower alkyl is added; For 30 minutes to 10 hours.

<Production method 2> In compound (I), R ^2-
Compound (Id) in which at least one of R ⁶ is hydroxy is prepared from compound (II) and compound (IIIb) shown below as a starting material, from compound (V) which can be synthesized according to Production method 1, by the following steps: 5 can be manufactured.

[0025]

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[0026] (wherein, R ^2b to R ^6b represents the same or different and each is hydrogen, lower alkoxy or benzyloxy group, R ^2c to R ^6c represents hydrogen, lower alkoxy or hydroxy same or different, R ^1, R ^7a , R ^7b ,
R ^7c and R ^7d are as defined above. However,
At least one of R ^{2b to} R ^6b represents a benzyloxy group and at least one of the corresponding R ^{2c to} R ^6c represents hydroxy) wherein R ^{2b to} R ^6b and R ^{2c to} R ^6c are defined Has the same meaning as defined above.

(Step 5) Compound (Id) is prepared by dissolving compound (V) in a solvent such as ethyl acetate or methanol, and heating at 0-60 ° C. in the presence of a catalyst such as palladium on carbon.
It can be produced by hydrogenating for minutes to 50 hours.

The compound (I) having a desired functional group at a desired position can be obtained by carrying out the reaction by appropriately combining the above-described method and other methods commonly used in organic synthetic chemistry.

The intermediates and target compounds in each of the above production methods are isolated by a purification method commonly used in synthetic organic chemistry, for example, filtration, extraction, washing, drying, concentration, recrystallization, various types of chromatography and the like. It can be purified.

The compound (I) may exist in the form of adducts with water or various solvents, and these adducts are also included in the flavone derivative used in the present invention. Although some of the compounds (I) may exist in the form of optical isomers, all possible isomers, including these, and mixtures in any ratio thereof are included in the flavone derivative used in the present invention.

Compound (I) can be used as it is or in various pharmaceutical forms depending on its pharmacological action and the purpose of its administration. The pharmaceutical composition of the present invention can be produced, for example, by uniformly mixing an effective amount of the compound (I) or a pharmaceutically acceptable salt thereof as an active ingredient with a pharmaceutically acceptable carrier. The carrier may take a wide variety of forms depending on the form of preparation desired for administration. Desirably, these pharmaceutical compositions are in unit dosage form suitable for parenteral administration, such as orally or intravenously. Dosage forms include tablets, granules, powders, capsules, injections and the like.

In preparing tablets, excipients such as lactose, glucose, sucrose, mannitol, methylcellulose, etc., disintegrants such as starch, sodium alginate, calcium carboxymethylcellulose, crystalline cellulose, magnesium stearate, talc, etc. Lubricants, binders such as gelatin, polyvinyl alcohol, polyvinylpyrrolidone, hydroxypropylcellulose, and methylcellulose, and surfactants such as sucrose fatty acid esters and sorbite fatty acid esters may be used in accordance with a conventional method. Tablets containing 1 to 300 mg of active ingredient per tablet are preferred.

In preparing granules, for example, lactose,
Excipients such as sucrose, disintegrating agents such as starch, binders such as gelatin, and the like may be used in a conventional manner. In preparing the powder, for example, excipients such as lactose and mannitol may be used in a conventional manner. In preparing capsules,
For example, gelatin, water, sucrose, gum arabic, sorbite, glycerin, crystalline cellulose, magnesium stearate, talc and the like may be used in a conventional manner. Capsules containing 1 to 300 mg of active ingredient per capsule are preferred.

In preparing the injection, water, physiological saline, vegetable oils (eg, olive oil, peanut oil, etc.), solvents such as ethyl oleate and propylene glycol, solubilizing agents such as sodium benzoate, sodium salicylate, urethane, etc. Isotonic agents such as salt and glucose, preservatives such as phenol, cresol, p-hydroxybenzoate, and chlorobutanol, and antioxidants such as ascorbic acid and sodium pyrosulfite may be used in a conventional manner.

Compound (I) or a pharmaceutically acceptable salt thereof can be administered orally or parenterally, such as by injection. Although the effective dose and the number of administrations vary depending on the administration form, the age, weight, and symptoms of the patient, it is usually preferable to administer 0.01 to 300 mg / kg one to four times per day.

The inhibitory activity of a compound on the drug efflux is determined by inhibiting the inhibitory activity on the drug efflux of P-gp in cells where P-gp is involved in the accumulation of the drug in cells and target cells in the presence of the compound. It can be measured by examining the rate of increase in intracellular accumulation of a drug that is a substrate of P-gp as an index. In the measurement, a labeled compound or the like can be used.

Specifically, for example, it can be examined as described in the following test examples. <Test Example 1> Incorporation experiment with Caco-2 cells Using human colon cancer-derived cultured epithelial cells (hereinafter, Caco-2 cells) as a gastrointestinal tract model, a label of vinblastine, a substrate of P-gp ([ ³ H Vinblastine), and the inhibitory activity of P-gp on the drug efflux function was examined based on the increase in the uptake equilibrium value.

Caco-2 cells were cultured at 1.26 × 10 ⁵ cells / well for 4 hours.
The cells were seeded on a well multidish (Nunc, Denmark) and cultured. Culture conditions are as follows. Reference [Pharmaceutical researc
h), Volume 11, page 30], the medium to be used, culture conditions, experimental conditions, and the like were set.

10% fetal bovine serum (GIBCO BRL Life Techno
logies), 1% non-essential amino acids (GIBCO BRL Life Technol
ogies), 2 mmol / L L-glutamine (Wako Pure Chemical Industries), 100 un
Dulbecco's Modified containing its / mL penicillin G (Wako Pure Chemical Industries) and 100 μg / mL streptomycin (Nacalai Tesque)
3 in Eagle's medium (GIBCO BRL Life Technologies)
13-15 days at 7 ° C, 5% CO ₂ -95% air.

After the cells reached confluence 5 to 6 days after the seeding, the medium was replaced every day. In the uptake experiment, after cultivation, the medium of Caco-2 cells was removed and Hanks' balanced salt solutio
n (hereinafter, referred to as HBSS), 136.7 mmol / L NaCl, 5.4 mmol
/ L KCl, 0.95 mmol / L CaCl ₂・ 2H ₂ O, 0.81 mmol / L MgSO _{4
· 7H 2 O, 0.44 mmol /} L KH 2 PO 4, 0.39 mmol / L Na 2 HPO 4 · 1
And washed 1-2 times with _{pH 6.5); 2H 2 O,} 25 mmol / L D- glucose, 10 mmol / L 2- (N- morpholino) ethanesulfonic acid (MES). Orange juice extract components or methoxyflavones (3,5,6,7,8,3 ′, 4′-heptamethoxyflavone (hereinafter referred to as HMF) or tangeretin (5,6,7,8,
250 mL of HBSS containing 10 nmol / L [ ³ H] vinblastine in the presence or absence of 4'-hexamethoxyflavone))
Was used to perform an uptake experiment. After the incubation for the set time, the cells were washed with HBSS at 4 ° C. to terminate the intracellular uptake. Furthermore, it was solubilized with 1 mol / L NaOH (250 μL) and neutralized with 1 mol / L HCl (250 μL). Then add scintillation cocktail (Clearsol I, Nakarai Tesque, Kyoto) and add liquid scintillation counter (LS6500, Beckman Instruments, Inc., C
A, USA). The amount of protein in the cells was determined by the Lowry method, and the uptake of [ ³ H] vinblastine into cells was expressed as the amount of drug solution taken up per unit protein.

<Test Example 2> Measurement of testosterone oxidation in CYP3A4 reconstituted system and human liver microsomes CYP3A4 activity was measured by the amount of 6β-hydroxytestosterone produced from testosterone using the CYP3A4 reconstituted system or human liver microsomes.

A CYP3A4 reconstitution system was used to examine the effect of the orange juice extract. Methoxyflavones (HM
F and tangeretin) were examined using both the CYP3A4 reconstitution system and human liver microsomes. 100 mmol / L
1.3 mmol / L NADP ⁺ , 3.3 mmol / L in calcium phosphate
A reaction solution (500 μL) containing glucose-6-phosphate, 0.4 U / mL glucose-6-phosphate dehydrogenase, 3.3 mmol / L magnesium chloride and 0.2 mmol / L testosterone in the presence of orange juice extract components or methoxyflavones Alternatively, preincubation was carried out at 37 ° C. for 5 minutes in the absence. The reaction was started by adding 1.25 pmol of P450 when using the CYP3A4 reconstitution system, and by adding 0.05 mg of protein when using human liver microsomes. After incubation for 15 minutes, methylene chloride (470 μL) was added to terminate the reaction, and when the CYP3A4 reconstitution system was used, 20 μmol / L corticosterone (30 μL) was added as an internal standard substance, and the mixture was shaken for 3 minutes. 10,000 ×
After centrifugation at g for 3 minutes, the organic layer was dried and dissolved in methanol.
Quantification was performed by HPLC. 4.6 × 250 mm 5C18 for column
(Senshu Pak ODS-H-1251) was used for separation at 45 ° C. A 60% aqueous methanol solution was used for the mobile phase, and the flow rate was
It was 1.2 mL / min. Metabolites were detected by UV at 242 nm and CYP3A4
In the case of using the reconstituted system, 6β-hydroxytestosterone was quantified in comparison with the internal standard, and in the case of using human liver microsomes, quantification was performed by the absolute calibration method.

[0043] <Results A> Caco-2 when ^[3 H] vinblastine uptake drug absence in ^[3 H] Orange juice extract component effects Caco-2 cells on the vinblastine uptake by cells (control) Increases in a time-dependent manner, 60
A steady state was reached by the minute (o in FIG. 1).

Examination of the effect of 50% orange juice (100% orange juice adjusted with pH and osmotic pressure while diluting 2-fold using purified water and HBSS) on the equilibrium value of [ ³ H] vinblastine uptake for 60 minutes did. In the presence of 50% orange juice, the equilibrium value of [ ³ H] vinblastine uptake was significantly increased to 178 ± 5.5% compared to the control. From this, Caco-2 in orange juice
It was found that there was a component that increased the equilibrium value of [ ³ H] vinblastine uptake in cells.

Then, orange juice was extracted with three kinds of organic solvents, ethyl acetate, diethyl ether and methylene chloride, and the same experiment was carried out using the extracted components in each solvent. [ ³ H] vinblastine uptake equilibrium values were 238 ± 2.8, 203 ± 14, 198 ± 4.5% in the presence of ethyl acetate, diethyl ether, and methylene chloride extracts corresponding to 50% orange juice, respectively, as compared to the control. And all showed significant increases. In particular, the effect of the ethyl acetate extract was the highest. Furthermore, the components to be separated into an aqueous layer in the orange juice ethyl acetate extracts, was examined the effect on of ^[3 H] vinblastine uptake in Caco-2 cells, and the aqueous layer equivalent to 50% orange juice extraction In the presence of the substance, it was 100 ± 4.33% relative to the control, and no change was observed. Therefore, in orange juice
It was confirmed that the component that increases the equilibrium value of [ ³ H] vinblastine was almost completely extracted into the organic layer by extraction with ethyl acetate.

In addition, the uptake equilibrium value of [ ³ H] vinblastine was significantly increased in the presence of an orange juice ethyl acetate extract, as in the presence of 20 μmol / L cyclosporin A, a specific inhibitor of P-gp. (FIG. 1). On the other hand, the initial rate of [ ³ H] vinblastine uptake was not affected by the presence of cyclosporin A or the orange juice ethyl acetate extract. This suggests that the effect of the orange juice ethyl acetate extract is due to inhibition of the mechanism of [ ³ H] vinblastine efflux specific for P-gp.

<Result B> Effect of orange juice ethyl acetate extract on the equilibrium value of [ ³ H] vinblastine uptake in Caco-2 cells and 6β-hydroxylation of testosterone in human CYP3A4 reconstituted system. FIG. 2A shows the effect of a methanol aqueous solution eluted fraction of the orange juice ethyl acetate extract on the equilibrium value of [ ³ H] vinblastine uptake in Caco-2 cells. In the presence of the fractions eluted with 60, 70, and 80% aqueous methanol, the equilibrium value of [ ³ H] vinblastine was increased, and the most remarkable effect was observed in the fraction eluted with 70% aqueous methanol. It was suggested that a component inhibiting the efflux function of P-gp in Caco-2 cells was present in the 70% methanol aqueous solution eluted fraction. On the other hand, these methanol aqueous solution elution fractions had no effect on the 6β-hydroxylation activity of testosterone (FIG. 2B).

Accordingly, it was shown that orange juice contains a component that does not affect CYP3A4 metabolism and specifically inhibits the excretion mechanism.

<Result C> The fraction obtained by purifying and separating the fraction eluted with the aqueous methanol solution using a silica gel column was purified using Caco-2 cells.
^[3 H] in Caco-2 cells with the purified fraction by obtained silica gel column with effect reference example on the uptake equilibrium value of vinblastine ^[3 H] result of examining the effect on the uptake equilibrium value of vinblastine, 3 As shown in the figure, the fourth eluted fraction (2-4) eluted with a hexane: acetone = 3: 1 mixture gave the highest [ ³ H] vinblastine uptake equilibrium value.

[0050] The <Results D> HMF and tangeretin, the effect diagram 4 on the uptake equilibrium value of ^[3 H] vinblastine in human liver microsomes and human CYP3A4 testosterone 6β hydroxide and Caco-2 cells in reconstituted system The effects of HMF and tangeretin on testosterone 6β hydroxylation and [ ³ H] vinblastine uptake equilibrium values were demonstrated.

Both HMF and tangeretin have [ ³ H] in a concentration-dependent manner.
Increase the equilibrium value of vinblastine up to 50 μmol /
In the presence of L, 578 ±
It showed an increase to 15.9% and 906 ± 23.63%, suggesting that the effect was more pronounced for tangeretin. Using human liver microsomes and the human CYP3A4 reconstitution system,
The effects on CYP3A4 activity were examined, but none showed any change. In addition, a similar study was conducted for nobiletin, which has a flavone skeleton similar to HMF and tangeretin and is contained in orange juice. The uptake equilibrium value of [ ³ H] vinblastine increased to a lesser extent but in a concentration-dependent manner compared to the case of using HMF and tangeretin. On the other hand, no significant inhibitory effect was observed on the hydroxylation of 6β-position of testosterone in human liver microsomes and human CYP3A4 reconstituted system in the concentration range examined.

[0052]

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The flavone derivatives disclosed in the present invention are flavone derivatives in which the 5-, 6-, 7- and 8-positions of the flavone skeleton are all lower alkoxy (hereinafter referred to as tetrasubstituted flavone derivatives). Table 1 shows the inhibitory effect on the action of P-gp and the selectivity of the action of P-gp and the inhibitory effect on CYP3A4 with non-tetrasubstituted flavanone derivatives such as naringin, hesperetin and hesperidin. . In Table 1, the activity of the compound is indicated by four levels of "++, ++, +,-".

[0054]

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[0055]

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[0056]

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[0057]

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[0058]

[Table 1]

According to Table 1, the tetrasubstituted flavone derivative is
Compared to the non-tetrasubstituted flavanone derivative, the inhibitory activity against the action of P-gp was strong and did not show CYP3A4 inhibitory activity (has high selectivity for P-gp). Therefore, the tetrasubstituted flavone derivative was shown to have a specific and strong inhibitory activity on the action of P-gp.

[0060]

EXAMPLES Reference Example Separation of HMF and Tangeretin from Orange Juice Ethyl acetate was added to orange juice and shaken for 10 minutes.
After centrifugation at 900 × g for 10 minutes, the organic layer was dried to remove the aqueous layer. Purified water is added to the residue of the organic layer (orange juice ethyl acetate extract) to suspend the mixture, and a Cosmosil column (Co
smosil 75C18-OPN, Nakarai Tesqu
e), Kyoto).

For elution, an aqueous solution in which methanol was gradually increased from 0 to 100% by 10% was used, divided into 11 fractions, and each fraction was dried. The fraction active in the above-mentioned Test Example 1 was further subjected to a silica gel column (Silica gel 60, Kissel gel 60, Gel de silica 60,
(MERCK) and purified. For elution, use hexane: acetone system (5: 1,3: 1,1: 1) and chloroform: methanol system (1: 1) sequentially, and total 4 fractions in each solvent system.
After dividing into 16 fractions, each fraction was dried. Confirmation of target components in each fraction is performed by thin-layer chromatography (TLC, silica gel 60, F254, MERCK)
And a hexane: acetone system was used as a developing solvent.

In this eluted fraction, a component corresponding to the activity of increasing [ ³ H] vinblastine uptake into cells was further separated and purified by a column, and finally confirmed by TLC to confirm the two compounds (compounds 1 and 2). Obtained. The structure was determined by dissolving two compounds (compounds 1 and 2) in deuterated chloroform and using 500 MHz ¹ H-NMR using tetramethylsilane as an internal standard.
(UNITY-plus, Varian).

The obtained spectrum is shown below. Compound 1: δ 3.87 (3H, s, CH ₃ ), 3.93 (3H, s, CH ₃ ),
3.95 (6H, s, CH ₃ ), 3.96 (3H, s, CH ₃ ), 3.98 (3H,
s, CH ₃ ), 4.08 (3H, s, CH ₃ ), 6.99 (1H, d, J = 8), 7.
79 (1H, d, J = 2), 7.82 (1H, dd, J = 2, 8) Compound 2: δ 3.87 (3H, s, CH ₃ ), 3.93 (6H, s, CH ₃ ),
4.00 (3H, s, CH ₃ ), 4.08 (3H, s, CH ₃ ), 6.58 (1H,
d, J = 8), 7.01 (2H, d, J = 9), 7.85 (2H, d, J = 8) From these spectra, compound 1 was identified as HMF, and compound 2 was identified as tangeretin.

[0064]

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[0065]

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The active ingredient can be separated using diethyl ether or methylene chloride as an extraction solvent instead of ethyl acetate.

<Formulation Example> Formulation Example 1 Tablet A tablet having the following composition is prepared by a conventional method.

Formulation HMF 20 mg Lactose 143.4 mg Potato starch 30 mg Hydroxypropyl cellulose 6 mg Magnesium stearate 0.6 mg Total 200 mg

Formulation Example 2 Capsules A capsule having the following composition is prepared by a conventional method.

Formulation HMF 20 mg Avicel 99.5 mg Magnesium stearate 0.5 mg Total 120 mg

Formulation Example 3 Injection An injection having the following composition is prepared by a conventional method.

Formulation Tangeretin 2 mg Purified soybean oil 200 mg Purified egg yolk lecithin 24 mg Glycerin for injection 50 mg Distilled water for injection 1.72 ml Total 2.00 ml

[0073]

Industrial Applicability According to the present invention, a drug efflux inhibitor containing a flavone derivative as an active ingredient, an agent for improving and / or promoting gastrointestinal absorption of a drug containing a flavone derivative as an active ingredient, and a flavone derivative as an active ingredient are provided. An agent for overcoming multidrug resistance can be provided.

[Brief description of the drawings]

Fig. 1 Caco- of ethyl acetate extract of orange juice
FIG. 3 is a diagram showing the effect on [ ³ H] vinblastine uptake in two cells.

[Explanation of symbols]

-○-: [ ³ H] vinblastine uptake in the absence of drug (control). -●-: Incorporation of [ ³ H] vinblastine in the presence of an orange juice ethyl acetate extract (corresponding to 50% orange juice). -△-: [ ³ H] vinblastine uptake in the presence of cyclosporin (20 μM). *: Indicates that there is a significant difference of P <0.05 compared to [ ³ H] vinblastine uptake in the absence of drug.

[Figure 2] ethyl acetate orange juice extract purified by Cosmo Sil column (eluted with an aqueous solution of increasing methanol from 0 stepwise by 10% to 100%) were of the components, ^[3 in Caco-2 cells H] Vinblastine uptake equilibrium value (FIG. 2A) and CYP3A4 in human CYP3A4 reconstitution system
FIG. 2B shows the effect on the activity (FIG. 2B).

[Fig. 3] Equilibrium value of [ ³ H] vinblastine uptake in Caco-2 cells by fractionation of a component obtained by separating and purifying an ethyl acetate extract of orange juice with a cosmosil column (eluted with 70% aqueous methanol) using a silica gel column. It is a figure showing the effect to.

FIG. 4: [ ³ H] vinblastine uptake equilibrium value in Caco-2 cells and HM for CYP3A4 activity in human liver microsomes and CYP3A4 activity in human CYP3A4 reconstituted system
It is a figure showing the action of F and tangeretin.

[Explanation of symbols]

a: Effect of HMF on equilibrium value of [ ³ H] vinblastine uptake in Caco-2 cells. b: Effect of tangeretin on the equilibrium value of [ ³ H] vinblastine uptake in Caco-2 cells. c: Effect of HMF on CYP3A4 activity in human liver microsomes. d: Effect of tangeretin on CYP3A4 activity in human liver microsomes. e: Effect of HMF on CYP3A4 activity in human CYP3A4 reconstituted system. f: Effect of tangeretin on CYP3A4 activity in the human CYP3A4 reconstitution system.

Claims (17)

[Claims] 1. A compound of the formula (I) Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are the same or different and represent hydrogen, hydroxy or lower alkoxy, and R ^7a , R ^7b , R ^7c and R ^7d are the same or different Differently represents lower alkoxy) or a pharmacologically acceptable salt thereof as an active ingredient. 2. The drug efflux inhibitor according to claim 1, wherein R ⁴ is lower alkoxy. 3. R ¹ and R ³ are lower alkoxy,
^3. The drug efflux inhibitor according to claim 1, wherein R ² , R ⁵ and R ⁶ are hydrogen. 4. The drug efflux inhibitor according to claim ¹ , wherein R ¹ , R ² , R ³ , R ⁵ and R ⁶ are hydrogen. 5. The drug elimination inhibitor according to claim 1, wherein the lower alkoxy is methoxy. 6. An agent for improving and / or promoting gastrointestinal absorption of a drug, comprising the flavone derivative according to claim 1 or a pharmacologically acceptable salt thereof as an active ingredient. 7. The agent for improving and / or promoting gastrointestinal absorption of a drug according to claim 6, wherein R ⁴ is lower alkoxy. 8. R ¹ and R ³ are lower alkoxy,
The agent for improving and / or promoting gastrointestinal absorption of a drug according to claim 6 or 7, wherein R ² , R ⁵ and R ⁶ are hydrogen. 9. The agent for improving and / or promoting gastrointestinal absorption of a drug according to claim ⁶ , wherein R ¹ , R ² , R ³ , R ⁵ and R ⁶ are hydrogen. 10. The agent for improving and / or promoting gastrointestinal absorption of a drug according to any one of claims 6 to 9, wherein the lower alkoxy is methoxy. 11. An agent for overcoming multidrug resistance comprising the flavone derivative according to claim 1 or a pharmacologically acceptable salt thereof as an active ingredient. 12. The method according to claim 11, wherein R ⁴ is lower alkoxy.
The agent for overcoming multidrug resistance according to the above. 13. The agent for overcoming multidrug resistance according to claim 11, wherein R ¹ and R ³ are lower alkoxy and R ² , R ⁵ and R ⁶ are hydrogen. 14. The agent for overcoming multidrug resistance according to claim 11, wherein R ¹ , R ² , R ³ , R ⁵ and R ⁶ are hydrogen. 15. The agent for overcoming multidrug resistance according to claim 11, wherein the lower alkoxy is methoxy. 16. The agent for overcoming multidrug resistance according to any one of claims 11 to 15, wherein the subject expressing multidrug resistance is an antitumor agent. 17. The agent for overcoming multidrug resistance according to any one of claims 11 to 15, wherein the cell that expresses multidrug resistance is a cancer cell.