KR20040014007A - Chemosensitizer containing flavonoid derivatives as an effective ingredient - Google Patents

Abstract

PURPOSE: Provided is a chemosensitizer containing, as an active ingredient, flavonoid derivatives or their pharmaceutically acceptable salt. It increases the activity of anticancer agents and thus reduces dosage thereof. CONSTITUTION: A chemosensitizer is characterized by containing, as an active ingredient, flavonoid derivatives or their pharmaceutically acceptable salt which are represented by the formula(1) and show high chemosensitizing effects. In the formula, R1 is H or OCH3 and R2 is H or OCH3.

Description

Chemosensitizer containing flavonoid derivatives as an effective ingredient

The present invention relates to a chemical sensitizer (chemosensitizer) containing a compound represented by the formula (1) as an active ingredient, more specifically, a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof exhibiting a high chemical sensitizing effect It relates to a multi-drug resistance inhibitor mediated by a chemosensitizer or P-glycoprotein containing as an active ingredient.

<Formula 1>

In Formula 1, R ₁ is H or OCH ₃ , R ₂ is H, OH or OCH ₃ .

Multidrug resistance (MDR) is one of the major causes that make cancer treatment by chemotherapy difficult. Many mechanisms for multidrug resistance have been reported, the most intensively studied mechanism being due to the expression of P-glycoprotein or P-170, a multidrug resistant gene ( MDR1 ) product.

P-glycoprotein (Pgp), the product of multidrug resistance-related protein (MRP1) and breast cancer resistant protein / mitoxanthrone resistance / placental-specific product of multidrug resistance gene ( MDR 1) related to multidrug resistance Proteins such as the ATP binding cassette (BCRP / MCR / ABCP) are a type of ATP-binding cassette (ABC) superfamily of membrane transporters (Riordan et al., 1985 Nature 316 (6031): 817- 819; Cole et al., Science , 1992, 258: 1650-1654; Maliepaard et al., Cancer Res . 1999, 59 (18): 4559-4563) .The proteins are energy-dependent for chemotherapeutic agents of varying structure. It acts as an evacuation pump and thus reduces intracellular drug accumulation. As a result, tumor cells can avoid the effects of drug cytotoxicity.

Overexpression of P-glycoprotein makes tumor cells resistant to various drugs. These proteins act as drain pumps for commonly used cytotoxic agents such as doxorubicin, vincristine, vinblastine, paclitaxel, colchicine, actinomycin D and mitomycin C (Endicott and Ling., Annu Rev. Biochem . 1989, 58: 137-171; Gottesman and Pastan., Annu. Rev. Biochem . 1993, 62: 385-427). Meanwhile, calcium channel antagonists (verapamil; Tsuruo et al., Cancer Res . 1983, 43 (2): 808-813), calmodin inhibitors (phenothiazine; Ford et al., Mol. Pharmacol . 1989, 35: 105- 115) and compounds such as cyclosporin A are known to inhibit multi-drug resistance mediated by P-glycoproteins.

Treatment of P-glycoprotein inhibitors, known as chemosensitizers, in multidrug resistant cells simultaneously with anticancer drugs increases the effectiveness of anticancer drugs. However, the use of such first-generation chemical sensitizers has been limited in clinical practice due to the toxic side effects of high doses of chemical sensitizers to suppress multi-drug resistance (Raderer and Scheithauer., Cancer , 1993, 72: 3553-3563). Therefore, there is an urgent need for the development of a second-generation chemical sensitizer having high selectivity and efficacy for the main purpose of chemical sensitization.

Currently, a lot of research has been conducted on second-generation chemical sensitizers. Second-generation chemical sensitizers that reverse P-glycoprotein-mediated multidrug resistance include PSC833 (Boesch et al., Cancer Res . 1991, 51 (16): 4226-4233), VX710 (Germann et al., Anticancer Drugs , 1997, 8 (2): 125-140; Rowinsky et al., J. Clin.Oncol. 1998, 16 (9): 2964-2976; Yanagisawa et al., J. Cancer , 1999, 80 (8): 1190 -1196), LY335979 (Dantzig et al., Cancer Res . 1996, 56 (18): 4171-4179; Dantizig et al., J. Pharmacol.Exp.Ther . 1999, 290 (2): 854-862), CR9051 (Dale et al, Br.J. Cancer , 1998, 78 (7):. 885-892; Mistry et al, Br J. Cancer, 1999, 79 (11-12):.. 1672-1678), XR9576 (Mistry et al., Cancer Res . 2001, 61 (2): 749-758) and SCH66336 (Wang et al., Cancer Res . 2001, 61 (20): 7525-7529). Some of the second-generation chemosensitizers are currently in clinical trials.

Many chemical sensitizers, including verapamil, cyclosporin A, FK506, are known to be carried by P-glycoproteins. In other words, they are not actual P-glycoprotein inhibitors, but have been reported to act as pseudosubstrates that reduce the rate of release of anticancer drugs by competitive methods (Sikic, Semin. Hematol . 1997, 34 (4 Suppl5): 40-47).

Investigating hydrophobic steroids and flavonoids to find chemical sensitizers that are not carried by P-glycoprotein, hydrophobic steroid sensitizers include progesterone (Barnes et al., Biochemistry, 1996, 35 (15): 4820-4827) and medroxyprogesterone acetate (Zibera et al., Anticancer Res. 1995, 15 (3): 745-749). Flavonoids are considered to be a new class of chemosensitizers and are known to interact with the cytoplasmic domains of P-glycoproteins and their ATP binding sites (Conseil et al., Proc. Natl. Acad. Sci. USA , 1998, 95 (17). ): 9831-9836).

Flavonoids are known to have different mechanisms for the release of cytotoxic drugs in cancer cells. Hydroxyl compounds such as galanin, caperol and quercetin have been found to increase adriamycin release (Critchfield et al., Biochem. Pharmacol . 1994, 48 (7): 1437- 1445), quercetin and hydrophobic derivatives have been reported to inhibit Rhodamine-123 and Hoechst 33345 emissions (Scambia et al., Cancer Chemother Pharmacol . 1994, 34 (6): 459-464; Shapiro and Ling, Eur. J.) .. Biochem 1997, 250 (1 ): 130-137; Shapiro and Ling, Biochem Pharmacol, 1997, 53 (4):.. 587-596). However, these first generation flavonoid chemosensitizers have been reported to exhibit weak efficacy in P-glycoprotein-mediated multidrug resistant cells. In addition, until now, researches for screening novel flavonoid chemical sensitizers having chemical sensitizer activity from plants for the purpose of developing chemical sensitizers have not been conducted.

In order to develop a chemical sensitizer for P-glycoprotein-mediated multidrug resistant cells, the present inventors have isolated and purified flavonoid compounds exhibiting chemical sensitizing effects in citrus extracts to reveal their chemical structures, chemical sensitizing effects and By determining the toxicity, the present invention was completed by revealing that the flavonoid chemical sensitizer is superior to the conventional chemical sensitizer and has no toxicity.

An object of the present invention is to provide a chemical sensitizer containing a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

Figure 1 shows the cytotoxicity and chemosensory effect of the compound of formula 1 in P-glycoprotein overexpressing AML-2 / D100 cells,

●: AML-2 / D100 cells (vincristine-), Δ:: AML-2 / D100 cells (vincristine +),

A: quercetin B: 3,7-dihydroxy-3 ', 4'-dimethoxyflavone,

C: 3,5,7, -trihydroxy-3 ', 4', 5'-trimethoxyflavone,

D: 5,7,3 ', 4'-tetramethoxyflavone, E: 5,6,7,3', 4'-pentamethoxyflavone,

F: 3,6,3 ', 4',-tetramethoxyflavone,

2 is a graph comparing hemolytic activity by the compound of Formula 1,

●: 3,6,3 ', 4',-tetramethoxyflavone,

■: 3,7-dihydroxy-3 ', 4'-dimethoxyflavone,

▲: 3,5,7, -trihydroxy-3 ', 4', 5'-trimethoxyflavone,

◆: 5,7,3 ', 4'-tetramethoxyflavones,

▽: 5,6,7,3 ', 4'-pentamethoxyflavone,

3 is a graph comparing the drug accumulation effect of the compound of formula 1 in P-glycoprotein overexpressing AML-2 / D100 cells,

CTL: control, VP: verapamil (5 uM), CSA: cyclosporin A (5 uM),

1: quercetin, 2: 5,6,7,3 ', 4'-pentamethoxyflavone,

3: 5,7,3 ', 4'- tetramethoxy flavone, 4: 3,6,3', 4 ',-tetramethoxy flavone,

5: 3,5,7, -trihydroxy-3 ', 4', 5'-trimethoxyflavone,

FIG. 4 is a graph showing concentration dependent 5,6,7,3 ', 4'-pentamethoxyflavone (sinensetin) -induced drug accumulation in AML-2 / D100 cells, FIG.

CTL: control, VP: verapamil (5 uM), CSA: cyclosporin A (5 uM),

Figure 5 A is a 20 uM 5,6,7,3 ', 4'- penta-methoxy flavone and 5 uM Verapamil aftertreatment graph showing the accumulation rate of Rhodamine -123,

B of FIG. 5 is a graph showing a recovery rate of Rhodamine -123 5,6,7,3 ', 4'-penta-methoxy flavone after processing verapamil and after 1 hour,

VP: verapamil (5 uM),

Sinencetin: 5,6,7,3 ', 4'-pentamethoxyflavone (20 uM),

A in Fig. 6 is a graph showing the cytotoxicity of 10 uM Verapamil in daunorubicin or AML-2 / D100 cells in the presence of vincristine,

CTL: control, DNR: daunorubicin, VP: verapamil, VCR: vincristine,

B of FIG. 6, 5,6,7,3 ', 4'-penta-methoxy flavone or 5,6,7,3', 4'-penta is methoxy isoflavone and a graph showing the cytotoxicity under the verapamil is present,

Sinencetin: 5,6,7,3 ', 4'-pentamethoxyflavone, VP: verapamil,

7 is a photograph of Western blot analysis of the effects of various sensitizers on the expression level of P-glycoprotein,

CTL: control, CSA: cyclosporine A, VP: verapamil,

1: quercetin (25 uM), 2: 5,6,7,3 ', 4'-pentamethoxyflavone (25 uM),

3: 5,7,3 ', 4'-tetramethoxyflavone (45 uM),

4: 3,6,3 ', 4',-tetramethoxyflavone (8 uM),

5: 3,5,7, -trihydroxy-3 ', 4', 5'-trimethoxyflavone (2 uM),

FIG. 8 is a photograph showing the effect of a chemical sensitizer on the [ ³ H] azidopine photolabel of P-glycoprotein.

Sinencetin: 5,6,7,3 ', 4'-pentamethoxyflavone,

CSA: cyclosporine A, VP: verapamil

In order to achieve the above object, the present invention provides a multi-drug resistance inhibitor mediated by a chemical sensitizer or P-glycoprotein containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. .

In Formula 1, R ₁ is H or OCH ₃ , R ₂ is H, OH or OCH ₃ .

Hereinafter, the present invention will be described in detail.

The chemical sensitizer of the present invention is a 5,6,7,3 ', 4'-pentamethoxyflavone (cenencetin), 3,5,7-trihydroxy 3', 4 'in the compound represented by the formula (1) , 5'-trimethoxyflavone, 5,7,3 ', 4'-tetramethoxyflavone, 3,6,3', 4'-tetramethoxyflavone or pharmaceutically acceptable salts thereof as active ingredients Preference is given to containing 5,7,3 ', 4'-tetramethoxyflavones, 3,6,3', 4'-tetramethoxyflavones and 5,6,7,3 ', 4'-pentame It is more preferable that it is oxyflavone, and it is most preferable that it is 5,6,7,3 ', 4'- pentamethoxyflavone represented by General formula (2).

The present invention includes both the compound represented by Formula 1 and pharmaceutically acceptable salts thereof, as well as possible solvates and hydrates that can be prepared therefrom.

The compound of formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt, and as the salt, acid addition salts formed by pharmaceutically acceptable free acid are useful. Organic acids and inorganic acids may be used as the free acid, and hydrochloric acid, phosphoric acid, sulfuric acid, and nitric acid may be used as the inorganic acid, and methanesulfonic acid, p -toluenesulfonic acid, acetic acid, trifluoroacetic acid, citric acid, and maleic acid (maleic) may be used as the organic acid. acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutamic acid Tartaric acid (glutaric acid), glucuronic acid (glucuronic acid), aspartic acid, ascorbic acid, carboxylic acid, vanillic acid, iodic acid and the like can be used.

The compound represented by the formula (1) of the present invention or a pharmaceutically acceptable salt thereof is a chemosensitizer or a strengthening agent and is also useful as a resistance improving agent. In addition, in the case of obtaining resistance due to initial chemotherapy and repeated administration in multi-drug resistant cells mediated by P-glycoprotein, it may be administered simultaneously with an anticancer agent to improve the activity of the anticancer agent.

The compound of formula 1 of the present invention may be administered simultaneously with a cancer chemotherapeutic agent in a therapeutically effective amount, such as a dose sufficient to provide treatment for a disease state. In the case of resistant cancer cells mediated by P-glycoproteins, the anticancer agents that can be administered include vinca alkaloids (vincristine), anthracyclines (daonorubicin, doxorubicin and adriamycin), etoposide, teniposide and paclitaxel (taxol). ) And less chemosensitizing effect is observed for the anti-metabolizer and alkylating agent.

5,6,7,3 ', 4'-pentamethoxyflavones (cynencetin) have several effects besides the application of P-glycoprotein inhibition and the effect of reversing resistance mediated by P-glycoprotein. In particular, drugs that are substrates of P-glycoprotein (anticancer drugs or central nervous system drugs) may have lower bioavailability by P-glycoproteins present in the intestinal and cerebrovascular barriers (Britten CD et al., 2000 Clin. Cancer Res. 6 (9): 3459-3468; Schellens JH et al., 2000 Eur. J. Pharm. Sci. 12 (2): 103-110; Sparreboom A, et al., 1997 Proc. Natl. Acad. Sci . USA 94 (5): 2031-2035 ; Schinkel AH et al, Cell 1994 77 (4):. 491-502). Therefore, the bioavailability is low when orally administered a drug that is a substrate of P-glycoprotein, it is possible to increase the bioavailability when administered simultaneously with the P-glycoprotein inhibitor of the present invention. Specifically, by concurrently administering a clinically problematic drug (taxol, etc.) and cysenetine as a substrate of P-glycoprotein, not only can the bioavailability of oral drugs be increased, but also the delivery to the central nervous system is problematic. It can be applied to the delivery of drugs (antiviral agents, etc.) that are the substrate of the P-glycoprotein. In addition, the flavonoids have a mutation inhibitory effect (Miyazawa M et al., 1999 J. Agric. Food Chem. 47 (12): 5239-5244), cytochrome P-450 inhibitory effect (Zhang H et al., 2000 Drug Metabol.Drug Interact. 17 (1-4): 351-363), antioxidant efficacy (van Acker FA et al., 2001 Clin. Cancer Res. 7 (5): 1378-84; Sekher Pannala A et al., B .. 2001 Biochem Biophys Res Commun 282 (5):. 1161-1168), platelet aggregation inhibitory effects (Robbins RC et al, 1988 Int J. Vitam Nutr Res 58 (4.....): 418-421) and It is highly likely to have efficacy such as matrix metalloproteinase inhibitory effect (Ishiwa J, et al., 2000 J. Rheumatol. 27 (1): 20-55), It can be usefully used for the prevention or treatment of cancer, aging, thrombosis or rheumatism.

When MTT analysis is performed to determine the cytotoxic and chemosensory effects of the compound of Formula 1, the compound of Formula 1 decreases resistance by anticancer agents (see FIG. 1 ). The chemical sensitizing effect of the compound of Formula 1 is as follows. 5,6,7,3 ', 4'-pentamethoxyflavones> 3,5,7-trihydroxy 3', 4 ', 5'-trimethoxyflavones>5,7,3',4'-Tetramethoxyflavones> 3,6,3 ', 4'-tetramethoxyflavones>3,7-dihydroxy-3',4'-dimethoxyflavones. In addition, 5,6,7,3 ', 4'-pentamethoxyflavones exhibit relatively low cytotoxicity and high chemosensitizing effects compared to conventional P-glycoprotein inhibitors such as verapamil and cyclosporin A ( Table 1). 1 ). The sensitizing effect of 5,6,7,3 ', 4'-pentamethoxyflavone is at least 10 to 100 times higher than other flavonoids such as hesperidine, flavone, quercetin and ferulic acid. In addition, 5,6,7,3 ', 4'-pentamethoxyflavone has very low cell membrane toxicity compared to other flavonoids in the hemolytic effect on red blood cells (see FIG. 2 ).

5,6,7,3 ', 4'-pentamethoxyflavones and 3,6,3', 4'-tetramethoxyflavones increase rhodamine-123 accumulation, 5,6,7,3 ', 4'-pentamethoxyflavones increase rhodamine-123 accumulation in a concentration-dependent manner (see Figure 4 ). In addition, the rate and amount of rhodamine-123 accumulation by 5,6,7,3 ', 4'-pentamethoxyflavone is lower than that induced by verapamil, while 5,6, Recovery of the effect of 7,3 ', 4'-pentamethoxyflavones is faster than verapamil (see Figure 5 ).

One problem with first-generation chemosensitizers is that P-glycoprotein inhibitors themselves are substrates for P-glycoproteins (Mistry et al., Cancer Res . 2001, 61 (2): 749-758). However, 5,6,7,3 ', 4'-penta does not seem to increase the toxicity of 5,6,7,3', 4'-pentamethoxyflavone even when P-glycoprotein is completely inhibited. Methoxyflavones do not appear to be a substrate of P-glycoproteins (see FIG. 6 ).

Another problem with chemosensitizers is the effect of increasing the transporter content. The compound of formula 1 varied the content of P-glycoprotein by varying maximum noncytotoxic concentrations. 5,6,7,3 ', 4'-pentamethoxyflavones rather reduced the content of P-glycoproteins, as opposed to verapamil and cyclosporin A (see FIG. 7 ).

The compound of formula 1 of the present invention may be administered in various oral and parenteral dosage forms at the time of clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used Is prepared using. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which solid preparations comprise at least one excipient such as starch, calcium carbonate , Sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solution solutions, emulsion syrups, and the like, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used.

The dosage of the active ingredient according to the present invention may be appropriately used depending on the absorbency of the active ingredient in the body, the rate of water activation and excretion, the age, sex and condition of the patient, and the severity of the disease to be treated.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Preparation Example 1 Manufacturing Method of Syrup

A syrup containing the compound of Formula 1 of the present invention and a pharmaceutically acceptable salt thereof as an active ingredient of 2% (weight / volume) is prepared by the following method.

Acid addition salts, saccharin and sugars of 5,6,7,3 ', 4'-pentamethoxyflavone were dissolved in 80 g of warm water. After the solution was cooled, a solution consisting of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water was prepared and mixed thereto. Water was added to this mixture to 100 ml. The addition salt can be replaced with other salts according to the examples.

The components of the syrup are as follows.

5,6,7,3 ', 4'-pentamethoxyflavones hydrochloride 2 g

Saccharin: 0.8 g ··············

25.4 g of sugar

Glycerin ... 8.0 g

Spices ··················· 0.04 g

Ethanol 4.0 g

0.4 g of sorbic acid

Distilled water ·····················

Preparation Example 2 Preparation of Tablet

A tablet containing 15 mg of active ingredient is prepared by the following method.

250 g of 5,6,7,3 ', 4'-pentamethoxyflavone hydrochloride were mixed with 175.9 g lactose, 180 g potato starch and 32 g colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

The components of the tablet are as follows.

5,6,7,3 ', 4'-pentamethoxyflavone hydrochloride 250 g

Lactose ............... 175.9 g

Potato starch ··············· 180 g

Colloidal silicic acid 32 g

10% gelatin solution

Potato starch ... 160 g

Talc · 50 g

Magnesium stearate 5 g

Preparation Example 3 Manufacturing Method of Injection Solution

Injection solution containing 10 mg of the active ingredient was prepared by the following method.

5,6,7,3 ', 4'-pentamethoxyflavone hydrochloride One g, 0.6 g sodium chloride and 0.1 g ascorbic acid were dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

The components of the injection solution are as follows.

5,6,7,3 ', 4'-pentamethoxyflavone hydrochloride 1 g

Sodium Chloride ・ ・ ・ ・ 0.6 g

0.1 g of ascorbic acid

Distilled water ·················

Example 1 Analysis of Cytotoxicity and Chemical Sensitization Effect of Compounds of Formula 1

The inventors performed an MTT assay to determine the cytotoxic and chemical sensitizing effects of the compound of Formula 1 (Indofine Chemical, USA) in AML-2 / D100. Specifically, the OCI-AML-2 cell line was purchased from the Ontario Cancer Institute in Canada. Daunorubicin-resistant AML-2 (AML-2 / D100) and doxorubicin-resistant AML-2 (AML-2DX100) were used as positive cell lines expressing P-glycoprotein and MRP, respectively (Choi and Ling., Mol Cells, 1997, 7: 170-177; Choi et al., Mol. Cells , 1999, 9: 314-319). All cells were incubated at 37 ° C. in a 5% CO ₂ environment using α-MEM medium (Gibco) containing 10% heat inert fetal bovine serum (FBS). Cells were maintained in suspension culture and subcultured in confluence state.

In vitro cytotoxicity of drugs was analyzed by MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide, Sigma) assay (Pieters et al., Cancer Lett. 1988, 41). : 323-332). The 50% inhibitory concentration (IC ₅₀ ) for a particular formulation was defined as the drug concentration when the cell number was reduced by 50% compared to the untreated control. IC ₅₀ values were determined directly from the half-log drug-response curves. The resistant cell line AML-2 / D100, which overexpresses P-glycoprotein, also grows well at 120 ng / ml vincristine, a suitable substrate for P-glycoprotein. The chemosensitizing effect was therefore observed in the presence of vincristine 120 ng / ml (Choi et al., Mol. Cells , 1999, 9: 314-319).

As a result, the average IC ₁₀ value (concentration indicating 10% inhibition of cell growth) for 5,6,7,3 ', 4'-pentamethoxyflavone was found to be above 20 uM in AML-2 / WT cells. Whereas IC ₉₀ values in P-glycoprotein expressing cells (AML-2 / D100) in the presence of vincristine are 2 uM or less ( E in FIG. 1 ). This indicates that 5,6,7,3 ', 4'-pentamethoxyflavones have minimal toxicity at the concentrations necessary to completely change drug resistance. The chemical sensitization effect of 5,6,7,3 ', 4'-pentamethoxyflavones was found to be at least 10 to 100 times higher than other flavonoids such as hesperidine, flavone, quercetin and ferulic acid. .

Compounds of Formula 1 exhibit cytotoxicity in AML2 / D100 cell lines in the absence of vincristine (IC ₅₀ = 10-200 uM) as well as AML2 / D100 in the presence of vincristine (IC ₅₀ = 1-18 uM) Cell line resistance was lost ( FIG. 1 ). Of the flavonoids tested, 5,6,7,3 ', 4'-pentamethoxyflavones showed the strongest chemical sensitizing effect, almost identical to that of verapamil or cyclosporin A ( Table 1 ). The chemical sensitizing effect of the compound is high in the following order. 5,6,7,3 ', 4'-pentamethoxyflavones> 3,5,7-trihydroxy 3', 4 ', 5'-trimethoxyflavones>5,7,3',4'-Tetramethoxyflavones> 3,6,3 ', 4'-tetramethoxyflavones>3,7-dihydroxy-3',4'-dimethoxyflavones.

The chemical sensitization effect of 5,6,7,3 ', 4'-pentamethoxyflavone is 5,7,3', 4'-tetramethoxyflavone and 3,7-dihydroxy-3 ', 4'- It was 10 times and 18 times higher than the effect of dimethoxyflavones, respectively. In addition, 5,6,7,3 ', 4'-pentamethoxyflavones exhibited relatively low cytotoxicity and high chemosensitization indices compared to conventional P-glycoprotein inhibitors such as verapamil and cyclosporin A ( Table 1 ).

Chemical sensitizer IC ₅₀ (Vincristine) IC ₅₀ (Vincristine +) CI ^a 5,6,7,3 ', 4'-pentamethoxyflavones 82.4 uM 1.14 uM 72.28 5,7,3 ', 4'-tetramethoxyflavone 200 uM 4.95 uM 40.42 3,6,3 ', 4'-tetramethoxyflavone 200 uM < 11.40 uM 17.5 < 3,5,7-trihydroxy-3 ', 4', 5'-trimethoxyflavone 9.8 uM 3.90 uM 2.51 3,7-dihydroxy-3 ', 4'-dimethoxyflavone 15.9 uM 18.14 uM 0.88 Verapamil 50.9 uM 1.07 uM 47.57 Cycloporin A 14.53 uM 0.87 uM 16.70

a: CI (chemical sensitization index) = IC ₅₀ (Vincristine-) / IC ₅₀ (Vincristine +)

Example 2 Hemolytic Activity of the Compound of Formula 1

We measured the hemolytic activity of the compound of Formula 1 using the method of Subbaraksmi et al. (Subbalaksmi et al., 1996, FEBS Lett 395 (1): 48-52). Hemolytic activity of the compounds was determined by determining hemoglobin secretion of 4% suspension of fresh human erythrocytes at 414 nm. Human erythrocytes were centrifuged and washed three times with PBS (phosphate-buffered saline; 35 mM Phosphate buffer / 0.15 M NaCl, pH 7.0). 100 ml of human red blood cells were inoculated in 96-well plates with 8% (v / v) suspension in PBS and flavonoids were added to each well. The plate was left at 37 ° C. for 1 hour and then centrifuged at 150 g for 5 minutes. 100 μl of supernatant was transferred to a flat-bottom 96-microtiter plate. Hemolysis was measured with an ELISA plate reader (Molecular Devices Emax, Sunnyvale, CA, USA) at 414 nm. 0% and 100% hemolysis rates were determined in PBS and 0.1% Triton-100, respectively. Hemolysis percentage was calculated by the following equation.

Hemolysis (%) = ((Abs 414 nm in compound solution-Abs 414 nm in PBS) / (Abs 414 nm in 0.1% Triton-100-Abs 414 nm in PBS)) X 100

As a result, 5,6,7,3 ', 4'-pentamethoxyflavone was found to be low toxicity compared to other flavonoids in the hemolytic effect on red blood cells ( Fig. 2 ). However, 5,6,7,3 ', 4'-pentamethoxyflavone had no chemical sensitizing effect on AML-2 / DX100, MRP-overexpressing cells.

Example 3 Effect of 5,6,7,3 ', 4'-pentamethoxyflavone on Rhodamine-123 Accumulation

Multidrug-resistant phenotypes are often caused by reduced intracellular accumulation of anticancer drugs by pumping anticancer agents out of cells by transporters such as P-glycoproteins. This effect of P-glycoprotein is reversed by the inhibitors verapamil and cyclosporin, increasing the intracellular accumulation of anticancer agents. The inventors have investigated the effect of Rhodamine-123 accumulation of the compound of Formula 1. Specifically, cell suspension (1 × 10 ⁶ cells / ml) in PBS was exposed to rhodamine-123 (5 uM) with or without verapamil or cyclosporin A. The cells were analyzed for their cytoplasmic drug fluorescence by flow cytometry (Becton Dickinson, USA), where the focused argon laser beam (485 nm) excited the cells in laminar sheath flow and their fluorescence The divergence was collected and represented by a bar graph. Accumulation of Rhodamine-123, a drug carried by P-glycoprotein, was determined using flow cytometry on AML-2 / D100 cells, P-glycoprotein overexpressing cells.

As a result, 5 uM of Verapamil or cyclosporin A and Quercetin, 5,6,7,3 ', 4'-pentamethoxyflavone, 5,7,3', 4'-tetramethoxyflavone (luteolin tetrameth Oxyether), 3,6,3 ', 4'-tetramethoxyflavone and 3,5,7-trihydroxy 3', 4 ', 5'-trimethoxyflavone (mercetin trimethylether) Comparison of their drug accumulation effects with flavonoids of 20 uM ( FIG. 3 ) showed 5,6,7,3 ', 4'-pentamethoxyflavones and 3,6,3', 4 'by flow cytometry. It has been shown to increase rhodamine-123 accumulation only in tetramethoxyflavones. 5,6,7,3 ', 4'-pentamethoxyflavones increased rhodamine-123 accumulation in a concentration-dependent manner ( FIG. 4 ). However, the effects of 5,6,7,3 ', 4'-pentamethoxyflavone on drug accumulation in AML-2 / D100 were 4 compared to verapamil and cyclosporine A, respectively, when compared at concentrations that led to the same effect. Pear and 16 times lower ( FIG. 3 ).

Example 4 Accumulation and Recovery of Rhodamine-123 After Treatment and Removal of 5,6,7,3 ', 4'-pentamethoxyflavone and Verapamil

Accumulation and recovery of rhodamine-123 after treatment with chemosensitizers was compared with 20 uM 5,6,7,3 ', 4'-pentamethoxyflavone and 5 uM verapamil in AML-2 / D100. Intracellular levels of rhodamine-123 were analyzed by flow cytometry.

As a result, the rate and amount of rhodamine-123 accumulation by 5,6,7,3 ', 4'-pentamethoxyflavone was lower than that induced by verapamil, while 5,6, The recovery of the effect of 7,3 ', 4'-pentamethoxyflavones was faster than verapamil ( FIG. 5 ).

Example 5 P-glycoprotein Inhibitory Effects on 5,6,7,3 ', 4'-Pentamethoxyflavone Cytotoxicity

One problem with first-generation chemosensitizers is that P-glycoprotein inhibitors themselves are substrates for P-glycoproteins (Mistry et al., Cancer Res . 2001, 61 (2): 749-758). If 5,6,7,3 ', 4'-pentamethoxyflavone is the substrate for P-glycoprotein, inhibition of P-glycoprotein by other P-glycoprotein inhibitors is intracellular in P-glycoprotein cells. It can increase the amount of 5,6,7,3 ', 4'-pentamethoxyflavone accumulation and thus increase the 5,6,7,3', 4'-pentamethoxyflavone cytotoxicity. Induction of cytotoxicity on AML-2 / D100 cells as a result of cytotoxicity measured by MTT method with 10 uM verapamil treated to completely block P-glycoprotein function in AML-2 / D100 does not, daunorubicin or AML-2 / D100 cells were treated with vincristine when could not survive (a in Fig. 6). These results show that these anticancer agents are substrates of P-glycoprotein. The same experiment was conducted to determine whether 5,6,7,3 ', 4'-pentamethoxyflavone was the substrate of P-glycoprotein on the same basis. 5,6,7,3', 4'-penta methoxy-2 AML / D100 cytotoxicity by flavone has not changed by 10 uM verapamil (B in Fig. 6). From the above results, it can be seen indirectly that 5,6,7,3 ', 4'-pentamethoxyflavone is not a substrate of P-glycoprotein.

Example 6 Effect of Chemosensitizers on P-Glycoprotein Levels

Another concern for chemosensitizers is the increased level of target transporter expression. Western blot analysis was performed to determine if the flavonoid chemosensitizer could increase the expression level of P-glycoprotein in AML-2 / D100 cells.

Specifically, AML2 / D100 cells were treated for 72 hours with the maximum non-cytotoxic concentration (90% or more survival) of the chemosensitizer. Isolation of plasma membrane proteins from drug-sensitive and drug-resistant cells was performed according to conventional methods (Riordan et al., J. Biol. Chem. 1979, 254: 12701-705). Membrane proteins were thawed and fractionated on SDS-PAGE. Western blotting was performed with a slight modification of Tobin's method (Towbin et al., Proc. Natl. Acad. Sci. USA, 1979, 76: 4350-4354). Proteins were transferred to nitrocellulose membranes overnight by electroluting with a current of 60 V. The membrane was placed in a blocking solution (5% skim milk) at room temperature for 1 hour, washed, and then added C219 (Signet), a primary antibody against P-glycoprotein diluted at 1: 1,000. The membranes were washed and left for 1 hour with Horseradish peroxidase-conjugated rabbit-antibiotic IgG (Sigma) diluted 1: 2,000. Finally, the membrane was stained using the detection reagent of the ECL detection kit (Amersham). Protein concentration was determined using a protein analysis kit (Bio-Rad) and based on BSA (Bovine serum albumin).

As a result, the expression level of P-glycoprotein was changed at various levels by the maximum non-cytotoxic concentration of the chemosensitizer including flavonoids. 5,6,7,3 ', 4'-pentamethoxyflavones have been shown to reduce P-glycoprotein levels, as opposed to verapamil and cyclosporin A ( FIG. 7 ).

Example 7 Effect Analysis of Azidopine Photolabels of P-Glycoproteins by 5,6,7,3 ', 4'-pentamethoxyflavones

Azidopine, a photoactive analog of dihydropyridine, photolabels P-glycoproteins in multidrug resistant cell plasma membranes. Such labels are inhibited by vinblastine and some calcium channel blockers (Safa et al., J. Biol. Chem. 1987, 262: 7884-7888; Safa, Proc . Natl . Acad . Sci . USA, 1988, 85 (19): 7187-7191). We investigated whether 5,6,7,3 ', 4'-pentamethoxyflavones inhibit the [ ³ H] -azidopine photolabel of P-glycoprotein.

Azidopin binding assays were performed with a slight modification of Gutailil's method (Gutheil et al., 1994 J. Biol. Chem . ; 269 (11): 7976-7981). Plasma membranes of AML-2 / D1000 cells (Choi and Ling, 1997 Mol. Cells 7 (2): 170-177) were isolated as in the previous method (Riordan and Ling, J. Biol. Chem . 1979, 254: 12701 -12705). Membrane vesicles (50 μg protein) were with or without 1 mM Tris-HCl buffer (pH 7.4) and 2 uM [ ³ H] -azidopine (50 Ci / mmol, Amersham Corp.) with or without test drug. Photolabeled to a final volume of 20 μl. Cyclosporin A or verapamil was added to the control plate to a final concentration of 100 uM 1 hour prior to the addition of azidopine [ ³ H] azidopine as a positive control for inhibition of binding to P-glycoprotein. The plate on ice was placed in a 315 nm UV lamp for 30 minutes directly to allow azidopine to cross-link P-glycoprotein. The medium was removed and the membrane protein dissolved in lysis buffer solution (0.175 mg / ml phenylmethylsulfonyl fluoride, 0.012 mg / ml aprotinin, 10.3 mM H2PO4, 1% Triton X-100, 0.1% SDS, 15 mM sodium azide, 100 mM NaCl, 5 mg / ml sodium deoxycholate. Insoluble debris was removed by simple centrifugation, and the supernatant was loaded on a 7% SDS-polyacrylamide gel electrophoresis gel without heating and electrophoresed at 200 V for 35 minutes. The gel was placed in a fixed solution (isopropanol: water: acetic acid = 25%: 65%: 10%) for 30 minutes and then treated with an amplify solution (Amersham Corp. USA) for 30 minutes. The gel was exposed to film (Kodak X-Omat film) for 14 days after drying at 60-80 C with a vacuum dryer on 3 mm Whatman filter paper.

As a result, [ ³ H] -azidopine specifically labeled P-glycoprotein by ultraviolet rays above 100 uM concentration. Photolabeling of P-glycoprotein by [ ³ H] -azidopin is partially inhibited by cyclosporin A and verapamil but not by 5,6,7,3 ', 4'-pentamethoxyflavone ( FIG. 8 ). These results indicate that 5,6,7,3 ', 4'-pentamethoxyflavone does not interact with P-glycoprotein at the azidopine binding site.

As described above, the compound represented by Chemical Formula 1 of the present invention not only has excellent chemical sensitization effect as compared to conventional chemical sensitizers, but is not a substrate for P-glycoprotein, which is a problem of first-generation chemical sensitizers, but P-sugars. By acting to reduce the expression of the protein, a chemosensitizer including the same can be usefully used for the prevention or treatment of multi-drug resistance.

Claims (6)

A chemical sensitizer containing the compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. <Formula 1> In Formula 1, R ₁ is H or OCH ₃ , R ₂ is H, OH or OCH ₃ . The compound of claim 1, wherein the compound is 5,6,7,3 ', 4'-pentamethoxyflavones, 3,5,7-trihydroxy 3 ', 4', 5'-trimethoxyflavone, 5,7,3 ', 4'-tetramethoxyflavones, or A chemical sensitizer, characterized in that 3,6,3 ', 4'-tetramethoxyflavones. The chemical sensitizer according to claim 2, wherein the compound is 5,6,7,3 ', 4'-pentamethoxyflavone. The chemical sensitizer according to any one of claims 1 to 3, wherein the sensitizer inhibits multi-drug resistance mediated by P-glycoprotein. The method according to any one of claims 1 to 3, wherein the chemosensitizer is a substrate of P-glycoprotein or cytochrome 450, so that the substrate of P-glycoprotein is low in oral bioavailability drug or cerebrovascular barrier. And a chemical sensitizer, which is administered together with the drug that is released. The chemosensitizer according to any one of claims 1 to 3, wherein the chemosensitizer is used for the prevention or treatment of cancer, aging, thrombosis or rheumatism.