JP2004043401A - Multidrug resistance suppressor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a multidrug resistance suppressor which is safe, is easy in preparation and contains a new component having an action inhibiting the function of P-glycoprotein. <P>SOLUTION: This multidrug resistance suppressor contains, as an effective component, a substance which is obtained by extracting from Momordica Charantia and/or a treated material thereof using water and/or an organic solvent and has the action inhibiting the function of P-glycoprotein. <P>COPYRIGHT: (C)2004,JPO

Description

＜評価２＞
上記試料抽出方法において、メタノールの濃度を変えて０％から１００％のメタノール水溶液で抽出したニガウリ抽出物の試料を用意した。
【００４１】
上記方法に従って、０％から１００％のメタノール水溶液で抽出したニガウリ抽出物、２ｍｇ／ｍｌの濃度の試料を用いて評価を行った。
【００４２】
その結果、表２に示すように、各抽出物とも強い［^３Ｈ］−ダウノマイシン取り込み作用が認められた。
【００４３】
【表２】

＜評価３＞
上記試料抽出方法（４０％メタノールで抽出）により抽出した２ｍｇ／ｍｌの濃度のニガウリ抽出物の試料を用いた。
【００４４】
Ｃａｃｏ−２細胞の培養は、上記の方法に準じて行った。
【００４５】
Ｃａｃｏ−２細胞を１２穴透過性プレートに１×１０^５ずつまき、３７℃で１４日間培養した。その後、ｕｐｔａｋｅ　ｂｕｆｆｅｒで洗浄後、ｕｐｔａｋｅ　ｂｕｆｆｅｒを５００μｌ添加し、３０分間平衡化を行った。
【００４６】
反応液（２ｍｇ／ｍｌの試料および１００ｎＭ（０．５　μＣｉ／ｍｌ）の［^３Ｈ］−ダウノマイシンをｕｐｔａｋｅ　ｂｕｆｆｅｒに溶解したもの）を細胞の先端側（Ａｐｉｃａｌ側）に添加し、一定時間（１５、３０、６０、９０、１２０分）後に細胞の基底側（Ｂａｓｏｌａｔｅｒａｌ側）の溶液を５０μｌ回収し、クリアゾル３ｍｌに溶解後、液体シンチレーションカウンター（ＡＬＯＫＡ）により、細胞の基底側へ透過した［^３Ｈ］−ダウノマイシン量を測定した。
【００４７】
上記試料抽出方法（４０％メタノールで抽出）により抽出した２ｍｇ／ｍｌの濃度のニガウリ抽出物の試料を用いて上記方法に従い各時間経過後に評価した結果を表３に示す。
【００４８】
その結果、表３に示すように、ニガウリ抽出物に［^３Ｈ］−ダウノマイシンの細胞の先端側から細胞の基底側への透過を促進させる作用が認められた。
【００４９】
【表３】

＜評価４＞
上記試料抽出方法（４０％メタノールで抽出）により抽出した２ｍｇ／ｍｌの濃度のニガウリ抽出物の試料を用いた。
【００５０】
２ｍｇ／ｍｌの試料をｕｐｔａｋｅ　ｂｕｆｆｅｒに溶解したものを細胞の先端側に、１００ｎＭ（０．５　μＣｉ／ｍｌ）の［^３Ｈ］−ダウノマイシンをｕｐｔａｋｅ　ｂｕｆｆｅｒに溶解したものを細胞の基底側に添加した他は、上記した評価３の方法に準じて行った。
【００５１】
試料と［^３Ｈ］−ダウノマイシンを上記のようにそれぞれ細胞の先端側と細胞の基底側に添加し、一定時間（１５、３０、６０、９０、１２０分）後に細胞の先端側の溶液を５０μｌ回収し、クリアゾル３ｍｌに溶解後、液体シンチレーションカウンター（ＡＬＯＫＡ）により、細胞の先端側へ透過した［^３Ｈ］−ダウノマイシン量を測定した。
【００５２】
上記試料抽出方法（４０％メタノールで抽出）により抽出した２ｍｇ／ｍｌの濃度のニガウリ抽出物の試料を用いて上記方法に従い各時間経過後に評価した結果を表４に示す。
【００５３】
その結果、表４に示すように、ニガウリ抽出物に［^３Ｈ］−ダウノマイシンの細胞の基底側から細胞の先端側への透過を抑制させる作用が認められた。
【００５４】
【表４】

評価３と評価４において、腸管上皮細胞であるＣａｃｏ−２細胞は培養液中で上下規則正しく配列しており、ここで、Ｃａｃｏ−２細胞の先端側（Ａｐｉｃａｌ側）は、生体内での腸管内腔側に相当し、Ｃａｃｏ−２細胞の基底側（Ｂａｓｏｌａｔｅｒａｌ側）は、生体内の腸管壁の内部側に相当する。栄養成分や薬物は、細胞の先端側（Ａｐｉｃａｌ側）から取り込まれる。
【００５５】
上記の評価３と評価４の実験では、ニガウリ抽出物の添加により、細胞の先端側から細胞の基底側への薬剤の取り込みが維持されること、細胞の基底側から細胞の先端側への薬剤の排出が抑制されることが明らかとなった。
【００５６】
これにより、薬剤を投与すると、腸管上皮細胞上のＰ−糖タンパク質の働きにより薬剤は腸管内腔側に戻されるが、ニガウリ抽出物を添加することにより、Ｐ−糖タンパク質の働きが阻害され（多剤耐性が改善され）、投与された薬剤の腸管内腔側への移行が抑制されたといえる。
【００５７】
以上、上記評価実験から明らかなように、ニガウリ植物及び／又はその処理物から得られる抽出物にＰ−糖タンパク質を阻害する作用が認められた。よって、ニガウリ植物及び／又はその処理物から水及び／又は有機溶媒を用いて抽出することにより得られる物質を有効成分として含有する剤は、多剤耐性抑制剤として好適に使用することができる。
【００５８】
【発明の効果】
本発明により、安全かつ調製が容易で、新規なＰ−糖タンパク質の機能を阻害する作用を有する成分を含有する多剤耐性抑制剤を提供することができた。[0001]
TECHNICAL FIELD OF THE INVENTION
TECHNICAL FIELD The present invention relates to a multidrug resistance inhibitor containing, as an active ingredient, a substance having a P-glycoprotein inhibitory action.
[0002]
[Prior art]
Overcoming cancer is a common problem for human beings, and many cancer drugs have been developed to date. On the other hand, the fact that cancer cells have resistance to a plurality of anticancer drugs and other drugs (multidrug resistance) has become a major problem of treatment with anticancer drugs.
[0003]
There are still many unclear points about the mechanism by which cancer cells develop multidrug resistance, but basically, many multidrug-resistant cancer cells produce excessive amounts of P-glycoprotein. Is thought to be responsible for transporting anticancer drugs out of cells. As a result, the concentration of the anticancer agent in the cell is reduced.
[0004]
Since P-glycoprotein has low substrate specificity and binds to many compounds having dissimilar structures to transport drugs out of the cell, once P-glycoprotein is expressed in cancer cells, it can be used for many anticancer drugs. Will also be resistant. In fact, it is known that many structurally different anticancer drugs such as adriamycin, vinblastine, vincristine, actinomycin D, and colchicine are excreted extracellularly by P-glycoprotein. Therefore, it is thought that inhibiting the function of P-glycoprotein leads to overcoming multidrug resistance.
[0005]
In order to overcome such multidrug resistance, various drugs have been developed which inhibit the function of P-glycoprotein. For example, a method of blocking the function of P-glycoprotein itself using an anti-P-glycoprotein monoclonal antibody (Jpn. J. Cancer Res., 80, 627-631 (1989); BIO medical 6 (5), 442) 445 (1991)).
[0006]
However, in the method using the above monoclonal antibody, since such an antibody is derived from another animal (for example, mouse), its own antigenicity is high and it is not always easy to use it in clinical practice.
[0007]
It has also been reported that calcium antagonists such as verapamil and immunosuppressants such as cyclosporin A and FK506 inhibit the function of P-glycoprotein.
[0008]
However, when these are administered for the purpose of overcoming multidrug resistance, their inherent effects of calcium antagonism and immunosuppression appear as strong side effects. For example, a calcium antagonist such as verapamil has a strong vasodilator effect and may induce a shock such as hypotension.
[0009]
Therefore, a new substance with less possibility of side effects has been demanded.
[0010]
On the other hand, for bitter melon, although it has been reported that the pulverized product or extract improves lipid metabolism (Japanese Patent Application Laid-Open No. 2001-278804), it has a multidrug resistance inhibitory effect and furthermore, it has an effect of inhibiting P-glycoprotein. Was not known at all.
[0011]
[Problems to be solved by the invention]
The present invention has been made in view of the above, and an object of the present invention is to provide a multidrug resistance inhibitor containing a component that is safe and easy to prepare and has a function of inhibiting the function of a novel P-glycoprotein. .
[0012]
[Means for Solving the Problems]
The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, it has been found that the function of P-glycoprotein is added to a substance obtained by extracting watermelon plants and / or processed products thereof with water and / or an organic solvent. Have been found to have the effect of inhibiting the present invention, and have completed the present invention.
[0013]
That is, the present invention is as follows.
(1) A multidrug resistance inhibitor comprising, as an active ingredient, a substance having an action of inhibiting a P-glycoprotein obtained by extracting from a bitter melon plant and / or a processed product thereof with water and / or an organic solvent.
(2) The multidrug resistance inhibitor according to (1), wherein the organic solvent is methanol.
(3) A pharmaceutical composition comprising the multidrug resistance inhibitor according to (1) or (2).
(4) A food composition comprising the multidrug resistance inhibitor according to (1) or (2).
[0014]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
<1> Multidrug resistance inhibitor of the present invention The multidrug resistance inhibitor of the present invention refers to a drug capable of suppressing the resistance to a drug (hereinafter, also referred to as “target drug”). In other words, the target drug It is a drug that can suppress the excretion of extracellular. In particular, in the present invention, a drug that can specifically inhibit the function of P-glycoprotein can be used as a multidrug resistance inhibitor.
[0015]
Here, as the target drug to be suppressed, cyclophosphamide, alkylated anticancer drugs such as melphalan, antimetabolic anticancer drugs such as fluorofuracyl, daunomycin, mitomycin C, etc. Examples include plant-derived alkaloid anticancer agents such as anticancer antibiotics, vincristine, and vinblastine.
[0016]
The multidrug resistance inhibitor of the present invention is one in which at least one of the above target drugs has a drug resistance inhibitory effect.
[0017]
The multidrug resistance inhibitor of the present invention contains, as an active ingredient, a substance having an action of inhibiting P-glycoprotein obtained by extracting a bitter melon plant and / or a processed product thereof with water and / or an organic solvent. I do.
[0018]
A substance obtained by extracting a bitter melon plant and / or a processed product thereof with water and / or an organic solvent has an action of inhibiting a P-glycoprotein as apparent from the following examples. Therefore, an agent containing a substance obtained by extracting a bitter melon plant and / or a processed product thereof by using water and / or an organic solvent as an active ingredient and containing an effective amount to show an effect is suitable as a multidrug resistance inhibitor. Can be used for
[0019]
Here, the processed product of the bitter melon plant refers to one obtained by subjecting the bitter melon plant to processing such as crushing, grinding, drying, squeezing, and concentration.
[0020]
As an organic solvent, for example, methanol, dimethyl sulfoxide, ethanol, n-propyl alcohol, i-propyl alcohol, n-butyl alcohol, i-butyl alcohol, sec-butyl alcohol, t-butyl alcohol, acetone, tetrahydrofuran, dimethylformamide and the like An organic solvent having high polarity can be used. Then, one or more of these organic solvents can be selected and used. Among them, particularly preferred is methanol.
[0021]
An example of extracting a substance having an action of inhibiting P-glycoprotein from the aforementioned bitter melon plant and / or its processed product using water and / or an organic solvent is described below, but the present invention is limited to this extraction example. It is not done.
[0022]
An organic solvent (preferably methanol) is added to the freeze-dried powder of the bitter melon plant, shaken, and then separated into a supernatant and a precipitate by centrifugation. The supernatant is concentrated under reduced pressure and freeze-dried to obtain an extract.
[0023]
As a method for confirming the P-glycoprotein inhibitory effect of the extract thus obtained, an evaluation method described in Examples described later can be used.
[0024]
Combining the multidrug resistance inhibitor with other optional ingredients commonly used for medicine or food, a multidrug resistance-suppressing pharmaceutical composition capable of inhibiting P-glycoprotein or A food composition can be provided.
<2> A pharmaceutical composition containing the multidrug resistance inhibitor of the present invention.
[0025]
The pharmaceutical composition of the present invention contains the above-mentioned multidrug resistance inhibitor according to a conventional method, and is not particularly limited as long as the action of inhibiting a P-glycoprotein can be expected. .
[0026]
The dosage form of the pharmaceutical composition of the present invention is not particularly limited, but is generally mixed with one or more pharmaceutically acceptable carriers, excipients, integrating agents, preservatives, stabilizers, flavoring agents, and the like. In addition, it is preferable to use an internal dosage form such as a tablet, a granule, a capsule, a liquid medicine, and a drink. Such a formulation can be generally formulated according to a method used for manufacturing a medicament.
[0027]
The dosage of the pharmaceutical composition varies depending on the type and condition of the disease, the age and weight of the patient, etc., but is extracted from a bitter melon plant and / or its treated product using water and / or an organic solvent per day for an adult. It is preferable to administer a pharmaceutical composition containing a multidrug resistance inhibitor containing 50 mg to 10000 mg of a substance having an action of inhibiting a P-glycoprotein obtained by the above method in one or several divided doses.
<3> Food Composition Containing the Multidrug Resistance Inhibitor of the Present Invention The food composition of the present invention is obtained by blending the above multidrug resistance inhibitor according to a conventional method.
[0028]
The food composition of the present invention is not particularly limited as long as the action of inhibiting the above-mentioned P-glycoprotein can be expected, but it is an optional ingredient commonly used as a food in various foods. At the same time, a required amount of the extract can be blended with the food material. It is not necessary to pay special attention when blending this extract, and it is possible to manufacture health foods and functional foods by processing and manufacturing by a normal manufacturing method.
[0029]
The compounding amount varies depending on the type of food, but in order to obtain the effect of inhibiting the P-glycoprotein without deteriorating the taste of the food, P-glycoprotein is inhibited with respect to the total amount of the food composition. It is preferable to mix a substance having an action of 0.1 to 20% by weight, more preferably 0.5 to 5% by weight.
[0030]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples.
[0031]
Example 1 <Sample extraction method>
The sample was extracted as follows.
[0032]
After washing a commercially available bittern, the seed part and both ends were removed, crushed, and freeze-dried to powder.
[0033]
40% methanol (50 ml) was added to 5 g of the freeze-dried bitter melon and shaken at room temperature for 20 minutes. Thereafter, the mixture was separated into a supernatant and a precipitate by centrifugation (3,000 rpm × 15 minutes), and the supernatant was concentrated under reduced pressure and lyophilized to obtain 2.3 g of an extract.
[0034]
Example 2 <Evaluation of inhibitory activity of P-glycoprotein>
The evaluation of the inhibitory activity of P-glycoprotein was performed using the following in vitro evaluation system.
<Evaluation 1>
Using the bitter gourd extract extracted by the above-mentioned sample extraction method (extraction with 40% methanol), a sample of bitter gourd extract whose concentration was changed in the range of 0.1 to 5.0 mg / ml was prepared.
[0035]
Caco-2 cells were obtained from the American Type Culture Collection (Rockville, MD, USA) (ATCC HTB-37), 10% fetal bovine serum, 1% non-essential amino acids, 2 mM L-glutamine, 100 units. The cells were cultured in a Dulbecco's modified Eagle's medium supplemented with 100 μg / ml penicillin G and 100 μg / ml streptomycin at 37 ° C. in a 5% carbon dioxide / 95% air atmosphere.
[0036]
Caco-2 cells were seeded on a 24-well plate at 1.4 × 10 ⁵ cells each and cultured at 37 ° C. for 14 days. Thereafter, the medium was removed and washed with a phosphate buffered saline. Then, HBSS (136.7 mM sodium chloride, 5.4 mM potassium chloride, 0.95 mM calcium chloride dihydrate, 0.81 mM magnesium sulfate heptahydrate, 0.44 mM potassium dihydrogen phosphate, 0.39 mM phosphate 700 μl of a solution prepared by adding 4 mM sodium bicarbonate and 10 mM Hepes to disodium dodecahydrate (25 mM D-glucose) and correcting the pH to 7.4 (hereinafter also referred to as “uptake buffer”) was added thereto, and equilibrated for 10 minutes. went.
[0037]
300 μl of a reaction solution (sample and 40 nM (0.2 μCi / ml) [ ³ H] -daunomycin (Daunomycin) dissolved in uptake buffer) were added, and after standing for 2 hours, 300 μl of 0.1% Triton X-100 was added. After solubilizing the cells, the amount of [ ³ H] -daunomycin incorporated into Caco-2 cells was measured by a liquid scintillation counter (ALOKA).
[0038]
Table 1 shows the results of evaluation using samples of bitter melon extract having different concentrations of 0.1 to 5.0 mg / ml according to the above method.
[0039]
As is clear from Table 1, the bittern extract had a concentration-dependent [ ³ H] -daunomycin uptake action.
[0040]
[Table 1]

<Evaluation 2>
In the above-mentioned sample extraction method, a sample of bitter melon extract extracted with a 0% to 100% aqueous methanol solution while changing the concentration of methanol was prepared.
[0041]
According to the above method, evaluation was carried out using a bitter melon extract extracted with a 0% to 100% aqueous methanol solution and a sample having a concentration of 2 mg / ml.
[0042]
As a result, as shown in Table 2, a strong [ ³ H] -daunomycin incorporation effect was observed in each extract.
[0043]
[Table 2]

<Evaluation 3>
A sample of bitter melon extract having a concentration of 2 mg / ml extracted by the sample extraction method (extraction with 40% methanol) was used.
[0044]
The Caco-2 cells were cultured according to the method described above.
[0045]
Caco-2 cells were seeded at 1 × 10 ⁵ on a 12-well permeable plate and cultured at 37 ° C. for 14 days. Then, after washing with an uptake buffer, 500 μl of the uptake buffer was added, and equilibration was performed for 30 minutes.
[0046]
A reaction solution (2 mg / ml sample and 100 nM (0.5 μCi / ml) of [ ³ H] -daunomycin dissolved in uptake buffer) was added to the tip side (Apical side) of the cells, and the mixture was added for a certain period (15 minutes). , 30, 60, 90, and 120 minutes), 50 μl of a solution on the basal side of the cells (Basolateral side) was recovered, dissolved in 3 ml of Clearsol, and then permeated to the basal side of the cells by a liquid scintillation counter (ALOKA) [ ³ H]. ] -The amount of daunomycin was measured.
[0047]
Table 3 shows the results of the evaluation of the samples of bitter melon extract at a concentration of 2 mg / ml extracted by the above-described sample extraction method (extraction with 40% methanol) after each elapse of time according to the above method.
[0048]
As a result, as shown in Table 3, the action of promoting the permeation of [ ³ H] -daunomycin from the apical side of the cells to the basal side of the cells was observed in the bitter melon extract.
[0049]
[Table 3]

<Evaluation 4>
A sample of bitter melon extract having a concentration of 2 mg / ml extracted by the sample extraction method (extraction with 40% methanol) was used.
[0050]
A 2 mg / ml sample dissolved in an uptake buffer was added to the tip of the cells, and a 100 nM (0.5 μCi / ml) [ ³ H] -daunomycin dissolved in an uptake buffer was added to the basal side of the cells. Others were performed according to the method of the above-mentioned evaluation 3.
[0051]
The sample and [< ³ > H] -daunomycin were added to the cell tip side and the cell base side, respectively, as described above, and after a certain period of time (15, 30, 60, 90, 120 minutes), 50 [mu] l of the solution at the cell tip side was added. After collecting and dissolving in 3 ml of Clearsol, the amount of [ ³ H] -daunomycin permeated to the cell tip side was measured by a liquid scintillation counter (ALOKA).
[0052]
Table 4 shows the results of the evaluation after each elapse of time according to the above method using a sample of the bitter melon extract having a concentration of 2 mg / ml extracted by the above-described sample extraction method (extraction with 40% methanol).
[0053]
As a result, as shown in Table 4, the action of inhibiting bitter gourd extract from inhibiting permeation of [ ³ H] -daunomycin from the basal side of the cell to the tip side of the cell was observed.
[0054]
[Table 4]

In Evaluation 3 and Evaluation 4, Caco-2 cells, which are intestinal epithelial cells, are arranged regularly in the culture solution, and the tip side (Apical side) of Caco-2 cells is intestinal tract in vivo. The basal side of the Caco-2 cells corresponds to the inner side of the intestinal wall in the living body. Nutrient components and drugs are taken in from the tip side (Apical side) of the cell.
[0055]
In the experiments of Evaluations 3 and 4 described above, the addition of the bitter gourd extract maintained the uptake of the drug from the tip side of the cell to the basal side of the cell, and the drug from the basal side of the cell to the tip side of the cell. It was clarified that the emission of water was suppressed.
[0056]
Thus, when the drug is administered, the action of the P-glycoprotein on the intestinal epithelial cells returns the drug to the lumen of the intestinal tract, but the action of the P-glycoprotein is inhibited by adding the bitter melon extract ( It can be said that the multidrug resistance was improved) and the transfer of the administered drug to the intestinal lumen side was suppressed.
[0057]
As described above, as is clear from the above evaluation experiment, the action of inhibiting the P-glycoprotein was observed in the extract obtained from the bitter melon plant and / or the processed product thereof. Therefore, an agent containing, as an active ingredient, a substance obtained by extracting a bitter melon plant and / or a processed product thereof with water and / or an organic solvent can be suitably used as a multidrug resistance inhibitor.
[0058]
【The invention's effect】
According to the present invention, a novel multidrug resistance inhibitor containing a component that is safe and easy to prepare and has a function of inhibiting the function of a novel P-glycoprotein can be provided.

Claims (4)

A multidrug resistance inhibitor comprising, as an active ingredient, a substance having an action of inhibiting P-glycoprotein obtained by extracting watermelon plants and / or processed products thereof with water and / or an organic solvent. The multidrug resistance inhibitor according to claim 1, wherein the organic solvent is methanol. A pharmaceutical composition comprising the multidrug resistance inhibitor according to claim 1. A food composition comprising the multidrug resistance inhibitor according to claim 1.