JP4324351B2 - Multidrug resistance inhibitor - Google Patents

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１×１０⁵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]
BACKGROUND OF THE INVENTION
The present invention relates to a multidrug resistance suppressor containing a substance having an action of inhibiting P-glycoprotein as an active ingredient.
[0002]
[Prior art]
Overcoming cancer is a common problem for humankind, and many anticancer drugs have been developed to date. On the other hand, the fact that cancer cells are resistant to a plurality of anticancer drugs and other drugs (multi-drug resistance) is a major problem in the treatment with anticancer drugs.
[0003]
Although there are still many unclear points regarding the mechanism by which cancer cells develop multidrug resistance, basically, many of the multidrug resistant cancer cells overproduce P-glycoprotein, and this P-glycoprotein Are thought to be responsible for the transport of anticancer drugs out of the cell. 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 whose structures are not similar and transports drugs out of the cell, once P-glycoprotein is expressed in cancer cells, Will also be resistant. In fact, it is known that many structurally different anticancer agents such as adriamycin, vinblastine, vincristine, actinomycin D, colchicine and the like 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 that inhibit the function of P-glycoprotein have been developed. 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 medica 6 (5), 442- 445 (1991)).
[0006]
However, in the method using the above monoclonal antibody, such an antibody is an antibody derived from another animal (for example, mouse), so that its own antigenicity is strong and its clinical use is not always easy.
[0007]
In addition, calcium antagonists such as verapamil, and immunosuppressive agents such as cyclosporin A and FK506 have been reported to inhibit the function of P-glycoprotein.
[0008]
However, when these are administered for the purpose of overcoming multidrug resistance, calcium antagonistic action and immunosuppressive action, which are their original actions, appear as powerful side effects. For example, calcium antagonists such as verapamil have strong vasodilatory effects and may induce shocks such as hypotension.
[0009]
Therefore, a new substance with a low possibility of side effects has been demanded.
[0010]
On the other hand, for bitter gourd, there is a report that the pulverized product or extract improves lipid metabolism (Japanese Patent Laid-Open No. 2001-278804). Was not known at all.
[0011]
[Problems to be solved by the invention]
The present invention has been made from the above viewpoint, and it is an object of the present invention to provide a multidrug resistance inhibitor containing a component that has a function of inhibiting the function of a novel P-glycoprotein that is safe and easy to prepare. .
[0012]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventor has developed a function of P-glycoprotein on a substance obtained by extraction from a bitter melon plant and / or a processed product thereof using water and / or an organic solvent. As a result, the present invention was completed.
[0013]
That is, the present invention is as follows.
(1) Multidrug resistance suppression for anticancer agents containing as an active ingredient a substance having an action of inhibiting P-glycoprotein obtained by extraction from a bitter melon plant and / or its treated product using water and / or an organic solvent Agent.
(2) The multidrug resistance inhibitor according to (1), wherein the organic solvent is methanol.
(3) A pharmaceutical composition for suppressing multidrug resistance against an anticancer agent, comprising the multidrug resistance suppressor according to (1) or (2) .
[0014]
DETAILED DESCRIPTION OF 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 resistance to a drug (hereinafter also referred to as “target drug”), in other words, a target drug. It is a drug that can suppress the excretion of cells from the cell. Particularly in the present invention, a drug that can specifically inhibit the function of P-glycoprotein can be used as a multidrug resistance suppressor.
[0015]
Here, the target drugs to be suppressed include alkylated anticancer agents such as cyclophosphamide and melphalan, antimetabolite anticancer agents such as fluorofuracil, daunomycin, mitomycin C and the like. 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 a drug resistance suppressing effect is recognized in at least one of the above target drugs.
[0017]
The multidrug resistance suppressor of the present invention contains, as an active ingredient, a substance having an action of inhibiting P-glycoprotein obtained by extraction from a bitter melon plant and / or a processed product thereof using water and / or an organic solvent. To do.
[0018]
A substance obtained by extraction from a bitter melon plant and / or its treated product using water and / or an organic solvent has an action of inhibiting P-glycoprotein, as is apparent from the following examples. Therefore, an agent containing, as an active ingredient, a substance obtained by extraction from a bitter melon plant and / or its treated product using water and / or an organic solvent, an agent containing an effective amount for showing an effect is suitable as a multidrug resistance inhibitor. Can be used for
[0019]
Here, the processed product of a bitter gourd plant means what processed the bitter gourd plant, such as crushing, grinding, drying, squeezing, concentration, etc.
[0020]
Examples of the organic solvent include 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. A highly polar organic solvent can be mentioned. And 1 type (s) or 2 or more types can be selected and used among these organic solvents. Among these, methanol can be particularly preferably used.
[0021]
Although the example which extracts the substance which has the effect | action which inhibits P-glycoprotein using water and / or an organic solvent from said bittern plant and / or its processed material is illustrated below, this invention is limited to this extraction example. Is not to be done.
[0022]
An organic solvent (preferably methanol) is added to the lyophilized powder of bitter melon plant, shaken, and then separated into a supernatant and a precipitate by centrifugation. The supernatant is concentrated under reduced pressure and lyophilized to obtain an extract.
[0023]
As a method for confirming the P-glycoprotein inhibitory action of the extract thus obtained, the evaluation methods shown in Examples below can be used.
[0024]
A pharmaceutical composition for suppressing multidrug resistance, which can inhibit P-glycoprotein by combining the multidrug resistance suppressor with other optional components usually used for pharmaceuticals or foods. 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 is not particularly limited as long as the above-mentioned multidrug resistance inhibitor is formulated according to a conventional method and can be expected to inhibit P-glycoprotein. .
[0026]
The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and is generally mixed with one or more pharmaceutically acceptable carriers, excipients, integrating agents, preservatives, stabilizers, flavoring agents and the like. Thus, it is preferable to adopt an internal dosage form such as a tablet, granule, capsule, liquid medicine, or drink. Such formulation can be formulated usually according to a method used for production of a medicine.
[0027]
The dosage of the above pharmaceutical composition varies depending on the type of disease, symptoms, patient age, body weight, etc., but is extracted from a bitter melon plant and / or its treated product using water and / or an organic solvent per day for adults. It is preferable to administer a pharmaceutical composition containing a multidrug resistance suppressant containing 50 mg to 10000 mg of a substance having an action of inhibiting P-glycoprotein obtained by dividing into 1 to several times.
<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 it can be expected to inhibit the above-mentioned P-glycoprotein, but it is an arbitrary component that is usually used as a food for various foods. At the same time, the required amount of the extract can be added to the food material. When blending this extract, there is no particular attention, and health foods and functional foods can be produced by processing and producing them by a normal production method.
[0029]
The blending amount varies depending on the type of food, but in order to obtain the effect of inhibiting the P-glycoprotein without impairing the taste of the food, the P-glycoprotein is inhibited with respect to the total amount of the food composition. It is preferable to add 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 extraction method was performed as follows.
[0032]
After washing commercially available bitter gourd, the seed part and both ends were removed, crushed and freeze-dried to be powdered.
[0033]
40% methanol (50 ml) was added to 5 g of bitter gourd freeze-dried and shaken at room temperature for 20 minutes. Thereafter, the supernatant and the precipitate were separated by centrifugation (3,000 rpm × 15 minutes), and the supernatant was concentrated under reduced pressure and then lyophilized to obtain 2.3 g of an extract.
[0034]
[Example 2]
<Evaluation of inhibitory activity of P-glycoprotein>
Inhibitory activity evaluation of P-glycoprotein was measured in the following in vitro evaluation system.
<Evaluation 1>
Using the bitter gourd extract extracted by the above sample extraction method (extracted with 40% methanol), a sample of bitter gourd extract having a concentration varied from 0.1 to 5.0 mg / ml was prepared.
[0035]
Caco-2 cells were obtained from American Type Culture Collection (Rockvile, MD, USA) (ATCC HTB-37), 10% fetal bovine serum, 1% non-essential amino acid, 2 mM L-glutamine, 100 unit / ml penicillin G, The cells were cultured in Dulbecco's modified Eagle medium supplemented with 100 μg / ml streptomycin at 37 ° C. in a 5% carbon dioxide / 95% air atmosphere.
[0036]
Caco-2 cells were seeded at 1.4 × 10 ⁵ in a 24-well plate and cultured at 37 ° C. for 14 days. Thereafter, the medium was removed and washed with phosphate buffered saline. 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 disodium phosphate dodecahydrate Product, 25 mM D-glucose) and 700 μl of 4 mM sodium bicarbonate and 10 mM Hepes added to pH 7.4 (hereinafter also referred to as “uptake buffer”) were added and equilibrated for 10 minutes.
[0037]
Add 300 μl of the reaction solution (sample and 40 nM (0.2 μCi / ml) [ ³ H] -Daunomycin dissolved in uptake buffer) and leave it for 2 hours, then add 300 μl of 0.1% Triton X-100. After solubilizing the cells, the amount of [ ³ H] -daunomycin incorporated into Caco-2 cells was measured with a liquid scintillation counter (ALOKA).
[0038]
Table 1 shows the results of evaluation according to the above method using bitter melon extract samples having different concentrations of 0.1 to 5.0 mg / ml.
[0039]
As apparent from Table 1, the concentration-dependent [ ³ H] -daunomycin uptake action was observed in the bitter gourd extract.
[0040]
[Table 1]

<Evaluation 2>
In the above sample extraction method, a sample of bitter gourd extract extracted with 0% to 100% methanol aqueous solution at different concentrations of methanol was prepared.
[0041]
According to the above method, evaluation was performed using a bitter gourd 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, each extract had a strong [ ³ H] -daunomycin uptake action.
[0043]
[Table 2]

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

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

In Evaluation 3 and Evaluation 4, Caco-2 cells, which are intestinal epithelial cells, are regularly arranged in the culture medium, and the tip side (Apical side) of Caco-2 cells is in the intestinal tract in vivo. The basal side (Basolateral side) of Caco-2 cells corresponds to the inner side of the intestinal tract wall in the living body. Nutritional components and drugs are taken in from the tip side (Apical side) of the cell.
[0055]
In the experiments of Evaluations 3 and 4 above, the addition of the bitter melon extract maintains the uptake of the drug from the cell tip side to the cell basal side, and the drug from the cell basal side to the cell tip side. It has become clear that the emission of is suppressed.
[0056]
Thus, when a drug is administered, the drug is returned to the intestinal lumen side by the action of P-glycoprotein on the intestinal epithelial cells, but the action of P-glycoprotein is inhibited by adding bittern extract ( Multidrug resistance was improved), and it can be said that the administration of the administered drug to the intestinal lumen side was suppressed.
[0057]
As described above, as apparent from the above evaluation experiment, the action of inhibiting P-glycoprotein was observed in the extract obtained from the bitter melon plant and / or its treated product. Therefore, the agent which contains as an active ingredient the substance obtained by extracting from a bitter melon plant and / or its processed material using water and / or an organic solvent can be used suitably as a multi-drug resistance inhibitor.
[0058]
【The invention's effect】
According to the present invention, it was possible 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.

Claims (3)

The multidrug resistance inhibitor with respect to the anticancer agent which contains as an active ingredient the substance which has the effect | action which inhibits P-glycoprotein obtained by extracting from a bitter melon plant and / or its processed material using water and / or an organic solvent.   The multidrug resistance inhibitor according to claim 1, wherein the organic solvent is methanol. A pharmaceutical composition for suppressing multidrug resistance against an anticancer agent, comprising the multidrug resistance suppressor according to claim 1 or 2.