JP2694701B2 - Liquid film cancer drug - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention relates to a water-soluble anticancer agent except for phospholipids, micelle surfactants and hormones, or a liquid film anticancer agent comprising a functional hybrid molecular assembly composed of flavonoids. is there.

(Prior art) Alkylating agents (nitrogen mustards, ethyleneimines, alkylsulfonic acids, nitrosoureas) as cancer chemotherapeutic agents, antimetabolites (folic acid antagonists, purine antagonists, pyrimidine antagonists) , Plant fission toxins (colcemid, vinblastine, etc.), antibiotics (sarcomycin, carcinophylline, mitomycin, etc.),
Hormones (adrenal steroids, male hormones, female hormones), porphyrin complexes (murphyrin, cobalt protoporphyrin), etc. have been used.

(Problems to be Solved by the Invention) However, most of them are cytotoxic substances and exhibit serious side effects. Therefore, development of an antitumor agent having low toxicity and excellent antitumor activity has been strongly desired.

Therefore, the present inventors searched for and studied a substance having low toxicity and anticancer activity, and found a functional hybrid molecular assembly consisting of a phospholipid, a water-soluble anticancer agent except for micelle surfactant and hormones, or a flavonoid. It was found that it exhibits excellent antitumor activity against human tumor cells.
Therefore, an object of the present invention is to provide a water-soluble anticancer agent excluding this active agent and hormones, or a liquid film anticancer agent comprising flavonoids.

Another object of the present invention is to provide an antitumor agent which can be an excellent cancer chemotherapeutic agent not only for humans but also for warm-blooded animals such as domestic animals, dogs and cats.

(Means for Solving the Problems) The present invention is a liquid film anticancer agent in which a water-soluble anticancer agent excluding hormones or a flavonoid is added to a hybrid molecular assembly formed by a phospholipid and a micelle surfactant.

A water-soluble anticancer agent other than hormones or a flavonoid is added to a liquid membrane of a phospholipid-micelle mixed molecular assembly, and an excellent anticancer effect is sought by this liquid membrane. That is, even if an aqueous solution cannot be obtained with a water-soluble anti-cancer agent except flavours or flavonoids alone, or even if the anti-cancer effect is small, the anti-cancer effect of the liquid film itself based on the fusion of the liquid film to cancer cells can be obtained by forming a liquid film. In addition to the effect, a synergistic anti-cancer effect of a water-soluble anticancer agent or flavonoid excluding hormones that are encapsulated in a liquid film can be expected.

The active ingredient constituting the liquid film antitumor agent of the present invention is a surfactant that forms vesicle-forming phospholipids and micelles, and a water-soluble anticancer agent or flavonoid excluding hormones, and these compounds include, for example: Can be mentioned.

The phospholipids for making vesicles are represented by the following general formula (I)
An L-form diacylphosphatidylcholine represented by is preferably mentioned.

(However, in the formula, n represents an even number of 12 to 18.) Other examples include phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and the like. The phospholipid may be a mixture, and lecithin and the like are preferable. Can be used.

As a surfactant for forming micelles, the following general formula (II) is used.
Examples thereof include a methyl-type cationic surfactant represented by and a polyoxyethylene-type neutral surfactant represented by the general formulas (III) and (IV).

_{CH 3 (CH 2) n N} + (CH 3) 3 X - (II) ( In the formula, n represents an even number of 12 to 18, X is a halogen atom.) (However, in the formula, n represents 10 to 40.) (However, in the formula, a + b + c = 20, R = H (CH ₂ ) _n-1 ,
n is an even number from 12 to 18).

Alkylating agents, antimetabolites as water-soluble anticancer agents,
Plant fission toxins, antibiotics, and porphyrin complexes, and the like, adriamycin, actinomycete-producing antibiotics, aclarubicin, etc., plant fission toxins vinblastine, vincrustine, vindesine, etc., and flavonoids, chalcones, flavanones, Flavone,
Flavonols, flavanonols, flavanols, isoflavones, anthocyans, catechins, flavans 3,4-diols, and the like. Quercetin, quercitrin, rutin having a hydroxyl group or a substituent at the 3-position, such as a hydroxyl group or a substituent at the 7-position. Specific examples thereof include baicalein and baicalin.

As the constitution of the liquid membrane, it is desirable that the phospholipids forming vesicles have a molar concentration of (1-10) × 10 ^-5 and the surfactant forming micelles have a concentration of 3 × 10 ^-6 -5 × 10 ^-5 . If the concentration is too low, there is no effect. If the concentration is too high, the solution is not uniform and the cytotoxicity is too strong, and it is economically disadvantageous.

The method for producing the liquid membrane is as follows. After dissolving cholesterol or the like in an organic solvent in a phospholipid and distilling off the solvent under reduced pressure,
There is a method of obtaining a liquid film by subjecting an emulsion containing an aqueous buffer solution to ultrasonic treatment, followed by column treatment and centrifugation, and this method can be applied. Furthermore, as shown in the examples, in the method of adding a synthetic surfactant having low toxicity to phospholipids to an aqueous buffer solution at an appropriate mixing ratio and obtaining a liquid film of any size by one ultrasonic treatment,
It is easy and safe with no fear of mixing organic solvent.

The water-soluble anticancer agent or flavonoid excluding hormones is preferably in the range of (1-8) × 10 ⁻⁶ molar concentration. If the concentration is too low, there is no anti-cancer activity, and if it is too high, it becomes insoluble in the liquid film, which is uneconomical.

For the toxicity test on the cultured cells, for example, a hybridoma of human lymphoma-human lymphoid B cell fusion cells (HF-323) can be used. That is, a liquid membrane prepared by adding a water-soluble anticancer drug except hormones or flavonoids to a hybrid molecular assembly formed by a phospholipid and a micellar surfactant is sterilized by filtration with a 0.2 μm filter, and then added to a hybridoma to obtain sterilized serum. Medium (FCS: Grando Island
Biological Company) and culture at 36.8 ° C. for 4 days.

The number of cells can be measured by a hemocytometer, and the survival rate can be measured by observing cells stained with trypan blue with a microscope.

Human lymphoma-human lymphoid B cell fusion cell (HF-323)
When the antitumor activity of the liquid film added to the hybridoma was examined, the liquid film itself containing the phospholipid-micelle surfactant showed the anticancer effect of reducing the number of cells.
The anticancer effect was more remarkable in the liquid film containing the water-soluble anticancer drug except the hormones or the flavonoid.

(Effect of the Invention) According to the present invention, it is possible to provide a liquid film anticancer agent in which a water-soluble anticancer agent excluding hormones or a flavonoid is added to a mixed molecular assembly formed by a phospholipid and a micelle surfactant. Is a substance with low toxicity and excellent anticancer activity. As a result, a carcinostatic agent having a functional hybrid molecular assembly consisting of a water-soluble anticancer agent excluding phospholipids, micellar surfactants, and hormones or flavonoids can be obtained. A cancer chemotherapeutic agent having an excellent effect on blood animals was obtained.

(Examples) In Examples 1, 2 and 3, flavonoids were added to a liquid film formed by two kinds of surfactants, a phospholipid molecule and a micelle molecule, and a toxicity test was conducted. Table 1 shows the results.
It was shown to.

In Comparative Examples 1 to 5, toxicity tests were conducted on phosphate buffer, quercitrin and phosphate buffer as flavonoids, quercetin and phosphate buffer, and surfactant and phosphate buffer.

 The results are shown in Table 1.

Example 1 The following formula in which n in the formula (I) is 12: L-α-dilauroylphosphatidylcholine (manufactured by NOF CORPORATION) 0.0312 g (1 × 10 ⁻³ mol), CH ₃ C
_{(CH 3) 2 CH 2 C} (CH 3) 2 -Ph-O- (CH 2 CH 2 O) 10 polyethylene glycol represented by -H (10) p-1,1,3,3-
Tetramethyl butyl phenyl ether (NOF Corporation)
Made) 0.0420 g (1.3 × 10 ^-3 mol), and quercitrin 0.
0022 g (1 × 10 ^-4 mol) of 1 × 10 ^-4 mol phosphate buffer 50 m
The liquid film was manufactured by placing the tube in a 1-hour chamber and sonicating at 50 ° C. (Bransonic Model B 3200 apparatus (manufactured by Branson)) for 1 hour.

In order to examine the antitumor effect of the obtained liquid film, the liquid film was sterilized by filtration through a 0.2 μm filter, and then 50 μl of the liquid film was mixed with human lymphoma-human lymphocyte B cell fusion cells (HF-323) having an initial cell number of 13 × 10 ⁴ cells / ml. 1) was added to 1 ml of a medium containing the hybridoma and cultured at 36.8 ° C. for 4 days.

The cell number was measured with a hemocytometer, and the viability was measured by observing cells stained with trypan blue with a microscope.

Example 2 A liquid film was produced in the same manner as in Example 1 except that 0.0015 g (1 × 10 ⁻⁴ mol) of quercetin was used as the flavonoid, and the carcinostatic effect was examined in the same manner as in Example 1.

Example 3 (Hexadecyltrimethylammonium chloride, manufactured by NOF CORPORATION) 0.0048 g (3 x 10 ^-4 mol) and quercitrin 0.0029 g (1.3 x 10 ^-4 mol) were used, and L-α-
The same amount of dilauroylphosphatidylcholine was used as in Example 1, a liquid film was produced in the same manner as in Example 1, and the anticancer effect was examined in the same manner as in Example 1 except that 25 μl of the liquid film was added to 1 ml of the medium.

Comparative Examples 1 to 5 50 ml (3 × 10 ⁻³ mol) of the phosphate buffer used in Examples 1 to ³ (Comparative Example 1), 0.0034 g of quercitrin (1.5 × 10 ⁻⁴ mol) (Comparative Example 2), quercetin 0.0023 g (1.5 × 10 ⁻⁴ mol) (Comparative Example 3), L-α-dilauroylphosphatidylcholine 0.0312 g (1 × 10 ⁻³ mol) and polyethylene glycol (10) p-1,1,3,3-tetra Hybrid liquid membrane of 0.0420 g (1.3 × 10 ⁻³ mol) of methylbutyl phenyl ether (Comparative Example 4), 0.0312 g (1 × 10 ⁻³ mol) of L-α-dilauroylphosphatidylcholine and 0.0048 g of hexadecyltrimethylammonium chloride. (3 x 1
A hybrid liquid membrane (0 ⁻⁴ mol) (Comparative Example 5) was prepared in the same manner as in Example 1 and then subjected to the same toxicity test as in Example 1. However, in Comparative Example 5, 25 μm of the liquid film was added to 1 ml of the medium.

Next, in Examples 4, 5 and 6, a water-soluble anticancer agent was added to a liquid film formed by two kinds of surfactants of a phospholipid and a micelle molecule, and a toxicity test was conducted. Table 2 shows the results.
It was shown to.

In Comparative Examples 6 to 8, toxicity tests were conducted on a phosphate buffer solution, peplomycin sulfate as a water-soluble anticancer agent and a phosphate buffer solution, and a surfactant and a phosphate buffer solution.

 The results are shown in Table 2.

Example 4 The following formula in which n in the formula (I) is 12: L-α-dilauroylphosphatidylcholine (manufactured by NOF CORPORATION) 0.0312 g (1 × 10 ⁻³ mol), CH ₃ C
_{(CH 3) 2 CH 2 C} (CH 3) 2 -Ph-O- (CH 2 CH 2 O) 10 Polyoxyethylene (10) represented by -H p-1,1,3,3-tetramethyl Butylphenol (manufactured by NOF CORPORATION) 0.0420
g (1.3 × 10 ^-3 mol) and 0.0204 g (2.6 × 10 ^-4 mol) of peplomacinsulfate (Nippon Kayaku Co., Ltd.) were placed in 50 ml of 1 × 10 ^-4 mol phosphate buffer and sonicated at 40 ° C. Bransonic Mo
A del B 3200 apparatus (manufactured by Branson) was used for 1 hour to produce a liquid film.

In order to examine the antitumor effect of the obtained liquid film, the liquid film was sterilized by filtration through a 0.2 μm filter, and then 50 μl of the liquid film was mixed with human lymphoma-human lymphocyte B cell fusion cells (HF-323) having an initial cell number of 13 × 10 ⁴ cells / ml. 1) was added to 1 ml of a medium containing the hybridoma and cultured at 36.8 ° C. for 4 days.

The cell number was measured with a hemocytometer, and the viability was measured by observing cells stained with trypan blue with a microscope.

Examples 5 and 6 As water-soluble anticancer agent, doxorubicin hydrochloride (manufactured by Kyowa Hakko Co., Ltd.) 0.0075 g (2.6 x 10 ^-4 mol) (Example 5), aclarubicin hydrochloride (manufactured by Sanraku) 0.0110 g (2.6 x 10 ^-4 mol) )
The anticancer effect was examined in the same manner as in Example 4 except that (Example 6) was used.

Comparative Examples 6-8 50 ml (3 × 10 ⁻³ mol) of the phosphate buffer used in Examples 4-6 (Comparative Example 6), 0.0204 g of peplomycin sulfate (2.6 ×)
10 ⁻⁴ mol) (Comparative Example 7), 0.0312 g (1 × 10 ⁻³ mol) of L-α-dilauroylphosphatidylcholine, and polyoxyethylene (10) p-1,1,3,3-
A hybrid liquid membrane (Comparative Example 8) containing 0.0420 g (1.3 × 10 ⁻³ mol) of tetramethylbutylphenol was prepared in the same manner as in Example 4 and then subjected to the same toxicity test as in Example 4.

Claims (1)

(57) [Claims] 1. A liquid film antitumor agent in which a water-soluble anticancer agent excluding hormones or flavonoids is added to a mixed molecular assembly formed of a phospholipid and a micellar surfactant.