JP2006206607A - Intra-cancer-cell nuclease activator - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new drug efficacious for cancer therapy and containing poly(I)-poly(C). <P>SOLUTION: The intra-cancer-cell nuclease activator contains a composite comprising a carrier prepared from 2-O-(2-diethylaminoethyl)-carbamoyl-1,3-O-dioleoylglycerol and a phospholipid as essential components and poly(I)-poly(C) or mismatched poly(I)-poly(C). <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a cancer intracellular nuclease activator. Here, the “cancer cell nuclease activator” refers to a drug that can activate a nuclease in cancer cells to induce apoptosis in the cancer cells and kill the cancer cells. “I” means inosinic acid, “C” means cytidylic acid, “A” means adenylic acid, and “U” means uridylic acid.

  Mismatched poly (I) / poly (C) and mismatched poly (A) / poly (U) are well known to those skilled in the art, but are partially complementary to the nucleobases constituting the double strand. It means poly (I) · poly (C) and poly (A) · poly (U) containing unfavorable bases.

  Poly (I) · poly (C) is a double-stranded RNA of a polyribonucleotide copolymer composed of polyinosinic acid and polycytidylic acid, and is widely known as a drug having a strong interferon-inducing ability and an immunostimulatory action. And since such poly (I) and poly (C) have an immunostimulatory action, it is considered that the proliferation of cancer cells can be indirectly suppressed by an immune reaction, and the possibility as a cancer therapeutic agent has been sought so far. It has been. However, the indirect action by the immune reaction of poly (I) / poly (C) cannot effectively suppress the growth of cancer cells, and poly (I) / poly (C) is developed as a cancer therapeutic agent. Not reached. Poly (I) and poly (C) have not yet been developed for other indications based on interferon-inducing ability and immunostimulatory action.

  For poly (A) / poly (U), mismatched poly (I) / poly (C), and mismatched poly (A) / poly (U), which are polyribonucleotide copolymers composed of polyadenylic acid and polyuridylic acid, It is thought that it has the effect | action similar to poly (I) * poly (C) to some extent.

  On the other hand, as an effective carrier for transferring a drug into a cell, for example, Lipofectin (registered trademark) generally called a cationic liposome, or 2-0- (2 related to the following structural formula [I] Carriers formed from glycerol derivatives such as -diethylaminoethyl) carbamoyl-1,3-0-dioleoylglycerol and phospholipids as essential constituents are known (for example, see Patent Document 1 and Patent Document 2).

上記カチオニック・リポソームは、脂質二分子膜からなる、水溶液中で正電荷を持った小胞体であると考えられる。かかるカチオニック・リポソームは水溶液中で正電荷を帯び、ｐｏｌｙ（Ｉ）・ｐｏｌｙ（Ｃ）等の二本鎖ＲＮＡは水溶液中で負電荷を帯びることから、カチオニック・リポソームとｐｏｌｙ（Ｉ）・ｐｏｌｙ（Ｃ）等とは容易に複合体を形成することができる。

The cationic liposome is considered to be a vesicle made of a lipid bilayer and having a positive charge in an aqueous solution. Such cationic liposomes are positively charged in an aqueous solution, and double-stranded RNAs such as poly (I) · poly (C) are negatively charged in an aqueous solution, so that cationic liposomes and poly (I) · poly ( A complex can be easily formed with C) and the like.

However, double-stranded RNAs such as poly (I) / poly (C) themselves and complexes of them with cationic liposomes activate nucleases in cancer cells to induce apotones in cancer cells and kill them. Whether or not was known at all.
WO91 / 17424 pamphlet International Publication No. 94/19314 Pamphlet

  An object of the present invention is to provide a drug effective for cancer treatment. Moreover, the objective of this invention is providing the novel chemical | medical agent containing double stranded RNA, such as poly (I) * poly (C).

  As a result of intensive studies, the present inventors have found that a cancer cell nuclease activator is effective for cancer treatment and completed the present invention.

  Accordingly, the present invention relates to a cancer cell nuclease activator. Whether or not it is a cancer cell nuclease activator can be easily determined by conducting an experiment or the like for observing fragmentation of DNA or RNA as in Test Example 2 described later. For example, in the present invention, a carrier effective for transferring a drug into cells and poly (I) · poly (C) or mismatched poly (I) · poly (C) or poly (A) · poly (U) Alternatively, a nuclease activity in a cancer cell containing a complex with mismatched poly (A) / poly (U) (hereinafter, these double-stranded RNAs are collectively referred to as “poly (I) / poly (C) etc.”). And a cancer cell nuclease activator containing a complex of an agent, cationic liposome and poly (I) / poly (C).

  Specific examples of the present invention preferably include 2-0- (2-diethylaminoethyl) carbamoyl-1,3-0-dioleoylglycerol (hereinafter referred to as “the present glycerol derivative”) and phospholipid as essential components. Cancer cell nuclease activator (hereinafter “this activation”) containing a complex (hereinafter referred to as “the present complex”) of a carrier (hereinafter referred to as “the present carrier”) and poly (I) / poly (C), etc. An agent)).

Hereinafter, the activator which is a preferred embodiment of the present invention will be described in detail.
Although this carrier can generally be considered as a kind of cationic liposome, it does not necessarily have a form like a cationic liposome as long as it has a function of transferring a drug into cells.

The chain length of poly (I) · poly (C) and the like according to the present invention is not particularly limited. For example, in the case of poly (I) · poly (C), 50 to 2,000 bp (bp: base pair, Base pair number) is suitable, preferably 100 to 500 bp, more preferably 200 to 400 bp. If it is less than 50 bp, there may be a problem in effectiveness, and if it is longer than 2,000 bp, there may be a problem in safety. Poly (I) · poly (C) in the range of 100 to 500 bp is considered to be a chain length region in which effectiveness and safety are balanced in the present invention. Since poly (I) / poly (C) and the like usually exist with a certain distribution of various chain lengths, the chain lengths of poly (I) / poly (C) on the left mean the average distributed chain length. .
The phospholipid according to the present invention is not particularly limited as long as it is a pharmaceutically acceptable phospholipid. Specific examples include phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin, lecithin and the like. Mention may also be made of hydrogenated phospholipids. Preferred phospholipids include egg yolk phosphatidylcholine, egg yolk lecithin, soybean lecithin, and egg yolk phosphatide. Two or more phospholipids can also be used. It should be noted that phosphatidylcholine or lecithin can significantly reduce toxicity without a decrease in activity as compared with phosphatidylethanolamine generally used in cationic liposomes.

  The composition ratio of the present carrier to poly (I) / poly (C) in the complex varies depending on the type of phospholipid, the type of poly (I) / poly (C), the type of cancer, and the like. In addition, 0.05 to 10 parts by weight, such as poly (I) · poly (C), is suitably 0.1 to 4 parts by weight, and 0.5 to 2 parts by weight with respect to 10 parts by weight of the carrier. More preferred.

  The composition ratio of the present glycerol derivative and phospholipid in this carrier varies depending on the type and amount of poly (I) and poly (C) used, the type of phospholipid, etc., but relative to 1 part by weight of the present glycerol derivative. Thus, 0.1 to 10 parts by weight of phospholipid is suitable, 0.5 to 5 parts by weight is preferable, and 1 to 2 parts by weight is more preferable.

  The activator can take the form of, for example, a liquid (injection, instillation, etc.) in which the complex is dispersed in an aqueous solution or a lyophilized preparation thereof. In the case of a liquid agent, it is appropriate that the present complex is present within a concentration range of 0.001 to 25% (w / v), and within a concentration range of 0.01 to 5% (w / v). Is preferably present, and more preferably within a concentration range of 0.1 to 1% (w / v).

  The activator may contain an appropriate amount of any pharmaceutically acceptable additive such as an emulsification aid, a stabilizer, an isotonic agent, and a pH adjuster. Specifically, fatty acids having 6 to 22 carbon atoms (eg, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, arachidonic acid, docosahexaenoic acid) and pharmaceutically acceptable products thereof Salts (eg, sodium salts, potassium salts, calcium salts), emulsifying aids such as albumin and dextran, stabilizers such as cholesterol and phosphatidic acid, sodium chloride, glucose, maltose, lactose, sucrose, trehalose, etc. Examples include tonicity agents, hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, sodium hydroxide, potassium hydroxide, pH adjusters such as triethanolamine, and the like.

  This activator can be produced, for example, in the same manner as a general production method of liposomes. For example, first, a predetermined amount of water (water for injection, distilled water for injection, physiological saline, etc.) is added to a predetermined amount of the present glycerol derivative and phospholipid, and these are stirred and mixed, and this mixture is mixed with an appropriate disperser, for example, Dispersion using homomixer, homogenizer, ultrasonic disperser, ultrasonic homogenizer, high-pressure emulsifying disperser, microfluidizer (trade name), nanomizer (trade name), optimizer (trade name), Manton-Gaurin type high-pressure homogenizer To process. Then, a predetermined amount of poly (I) / poly (C) or the like is added thereto, and the mixture is again dispersed with an appropriate disperser to produce the present activator as an injection. Other optional additives can be added in an appropriate step before or after dispersion. In addition, the activator can be produced by adding water to a mixture of the glycerol derivative, phospholipid, and poly (I) / poly (C), etc. It can also be produced via coarse dispersion.

  Next, if the activator obtained by the above dispersion treatment is freeze-dried, a freeze-dried preparation of the activator can be prepared. The freeze-drying process can be performed by a conventional method. For example, after sterilizing the present activator obtained by the above dispersion treatment, a predetermined amount is dispensed into a vial. Pre-freeze for about 2 hours at about −40 to −20 ° C., perform primary drying under reduced pressure at about 0 to 10 ° C., and then secondary dry under reduced pressure at about 15 to 25 ° C. dry. In general, the inside of the vial can be replaced with nitrogen gas and stoppered to obtain a freeze-dried preparation of the present activator.

  In general, the lyophilized preparation of the activator can be re-dissolved and used by adding any appropriate solution (re-dissolved solution). Examples of such redissolved solution include water for injection, physiological saline, and other general infusion solutions. The amount of the redissolved solution varies depending on the use and the like and is not particularly limited. However, an amount of 0.5 to 2 times the amount of the solution before lyophilization or 500 mL or less is appropriate.

  This activator can activate nuclease in cancer cells to induce apoptosis in cancer cells and kill cancer cells, and has low toxicity, so it is useful for cancer treatment of mammals including humans, for example, liver cancer treatment. Useful. In particular, the present activator containing a complex of the present carrier and poly (I) / poly (C) is excellent because it is extremely effective but extremely low in toxicity.

  This activator can be administered intravenously for cancer treatment, local administration within cancer, transmucosal administration, etc. When this activator is used for liver cancer treatment, intravenous administration, intrahepatic artery Administration and intraportal administration are appropriate.

  The dose of this activator for cancer treatment depends on poly (I) / poly (C), etc., phospholipid type, cancer type, cancer progression, age, species difference, administration route, administration method, etc. Although different, the dose of poly (I), poly (C), etc. is usually 50 μg to 50 mg / human per time, preferably 100 μg to 2 mg / human. As a dose of poly (I) · poly (C), 50 μg to 50 mg / human is usually appropriate per dose, and 100 μg to 2 mg / human is preferable. This activator can be administered 1 shot or drip, etc., once to 3 times a day, every day, every other day, every week, every two weeks or the like.

Hereinafter, the present invention will be described in more detail with reference to examples and test examples. In each example and each test example, the concentration of the activator is expressed as the concentration of the poly (I) / poly (C) in the activator.
Example 1
40 g of maltose dissolved in 100 mL of water for injection was added to 2 g of this glycerol derivative and 2 g of purified egg yolk lecithin, mixed with stirring, and dispersed for 5 minutes using a homogenizer to obtain a crude dispersion of this carrier. The crude dispersion was further dispersed for 1 hour using a small experimental emulsifying disperser, and the volume was fixed to 250 mL with water for injection to recover the carrier dispersion. To this carrier dispersion 250 mL, 150 mL of an aqueous solution containing 500 mg of poly (I) · poly (C) [average chain length is approximately 200 bp] was added with stirring, and further for 1 hour using a small-sized emulsifying disperser for experiments. This activator was obtained by dispersion treatment. Thereafter, the activator was dispensed in 1 mL vials to prepare a freeze-dried preparation according to a conventional method.
Example 2
50 g of this glycerol derivative and 30 g of egg yolk phosphatide are added with 4 kg of sucrose dissolved in 10 L of water for injection, and dispersed for 10 minutes using a high pressure homogenizer of Manton-Gaurin type. did. To this carrier dispersion 20L, a 12 L aqueous solution containing 10 g of poly (I) · poly (C) [average chain length is approximately 200 bp] is added without stirring, and the pH is adjusted to 5.5 using hydrochloric acid. The mixture was further dispersed for 30 minutes using a Manton-Gaurin type high pressure homogenizer to obtain the activator. Then, 20 mL of this activator was dispensed into vials to prepare a freeze-dried preparation according to a conventional method. A commercially available 5% dextrose solution (500 mL) was added to the lyophilized preparation and dissolved.
Example 3
20 g of glucose dissolved in 100 mL of water for injection was added to 2 g of this glycerol derivative and 2 g of soy lecithin, mixed with stirring, and dispersed for 5 minutes using a homogenizer to obtain a crude dispersion of this carrier. The crude dispersion was further dispersed for 1 hour using a small experimental high-pressure emulsifying dispersion vessel, and the volume of the dispersion was made up to 250 mL with water for injection to recover the carrier dispersion. A 250 mL aqueous solution containing 50 mg of poly (I) · poly (C) [average chain length is approximately 200 bp] is added to 250 mL of this carrier dispersion without stirring, and a small high-pressure emulsifying disperser for experiment is further added for 1 hour. This activator was obtained by dispersion treatment.
Example 4
40 g of maltose dissolved in 100 mL of water for injection is added to 1.2 g of this glycerol derivative and 2.0 g of purified egg yolk lecithin, mixed with stirring, dispersed for 30 minutes using a small emulsifying disperser for experiments, and fixed to 250 mL with water for injection. The carrier dispersion was recovered. To this carrier dispersion 250 mL, 150 mL of an aqueous solution containing 200 mg of poly (I) · poly (C) [average chain length is approximately 200 bp] was added with stirring, and further for 2 hours using a small-sized emulsifying disperser for experiments. The activator was obtained by dispersion treatment.
Example 5
40 g of maltose dissolved in 100 mL of water for injection is added to 1.2 g of this glycerol derivative and 2.0 g of purified egg yolk lecithin, mixed with stirring, dispersed for 30 minutes using a small emulsifying disperser for experiments, and fixed to 250 mL with water for injection. The carrier dispersion was recovered. To 250 mL of this carrier dispersion, 150 mL of an aqueous solution containing 100 mg of poly (I) having an average chain length of 360 and 100 mg of 318 based poly (C) was added with stirring. This activator was obtained by dispersion treatment.
Example 6
40 g of maltose dissolved in 100 mL of water for injection was added to 2 g of this glycerol derivative and 2 g of purified egg yolk lecithin, mixed with stirring, and dispersed for 5 minutes using a homogenizer to obtain a crude dispersion of this carrier. The crude dispersion was further dispersed for 1 hour using a small experimental emulsifying disperser, and the volume was fixed to 250 mL with water for injection to recover the carrier dispersion. 150 mL of aqueous solution containing 250 mg of poly (I) having an average chain length of 1419 base and 250 mg of 1491 base is added to 250 mL of this carrier dispersion without stirring, and then a small emulsification disperser for 1 hour experiment. This activator was obtained by a dispersion treatment using Thereafter, the activator was dispensed in 1 mL vials to prepare a freeze-dried preparation according to a conventional method.
Example 7
40 g of maltose dissolved in 100 mL of water for injection is added to 1.2 g of this glycerol derivative and 2.0 g of purified egg yolk lecithin, mixed with stirring, dispersed for 30 minutes using a small emulsifying disperser for experiments, and fixed to 250 mL with water for injection. The carrier dispersion was recovered. To 250 mL of this carrier dispersion, 150 mL of an aqueous solution containing 100 mg of poly (I) having an average chain length of 84 base and 100 mg of 76 (base) poly (C) was added with stirring, and a small-sized emulsification disperser for experiment was further added for 2 hours. This activator was obtained by dispersion treatment.
Example 8
In the same manner as in Example 4, the activator containing poly (I) · poly (C) having an average chain length of about 350 bp was obtained.
Example 9
In the same manner as in Example 4, the activator containing poly (I) · poly (C) having an average chain length of about 1450 bp was obtained.
Example 10
In the same manner as in Example 4, the activator containing poly (I) · poly (C) having an average chain length of about 80 bp was obtained.
Test Example 1 Growth inhibitory effect on various cell lines (in vitro)
Each cell was seeded in a 96-well plate at a density of 10 ⁴ cells / well, and the following day, the activator according to Example 4 or adriamycin was added to continue the culture. Three days later, the number of viable cells was measured by the MTT method. The results are shown in Tables 1 and 2.

表１及び表２から明らかなように、数多くの上皮系及び繊維芽細胞由来の癌細胞株に対し、０．０１〜５００ｎｇ／ｍＬの濃度で強い増殖抑制作用が見られた。核酸合成阻害により抗癌作用を示すアドリアマイシンと比較しても遜色のない強さであった。どの臓器の癌細胞に対しても効果があり、臓器特異性は見られなかった。一方、非癌細胞である肝臓由来株化細胞４株及び株化繊維芽細胞７株に対して本活性化剤は１０００ｎｇ／ｍＬの濃度でも増殖抑制作用を示さなかった。なお、このｉｎ ｖｉｔｒｏ癌細胞増殖抑制効果は、ｐｏｌｙ（Ｉ）・ｐｏｌｙ（Ｃ）単独添加及び本担体単独では全く見られず、また単に細胞内へｐｏｌｙ（Ｉ）・ｐｏｌｙ（Ｃ）が移入されただけでは想像のつかなかった現象である。
試験例２ アポトーシス現象の観察
（１）ＤＮＡ及びＲＮＡフラグメンテーション
1)ＤＮＡフラグメンテーション
Ａ４３１細胞及びＫＭ１２−ＨＸ細胞の各々にｐｏｌｙ（Ｉ）・ｐｏｌｙ（Ｃ）の濃度として１μｇ／ｍＬの実施例４に係る本活性化剤を添加し、Ａ４３１細胞については５時間後に、ＫＭ１２−ＨＸ細胞については７．５時間後に細胞を回収した。５ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）・１０ｍＭ ＥＤＴＡ・０．５％（ｖ／ｖ）Ｔｒｉｔｏｎ Ｘ−１００で細胞を溶解した後、１３０００×ｇで２０分間遠心してフラグメント化したＤＮＡ（上清）とクロマチン画分（沈殿）を分離した。上清に１００μｇ／ｍｌ ＲＮａｓｅ Ａを加え３７℃で１時間作用させた後、２００μｇ／ｍＬ Ｐｒｏｔｅｉｎａｓｅ Ｋ及び１％（ｗ／ｖ）ＳＤＳ（Ｓｏｄｉｕｍ ｄｏｄｅｃｙｌ ｓｕｌｆａｔｅ）を５０℃で１．５時間反応させた。さらにフェノール／クロロホルムによりフラグメント化ＤＮＡを抽出し、１．８％アガロースゲルで電気泳動した。その結果、いずれの細胞においてもＤＮＡフラグメンテーションが観察された。

As is clear from Tables 1 and 2, a strong growth inhibitory action was observed at a concentration of 0.01 to 500 ng / mL against many epithelial and fibroblast-derived cancer cell lines. Compared with adriamycin, which exhibits anticancer activity due to inhibition of nucleic acid synthesis, the strength was comparable. It was effective against cancer cells in any organ, and no organ specificity was observed. On the other hand, this activator did not show growth-inhibiting action even at a concentration of 1000 ng / mL against 4 liver-derived cell lines and 7 cell line fibroblasts that are non-cancer cells. This in vitro cancer cell growth inhibitory effect was not observed when poly (I) / poly (C) alone was added or the carrier alone, and poly (I) / poly (C) was simply transferred into the cells. This is a phenomenon that could not have been imagined.
Test Example 2 Observation of apoptosis phenomenon (1) DNA and RNA fragmentation
1) DNA fragmentation The present activator according to Example 4 at a concentration of poly (I) · poly (C) of 1 μg / mL was added to each of A431 cells and KM12-HX cells, and after 5 hours for A431 cells, KM12-HX cells were collected after 7.5 hours. The cells were lysed with 5 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.5% (v / v) Triton X-100, and then centrifuged at 13000 × g for 20 minutes to fragment the DNA (supernatant). The chromatin fraction (precipitate) was separated. 100 μg / ml RNase A was added to the supernatant and allowed to act at 37 ° C. for 1 hour, and then 200 μg / mL Proteinase K and 1% (w / v) SDS (Sodium dodecyl sulfate) were reacted at 50 ° C. for 1.5 hours. . Further, the fragmented DNA was extracted with phenol / chloroform and electrophoresed on a 1.8% agarose gel. As a result, DNA fragmentation was observed in all cells.

In addition, the time course of A431 cells was examined. A431 cells were seeded in a 6-well plate at a density of 2.8 × 10 ⁵ cells / well, and 2 μCi [ ³ H] Thymidine was added the next day to label the cell DNA. Thereafter, the activator according to Example 4 (1 μg / mL) was added, and cells were collected at each time. The cells were lysed with 5 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.5% (v / v) Triton X-100, and then centrifuged at 13000 × g for 20 minutes to fragment the DNA (supernatant). The chromatin fraction (precipitate) was separated. The ratio of fragmented DNA to total DNA was calculated from the amount of radioactivity in the supernatant and precipitate. The result is shown in FIG.

Fragmentation of about 30% of the total DNA occurred in 3 hours after addition and 55% or more in 5 hours, and it was found that this degradation occurred immediately after the activator entered the cells.
2) RNA fragmentation

The activator according to Example 4 having a concentration of poly (I) · poly (C) of 1 μg / mL in each of A431 cells, MDA-MB-468 cells, KB cells, HeLaS3 cells, and MCF-7 cells. The cells were harvested after 4 hours. The ribosome fraction was fractionated from each cell, and total RNA was extracted by the AGPC (Acid-Guanidinium-Phenol-Chloroform) method. RNA was electrophoresed on a formaldehyde-denaturing gel (1.8% agarose gel) and then stained with ethidium bromide. As a result, fragmentation of 28S and 18S ribosomal RNA was observed in all cells.
(2) Effect of nuclease inhibitor HeLaS3 cells were seeded in a 96-well plate at a density of 10 ⁴ cells / well, and the following day, nuclease inhibitor ATA (aurintricboxylic acid) 10 μM and the activator according to Example 4 were added simultaneously. . The culture was further continued for 3 days, and the number of viable cells was measured by the MTT method. The result is shown in FIG.

３０μｇ／ｋｇ投与群では、コントロール群（１０％マルトース投与）に比較して７２％、１００μｇ／ｋｇ投与群では９１％の肝臓癌細胞の増殖抑制効果が見られた。また、１００μｇ／ｋｇ投与に関しては週１回投与でも７７％の肝臓癌細胞の増殖抑制効果が見られた。 As is clear from FIG. 2, when ATA was added to stop the function of intracellular nuclease, this activator did not show the cancer cell growth inhibitory action. Then, the added ATA is removed from the medium after 8 hours, and the activator does not act even if the activator is added thereafter. Therefore, the effect of the ATA is that the activator is taken into the cells. It is thought to be a result of working as a nuclease inhibitor.
(3) From the above test results, it was found that this activator activates intracellular nuclease, thereby causing apoptosis in cancer cells.
Test Example 3 Effect in mouse metastatic liver cancer model (in vivo)
KM12-HX (human colon cancer cells in the spleen of nude mice Balb / c, nu / nu (5 weeks old, male); when transplanted into the spleen of nude mice, it efficiently metastasizes to the liver and forms liver cancer lesions Strain) was injected at 10 ⁶ cells / mouse, and 10 minutes later, the spleen was removed. Three days later, the activator according to Example 4 was intravenously administered twice at a regular interval twice a week for 5 weeks. Two days after the final administration, the liver was removed, and the number and area of cancer nodules formed in the liver were measured. The results are shown in Table 3.

In the 30 μg / kg administration group, 72%, and in the 100 μg / kg administration group, 91% of the liver cancer cell growth inhibitory effect was observed as compared to the control group (10% maltose administration). In addition, regarding the administration of 100 μg / kg, 77% of the liver cancer cell growth inhibitory effect was observed even once a week.

  Moreover, when specimens of each liver tissue were prepared and pathological analysis was performed, the cancer formed in the liver of the control group was an epithelial poorly differentiated adenocarcinoma. Tumor blood vessels had developed to a normal extent. In particular, no infiltration of immunocompetent cells was observed. In this cancer tissue, calcification was accompanied in some places. No obvious cancer cells were found in this activator-administered group, and only calcification remaining after cancer treatment by drug administration was observed.

Thus, the present activator had a significant effect in continuous intravenous administration of 10 μg / kg to 100 μg / kg · twice a week in liver cancer model animals.
Test Example 4 Toxicity test (1) Expression of liver toxicity after single administration to rats (acute toxicity test)
A single intravenous administration of the activator according to Example 4 was performed on 8 6-week-old SD male rats, and the serum aminoacyltransferase activity 20 hours later was measured. As a result, no death was observed up to 5 mg / kg, and a slight increase in serum aminoacyltransferase was observed at 5 mg / kg. There was almost no increase in serum aminoacyltransferase at 1 mg / kg.
(2) Rat 2-week limited subacute toxicity test The activator according to Example 4 was intravenously administered to 6 SD male rats (6 weeks old) daily for 14 days, which was particularly problematic at 1 mg / kg or less. No toxic effects were seen.
(3) Antigenicity test The antigenicity test was conducted on the activator of Example 4 using male guinea pigs (hartley, 5 weeks old). No antigenicity was observed at 50 μg / guinea pig.
(4) Simple mutagenicity test The present activator according to Example 4 was subjected to a simple reverse mutation test and a simple chromosome aberration test, and no mutagenicity was observed at 10 µg / mL.

The percentage of DNA fragmentation is shown. The vertical axis represents the percentage (%) of DNA fragmentation, and the horizontal axis represents time (hour). The influence of the nuclease inhibitor by ATA addition is shown. The vertical axis represents the inhibition rate (%), and the horizontal axis represents the concentration (ng / ml) of the activator according to Example 4. -○-represents the result in the system without addition of ATA, and-●-represents the result in the system to which ATA was added.

Claims (5)

Carrier formed with 2-O- (2-diethylaminoethyl) carbamoyl-1,3-O-dioleoylglycerol and phospholipid as essential components and poly (I) · poly (C) or poly (A) · A cancer cell nuclease activator comprising a complex with poly (U). The cancer cell nuclease activator according to claim 1, wherein the phospholipid is lecithin. The cancer cell nuclease activator according to claim 1 or 2, wherein the average chain length of poly (I) · poly (C) or poly (A) · poly (U) is in the range of 100 to 500 bp. The cancer intracellular nuclease activator according to any one of claims 1 to 3, wherein the cancer is liver cancer. A cancer therapeutic agent comprising the cancer cell nuclease activator according to claim 1.

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