JPH11228451A - Anticancer action-enhancing medicine composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an antitumor action-enhancing medicine capable of exhibiting a sufficient antitumor action against cancer cells at a lower dose than a usual administration dose, and to provide an antitumor composition. SOLUTION: This antitumor action-enhancing medicine contains an oligonucleotide capable of hybridizing with a template region in a RNA portion contained in telomerase as an active ingredient. The antitumor composition contains the oligonucleotide and an antitumor medicine as active ingredients.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antitumor effect enhancer of an antitumor agent and an antitumor composition.

[0002]

2. Description of the Related Art Telomerase is an enzyme that has recently attracted attention in connection with canceration and aging of cells, and has an action of elongating telomere DNA. Telomere consists of a short repeating base sequence, and with this base sequence as the axis,
It forms a high-order complex containing many proteins, and has a function to prevent chromosome degradation and chromosome fusion.
rc. Natl. Acad. Sci. USA, 85 ,
6622-6626 (1988)]. Human telomere DN
A is composed of several hundred repetitive nucleotide sequences of TTAGGG, and this nucleotide sequence is shortened at the 5 ′ end every time cell division replicates [Nature, 345 , 458-5].
60 (1990)]. It is thought that cell division arrest occurs when telomere DNA is shortened to a certain length, and telomere DNA length is estimated to play a role as a "clock" for division aging [Nature, 346]. , 8
66-868 (1990)]. Telomerase is an enzyme composed of ribonucleoprotein, and synthesizes telomere DNA at the end of chromosome via template RNA [Na
cure, 344 , 126-132 (1990)].

In humans, the germline (germl)
ine), telomerase activity is generally not detected in normal adult tissues [Dev. Genet, 18 , 173-1
79 (1996)]. On the other hand, in many malignant tumors, high telomerase activity is observed along with telomere elongation. In order for cells to divide indefinitely, it is necessary to compensate for telomere shortening that occurs with each replication, and it is presumed that telomere length is often maintained by telomerase activity that extends telomeres [Sci.
ence, 266 , 2011-2015 (1994);
Proc. Natl. Acad. Sci. USA, 9
1 , 2882-2885 (1994); Nature.
Med. , 1 , 249-255 (1995); Canc.
er Res. , 55 , 2734-2736 (199).
5); Cancer Res. , 55 , 3258-32
62 (1995); Cancer Res. , 56 , 6
45-650 (1996); Natl. Can
cer Inst. , 88 , 116-122 (199
6)]. Recently, findings have been obtained indicating that telomerase activity is regulated by the Bcl-2 protein, an antagonist of programmed death (apoptosis), and the anti-apoptotic pathway (anti-apoptotic) has been obtained.
c pathway) has been shown to potentially activate telomerase in many cancer cells [J. Biol. Chem. , 272 , 14183-1
4187 (1997)]. From these series of information, etc.,
The possibility of anticancer therapy targeting telomerase has been considered [Cancer Res. , 5
6 , 645-650 (1996)].

[0004]

As such antisense therapy targeting telomerase, for example, telomerase activity is increased by introducing antisense DNA to an RNA portion contained in human telomerase into human cancer cells (HeLa cells). Have been reported to decrease, telomere DNA shortens with cell division, and cells die after about 25 cell divisions [Scienc].
e, 269 , 1236-1241 (1995)]. However, in this method, cancer cells should continue to proliferate until about 25 divisions have been completed, during which time they continue to grow as cancerous tissues, and when used as an anticancer drug, the period until the effect appears. Has the disadvantage of being too long.

On the other hand, in the chemotherapy using an anticancer drug which is generally used at present, in many cases, DNA damage or transcriptional inhibition occurs nonspecifically in cells at an effective dose of the drug, and the drug dose or cytotoxicity is high. In many cases, it is necessary to limit the dose in consideration of the above. The present inventor has found that when an antitumor agent is administered, when an antisense DNA against a base sequence of a specific region of an RNA portion contained in telomerase is administered in combination, a sufficient antitumor effect can be obtained with a smaller amount of an antitumor agent than before. It has been found that it can be shown. The antisense DNA enhances the antitumor effect of the antitumor agent,
The present invention is not known at all, and the present invention is based on such knowledge.

[0006]

DISCLOSURE OF THE INVENTION The present invention provides an agent for enhancing the antitumor activity of an antitumor agent, which comprises, as an active ingredient, an oligonucleotide capable of hybridizing to a template region in an RNA portion contained in telomerase. About. The present invention also relates to an antitumor composition comprising an oligonucleotide capable of hybridizing to a template region in an RNA portion contained in telomerase and an antitumor agent as active ingredients.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. The antitumor effect enhancer according to the present invention or the oligonucleotide used as an active ingredient in the antitumor composition according to the present invention is an oligonucleotide capable of hybridizing to a template region in an RNA portion (hereinafter referred to as template RNA) contained in telomerase. That is, it is an oligonucleotide containing a base sequence complementary to the base sequence of the template region. As used herein, the term "template region in template RNA" refers to RNA contained in telomerase.
It means the region that is complementary to the telomere sequence. The telomere sequence means a nucleotide sequence which is present in a terminal region of a chromosome and in which a short repetitive sequence is repeated several tens to several hundred times. Telomerase is an enzyme generally present in organisms ranging from microorganisms to higher animals (including humans). At present, the sequence of the repetitive sequence in the telomere sequence and the sequence of the template region are known in various organisms. For example, in mammals, human and mouse sequences are known, the repeat sequence in the human or mouse telomere sequence is 5′-TTAGGG-3 ′, and the template region in human or mouse is '-CAAUCCCAAUC-3'. At present, each of the above-mentioned sequences of an organism whose sequence has not been determined can also be easily determined by known means.

[0008] The number of bases of the oligonucleotide used as an active ingredient in the present invention is not particularly limited, but is preferably at least the number of bases capable of specifically hybridizing to the template region in the template RNA,
It is preferable that the number of bases is equal to or less than the number of bases that can pass through a cell membrane or a nuclear membrane. The number of bases capable of specifically hybridizing to the template region in the template RNA is preferably 1
It has 0 bases or more, more preferably 13 bases or more. In order to ensure membrane permeability, it is preferably 100 bases or less, more preferably 50 bases or less. Therefore,
The oligonucleotide used in the present invention is preferably 10
-100 bases, more preferably 13-50 bases.

The oligonucleotide used in the present invention includes:
It is not necessary to continuously include a base sequence complementary to the template region in the template RNA, as long as it can specifically bind to the template region in the template RNA to form a duplex, and may include the non-complementary base 1 or The above can be deleted, inserted and / or substituted at one or more points. Oligonucleotides used in the present invention may be used under conditions of high stringency with DNA consisting of the base sequence of the template region in the template RNA [for example, 2 × SSC (0.
In 3M sodium chloride and 0.03M sodium citrate), and an oligonucleotide containing a continuous base sequence complementary to the template region in the template RNA is particularly preferred.

[0010] In the oligonucleotide used in the present invention, the internucleotide bond between each nucleoside is independently a phosphodiester bond or a modified phosphodiester bond. Examples of the modified phosphodiester bond include a methylphosphonate-type bond in which one oxygen atom of two non-crosslinking oxygen atoms of a phosphodiester bond is substituted with a methyl group, and two non-crosslinking oxygen atoms of a phosphodiester bond A phosphoramidate-type bond in which one oxygen atom is substituted with an amino group or a substituted amino group, and a phosphorothioate type in which one oxygen atom of two non-crosslinked oxygen atoms in a phosphodiester bond is substituted with a sulfur atom Or a phosphorodithioate-type bond in which two oxygen atoms of two non-bridging oxygen atoms of a phosphodiester bond are substituted with two sulfur atoms, and one or more of these are bonded. , One of the bonds between nucleosides
It can be introduced at a point or more. From the viewpoint of nucleotide sequence specificity, double-stranded stability, antinuclease resistance, cell membrane permeability, low cytotoxicity and moderate metabolism, and a simple preparation method, it is preferable that the modified phosphodiester bond is a modified phosphodiester bond. , Because of its good stability in vivo
Particularly preferred are phosphorothioate-type bonds.
Further, it is particularly preferable that at least half (particularly all) of the bonds between nucleosides are modified phosphodiester bonds (particularly, phosphorothioate type bonds).

The oligonucleotide used in the present invention is a deoxynucleoside, a ribonucleoside, and / or a modified ribonucleoside thereof, for example, as long as it can specifically bind to the template region of the template RNA to form a duplex. , 2'-O-modified ribonucleosides. As a modified ribonucleoside, from the viewpoint of the strength of binding to the target base sequence,
2'-O-methyl ribonucleoside is preferred. Therefore, the oligonucleotide used in the present invention is an oligoribonucleotide (RNA) composed of ribonucleoside and / or modified ribonucleoside, an oligodeoxyribonucleotide (DNA) composed only of deoxyribonucleoside, or a ribonucleoside (and / or modified ribonucleoside). And chimeric oligoribo / deoxyribonucleotides (RNA / DNA) consisting of both oxynucleotides and deoxyribonucleosides.

Oligonucleotides that can hybridize to the template region in human template RNA include, for example,
An oligonucleotide having a base sequence represented by SEQ ID NO: 1 in the sequence listing can be mentioned.

The oligonucleotide used in the present invention can be synthesized by a known method. Except for the site for introducing a 2′-O-methyl ribonucleotide or phosphorothioate bond, for example, DNA / RN by the usual phosphodiester method or phosphotriester method, for example, H-phosphonate method or phosphoramidite method
A It can be synthesized using an automatic synthesizer. 2'-
Oligonucleotides having O-methylribonucleotides are, for example, 5'-dimethoxytrityl-2'-O-
Using a phosphoramidite method, methylribonucleoside-3 ′-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite unit is used.
It can be synthesized using an NA / RNA automatic synthesizer. Oligonucleotides having a phosphorothioate linkage can be used, for example, instead of a water / iodine / pyridine solution which is an oxidizing agent used for ordinary polynucleotide synthesis.
It can be synthesized using a 15% N, N, N ', N'-tetraethylthiolamb disulfide / acetonitrile solution.

In order to improve the membrane permeability of the oligonucleotide used in the present invention, various known methods can be used. Known methods include, for example, a method of embedding in liposomes [Cancer Res. , 5
0 , 7826 (1991)], a method of introducing cholesterol to the terminal of an oligomer [Nucleic Acid]
s Res. , 20 , 533 (1993)], transferrin-poly-L-lysine complex [Proc. Nat
l. Acad. Sci. USA, 89 , 7031 (19
92)] in a non-covalent manner to specifically incorporate oligomers into hepatocytes expressing the galactose receptor or cancer cells expressing the transferrin receptor, respectively, or by using a cationic lipid (for example,
Lipofectin) interacts with oligomers and is added to target cells to increase intracellular approach, translocation into the nucleus, and enhance antisense effects [J. Biol. Chem. , 266 , 18162 (1
991)]. The oligonucleotide used in the present invention is preferably embedded in a liposome [for example, a positively charged liposome (Coatsome EL-C-01, Nippon Oil & Fat) and the like] to improve membrane permeability.

The antitumor effect enhancer according to the present invention
The antitumor agent whose antitumor activity is enhanced, or the antitumor agent used as an active ingredient in the antitumor composition according to the present invention is particularly limited as long as it is a compound having an antitumor effect by itself. But, for example,
Examples include chemotherapeutic agents that can be used for treating cancer. Examples of the chemotherapeutic agent include alkylating agents (eg, melphalan, carbone, or cyclophosphamide), anticancer metal complexes [eg, cisplatin (CDDP), etc.], antibiotics [eg, adriamycin, Or mitomycin C (MMC)), antimetabolites (eg, cytarabine, fluorouracil, or methotrexate), plant preparations [eg, vincristine, vindesine (VDS) or vinblastine, etc.], or DNA topoisomerase II inhibitors [eg,
Etoposide (VP-16)].

The antitumor effect enhancer according to the present invention contains, as an active ingredient, an oligonucleotide capable of hybridizing to the template region of the template RNA, and if necessary, can be pharmaceutically or veterinarily acceptable. May be contained. In addition, the antitumor composition according to the present invention contains the oligonucleotide and the antitumor agent as active ingredients, and, if desired, contains a usual carrier that is pharmaceutically or veterinarily acceptable. Can be.

The dosage form of the antitumor effect enhancer of the present invention or the antitumor composition of the present invention is not particularly limited, and examples thereof include powders, fine granules, granules, tablets, capsules, and suspensions. Liquids, emulsions, syrups, extracts,
Oral preparations such as pills, or parenteral preparations such as injections, external solutions, ointments, suppositories, creams for topical administration, or eye drops can be mentioned.

These oral preparations include, for example, gelatin, sodium alginate, starch, corn starch, sucrose, lactose, glucose, mannitol, carboxymethylcellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soy lecithin, sucrose, fatty acid ester, talc Excipients, such as magnesium stearate, polyethylene glycol, magnesium silicate, silicic anhydride, or synthetic aluminum silicate, binders, disintegrants, surfactants, lubricants, fluidity promoters, diluents, and preservatives Agents, coloring agents,
It can be produced according to a conventional method using a flavor, a flavoring agent, a stabilizer, a humectant, a preservative, an antioxidant and the like.

Examples of parenteral administration methods include injection (subcutaneous, intravenous, etc.) and rectal administration. Of these, injections are most preferably used. For example, in the preparation of an injection, as an active ingredient, in addition to an oligonucleotide capable of hybridizing to a template region in a template RNA, or the oligonucleotide and an antitumor agent,
For example, water-soluble solvents such as saline or Ringer's solution, water-insoluble solvents such as vegetable oils or fatty acid esters,
An isotonic agent such as glucose or sodium chloride, a solubilizer, a stabilizer, a preservative, a suspending agent, an emulsifier, or the like can be optionally used.

The antitumor effect enhancer of the present invention or the antitumor composition of the present invention may be administered by using a sustained-release preparation technique using a sustained-release polymer or the like. For example,
The antitumor effect enhancer according to the present invention or the antitumor composition of the present invention can be incorporated into a pellet of ethylene vinyl acetate polymer, and the pellet can be surgically implanted into a tissue to be treated.

The antitumor composition according to the present invention can be administered alone, orally or parenterally to an animal, preferably a mammal, especially a human. The dosage in the case of using the antitumor composition of the present invention differs depending on the type, age, sex, or weight of the animal to be administered, the type of disease, the administration method, and the like, and can be determined as appropriate. Furthermore, the form is not limited to pharmaceuticals, and it can be provided in various forms, for example, in the form of food or drink as a functional food, a health food, or a feed.

The antitumor agent enhancer according to the present invention comprises (1) an oligonucleotide capable of enhancing the antitumor effect of the antitumor agent and capable of hybridizing to the template region of the template RNA, and an antitumor agent. A template that can be administered as a single formulation (ie, an anti-tumor composition according to the present invention) that contains together as an active ingredient, or (2) an anti-tumor effect of an anti-tumor agent can be enhanced An antitumor effect enhancer according to the present invention was prepared as a preparation containing only an oligonucleotide capable of hybridizing to a template region in RNA as an active ingredient, and together with a preparation containing an antitumor agent separately prepared, Can be simultaneously administered.

In the antitumor effect enhancer according to the present invention,
When the antitumor effect enhancer and the antitumor agent are prepared as separate preparations, a preparation containing the antitumor agent in an animal, preferably a mammal (particularly a human), and an antitumor effect according to the present invention are prepared. The enhancer can be administered together orally or parenterally. In this case, the dosage of the antitumor agent and the antitumor effect enhancer of the present invention varies depending on the type, age, gender, or weight of the animal to be administered, the type of disease, or the method of administration, and is determined as appropriate. can do. Furthermore, the form is not limited to pharmaceuticals, and it can be provided in various forms, for example, in the form of food or drink as a functional food, a health food, or a feed.

Animals, preferably mammals (especially humans)
When the antitumor composition according to the present invention is administered alone, or when the antitumor effect enhancer according to the present invention is administered together with an antitumor agent, the antitumor agent can be administered at a lower dose than a normal antitumor agent dose. Even when administered, it shows a sufficient antitumor effect on cancer cells. That is, the oligonucleotide capable of hybridizing to the template region of the template RNA used as an active ingredient in the present invention can synergistically enhance the antitumor effect of the antitumor agent.

The antitumor composition or the antitumor effect enhancer according to the present invention can be used for the treatment of any tumor, especially any malignant tumor (for example, lung cancer, stomach cancer, colon cancer, leukemia, etc.).

[0026]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention.

Example 1 In this example, a human lung squamous cell carcinoma cell line RERF-LC-A1 (RIKEN, RCB044) was used.
4; hereinafter referred to as RERF cells) to confirm the tumor growth inhibitory effect of the antitumor effect enhancer of the present invention.
In this example, a DNA consisting of 13 bases represented by the sequence of SEQ ID NO: 1 in the sequence listing was used as an oligonucleotide capable of hybridizing to the template region of the template RNA, and CDDP (trade name) was used as an antitumor agent. = Landa, Nippon Kayaku), etoposide (trade name = Lastet, Nippon Kayaku; hereinafter sometimes referred to as VP-16), mitomycin C (trade name = mitomycin, Kyowa Hakko;
MMC) and vindesine (trade name: fildesine, Shionogi & Co., Ltd .; hereinafter, sometimes referred to as VDS) were used.

DN represented by the sequence of SEQ ID NO: 1 in the sequence listing
A (hereinafter referred to as antisense DNA) 131.3n
mol was dissolved in 1.313 ml of physiological saline (100 μl).
M), and the whole amount of the obtained antisense DNA solution was added to one vial of coatsome EL-C-01 powder (Nippon Oil & Fat), and the mixture was stored at 4 ° C. after inverting and stirring (number of inversions = 3 times).

10% fetal bovine serum, 100 units / m
Using an MEM medium containing 1 penicillin and 100 unit / ml streptomycin (hereinafter referred to as a culture medium), the RERF cells were cultured under the conditions of 37 ° C. and 5% CO ₂ aeration. Seeding into well plate (2 × 10 ⁴ cells / well, 99 μl)
did. 1 μl of the 100 μM liposome-encapsulated antisense DNA prepared above was added to each well, and after culturing for 24 hours, the cells were washed with a phosphate buffer solution (phosphate).
buffered saline; hereinafter, referred to as PBS), added with 100 μl of a culture medium containing the following anti-tumor agent at a predetermined concentration, and further cultured for 48 hours. MT
Tumor growth inhibitory activity was measured by T assay.

The concentrations of the antitumor agents were set at three levels (ie, low level, medium level and high level), and the concentrations when each antitumor agent was administered alone are shown in Table 1. In Table 1, the unit of the concentration of each antitumor agent is μg / ml. In addition, as simultaneous administration of a plurality of antitumor agents, (1) C
Combination of DDP, VDS and MMC, and (2) C
Combinations of DDP, VP-16, and MMC were performed, and also in these combinations, the concentration was three levels (ie, low, medium, and high). As shown in Table 2, in the simultaneous administration of a plurality of antitumor agents, the concentration of each antitumor agent at each level was set to be the same as that in the single administration of the antitumor agent. Also in Table 2, the unit of the concentration of each antitumor agent is μg / ml.

[0030]

Table 1 Antitumor agent Low level Medium level High level CDDP 0.132 0.66 3.3 VP-16 0.106 0.532 2.66 MMC 0.008 0.04 0.2 VDS 0.0030 .016 0.08

[0031]

[Table 2] Antitumor agent Low level Medium level High level (1) Combination of CDDP, VDS and MMC CDDP 0.132 0.66 3.3 VDS 0.003 0.016 0.08 MMC 0.008 04 0.2 (2) Combination of CDDP, VP-16 and MMC CDDP 0.132 0.66 3.3 VP-16 0.106 0.532 2.66 MMC 0.008 0.04 0.2

On the other hand, liposome-encapsulated antisense DNA
In order to examine the effect of the administration of the antitumor agent alone without the addition of
After seeding in a well plate, the cells were cultured for 72 hours in 100 μl of a culture medium containing the antitumor agent at a predetermined concentration, and the tumor growth inhibitory activity was measured in the same manner.

FIG. 1 shows the results. Symbols 1 to 7 shown in FIG.
And symbols a to c are as follows. 1: Administration of antisense DNA alone (control). 2: CDDP alone administration. 3: VP-16 alone administration. 4: MMC alone administration. 5: VDS alone administration. 6: Co-administration with a combination of CDDP, VDS and MMC. 7: Co-administration with a combination of CDDP, VP-16 and MMC. a: Low level. b: Medium level. c: High level. In addition, in the bar graph of FIG. 1, “solid bars” indicate the results when liposome-encapsulated antisense DNA was added, and “open bars” indicate liposome-encapsulated antisense D.
The result when no NA is added is shown. “*” Indicates p
<0.001. When the antitumor agent and the liposome-encapsulated antisense DNA are administered in combination, a sufficient concentration of any antitumor agent, even if the antitumor agent alone does not show a sufficient tumor growth inhibitory effect, is sufficient. (90% or more) showed tumor growth inhibitory activity.

[0034]

Example 2 Instead of RERF cells, a human lung adenocarcinoma-derived cell line A427 (ATCC HTB53; hereinafter, A42
7 cells) or human squamous cell carcinoma-derived cell line A54
Example 9 was repeated except that No. 9 (RIKEN, RCB0098; hereinafter, referred to as A549 cells) was used.

The results for A549 cells are shown in FIG.
The results for A427 cells are shown in FIG. FIG. 2 or FIG.
1 to 7 and symbols a to c have the same meanings as those shown in FIG. 2 and 3, "black rectangles" indicate the results when the liposome-encapsulated antisense DNA was added, and "white rectangles" indicate the results when the liposome-encapsulated antisense DNA was not added. "*" Indicates that p <0.001, and "*"
“*” Indicates that p <0.01, and “***” indicates p
<0.05. When the antitumor agent and the liposome-encapsulated antisense DNA are administered in combination, a sufficient concentration of any antitumor agent, even if the antitumor agent alone does not show a sufficient tumor growth inhibitory effect, is sufficient. It showed tumor growth inhibitory activity (except when the concentration of CDDP in A427 cells was 0.132 μg / ml).

[0036]

EFFECT OF THE INVENTION An animal, preferably a mammal (particularly a human) is administered with the antitumor composition according to the present invention alone,
Alternatively, when the antitumor effect enhancer according to the present invention is administered together with an antitumor agent, even if the antitumor agent is administered at a lower dose than a normal antitumor agent dose, sufficient antitumor activity against cancer cells is obtained. Show action.

[0037]

[Sequence list]

SEQ ID NO: 1 Sequence length: 13 Sequence type: nucleic acid sequence TAGGGTTAGA CAA 13

[Brief description of the drawings]

FIG. 1 is a graph showing the tumor growth inhibitory effect on RERF cells of the antitumor effect enhancer according to the present invention.

FIG. 2 is a graph showing the inhibitory effect of the antitumor effect enhancer of the present invention on tumor growth in A549 cells.

FIG. 3 is a graph showing the tumor growth inhibitory effect of A427 cells of the antitumor effect enhancer of the present invention.

Continued on the front page (72) Inventor Yukiko Ishida 4-6-1 Nishinogawa, Komae-shi, Tokyo Keio Shibasaki Corporus 508

Claims (2)

[Claims] 1. An antitumor agent-enhancing agent for an antitumor agent, comprising an oligonucleotide capable of hybridizing to a template region in an RNA portion contained in telomerase as an active ingredient. 2. An antitumor composition comprising an oligonucleotide capable of hybridizing to a template region in an RNA portion contained in telomerase and an antitumor agent as active ingredients.