JPH04235919A - Anticancer drug - Google Patents

Abstract

PURPOSE:To provide the subject anticancer drug containing, as the active component, a ribozyme having a specific breaking activity to mRNA transcribed by oncogene. CONSTITUTION:A hammer head type ribozyme having a nucleotide sequence of formula I or II (A is adenine nucleotide; U is uracil nucleotide; C is cytosine nucleotide; G is guanine nucleotide; 3' and 5' show 3' and 5' terminal respectively) is contained as the active component. The above-mentioned ribozyme can break only activated ras-gene mRNA molecule and can not break normal m-RNA molecule.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antitumor agent containing as an active ingredient a ribozyme that has specific cleaving activity against mRNA transcribed from cancer genes.

0002

[Prior Art] RNA not only plays a role as a genetic information transmitter, but also has the function of catalytically acting to cleave and link RNA (hereinafter referred to as
RNA having such a function is called a "ribozyme." )It is known. Based on the base sequence near the cleavage site and the nature of the reaction, ribozymes can be further classified into: (1) hammerhead RNA, (2) hairpin RNA, and (2) cloverhead RNA.
It can be classified into four types: leaf-like RNA, and other self-cleavage reaction RNA (Protein, Nucleic Acids, Enzymes, vol. 3).
5. No. 13, 2191-2200, (199
0)).

[0003] The present inventors have discovered that a hammerhead ribozyme can be used as an endonuclease that cleaves target RNA in a sequence-specific manner, and this content has already been reported and will be described in detail below. (Nu
cleic acid Res. , vol. 17,
7059-7071, (1989)). c-Har-r
The as gene is a protein with a molecular weight of 21,000 (p21
) is coded. Activated r detected from human cancer
The as gene has several mutations in the p21 amino acids due to point mutations in the bases. We investigated whether ribozymes could recognize differences in one of these bases. Ribozyme is an activated ras gene mR in which Gly (GGU) at position 12 of p21 is mutated to Val (GUU).
It was designed to cleave NA but not normal mRNA. In vitro experiments confirmed that this ribozyme only cleaves activated ras gene mRNA molecules, but not normal mRNA molecules.

0004

[Problems to be Solved by the Invention] The present inventors have developed a ribozyme that specifically cleaves mRNA transcribed from tumor genes by utilizing the ability of ribozyme's catalytic activity to recognize even a single base difference. The present invention was completed based on the discovery that it exhibits an effective antitumor effect.

[0005]

[Means for Solving the Problems] The present invention provides the general formula

00
06]

[Chemical formula 3]

[0007] or

[0008]

[C4]

"In the formula, A is an adenine nucleotide, U is a uracil nucleotide, C is a cytosine nucleotide, G
represents a guanine nucleotide, and 3' and 5' represent the 3' and 5' ends, respectively. The present invention relates to an antitumor agent containing a compound represented by the following as an active ingredient.

[0010] In the present invention, the term "tumor" refers to a disease caused by an activated oncogene, and particularly preferably refers to a disease caused by an activated c-Ha-ras gene. .

Compounds (I) and (II) of the present invention can be prepared by a known method (Nucleic Acid Res.,
vol. 17, 7059-7071).

[0012] In the present invention, the antitumor effect of ribozyme can be confirmed by the following method.

Specifically, NIH-3T3 cells are transformed with a plasmid expressing the ribozyme of interest, and then activated c-Ha-
This method involves transducing the ras gene and comparing the focus formation rate with cells transformed with a plasmid that does not express ribozyme.

[0014] The ribozyme obtained according to the present invention exhibits extremely effective antitumor effects when administered in various ways.

[0015] The method for administering the ribozyme of the present invention includes oral administration or parenteral administration (eg, intravenous injection, intramuscular injection,
(subcutaneous or intraperitoneal injection) and the administered composition includes a therapeutically effective amount of the product in a pharmaceutically acceptable diluent,
It is usually included together with a carrier and/or an adjuvant.

Standard diluents such as human serum albumin are contemplated for the pharmaceutical compositions of the present invention, as are standard carriers such as physiological saline.

[0017] As for the administration form, subcutaneous injection, intravenous injection, intramuscular injection, parenteral administration methods such as suppositories, tablets, etc.
Oral administration methods include capsules, powders, granules, and the like.

[0018] It is also possible to encapsulate the ribozyme molecule in a liposome and administer it orally or parenterally.

The dosage varies depending on the route of administration and the number of administrations, but for adults, it is usually 1 to 1,000 μg/kg body weight per day or divided into several doses per day. It is preferable to administer the drug.

The present invention will be explained in more detail below with reference to reference examples and examples, but these are not intended to limit the scope of the present invention.

In the following Reference Examples and Examples, adenine nucleotides are indicated as A, guanine nucleotides as G, cytosine nucleotides as C, and uracil nucleotides as U.

[Reference Example] The oligodeoxyribonucleotide is 380A manufactured by Applied Biosystems.
It was synthesized by the phosphoramidite method using a DNA synthesizer. Oligoribonucleotides can be prepared using the phosphoramidite method (using a dimethoxytrityl group as the protecting group for the 5'-hydroxyl group and a tetrahydropyranyl group as the protecting group for the 2'-hydroxyl group).
Nucleic Acid Res. , vol. 17
7059-7071, (1989)).

[0023] pRSV-rv12, pRSV-r16
The construction of 1 is described in the literature (Cancer Res., vo
l. 80, 200-203, (1989)).

Reference Example 1 Ribozyme CUACACCC
UGAUGAAGGGGUGAUACCCUGAAACA
Preparation of GCGCp oligoribonucleotide (UACCC
UGAAACAGCGCG, 5 nmol), polynucleotide kinase (10 units, manufactured by Takara Shuzo Co., Ltd.),
0.67mM ATP, 50mM Tris-H
Cl (pH 7.6), 10 mM MgCl2,
Add 10 mM mercaptoethanol and
It was heated at ℃ for 1 hour. Treated with phenol chloroform, Sephadex G-25 (manufactured by Pharmacia: φ
1.8 x 37 cm, the solvent was 0.1 M triethylammonium bicarbonate (hereinafter referred to as "TEA").
B”. ) to remove ATP and ADP.

The obtained 5'-phosphorylated oligoribonucleotide (pUACCCUGAAACAGCGCG) was dissolved in sterile water (20 μl), and 0.1 M Na
Add IO4 (2.5 μl) and incubate at 0 °C in the dark for 70 min.
Leave for 0.1 M ethylene glycol (25
μl) was added and reacted at 0°C for 30 minutes. To this was added 2 M lysine-HCl (pH 8.2, 50 μl
1) was added, heated at 45°C for 2 hours, and desalted with Sephadex G-25 (φ1.8 x 37 cm, solvent: 0.1 M TEAB). This was subjected to anion exchange HPLC (TSK gel DEAE-2SW,
φ0.46 x 25 cm: manufactured by Tosoh Corporation) to obtain an oligoribonucleotide having 5',3'-diphosphoric acid (pUACCCUGAAACAGCG).
Cp) was obtained.

[0026] Separately prepared oligoribonucleotide (C
UACACCCUGAUGAAGGGUGA, 2.5
nmol) and the above oligoribonucleotides (
pUACCCUGAAACAGCGCp, 2.5 n
mol) to 50mM HEPES-NaOH (pH
7.5), 20mM MgCl2, 3.3mM
DTT, 0.001% BSA, 10% (v/v
) DMSO, 1.38 units/μl RNA ligase (manufactured by Takara Shuzo Co., Ltd.), and 50 μl of a solution of 100 μM ATP was heated at 6° C. for 13.5 hours. Phenol chloroform treatment and anion exchange HPL
Ribozyme CUACACCCUGAU of desired chain length in C
GAAGGGUGAUACCCUGAAACAGCGC
I got p.

Reference Example 2 Activated c-Ha-ras
Preparation of DNA template A point mutation was made in the codon encoding the 12th amino acid (Gly12 (GGU) → Val12 (GUU
)) Activated c-Ha-ras DNA was prepared.

Seven synthetic oligodeoxyribonucleotides (1 nmol) were each mixed with T4 polynucleotide kinase (1 μl, 10 units) and 1 mM ATP (1 nmol).
5 μl), buffer (4 μl, 250 mM Tr
is-HCl (pH 7.6), 50 mM Mg
Cl2, 50 mM mercaptoethanol) was added, and the mixture was heated at 37°C for 1 hour.

[0029] Combine these and add buffer (40 μl,
330 mM Tris-HCl (pH 7.6)
, 33mM MgCl2), 2.5mM ATP
(48 μl) was added, heated at 75°C for 5 minutes, and slowly cooled to room temperature. 0.2 M 2-mercaptoethanol (10 μl), T4 DNA ligase (
Add 2 μl, 700 units, manufactured by Takara Shuzo Co., Ltd., and add 15
The reaction was carried out at ℃ for 15 hours. After ethanol precipitation, approximately 3 μg was fractionated by 10% polyacrylamide gel electrophoresis.
An activated c-Ha-ras DNA template was obtained (
Figure 1).

[0030] A normal c-Ha-ras DNA template was also prepared in the same manner.

Reference Example 3 Preparation of activated c-Ha-ras mRNA using T7 polymerase The transcription reaction was performed using a known method (Jikken Igaku, Vol. 8, (13)
, pp. 1685-1689, (1990)) under the following conditions. The obtained activated c-Ha-rasDN
A template (30 pmol), buffer solution (40 pmol)
mM Tris-HCl (pH 8.1), 20
mM MgCl2, 5 mM DTT, 1 mM spermidine, 0.01% (v/v) Triton
X-100, 0.05 μg/μl Bovine serum albumin, 4mM 4NTPs (ATP, GTP, CTP,
and UTP: hereinafter referred to as "4NTPs". ), 8%
(W/V) dissolved in polyethylene glycol), T7
A transcription reaction was performed by adding RNA polymerase (840 units, manufactured by Pharmacia) and heating at 37°C for 2 hours (Fig. 1). After treatment with phenol chloroform, Sephadex G-50 (φ1.8 x
After desalting with 0.1 M TEAB (solvent: 0.1 M TEAB) to remove unreacted 4NTPs, it was purified by electrophoresis on a 10% polyacrylamide gel containing 8 M urea.

[0032] Normal c-Ha-ras mRNA
was also prepared in a similar manner.

Reference Example 4 Activated c-Ha-ras
5'-end labeled activated c-Ha-rasRNA (20 pmol) of mRNA was dissolved in 0.1 M Tris-HCl (pH 8.0), and E. coli alkaline phosphatase (0.0
2 units of Takara Shuzo Co., Ltd.) were added, heated at 37°C for 1 hour, and then treated with phenol chloroform twice.
γ-32P]ATP (5 μCi), 50 mM T
ris-HCl (pH 7.6), 10 mM MgC
l2, 10mM 2-mercaptoethanol, T4
- Polynucleotide kinase (1 unit, manufactured by Takara Shuzo Co., Ltd., phosphorylates the 5' end, and NENSORB 2
0 (manufactured by DuPont) and was subjected to desalting and protein removal.

[0034] Note that normal c-Ha-ras mRNA
were also labeled in the same manner.

Reference Example 5 In vitro cleavage reaction Activated c-Ha-ras obtained in Reference Example 4
The ribozyme (7.5 pmol) obtained in Reference Example 1 was added to mRNA (5 pmol), and 40 mM Tr
is-HCl (pH 7.5), 20 mM NaCl
, add a solution of 25 mM MgCl2 and heat to 37 °C.
The reaction was stopped by adding 50 mM EDTA. The mixture was rapidly cooled at 55° C. for 33 minutes and analyzed by electrophoresis on a 10% polyacrylamide gel containing 8 M urea. Cutting rate is determined by cutting out the gel and cutting out the liquid scintillator.
It was determined by measuring with a tion counter.

As a control, normal c-Ha-ras m
Similar experiments were conducted with RNA.

As a result, it was confirmed that the ribozyme obtained in Reference Example 1 did not cleave normal c-Ha-rasm RNA, but only cleaved activated c-Ha-ras mRNA.

[0038]

[Example] Example 1 Construction of plasmid pKCS pR
SVneo (5 μl, 1.4 pmol) was added to H
The mixture was digested with indIII (12 units) at 37° C. overnight, and the digested product was obtained by precipitation with ethanol. To this, sterile water (10 μl) and buffer solution (4 μl, 3
30 mM Tris-HCl (pH 7.6), 3
3mM MgCl2, 2.5mM ATP),
0.2M 2-mercaptoethanol (1 μl),
40 mer ClaI-SalI linker (5μ
1,7 pmol (Fig. 2) and T4 ligase (10 units) were added, and the mixture was heated at 16°C for 2 hours. After ethanol precipitation, pKCS was obtained. This was transfected into Escherichia coli HB101, and a method known in the literature (Anal.
ocem. , vol. 113 34-42, (19
89)) from 250 ml of medium.
μg of pKCS was isolated (Figure 3).

Example 2 Preparation of plasmid pRZ1 p
KCS (7.5 μl, 2.5 pmol) was dissolved in Cl
After digestion with a I (15 units) at 37°C for 2 hours, the mixture was digested with Sal I (30 units) at 37°C overnight. 0.1 M Tris-HCl (pH
8.0.48 μl), added E. coli alkaline phosphatase (0.7 units), and incubated at 37°C.
The mixture was heated for 2 hours. After treatment with phenol chloroform,
A digestate was obtained by ethanol precipitation. Separately prepared ribozyme (CUACACCCUGAUGAAGGGUG
Similarly, the double-stranded oligoribonucleotide containing the AUACCCUGAAACAGCGCp) gene is Cla I,
Digested with Sal I (Figure 4). This pKCSCla
The above ribozyme gene was added to the I-Sal I digest and ligated with T4 DNA ligase to create pRZ1.
I got it. This was transfected into Escherichia coli HB101, and a method known in the literature (Anal. Biocem.,
vol. 113 34-42, (1989)), approximately 470 μg of pR was obtained from 250 ml of medium.
Z1 was isolated (Figure 3).

Example 3 Preparation of plasmid pRZ2 Ribozyme (UGCCUACA
CCCUGAUGAGUCGUGAUACGACGAA
A plasmid pRZ2 incorporating the ACAGCGCCAp) gene was prepared.

Example 4 Preparation of plasmid pRZ3 Ribozyme (CUACACCC
UGAUGAGGGGGGCUUUUCCCCCGAAA
A plasmid pRZ3 incorporating CAGCGCp) was prepared.

Example 5 pRZ1, pRZ2 or p
Preparation of NIH3T3 cells transformed with RZ3 pRZ1 (1 μg), NIH3T3 cell DNA (
30 μg) to 2 M CaCl2 (62.5
μl) was added, and the total volume was made up to 500 μl with sterile water. This was mixed with HBS solution (11.92 g HEPES,
16.36 g NaCl, 0.23 g NaH
2PO4 (pH 7.1) /1000ml)50
0 μl and left for 30 minutes to form a calcium phosphate precipitate. This was added to a petri dish in which NIH3T3 cells (8 x 105 cells) had been cultured, and cultured at 37°C for 5 hours (calcium phosphate method). Then, the DMEM medium containing 10% bovine serum (Dulbecco's modified Eagle's minimal medium) was removed, and 17% glycerin (2 ml) was added for 1 minute.
After standing for a second, DMEM medium (10 ml) was added. D
Replace with MEM medium (containing 10% bovine serum) and
The cells were cultured at 7°C for 15 hours. After trypsin treatment, the cells were peeled from the petri dish, spread on three petri dishes, and cultured in DMEM medium (containing 5% bovine serum). 4 days later, G.
The medium was replaced with DMEM medium (containing 5% bovine serum) supplemented with ENETICIN (300 mg/500 ml, manufactured by Gibco) and cultured for 7 days.
3 cells were obtained.

[0043] pKCS/NIH3T3 cells transformed with pKCS, pRZ2, and pRZ3, p
RZ2/NIH3T3 cells, pRZ3/NIH3T3
Cells were also obtained in the same manner.

Example 6 Transformation by activated c-Ha-ras gene (pRSV-rv12)
pRSV-rv12 (20 ng), NIH3T
pKCS/NIH3T3 cells, pRZ2/NIH3T3 cells, pRZ3/NIH3 cells were isolated using the same calcium phosphate method as above using 3 cell DNA (30 μg).
T3 cells were transformed. Then DMEM
The cells were cultured in a medium (containing 5% bovine serum) for about 2 weeks.

[0045] pRSV-rv12 is normal c-
A point mutation was caused in the codon encoding the 12th amino acid of Ha-ras (Gly 12 (GGU) →
Val 12 (GUU)) activated c-Ha-rasD
It incorporates NA.

As a control, normal c-Ha-ras 12
There is no change in the codon encoding the 6th amino acid, but
Activated c-Ha- with a point mutation in the codon encoding the first amino acid (Gln61→Leu61)
Each of the above cells was transformed using pRSV-rl61 incorporating ras DNA and cultured.

After culturing, the cells were stained with Giemsa, and the number of foci produced was counted.

The results are shown in FIG.

The graph in (A) shows that pKCS/NIH3T3 cells, pRZ1/NIH
3T3 cells, pRZ2/NIH3T3 cells, pRZ
This is the number of foci when transfecting 3/NIH3T3 cells.

The graph in (B) shows that pKCS/NIH3T3 cells, pRZ1/NIH
3T3 cells, pRZ2/NIH3T3 cells, pRZ
This is the number of foci when transfecting 3/NIH3T3 cells.

pRZ1/NIH3T3 cells, pRZ2
/NIH3T3 cells, pRSV-rv1
In transfection with 2, approximately 50%
Although inhibition of focus formation was observed in pRZ3/NIH
No inhibition was seen in 3T3 cells.

When transfected with pRSV-rl61, compared to pKCS/NIH3T3 cells,
The activated c-Ha-rasm transcribed by the plasmid showed no inhibition of focus formation.
It was confirmed that ribozyme had no effect on RNA. Therefore, the ribozymes of the present invention are suitable for normal c.
-A point mutation was caused in the codon encoding the 12th amino acid of Ha-ras (Gly 12 (GGU)
→Val 12 (GUU) ) activated c-Ha-ras
It was concluded that it specifically cleaves mRNA.

[0053]

Effect of the invention: mR transcribed from oncogenes of the present invention
Ribozymes with specific cleaving activity for NA are
It exhibits an effective antitumor effect, and can be expected to be used as an antitumor agent containing it as an active ingredient.

[Brief explanation of the drawing]

Figure 1 shows activated c-Ha-ras DNA template and activated c-Ha-ras m transcribed from it.
Figure 2 shows Cla I-Sal I
The nucleotide sequence of the linker is shown, FIG. 3 shows the method of construction of plasmid pRZ1, FIG. 4 shows the oligoribonucleotide containing the ribozyme gene, and FIG. 5 shows the cells transfected with the plasmid carrying the ribozyme gene. Figure 2 shows the inhibition rate of focus formation by oncogenes.

Claims (2)

[Claims] Claim 1: General formula: [Formula 1] In the formula, A represents an adenine nucleotide, U represents a uracil nucleotide, C represents a cytosine nucleotide, and G represents a guanine nucleotide, and 3' and 5' represent the 3' end and 5', respectively. An antitumor agent whose active ingredient is a compound represented by the following formula: [Claim 2] General formula: [Formula 2] In the formula, A represents an adenine nucleotide, U represents a uracil nucleotide, C represents a cytosine nucleotide, and G represents a guanine nucleotide, and 3' and 5' represent the 3' end and 5', respectively. An antitumor agent whose active ingredient is a compound represented by the following formula: