JP2011251912A - Pharmaceutical preparation comprising micro-rna-143 derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a benzene-pyridine derivative of micro-RNA-143 less susceptible to degradation by a degrading enzyme (RNase) and a pharmaceutical use thereof.SOLUTION: The micro-RNA-143 is matched at two positions of 3' terminal of antisense strand among mismatch sequences of four positions of micro-RNA-143, modified with benzene-pyridine (BP) at 3' terminal and is less susceptible to degradation by a degrading enzyme (RNase). The therapeutic agent for digestive system cancer contains the micro-RNA-143.

Description

  The present invention relates to a medicament containing a microRNA-143 derivative. More specifically, the present invention relates to a benzene-pyridine derivative of microRNA-143 that is not easily degraded by a degrading enzyme (RNase) and its pharmaceutical use.

  Measurement of microRNAs-143 and -145 expression levels using colorectal cancer and various types of established cancer cell lines revealed decreased expression levels of microRNAs-143 and -145 However, it has been found that the expression level of microRNAs is strongly related to carcinogenesis (Non-patent Document 1).

  In colorectal tumors, it has been reported that the expression of microRNA-143 and -145 decreases very frequently. As a cause of the decrease in the expression of microRNA-143 and microRNA-145, deletion of genes in the localized region and epigenetic changes are considered (Non-patent Document 2).

  Patent Document 1 discloses a chemically modified nucleic acid characterized by a chemically modified small interfering nucleic acid (siRNA) construct having specificity for a target nucleic acid molecule in a cell. Examples of chemical modifications include phosphorothioate internucleotide linkages, 2′-O-methylribonucleotides, 2′-deoxy-2′-fluororibonucleotides, 2′-deoxyribonucleotides, “universal base” nucleotides, 5-C-methyl nucleotides, And incorporating an inverted deoxy abasic residue. These chemical modifications have been shown to preserve RNAi activity in cells and at the same time dramatically increase the serum stability of these compounds when used in various siRNA constructs.

  Patent Document 2 discloses a chemically modified small interfering nucleic acid (siRNA) structure having specificity for a target nucleic acid molecule in a cell. Examples of chemical modifications include phosphorothioate internucleotide linkages, 2′-O-methylribonucleotides, 2′-deoxy-2′-fluororibonucleotides, 2′-deoxyribonucleotides, “universal base” nucleotides, 5-C-methyl nucleotides, And the attachment of an inverted deoxyabasic residue. It has been disclosed that multiple (more than one) phosphorothioate substitutions exhibit excellent resistance and greatly improve serum stability against modified siRNA structures.

  Furthermore, in Patent Document 3, as a result of analyzing the correlation between the decrease in let-7 miRNA expression and the survival time of the patient using the Kaplan-Meier method for lung cancer patients after surgery, the expression of let-7 miRNA We found that there was a significant correlation between the decrease and the patient's survival time, and analysis by Cox regression analysis showed that reduced expression of let-7 miRNA is an independent prognostic factor for the survival of lung cancer patients The knowledge is reported. And it is disclosed that the prognosis of a cancer patient can be determined by measuring the expression level of a specific mature miRNA, pre-miRNA, pri-miRNA or the like in a biological sample derived from a cancer patient. Yes.

As described above, it was found that the expression level of microRNA was decreased in cancer patients, and that cell proliferation was suppressed when the reduced microRNA was introduced into cancer cells. However, even when wild-type microRNA is administered to a living body, it is easily degraded by a degrading enzyme (RNase) and cannot sufficiently exert an antitumor effect. Therefore, the appearance of microRNA that is not degraded by enzymes, has low toxicity, and has high antitumor activity is desired.
JP 2006-271387 A Special table 2007-525192 gazette JP 2008-000137 A Japanese Patent Application No. 2006-266918 Y. Akao, N. Nakagawa, Y. Kitade, T. Kinoshita and T. Naoe; Cancer Sci., 2007,98 (12), 1914-1920 Nakagawa Yoshihito, Akao Yukihiro, Naoe Tomoki; Gastroenterology, 2007,44 (5), 484-492

  It is known that the expression level of microRNA-143 is decreased in tumor cells, and that cell proliferation is suppressed when this microRNA-143 is introduced into cells. However, even if microRNA-143 is administered in vivo, it may be decomposed before exhibiting an antitumor effect and a sufficient effect may not be obtained. Therefore, the present invention is to provide a modified microRNA-143 having high anti-tumor effect against degradation enzyme (RNase), which has high activity in vivo and is hardly susceptible to degradation.

  It has been found that the expression level of microRNA-143 is reduced in tumor cell lines, and when microRNA is introduced into tumor cells, the growth of tumor cells is suppressed, so that microRNA-143 has an antitumor effect (Japanese Patent Application No. 2006-266918). However, even if wild type microRNA-143 is administered, it is easily degraded by a degrading enzyme (RNase) before exerting an antitumor effect, so that microRNA-143 that is not easily degraded in vivo is provided. Researched earnestly. As a result, the present inventors succeeded in providing a BP microRNA-143 derivative in which an antisense strand which is hardly degraded by a degrading enzyme and has an antitumor effect higher than that of the wild type is modified.

  A feature of the present invention is that the 3 ′ end is modified with benzene-pyridine (BP), which is characterized in that two 3 ′ ends of the antisense strand of four mismatch sequences of microRNA-143 are matched. MicroRNA-143 (3,4 M-miR-143).

  Another feature of the present invention is that the 3 ′ end is modified with benzene-pyridine (BP), characterized in that two 3 ′ ends of the antisense strand represented by SEQ ID No. 1 are matched. MicroRNA-143 (3,4 M-miR-143).

  Another feature of the present invention is a microRNA-143 (3,4M-miR) modified at the 3 ′ end with benzene-pyridine (BP), characterized in that one 3 ′ end of the antisense strand is matched. -143).

  Another feature of the present invention is that the 3 ′ end is modified with benzene-pyridine (BP), which is characterized in that one 3 ′ end of the antisense strand represented by the sequence listing SEQ ID No. 2 is matched. MicroRNA-143 (1,2,4M-miR-143).

  Another feature of the present invention is that the 3 ′ end of the antisense strand is matched at two or one site, or the 3 ′ end is modified with benzene-pyridine (BP) -modified microRNA-143, or A pharmaceutical comprising the compound described in Sequence Listing SEQ ID No. 2 or SEQ ID No. 1 in Sequence Listing.

  Another feature of the present invention is that the 3 ′ end of the antisense strand is matched at two or one site, or the 3 ′ end is modified with benzene-pyridine (BP) -modified microRNA-143, or A therapeutic agent for gastrointestinal cancer comprising a compound described in SEQ ID NO. 2 of Sequence Listing or SEQ ID No. 1 of Sequence Listing.

  Provided is a microRNA-143 in which two or three sites on the 3 ′ end of an antisense strand modified with benzene-pyridine (BP), which is difficult to undergo enzymatic degradation in vivo, are matched. That is, microRNAs that match two or three 3 ′ ends of an antisense strand in which the 3 ′ end is modified with benzene-pyridine and has an antitumor effect that is difficult to be degraded by a degrading enzyme (RNase) in vivo— 143 is provided.

I. Monomer Synthesis In this example, compounds 2 to 5 shown in the following scheme I were synthesized. That is, dimethyl isophthalate was reduced to obtain compound 2 in a yield of 75%, followed by DMTr conversion to obtain compound 3 in a yield of 54%. DTMr body 3 was amidated to obtain compound 4 in a yield of 88%. DMTr body 3 was succinylated and bound to CPG resin to obtain compound 5 with an activity of 106 μmol / g. Production examples of compounds 2 to 5 are shown below.
I-I Synthesis of benzene unit Using dimethyl isophthalate (Compound 1) as a starting material, a reduction reaction was performed with LiBH ₄ to obtain Compound 2. Further, one hydroxyl group was DMTr protected to obtain a trityl compound, which was compound 3, and then phosphorylated to obtain an amidite unit, which was compound 4. Further, Compound 5 which is a CPG carrier was obtained from Compound 3 through succinylation.

<Scheme1>

(Compound 2: Production example of 1,3-bis-hydroxymethylbenzene)
In THF, dry THF (51.5 ml, 0.2 M solution) was added to dimethyl isophthalate (2.00 g, 10.30 mmol), and lithium borohydride (1.12 g, 51.1 mmol, 5 eq) was added. After stirring for 23 hours, several drops of acetic acid were added in an ice bath to neutralize the reaction solution, and the reaction was stopped. After stirring for a while, the precipitated crystals were dissolved with MeOH. In TLC during the reaction (Hex: EtOAc = 1: 1), the product was one spot, but when the reaction was stopped, it was divided into two spots. After evaporating the solvent under reduced pressure, the residue was isolated by silica gel chromatography (EtOAc only) to obtain Compound 2 (75%).
(Compound 3: Production Example of 1- (4,4'-dimethoxytrityloxy) methyl-3-hydroxymethylbenzene) Compound 2 (0.5 g, 3.62 mmol), which had been dried in advance, was dissolved in pyridine (18 mL) and DMAP ( 22.1 mg, 0.18 mmol, 0.05 eq) and 4,4′-Dimethoxytrityl chloride (1.23 g, 3.62 mmol, 1 eq) were added, and the mixture was stirred under Ar atmosphere for 17 hours. The disappearance of the starting material was confirmed by TLC (Hex: EtOAc = 3: 1). Extraction was performed with EtOAc and satNaHCO ₃ aq, and the organic layer was washed with sat NaCl aq, dried over anhydrous Na ₂ SO ₄ . After evaporating the solvent under reduced pressure, the residue was isolated by silica gel chromatography (Hex: EtOAc = 4: 1) to obtain Compound 3 (54%).
(Compound 4: 1- (4,4'-dimethoxytrityloxy) methyl-3-O-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoamidiomethyl-hydroxymethylbenzene
Production example)
Compound 3 (0.35 g, 0.80 mmol), which had been vacuum-dried in advance, was dissolved in dry THF (8 mL), and DIPEA (0.4 mL, 4.00 mmol, 5 eq) and phosphorylation reagent (0.29 mL, 1.60 mmol, 2 eq) And stirred for 1.5 hours under Ar atmosphere. The disappearance of the raw material was confirmed by TLC (EtOAc only). Extraction was performed with EtOAc and satNaHCO ₃ aq, and the organic layer was washed with sat NaCl aq, dried over anhydrous Na ₂ SO ₄ . After evaporating the solvent under reduced pressure, the residue was isolated by silica gel chromatography (Hex: EtOAc = 1: 1) to obtain Compound 4 (88%).
(Production Example of Compound 5 (CPG resin of isophthalic acid derivative))
Compound 3 (0.30 g, 0.68 mmol) is dissolved in pyridine (6.8 mL), and DMAP (3.7 mg, 0.03 mmol, 0.02 eq) and succinic anhydride (204 mg, 2.04 mmol, 3 eq) are added thereto under Ar atmosphere. Stir. After stirring for 24 hours, the progress of the reaction was confirmed by TLC (Hex: EtOAc = 3: 1), extracted with EtOAc and satNaHCO ₃ aq, the organic layer was washed with sat NaCl aq, dried by adding anhydrous Na ₂ SO ₄ I let you. The solvent was distilled off under reduced pressure, followed by vacuum drying. To this concentrate (5) (0.04 g, 0.74 mmol, 109%), dry DMF (10 mL) was added and dissolved, CPG (500 mg, 0.11 mmol) was added, and the mixture was allowed to stand for 30 minutes to allow it to blend with the reaction solution. Thereafter, WSC (110 mg, 0.57 mmol, 4.9 eq) was added, and the mixture was shaken at room temperature for one day. As a post-treatment, after washing with pyridine, 0.1M DMAP in pyridine: acetic anhydride (9: 1) solution (6 mL) was added and shaken for 16 hours. This was washed with MeOH and acetone, dried, and the activity was measured. The activity of Compound 5 was 108.6 μmol / g. For activity, 6 mg of dried CPG resin was placed on a glass filter, a solution of HClO4: EtOH = 3: 2 was poured, and the absorbance of the filtrate at a wavelength of UV 498 nm (wavelength of DMTr group) was determined. Calculated by substituting into the equation.
Activity (μmol / g) = Abs, (498 nm) × Vol. (Solution) (mL) × 14.3 / Weight (support) (mg)


Synthesis of I-II pyridine unit

<Scheme2>

(Synthesis of dimethyl pyridinecarboxylate for the synthesis of 3 'terminal down-gring end)
Compound 7 was obtained by carrying out a reduction reaction with LiBH ₄ using dimethyl 2,6-pyridinecarboxylate (compound 6) as a starting material. Further, one hydroxyl group was DMTr protected to obtain a trityl compound, which was compound 8, and then phosphorylated to obtain an amidite unit, which was compound 9. Moreover, the compound 10 which is a CPG carrier was obtained from this compound through succinylation.
(Compound 7: Production example of 2,6-bis-hydroxymethylpyridine)
2.6 Anhydrous THF (51.3 mL, 0.2 M solution) was added to dimethyl pyridinecarboxylate (2.00 g, 10.25 mmol) under an Ar atmosphere, and lithium borohydride (1.16 g, 51.3 mmol,
5 eq) was added. After stirring for 16 hours, several drops of acetic acid were added in an ice bath to neutralize the reaction solution, and the reaction was stopped. After stirring for a while, the precipitated crystals were dissolved in methanol. In TLC during the reaction (chloroform: methanol = 3: 1), the product was one spot, but when the reaction was stopped, it was divided into two spots. After the solvent was removed under reduced pressure, the residue was isolated by silica gel chromatography (chloroform: methanol = 10: 1 to 3: 1) to obtain Compound 7 (91%).
(Compound 8: Production example of 2- (4,4'-dimethoxytrityloxy) methyl-6-hydroxymethylpyridine)
Compound 7 (0.5 g, 3.62 mmol), which had been vacuum-dried in advance, was dissolved in pyridine (18 mL), and DMAP (22.1 mg, 0.18 mmol, 0.05 eq) and 4,4'-Dimethoxytrityl chloride
-22 g, 3.60 mmol, 1 eq) was added, and the mixture was stirred for 16 hours under an Ar atmosphere.
(TLC (Hex: EtOAc = 1: 1) confirmed the disappearance of the starting material EtOAc and satNaHCO _3.
Extracted with aq, the organic layer was washed with sat NaCl aq, dried over anhydrous Na ₂ SO ₄ . After evaporating the solvent under reduced pressure, the residue was isolated by silica gel chromatography (Hex: EtOAc = 4: 1 to 3: 1) to obtain Compound 8 (44%).
(Compound 9: Production Example of 2- (4,4'-dimethoxytrityloxy) methyl-6-0-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoamidiomethyl-hydroxymethylbenzene)
Compound 8 (0.20 g, 0.45 mmol), which had been vacuum-dried in advance, was dissolved in anhydrous THF (4.5 mL), and DIPEA (0.23 mL, 2.25 mmol, 5 eq) and phosphite reagent (0.16 mL, 0.90 mmol, 2 eq) were dissolved. And stirred for 15 hours under Ar atmosphere. The disappearance of the raw material was confirmed by TLC (EtOAc only). Extraction was performed with EtOAc and satNaHCO ₃ aq, and the organic layer was washed with sat NaCl aq, dried over anhydrous Na ₂ SO ₄ . After evaporating the solvent under reduced pressure, the residue was isolated by silica gel chromatography (EtOAc only) to obtain 0.27 g (88%) of Compound 9.
(Compound 10: Production example of CPG resin of dimethyl pyridylcarboxylate derivative)
Compound 8 (0.20 g, 0.45 mmol) is dissolved in pyridine (4.5 mL), and DMAP (1.1 mg, 0.009 mmol, 0.02 eq) and succinic anhydride (136 mg, 1.36 mmol, 3 eq) are added thereto under Ar atmosphere. Stir. After stirring for 17 hours, the progress of the reaction was confirmed by TLC (Hex: EtOAc = 1: 1), extracted with EtOAc and satNaHCO ₃ aq, the organic layer was washed with sat NaCl aq, dried by adding anhydrous Na ₂ SO ₄ I let you. The solvent was distilled off under reduced pressure, followed by vacuum drying. To this concentrate (0.16 g, 0.30 mmol, 66%), dry DMF (7.5 mL) was added and dissolved, CPG (338 mg, 0.075 mmol) was added, and the mixture was allowed to stand for 30 minutes to allow it to blend with the reaction solution. Thereafter, WSC (71 mg, 0.37 mmol, 4.9 eq) was added and the mixture was shaken at room temperature for one day. As a post-treatment, after washing with pyridine, 0.1M DMAP in pyridine: acetic anhydride (9: 1) solution (6 mL) was added and shaken for 16 hours. This was washed with pyridine, EtOH and acetone and dried to measure the activity. The activity of Compound 10 was 31.4 μmol / g.
(Synthesis of microRNA (miRNA) with chemically modified dangling ends)
An RNA oligonucleotide having a 3 ′ terminal dangling end was synthesized by an automatic nucleic acid synthesizer according to the solid phase phosphoramidite method.

For solid phase synthesis of RNA, 2 mL of EtOH: NH ₃ = 3: 1 aqueous solution was added to the oligonucleotide bound to the CPG resin and shaken at room temperature for 12 hours to cleave from the resin and deprotect. The condensation time was 15 minutes. The filtrate after the reaction was transferred to an Eppendorf tube and dried under reduced pressure. 1 mL of 1M TBAF in THF solution was added to the residue and shaken for 12 hours. This reaction solution was diluted with 0.1 M TEAA buffer to 30 mL. This reaction solution was passed through an equilibrated C-18 column (Sep-Pak) and adsorbed on the column. Here, the column was washed with sterilized water to remove salt, and eluted with 3 mL of 50% CH ₃ CN in H ₂ O, and dried under reduced pressure. To the residue, 100 μL of loading solution (1 × TBE in 90% formamide) was added, and the target oligonucleotide was isolated by 20% PAGE (600 V, 20 mA). The target oligonucleotide was cut out, 20 mL of 0.1 M TEAA buffer 1 mM EDTA aqueous solution was added, and the mixture was shaken for 12 hours. The filtrate was passed through an equilibrated C-18 reverse phase or ram (Sep-Pak) and adsorbed onto the column. Here, the column was washed with sterilized water to remove salts, and eluted with 3 mL of 50% CH ₃ CN in H ₂ O, and dried under reduced pressure.

Table 1. Sequence of synthesized microRNA-143 derivatives

P represents a pyridine unit and B represents a benzene unit.

As shown in Table 1, wild type microRNA-143 (wild) has four mismatches indicated by (1), (2), (3), and (4). A compound in which each mismatch position was matched was synthesized and its antitumor effect was examined.

Inhibition of DLD-1 cells Human cancer cells are 95% air and 5% carbon dioxide in RPMI-1640 medium containing 2 mM L-glutamine, 10% heat inactivated FBS (Sigma, St. Louis, Mo, USA). In an atmosphere of 37 ° C. The number of viable cells was measured by trypan blue staining.

  FIG. 1 shows the results of examining the effect of a microRNA-143 derivative on the proliferation of colon cancer cells DLD-1. Match 1,4,4,4M-miR-143 matched with mismatch at 4 locations (1), (2), (3), (4), and (1), (2) at 5 'end In addition, 1,2M-miR-143 tended to promote proliferation compared to wild-type microRNA-143 (Wild-miR-143) having four mismatches. On the other hand, the 3 'end 2 locations (3) and 3,4M-miR-143 matched (4), and 1 location (4) matched 1,2,4M-miR-143 are significant. In addition, the growth of colon cancer cells was suppressed.

As a result, the antitumor effect of microRNA-143 can be made higher than that of the wild type by matching the mismatch at the 3 ′ end in two or one place, and the 3 ′ end is further modified with benzene-pyridine. As a result, microRNA-143 resistant to degrading enzyme (RNase) can be obtained.
[Pharmaceutical composition]
The microRNA-143 of the present invention may be given as microRNA-143 itself when administered to humans and animals as a medicine, or together with a pharmaceutically acceptable carrier, for example, 0.1 to 99. It may be given as a pharmaceutical composition containing 5% (more preferably 0.5-90%) of the active ingredient.

  The medicament containing the microRNA-143 of the present invention includes other compositions adapted for therapeutic purposes (including compounds such as stabilizers, degrading enzyme inhibitors), and the use of such compositions in medical treatment methods. A method comprising administering such a composition to a patient, eg for the treatment of cancer (which may include prophylactic treatment), a composition, pharmaceutical or pharmaceutical administered for any such purpose There is provided a process for the manufacture of a pharmaceutical composition comprising the use of microRNA-143 in the manufacture of and a compound of the pharmaceutically acceptable excipient, vehicle or carrier and optionally other ingredients and the compound.

In one embodiment, the method for providing a pharmaceutical composition comprises:
(A) producing the microRNA-143 of the present invention,
(B) formulating the so-produced microRNA-143 with a pharmaceutically acceptable excipient.

  The pharmaceutical composition of the present invention may comprise the microRNA-143 of the present invention and a pharmaceutically acceptable excipient. The microRNA-143 can be used as a single active agent, in combination with each other, or any other active agent, such as an anti-tumor therapeutic agent, other anti-tumor compounds or radiotherapy or chemotherapy It can be used in combination with therapy.

  In using the microRNA-143 of the present invention, administration is preferably “prophylactically effective amount” or “therapeutically effective amount” (prophylaxis can be considered a therapy, but will be determined accordingly). This is an amount sufficient to show benefit to the individual. The actual dose and the rate and time course of administration will depend on the nature and severity of the subject being treated. Decisions regarding treatment prescriptions, such as dosages, are made at the responsibility of general practitioners and other physicians.

  The microRNA-143 or composition of the invention can be administered alone or in combination with other treatments, either simultaneously or sequentially, eg, depending on the condition to be treated as described above.

  Pharmaceutical compositions using the microRNA-143 of the present invention include, in addition to the active ingredient, pharmaceutically acceptable excipients, carriers, buffers, stabilizers or other formulations well known in the art. May be included. In particular, they can contain pharmaceutically acceptable excipients. Such formulations should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other formulation will depend on the route of administration, which may be oral administration or injection, eg, skin, subcutaneous or intravenous injection.

  For intravenous, dermal or subcutaneous injection, or injection into the affected site, microRNA-143 is parenterally acceptable with appropriate pH, isotonicity and stability and does not contain pyrogens It will be in the form of an aqueous solution. Those skilled in the art are well able to produce suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. If necessary, preservatives, stabilizers, buffers, antioxidants and / or other additives may be included.

  In the preparation for transporting the microRNA-143 of the present invention to the affected area, it is possible to use liposomes, particularly cationic liposomes. Specific examples of such techniques and protocols are described in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.), 1980.

  The microRNA-143 of the present invention can be administered locally at a specific site, or transported so that it targets a specific cell or tissue, for example using intraarterial stent based transport Is possible. In order to deliver the compound of the present invention more specifically to a certain type of cell, a targeting therapy using a targeting system such as an antibody or a cell-specific ligand can be used. Targeting is desirable to allow more compounds to enter the target cell accurately.

Examination of the stability of synthetic double-stranded RNA in the presence of serum Investigate the effects of degradation by blood components (various components contained in serum) when intravenously administering synthetic double-stranded RNA, and examine various modifications of synthetic RNA RNA stabilization was investigated.

  Each synthetic double-stranded RNA of hsa-pre-143 (manufactured by Ambion), 3,4M-miR-143 (BP), 3,4M-miR-143 (TTP), 5% fetal calf at a concentration of 20 nM each Add to serum-containing RPMI-1640 medium, incubate at 37 ° C for 1, 2, 5, 10, 15, 30 minutes, immediately add RNase inhibitor (RNase OUT 40U, Invitrogen) and add RNA Suppressed decomposition. Prepare cDNA using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) using 2 μl of the diluted reaction product as a template, PreMix Ex Taq (TAKARA), TaqMan MicroRNA Assay (Applied Biosystems) and Thermal Cycler Real-Time RT-PCR was performed using Dice TP800 (manufactured by TAKARA), and the residual RNA in the reaction was quantified. The quantification was analyzed by the 2nd Derivative Maximum method and calculated as a Ct value (the Ct value at time 0 was set to 0. The larger the Ct value, the smaller the amount).

  As a result, as shown in Fig. 2, degradation of RNA generally progressed within 5 minutes when comparing ATP's hsa-pre-143, 3,4M-miR-143 BP, and TTP modified TTP. However, the Ct values after 30 minutes were 8.14, 5.38, and 6.51, respectively, and it was found that BP-modified synthetic 3,4M-miR-143 had the largest amount of residual RNA and was stable in the presence of serum.

Anti-tumor effect of benzene-pyridine modified miR-143 using animal model The anti-tumor effect of benzene-pyridine modified miR-143 was examined using an animal tumor model. Human colon cancer cell line DLD-1 2 × 10 ⁶ cells were implanted subcutaneously in 4-5 week old nude mice. Tumors that appeared and the tumor diameter exceeded 1 x 0.5 cm were divided into 3 groups (1 group: 8 animals). Renilla (sea shiitake) luciferase (R), benzene-pyridine modified miR-143 3 , 4M-miR-143 and 1,2,4-miR-143 were administered locally to the tumor only 0.2 mg each. After 4 weeks, the tumor was removed and its weight was measured.

  As a result, the 3,4M-miR-143 group of benzene-pyridine modified miR-143 significantly decreased tumor weight compared to the control R group, and 1,2,4-miR-143 was halved by anatomical findings. Although the effectiveness was recognized, the variation was large and there was no significant difference. However, as shown in FIG.

FIG. 1 shows the survival rate of microRNA-143 against colon cancer cell DLD-1. FIG. 2 shows the results of studying the stability of synthetic double-stranded RNA in the presence of serum. FIG. 3 shows the results of the antitumor effect of modified miR-143 using an animal model.

SEQUENCE LISTING

<110> Gifu International Institute of Biotechnology
Gifu University
Hokkaido System Sience KK

<120> Chemical modified Micro RNA

<130> I00104

<150> Akao Yukihiro
<151> 2008-08-10

<150> Kitade Yukio
<151> 2008-08-10

<150> Takenishi Shouichirou
<151> 2008-08-10

<150> Susa Tomoe
<151> 2008-08-10

<160> 5

<170> PatentIn version 3.3

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<212> RNA
<213> colon


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<222> (1) .. (22)

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<210> 2
<211> 22
<212> RNA
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<220>
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<222> (1) .. (22)
<223>3'-benzene, pyridine

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acucuacuuc gugacgucga gu 22


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<213> colon


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acucuacuuc gugacaucga gu 22

Claims (6)

A microRNA-143 whose 3 'end is modified with benzene-pyridine, characterized in that two 3' ends of the antisense strand are matched. The microRNA-143 modified at the 3 'end with benzene-pyridine, characterized in that two 3' ends of the antisense strand according to claim 1 represented by the sequence listing SEQ ID No. 1 are matched. . A microRNA-143 in which the 3 'end is modified with benzene-pyridine, wherein one 3' end of the antisense strand is matched. A microRNA-143 modified at the 3 'end with benzene-pyridine, wherein one 3' end of the antisense strand according to claim 3 represented by the sequence listing SEQ ID No. 2 is matched. . A pharmaceutical comprising the microRNA-143 according to any one of claims 1 to 4. A therapeutic agent for digestive organ cancer comprising the microRNA-143 according to any one of claims 1 to 4.