JP2011032269A - Treating or prophylactic agent of inflammatory bowel disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new prophylactic or treating agent of inflammatory bowel diseases including a decoy oligonucleotide as an effective component, and efficacious even by intravenous administration. <P>SOLUTION: A hairpin type double-stranded oligonucleotide having a structure, in which an oligonucleotide having a specific base sequence and its complementary strand are linked together through a specific linker, exerts NF-κB binding affinity higher than that of heretofore known NF-κB decoy. The oligonucleotide having the specific structure, among such oligonucleotides, can prominently suppress inflammation in bowel tissue even by intravenous administration in a naked form, without necessitating special delivery technique such as virus envelope vector. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an agent for treating or preventing inflammatory bowel disease.

  NF-κB (nuclear factor kappa B) is a collective term for a group of transcription factors that regulate the expression of genes related to immune responses, such as cytokines and adhesion factors, and NF-κB binds to the binding site on the genomic gene. Then, genes related to immune responses are overexpressed. For this reason, NF-κB is involved in allergic diseases such as atopic dermatitis and rheumatoid arthritis caused by immune reactions, autoimmune diseases, ischemic diseases such as myocardial infarction, and various diseases such as arteriosclerosis. It is known.

  One of the diseases involving NF-κB is inflammatory bowel disease. Inflammatory bowel disease is a chronic disease in which ulcerative colitis and Crohn's disease are the two major diseases, and is an intractable disease whose cause is unknown due to long-term diarrhea and bloody stool. Unlike normal food poisoning etc., it does not improve in a few days, and symptoms continue with remission and deterioration over a long period of time (many lifetimes). Although it does not kill lives, it is a characteristic of this disease that life is greatly sacrificed for the disease, especially in young patients. The number of patients in Japan exceeds 100,000 and is still increasing. The relationship with Western diet and autoimmunity has been studied, but the cause has not yet been investigated. For this reason, radical cure is difficult, and treatment is aimed at early remission introduction and long-term remission maintenance.

  On the other hand, it is known to administer a decoy for a transcription factor to reduce the activity of the transcription factor to be treated and to treat or prevent a disease caused by the transcription factor. Decoy means “decoy” in English, and a substance having a structure resembling that which a substance should originally bind to or act on is called decoy. As a decoy of a transcription factor, a double-stranded oligonucleotide having the same base sequence as the binding region on the transcription factor genomic gene is mainly used (Patent Documents 1 to 3). In the coexistence of such an oligonucleotide decoy, a part of the transcription factor molecule binds to the decoy oligonucleotide without binding to the binding region on the genome gene to be originally bound. For this reason, the number of molecules of the transcription factor that binds to the binding region on the genomic gene to be originally bound decreases, and as a result, the activity of the transcription factor decreases. In this case, the oligonucleotide is called a decoy because it functions as a fake (bait) of the binding region on the real genomic gene and binds a transcription factor. Various decoy oligonucleotides for NF-κB are also known, and their pharmacological effects are also known (Patent Documents 4 to 12).

  Among the medical uses of decoy oligonucleotides, the following are reports on the use of inflammatory bowel disease (IBD). Non-Patent Document 11 discloses the effect of intra-colonic administration of a double-chain NF-κB decoy without using a viral envelope in a mouse IBD model created by dextran sulfate sodium administration. Non-Patent Document 12 discloses the effect of intra-colonizing NF-κB (p65) antisense in a mouse IBD model. Patent Document 17 discloses that a mouse colitis model induced by 2,4,6-Trinitrobenzene sulphonic acid (TNBS) is mixed with a double-stranded NF-κB decoy encapsulated in an HVJ-E envelope vector in the rectum ( the effects of intrarectal) or intraperitoneal administration are disclosed. However, these documents do not describe or suggest decoys other than double-stranded decoys. Moreover, all are administered by administration routes, such as intracolonic and rectal administration, and there is no description or suggestion that they are effective when administered intravenously. Inflammatory bowel disease is a disease that repeats recurrence for a long time even if it recovers once, and it is too heavy for the patient to administer locally to the affected area each time. Therefore, a drug that can be administered intravenously is strongly desired for the treatment of inflammatory bowel disease.

No. 96/035430 gazette Japanese Patent No. 3392143 International Publication No. WO 95/11687 JP 2005-160464 A International Publication WO 96/35430 International Publication WO 02/066070 International Publication WO 03/043663 International Publication WO 03/082331 International Publication WO 03/099339 International Publication WO 04/026342 International Publication WO 05/004913 International Publication WO 05/004914 Japanese Translation of National Publication No. 08-501928 International Publication WO 93/06122 U.S. Patent No. 5,495,006 U.S. Pat.No. 5,556,752 International Publication WO2006 / 122242

Milligan et al. Med. Chem. 1993, 36, 1923 Marwick, C., (1998) J. Am. Med. Assoc., 280, 871 Stein & Cheng, Science 1993, 261, 1004 Levin et al., Biochem. Biophys. Acta, 1999, 1489, 69 Neish AS et al., J. Exp. Med. 1992, Vol. 176, 1583-1593. Leung K et al., Nature. 1988 Jun 23; 333 (6175): 776-778. Marina A. et al., The Journal of Biological Chemistry, 1995, Vol.270, Number 6, pp. 2620-2627 M. Durand et al., Nucleic Acids Res., 1990, 18 (21), 6353-6359 P.E.VOROBJEV et al., Biopolymers. 1993 Dec; 33 (12): 1765-77 Squire Rumney, IV et al., J. Am. Chem. Soc., 117 (21), 5635-5646, 1995 Gut. 2007 Apr; 56 (4): 524-33. Clin Exp Immunol. 2000 Apr; 120 (1): 51-8.

  An object of the present invention is to provide a novel preventive or therapeutic agent for inflammatory bowel disease, which comprises a decoy oligonucleotide as an active ingredient and can be effective even by intravenous administration.

  As a result of earnest research, the inventors of the present application have found that a hairpin type double-stranded oligonucleotide having a structure in which an oligonucleotide having a specific base sequence and its complementary strand are bound by a specific linker is a known NF-κB. It has been found that it exhibits higher NF-κB binding affinity than decoy. Furthermore, among the oligonucleotides, it has been found that oligonucleotides having a specific structure can remarkably suppress intestinal tissue inflammation even when intravenously administered in naked form without the need for special delivery techniques such as viral envelope vectors. The present invention has been completed.

That is, the present invention provides a therapeutic or preventive agent for inflammatory bowel disease comprising an oligonucleotide derivative represented by the general formula (I) as an active ingredient.
5'-aggggatttcccc- (CH ₂ CH ₂ O) _n -ggggaaatcccct-3 '(I)
(However, at least a part of the bond between nucleotides and the bond between nucleotides and ethylene glycol units may be nuclease-resistant modified. N represents an integer of 4 to 8.)

  According to the present invention, a novel therapeutic or preventive agent for inflammatory bowel disease comprising an oligonucleotide derivative having a high binding affinity for NF-κB as an active ingredient has been provided. The therapeutic or prophylactic agent of the present invention can remarkably improve symptoms even when administered intravenously. Inflammatory bowel disease is a disease that repeats recurrence for a long time even if it recovers once, and it is too heavy for the patient to administer locally to the affected area each time. According to the therapeutic or prophylactic agent of the present invention, since a therapeutic and prophylactic effect can be exhibited by a simple administration route called intravenous administration, the burden on the patient can be reduced. Moreover, the therapeutic or prophylactic agent of the present invention does not require a special delivery technique such as a viral envelope, and can administer the oligonucleotide derivative in a naked form without using a vector. Therefore, the preparation process is simple and the manufacturing cost can be reduced.

The oligonucleotide derivative used as an active ingredient in the present invention includes 5′-aggggatttcccc-3 ′ (SEQ ID NO: 1) and 5′-ggggaaatcccct-3 ′ (SEQ ID NO: 2) as represented by the general formula (I). It has a structure connected by a linker (CH ₂ CH ₂ O) _n (n is an integer of 4 to 8) consisting of repeating ethylene glycol units. The 5'-side aggggatttcccc and the 3'-side ggggaaatcccct are complementary to each other, and form a double strand when the linker is folded in half as a loop part. Specifically, it has a structure represented by the following formula (II) (L represents a linker (CH ₂ CH ₂ O) _n ). A structure in which two complementary oligonucleotides whose ends are linked via a linker in this way forms a double strand may be referred to as a “hairpin type” in this specification for convenience. It is clear from the melting temperature and the binding activity to NF-κB that are specifically shown in the following examples that the oligonucleotide derivative has a hairpin structure (that is, the oligonucleotide part becomes a double strand).

The linker (CH ₂ CH ₂ O) _n is particularly preferably n = 6.

  In the oligonucleotide used in the present invention, nuclease resistance modification such as phosphorothioation may be applied to all or part of the internucleotide linkage for the purpose of providing appropriate biochemical stability. That is, it is preferable that the oligonucleotide part in the oligonucleotide derivative is basically DNA, but the bond between at least two adjacent nucleotides (and / or the nucleotide adjacent to the linker part, the nucleotide and the linker). May be modified with nuclease resistance to increase nuclease resistance. Here, “nuclease resistance modification” means a modification that makes degradation by nuclease more difficult than natural DNA, and such modification of DNA itself is well known. Examples of the nuclease resistance modification include phosphorothioation (sometimes referred to herein as “Sation”), phosphorodithioation, phosphoramidateation, and the like. Of these, S is preferred. As described above, conversion to S means that one of two non-bridging oxygen atoms bonded to a phosphorus atom constituting a phosphodiester bond between adjacent nucleotides is converted to a sulfur atom. The technique of converting the bond between any adjacent nucleotides to S is well known, and can be carried out, for example, by the method described in Non-Patent Document 7, and the S-modified oligonucleotide is also synthesized commercially. In the present specification and claims, a simple base sequence is S-modified in part or all of the bond between nucleotides and the bond between the linker part and the nucleotide unless the context clearly indicates otherwise. And those that are not S at all. However, when the S site is clearly described in one sequence, the site where S site is not described is not S site.

Among (I), at least a part of the bond between nucleotides and the bond between nucleotides and ethylene glycol units, preferably about 12 or more, more preferably about 15 are S-modified. Specific examples of such preferable derivatives include the following (a) to (h). Among these, (c) is particularly preferable. The oligonucleotide (c) was prepared as derivative 5 in the following examples.
(a) 5'-ag _s gg _s ga _s tt _s tc _s cc _s c- (CH ₂ CH ₂ O) ₆ -gg _s gg _s aa _s at _s cc _s cc _s t-3 '
(b) 5'-ag _s gg _s ga _s tt _s tc _s cc _s c _s- (CH ₂ CH ₂ O) ₆ - _s gg _s gg _s aa _s at _s cc _s cc _s t-3 '
(c) 5'-ag _s g _s gg _s a _s tt _s t _s cc _s c _s c- (CH ₂ CH ₂ O) ₆ -ggg _s g _s aa _s a _s tc _s c _s cc _s t-3 '
(d) 5'-ag _s g _s g _s ga _s t _s t _s tc _s c _s c _s c- (CH ₂ CH ₂ O) ₆ -ggg _s g _s a _s aa _s t _s c _s cc _s c _s t-3 '
(e) 5'-ag _s g _s g _s ga _s t _s t _s tc _s c _s c _s c _s- (CH ₂ CH ₂ O) ₆ - _s ggg _s g _s a _s aa _s t _s c _s cc _s c _s t-3 '
(f) 5'-a _s g _s g _s g _s g _s a _s t _s t _s t _s c _s c _s c _s c- (CH ₂ CH ₂ O) ₆ -g _s g _s g _s g _s a _s a _s a _s t _s c _s c _s c _s c _s t-3 '
(g) 5'-a _s g _s g _s g _s g _s a _s t _s t _s t _s c _s c _s c _s c _s- (CH ₂ CH ₂ O) ₆ - _s g _s g _s g _s g _s a _s a _s a _s t _s c _s c _s c _s c _s t-3 '
(h) 5'-ag _s g _s gg _s a _s tt _s t _s cc _s c _s c _s- (CH ₂ CH ₂ O) ₆ - _s ggg _s g _s aa _s a _s tc _s c _s cc _s t -3 '
(In (a) to (h), the subscript s indicates that the nucleotides on both sides of s or the nucleotides on both sides of s and the ethylene glycol unit are linked by phosphorothioate).

  The oligonucleotide derivative used in the present invention first synthesizes a single-stranded oligonucleotide (that is, ggggaaatcccct) whose linker is bound to its 5 ′ end by a conventional method, and a linker is bound to its 5 ′ end, Furthermore, the 3′-position of cytosine is linked to the other end of the linker so that 5′-aggggatttcccc-3 ′ is linked to the other end of the linker, and a predetermined oligonucleotide 5 It can be synthesized by connecting them one by one. Such linkers as defined in the present invention are known per se, and it is also known that the ends of complementary oligonucleotides are linked with such linkers to make the oligonucleotide part a double-stranded oligonucleotide ( Non-patent document 8, Non-patent document 9, Non-patent document 10, Patent document 14, Patent document 15). In addition, since a reagent (linker derivative) for introducing a linker composed of repeating ethylene glycol units for preparing a hairpin type double-stranded oligonucleotide is commercially available, these commercially available linker reagents are used. According to the instructions, a hairpin type double-stranded oligonucleotide can be easily prepared. One example of the preparation method is also specifically described in the following examples.

  Examples of commercially available reagents for introducing a linker consisting of repeating ethylene glycol units as defined in the present invention that can be used to prepare hairpin double-stranded oligonucleotides include the following: it can.

(1) 18-O-Dimethoxytritylhexaethyleneglycol, 1-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite {18-O-Dimethoxytritylhexaethyleneglycol, 1-[(2-cyanoethyl)- (N, N-diisopropyl)]-phosphoramidite}
(Product name: Spacer Phosphoramidite 18, Glen Research, USA)
(Links a linker part consisting of 6 ethylene glycol units)
(2) 9-O-dimethoxytrityltriethylene glycol, 1-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite
{9-O-Dimethoxytrityl-triethylene glycol, 1-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite}
(Product name: Spacer Phosphoramidite 9, Glen Research, USA)
(Links a linker part consisting of 3 ethylene glycol units)
(3) 12-O-dimethoxytrityltetraethylene glycol, 1-[(2-cyanoethyl)-(N, N-diisopropyl)] phosphoramidite
12-O-Dimethoxytrityl-tetraethyleneglycol, 1-[(2-cyanoethyl)-(N, N-diisopropyl)] phosphoramidite
(Product name: Spacer 12, USA ChemGenes)
(Linker unit consisting of 4 ethylene glycol units is connected)

  The disease targeted by the therapeutic or prophylactic agent of the present invention is inflammatory bowel disease, and specific examples include ulcerative colitis and Crohn's disease. It is recognized in this field that a medicine using NF-κB decoy as an active ingredient is effective for the treatment and prevention of inflammatory bowel disease. Therefore, any of the above hairpin type oligonucleotide derivatives having high NF-κB transcription factor binding inhibitory activity is effective in the treatment and prevention of inflammatory bowel disease. The oligonucleotide derivative which is an active ingredient of the present invention functions as an NF-κB decoy because ggattttcc in the 5′-side sequence is a consensus sequence of NF-κB. The following examples show that these derivatives have a higher NF-κB inhibitory action and a potential of about 20 to 500 times that of known NF-κB decoys that do not have a hairpin structure (Comparative Example 1 below). As shown.

  The route of administration is not particularly limited, but parenteral administration such as intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, direct administration to the target organ or tissue is preferred. In particular, the therapeutic or prophylactic agent of the present invention can significantly suppress intestinal tissue inflammation in a mouse enteritis model even when administered intravenously. In addition, no special delivery technology such as a viral envelope is required for administration. That is, the oligonucleotide derivative can be formulated and administered in naked form without being incorporated into a vector. The preparation can be performed by a conventional method. For example, in the case of an injection, it can be in the form of a solution in which the oligonucleotide of the present invention is dissolved in physiological saline. In the preparation, additives commonly used in the field of preparation, such as preservatives, buffers, solubilizers, emulsifiers, diluents, and isotonic agents, may be appropriately mixed. Moreover, the other medicinal component may be included.

  The dose is appropriately set according to the patient's symptoms, administration route, etc., but usually 2.5 mg / kg body weight or more per day for an adult, more preferably 5.0 mg / kg body weight or more can be administered. The upper limit of the dose is not particularly limited, but is usually about 10.0 g / kg body weight.

  As specifically shown in the Examples below, the oligonucleotide derivative that is the active ingredient of the present invention has a higher binding affinity for NF-κB than the known NF-κB decoy, and therefore, the conventional NF-κB decoy It exhibits a medicinal effect superior to the therapeutic or prophylactic agent for inflammatory bowel disease containing as an active ingredient. In particular, it has a smaller molecular structure than the known NF-κB decoy and has a higher binding activity, so that a small dose is required and the production cost is low. Moreover, as specifically described in the following Examples, the oligonucleotide derivative which is an active ingredient of the present invention has lower cytotoxicity than the known NF-κB decoy. Furthermore, since the oligonucleotide derivative has a hairpin type structure in which the ends of double-stranded oligonucleotides are linked by a linker, the melting temperature is much higher than that of ordinary double-stranded oligonucleotides. High stability in time and in vivo. In addition to these features, the oligonucleotide derivative according to the present invention also has excellent nuclease resistance, so that the effect can be exerted even by systemic administration such as intravenous injection.

  Hereinafter, the present invention will be described more specifically based on examples, comparative examples, and reference examples. However, the present invention is not limited to the following examples. Prior to the description of each example, a measurement method and an evaluation method for each characteristic will be described.

1. Melting temperature (Tm value) measurement test (measures decoy stability as a double strand)
Using a Tm analysis system (UV-1650PC / TMSPC-8: manufactured by SHIMADZU), the absorbance of each decoy in PBS solution was measured multiple times in the range of 1 ° C to 99 ° C, and double-stranded at each temperature. The dissociation of was monitored. Each Tm value (° C.) was calculated from the temperature-absorbance curve by a differential method.

2. Binding activity test (decoy activity stability against mouse plasma measured by cell-free experiment)
Mouse plasma was added to each decoy solution (final nucleic acid concentration 10 μmol / L, mouse plasma 90%), and reacted at 37 ° C. for 0-24 hours. After the reaction, using a commercially available transcription factor assay kit (TransAM (trade name) NF-κB p65: Cat. No. 40096: Active Motif, Inc), the above plate was placed on the plate on which the NF-κB consensus sequence was immobilized. Nucleic acid reaction solution and Jurkat cell (human T lymphoma derived) nuclear extract were added and reacted (room temperature, 1 hour). After washing, a primary antibody (anti-NF-κB p65 antibody) and a secondary antibody (HRP-anti-IgG antibody) were added according to the kit instructions, washed, and measured using a chemiluminescence method.

Evaluation method The concentration value of each decoy solution is converted to logarithm, the horizontal axis is converted to logarithm (5 to 10 points within the concentration of 0.005 to 167 nmol / L), and the vertical value is plotted as the percentage value of each group. Then, an approximate curve was prepared, and IC ₅₀ (inhibition concentration 50%) at each reaction time was calculated.

3. Denaturing polyacrylamide gel electrophoresis test (determining the structural stability of decoy against mouse plasma using gel electrophoresis)
Mouse plasma was added to each decoy solution (final nucleic acid concentration 10 μmol / L, mouse plasma 90%), and reacted at 37 ° C. for 0-24 hours. After the reaction, separation was performed at 100 V for 100 minutes by 20% non-denaturing polyacrylamide gel electrophoresis. After staining with 2.5 μg / mL ethidium bromide aqueous solution for 20 minutes, it was destained with deionized water for 15 minutes and visualized as a fluorescent band with a UV transilluminator. The obtained gel image was photographed with an image analyzer (LAS-3000 UVmini: FUJI FILM), and the visualized full-length decoy nucleic acid compound was quantified with software attached to the device.

4). Intracellular binding activity test (decoy NF-κB transcription factor binding inhibitory activity measured in an experimental system using cells)
Mouse macrophage-derived RAW264.7 cells were seeded (6 well plate: 6.0 to 9.0 × 10 ⁵ cells / well / 2 mL) and cultured at 37 ° C. and 5% CO ₂ for 24 hours (RPMI 1640 containing 10% FBS). Each decoy nucleic acid (final concentration: double-stranded DNA of Comparative Example 1: 0.12 to 4 μmol / L, other decoy: 0.00012 to 1.2 μmol / L) is introduced into the cells using a DMRIE-C gene introduction reagent (Invitrogen) (4 hours and 24 hours were introduced for 4 hours). In the case of 24Hr, it was washed after introduction for 4 hours and cultured in RPMI1640 culture solution for 20 hours. Cells were then washed and LPS stimulation (100 ng / mL, 1 hour) was applied. After cell washing, a nuclear extract of cells is prepared, and each nucleus is prepared using a commercially available transcription factor assay kit (TransAM (trade name) NF-κB p65: Cat. No. 40096: Active Motif, Inc). The binding inhibitory activity in the extracted sample was measured. (See the binding activity test above)

Evaluation method The calculation used Microsoft Excel 2002 (trade name, Microsoft Corporation). The average value was calculated using the Excel function AVERAGE, and the standard deviation (SD) was calculated using the function STDEV. Data were expressed as the mean ± standard deviation of 3 cases.

5). Cytotoxicity test
HeLa cells were detached once by Trypsin-EDTA treatment and seeded (96 well plate: 1.0 × 10 ⁴ cells / well / 50 μL, RPMI1640 containing 10% FBS). Next, 50 μL of each 6, 20, 60 and 200 μmol / L decoy prepared in 10% FBS MEM medium was added, cultured at 37 ° C. and 5% CO ₂ for 24 hours, and then live cells were measured by the WST-1 method. Cell proliferation was measured using mitochondrial metabolic activity as an index.

Evaluation method Concentration values (3, 10, 30, 100 μmol / L) of each decoy are plotted on the horizontal axis, and the percentage value when the measured value of the untreated group at each concentration is 100% is plotted on the vertical axis. calculating the LC ₅₀ from two points sandwiching (median lethal concentration) was determined the ratio between the ₅₀ value LC of Comparative example 1 and LC ₅₀ values of each group.

6). UV absorption analysis
Using a NanoDrop ND-1000 Spectrophotometer (trade name, NanoDrop Technologies, LLC), the UV (water) λmax of each sample was measured.
7). HPLC retention time Each sample was analyzed by reversed-phase ion-pair HPLC under the following conditions, and the retention time was measured.
Device: SHIMADZU prominence (trade name, Shimadzu Corporation)
Column: Waters Xbridge C18 (trade name, Nihon Waters, 2.5μm, 4.6 x 75mm)
Column temperature: 50 ° C
Solution A: 5% acetonitrile, 0.1M triethylamine acetate buffer (pH 7.0)
Solution B: 90% acetonitrile, 0.1M triethylamine acetate buffer (pH 7.0)
Gradient B concentration: 0% -30% (30min)
Flow rate: 1mL / min
Detection wavelength: 260nm

Example 1 Synthesis of Oligonucleotide Derivatives Thirty kinds of oligonucleotide derivatives having the following structures were prepared according to the following method and synthesis examples. Of the following derivatives 1-30, derivatives 1, 3, 5, 8, 10, 12, 13 and 16 are derivatives corresponding to the active ingredients of the present invention, and the others show the stability of the hairpin structure, etc. This is a reference example.
(1) 5'-ag _s gg _s ga _s tt _s tc _s cc _s c- (CH ₂ CH ₂ O) ₆ -gg _s gg _s aa _s at _s cc _s cc _s t-3 '(derivative 1)
(2) 5'-gg _s gg _s aa _s at _s cc _s cc _s t- (CH ₂ CH ₂ O) ₆ -ag _s gg _s ga _s tt _s tc _s cc _s c-3 '(derivative 2)
(3) 5'-ag _s gg _s ga _s tt _s tc _s cc _s c _s- (CH ₂ CH ₂ O) ₆ - _s gg _s gg _s aa _s at _s cc _s cc _s t-3 '(derivative 3 )
(4) 5'-gg _s gg _s aa _s at _s cc _s cc _s t _s- (CH ₂ CH ₂ O) ₆ - _s ag _s gg _s ga _s tt _s tc _s cc _s c-3 '(derivative 4 )
(5) 5'-ag _s g _s gg _s a _s tt _s t _s cc _s c _s c- (CH ₂ CH ₂ O) ₆ -ggg _s g _s aa _s a _s tc _s c _s cc _s t-3 '(Derivative 5)
(6) 5'-ggg _s g _s aa _s a _s tc _s c _s cc _s t- (CH ₂ CH ₂ O) ₆ -ag _s g _s gg _s a _s tt _s t _s cc _s c _s c-3 '(Derivative 6)
(7) 5'-ggg _s g _s aa _s a _s tc _s c _s cc _s t _s- (CH ₂ CH ₂ O) ₆ - _s ag _s g _s gg _s a _s tt _s t _s cc _s c _s c -3 '(derivative 7)
(8) 5'-ag _s g _s g _s ga _s t _s t _s tc _s c _s c _s c- (CH ₂ CH ₂ O) ₆ -ggg _s g _s a _s aa _s t _s c _s cc _s c _s t-3 '(derivative 8)
(9) 5'-ggg _s g _s a _s aa _s t _s c _s cc _s c _s t- (CH ₂ CH ₂ O) ₆ -ag _s g _s g _s ga _s t _s t _s tc _s c _s c _s c-3 '(derivative 9)
(10) 5'-ag _s g _s g _s ga _s t _s t _s tc _s c _s c _s c _s- (CH ₂ CH ₂ O) ₆ - _s ggg _s g _s a _s aa _s t _s c _s cc _s c _s t-3 '(derivative 10)
(11) 5'-ggg _s g _s a _s aa _s t _s c _s cc _s c _s t _s- (CH ₂ CH ₂ O) ₆ - _s ag _s g _s g _s ga _s t _s t _s tc _s c _s c _s c-3 '(derivative 11)
(12) 5'-a _s g _s g _s g _s g _s a _s t _s t _s t _s c _s c _s c _s c- (CH ₂ CH ₂ O) ₆ -g _s g _s g _s g _s a _s a _s a _s t _s c _s c _s c _s c _s t-3 '(derivative 12)
(13) 5'-a _s g _s g _s g _s g _s a _s t _s t _s t _s c _s c _s c _s c _s- (CH ₂ CH ₂ O) ₆ - _s g _s g _s g _s g _s a _s a _s a _s t _s c _s c _s c _s c _s t-3 '(derivative 13)
(14) 5'-g _s g _s g _s g _s a _s a _s a _s t _s c _s c _s c _s c _s t- (CH ₂ CH ₂ O) ₆ -a _s g _s g _s g _s g _s a _s t _s t _s t _s c _s c _s c _s c-3 '(derivative 14)
(15) 5'-g _s g _s g _s g _s a _s a _s a _s t _s c _s c _s c _s c _s t _s- (CH ₂ CH ₂ O) ₆ - _s a _s g _s g _s g _s g _s a _s t _s t _s t _s c _{s s} c _s c _s c-3 '(derivative 15)
(16) 5'-ag _s g _s gg _s a _s tt _s t _s cc _s c _s c _s- (CH ₂ CH ₂ O) ₆ - _s ggg _s g _s aa _s a _s tc _s c _s cc _s t -3 '(derivative 16)
(17) 5'-g _s gg _s ga _s tt _s tc _s cc _s c- (CH ₂ CH ₂ O) ₆ -gg _s gg _s aa _s at _s cc _s cc -3 '(derivative 17)
(18) 5'-gg _s gg _s aa _s at _s cc _s cc- (CH ₂ CH ₂ O) ₆ -g _s gg _s ga _s tt _s tc _s cc _s c-3 '(derivative 18)
(19) 5'-g _s gg _s ga _s tt _s tc _s cc _s c _s- (CH ₂ CH ₂ O) ₆ - _s gg _s gg _s aa _s at _s cc _s cc -3 '(derivative 19)
(20) 5'-gg _s gg _s aa _s at _s cc _s cc _s- (CH ₂ CH ₂ O) ₆ - _s g _s gg _s ga _s tt _s tc _s cc _s c-3 '(derivative 20)
(21) 5'-g _s g _s gg _s a _s tt _s t _s cc _s c _s c- (CH ₂ CH ₂ O) ₆ -ggg _s g _s aa _s a _s tc _s c _s cc-3 '( Derivative 21)
(22) 5'-gg _s gg _s a _s tt _s t _s cc _s c _s c- (CH ₂ CH ₂ O) ₆ -ggg _s g _s aa _s a _s tc _s c _s c _s c-3 '( Derivative 22)
(23) 5'-ggg _s g _s aa _s a _s tc _s c _s cc- (CH ₂ CH ₂ O) ₆ -g _s g _s gg _s a _s tt _s t _s cc _s c _s c-3 '( Derivative 23)
(24) 5'-g _s g _s gg _s a _s tt _s t _s cc _s c _s c _s- (CH ₂ CH ₂ O) ₆ - _s ggg _s g _s aa _s a _s tc _s c _s cc-3 '(Derivative 24)
(25) 5'-ggg _s g _s aa _s a _s tc _s c _s cc _s- (CH ₂ CH ₂ O) ₆ - _s g _s g _s gg _s a _s tt _s t _s cc _s c _s c-3 '(Derivative 25)
(26) 5'-g _s g _s g _s ga _s t _s t _s tc _s c _s c _s c- (CH ₂ CH ₂ O) ₆ -ggg _s g _s a _s aa _s t _s c _s cc _s c -3 '(derivative 26)
(27) 5'-ggg _s g _s a _s aa _s t _s c _s cc _s c- (CH ₂ CH ₂ O) ₆ -g _s g _s g _s ga _s t _s t _s tc _s c _s c _s c -3 '(derivative 27)
(28) 5'-g _s g _s g _s ga _s t _s t _s tc _s c _s c _s c _s- (CH ₂ CH ₂ O) ₆ - _s ggg _s g _s a _s aa _s t _s c _s cc _s c-3 '(derivative 28)
(29) 5'-ggg _s g _s a _s aa _s t _s c _s cc _s c _s- (CH ₂ CH ₂ O) ₆ - _s g _s g _s g _s ga _s t _s t _s tc _s c _s c _s c-3 '(derivative 29)
(30) 5'-g _s gg _s a _s tt _s t _s cc _s c- (CH ₂ CH ₂ O) ₆ -gg _s g _s aa _s a _s tc _s c _s c-3 '(derivative 30)
(In (1) to (30), the subscript s indicates that the nucleotides on both sides of s or the nucleotides on both sides of s and the ethylene glycol unit are phosphorothioate-bonded).

  Each of the above-mentioned oligonucleotide derivatives basically uses a commercially available solid phase support for DNA synthesis, DNA cyanoethyl phosphoramidite, and an appropriate reaction / modification reagent, and all or partly phosphorothioate-modifies the phosphodiester bond. The oligonucleotide subjected to was synthesized in the 3 ′ → 5 ′ direction with an automatic DNA synthesizer (trade name: ABI394, manufactured by Applied Biosystems, USA). In producing a hairpin type NF-κB decoy into which a polyethylene glycol chain has been introduced, firstly, a 3 ′ side sequence (a sequence to which a linker is bonded to the 5 ′ end) is synthesized in the 3 ′ → 5 ′ direction. Following the 5 ′ end, 18-O-dimethoxytritylhexaethylene glycol, 1-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite (trade name: Spacer Phosphoramidite 18, Glen Research, USA) ) 1 molecule is coupled, and then the 5 ′ side sequence (the one to which the linker is bound to the 3 ′ end) is synthesized in the 3 ′ → 5 ′ direction, so that the 3 ′ side sequence and the 5 ′ side are synthesized. An oligonucleotide derivative having a side sequence linked with hexaethylene glycol was obtained. It was manufactured by intramolecular annealing by heating and rapid cooling.

  More specifically, the above synthesis was performed as follows. DMT-deoxyadenosine (bz) β-cyanoethyl phosphoramidite, DMT-deoxycytidine (bz) β-cyanoethyl phosphoramidite, DMT-deoxyguanosine (bz) β-cyanoethyl phosphoramidite and DMT-deoxythymidine β-cyanoethylphosphomid Loamidite from Sigma-Aldrich, Spacer18 phosphoramidite (trade name), S-reagent (CPRII, 3H-1,2-benzodithiol-3-one-1,1-dioxide) and synthesis column from Glen Research A deprotection solution, an activation solution, an oxidation solution, and a capping solution (CapA solution and CapB solution) for oligo DNA synthesis were purchased from Wako Pure Chemical, respectively. (Bz means a benzoyl group.)

Synthesis Example 1 (Synthesis of Derivative 1)
Using an automatic DNA synthesizer ABI394 (Applied Biosystems), a desired oligonucleotide was synthesized on a solid support by the phosphoramidite method as follows. The synthesizer is equipped with a synthesis column containing porous glass (Controlled Pore Glass: CPG) pre-bound with a protected 3 'terminal nucleotide (dT), next to the deprotection solution, activation solution and 3' end. Β-cyanoethyl phosphoramidite (acetonitrile solution) of nucleotide (dC) adjacent to, oxidation solution, capping solution (1: 1 mixture of CapA solution and CapB solution) are passed through the synthesis column in this order. An inter-phosphodiester bond was formed. When the internucleotide linkage was phosphorothioate, an S reagent dissolved in anhydrous acetonitrile was used instead of the oxidizing solution. The concentration and amount of each reagent were in accordance with the manufacturer's instructions. In the same manner, the bases were sequentially extended one by one in the 3 ′ → 5 ′ direction. Spacer 18 phosphoramidite (trade name) is bound to the 5 ′ end of the synthesized oligonucleotide according to the instructions attached to the product, and then bound to the other end of Spacer 18 phosphoramidite (trade name) as described above. The oligonucleotides to be synthesized were extended and synthesized one base at a time in the 3 ′ → 5 ′ direction.

Cutting out and deprotecting from solid support
The oligonucleotide derivative was cut out from CPG by treatment with 28% aqueous ammonia at room temperature for 2 hours. Further, the solution was treated at 65 ° C. for 6 hours to remove the protecting group for the base moiety and the protecting group for the phosphoric acid moiety.

Cartridge purification
0.1M triethylamine acetate buffer (pH 7.0) is added to a solid-phase extraction column packed with Oligo R3 support (Applied Biosystems), equilibrated, and then the oligonucleotide before purification is added and adsorbed to it. Acetate buffer (pH 7.0): Acetonitrile (9: 1) was added to wash out incomplete oligonucleotides, and then 5% dimethoxytrityl was removed from the full-length oligonucleotide by adding 2% aqueous trifluoroacetic acid. The group was removed and washed once with water. Next, 20% acetonitrile was added to elute the oligonucleotide. Subsequently, the solvent was evaporated from the eluate by lyophilization to obtain an oligonucleotide derivative.

Comparative Example 1
S-modified normal double-stranded DNA having the following structure was synthesized by a conventional method (the base sequence is shown in SEQ ID NO: 3).

5'-CsCsTsTsGsAsAsGsGsGsAsTsTsTsCsCsCsTsCsC-3 '
3'-GsGsAsAsCsTsTsCsCsCsTsAsAsAsGsGsGsAsGsG-5 '
(The subscript s indicates that the nucleotides on both sides of s are phosphorothioate-bonded).

Example 2
For the oligonucleotide derivatives (derivatives 1 to 30) prepared in the examples and the double-stranded DNA of Comparative Example 1, the above-described measurement methods or the properties that have not been described in the measurement method are variously measured according to conventional methods. It was measured. The results are shown in Table 1-1 and Table 1-2 below.

  The pharmacological meaning of each characteristic shown in Table 1-1 and Table 1-2 will be described below.

(1) Binding activity
(i) 0Hr
IC ₅₀ value for NF-κB binding of each compound in a cell-free system (cell-free system) in the presence of 90% of mouse plasma at a reaction time of 0 hr (0 hr, the same applies hereinafter). Actually, the value measured within 3 seconds after adding mouse plasma to each decoy solution was taken as 0Hr for convenience.

(ii) 24Hr, 24Hrs / 0Hr ratio IC ₅₀ value for NF-κB binding of each compound at a reaction time of 24Hr in the presence of 90% mouse plasma in a cell-free system. The IC ₅₀ values of Comparative Example 1 whereas almost no change between 0Hr and 24Hr, in each compound derivatives 1～16,21 and 23-25 has been an increase in the IC ₅₀ of 24Hr. This indicates that each compound is degraded by mouse plasma. The oligonucleotide derivative synthesized above is intended to be applied to a living body, and it is also desirable to undergo moderate metabolism in consideration of side effects as well as resistance to plasma components. In the living body, any natural substance having physiological activity is maintained at an appropriate concentration by the balance between decomposition and biosynthesis, and the homeostasis of the living body is maintained. It is clear that the oligonucleotide derivatives 1 to 30 have characteristics that are easily metabolized as compared with Comparative Example 1.

(2) 50% remaining time It is data showing the biochemical stability of the above oligonucleotide derivative to nucleic acid as a nucleic acid in a cell-free system.

(3) Intracellular binding activity
(i) 4Hrs
This data shows the NF-κB inhibitory action in cultured cell lines, and includes the intracellular / external resistance and cell membrane permeability of each compound. It is clear that the oligonucleotide derivative has a potential of about 20 to 500 times that of the double-stranded DNA of Comparative Example 1.

(ii) 24Hrs
As described above, the oligonucleotide derivative has a feature that it is more easily decomposed than the double-stranded DNA of Comparative Example 1, but it is still 3 to 14 times more than the double-stranded DNA of Comparative Example 1 after 24 hours. It is clear that it has potential.

(4) Cytotoxicity The oligonucleotide derivative was superior to the double-stranded DNA of Comparative Example 1 in that the LC ₅₀ was about 1.5 to 3.8 times higher.

(5) Tm value This data shows the physical (thermodynamic) stability of oligonucleotide derivatives. The double-stranded DNA of Comparative Example 1 has only a thermodynamic stability that is slightly higher than the body temperature of mammals including humans, and can hardly be heated in a formulation step such as an ointment. Oligonucleotide derivatives are stable under temperature conditions in a general pharmaceutical process.

(6) UV λmax and HPLC retention time Physical property value data of each compound.

  As shown in Table 1-1 and Table 1-2, the oligonucleotide derivative has a binding activity much higher than that of the double-stranded DNA of Comparative Example 1 (0Hr binding activity), and is metabolic in cells. Is much higher than the double-stranded DNA of Comparative Example 1 (24Hrs / 0Hr ratio), but the oligonucleotide derivative still has higher binding activity after 24 hours. In addition, the cytotoxicity of the oligonucleotide derivative is lower. In particular, when the derivative 15 in which all internucleotide linkages are converted to S is compared with Comparative Example 1, the toxicity resulting from the conversion to S can be compared equally, but the cytotoxicity of the oligonucleotide derivative is compared. About 57% of the cytotoxicity of the double-stranded DNA of Example 1.

Example 3 Pharmacological Effect of Oligonucleotide Derivative on Inflammatory Bowel Disease Model The pharmacological effect of the oligonucleotide derivative prepared above on inflammatory bowel disease was examined as follows using a mouse model.

(Creation of inflammatory bowel disease model)
Using C57BL / 6 mice 8 weeks old, dextran sulfate sodium (DSS) mixed in the drinking water to give a 5% concentration, and allowed to drink freely and cultivate inflammatory bowel disease model for 10 days did.

(Test substance administration)
Substance administered: oligonucleotide derivative (derivative 5) of Example or double-stranded DNA of Comparative Example 1 dissolved in 100 μL of physiological saline, or physiological saline as a control.
Route of administration; timing and frequency of intravenous administration; immediately before inflammation (DSS administration started). Single dose.
Administration method: Administered from the caudal vein of mice under awakening using a syringe with a 27G needle.

(Experimental animal group)
1 Untreated group (normally drinking water. Healthy rat model) n = 10
2 DSS group (enteritis model group) n = 9
3 DSS + Example derivative 5 (5.0 mg / kg body weight) group n = 6
4 DSS + double stranded DNA of Comparative Example 1 (5.0 mg / kg body weight) group n = 6

(Evaluation item)
As an evaluation of disease improvement, the rate of change in body weight and disease activity index (DAI) after 3, 7, and 10 days after DNA administration were examined.
(i) The rate of change in body weight was calculated assuming that the body weight just before inflammation was induced was 100%.
(ii) DAI was calculated in accordance with Clin Exp Immunol. 2000 Apr; 120 (1): 51-8. The score was calculated by adding the scores of the following items (weight loss rate score + fecal property score + blood stool score, lowest 0 to max 12).

(result)
The results of changes in weight and DAI are shown in Tables 3 and 4 below. The oligonucleotide derivative 5 of the example markedly suppressed the decrease in body weight and the increase in DAI of enteritis model mice by intravenous administration.

(Histological examination)
On day 10, the animals were sacrificed, and light microscopy of colon tissue sections was performed (magnification: 40x, 100x). As a result, disappearance of goblet cells and infiltration of inflammatory cells were observed in the DSS group. On the other hand, in the group 5 of DSS + derivatives administered with the oligonucleotide derivative of the example, the colon tissue maintained almost normal structure. In DSS + Comparative Example 1 group to which the double-stranded DNA of Comparative Example was administered, partial disappearance of goblet cells and infiltration of inflammatory cells were observed.

  The above result has shown that the oligonucleotide derivative 5 synthesize | combined in the Example can suppress intestinal tissue inflammation by intravenous administration.

Claims (7)

A therapeutic or prophylactic agent for inflammatory bowel disease comprising an oligonucleotide derivative represented by the following general formula (I) as an active ingredient.
5'-aggggatttcccc- (CH ₂ CH ₂ O) _n -ggggaaatcccct-3 '(I)
(However, at least a part of the bond between nucleotides and the bond between nucleotides and ethylene glycol units may be nuclease-resistant modified. N represents an integer of 4 to 8.) The therapeutic or prophylactic agent according to claim 1, wherein the oligonucleotide derivative has a structure selected from the group consisting of the following (a) to (h).
(a) 5'-ag _s gg _s ga _s tt _s tc _s cc _s c- (CH ₂ CH ₂ O) ₆ -gg _s gg _s aa _s at _s cc _s cc _s t-3 '
(b) 5'-ag _s gg _s ga _s tt _s tc _s cc _s c _s- (CH ₂ CH ₂ O) ₆ - _s gg _s gg _s aa _s at _s cc _s cc _s t-3 '
(c) 5'-ag _s g _s gg _s a _s tt _s t _s cc _s c _s c- (CH ₂ CH ₂ O) ₆ -ggg _s g _s aa _s a _s tc _s c _s cc _s t-3 '
(d) 5'-ag _s g _s g _s ga _s t _s t _s tc _s c _s c _s c- (CH ₂ CH ₂ O) ₆ -ggg _s g _s a _s aa _s t _s c _s cc _s c _s t-3 '
(e) 5'-ag _s g _s g _s ga _s t _s t _s tc _s c _s c _s c _s- (CH ₂ CH ₂ O) ₆ - _s ggg _s g _s a _s aa _s t _s c _s cc _s c _s t-3 '
(f) 5'-a _s g _s g _s g _s g _s a _s t _s t _s t _s c _s c _s c _s c- (CH ₂ CH ₂ O) ₆ -g _s g _s g _s g _s a _s a _s a _s t _s c _s c _s c _s c _s t-3 '
(g) 5'-a _s g _s g _s g _s g _s a _s t _s t _s t _s c _s c _s c _s c _s- (CH ₂ CH ₂ O) ₆ - _s g _s g _s g _s g _s a _s a _s a _s t _s c _s c _s c _s c _s t-3 '
(h) 5'-ag _s g _s gg _s a _s tt _s t _s cc _s c _s c _s- (CH ₂ CH ₂ O) ₆ - _s ggg _s g _s aa _s a _s tc _s c _s cc _s t -3 '
(In (a) to (h), the subscript s indicates that the nucleotides on both sides of s or the nucleotides on both sides of s and the ethylene glycol unit are linked by phosphorothioate).   The therapeutic or prophylactic agent according to claim 2, which has the structure shown in (c).   The therapeutic or prophylactic agent according to any one of claims 1 to 3, which is for intravenous administration.   The therapeutic or prophylactic agent according to any one of claims 1 to 4, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.   The therapeutic or prophylactic agent according to any one of claims 1 to 5, wherein the oligonucleotide derivative is naked.   The therapeutic or prophylactic agent according to any one of claims 1 to 6, wherein the dosage is 5.0 mg / kg or more as the amount of the oligonucleotide derivative.