JP5601777B2 - Novel oligonucleotide derivative and NF-κB decoy comprising the same - Google Patents

Description

  The present invention relates to a novel oligonucleotide derivative and an NF-κB decoy comprising the same. The NF-κB decoy of the present invention is useful for prevention, improvement or treatment of ischemic diseases, allergic diseases, autoimmune diseases, cancer metastasis, infiltration and the like.

  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.

  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).

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

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

  An object of the present invention is to provide a novel oligonucleotide derivative having a higher binding affinity for NF-κB than that of known NF-κB decoy when used as NF-κB decoy, NF-κB decoy comprising the same, and It is to provide a medicament containing as an active ingredient.

  As a result of earnest research, the inventors of the present application have found that an oligonucleotide having a specific base sequence and a hairpin type double-stranded oligonucleotide in which its complementary strand is bound by a specific linker are known from the known NF-κB decoy. Was found to exhibit a high affinity for NF-κB, and the present invention was completed.

That is, the present invention provides an oligonucleotide derivative represented by the following general formula (I) or (II).
Nn ₁ -X ₁ -Nn ₂ -LX ₂ (I)
X ₂ -L-Nn ₁ -X ₁ -Nn ₂ (II)
(In these formulas,
Nn ₁ -X ₁ -Nn ₂ is aggggatttcccc or ggggatttcccc ,
X ₂ represents a base sequence that is complementary to Nn ₁ -X ₁ -Nn ₂ when L is folded in half as a loop part,
L represents -OPO _2- (OCH ₂ CH ₂ ) n ₃ -OPO ₂ O-
n ₃ represents an integer of 5 to 7 ).

  The present invention also provides an NF-κB decoy comprising the above-described oligonucleotide derivative of the present invention. Furthermore, this invention provides the pharmaceutical which contains the oligonucleotide derivative of the said invention as an active ingredient.

  According to the present invention, a novel oligonucleotide derivative having a higher binding affinity for NF-κB than known NF-κB decoys, an NF-κB decoy comprising the same, and a medicament containing the same as an active ingredient are provided. Since the oligonucleotide derivative of the present invention has an extremely high binding affinity for NF-κB compared to known NF-κB decoys, it exhibits superior medicinal effects than a pharmaceutical containing a conventional NF-κB decoy as an active ingredient. Moreover, the oligonucleotide derivative of the present invention is less cytotoxic than the known NF-κB decoy. Furthermore, since the oligonucleotide derivative of the present invention has a hairpin 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 during storage and in vivo. Furthermore, since the molecular structure is smaller than that of the known NF-κB decoy, the manufacturing cost is also reduced.

It is a figure which shows the IL-6 production amount in a mouse | mouth body when the oligonucleotide derivative of Example 5 is administered to a mouse | mouth LPS induction acute lung injury model. It is a figure which shows the IL-6 production amount in a mouse | mouth body when the double stranded DNA of the comparative example 1 is administered to a mouse | mouth LPS induction acute lung injury model. It is a figure which shows the IL-6 production amount in a mouse | mouth body when the double stranded DNA of the comparative example 1 and the oligonucleotide derivative of Example 10 and 16 are administered to a mouse | mouth LPS induction acute lung injury model.

As described above, the oligonucleotide derivative of the present invention is represented by the above general formula (I) or (II). In the general formulas (I) and (II), Nn ₁ -X ₁ -Nn ₂ is 5′-aggggatttcccc-3 ′ (SEQ ID NO: 1) or 5′-ggggatttcccc-3 ′ (SEQ ID NO: 3), and X ₁ Is ggatttcc . X ₂ represents a base sequence that is complementary to Nn ₁ -X ₁ -Nn ₂ when L (linker) is folded in half as a loop part. Here, “the base sequence that is complementary to Nn ₁ -X ₁ -Nn ₂ when L is folded in half with the loop portion” means, for example, that Nn ₁ -X ₁ -Nn ₂ is 5′-aggggatttcccc In the case of -3 ′ (SEQ ID NO: 1), as shown in the following formula (III) or (IV), the base of Nn ₁ -X ₁ -Nn _{2 and} the base of X ₂ face each other with L as a loop part Means complementary to each other.

In the general formulas (I) and (II), L is —OPO ₂ — (OCH ₂ CH ₂ ) n ₃ —OPO ₂ O— . Here, n ₃ represents an integer of 5 to 7, preferably 6 . Note that —OPO ₂ O— at both ends of the linker L represents a phosphodiester bond that binds to the 3′-position or 5′-position of the sugar of the adjacent nucleotide. In addition, a portion excluding —OPO ₂ O— at both ends of the linker may be referred to as “linker portion”. In addition, a structure in which two complementary oligonucleotides whose ends are linked via a linker form a double strand (a structure like the above formulas (III) and (IV)) is conveniently used in this specification. It is sometimes called “hairpin type”. In addition, the oligonucleotide derivative of the present invention has a hairpin structure (that is, the oligonucleotide part becomes a double strand) that the melting temperature and the binding activity to NF-κB specifically shown in the examples below. It is clear from

  In the oligonucleotide according to 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 of the present invention is basically DNA, but the bond between at least two adjacent nucleotides (and / or the nucleotide adjacent to the linker part) And the linker part) may be modified with nuclease resistance to increase resistance to nuclease. 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.

  The oligonucleotide derivative of the present invention is prepared by first synthesizing a single-stranded oligonucleotide whose linker is bound to the 5 ′ end thereof by a conventional method, binding the linker to the 5 ′ end, and further, the other end of the linker. Can be synthesized by binding the 3′-position of a predetermined nucleotide to the nucleotide, and then binding a predetermined oligonucleotide one by one to the 5 ′ side by a conventional method. 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 reagents (linker derivatives) for introducing various linkers for preparing hairpin-type double-stranded oligonucleotides are commercially available, it is easy to use these commercially available linker reagents and follow their instructions. Hairpin-type double-stranded oligonucleotides can be prepared. One example of the preparation method is also specifically described in the following examples.

  Examples of commercially available reagents for introducing the linkers defined in the present invention that can be used to prepare hairpin double-stranded oligonucleotides include the following.

1. Reagent for introducing a linker part consisting of repeating ethylene glycol units
(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) DMT-dodecane-diol phosphoramidite
DMT-dodecane-Diol phosphoramidite
(Product name: Spacer 12, USA ChemGenes)
(Linker unit consisting of 4 ethylene glycol units is connected)

The oligonucleotide derivative of the present invention functions as an NF-κB decoy because X ₁ in the general formulas (I) and (II) is a consensus sequence of NF-κB. Therefore, the oligonucleotide derivative of the present invention can be used as an active ingredient of a medicine in which NF-κB decoy is used as an active ingredient. Such medicaments are recognized to be effective for the treatment and prevention of the following various diseases.

Vascular restenosis, acute coronary syndrome, cerebral ischemia, myocardial infarction, reperfusion injury of ischemic disease, atopic dermatitis, psoriasis vulgaris, contact dermatitis, keloid, wound, ulcerative colitis, Crohn's disease, Nephropathy, glomerulosclerosis, albuminuria, nephritis, renal failure, rheumatoid arthritis, osteoarthritis, intervertebral disc degeneration, asthma, chronic obstructive pulmonary disease, cystic fibrosis, aortic aneurysm, cerebral aneurysm. Moreover,
1. Immune system disease Aortitis syndrome (Takayasu arteritis)
Buerger's disease (Bürger's disease)
Nodular periarteritis Wegener's granulomatosis Allergic granulomatous vasculitis (Charg-Strauss syndrome)
1. Systemic lupus erythematosus Polymyositis / dermatomyositis Sjogren's syndrome Adult Still's disease Neurological and muscular diseases Parkinson's disease Amyotrophic lateral sclerosis (ALS)
Multiple sclerosis (MS)
3. Respiratory system disease Idiopathic interstitial pneumonia Sarcoidosis Primary pulmonary hypertension 4. Gastrointestinal diseases Autoimmune hepatitis Fulminant hepatitis Severe acute pancreatitis Skin / connective tissue disease scleroderma Bone and joint diseases Broad spinal stenosis 7. Renal and urological diseases
IgA nephropathy Rapidly progressive glomerulonephritis

  When used for these pharmaceutical applications, the administration route of the oligonucleotide 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 preferable. . The dose is appropriately set according to the target disease, patient symptoms, administration route, etc., but usually 0.1 to 10,000 nmol, preferably 1 to 1000 nmol, more preferably 10 to 100 nmol per day for an adult can be administered. . 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.

  As specifically shown in the examples below, the oligonucleotide derivative of the present invention has a higher binding affinity for NF-κB than the known NF-κB decoy, so that the conventional NF-κB decoy is used as an active ingredient. Demonstrates better medicinal properties than the drug it contains. 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 of the present invention is less cytotoxic than the known NF-κB decoy. Furthermore, since the oligonucleotide derivative of the present invention has a hairpin 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 during storage 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 and comparative 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 value of each decoy (3, 10, 30, 100 μmol / L) is 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 the (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

Examples 1 to 29 and Reference Example 1
Thirty kinds of oligonucleotide derivatives having the following structures were prepared according to the following methods and synthesis examples.
(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 '(Example 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 '(Example 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 '(Example) 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 '(Example) 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 '(Example 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 '(Example 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 ′ (Example 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 '(Example 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 '(Example 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 ′ (Example 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 ′ (Example 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 s} c _s t-3 '(Example 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 '(Example 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 s} c _s c-3 ′ (Example 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 c _s c _s c-3 '(Example 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 '(Example 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 '(Example 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 '(Example 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 '(Example 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 '( Example 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 '( Example 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 '( Example 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 '( Example 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 '(Example 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 '(Example 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 '(Example 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 '(Example 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 '(Example 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 '(Example 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 '( Reference Example 1 )
(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 (sequence compound of Example 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: 2).

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

Example 31
For the above-mentioned oligonucleotide derivatives (Examples 1 to 29 and Reference Example 1 ) and the double-stranded DNA of Comparative Example 1, the above-described measurement method or the characteristics for which the measurement method has not been described previously can be determined by various methods. 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. While the IC ₅₀ value of Comparative Example 1 hardly changes between 0Hr and 24Hr, the compounds of Examples 1 to 16, 21 and 23 to 25 have increased IC ₅₀ of 24Hr. This indicates that each compound is degraded by mouse plasma. The oligonucleotide derivative of the present invention 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 derivative of the present invention has a feature that it is easily metabolized as compared with Comparative Example 1.

(2) 50% remaining time It is data showing the biochemical stability of the oligonucleotide derivative of the present invention against 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 apparent that the oligonucleotide derivative of the present invention 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 of the present invention has a feature that it is more easily decomposed than the double-stranded DNA of Comparative Example 1. However, even after 24 hours, the oligonucleotide derivative is 3 to 14 more than the double-stranded DNA of Comparative Example 1. It is clear that it has double the potential.

(4) Cytotoxicity The oligonucleotide derivative of the present invention 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 the oligonucleotide derivative of the present invention. 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 process such as an ointment. The oligonucleotide derivative of the invention is 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 of the present invention has a binding activity much higher than that of the double-stranded DNA of Comparative Example 1 (0Hr binding activity). Although the metabolic properties are much higher than the double-stranded DNA of Comparative Example 1 (24Hrs / 0Hr ratio), the binding activity of the oligonucleotide derivative of the present invention is still higher after 24 hours. In addition, the cytotoxicity of the oligonucleotide derivative of the present invention is lower. In particular, when Example 15 in which all the 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 cells of the oligonucleotide derivative of the present invention can be compared. Toxicity was about 57% of the cytotoxicity of the double-stranded DNA of Comparative Example 1.

Example 32 Effects in mouse LPS-induced acute lung injury model (1)
The oligonucleotide derivative of Example 5 was dissolved in physiological saline, adjusted to 2.5 μg / mL and 5 μg / mL, and then infused into BALB / c mice (7 weeks old, female, 10 per group) at 100 μL / mouse. Intraductal administration. Three days after administration, lipopolysaccharide (LPS, 500 μg / mL) was intratracheally administered at 100 μL / mouse, and the lungs were washed with 2 mL of physiological saline / mouse 24 hours after administration. The alveolar lavage fluid was collected and the concentration of IL-6 contained was measured by ELISA. The alveolar lavage fluid was diluted with physiological saline, and experiments were conducted according to the attached document using a commercially available mouse IL-6 immunoassay kit (Mouse IL-6 Immunoassay (trade name) (R & D systems)). . Wallac1420ARVOsx (trade name, Perkin Elmer Japan Co., Ltd.) was used for measuring the absorbance (450/650 nm). In the control group, physiological saline containing no oligonucleotide derivative was administered.

Analysis method Microsoft Excel 2002 (trade name, Microsoft) was used for the calculation. The average value was calculated using the Excel (product name) function AVERAGE, and the standard error (SE) was calculated using the functions STDEV, SQRT, and COUNT. The significance test was performed using statistical analysis software SAS preclinical package ver5.0 (SAS Institute Japan), and a risk rate of less than 5% was considered statistically significant. For comparison between other groups, analysis of variance was performed, and parametric Dunnett test was performed in the case of equal variance, and nonparametric Dunnett test was performed in the case of unequal variance.

  The results are shown in FIG. When the dosage of the oligonucleotide derivative was 0.5 μg / animal, the IL-6 production was statistically significantly reduced compared to the control group.

Example 33 Effect in mouse LPS-induced acute lung injury model (2)
(1) Action of the double-stranded DNA of Comparative Example 1 The double-stranded DNA of Comparative Example 1 was dissolved in physiological saline and adjusted to 1, 10, 100 and 1000 μg / mL, and BALB / c mice (7 weeks old) , Female, 6-16 mice per group) was administered intratracheally at 100 μL / mouse. A physiological saline containing no double-stranded DNA was administered to the control group. The next day, LPS 500 μg / mL was intratracheally administered at 100 μL / animal. Twenty-four hours after LPS administration, the lung was washed with 2 mL of physiological saline / animal (alveolar lavage fluid), and IL-6 production in the alveolar lavage fluid was measured by ELISA. Data analysis was performed in the same manner as in Example 32.

  The results are shown in FIG. Although IL-6 production tended to decrease in a double-stranded DNA dose-dependent manner, no significant difference was observed in any of the administration groups relative to the control group.

(2) Action of oligonucleotide derivative (Examples 10 and 16) The double-stranded DNA of Comparative Example 1 and the oligonucleotide derivative (Examples 10 and 16) were dissolved in physiological saline to adjust to 5 μg / mL, and BALB / c mice (7 weeks old, female, 9 mice per group) were intratracheally administered at 100 μL / mouse. Saline was administered to the control group. Three days after administration, LPS 500 μg / mL was intratracheally administered at 100 μL / animal. Twenty-four hours after LPS administration, the lung was washed with 2 mL of physiological saline / animal (alveolar lavage fluid), and IL-6 production in the alveolar lavage fluid was measured by ELISA. Data analysis was performed in the same manner as in Example 32.

  The results are shown in FIG. In the oligonucleotide derivative administration group of the present invention, IL-6 production was statistically significantly reduced compared to the control group.

Claims (8)

An oligonucleotide derivative represented by the following general formula (I) or (II).
Nn ₁ -X ₁ -Nn ₂ -LX ₂ (I)
X ₂ -L-Nn ₁ -X ₁ -Nn ₂ (II)
(In these formulas,
Nn ₁ -X ₁ -Nn ₂ is aggggatttcccc or ggggatttcccc ,
X ₂ represents a base sequence that is complementary to Nn ₁ -X ₁ -Nn ₂ when L is folded in half as a loop part,
L represents -OPO _2- (OCH ₂ CH ₂ ) n ₃ -OPO ₂ O-
n ₃ represents an integer of 5 to 7 ). Represented by the general formula (I), the 5′-end of X ₂ and the 3′-end of Nn ₂ are represented by the formula —OPO ₂ — (OCH ₂ CH ₂ ) n ₃ —OPO ₂ O—. They are joined by the structure, an oligonucleotide derivative according to claim 1, wherein. Represented by the general formula (II), the 5′-end of Nn ₁ and the 3′-end of X ₂ are represented by the formula —OPO ₂ — (OCH ₂ CH ₂ ) n ₃ —OPO ₂ O—. They are joined by structure, an oligonucleotide derivative according to claim 1, wherein. In the general formula (I) or (II), L is _{- (CH 2 CH 2 O)} 6 - oligonucleotide derivative according to any one of claims 1 to 3 is. The oligonucleotide derivative according to claim 4, which has a structure selected from the group consisting of the following (1) to ( 29 ).
(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 '
(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 '
(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 '
(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 '
(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 '
(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 '
(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 '
(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 '
(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 '
(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 '
(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 '
(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 '
(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 '
(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 '
(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 c _s c _s c-3 '
(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 '
(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 '
(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 '
(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 '
(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 '
(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 '
(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 '
(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 '
(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 '
(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 '
(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 '
(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 '
(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 '
(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 '
(In (1) to ( 29 ), 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). An NF-κB decoy comprising the oligonucleotide derivative according to any one of claims 1 to 5 . A pharmaceutical comprising the oligonucleotide derivative according to any one of claims 1 to 5 as an active ingredient. The medicament according to claim 7 , which is for intravenous administration.

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