JPH09118623A - Human interleukin-6 receptor-developing inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an expression inhibitor against human interleukin-6 receptor (HIL-6R) which contains antisense oligonucleotide derivative and is useful as an HIL-6R inhibitory agent. SOLUTION: This inhibitor contains, as an active ingredient, an antisense oligonucleotide derivative which hybridize in the region having at least 9, preferably 12-25 base sequence (for example, AGCCTCCTTCCCATGCCAGC), containing the base sequence of the part forming the loop structure of mRNA coding HIL-6R. This derivative acts on the cells producing HIL-6R to bond to the DNA or mRNA coding HIL-6R to inhibit the transcription and translation and inhibit the expression of HIL-6R. The actions of this derivative are, increase in blood platelets, enhancement of antibody production, protein induction in the acute stage, proliferation of tumor cells and differentiation of nerve cells and the like. The dose is 0.1-100mg/kg, preferably 0.1-50mg/kg.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antisense oligonucleotide derivative useful as a drug for inhibiting the expression of human interleukin-6 receptor (human IL-6R).

[0002]

2. Description of the Related Art Human interleukin-6 (human IL-
6) is a cytokine that has been cloned as a factor that induces the final stage of differentiation of B cells into antibody-producing cells (Kishimoto T et al., Blood 74, 1-10, 1989), and currently induces acute phase proteins in the liver. (Kishimoto T et al., Blood 74, 1-
10, 1989).

Human IL-6 is not limited to only lymphoid cells, but fibroblasts, vascular endothelial cells, and bladder cancer cell line T2.
4 and glioblastoma have been reported (Kohase M. et al., J. Cell Physiol. 13
2, 271-278, 1978; Meir EV et al., Cancer Res. 50, 6683-.
6688, 1990) and its target cells are diverse (Kishimoto T et al., Blood 74, 1-10, 1989).

Recently, Kawano M et al. Reported that human IL-6 functions as an autocrine growth factor in myeloma cells (Kawano M et al., Nature, 332, 83-85,
1988), and the same was also reported in renal cell carcinoma (Miki S et al., FEBS Letter 250, 607-610, 1989).

On the other hand, signals for cell proliferation or differentiation by human IL-6 are present on the cell surface.
It is known to be transmitted to cells via R and glycoprotein gp130. (Taga T. et al., Cell 58, 573-581,
1989; Hibi M et al., Cell 68, 1149-1157, 1990). recent years,
As a method of suppressing the action of the gene causing the pathological condition, it has been proposed to suppress the expression of the protein by using an oligonucleotide (antisense oligonucleotide) complementary to mRNA transcribed from DNA. (Murakami, Kagaku, 46, 681-684, 1991).

[0006] Furthermore, as a method for solving problems such as lifespan, stability, and cell uptake efficiency of antisense oligonucleotides, a methylphosphonate derivative or sulfur in which the oxygen of the phosphate group of the nucleotide is replaced with a methyl group is used. Modified antisense oligonucleotides such as substituted phosphorothioate derivatives are known (Murakami,
In fact, these antisense oligonucleotides were found to inhibit viral protein synthesis (Agris, CH et al., Biochemistry, 25, 6268-6275, 1).
986).

[0007] Based on this idea, Levy Y et al. Proliferate myeloma cell lines using human IL-6 as a growth factor by inhibiting the translation of human IL-6 mRNA with an antisense oligonucleotide. Were confirmed to be suppressed (Levy Y et al., J. Clin. Invest., 88, 696-69).
9, 1991). However, an antisense oligonucleotide derivative that significantly suppresses IL-6R expression in various cells expressing human IL-6R is not known.

[0008]

Accordingly, the present invention is intended to provide an antisense oligonucleotide derivative that inhibits the expression of human IL-6R.

[0009]

More specifically, the present invention provides at least 9 contiguous base sequences containing a base sequence of a part that is likely to form a loop structure of mRNA encoding human IL-6R. An expression inhibitor of human IL-6R comprising an antisense oligonucleotide derivative against

[0010]

[Detailed Description] The present inventor used human IL- using commercially available computer software for free energy calculation (eg, Genetyx, Secondary Structure Search and Hairpin Loop-Stem Parts Search manufactured by Software Development Co., Ltd.). In 6RmRNA, a base sequence that is considered to easily form a loop structure was sought from the viewpoint of free energy and structure, and an antisense oligonucleotide derivative against the base sequence containing the loop structure was synthesized. As a result, human soluble IL-6R (sIL- The inventors have found that the expression of 6R) is suppressed and completed the present invention. In a preferred embodiment of the present invention, an antisense oligonucleotide derivative for 9 to 30, more preferably 12 to 25 consecutive base sequences containing a base sequence capable of forming a loop structure of mRNA encoding human IL-6R To use.

The term "antisense oligonucleotide" as used herein means not only those in which nucleotides corresponding to nucleotides constituting a predetermined region of DNA or mRNA are all complementary, but also DNA or mRNA and oligonucleotide are stable. Some mismatch may be present as long as it can hybridize to Human IL-6R cd
The base sequence of NA is as follows (for example, JP-A-2-
288,898, or Science, 24
1,825-828 (1988)) (SEQ ID NO:
1).

Of these, the base sequence of the antisense oligonucleotide derivative of the present invention can be appropriately selected from a continuous base sequence capable of forming the loop structure found by the above method. The term "portion capable of forming a loop structure of mRNA" as used herein refers to a portion which is unlikely to have intramolecular hydrogen bonds of mRNA and is likely to be in a single-stranded state. Specifically, the above-mentioned computer software can be used to find a portion that tends to be in a single-stranded state even when the stable structure of mRNA is taken. In the present invention, the portion capable of forming the loop structure is
In the base sequence shown in SEQ ID NO: 1, for example, 640-
685, 770-810, 1340-1375, 460
-475,535-560 and 925-960,815
Around -840 can be mentioned.

Therefore, the oligonucleotide of the present invention is
For example, an oligonucleotide consisting of at least 9 consecutive nucleotides in the above region. 1 of the present invention
According to one embodiment, the expression-inhibiting oligonucleotide is a nucleotide sequence complementary to the nucleotide sequence of the codon encoding, for example, Ala at position 75 to Leu at position 81 in SEQ ID NO: 1, that is, 5'-AGCCTCCTTCCCATGCCA.
It has GC-3 ′ (SEQ ID NO: 2).

No. 12 as an example of another oligonucleotide
A base sequence complementary to the base sequence of the codon encoding from Leu at position 7 to Glu at position 133, ie, 5 '
-Having a base of CTCACAAACAACATTGCTGA-3 '(SEQ ID NO: 3), a base sequence complementary to the base sequence of the codon encoding His at position 70 to Met at position 77, that is, 5'-TGCCAGCCCATCTGCTGGGG-3 ′ (SEQ ID NO:
With 4), Val from 34th to Asp of 41st
Nucleotide sequences complementary to the nucleotide sequences of the codons encoding up to, ie, 5'-CTCCTGGCAGACTGGTCAGC-3 '
Having (SEQ ID NO: 5),

The nucleotide sequence complementary to the nucleotide sequence of the codon encoding Gln at the 118th position to Lys at the 124th position, ie, having 5'-TTCCGGAAGCAGGAGAGCTG-3 '(SEQ ID NO: 6) and 164th 1st from Pro in rank
A nucleotide sequence complementary to the nucleotide sequence of the codon encoding up to 70th Glu, that is, 5'-TCCTGGGAATACTGGC
Having ACGG-3 ′ (SEQ ID NO: 7), at position 75
A nucleotide sequence complementary to the base sequence of the codon encoding from Ala to Leu at position 82, ie, 5'-GC
Those having AGCCTCCTTCCCATGCCA-3 '(SEQ ID NO: 9), a nucleotide sequence complementary to the nucleotide sequence of the codon encoding Trp at the 74th position to Arg at the 80th position, that is, 5'-CTCCTTCCCATGCCAGCCCA-3' ( Sequence number:
10) are mentioned.

When the oligonucleotide derivative used in the present invention is a deoxyribonucleotide, each structure is as shown in Chemical formula 1, wherein X is independently oxygen (O), sulfur (S), a lower alkyl group. Alternatively, it may be either a primary amine or a secondary amine. Y may independently be either oxygen (O) or sulfur (S). B is selected from any of adenine, guanine, thymine, or cytosine, and is a complementary oligonucleotide of DNA or mRNA mainly encoding human IL-6R. R is independently hydrogen or a dimethoxytrityl group or a lower alkyl group. n is 7-28.

Preferred oligonucleotide derivatives are not only unmodified oligonucleotides but also modified oligonucleotides. Examples of such modified products include lower alkyl phosphonate modified products such as the above-mentioned methyl phosphonate type or ethyl phosphonate type, and other phosphorothioate modified products or phosphoroamidate modified products (see Chemical Formula 2).

[0018]

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[0019]

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These oligonucleotide derivatives can be obtained by a conventional method as follows. X and Y in formula 1
The oligonucleotide in which O is O is easily synthesized by a commercially available DNA synthesizer (for example, manufactured by Applied Biosystems). The synthesis method can be obtained by a solid phase synthesis method using phosphoramidite, a solid phase synthesis method using hydrogen phosphonate, and the like.

For example, T. Atkinson, M. Smith, in Olig
nucleotide Synthesis: A Practical Approach, ed.
MJ Gait, IRL Press, 35-81 (1984); MH Caruther
s, Science, 230, 281 (1985); A. Kume, M. Fujii, M.
Sekine, M. Hata, J. Org. Chem., 49, 2139 (1984); B.
C. Froehler, M. Matteucci, Tetrahedron Lett., 27,
469 (1986); PJ Garegg, I. Lindh, T. Regberg, J.
Stawinski, R. Stromberg, C. Henrichson, ibid., 2
7, 4051 (1986);

BS Sproat, MJ Gait, in Oligonucl
eotide Synthesis: A Practical Approach, ed. MJ
Gait. IRL Press, 83-115 (1984); SL Beaucage and
MH Caruthers, Tetrahedron Lett., 22, 1859-1862
(1981); MD Matteucci and M. H. Caruthers, Tetrah
edron Lett., 21, 719-722 (1980); MD Matteucciand
MH Caruthers, J. Am. Chem. Soc., 103, 3185-319
See 1 (1981).

The modified phosphate triester in which X is a lower alkoxy group can be obtained by a conventional method, for example, by treating the oligonucleotide obtained by chemical synthesis with a solution of tosyl chloride in DMF / methanol / 2,6-lutidine. Can (Moody HM, et al., Nucleic Acids Res.,
17, 4769-4782 (1989)). A modified alkylphosphonate in which X is an alkyl group can be obtained by a conventional method, for example, using a phosphoamidite (MA Dorman, et. Al.,
Tetrahedron, 40, 95-102 (1984); KL Agarwal and
F. Riftina, Nucleic Acids Res., 6, 3009-3024 (197
9)).

The phosphorothioate modified product in which X is S can be prepared by a conventional method, for example, a solid phase synthesis method using sulfur (CA St
ein, et.al., Nucleic Acids Res., 16, 3209-3221 (19
88)) or tetraethylthiuram disulfide, and can be obtained by solid phase synthesis (H. Vu and
BL Hirschbein, Tetrahedron Letters, 32, 3005-30
08 (1991)). The phosphorodithioate modified product in which both X and Y are S can be obtained by a solid phase synthesis method, for example, by converting a bisamidite into a thioamidite and reacting with sulfur (WK-D. Brill, et. Al. ., J. A
m. Chem. Soc., 111, 2321-2322 (1989)).

The phosphoramidate modified product in which X is a primary amine or a secondary amine can be obtained by a solid phase synthesis method, for example, by treating a hydrogenphosphonate with a primary or secondary amine (B. Froehler, et.
al. Nucleic Acids Res., 16, 4831-4839 (1988)). Alternatively, it can also be obtained by oxidizing amidite with tert-butyl hydroperoxide (H. Ozaki, e.
t. al., Tetrahedron Lett., 30, 5899-5902 (1989)).

Purification and confirmation of purity can be carried out by high performance liquid chromatography or polyacrylamide gel electrophoresis. Confirm molecular weight by Electrospray Ionizatio
n Mass Spectrometry or Fast Atom Bonbardment-Mass
It can be done with Spectrometry. The antisense oligonucleotide derivative of the present invention may be synthesized or derived from any method as long as it has a sequence that hybridizes to the base sequence of DNA or mRNA encoding human IL-6R.

The antisense oligonucleotide derivative of the present invention acts on human IL-6R-producing cells and binds to human IL-6R-encoding DNA or mRNA to inhibit its transcription or translation, IL-6
Suppressing the expression of R results in human IL-6
Has the effect of suppressing the action of. Human IL-suppressed by the antisense oligonucleotide derivative of the present invention
Examples of the action of 6 include a platelet increasing action, an antibody production enhancing action, an acute phase protein inducing action, a tumor cell proliferation action, a nerve cell differentiation action and the like.

Therefore, diseases caused by these effects, for example, cancers such as renal cancer, myeloma, Rennelt T lymphoma, Kaposi's sarcoma, autoimmune diseases such as rheumatoid arthritis, mesangial proliferative nephritis, psoriasis, cancer cachexia, The antisense oligonucleotide derivative of the present invention is considered to be effective in treating endotoxin shock in infectious diseases. The antisense oligonucleotide derivative of the present invention can be mixed with a suitable base material that is inactive against them to be used as an external preparation such as a coating agent and a poultice.

If necessary, an excipient, a tonicity agent,
Tablets, powders, granules, capsules, liposome capsules, injections, solutions, nasal drops, etc., and lyophilization agents can be prepared by adding solubilizers, stabilizers, preservatives, soothing agents, etc. it can. These can be prepared according to a conventional method. The antisense oligonucleotide derivative of the present invention is applied to a patient's affected area directly, or is administered to a patient so as to be able to reach the affected area as a result of intravascular administration. Furthermore, an antisense encapsulating material that enhances durability and membrane permeability can also be used. Examples thereof include liposomes, poly-L-lysine, lipids, cholesterol, lipofectin or derivatives thereof.

The dose of the antisense oligonucleotide derivative of the present invention can be adjusted appropriately according to the patient's condition and used in a preferable amount. For example, 0.1-100
The dose can be administered in the range of mg / kg, preferably 0.1 to 50 mg / kg. Hereinafter, the present invention will be described in detail with reference to Examples.

[0031]

EXAMPLES Synthesis Example 1 5'-AGCCTCCTTCCCATGCCAGC-3 '(Sequence number
No. 2) (Synthesis of phosphorothioate modified product ) The dimethoxytrityl group of 5′-dimethoxytrityl-2′-deoxycytidine (1 μmol) having a 3′-hydroxyl group bound to a support is deprotected with trichloroacetic acid, and then 5 ′. -Condensation of 5'-dimethoxytrityl-2'-deoxyguanosine β-cyanoethylphosphoamidite derivative with hydroxyl group with tetrazole, sulfurization of phosphorus with tetraethylthiuram disulfide, and reaction of unreacted 5'-hydroxyl group with acetic anhydride and dimethylamino Acetylate with pyridine.

Similarly, deprotection, condensation, sulfurization and acetylation are repeated. The last 5'-dimethoxytrityl-2 '
20-me obtained by condensing a deoxyadenosine β-cyanoethylphosphoamidite derivative and converting to sulfur
Modified phosphorothioate of r
It was carried out by a 381A type DNA synthesizer manufactured by ied Biosystems. ) Is cleaved from the support with 2 ml of concentrated aqueous ammonia, the cyanoethyl group is removed from phosphorus, and the protective groups attached to adenine, guanine and cytosine are removed.

The resulting 5'-dimethoxytrityl oligonucleotide phosphorothioate is unpurified, or after purification by high performance liquid chromatography, or a synthetic DNA purification cartridge column (for example,
After holding it on an oligo pack SP manufactured by Millipore Japan, etc., the 5'-dimethoxytrityl protecting group is removed with trifluoroacetic acid. If necessary, the obtained oligonucleotide phosphorothioate is purified by high performance liquid chromatography to obtain the desired 5′-AGCCTCCTTCCCATGCCAGC-3 ′.
(SEQ ID NO: 2) (phosphorothioate modified product) About 1.
37 mg are obtained.

Synthesis Example 2 5'-CTCACAAACAACATTGCTGA-
3 '(SEQ ID NO: 3) (modified phosphorothioate)
Synthesis In the same manner as in Synthesis Example 1, the desired 5'-CTCACAAACAACATTG
About 0.65 mg of CTGA-3 '(SEQ ID NO: 3) (phosphorothioate modification) is obtained.

Synthesis Example 3 5'-TGCCAGCCCATCTGCTGGGG-
3 '(SEQ ID NO: 4) (phosphorothioate modified)
Synthesis In the same manner as in Synthesis Example 1, the desired 5'-TGCCAGCCCATCTGCT
About 0.98 mg of GGGG-3 '(SEQ ID NO: 4) (phosphorothioate modification) is obtained.

Synthesis Example 4 5'-CTCCTGGCAGACTGGTCAGC-
Of 3 '(SEQ ID NO: 5) (modified phosphorothioate)
Synthesis In the same manner as in Synthesis Example 1, the target 5'-CTCCTGGCAGACTGGT
About 1.61 mg of CAGC-3 ′ (SEQ ID NO: 5) (phosphorothioate modified product) is obtained.

Synthesis Example 5 5'-TTCCGGAAGCAGGAGAGCTG-
Of 3 '(SEQ ID NO: 6) (modified phosphorothioate)
Synthesis In the same manner as in Synthesis Example 1, the target 5′-TTCCGGAAGCAGGAGA
About 1.47 mg of GCTG-3 ′ (SEQ ID NO: 6) (phosphorothioate modified product) is obtained.

Synthesis Example 6 5'-TCCTGGGAATACTGGCACGG-
Of 3 '(SEQ ID NO: 7) (modified phosphorothioate)
Synthesis In the same manner as in Synthesis Example 1, the desired 5'-TCCTGGGAATACTGGC
About 1.64 mg of ACGG-3 '(SEQ ID NO: 7) (phosphorothioate modification) is obtained.

Synthesis Example 7 5'-GCAGCCTCCTTCCCATGCCA-
Of 3 '(SEQ ID NO: 9) (modified phosphorothioate)
Synthesis In the same manner as in Synthesis Example 1, the desired 5′-GCAGCCTCCTTCCCAT
About 2.47 mg of GCCA-3 ′ (SEQ ID NO: 9) (phosphorothioate modified product) is obtained.

Synthesis Example 8 5'-CTCCTTCCCATGCCAGCCCA-
3 '(SEQ ID NO: 10) (modified phosphorothioate)
Synthesis of the target 5′-CTCCTTCCCATGCCAG in the same manner as in Synthesis Example 1
About 2.06 mg of CCCA-3 ′ (SEQ ID NO: 10) (phosphorothioate modification) is obtained.

Reference Example 1 5'-CCCCAGCAGATGGGCTGGCA-
3 '(SEQ ID NO: 8) (phosphorothioate modified product:
Synthesis of (sense sequence of column number: 4) The desired 5'-CCCCAGCAGATGGGCTGG was prepared in the same manner as in Synthesis Example 1.
About 0.53 mg of CA-3 '(SEQ ID NO: 8) (phosphorothioate modification) is obtained.

Reference Example 2 5'-GCTGGCATGGGAAGGAGGCT-
3 '(SEQ ID NO: 11) (modified phosphorothioate:
Synthesis of (sense sequence of SEQ ID NO: 2) In the same manner as in Synthesis Example 1, the desired 5'-GCTGGCATGGGAAGGA
About 1.91 mg of GGCT-3 ′ (SEQ ID NO: 11) (phosphorothioate modified product) is obtained.

Experimental Example 1 Human soluble IL-6R (sIL
-6R) expression suppression effect (1) COH. Preparation of SR344 cells pBSF2R. 236 (Science, 241, 825-828 (1988))
Was cleaved with SphI and 1205 bp of IL-6RcDN
The A fragment was inserted into mp18 (Amersham). sIL-6R cDNA is 5'-ATATTCTAGAGAGCTTCT
-3 'synthetic oligonucleotide was prepared and
It was prepared using an utagenesis system (manufactured by Amersham). As a result, the termination cordon was the 345th amino acid sequence.

Dhfr-cDNA is a plasmid pECE (Cel
l, 45, 721-735 (1986)) Pvu II site,
Plasmid pECEdhfr was prepared. Hind of sIL-6R
The III-SalI fragment was inserted into plasmid pECEdhfr to prepare a soluble IL-6R expression vector plasmid pECEdhfr344.

PECEdhfr344 was replaced with dhfr-CH
O cell DXB-11 (Pro. Natl. Acad. Sci. USA,
77, 4216-4220 (1980)) by the calcium phosphate method and amplified by MTX. Finally, 200 nMMTX resistant sIL-6R producing CHO cells (CHO.SR344)
Was prepared (J. Biochem., 108, 673-676 (1990)). Normal cell culture is 5% FCS (Xavier Investments)
And IMDM medium containing 200 nMMTX (Gibc
o).

(2) CHO. SIL-of SR344 cells
IL-6R antisense oligonucleotides for 6R production
Effect of Ochid CHO. SR344 cells are trypsin-EDTA
It was peeled from the culture dish with (Gibco), washed with a culture solution, further washed with serum-free medium non-serum (manufactured by Nippon Zenyaku Kogyo), and suspended in serum-free medium non-serum containing 200 nMMTX. Add CHO. S
2 μM of IL-6R antisense oligonucleotide 1 in 100 μl of R344 cell suspension (5 × 10 ⁴ cells / ml)
00 μl was added, and the cells were cultured in an incubator at 37 ° C. under 5% CO ₂ .

After culturing for 24 hours, the soluble I of the culture supernatant was
Sandwich E using the amount of L-6R as a mouse anti-IL-6R monoclonal antibody (MT18) (JP-A-2-288898) and a rabbit anti-IL-6R polyclonal antibody
It was measured by the LISA method. As a control, the sense oligonucleotide derivative for SEQ ID NO: 4 (SEQ ID NO: 8) or the sense oligonucleotide derivative for SEQ ID NO: 2 (SEQ ID NO: 1)
It measured using 1). Human IL-6R antisense oligonucleotide showed the effect of suppressing the expression of soluble IL-6R (Figs. 1 and 2).

[0048]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 3319 Sequence type: Nucleic acid Number of strands: Single-stranded Sequence type: cDNA sequence GGCGGTCCCC TGTTCTCCCC GCTCAGGTGC GGCGCTGTGG CAGGAAGCCA CCCCCTCGGT 60 CGGCCGGTGCCGCGGGGGGCTGT CCGCGACTGCTCCGGACGTCTCCGGACGTCTCCGACACCTC ACGGACGTCTCCGGACGTCTCCGGACGTCTCCGACACCGC ACTC GTCATGTGCG AGTGGGAAGT CGCACTGACA CTGAGCCGGG CCAGAGGGAG 240 AGGAGCCGAG CGCGGCGCGG GGCCGAGGGA CTCGCAGTGT GTGTAGAGAG CCGGGCTCCT 300 GCGGATGGGG GCTGCCCCCG GGGCCTGAGC CCGCCTGCCC GCCCACCGCC CCGCCCCGCC 360 CCTGCCACCC CTGCCGCCCG GTTCCCATTA GCCTGTCCGC CTCTGCGGGA CCATGGAGTG 420 GTAGCCGAGG AGGAAGC ATG CTG GCC GTC GGC TGC GCG CTG CTG GCT GCC 470 Met Leu Ala Val Gly Cys Ala Leu Leu Ala Ala 1 5 10 CTG CTG GCC GCG CCG GGA GCG GCG CTG GCC CCA AGG CGC TGC CCT GCG 518 Leu Leu Ala Ala Pro Gly Ala Ala Leu Ala Pro Arg Arg Cys Pro Ala 15 20 25 CAG GAG GTG GCA AGA GGC GTG CTG ACC AGT CTG CCA GGA GAC AGC GTG 566 Gln Glu Val Ala Arg Gly Val Leu Thr Ser Leu Pro Gly Asp Ser Val 30 35 40 ACT CTG ACC TGC CCG GGG GTA GAG CCG GAA GAC AAT GCC ACT GTT CAC 614 Thr Leu Thr Cys Pro Gly Val Glu Pro Glu Asp Asn Ala Thr Val His 45 50 55

TGG GTG CTC AGG AAG CCG GCT GCA GGC TCC CAC CCC AGC AGA TGG GCT 662 Trp Val Leu Arg Lys Pro Ala Ala Gly Ser His Pro Ser Arg Trp Ala 60 65 70 75 GGC ATG GGA AGG AGG CTG CTG CTG AGG TCG GTG CAG CTC CAC GAC TCT 710 Gly Met Gly Arg Arg Leu Leu Leu Arg Ser Val Gln Leu His Asp Ser 80 85 90 GGA AAC TAT TCA TGC TAC CGG GCC GGC CGC CCA GCT GGG ACT GTG CAC 758 Gly Asn Tyr Ser Cys Tyr Arg Ala Gly Arg Pro Ala Gly Thr Val His 95 100 105 TTG CTG GTG GAT GTT CCC CCC GAG GAG CCC CAG CTC TCC TGC TTC CGG 806 Leu Leu Val Asp Val Pro Pro Glu Glu Pro Gln Leu Ser Cys Phe Arg 110 115 120 AAG AGC CCC CTC AGC AAT GTT GTT TGT GAG TGG GGT CCT CGG AGC ACC 854 Lys Ser Pro Leu Ser Asn Val Val Cys Glu Trp Gly Pro Arg Ser Thr 125 130 135 CCA TCC CTG ACG ACA AAG GCT GTG CTC TTG GTG AGG AAG TTT CAG AAC 902 Pro Ser Leu Thr Thr Lys Ala Val Leu Leu Val Arg Lys Phe Gln Asn 140 145 150 155 AGT CCG GCC GAA GAC TTC CAG GAG CCG TGC CAG TAT TCC CAG GAG TCC 950 Ser Pro Ala Glu Asp Phe Gln Glu Pro Cys Gln Tyr S er Gln Glu Ser 160 165 170 CAG AAG TTC TCC TGC CAG TTA GCA GTC CCG GAG GGA GAC AGC TCT TTC 998 Gln Lys Phe Ser Cys Gln Leu Ala Val Pro Glu Gly Asp Ser Ser Phe 175 180 185 TAC ATA GTG TCC ATG TGC GTC GCC AGT AGT GTC GGG AGC AAG TTC AGC 1046 Tyr Ile Val Ser Met Cys Val Ala Ser Ser Val Gly Ser Lys Phe Ser 190 195 200

AAA ACT CAA ACC TTT CAG GGT TGT GGA ATC TTG CAG CCT GAT CCG CCT 1094 Lys Thr Gln Thr Phe Gln Gly Cys Gly Ile Leu Gln Pro Asp Pro Pro 205 210 215 GCC AAC ATC ACA GTC ACT GCC GTG GCC AGA AAC CCC CGC TGG CTC AGT 1142 Ala Asn Ile Thr Val Thr Ala Val Ala Arg Asn Pro Arg Trp Leu Ser 220 225 230 235 GTC ACC TGG CAA GAC CCC CAC TCC TGG AAC TCA TCT TTC TAC AGA CTA 1190 Val Thr Trp Gln Asp Pro His Ser Trp Asn Ser Ser Phe Tyr Arg Leu 240 245 250 CGG TTT GAG CTC AGA TAT CGG GCT GAA CGG TCA AAG ACA TTC ACA ACA 1238 Arg Phe Glu Leu Arg Tyr Arg Ala Glu Arg Ser Lys Thr Phe Thr Thr 255 260 265 TGG ATG GTC AAG GAC CTC CAG CAT CAC TGT GTC ATC CAC GAC GCC TGG 1286 Trp Met Val Lys Asp Leu Gln His His Cys Val Ile His Asp Ala Trp 270 275 280 AGC GGC CTG AGG CAC GTG GTG CAG CTT CGT GCC CAG GAG GAG TTC GGG 1334 Ser Gly Leu Arg His Val Val Gln Leu Arg Ala Gln Glu Glu Phe Gly 285 290 295 CAA GGC GAG TGG AGC GAG TGG AGC CCG GAG GCC ATG GGC ACG CCT TGG 1382 Gln Gly Glu Trp Ser Glu Trp Ser Pro Gl u Ala Met Gly Thr Pro Trp 300 305 310 315 ACA GAA TCC AGG AGT CCT CCA GCT GAG AAC GAG GTG TCC ACC CCC ATG 1430 Thr Glu Ser Arg Ser Pro Pro Ala Glu Asn Glu Val Ser Thr Pro Met 320 325 330 CAG GCA CTT ACT ACT AAT AAA GAC GAT GAT AAT ATT CTC TTC AGA GAT 1478 Gln Ala Leu Thr Thr Asn Lys Asp Asp Asp Asn Ile Leu Phe Arg Asp 335 340 345

TCT GCA AAT GCG ACA AGC CTC CCA GTG CAA GAT TCT TCT TCA GTA CCA 1526 Ser Ala Asn Ala Thr Ser Leu Pro Val Gln Asp Ser Ser Ser Val Pro 350 355 360 CTG CCC ACA TTC CTG GTT GCT GGA GGG AGC CTG GCC TTC GGA ACG CTC 1574 Leu Pro Thr Phe Leu Val Ala Gly Gly Ser Leu Ala Phe Gly Thr Leu 365 370 375 CTC TGC ATT GCC ATT GTT CTG AGG TTC AAG AAG ACG TGG AAG CTG CGG 1622 Leu Cys Ile Ala Ile Val Leu Arg Phe Lys Lys Thr Trp Lys Leu Arg 380 385 390 395 GCT CTG AAG GAA GGC AAG ACA AGC ATG CAT CCG CCG TAC TCT TTG GGG 1670 Ala Leu Lys Glu Gly Lys Thr Ser Met His Pro Pro Tyr Ser Leu Gly 400 405 410 CAG CTG GTC CCG GAG AGG CCT CGA CCC ACC CCA GTG CTT GTT CCT CTC 1718 Gln Leu Val Pro Glu Arg Pro Arg Pro Thr Pro Val Leu Val Pro Leu 415 420 425 ATC TCC CCA CCG GTG TCC CCC AGC AGC CTG GGG TCT GAC AAT ACC TCG 1766 Ile Ser Pro Pro Val Ser Pro Ser Ser Leu Gly Ser Asp Asn Thr Ser 430 435 440 AGC CAC AAC CGA CCA GAT GCC AGG GAC CCA CGG AGC CCT TAT GAC ATC 1814 Ser His Asn Arg Pro Asp Ala Arg Asp Pr o Arg Ser Pro Tyr Asp Ile 445 450 455 AGC AAT ACA GAC TAC TTC TTC CCC AGA TAG CTGGCTGGGT GGCACCAGCA 1864 Ser Asn Thr Asp Tyr Phe Phe Pro Arg --- 460 465 CGCTGGACCCTGTGCATGCTGCTCACTACTGACCCT AAAGCAGCACT 1984 TCTACGCCAG GGGAAAATCA GCCTGCTCCA GCTGTTCAGC TGGTTGAGGT TTCAAACCTC 2044 CCTTTCCAAA TGCCCAGCTT AAAGGGGTTA GAGTGAACTT GGGCCACTGT GAAGAGAACC 2104

[0052] ATATCAAGAC TCTTTGGACA CTCACACGGA CACTCAAAAG CTGGGCAGGT TGGTGGGGGC 2164 CTCGGTGTGG AGAAGCGGCT GGCAGCCCAC CCCTCAACAC CTCTGCACAA GCTGCACCCT 2224 CAGGCAGGTG GGATGGATTT CCAGCCAAAG CCTCCTCCAG CCGCCATGCT CCTGGCCCAC 2284 TGCATCGTTT CATCTTCCAA CTCAAACTCT TAAAACCCAA GTGCCCTTAG CAAATTCTGT 2344 TTTTCTAGGC CTGGGGACGG CTTTTACTTA AACGCCAAGG CCTGGGGGAA GAAGCTCTCT 2404 CCTCCCTTTC TTCCCTACAG TTCAAAAACA GCTGAGGGTG AGTGGGTGAA TAATACAGTA 2464 TGTCAGGGCC TGGTCGTTTT CAACAGAATT ATAATTAGTT CCTCATTAGC AGTTTTGCCT 2524 AAATGTGAAT GATGATCCTA GGCATTTGCT GAATACAGAG GCAACTGCAT TGGCTTTGGG 2584 TTGCAGGACC TCAGGTGAGA AGCAGAGGAA GGAGAGGAGA GGGGCACAGG GTCTCTACCA 2644 TCCCCTGTAG AGTGGGAGCT GAGTGGGGGA TCACAGCCTC TGAAAACCAA TGTTCTCTCT 2704 TCTCCACCTC CCACAAAGGA GAGCTAGCAG CAGGGAGGGC TTCTGCCATT TCTGAGATCA 2764 AAACGGTTTT ACTGCAGCTT TGTTTGTTGT CAGCTGAACC TGGGTAACTA GGGAAGATAA 2824 TATTAAGGAA GACAATGTGA AAAGAAAAAT GAGCCTGGCA AGAATGCGTT TAAACTTGGT 2884 TTTTAAAAAA CTGCTGACTG TTTTCTCTTG AGAGGGTGGA ATATCCAATA TTC GCTGTGT 2944 CAGCATAGAA GTAACTTACT TAGGTGTGGG GGAAGCACCA TAACTTTGTT TAGCCCAAAA 3004 CCAAGTCAAG TGAAAAAGGA GGAAGAGAAA AAATATTTTC CTGCCAGGCA TGGAGGCCCA 3064 CGCACTTCGG GAGGTCGAGG CAGGAGGATC ACTTGAGTCC AGAAGTTTGA GATCAGCCTG 3124 GGCAATGTGA TAAAACCCCA TCTCTACAAA AAGCATAAAA ATTAGCCAAG TGTGGTAGAG 3184 TGTGCCTGAA GTCCCAGATA CTTGGGGGGC TGAGGTGGGA GGATCTCTTG AGCCTGGGAG 3244 GTCAAGGCTG CAGTGAGCCG AGATTGCACC ACTGCACTCC AGCCTGGGGT GACAGAGCAA 3304 GTGAGACCCT GTCTC 3319

SEQ ID NO: 2 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Sequence type: Synthetic DNA Sequence AGCCTCCTTC CCATGCCAGC 20

SEQ ID NO: 3 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Sequence type: Sequence CTCACAAACA ACATTGCTGA 20

SEQ ID NO: 4 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Sequence type: Synthetic DNA Sequence TGCCAGCCCA TCTGCTGGGG 20

SEQ ID NO: 5 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Sequence type: Sequence CTCCTGGCAG ACTGGTCAGC 20

SEQ ID NO: 6 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Sequence type: Synthetic DNA Sequence TTCCGGAAGC AGGAGAGCTG 20

SEQ ID NO: 7 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Sequence type: Synthetic DNA Sequence TCCTGGGAAT ACTGGCACGG 20

SEQ ID NO: 8 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Sequence type: Synthetic DNA Sequence CCCCAGCAGA TGGGCTGGCA 20

SEQ ID NO: 9 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Sequence type: Synthetic DNA Sequence GCAGCCTCCT TCCCATGCCA 20

SEQ ID NO: 10 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Sequence type: Synthetic DNA Sequence CTCCTTCCCA TGCCAGCCCA 20

SEQ ID NO: 11 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Sequence type: Synthetic DNA Sequence GCTGGCATGG GAAGGAGGCT 20

[Brief description of the drawings]

FIG. 1 is a graph showing that the antisense oligonucleotide derivatives of the present invention (SEQ ID NOS: 2 and 3) in Experimental Example 1 suppress the expression of soluble IL-6R.

FIG. 2 is a graph showing that the antisense oligonucleotides of the present invention (SEQ ID NOS: 2, 9 and 10) in Experimental Example 1 suppress the expression of soluble IL-6R.

Claims (4)

[Claims] 1. An antisense oligonucleotide derivative against at least 9 consecutive base sequences including a base sequence of a portion capable of forming a loop structure of mRNA encoding human interleukin-6 receptor (human IL-6R) is effective. A human IL-6R expression inhibitor as a component. 2. The expression inhibitor according to claim 1, which comprises an antisense oligonucleotide derivative against the base sequence of a portion of the base sequence of SEQ ID NO: 1 that can form a loop structure. 3. The expression inhibitor according to claim 2, wherein the nucleotide sequence is AGCCTCCTTCCCATGCCAGC (SEQ ID NO: 2). 4. The expression inhibitor according to claim 2, wherein the base sequence is CTCACAAACAACATTGCTGA (SEQ ID NO: 3).