JP3868021B2 - Human interleukin-6 receptor expression inhibitor - Google Patents

Description

【００１９】
【化２】

【００２０】
これらのオリゴヌクレオチド誘導体は次のとおり常法によって得ることができる。
化１のＸ及びＹがＯであるオリゴヌクレオチドは市販のＤＮＡ合成装置（例えばApplied Biosystems社製など）によって容易に合成される。
合成法はホスホロアミダイトを用いた固相合成法、ハイドロジェンホスホネートを用いた固相合成法などで得ることができる。
【００２１】
例えば、T. Atkinson, M. Smith, in Oligonucleotide Synthesis: A Practical Approach, ed. M. J. Gait, IRL Press, 35-81(1984); M. H. Caruthers, 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); P. J. Garegg, I. Lindh, T. Regberg, J. Stawinski, R. Stromberg, C. Henrichson, ibid., 27, 4051(1986);
【００２２】
B. S. Sproat, M. J. Gait, in Oligonucleotide Synthesis: A Practical Approach, ed. M. J. Gait. IRL Press, 83-115(1984); S. L. Beaucage and M. H. Caruthers, Tetrahedron Lett., 22, 1859-1862(1981); M. D. Matteucci and M. H. Caruthers, Tetrahedron Lett., 21, 719-722(1980); M. D. Matteucci and M. H. Caruthers, J. Am. Chem. Soc., 103, 3185-3191(1981)を参照のこと。
【００２３】
Ｘが低級アルコキシ基であるリン酸トリエステル修飾体は、常法、例えば化学合成で得られたオリゴヌクレオチドをトシルクロリドのＤＭＦ／メタノール／２，６−ルチジン溶液で処理することにより得ることができる（Moody H. M., et al., Nucleic Acids Res., 17, 4769-4782(1989)) 。
Ｘがアルキル基であるアルキルホスホネート修飾体は、常法、例えばホスホアミダイトを用いて得ることができる（M. A. Dorman, et. al., Tetrahedron, 40, 95-102(1984); K. L. Agarwal and F. Riftina, Nucleic Acids Res., 6, 3009-3024(1979)) 。
【００２４】
ＸがＳであるホスホロチオエート修飾体は、常法、例えばイオウを用いた固相合成法（C. A. Stein, et. al., Nucleic Acids Res., 16, 3209-3221(1988))あるいはテトラエチルチウラム ジスルフィドを用いて、固相合成法により得ることができる（H. Vu and B. L. Hirschbein, Tetrahedron Letters, 32, 3005-3008(1991)) 。
Ｘ，ＹがともにＳであるホスホロジチオエート修飾体は、例えばビスアミダイトをチオアミダイトに変換しイオウを作用させることにより固相合成法により得ることができる（W. K. -D. Brill, et. al., J. Am. Chem. Soc., 111, 2321-2322(1989))。
【００２５】
Ｘが一級アミンあるいは二級アミンであるホスホロアミデート修飾体は、例えばハイドロジェンホスホネートを一級あるいは二級アミンで処理することにより固相合成法で得ることができる（B. Froehler, et. al. Nucleic Acids Res., 16, 4831-4839(1988)) 。あるいは、アミダイトをｔｅｒｔ−ブチルハイドロパーオキサイドで酸化しても得ることができる（H. Ozaki, et. al., Tetrahedron Lett., 30, 5899-5902(1989))。
【００２６】
精製および純度確認は、高速液体クロマトグラフィー、ポリアクリルアミドゲル電気泳動で行うことができる。分子量の確認は、Electrospray Ionization Mass Spectrometry 又はFast Atom Bonbardment-Mass Spectrometry で行うことができる。本発明のアンチセンスオリゴヌクレオチド誘導体はヒトＩＬ−６ＲをコドするＤＮＡ又はｍＲＮＡの塩基配列にハイブリダイズする配列を有するものであれば、その合成法や由来はいずれでもよい。
【００２７】
本発明のアンチセンスオリゴヌクレオチド誘導体は、ヒトＩＬ−６Ｒの産生細胞に作用して、ヒトＩＬ−６ＲをコードするＤＮＡ又はｍＲＮＡに結合することにより、その転写又は翻訳を阻害し、ヒトＩＬ−６Ｒの発現を抑制することによって結果的にヒトＩＬ−６の作用を抑制する効果を有する。本発明のアンチセンスオリゴヌクレオチド誘導体により抑制されるヒトＩＬ−６の作用としては、血小板増多作用、抗体産生増強作用、急性期蛋白誘導作用、腫瘍細胞増殖作用、神経細胞分化作用等が挙げられる。
【００２８】
従って、これらの作用に起因する疾患、例えば腎癌、ミエローマ、レンネルトＴリンパ腫、カポジ肉腫などの癌、慢性関節リウマチ等の自己免疫疾患、メサンギウム増殖性腎炎、乾癬、癌性悪液質、感染症におけるエンドトキシンショック等の治療において本発明のアンチセンスオリゴヌクレオチド誘導体は有効であると考えられる。
本発明のアンチセンスオリゴヌクレオチド誘導体は、それらに対して不活性な適当な基剤と混和して塗布剤、パップ剤などの外用剤とすることができる。
【００２９】
また、必要に応じて、賦形剤、等張化剤、溶解補助剤、安定化剤、防腐剤、無痛化剤等を加えて錠剤、散剤、顆粒剤、カプセル剤、リポソームカプセル剤、注射剤、液剤、点鼻剤など、さらに凍結乾燥剤とすることができる。これらは常法に従って調製することができる。
本発明のアンチセンスオリゴヌクレオチド誘導体は患者の患部に直接適用するか、または血管内に投与するなどして結果的に患部に到達し得るように患者に適応させる。さらに持続性、膜透過性を高めるアンチセンス封入素材を用いることもできる。例えば、リポソーム、ポリ−Ｌ−リジン、リピッド、コレステロール、リポフェクチン又はこれらの誘導体が挙げられる。
【００３０】
本発明のアンチセンスオリゴヌクレオチド誘導体の投与量は、患者の状態に応じて適宜調整し好ましい量を用いることができる。例えば、０．１〜１００mg／kg好ましくは０．１〜５０mg／kgの範囲で投与することができる。
以下本発明を実施例において詳しく説明する。
【００３１】
【実施例】
合成例１．５′− AGCCTCCTTCCCATGCCAGC −３′（配列番号：２）（ホスホロチオエート修飾体）の合成
３′−水酸基が支持体に結合した５′−ジメトキシトリチル−２′−デオキシシチジン（１μmol ）のジメトキシトリチル基をトリクロロ酢酸によって脱保護し、その５′−水酸基に５′−ジメトキシトリチル−２′−デオキシグアノシンβ−シアノエチルホスホアミダイト誘導体をテトラゾールにより縮合し、テトラエチルチウラム ジスルフィドによってリンをイオウ化した後、未反応の５′−水酸基を無水酢酸とジメチルアミノピリジンでアセチル化する。
【００３２】
同様に脱保護、縮合、イオウ化、アセチル化を繰り返す。最後の５′−ジメトキシトリチル−２′−デオキシアデノシン β−シアノエチルホスホアミダイト誘導体を縮合し、イオウ化して得られた２０−ｍｅｒのホスホロチオエート修飾体（以上までの工程はApplied Biosystems社製３８１Ａ型ＤＮＡ合成装置により行った。）を濃アンモニア水２mlによって支持体から切り出すと共に、リンからシアノエチル基をはずし、さらにアデニン、グアニン、シトシンに付いている保護基をはずす。
【００３３】
得られた５′−ジメトキシトリチルオリゴヌクレオチドホスホロチオエートは未精製のまま、または高速液体クロマトグラフィーにより精製した後、あるいは、合成ＤＮＡ精製用カートリッジカラム（例えば、日本ミリポア社製オリゴパックＳＰなど）に保持した後、トリフルオロ酢酸によって５′−ジメトキシトリチル保護基をはずす。得られたオリゴヌクレオチドホスホロチオエートを必要があれば高速液体クロマトグラフィーで精製し、目的５′−AGCCTCCTTCCCATGCCAGC−３′（配列番号：２）（ホスホロチオエート修飾体）約１．３７mgを得る。
【００３４】
合成例２．５′− CTCACAAACAACATTGCTGA −３′（配列番号：３）（ホスホロチオエート修飾体）の合成
合成例１と同様にして、目的の５′−CTCACAAACAACATTGCTGA−３′（配列番号：３）（ホスホロチオエート修飾体）約０．６５mgを得る。
【００３５】
合成例３．５′− TGCCAGCCCATCTGCTGGGG −３′（配列番号：４）（ホスホロチオ エート修飾体）の合成
合成例１と同様にして、目的の５′−TGCCAGCCCATCTGCTGGGG−３′（配列番号：４）（ホスホロチオエート修飾体）約０．９８mgを得る。
【００３６】
合成例４．５′− CTCCTGGCAGACTGGTCAGC −３′（配列番号：５）（ホスホロチオエート修飾体）の合成
合成例１と同様にして、目的の５′−CTCCTGGCAGACTGGTCAGC−３′（配列番号：５）（ホスホロチオエート修飾体）約１．６１mgを得る。
【００３７】
合成例５．５′− TTCCGGAAGCAGGAGAGCTG −３′（配列番号：６）（ホスホロチオエート修飾体）の合成
合成例１と同様にして、目的の５′−TTCCGGAAGCAGGAGAGCTG−３′（配列番号：６）（ホスホロチオエート修飾体）約１．４７mgを得る。
【００３８】
合成例６．５′− TCCTGGGAATACTGGCACGG −３′（配列番号：７）（ホスホロチオエート修飾体）の合成
合成例１と同様にして、目的の５′−TCCTGGGAATACTGGCACGG−３′（配列番号：７）（ホスホロチオエート修飾体）約１．６４mgを得る。
【００３９】
合成例７．５′− GCAGCCTCCTTCCCATGCCA −３′（配列番号：９）（ホスホロチオエート修飾体）の合成
合成例１と同様にして、目的の５′−GCAGCCTCCTTCCCATGCCA−３′（配列番号：９）（ホスホロチオエート修飾体）約２．４７mgを得る。
【００４０】
合成例８．５′− CTCCTTCCCATGCCAGCCCA −３′（配列番号：１０）（ホスホロチオエート修飾体）の合成
合成例１と同様にして、目的の５′−CTCCTTCCCATGCCAGCCCA−３′（配列番号：１０）（ホスホロチオエート修飾体）約２．０６mgを得る。
【００４１】
参考例１．５′− CCCCAGCAGATGGGCTGGCA −３′（配列番号：８）（ホスホロチオエート修飾体：配列番号：４のセンス配列）の合成
合成例１と同様にして目的の５′−CCCCAGCAGATGGGCTGGCA−３′（配列番号：８）（ホスホロチオエート修飾体）約０．５３mgを得る。
【００４２】
参考例２．５′− GCTGGCATGGGAAGGAGGCT −３′（配列番号：１１）（ホスホロチオエート修飾体：配列番号：２のセンス配列）の合成
合成例１と同様にして、目的の５′−GCTGGCATGGGAAGGAGGCT−３′（配列番号：１１）（ホスホロチオエート修飾体）約１．９１mgを得る。
【００４３】
実験例１．ヒト可溶性ＩＬ−６Ｒ（ｓＩＬ−６Ｒ）の発現抑制効果
（１）ＣＯＨ．ＳＲ３４４細胞の作製
ｐＢＳＦ２Ｒ．２３６（Science, 241, 825-828(1988))をＳｐｈＩで切断し、１２０５ｂｐのＩＬ−６ＲｃＤＮＡ断片をｍｐ１８（Ａｍｅｒｓｈａｍ社製）に挿入した。ｓＩＬ−６ＲｃＤＮＡは５′−ATATTCTAGAGAGCTTCT−３′の合成オリゴヌクレオチドを作製し、in vitro mutagenesis system （Ａｍｅｒｓｈａｍ社製) を用いて調製した。その結果、termination cordonはアミノ酸配列の３４５番目となった。
【００４４】
ｄｈｆｒ−ｃＤＮＡはplasmid pECE（Cell, 45, 721-735(1986))のＰｖｕ II ｓｉｔｅに挿入し、plasmid pECEdhfrを作製した。ｓＩＬ−６ＲのＨｉｎｄ III−ＳａｌＩ断片をplasmid pECEdhfrに挿入し、可溶性ＩＬ−６Ｒ発現ベクターplasmid pECEdhfr３４４を作製した。
【００４５】
ｐＥＣＥｄｈｆｒ３４４をｄｈｆｒ−ＣＨＯ細胞ＤＸＢ−１１（Pro. Natl. Acad. Sci. U.S.A., 77, 4216-4220(1980))にリン酸カルシウム法により導入し、ＭＴＸにて増幅した。最終的に２００ｎＭＭＴＸ耐性ｓＩＬ−６Ｒ産生ＣＨＯ細胞（ＣＨＯ．ＳＲ３４４）を作製した（J. Biochem., 108, 673-676(1990))。細胞の通常の培養は５％ＦＣＳ（Xavier Investments社製）および２００ｎＭＭＴＸを含むＩＭＤＭ培地（Ｇｉｂｃｏ）で行った。
【００４６】
（２）ＣＨＯ．ＳＲ３４４細胞のｓＩＬ−６Ｒ産生に対するＩＬ−６Ｒアンチセンスオリゴヌクレオチドの効果
ＣＨＯ．ＳＲ３４４細胞はｔｒｙｐｓｉｎ−ＥＤＴＡ（Ｇｉｂｃｏ社製）で培養ｄｉｓｈから剥がし、培養液で洗浄後、更に無血清培地ノンセラム（日本全薬工業製）で洗浄し、２００ｎＭＭＴＸを含む無血清培地ノンセラムに懸濁した。９６穴の培養プレートにＣＨＯ．ＳＲ３４４細胞懸濁液１００μｌ（５ｘ１０⁴ 個／ml）に２μＭのＩＬ−６Ｒアンチセンスオリゴヌクレオチド１００μｌを加え、３７℃、５％ＣＯ₂ 下でインキュベーターで培養した。
【００４７】
２４時間培養後、その培養上清の可溶性ＩＬ−６Ｒ量をマウス抗ＩＬ−６Ｒモノクローナル抗体（ＭＴ１８）（特開平２−２８８８９８）及びウサギ抗ＩＬ−６Ｒポリクローナル抗体を用いたサンドイッチＥＬＩＳＡ法によって測定した。
なお、対照（コントロール）としては、配列番号：４に対するセンスオリゴヌクレオチド誘導体（配列番号：８）または配列番号：２に対するセンスオリゴヌクレオチド誘導体（配列番号：１１）を用いて測定した。
ヒトＩＬ−６Ｒアンチセンスオリゴヌクレオチドは可溶性ＩＬ−６Ｒの発現抑制効果を示した（図１、図２）。
【００４８】
【配列表】

【００４９】

【００５０】

【００５１】

【００５２】

【００５３】

【００５４】

【００５５】

【００５６】

【００５７】

【００５８】

【００５９】

【００６０】

【００６１】

【００６２】

【図面の簡単な説明】
【図１】図１は実験例１における本発明のアンチセンスオリゴヌクレオチド誘導体（配列番号２及び３）が可溶性ＩＬ−６Ｒの発現を抑制することを示すグラフである。
【図２】図２は実験例１における本発明のアンチセンスオリゴヌクレオチド（配列番号２，９及び１０）が可溶性ＩＬ−６Ｒの発現を抑制することを示すグラフである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an antisense oligonucleotide derivative useful as a pharmaceutical agent that inhibits the expression of human interleukin-6 receptor (human IL-6R).
[0002]
[Prior art]
Human interleukin-6 (human IL-6) is a cytokine cloned as a factor that induces the final differentiation of B cells into antibody-producing cells (Kishimoto T et al., Blood 74, 1-10, 1989), It is now known to have various effects such as induction of acute phase proteins in the liver (Kishimoto T et al., Blood 74, 1-10, 1989).
[0003]
Human IL-6 has been reported to be produced not only in lymphoid cells but also in fibroblasts, vascular endothelial cells, bladder cancer cell line T24, glioblastoma and the like (Kohase M. et al., J. Biol. Cell Physiol. 132, 271-278, 1978; Meir EV et al., Cancer Res. 50, 6683-6688, 1990), and its target cells are also diverse (Kishimoto T et al., Blood 74, 1-10, 1989) .
[0004]
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). (Miki S et al., FEBS Letter 250, 607-610, 1989).
[0005]
On the other hand, it is known that signals of cell proliferation or differentiation by human IL-6 are transmitted to cells via human IL-6R and glycoprotein gp130 present on the cell surface. (Taga T. et al., Cell 58, 573-581, 1989; Hibi M et al., Cell 68, 1149-1157, 1990).
In recent years, as a method for suppressing the action of a gene causing a disease state, it has been proposed to suppress the expression of the protein using an oligonucleotide (antisense oligonucleotide) complementary to mRNA transcribed from DNA. (Murakami, Kagaku, 46: 681-684, 1991).
[0006]
Furthermore, methylphosphonate derivatives in which the oxygen of the phosphate group of the nucleotide is substituted with a methyl group or phosphorothioate substituted with sulfur as a method of solving problems such as the lifetime, stability, and cellular uptake efficiency of antisense oligonucleotides. Modified antisense oligonucleotides such as type derivatives are known (Murakami, supra) and indeed these antisense oligonucleotides have been found to inhibit viral protein synthesis (Agris, CH et al., Biochemistry, 25 , 6268-6275, 1986).
[0007]
Based on this idea, Levy Y et al. Inhibited the growth of myeloma cell lines containing human IL-6 as a growth factor by inhibiting the translation of human IL-6 mRNA with an antisense oligonucleotide. (Levy Y et al., J. Clin. Invest., 88, 696-699, 1991).
However, there is no known antisense oligonucleotide derivative that significantly suppresses IL-6R expression in various cells expressing human IL-6R.
[0008]
[Problems to be solved by the invention]
Accordingly, the present invention seeks to provide antisense oligonucleotide derivatives that inhibit the expression of human IL-6R.
[0009]
[Means for Solving the Problems]
More specifically, the present invention includes an antisense oligonucleotide derivative for at least 9 consecutive nucleotide sequences including a nucleotide sequence of a portion that is likely to form a loop structure of mRNA encoding human IL-6R. The human IL-6R expression inhibitor is provided.
[0010]
[Specific explanation]
The present inventor uses commercially available computer software for free energy calculation (for example, Genetyx, Secondary Structure Search and Hairpin Loop-Stem Parts Search, etc., manufactured by Software Development Co., Ltd.) to free energy in human IL-6R mRNA. In addition, we searched for a base sequence that is likely to form a loop structure from the viewpoint of structure, and synthesized an antisense oligonucleotide derivative for the base sequence containing the loop structure, thereby suppressing the expression of human soluble IL-6R (sIL-6R) The present invention has been completed.
In a preferred embodiment of the present invention, an antisense oligonucleotide derivative for 9 to 30, more preferably 12 to 25 consecutive nucleotide sequences comprising a nucleotide sequence capable of forming a loop structure of mRNA encoding human IL-6R Is used.
[0011]
The term “antisense oligonucleotide” as used herein refers not only to 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 stably hybridized. There may be as few mismatches as possible.
The nucleotide sequence of human IL-6R cDNA is as follows (see, for example, JP-A-2-288,898 or Science, 241, 825-828 (1988)) (SEQ ID NO: 1).
[0012]
Among these, the base sequence of the antisense oligonucleotide derivative of the present invention can be appropriately selected from continuous base sequences that can form the loop structure found by the above method.
The part which can form the loop structure of mRNA here refers to a part where the intramolecular hydrogen bond of mRNA hardly exists and tends to be in a single-stranded state.
Specifically, it is possible to find a portion that tends to be in a single-stranded state even when the mRNA has a stable structure using the above-described computer software.
In the present invention, the portion capable of forming the loop structure is, for example, 640-685, 770-810, 1340-1375, 460-475, 535-560 and 925-960, in the base sequence shown in SEQ ID NO: 1. 815-840 vicinity is mentioned.
[0013]
Thus, the oligonucleotide of the present invention is an oligonucleotide consisting of at least 9 consecutive nucleotides in the above-mentioned region, for example.
According to one embodiment of the present invention, the expression-inhibiting oligonucleotide is a nucleotide sequence complementary to the base sequence of the codon encoding, for example, Ala at position 75 to Leu at position 81 in SEQ ID NO: 1, 5'-AGCCTCCTTCCCATGCCAGC-3 '(SEQ ID NO: 2).
[0014]
As an example of another oligonucleotide, a base sequence complementary to the base sequence of the codon encoding from Leu at position 127 to Glu at position 133, ie, 5′-CTCACAAACAACATTGCTGA-3 ′ (SEQ ID NO: 3) Having a base sequence complementary to the base sequence of the codon encoding from His at position 70 to Met at position 77, ie, having 5′-TGCCAGCCCATCTGCTGGGG-3 ′ (SEQ ID NO: 4), Having a base sequence complementary to the base sequence of the codon encoding Val from position 34 to Asp at position 41, ie, 5'-CTCCTGGCAGACTGGTCAGC-3 '(SEQ ID NO: 5);
[0015]
A nucleotide sequence complementary to the nucleotide sequence of the codon encoding Gln at position 118 to Lys at position 124, that is, one having 5'-TTCCGGAAGCAGGAGAGCTG-3 '(SEQ ID NO: 6) and Pro at position 164 Having a nucleotide sequence complementary to the nucleotide sequence of a codon encoding from Glu to Glu at position 170, ie, having 5′-TCCTGGGAATACTGGCACGG-3 ′ (SEQ ID NO: 7), Ala from position 75 to position 82 A nucleotide sequence complementary to the base sequence of the codon encoding up to Leu, ie, having 5′-GCAGCCTCCTTCCCATGCCA-3 ′ (SEQ ID NO: 9), coding from Trp at position 74 to Arg at position 80 And a nucleotide sequence complementary to the base sequence of the codon to be used, that is, one having 5′-CTCCTTCCCATGCCAGCCCA-3 ′ (SEQ ID NO: 10).
[0016]
When the oligonucleotide derivative used in the present invention is deoxyribonucleotide, each structure is as shown in Chemical Formula 1, but X is independently oxygen (O), sulfur (S), lower alkyl group or primary amine. Or any of a secondary amine may be sufficient. Y may independently be oxygen (O) or sulfur (S). B is selected from any one of adenine, guanine, thymine, or cytosine, and is mainly a complementary oligonucleotide of DNA or mRNA encoding human IL-6R. R is independently hydrogen, a dimethoxytrityl group or a lower alkyl group. n is 7-28.
[0017]
Preferred oligonucleotide derivatives include not only unmodified oligonucleotides but also modified oligonucleotides. Examples of such modifications include lower alkyl phosphonate modifications such as the aforementioned methyl phosphonate type or ethyl phosphonate type, and other phosphorothioate modifications or phosphoramidate modifications (see Chemical Formula 2).
[0018]
[Chemical 1]

[0019]
[Chemical 2]

[0020]
These oligonucleotide derivatives can be obtained by a conventional method as follows.
Oligonucleotides in which X and Y in Chemical Formula 1 are O are easily synthesized by a commercially available DNA synthesizer (for example, Applied Biosystems).
The synthesis method can be obtained by a solid phase synthesis method using phosphoramidite, a solid phase synthesis method using hydrogen phosphonate, or the like.
[0021]
For example, T. Atkinson, M. Smith, in Oligonucleotide Synthesis: A Practical Approach, ed. MJ Gait, IRL Press, 35-81 (1984); MH Caruthers, Science, 230, 281 (1985); A. Kume, M Fujii, M. Sekine, M. Hata, J. Org. Chem., 49, 2139 (1984); BC Froehler, M. Matteucci, Tetrahedron Lett., 27, 469 (1986); PJ Garegg, I. Lindh, T. Regberg, J. Stawinski, R. Stromberg, C. Henrichson, ibid., 27, 4051 (1986);
[0022]
BS Sproat, MJ Gait, in Oligonucleotide 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 MH Caruthers, Tetrahedron Lett., 21, 719-722 (1980); MD Matteucci and MH Caruthers, J. Am. Chem. Soc., 103, 3185-3191 (1981).
[0023]
A modified phosphate triester in which X is a lower alkoxy group can be obtained by a conventional method, for example, by treating an oligonucleotide obtained by chemical synthesis with a DMF / methanol / 2,6-lutidine solution of tosyl chloride. (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 such as phosphoramidite (MA Dorman, et. Al., Tetrahedron, 40, 95-102 (1984); KL Agarwal and F. Riftina, Nucleic Acids Res., 6, 3009-3024 (1979)).
[0024]
Phosphorothioate modified compounds in which X is S can be prepared by conventional methods such as solid phase synthesis using sulfur (CA Stein, et. Al., Nucleic Acids Res., 16, 3209-3221 (1988)) or tetraethylthiuram disulfide. And can be obtained by solid phase synthesis (H. Vu and BL Hirschbein, Tetrahedron Letters, 32, 3005-3008 (1991)).
A phosphorodithioate modified product in which X and Y are both S can be obtained by a solid phase synthesis method, for example, by converting bisamidite to thioamidite and allowing sulfur to act (WK-D. Brill, et. Al). , J. Am. Chem. Soc., 111, 2321-2322 (1989)).
[0025]
A phosphoramidate modified product in which X is a primary amine or a secondary amine can be obtained by a solid phase synthesis method by treating, for example, hydrogen phosphonate with a primary or secondary amine (B. Froehler, et. Al. Nucleic Acids Res., 16, 4831-4839 (1988)). Alternatively, amidites can be obtained by oxidation with tert-butyl hydroperoxide (H. Ozaki, et. Al., Tetrahedron Lett., 30, 5899-5902 (1989)).
[0026]
Purification and purity confirmation can be performed by high performance liquid chromatography or polyacrylamide gel electrophoresis. The molecular weight can be confirmed by Electrospray Ionization Mass Spectrometry or Fast Atom Bonbardment-Mass Spectrometry. The antisense oligonucleotide derivative of the present invention may have any synthesis method or origin as long as it has a sequence that hybridizes to the base sequence of DNA or mRNA encoding human IL-6R.
[0027]
The antisense oligonucleotide derivative of the present invention acts on human IL-6R-producing cells to bind to DNA or mRNA encoding human IL-6R, thereby inhibiting its transcription or translation, and human IL-6R As a result, the action of human IL-6 is suppressed. Examples of the action of human IL-6 suppressed by the antisense oligonucleotide derivative of the present invention include platelet increasing action, antibody production enhancing action, acute phase protein inducing action, tumor cell proliferation action, nerve cell differentiation action and the like. .
[0028]
Therefore, in diseases caused by these effects, for example, cancers such as renal cancer, myeloma, Rennert T lymphoma, Kaposi's sarcoma, autoimmune diseases such as rheumatoid arthritis, mesangial proliferative nephritis, psoriasis, cancer cachexia, infection The antisense oligonucleotide derivative of the present invention is considered to be effective in the treatment of endotoxin shock and the like.
The antisense oligonucleotide derivative of the present invention can be mixed with a suitable base that is inactive to them to prepare an external preparation such as a coating agent or a poultice.
[0029]
In addition, tablets, powders, granules, capsules, liposome capsules, injections with addition of excipients, isotonic agents, solubilizers, stabilizers, preservatives, soothing agents, etc. as necessary Furthermore, it can be made into a freeze-drying agent, such as a liquid preparation and a nasal drop. These can be prepared according to conventional methods.
The antisense oligonucleotide derivative of the present invention is applied directly to the affected area of the patient or is administered to the patient so that the affected area can be reached by administration into the blood vessel. Furthermore, an antisense encapsulating material that enhances durability and membrane permeability can also be used. For example, liposome, poly-L-lysine, lipid, cholesterol, lipofectin or derivatives thereof can be mentioned.
[0030]
The dosage of the antisense oligonucleotide derivative of the present invention can be appropriately adjusted according to the patient's condition and used in a preferable amount. For example, 0.1-100 mg / kg, preferably 0.1-50 mg / kg can be administered.
Hereinafter, the present invention will be described in detail in Examples.
[0031]
【Example】
Synthesis Example 1 Synthesis of 5′- AGCCTCCTTCCCATGCCAGC- 3 ′ (SEQ ID NO: 2) (phosphorothioate modified product) 5′-dimethoxytrityl-2′-deoxycytidine (1 μmol) dimethoxytrityl group in which 3′-hydroxyl group was bonded to the support After deprotection with acetic acid, the 5'-hydroxyl group was condensed with 5'-dimethoxytrityl-2'-deoxyguanosine β-cyanoethylphosphoamidite derivative with tetrazole, phosphorus was sulfurized with tetraethylthiuram disulfide, and unreacted 5 The '-hydroxyl group is acetylated with acetic anhydride and dimethylaminopyridine.
[0032]
Similarly, deprotection, condensation, sulfurization and acetylation are repeated. The final 5'-dimethoxytrityl-2'-deoxyadenosine β-cyanoethyl phosphoramidite derivative is condensed and sulfurized to give a 20-mer phosphorothioate modified product (the above steps are the DNA synthesis model 381A manufactured by Applied Biosystems). Was removed from the support with 2 ml of concentrated aqueous ammonia, the cyanoethyl group was removed from the phosphorus, and the protecting groups attached to adenine, guanine and cytosine were removed.
[0033]
The obtained 5'-dimethoxytrityl oligonucleotide phosphorothioate was kept unpurified or purified by high performance liquid chromatography or held in a cartridge column for synthetic DNA purification (for example, Oligopack SP manufactured by Nihon Millipore). Subsequently, 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 about 1.37 mg of the objective 5′-AGCCTCCTTCCCATGCCAGC-3 ′ (SEQ ID NO: 2) (phosphorothioate modified product).
[0034]
Synthesis Example 2 5'- CTCACAAACAACATTGCTGA -3 '(SEQ ID NO: 3) In the same manner as <br/> Synthesis Example 1 (phosphorothioate modifications), 5'-CTCACAAACAACATTGCTGA-3 object' (SEQ ID NO: 3) (phosphorothioate modified Body) about 0.65 mg is obtained.
[0035]
Synthesis Example 3 5'- TGCCAGCCCATCTGCTGGGG -3 '(SEQ ID NO: 4) In the same manner as <br/> Synthesis Example 1 (phosphorothioate benzoate modifications), 5'-TGCCAGCCCATCTGCTGGGG-3 object' (SEQ ID NO: 4) (phosphorothioates (Modified form) About 0.98 mg is obtained.
[0036]
Synthesis Example 4 5'- CTCCTGGCAGACTGGTCAGC -3 '(SEQ ID NO: 5) In the same manner as <br/> Synthesis Example 1 (phosphorothioate modifications), 5'-CTCCTGGCAGACTGGTCAGC-3 object' (SEQ ID NO: 5) (phosphorothioate modified Body) about 1.61 mg is obtained.
[0037]
Synthesis Example 5 5'- TTCCGGAAGCAGGAGAGCTG -3 '(SEQ ID NO: 6) In the same manner as <br/> Synthesis Example 1 (phosphorothioate modifications), 5'-TTCCGGAAGCAGGAGAGCTG-3 object' (SEQ ID NO: 6) (phosphorothioate modified Body) to obtain about 1.47 mg.
[0038]
Synthesis Example 6 5'- TCCTGGGAATACTGGCACGG -3 '(SEQ ID NO: 7) In the same manner as <br/> Synthesis Example 1 (phosphorothioate modifications), 5'-TCCTGGGAATACTGGCACGG-3 object' (SEQ ID NO: 7) (phosphorothioate modified Body) to obtain about 1.64 mg.
[0039]
Synthesis Example 7 5'- GCAGCCTCCTTCCCATGCCA -3 '(SEQ ID NO: 9) In the same manner as <br/> Synthesis Example 1 (phosphorothioate modifications), 5'-GCAGCCTCCTTCCCATGCCA-3 object' (SEQ ID NO: 9) (phosphorothioate modified Body) about 2.47 mg is obtained.
[0040]
Synthesis Example 8 5'- CTCCTTCCCATGCCAGCCCA -3 '(SEQ ID NO: 10) In the same manner as <br/> Synthesis Example 1 (phosphorothioate modifications), 5'-CTCCTTCCCATGCCAGCCCA-3 object' (SEQ ID NO: 10) (phosphorothioate modified Body) about 2.06 mg is obtained.
[0041]
Reference Example 1 Synthesis of 5'- CCCCAGCAGATGGGCTGGCA- 3 '(SEQ ID NO: 8) (phosphorothioate modified: sense sequence of SEQ ID NO: 4) 5'-CCCCAGCAGATGGGCTGGCA-3' (sequence) Number: 8) About 0.53 mg (modified phosphorothioate) is obtained.
[0042]
Reference Example 2 Synthesis of 5'- GCTGGCATGGGAAGGAGGCT- 3 '(SEQ ID NO: 11) (phosphorothioate modified: sense sequence of SEQ ID NO: 2) In the same manner as in Synthesis Example 1, the target 5'-GCTGGCATGGGAAGGAGGCT-3' ( SEQ ID NO: 11) (phosphorothioate modified form) about 1.91 mg is obtained.
[0043]
Experimental Example 1 Expression suppression effect of human soluble IL-6R (sIL-6R) (1) COH. Production of SR344 cells pBSF2R. 236 (Science, 241, 825-828 (1988)) was cleaved with SphI, and a 1205 bp IL-6R cDNA fragment was inserted into mp18 (Amersham). sIL-6R cDNA was prepared using an in vitro mutagenesis system (manufactured by Amersham) by preparing a synthetic oligonucleotide of 5'-ATATTCTAGAGAGCTTCT-3 '. As a result, termination cordon was 345th in the amino acid sequence.
[0044]
The dhfr-cDNA was inserted into Pvu II site of plasmid pECE (Cell, 45, 721-735 (1986)) to prepare plasmid pECEdhfr. The HindIII-SalI fragment of sIL-6R was inserted into plasmid pECEdhfr to prepare a soluble IL-6R expression vector plasmid pECEdhfr344.
[0045]
pECEdhfr344 was introduced into dhfr-CHO cells DXB-11 (Pro. Natl. Acad. Sci. USA, 77, 4216-4220 (1980)) by the calcium phosphate method and amplified with MTX. Finally, 200 nMMTX resistant sIL-6R-producing CHO cells (CHO. SR344) were prepared (J. Biochem., 108, 673-676 (1990)). Normal cell culture was performed in IMDM medium (Gibco) containing 5% FCS (Xavier Investments) and 200 nMMTX.
[0046]
(2) CHO. Effect of IL-6R antisense oligonucleotide on sIL-6R production in SR344 cells CHO. SR344 cells were detached from the culture dish with trypsin-EDTA (manufactured by Gibco), washed with the culture medium, further washed with serum-free medium nonserum (manufactured by Nippon Zenyaku Kogyo), and suspended in serum-free medium nonserum containing 200 nMMTX. . In a 96-well culture plate, CHO. 100 μl of SR344 cell suspension (5 × 10 ⁴ cells / ml) was added with 100 μl of 2 μM IL-6R antisense oligonucleotide and cultured in an incubator at 37 ° C. and 5% CO ₂ .
[0047]
After culturing for 24 hours, the amount of soluble IL-6R in the culture supernatant was measured by a sandwich ELISA method using a mouse anti-IL-6R monoclonal antibody (MT18) (JP-A-2-288898) and a rabbit anti-IL-6R polyclonal antibody. .
In addition, it measured using the sense oligonucleotide derivative (sequence number: 8) with respect to sequence number: 4 or the sense oligonucleotide derivative (sequence number: 11) with respect to sequence number: 2 as control (control).
Human IL-6R antisense oligonucleotide showed an inhibitory effect on the expression of soluble IL-6R (FIGS. 1 and 2).
[0048]
[Sequence Listing]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[Brief description of the drawings]
FIG. 1 is a graph showing that the antisense oligonucleotide derivatives (SEQ ID NOs: 2 and 3) of the present invention 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)

Antisense oligonucleotide derivatives against at least 9 consecutive nucleotide sequences including a nucleotide sequence of a portion capable of forming a loop structure of mRNA encoding human interleukin-6 receptor (human IL-6R) (however, oligonucleotide derivatives And a human IL-6R expression inhibitor comprising as an active ingredient ( excluding an oligonucleotide derivative in which at least one of phosphodiester bonds between nucleotides constituting the derivative is substituted with a carbamate bond or a thiocarbamate bond) . The expression inhibitor according to claim 1, comprising an antisense oligonucleotide derivative for the base sequence of a part capable of forming a loop structure in the base sequence of SEQ ID NO: 1. The expression inhibitor according to claim 2, wherein the base sequence is AGCCTCCTTCCCATGCCAGC (SEQ ID NO: 2). The expression inhibitor according to claim 2, wherein the base sequence is CTCACAAACAACATTGCTGA (SEQ ID NO: 3).

Images