JP2005314381A - Prophylactic/therapeutic/ameliorating agent for proliferative nephropathy - Google Patents

Prophylactic/therapeutic/ameliorating agent for proliferative nephropathy Download PDF

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JP2005314381A
JP2005314381A JP2005090496A JP2005090496A JP2005314381A JP 2005314381 A JP2005314381 A JP 2005314381A JP 2005090496 A JP2005090496 A JP 2005090496A JP 2005090496 A JP2005090496 A JP 2005090496A JP 2005314381 A JP2005314381 A JP 2005314381A
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decoy
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Masayoshi Aso
Shiro Jimi
Ryuichi Morishita
竜一 森下
至郎 自見
雅是 麻生
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Anges Mg Inc
アンジェスMg株式会社
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Abstract

PROBLEM TO BE SOLVED: To obtain a medicament useful for a therapeutic agent, and the like, for proliferative nephropathy, especially nephritis (diabetic nephritis or hypertensive nephritis).
SOLUTION: This invention relates to the medicament for prophylactic/therapeutic/ameliorating agent for proliferative nephropathy, comprising a decoy type nucleic acid medicine which inhibits coupling of a transcription factor to a coupling point, and is selected from decoy of NF-κB, decoy of STAT-6, decoy of AP-1, decoy of Ets and decoy of E2F. The medicament is suitable for prophylaxis/therapy/amelioration of nephritis (diabetic nephritis or hypertensive nephritis), glomerulosclerosis or albuminuria.
COPYRIGHT: (C)2006,JPO&NCIPI

Description

本発明は、デコイ(おとり)型核酸医薬を有効成分とする、腎症、糸球体硬化症又はアルブミン尿症等の増殖性腎疾患の予防・治療・改善剤に関する。 The present invention, as an active ingredient decoy type nucleic acid drugs, nephropathy, relates to the prevention, treatment and ameliorating agent for glomerular sclerosis or albuminuria like proliferative renal disease.

現在、日本の糖尿病患者は約700万人存在し、さらに潜在的糖尿病予備群は推定2000万人以上とされている。 Currently, Japan's diabetes patients is present about 700 million people, further potential diabetes preliminary group are an estimated 20 million or more people. 生活習慣病と位置付けされる糖尿病の原因は多彩で、食生活をはじめ、遺伝子、内分泌、炎症などの多くの要因が絡み、複雑な相互関係の上に形づくられている。 Cause of diabetes, which is positioned and lifestyle-related diseases is a versatile, including diet, genetic, endocrine, a number of factors, such as inflammation tangles, are shaped on top of the complex interrelationships.

糖尿病は慢性疾患で、その合併症性には細血管症、腎症、網膜症、神経症などがあり、患者個人としても多大な身体的、精神的な負荷があるが、医療福祉の面でも、現在から将来への日本社会が抱える社会的疾患へと進展している。 Diabetes is a chronic disease, its complications of the fine vasculopathy, nephropathy, retinopathy, include neurosis, also great physical as individual patients, there is a mental load, in terms of medical welfare , progress has been made to the social disease that Japanese society is facing from the current to the future. 最近の日本透析医学会の調査では、新規透析患者の内、糖尿病患者数は約35%に上っている。 In a recent Japanese Society for Dialysis Therapy of investigation, of the new dialysis patients, diabetes number of patients has risen to about 35 percent.

合併症性の内、糖尿病性腎症は、糸球体基底膜の肥厚、メサンジュウム細胞の増殖とKimmelstiel-Wilson 結節を代表とする線維化など細胞外基質の拡大を伴い、これら病理学的変化が糖尿病性腎症の直接的増悪因子となっている。 Among complications of diabetic nephropathy, thickened glomerular basement membrane, with the expansion of the extracellular matrix such as fibrosis typified proliferation and Kimmelstiel-Wilson nodules Mesanjuumu cells, these pathological changes diabetes and it has a direct aggravating factors of sexual nephropathy.

この糖尿病性腎症の病期は第1期から第5期に分類することができ、下記のとおり、それぞれの病期に応じた治療法が知られている。 This stage of diabetic nephropathy can be classified into the fifth phase from the first phase, as follows, treatment method is known according to the respective stage.

第1期(腎症前期):糸球体の中では高血圧になっていると考えられるが、現在の一般的な検査方法で検出できるような腎障害が認められない時期。 Phase 1 (nephropathy the previous fiscal year): It is considered that has become high blood pressure in the glomeruli, not observed renal disorders such as can be detected by the current general inspection method time. 厳格な血糖コントロールが有効。 Tight glycemic control is enabled.

第2期(早期腎症):精密な尿検査でわずかな蛋白尿(微量アルブミン尿)が認められる時期。 The second phase (early nephropathy): slight proteinuria (microalbuminuria) in precise urine test period for which it is recognized. 全身の血圧が上昇し始める。 Systemic blood pressure starts to rise. 厳格な血糖コントロールおよび血圧の管理が有効(血圧は130/85mmHg程度に)。 Effective management of strict glycemic control and blood pressure (blood pressure about 130/85 mmHg). アンギオテンシン変換酵素阻害薬が有用。 Useful angiotensin converting enzyme inhibitors.

第3期(顕性腎症):持続性蛋白尿(簡易蛋白尿検査で異常がわかる)が認められる時期(前期)。 Phase 3 (overt nephropathy): persistent proteinuria (abnormal is found in the simple proteinuria inspection) period for which it is recognized (the previous fiscal year). さらに進行すると、糸球体機能(老廃物排泄機能)が低下する(後期)。 More advanced glomerular function (waste excretory function) is reduced (late). 高血圧を伴いやすい。 Easy accompanied by high blood pressure. 厳格な血糖コントロールおよび高血圧の治療(血圧は130/85mmHg程度に)とともに食事中の塩分・蛋白質量を制限する(標準体重1キログラムあたり食事蛋白0.8グラム程度)必要がある。 Strict treatment of glycemic control and hypertension (blood pressure about 130/85 mmHg) is to limit the salt-protein content in the diet (standard weight dietary protein 0.8 g approximately per kilogram) required with. アンギオテンシン変換酵素阻害薬が有用。 Useful angiotensin converting enzyme inhibitors. この時期になると腎症の進行を完全に防止することは困難となる。 The progress of the time to become and nephropathy be prevented completely is difficult.

第4期(腎不全期):腎機能障害のため一般的な血液検査で異常が認められる時期。 Phase 4 (renal failure stage): renal function common blood time when the abnormality is observed in the inspection for the failure. 尿蛋白も増加し血液中の蛋白質が低下する(ネフローゼ症候群と呼ばれる)ことが多い。 Urinary protein also (known as nephrotic syndrome) increased protein decreases in the blood often. 降圧療法(140−150/90−95mmHg程度に)、塩分制限、低蛋白食が必要。 Antihypertensive therapy (about 140-150 / 90-95mmHg), salt restriction, requires low protein diet. 過激な運動は不可。 Extreme exercise is not allowed. 透析療法導入の準備も必要となる。 Preparation of dialysis therapy introduction is also required.

第5期(透析療法期):腎機能が廃絶し、慢性血液透析療法に導入された時期。 5th (dialysis treatment phase): timing of renal function and elimination, was introduced in chronic hemodialysis therapy.

糖尿病性腎症は、生活習慣の変化等を原因とするものと考えられ、腎不全により透析導入された患者の第一位の原因疾患ともなっており(2000年度)、より効果の高い治療法が求められている。 Diabetic nephropathy, believed to be caused by changes in lifestyle, such as, by renal failure has become both cause disease in the first place of a patient who has been introduced dialysis (fiscal 2000), more effective treatments It has been demanded.

非特許文献1では、組織の炎症性変化に起因する腎炎の治療にNF-κBのデコイ化合物である合成オリゴヌクレオチド(ODN)を使用したことが開示されているが、増殖性変化による疾患の治療についての開示はない。 In Non-Patent Document 1, although the treatment of nephritis caused by inflammatory changes in the tissue using synthetic oligonucleotide is decoy compound of NF-κB (ODN) have been disclosed, the treatment of a disease caused by proliferative changes There is no disclosure about.

本発明は、増殖性変化による増殖性腎疾患の予防・治療・改善剤を提供することを課題とする。 The present invention aims to provide a prevention, treatment, and ameliorating agent for proliferative kidney disease due to proliferative changes.

本発明者らは、2型糖尿病モデル動物として知られ、ヒト糖尿病性腎症の腎病変と酷似した病変ができる唯一のモデルとして知られるOtsuka-Long-Evance-Tokushima-Fatty(OLETF)ラットを用い、すでに腎病変形成期に入った動物の腎臓にデコイODNをトランスフェクトすることで、各種増殖因子やレセプターの遺伝子発現を抑制できることを見出し、本発明を完成した。 The present inventors have found that known as type 2 diabetes model animal, using Otsuka-Long-Evance-Tokushima-Fatty (OLETF) rats known as the only model that can lesions very similar to the renal pathology of human diabetic nephropathy the decoy ODN on animal kidney entering already renal lesion formation stage to transfect found that can suppress various gene expression of growth factors and receptors, and have completed the present invention.

即ち本発明は、課題の解決手段として、転写調節因子の結合部位への結合を阻害するデコイ(おとり)型核酸医薬(以下「デコイ」という)を有効成分とする、増殖性腎疾患の予防・治療・改善剤を提供するものである。 The present invention provides a solution to the problems, and transcriptional regulation decoy to inhibit binding to the binding site (decoy) factors type nucleic acid drugs (hereinafter "decoy" hereinafter) as an active ingredient, and prevention of proliferative kidney disease it is intended to provide a therapeutic and improving agents.

本発明で用いる「デコイ」又は「デコイ化合物」は、NF-κB、STAT-6、AP-1、Ets及びE2F等が結合する染色体上の部位、又はNF-κB、STAT-6、AP-1、Ets及びE2F等に制御される遺伝子の他の転写調節因子が結合する染色体上の部位(標的結合部位)に結合し、NF-κB、STAT-6、AP-1、Ets及びE2F等と、これらの標的結合部位への結合について拮抗する化合物をいう。 Used in the present invention "decoy" or "decoy compound", NF-κB, STAT-6, AP-1, a site on a chromosome that Ets and E2F etc. are bound, or NF-κB, STAT-6, AP-1 , binds to a site on chromosome other transcription regulatory factor for a gene controlled by Ets and E2F like are bound (the target binding site), and NF-κB, STAT-6, AP-1, Ets and E2F like, It refers to a compound which antagonizes the binding of these target binding site. 代表的なデコイ又はデコイ化合物は、核酸及びその類似体である。 Exemplary decoy or the decoy compound includes a nucleic acid and analogs thereof.

本発明の増殖性変化による増殖性腎疾患の予防・治療・改善剤は、増殖性変化による増殖性腎疾患の治療等において有効である。 Prevention, treatment and ameliorating agent for proliferative kidney disease due to proliferative changes in the present invention is effective in treating such a proliferative renal disease due to proliferative changes.

本発明の増殖性腎疾患の予防・治療・改善剤(以下「増殖性腎疾患用の薬剤」という)は、転写調節因子の結合部位への結合を阻害するデコイを有効成分とし、必要に応じて薬学的に許容できる担体を含むものである。 Prevention, treatment and ameliorating agent for proliferative renal diseases of the present invention (hereinafter referred to as "agents for proliferative renal disease") is an active ingredient a decoy to inhibit binding to the binding site of the transcriptional regulators, if necessary it is intended to include a pharmaceutically acceptable carrier Te.

デコイは、WO02/066070及びUS2002-0052333 A1等に開示されているものを用いることができ、中でも下記のデコイから選ばれる少なくとも1つのものが好ましい。 Decoy, can be used one disclosed in WO02 / 066070 and US2002-0052333 A1, etc., at least one is preferable selected from among others the following decoy.

NF-κBのデコイ(配列番号2)(WO02/066070参照) Decoy of NF-κB (SEQ ID NO: 2) (see WO02 / 066070)
STAT-6のデコイ(配列番号4)(WO02/066070参照) STAT-6 decoy (SEQ ID NO: 4) (see WO02 / 066070)
AP-1のデコイ(配列番号5)(WO02/066070参照) AP-1 decoy (SEQ ID NO: 5) (see WO02 / 066070)
Etsのデコイ(配列番号6)(WO02/066070参照) Ets decoy (SEQ ID NO: 6) (see WO02 / 066070)
E2Fのデコイ(配列番号7と配列番号8は、ずれて2本鎖を形成している。)(US2002-0052333 A1参照) Decoy E2F (SEQ ID NO: 7 and SEQ ID NO: 8 forms a double strand displaced.) (See US2002-0052333 A1)
デコイとしては、上記のデコイの相補体を含むオリゴヌクレオチド、これらの変異体、これらを分子内に含む化合物も用いることができる。 The decoy, an oligonucleotide containing a complement of the decoy, these mutants can also be used compounds containing these in the molecule. オリゴヌクレオチドは、DNAでもRNAでもよく、或いはそのオリゴヌクレオチド内に核酸修飾体及び/又は擬核酸を含むものであってもよい。 Oligonucleotides, DNA even better even RNA, or may comprise a nucleic acid modifications and / or 擬核 acids in the oligonucleotide.

オリゴヌクレオチドには、リン酸ジエステル結合部の酸素原子をイオウ原子で置換したチオリン酸ジエステル結合を持つオリゴヌクレオチド(S−オリゴ)、リン酸ジエステル結合を電荷を持たないメチルホスフェート基で置換したオリゴヌクレオチド等の生体内で分解を受けにくくするために改変されたものも含まれる。 The oligonucleotides, oligonucleotides with phosphorothioate linkages substituted phosphodiester bonds unit oxygen atom with a sulfur atom (S- oligos), oligonucleotides obtained by replacing the phosphodiester bond with methyl phosphate group with no charge also include those that have been modified in order to in vivo less susceptible to degradation and the like.

オリゴヌクレオチド、その変異体又はこれらを分子内に含む化合物は、1本鎖でも2本鎖でもよく、線状でも環状でもよい。 Oligonucleotides, variant or compounds containing them therein molecule may be single-stranded or double-stranded, may be cyclic or linear. 変異体とは、上記配列の一部が変異、置換、挿入、又は欠失しているもので、NF-κB等又はNF-κB等に制御される遺伝子のその他の転写調節因子が結合する核酸結合部位と特異的に拮抗する能力を有する核酸を意味する。 And variants, some of the sequence variations, substitutions, insertions, or those lacking, other transcription regulatory factor for a gene controlled by NF-[kappa] B, etc. or NF-[kappa] B and the like that bind nucleic acids means a nucleic acid having a binding site that specifically ability to antagonize.

デコイとしては、特にNF-κBのデコイが好ましい。 The decoy, especially decoy NF-[kappa] B is preferred. NF-κBは炎症・免疫、細胞増殖、癌化に関与する転写因子である。 NF-[kappa] B inflammation-immune cell proliferation, a transcription factor involved in carcinogenesis. NF-κBは、細胞外からのシグナルに応答すると、その阻害分子であるI-κBが分解され、遊離する。 NF-[kappa] B, when responding to signals from extracellular and I-[kappa] B is the inhibitory molecules are decomposed to free. 遊離したI-κBは、細胞質から核へと移行して行き、種々のDNA認識部位に結合することにより、細胞増殖や炎症に関係するインターロイキン(IL)-1、IL-6、IL-8、GM-CSF、TNFなどのサイトカイン、ケモカイン、インターフェロン及び種々の接着因子(E-selectin、VCAM-1、ICAM-1)などの発現のための重要な媒介物として働く。 Liberated I-[kappa] B is gradually shifted from the cytoplasm to the nucleus, by binding to various DNA recognition sites, interleukin (IL) -1 related to cell proliferation and inflammation, IL-6, IL-8 acts as GM-CSF, an important vehicle for the expression of such cytokines, such as TNF, chemokines, interferons, and various adhesion factors (E-selectin, VCAM-1, ICAM-1). NF-κBのデコイ化合物である合成オリゴヌクレオチド(ODN)は、NF-κBに高い親和性がある合成2本鎖DNAを有しており、目的遺伝子のプロモーター領域への核因子の結合を阻害することにより、遺伝子トランス活性化を阻止する。 A decoy compound of NF-[kappa] B synthetic oligonucleotides (ODN) has a synthetic double stranded DNA with high affinity to the NF-[kappa] B, inhibiting the binding of nuclear factors to the promoter region of the gene of interest it allows to block gene transactivation.

NF-κBのデコイは、塩基配列中に配列番号1を含むものが好ましく、特に配列番号2(SEQ1)及び配列番号3(SEQ2)からなる二重鎖核酸(NF-κBデコイオリゴヌクレオチド)(US6,262,033参照)が好ましい。 Decoy NF-[kappa] B is preferably those comprising SEQ ID NO: 1 in the nucleotide sequence, in particular SEQ ID NO: 2 (SEQ1) and double-stranded nucleic acid (NF-[kappa] B decoy oligonucleotide) consisting of SEQ ID NO: 3 (SEQ2) (US6, 262,033 reference) is preferred.

デコイは、WO02/066070に記載された方法により、製造することができる。 Decoy, by the method described in WO02 / 066.07 thousand, can be produced.

本発明の増殖性腎疾患用の薬剤は、デコイ単独、又は必要に応じて薬学的に許容できる担体とデコイとを用い、投与方法に応じた剤型にすることができ、例えば、WO02/066070に記載された投与方法(経口投与又は非経口投与)及び剤型にすることができる。 Agents for proliferative renal diseases of the present invention, decoy alone or with a pharmaceutically acceptable carrier and decoy if necessary, can be dosage forms according to the administration method, for example, WO02 / 066070 it can be manner of administration (oral or parenteral administration) and the dosage form described.

経口投与する場合の剤型は、錠剤、丸剤、糖衣剤、カプセル剤、液剤、シロップ剤等を挙げることができる。 Dosage forms for oral administration can include tablets, pills, dragees, capsules, solutions, syrups and the like.

非経口投与法としては、局所投与、皮膚塗布投与、動脈内投与、筋肉内投与、皮下投与、静脈内投与、鼻孔投与等を挙げることができ、注射剤を含む液剤にすることが好ましい。 Parenteral administration, topical administration, dermal application administration, intraarterial administration, intramuscular administration, subcutaneous administration, intravenous administration includes, for example, nasal administration and the like, it is preferable that the liquid containing the injection agent.

注射剤は、デコイをハンクスの溶液、リンゲル溶液、緩衝化生理食塩水等の生理学的に適合する緩衝液中に溶解乃至は懸濁させて得ることができる。 Injectables can decoys Hank's solution, Ringer's solution, dissolving or during physiologically compatible buffers such as buffered saline may be suspended.

注射剤を水性注射懸濁物にする場合には、懸濁物の粘度を安定させる物質、例えば、カルボキシメチルセルロースナトリウム、ソルビトール、デキストランを含有させることができる。 Injections when the aqueous injection suspensions may contain substances which stabilize the viscosity of the suspension, for example, may contain sodium carboxymethyl cellulose, sorbitol, dextran. また、注射剤は、ゴマ油のような脂肪酸、オレイン酸エチル又はトリグリセリドのような合成脂肪酸エステル、リポソームを含む油状注射剤懸濁物にすることができる。 The injection can also be prepared, fatty acids such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, can be oily injection suspensions containing liposomes.

本発明の増殖性腎疾患用の薬剤を注射剤とするときは、薬剤の注射と超音波照射を併用することが治療効果等を高める観点から望ましい。 When the medicament for proliferative renal diseases of the present invention an injection, it is desirable from the viewpoint of enhancing the therapeutic effect, such as a combination of injection and ultrasonic irradiation of the drug.

具体的には、NF-κB等を超音波心臓検査コントラスト剤Optison(商標名;米国Mallinkrodt社製の造影剤)と混合したものを循環血液中に投与し、目的臓器である腎臓にのみに循環血液側から導入するため、腎臓に超音波照射(例えば、実施例に記載の超音波遺伝子導入装置を使用する)を行い、腎糸球体支持細胞であるメサンジュウム細胞内に特異的にデコイODNをトランスフェクトする方法を適用する。 Specifically, the NF-[kappa] B, etc. echocardiographic contrast agent Optison; administering a mixture with (trade name US Mallinkrodt Co. contrast agent) in the circulating blood, circulating only in the kidney is a target organ to introduce the blood side, ultrasonic irradiation to the kidney (e.g., using ultrasound transgenic apparatus of example) performs a trans specifically decoy ODN into Mesanjuumu the cell is a renal glomeruli feeder cells the application of the method to effects. なお、腎臓への超音波照射は、開腹手術後、腎臓に直接照射してもよいし、開腹することなく体外から腹部に照射してもよい。 Note that ultrasonic irradiation to the kidney after laparotomy, may be directly irradiated to the kidney, it may be irradiated to the abdomen from outside without laparotomy.

超音波照射の条件は、実施例に記載の超音波遺伝子導入装置を使用するとき、1〜10W/cm 2 、好ましくは2〜5W/cm 2で、30秒〜30分、好ましくは5〜25分でよい。 Conditions of the ultrasonic irradiation, when using ultrasound transgenic apparatus of example, 1 to 10 W / cm 2, preferably at 2~5W / cm 2, 30 seconds to 30 minutes, preferably 5 to 25 it may be a minute. このときduty比を10〜100%、好ましくは50〜100%にすることができる。 10-100% duty ratio at this time, can preferably be 50-100%.

注射と超音波照射は、 Injection and the ultrasonic irradiation,
(1)注射をした後に超音波照射を開始する方法、 (1) how to start the ultrasonic irradiation after injection,
(2)超音波照射と注射を同時にする方法、 (2) a method of injecting at the same time as the ultrasonic irradiation,
(3)超音波照射をしながら注射をする方法、 (3) a method of injection while the ultrasonic irradiation,
のいずれかの方法を適用することができるが、本発明においては、治療効果等を高める観点から、特に(1)、(2)の方法が好ましい。 Can be applied to any of the methods, in the present invention, in view of enhancing the therapeutic effect and the like, in particular (1), preferably the method of (2).

本発明の増殖性腎疾患用の薬剤の投与量及び投与回数は、投与方法、治療期間、年齢、体重等により異なるが、注射剤では、有効成分であるデコイ量に換算して、通常1mg〜1g/日を1日1回又は複数回に分けて投与する。 The amount and frequency of administration of agents for proliferative renal disease of the invention, the method of administration, duration of treatment, age, varies body weight, etc., in the injection, in terms of decoy amount as an active ingredient, typically 1mg~ the 1 g / day administered in single or multiple doses per day.

本発明の増殖性腎疾患用の薬剤は、腎症(例えば、糖尿病性腎症又は高血圧性腎症)、糸球体硬化症又はアルブミン尿症の予防剤、治療剤及び改善剤として好適である。 Agents for proliferative renal disease of the invention, nephropathy (such as diabetic nephropathy or hypertensive nephropathy), prevention agent of glomerulosclerosis or albuminuria, is suitable as a therapeutic agent and improving agent.

実施例1〔灌流法による腎臓へのFITC標識NF-κB デコイ導入〕 Example 1 [FITC-labeled NF-[kappa] B decoy introduction into kidney by perfusion]
ラットは、2匹の雌のウイスター(リタイヤ)1年齢を用いた。 The rats, two females of Wistar (retired) was used as the 1-year-old. 開腹後、左腎の腎動脈を分離し、腎動脈へサーフローでカニュレーションを行い、ヘパリン液で腎臓を灌流した後、FITC標識NF-κB デコイ200μgとOptison(商標名)(10%)及び生理食塩水(合計で500μl)を混和した本発明の薬剤を注入した。 After laparotomy, separating the left renal renal arteries, performed cannulation in Safuro into the renal arteries, after perfusing the kidney with heparin solution, FITC-labeled NF-[kappa] B decoy 200μg and Optison (TM) (10%) and physiological agents of the invention admixed saline (500 [mu] l in total) was injected.

薬剤の注入直後に超音波照射を開始した。 It began ultrasonic irradiation immediately after infusion of the drug. 超音波の照射条件は、超音波遺伝子導入装置(Sonitoron 1000、ネッパジーン,千葉)で連続(100%)、1W/cm 2 、10分間とした。 Irradiation conditions of ultrasonic wave, ultrasonic gene introduction apparatus (Sonitoron 1000, Neppajin, Chiba) continuous (100%), was set to 1W / cm 2, 10 minutes.

腎臓組織は、超音波照射直後、4時間後、4日後に採取し、腎組織横断面をコンパウンドで包埋後、凍結し、DAPI(4',6-ジアミジノ-2-フェニルインドール)により核染し、蛍光顕微鏡下で観察した。 Kidney tissue, after ultrasonic irradiation, after 4 hours, were taken after 4 days after embedding the renal tissue cross section with compound, frozen, DAPI (4 ', 6- diamidino-2-phenylindole) by Kakusome and it was observed under a fluorescence microscope. 実験終了時、両腎とも梗塞は起こしていなかった。 At the end of the experiment, did not cause is infarction in both kidneys.

FITCの蛍光は、超音波照射を受けていない部分(写真左側)に比べ、右側では蛍光は強い。 FITC fluorescence as compared to the portion (photograph on the left) that is not subjected to ultrasonic irradiation, in the right side fluorescence is strong. 超音波照射腎半面にのみ認められ、非照射の裏半面にはほとんど蛍光はなかった(図1)。 Found only in the ultrasonic irradiation kidney half, the back half of the non-irradiated was little fluorescence (FIG. 1).

蛍光は腎組織内細動脈内には認められず、一部尿細管上皮細胞内に認められたが、主に糸球体細胞内に認められた(図2)。 Fluorescence was not observed in the renal tissue arterioles, was observed in a part tubular intraepithelial cells were mainly observed in the glomerular cells (Figure 2).

4時間後では、糸球体内に蛍光は残存するものの、照射直後より減少していた。 In 4 hours, although fluorescence remaining in the glomeruli was reduced immediately after irradiation. 逆に尿細管内の方が優位となり、上皮細胞内に顆粒状に分布していた(図3)。 Conversely becomes dominant it is more in the tubules, were distributed granular in epithelial cells (Fig. 3).

4日後でも糸球体内に蛍光はなお残存し、尿細管上皮細胞内では顆粒状に認められた。 Fluorescence still remains in the glomeruli even after 4 days was observed in the granular within tubular epithelial cells. 反対側腎臓には、直後および4時間、4日後ともほとんど蛍光は認められなかった。 On the opposite side of the kidney, immediately after and 4 hours, most fluorescence was not observed and after four days.

実施例2〔静注法による腎臓へのFITC標識NF-κBデコイ導入〕 Example 2 [FITC-labeled NF-[kappa] B decoy introduction into kidney by intravenous Method]
体重及び腎臓の大きさによる影響を見る目的で、マウス及びラットを用い、FITC標識NF-κB デコイの腎臓内導入効率を検討した。 In order to see the effect of the size of the body weight and kidney, using mice and rats, it was examined intrarenal introduction efficiency of FITC-labeled NF-[kappa] B decoy.

マウスは3匹のWBB6F1+/+で、雄、体重約25g、年齢2ヶ月齢を用いた。 Mice Three of WBB6F1 + / + and was used males, weighing about 25 g, the age 2 months. 麻酔下でマウスを開腹後、左腎を露出させ、FITC-NF-κBデコイ400μg/100μに超音波コントラスト剤Optison(商標名)100μlを超音波照射直前に混和した本発明の薬剤を静注した。 After laparotomy the mouse under anesthesia to expose the left kidney, was injected intravenously with agents of the invention admixed with FITC-NF-κB decoy 400 [mu] g / 100 microns in ultrasound contrast agents Optison (trade name) 100 [mu] l to ultrasonic irradiation immediately before .

薬剤の注入直後に超音波照射を開始した。 It began ultrasonic irradiation immediately after infusion of the drug. 腎への超音波の照射条件は、超音波遺伝子導入装置で2W/cm 2 、duty比を50%、100%、照射時間5分間、10分間とした。 Ultrasonic irradiation condition of the kidneys, 50% 2W / cm 2, duty ratio ultrasonic gene introduction apparatus, 100%, irradiation time of 5 minutes, was 10 minutes.

マウスの場合、照射時間を一定にし(10分間)、duty比を減少させると、腎細胞内導入率は減少した。 For mice, the irradiation time constant (10 min), reducing the duty ratio, the renal cell uptake efficiency was decreased. 超音波照射プローブを当てた場所にのみ、限局性に蛍光が見られ、その深さは皮質内にとどまっていた。 Only the location irradiated with ultrasonic irradiation probe, fluorescence was observed in localized, its depth was limited in the cortex. 蛍光は糸球体と尿細管内にも認められた(図4)。 Fluorescence was also observed in the glomeruli and tubules (Figure 4).

duty比を減少させると、細胞内導入率も減少した。 Reducing the duty ratio, also decreased cellular uptake efficiency. 逆に、出血斑などは連続照射より少なくなった。 On the contrary, such as bleeding spots was less than continuous irradiation. 超音波照射をしていない反対側の腎臓を見ると、糸球体内細胞には蛍光は見られないものの、尿細管上皮細胞内に顆粒状に蛍光が観察された。 Looking at the opposite side of the kidney is not the ultrasonic irradiation, although the glomerular intracellular fluorescence is not observed, fluorescence was observed in the granular in tubular epithelial cells.

一方、ラット(雄のウイスター2匹、8週齢、体重約300g)では、FITC-NF-κB デコイ600μgとOptison(商標名)(300μl)を投与直前に混和後、静注し、duty比50%、100%、2W/cm 2 、照射時間5分間、10分間、20分間の超音波照射を行い、プローブの径が5mm程度で小さいため、プローブをゆっくり動かしながら腎全体に照射した。 On the other hand, rats (male 2 rats Wistar the, 8 weeks old, weighing about 300 g) at, FITC-NF-κB decoy 600μg and Optison (TM) after mixing just prior to administration of (300 [mu] l), injected intravenously, duty ratio 50 %, 100%, 2W / cm 2, the irradiation time of 5 minutes, 10 minutes, subjected to ultrasonic irradiation for 20 minutes, since the diameter of the probe is smaller in the order of 5 mm, it was irradiated on the entire kidney gently rock the probe.

マウスでの最適条件(2W/cm 2 、duty比50%、照射時間10分間)では、ラットの導入効率は明らかに悪かった。 Optimal conditions in mice (2W / cm 2, duty ratio of 50%, irradiation time 10 min), the transfer efficiency of the rats was obviously poor. マウスとほぼ同一の遺伝子導入効率は、ラットでは2W/cm 2 、duty比50%、照射時間20分間だった(図5,6)。 Almost the same gene transfer efficiency as mice, in rats was 2W / cm 2, duty ratio of 50%, irradiation time 20 min (Fig. 5,6).

FITC-NF-κB デコイ導入後、蛍光は糸球体内に特異的に見られ、その蛍光分布は上皮内や内皮内ではなく、結合組織内にある細胞(メサンジュム細胞)内に存在している(図5,6)。 After FITC-NF-κB decoy introduced, fluorescence was observed specifically in the glomerulus, the fluorescence distribution is not within or within the endothelium epithelial, are present in the cell (Mesanjumu cells) within connective tissue ( FIGS. 5 and 6).

尿細管上皮細胞内の蛍光は、照射直後では顆粒状に認められたものの(図7A)、時間が経つにつれ徐々に減少し、6日目になるとほとんど認められなくなった(図7B)。 Fluorescence in tubular epithelial cells, although immediately after irradiation was observed in granular (Fig. 7A), and gradually decreased over time, no longer observed almost becomes day 6 (Fig. 7B).

実施例3〔糖尿病モデル動物の腎臓へのNF-κBデコイによる治療と遺伝子発現〕 Example 3 [Treatment and gene expression by NF-[kappa] B decoy into diabetic model animal kidney]
ヒト2型糖尿病モデル動物であるOLETFラット(2匹)、オス、20ヶ月齢(平均体重625g)を用いた。 OLETF rats (2 animals) is a human type 2 diabetes model animal, it was used a male, 20 months of age (average body weight 625 g). 麻酔後、正中線からやや右側を5cm程度、縦に切開し右腎を露出させた。 After anesthesia, 5 cm approximately slightly to the right from the midline, longitudinally dissected to expose the right kidney. 腸を注意深く片側に寄せ、麻酔下で、NF-κBデコイ15mg/生理食塩水1.5mlとOptison(商標名)500μlの混合液を投与した。 Asked carefully side intestines, under anesthesia was administered NF-[kappa] B decoy 15 mg / saline 1.5ml and Optison (TM) mixture of 500 [mu] l. 超音波の照射条件は、duty比(50%)、2W/cm 2 、20分間とし、注入直後より右腎臓全体に照射した(表1)。 Irradiation conditions of ultrasonic waves, duty ratio (50%), and between 2W / cm 2, 20 minutes were irradiated to the entire right kidney immediately after injection (Table 1). プローブの径が5mm程度で小さいため、プローブをゆっくり動かしながら全体に照射した。 Since the diameter of the probe is smaller in the order of 5 mm, it was irradiated on the entire Gently rock the probe.

20分間の超音波照射後、腎臓はやや腫れ、色は暗褐色になっていたが、徐々に色調は戻った。 After ultrasonic irradiation for 20 minutes, kidney slightly swollen, the color had turned dark brown, was gradually returned color tone. 腎臓を元の場所に戻し、切開創を縫合し、その後1ヶ月間、通常飼育した。 Kidney back to its original location, the incision was sutured, and then for one month, was usually breeding. 1ヶ月後、深麻酔下、左右の腎臓を採取し、腎皮質を分離後、即座に液体窒素内で皮質組織を凍結、遺伝子解析まで保存した。 One month later, deep under anesthesia, the left and right kidneys were harvested, after the separation of the renal cortex, immediately freeze the cortical tissue in liquid nitrogen, and stored until genetic analysis.

〔組織学的変化の検討〕 [Study of histological changes]
腎の組織学的障害を表す糸球体硬化と間質障害のどちらも、コントロール群に比べOLETFラットで明らかに進行していたものの、NF-κBデコイ投与後に超音波治療した右腎と対照の左腎の間には、両ラットとも、大きな差は認められなかった。 Both glomerulosclerosis and interstitial disorders representing histological disorders of kidney, but was in progress clearly in OLETF rats than in the control group, left control right kidney was ultrasonically treated after NF-[kappa] B decoy between the kidney, both rat, a large difference was not observed. 糸球体や尿細管の機能低下時に見られる尿円柱は、OLETFラットでは尿細管腔内にPAS陽性の無構造な尿円柱(*)が数多く見られたものの(図8C)、コントロール群ではごく少数しか認められなかった(図8A=左腎、図8B=右腎)。 Urinary cast seen when degraded glomerular or tubular, although no structures urinary cast of PAS positive tubular lumen (*) was observed numerous in OLETF rats (Fig. 8C), only the control group only a small number was observed (Fig. 8A = left kidney, Figure 8B = right kidney). しかし、OLETFラットの超音波処理した右腎(図8D)は対照の左腎(図8C)に比べ、尿円柱形成は明らかに減少していた。 However, the right kidney (FIG. 8D) sonicated in OLETF rats compared to the control of the left kidney (Fig. 8C), urinary cast formation was clearly reduced.

〔腎皮質内遺伝子発現の検討〕 [Study of the renal cortex in the gene expression]
採取した腎皮質内RNAをRT-PCRでcDNAとして増幅後、それぞれ左腎サンプルを対照とし(cy5)、右腎を治療サンプル(cy3)とし、DNAチップ(Atlas Glass Rat 1.0 Microarray, 1081遺伝子、CLONTECH, CA,USA)を用い、検討した。 After amplification the collected renal cortex in RNA as cDNA by RT-PCR, respectively as a control left renal samples (cy5), the right kidney and treatment samples (cy3), DNA chips (Atlas Glass Rat 1.0 Microarray, 1081 genes, CLONTECH , CA, USA) was used to study. 全1081遺伝子の中で増殖因子、増殖因子レセプターに関係するものは49遺伝子含まれている。 Growth factor among all 1081 genes, relate to growth factor receptors contain 49 genes. 標本による発現のばらつきはチップ内にある9種類のhose-keeping 遺伝子(Glyceraldehyde-3-phosphate dehydrogenase, alpha-tubulin, Ornitine decarboxylase, myosin IB, beta actin, Ribosomal protein S29, polyubiquitin, phospholipase A2, hypoxanthine-guanine phosphoribosyltransferase)の平均値で補正した。 Sample variation in expression by the nine hose-keeping gene (Glyceraldehyde-3-phosphate dehydrogenase in the chip, alpha-tubulin, Ornitine decarboxylase, myosin IB, beta actin, Ribosomal protein S29, polyubiquitin, phospholipase A2, hypoxanthine-guanine was corrected by the average value of phosphoribosyltransferase). 最終的に、1081遺伝子中検討できた遺伝子は1062個であった。 Finally, a gene that could be considered during the 1081 gene was 1062 pieces. 検討した遺伝子の度数分布を示す(図9)。 It shows the frequency distribution of the examined genes (Fig. 9).

図9から明らかなとおり、全遺伝子の発現分布はほぼ正規分布を示すが、全体の中心が1からやや左にずれている。 As apparent from FIG. 9, the expression distribution of all genes shows almost normal distribution, but the whole of the center is shifted to the left slightly from 1. 両端の棄却域を約10%とすると、減少した遺伝子は発現比0.75以下、減少傾向のある遺伝子を発現比0.75以上0.8未満、増加傾向のある遺伝子は発現比1.2以上1.5未満、増加した遺伝子は発現比1.5以上とし、それ以外のもの(0.8以上、1.5未満)を変動なしとして表2に示した。 When about 10% rejection region at both ends, decreased gene expression ratio of 0.75 or less, decreasing gene expression ratio of 0.75 to less than 0.8 with, the gene of increasing the expression ratio 1.2 less than 1.5 or more, increased gene and expression ratio of 1.5 or more, and the others (0.8 to less than 1.5) shown in Table 2 as without varying.

表2から明らかなとおり、全体として大きな変動のある遺伝子はきわめて少なかった。 As apparent from Table 2, the gene of large variations as a whole was very small. 変動のあった遺伝子の内、増殖因子、増殖因子レセプター、血圧に関係する遺伝子の変動率を表3に示す。 Of a gene fluctuation, showing growth factors, growth factor receptors, the variation rate of the genes involved in blood pressure in Table 3.

表3から明らかなとおり、最も減少したものはレニンで、その他増殖因子・サイトカインに関連するPDGFおよびVEGFレセプター遺伝子に減少を認めた。 As apparent from Table 3, the most reduced ones renin showed a decrease in PDGF and VEGF receptor gene associated with other growth factors, cytokines. 減少傾向を認めたものはTGFβとαおよびTNFであった。 Which showed a downward trend was TGFβ and α and TNF. 逆に増加したものは、増殖因子・サイトカインにはなかったものの、尿細管で発現のあるチトクロームP450に増加をみとめ、もっとも増加したものはニュウレキシン3であった。 Conversely to what has been increased, while there was no growth factor cytokine, we observed an increase in cytochrome P450 with expression in renal tubules, was Nyuurekishin 3 that increased most.

ラット腎動脈からのFITC-NF-κBデコイの灌流による投与後、超音波照射した腎臓内のFITC分布を示す蛍光顕微鏡写真(100倍)。 After administration by perfusion of FITC-NF-[kappa] B decoy from rat renal arteries, fluorescence micrographs (100 times) showing a FITC distribution in kidneys ultrasonic irradiation. 図1と同様の条件による、尿細管内のFITC分布を示す蛍光顕微鏡写真(100倍)。 Under the same conditions as FIG. 1, a fluorescent micrograph showing FITC distribution in tubule (100-fold). 図1と同様の条件でFITC-NF-κBデコイ投与後4時間の腎尿細管のFITC分布を示す蛍光顕微鏡写真(100倍)。 Fluorescence micrograph showing FITC distribution in renal tubules FITC-NF-[kappa] B decoy After 4 hours under the same conditions as FIG. 1 (100-fold). マウスへのFITC-NF-κBデコイの静注による全身投与後、超音波処理した腎組織のFITC分布を示す蛍光顕微鏡写真(100倍)。 After systemic administration by intravenous injection of FITC-NF-[kappa] B decoy into mice, fluorescence micrographs (100 times) showing a FITC distribution of kidney tissue was sonicated. ラットへのFITC-NF-κBデコイの静注による全身投与投与後、超音波処理した腎組織のFITC分布を示す蛍光顕微鏡写真(100倍)。 After systemic administration administration by intravenous injection of FITC-NF-[kappa] B decoy into rat fluorescence micrograph (100 times) showing a FITC distribution of kidney tissue was sonicated. ラットへのFITC-NF-κBデコイの静注による全身投与投与後、超音波処理した腎組織のFITC分布を示す蛍光顕微鏡写真(100倍)。 After systemic administration administration by intravenous injection of FITC-NF-[kappa] B decoy into rat fluorescence micrograph (100 times) showing a FITC distribution of kidney tissue was sonicated. 図5,6と同様の条件でFITC-NF-κBデコイの静注による全身投与投与後、超音波処理した尿細管上皮細胞内のFITC分布を示す蛍光顕微鏡写真(100倍)。 After systemic administration administration by intravenous injection of FITC-NF-[kappa] B decoy under the same conditions as FIG. 5 and 6, a fluorescent micrograph (100 times) showing a FITC distribution in tubular epithelial cells and sonicated. ラットへの静注によるNF-κBデコイの全身投与後、右腎に超音波照射し1ヶ月後の腎組織像(PAS染色)を示す蛍光顕微鏡写真(100倍)。 After systemic administration of NF-[kappa] B decoy by intravenous in rats, fluorescence micrographs (100 times) showing renal histology after 1 month ultrasonic irradiation to the right kidney a (PAS staining). 遺伝子発現度数を示す図。 It shows the gene expression frequency.

Claims (8)

  1. 転写調節因子の結合部位への結合を阻害するデコイ(おとり)型核酸医薬を有効成分とする、増殖性腎疾患の予防・治療・改善剤。 Prevention, treatment and amelioration agent for the decoy-type nucleic acid drugs as an active ingredient, proliferative renal diseases to inhibit the binding of the binding site of the transcriptional regulatory factor.
  2. デコイ(おとり)型核酸医薬が、NF-κB、STAT-6、AP-1、Ets及びE2Fのおとり型核酸医薬から選ばれる少なくとも1つのものである、請求項1記載の増殖性腎疾患の予防・治療・改善剤。 Decoy type nucleic acid drugs is at least one thing selected from NF-κB, STAT-6, AP-1, the Ets and E2F decoy type nucleic acid drugs, prevention of proliferative renal disease of claim 1, wherein , treatment and improving agent.
  3. デコイ(おとり)型核酸医薬が、NF-κBのおとり型核酸医薬である、請求項1又は2記載の増殖性腎疾患の予防・治療・改善剤。 Decoy type nucleic acid drugs is a decoy-type nucleic acid drugs of NF-[kappa] B, according to claim 1 or 2 prevention, treatment and ameliorating agent for proliferative kidney disease according.
  4. NF-κBのデコイ(おとり)型核酸医薬が、塩基配列中に配列番号1を含むものである、請求項1〜3のいずれかに記載の増殖性腎疾患の予防・治療・改善剤。 Decoy NF-[kappa] B (decoy) type nucleic acid drugs is those in the nucleotide sequence comprising SEQ ID NO: 1, prevention, treatment and ameliorating agent for proliferative kidney disease according to any one of claims 1 to 3.
  5. NF-κBのデコイ(おとり)型核酸医薬が、配列番号2及び配列番号3からなる二重鎖核酸である、請求項1〜4のいずれかに記載の増殖性腎疾患の予防・治療・改善剤。 Decoy NF-[kappa] B (decoy) type nucleic acid drugs is a double-stranded nucleic acid consisting of SEQ ID NO: 2 and SEQ ID NO: 3, prevention, treatment and improvement of proliferative kidney disease according to any one of claims 1 to 4 agent.
  6. 増殖性腎疾患が、腎症、糸球体硬化症又はアルブミン尿症である、請求項1〜5のいずれかに記載の増殖性腎疾患予防・治療・改善剤。 Proliferative kidney disease, nephropathy, and glomerulosclerosis or albuminuria, proliferative kidney disease prevention, treatment and improvement agent according to any one of claims 1 to 5.
  7. 腎症が、糖尿病性腎症又は高血圧性腎症である、請求項6記載の増殖性腎疾患予防・治療・改善剤。 Nephropathy is diabetic nephropathy or hypertensive nephropathy, proliferative kidney disease prevention, treatment and improvement agent according to claim 6.
  8. 剤型が注射剤を含む液剤である、請求項1〜7のいずれかに記載の増殖性腎疾患予防・治療・改善剤。 Dosage form is a liquid formulation containing the injections, proliferative kidney disease prevention, treatment and improvement agent according to any one of claims 1 to 7.
JP2005090496A 2004-03-30 2005-03-28 Prophylactic/therapeutic/ameliorating agent for proliferative nephropathy Pending JP2005314381A (en)

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