JP2001340080A - Searching system for insulin-resistant ameliorant without possibility of exacerbating diabetic retinopathy and without edema inducing action - Google Patents
Searching system for insulin-resistant ameliorant without possibility of exacerbating diabetic retinopathy and without edema inducing actionInfo
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- JP2001340080A JP2001340080A JP2001103391A JP2001103391A JP2001340080A JP 2001340080 A JP2001340080 A JP 2001340080A JP 2001103391 A JP2001103391 A JP 2001103391A JP 2001103391 A JP2001103391 A JP 2001103391A JP 2001340080 A JP2001340080 A JP 2001340080A
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- Japan
- Prior art keywords
- gene
- insulin
- screening
- diabetic retinopathy
- growth factor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
Description
【０００１】糖尿病、特に２型糖尿病は、膵β細胞から
のインスリン分泌低下とインスリン感受性の低下、すな
わちインスリン抵抗性を特徴とする。生活習慣の欧米化
に伴うインスリン抵抗性の増加は、現在、糖尿病が急増
している最大の原因となっている。インスリン抵抗性改
善剤には、日本ならびに海外で臨床使用されているピオ
グリタゾン（アクトス（登録商標））、海外で臨床使用
されているロシグリタゾン（ＢＲＬ−４９６５３）、日
本ならびに海外で臨床使用されていたが重篤な肝障害を
生じる副作用のために２０００年３月に販売が停止され
たトログリタゾン（ノスカール（登録商標））、ならび
に開発中のＭＣＣ−５５５、、ＫＲＬ−４９６５３、お
よびＫＲＰ２９７などがあり、これらはいずれもチアゾ
リジン系の化合物である。また、非チアゾリジン骨格を
有するものとしてＪＴＴ−５０１およびＹＭ４４０が開
発中であり、その他に、ビグアナイド系薬剤と総称され
るインスリン抵抗性改善剤として臨床使用されている塩
酸メトフォルミン（グリコラン、メルビン（登録商
標））、塩酸ブフォルミン（ジベトスＢ（登録商標））
がある。[0001] Diabetes, especially type 2 diabetes, is characterized by decreased insulin secretion from pancreatic β cells and decreased insulin sensitivity, ie insulin resistance. The increase in insulin resistance associated with the westernization of lifestyle is currently the largest cause of the rapid increase in diabetes. Insulin sensitizers include pioglitazone (Actos (R)) clinically used in Japan and overseas, rosiglitazone (BRL-49653) clinically used overseas, and clinically used in Japan and overseas. Troglitazone (Noscal®), which was discontinued in March 2000 due to side effects that cause severe liver damage, and MCC-555, KRL-49653, and KRP297 under development, and the like. These are all thiazolidine compounds. In addition, JTT-501 and YM440 are being developed as having a non-thiazolidine skeleton, and in addition, metformin hydrochloride (glycolane, Melvin (registered trademark)) clinically used as an insulin resistance improving agent collectively referred to as biguanides )), Buformin hydrochloride (Jibetos B (registered trademark))
There is.
【０００２】すでに市販されているチアゾリジン誘導体
系薬剤は浮腫（惹起作用）という副作用を有する。本発
明者はこの副作用はチアゾリン誘導体が、それを投与し
た患者において、強力な血管透過性亢進因子でもある血
管内皮増殖因子（vascular endothelial growth facto
r; ＶＥＧＦ）の血中濃度を増加させるためであるこ
と、さらに、これが血管内皮増殖因子をコードする遺伝
子の発現を増加させるためであることを見いだした。ま
た、ＶＥＧＦは糖尿病網膜症の発症・増悪の主要な因子
であることが示されている（Aiello L.P.ら、N. Engl.
J. Med. 331:1480-1487, 1994; Tanaka Y.ら、Lancet 3
49:1520, 1997; Adamis A.P.ら、Am. J. Ophthalmol. 1
18:445-450,1994; Miller J.W.ら、Am. J. Pathol. 14
5:574-584, 1994;およびTolentino M.J.ら、Ophthalmol
ogy 103:1820-1828, 1996参照）。[0002] Thiazolidine derivative drugs already on the market have the side effect of edema (causing action). The present inventor has found that this side effect is due to the fact that the thiazoline derivative is a vascular endothelial growth factor which is also a potent vascular permeability enhancer in patients receiving it.
r; VEGF) was found to increase blood levels of VEGF), and moreover, to increase expression of the gene encoding vascular endothelial growth factor. VEGF has also been shown to be a major factor in the onset and exacerbation of diabetic retinopathy (Aiello LP et al., N. Engl.
J. Med. 331: 1480-1487, 1994; Tanaka Y. et al., Lancet 3.
49: 1520, 1997; Adamis AP et al., Am. J. Ophthalmol. 1
18: 445-450, 1994; Miller JW et al., Am. J. Pathol. 14
5: 574-584, 1994; and Tolentino MJ et al., Ophthalmol
ogy 103: 1820-1828, 1996).
【０００３】チアゾリジン誘導体系薬剤が臨床使用され
てまだ数年しか経っておらず、糖尿病網膜症の経過が比
較的長いことから、該薬剤投与による糖尿病網膜症の悪
化に関する報告はまだなされていないが、ＶＥＧＦが糖
尿病網膜症を増悪させる主要な因子であり、その血中濃
度がチアゾリジン誘導体系薬剤の投与により増加するこ
と、さらに、糖尿病網膜症では症状の１つとして網膜に
浮腫がみられることが知られており（後藤ら編、最新医
学大事典、第２版（１９９６）、医歯薬出版、１２１３
頁「糖尿病性網膜症」の項参照）、チアゾリジン誘導体
系薬剤の副作用である浮腫自体も糖尿病網膜症悪化の一
因となり得ることからも、チアゾリジン誘導体系薬剤投
与による糖尿病網膜症の悪化は当然予期されることであ
る。[0003] Although thiazolidine derivative drugs have been clinically used for only a few years and the course of diabetic retinopathy is relatively long, no report has been made on the deterioration of diabetic retinopathy due to the administration of the drugs. VEGF is a major factor in exacerbating diabetic retinopathy, and its blood concentration is increased by administration of thiazolidine derivative drugs. In addition, edema in the retina is one of the symptoms of diabetic retinopathy. Known (edited by Goto et al., The latest medical encyclopedia, 2nd edition (1996), Dentistry Publishing, 1213
(See page "Diabetic retinopathy"), edema, which is a side effect of thiazolidine derivative drugs, can also contribute to the deterioration of diabetic retinopathy. Is to be done.
【０００４】したがって、血中ＶＥＧＦレベルの増加を
引き起こさないインスリン抵抗性改善剤は糖尿病網膜症
を増悪させる可能性がなく、浮腫という副作用を生じな
い優れた薬剤となることが期待される。そこで、本発明
者は、ＶＥＧＦ遺伝子発現を検出できる組換え遺伝子系
を作製し、この組換え遺伝子系を導入した細胞を用いれ
ば、糖尿病網膜症を増悪させる可能性がなく、浮腫とい
う副作用を生じない優れたインスリン抵抗性改善剤をス
クリーニングすることができることを見いだし、本発明
を完成した。[0004] Therefore, an insulin sensitizer that does not cause an increase in the blood VEGF level is expected to be an excellent drug that does not have the possibility of exacerbating diabetic retinopathy and does not cause the side effect of edema. Therefore, the present inventors have prepared a recombinant gene system capable of detecting VEGF gene expression, and using cells into which the recombinant gene system has been introduced, there is no possibility of exacerbating diabetic retinopathy, and the side effect of edema occurs. It has been found that no excellent insulin sensitizer can be screened, and the present invention has been completed.
【０００５】すなわち、本発明は、プロモーター領域を
含むヒト血管内皮増殖因子遺伝子にレポーター遺伝子を
結合させた組換え遺伝子を哺乳動物細胞に導入し、この
細胞のレポーター遺伝子発現によりヒト血管内皮増殖因
子遺伝子の発現を検出することを特徴とする、糖尿病網
膜症を増悪させる可能性がなく、浮腫惹起作用を有しな
いインスリン抵抗性改善薬のスクリーニング法を提供す
るものである。さらに本発明は、プロモーター領域を含
むヒト血管内皮増殖因子遺伝子にレポーター遺伝子を結
合させた組換え遺伝子を提供するものである。本発明の
別の目的は、プロモーター領域を含むヒト血管内皮増殖
因子遺伝子を、ベクターに組み込まれたレポーター遺伝
子の上流にクローニングして得られる発現ベクターを哺
乳動物細胞に導入し、この細胞のレポーター遺伝子の発
現によりヒト血管内皮増殖因子遺伝子の発現を検出する
ことを特徴とする、糖尿病網膜症を増悪させる可能性が
なく、浮腫惹起作用を有しないインスリン抵抗性改善薬
のスクリーニング法を提供することである。本発明のさ
らに別の目的は、プロモーター領域を含むヒト血管内皮
増殖因子遺伝子を、ベクターに組み込まれたレポーター
遺伝子の上流にクローニングして得られる発現ベクター
を提供することである。本発明はまた、上記スクリーニ
ング法を用いることにより得られるインスリン抵抗性改
善薬をも提供するものである。本発明のスクリーニング
法は、チアゾリジン系のみならずあらゆる種類のインス
リン抵抗性改善薬の開発に広く応用可能であり、より安
全なインスリン抵抗性改善薬を開発するためにはむしろ
ぜひ応用すべきものである。本明細書において「レポー
ター遺伝子」は、本発明の目的に使用可能なあらゆるレ
ポーター遺伝子を意味し、例えば、ルシフェラーゼ遺伝
子およびクロラムフェニコールアセチルトランスフェラ
ーゼ（ＣＡＴ）遺伝子などが含まれるがこれらに限定さ
れるものではない。これらレポーター遺伝子は一般に市
販されているものを使用することができる。好ましいレ
ポーター遺伝子はルシフェラーゼ遺伝子である。以下の
試験例および実施例において、本発明をさらに詳細に説
明するが、これらは単に例示であって、本発明の範囲を
何ら限定するものではない。[0005] That is, the present invention introduces a recombinant gene comprising a human vascular endothelial growth factor gene containing a promoter region and a reporter gene linked to a mammalian cell, and expresses the human vascular endothelial growth factor gene by expressing the reporter gene in this cell. It is intended to provide a method for screening an insulin sensitizer which does not have the potential to exacerbate diabetic retinopathy and has no edema-inducing action, characterized by detecting the expression of. Further, the present invention provides a recombinant gene in which a reporter gene is linked to a human vascular endothelial growth factor gene containing a promoter region. Another object of the present invention is to introduce, into a mammalian cell, an expression vector obtained by cloning a human vascular endothelial growth factor gene containing a promoter region upstream of a reporter gene incorporated into a vector, By detecting the expression of the human vascular endothelial growth factor gene by the expression of, there is no possibility of exacerbating diabetic retinopathy, by providing a method of screening for an insulin sensitizer having no edema-inducing action is there. Still another object of the present invention is to provide an expression vector obtained by cloning a human vascular endothelial growth factor gene containing a promoter region upstream of a reporter gene incorporated in the vector. The present invention also provides an insulin sensitizer obtained by using the above screening method. The screening method of the present invention can be widely applied to the development of not only thiazolidine-based but also all kinds of insulin sensitizers, and should be applied rather to develop safer insulin sensitizers. . As used herein, “reporter gene” means any reporter gene that can be used for the purpose of the present invention, and includes, but is not limited to, for example, the luciferase gene and the chloramphenicol acetyltransferase (CAT) gene. Not something. As these reporter genes, commercially available ones can be used. A preferred reporter gene is a luciferase gene. The present invention will be described in more detail in the following Test Examples and Examples, which are merely illustrative and do not limit the scope of the present invention in any way.
【０００６】試験例１ 本発明者は、薬剤投与で認められる浮腫の臨床像が、血
管透過性亢進でよく説明できることに着目し、チアゾリ
ジン系薬剤であるトログリタゾン投与糖尿病患者におい
て、強力な血管透過性亢進因子であるＶＥＧＦ（別名 V
ascular permeability factor) の血清値を測定したと
ころ、その平均値は１２０．１ ｐｇ／ｍＬ（ｎ＝３
０）であり、食事療法群（２９．２ｐｇ／ｍＬ、ｎ＝１
０）、スルホニル尿素剤投与（ＳＵ）群（２５．８ｐｇ
／ｍＬ、ｎ＝１０）、インスリン療法群（２４．６ｐｇ
／ｍＬ、ｎ＝１０）と比較して有意（ｐ＜０．００１）
に増加していた。５人の患者で投与前より経過を追った
ところ、投与により上昇し、投与中止により前値に復し
た。さらに、３Ｔ３−Ｌ１脂肪細胞中のＶＥＧＦｍＲＮ
Ａの発現は患者血中濃度に等しい濃度の、トログリタゾ
ン添加および同様の作用機構を有するロシグリダゾン添
加で増加した。Test Example 1 The present inventor focused on the fact that the clinical picture of edema observed by drug administration can be well explained by vascular hyperpermeability. In diabetic patients receiving troglitazone, a thiazolidine drug, strong vascular permeability was observed. VEGF (also known as V
When the serum value of the ascular permeability factor was measured, the average value was 120.1 pg / mL (n = 3
0) and the dietary treatment group (29.2 pg / mL, n = 1
0), sulfonylurea drug administration (SU) group (25.8 pg
/ ML, n = 10), insulin therapy group (24.6 pg)
/ ML, n = 10) significant (p <0.001)
Had increased. In five patients, progress was made before administration, and the level increased with administration, and returned to the previous value due to discontinuation of administration. Furthermore, VEGFmRN in 3T3-L1 adipocytes
The expression of A was increased by the addition of troglitazone and rosiglidazone, which has a similar mechanism of action, at a concentration equivalent to that in the patient's blood.
【０００７】実施例１ （１）ヒトVEGF遺伝子プロモーターの単離 既報のマウスVEGF mRNAの塩基配列（Claffey KPら、J.
Biol. Chem. 267: 16317-16322, 1992; GenBank access
on no. M95200）をもとにオリゴヌクレオチドプライマ
ーを作成し、次いでRT-PCR法により3T3-L1脂肪細胞RNA
よりマウスVEGFcDNA断片を得た。このcDNAをプローブと
してヒトゲノムDNAライブラリー（ストラタジーン社）
をスクリーニングし、ヒトVEGF遺伝子（その上流約9 kb
および下流約3 kbを含む）を単離した。Example 1 (1) Isolation of Human VEGF Gene Promoter The previously reported nucleotide sequence of mouse VEGF mRNA (Claffey KP et al., J. Am.
Biol. Chem. 267: 16317-16322, 1992; GenBank access
on no. M95200), and then prepare 3T3-L1 adipocyte RNA by RT-PCR.
Thus, a mouse VEGF cDNA fragment was obtained. Using this cDNA as a probe, a human genomic DNA library (Stratagene)
To screen the human VEGF gene (about 9 kb upstream
And about 3 kb downstream).
【０００８】（２）トログリタゾン応答領域を含むヒト
VEGF遺伝子プロモーター／ルシフェラーゼレポータープ
ラスミドの作成 上記（１）で単離したクローンから、ヒトVEGF遺伝子の
転写開始点より上流約2.2 kbを含むDNA断片（-2274〜+5
0, KpnI-NheI）を得た。このDNA断片をpGL3ベクター(プ
ロメガ社)のルシフェラーゼ遺伝子の上流にクローニン
グし、VEGF遺伝子プロモーター／ルシフェラーゼを作成
した。該レポータープラスミドphVEGF2.2LUCは、Escher
ichia coli JM109/phVEGF2.2LUCとして産業技術総合研
究所生命工学工業技術研究所（茨城県つくば市東1丁目
１番３号）に寄託された（受託番号：FERM P-18281、受
託日：平成１３年３月３０日）。phVEGF2.2LUCをβガラ
クトシダーゼの発現プラスミドpCMV-β（クロンテック
社）とともにA-172細胞（JCRB0228, JCRB Cell Bank）
にリポフェクション（lipofection）法を用いて導入し
た。導入６〜８時間後にトログリタゾン（20μM）添
加、または非添加培養液に移し、さらに２４時間培養
後、細胞を生理的食塩水で洗浄し、細胞溶解用緩衝液
（プロメガ社）に溶解し、上清のルシフェラーゼ活性及
びβガラクトシダーゼ活性を測定した。その結果、phVE
GF2.2LUCを導入した細胞で、トログリタゾン添加による
ルシフェラーゼ／βガラクトシダーゼ活性比の増加がみ
られ、phVEGF2.2LUCがトログリタゾン応答領域を含むVE
GF遺伝子プロモーター／ルシフェラーゼレポータープラ
スミドであることが確認された。図１に得られたphVEGF
2.2LUCの遺伝子マップを示す。(2) a human containing a troglitazone response region
Preparation of VEGF Gene Promoter / Luciferase Reporter Plasmid From the clone isolated in (1) above, a DNA fragment containing approximately 2.2 kb upstream of the transcription start site of the human VEGF gene (-2274 to +5
0, KpnI-NheI). This DNA fragment was cloned into the pGL3 vector (Promega) upstream of the luciferase gene to prepare a VEGF gene promoter / luciferase. The reporter plasmid phVEGF2.2LUC was from Escher
Deposited as ichia coli JM109 / phVEGF2.2LUC with the National Institute of Advanced Industrial Science and Technology (1-3 1-3, Higashi, Tsukuba, Ibaraki, Japan) (accession number: FERM P-18281, date of acceptance: 2001) March 30). phVEGF2.2LUC together with β-galactosidase expression plasmid pCMV-β (Clontech) in A-172 cells (JCRB0228, JCRB Cell Bank)
Was introduced using the lipofection method. After 6 to 8 hours from the introduction, the cells are transferred to a culture medium with or without troglitazone (20 μM). After further culturing for 24 hours, the cells are washed with physiological saline, lysed in a cell lysis buffer (Promega), and The luciferase activity and β-galactosidase activity of Qingdao were measured. As a result, phVE
In cells transfected with GF2.2LUC, the ratio of luciferase / β-galactosidase activity was increased by the addition of troglitazone, and phVEGF2.2LUC showed VE containing troglitazone-responsive region.
It was confirmed to be a GF gene promoter / luciferase reporter plasmid. PhVEGF obtained in FIG.
2 shows a genetic map of LUC.
【０００９】（３）被検薬剤によるヒトVEGF遺伝子転写
活性化のスクリーニング 上記（２）で得られたプラスミドが、広くインスリン抵
抗性改善薬のVEGF遺伝子転写活性化能のスクリーニング
に利用できることを確認するため以下の実験を行なっ
た。A-172細胞に上記（２）で作成したレポータープラ
スミドphVEGF2.2LUCおよびβガラクトシダーゼ発現プラ
スミドpCMV-βをリポフェクション法を用いて導入した
（以降、本明細書ではここで得られた細胞をA-172VEGF
細胞という）。レポータープラスミドおよびβガラクト
シダーゼ発現プラスミド導入６〜８時間後に被検薬剤
（ピオクリタゾン20μM）またはトログリタゾン（20μ
M）を含む培養液に移し、さらに２４時間後に細胞を生
理的食塩水で洗浄し、次いで細胞溶解用緩衝液に溶解し
た。細胞溶解液を遠心処理（12,000 g x 10秒）して得
られた上清のルシフェラーゼ及びβガラクトシダーゼ活
性を測定し、ルシフェラーゼ／βガラクトシダーゼ活性
比を算出した。結果を図２に示す。図２に示した結果か
ら明らかなように、トログリタゾンには強いVEGF遺伝子
転写活性化能が見られ、一方、ピオグリタゾンのVEGF遺
伝子転写活性化能はコントロールよりやや強いがトログ
リタゾンより弱かった。すなわち、この2剤を糖尿病網
膜症を増悪させる可能性および浮腫惹起作用が低いとい
う観点から比較すると、ピオグリタゾンのほうがトログ
リタゾンより優れていることがわかった。以上の結果か
ら、本発明のスクリーニング法を用いて候補化合物をス
クリーニングすることにより、VEGF遺伝子転写活性化能
がより低い、さらには、患者に投与した際に糖尿病網膜
症を増悪させる可能性がなく、浮腫惹起作用を有しない
インスリン抵抗性改善薬を得ることができるだけでな
く、既存のインスリン抵抗性改善薬の上記副作用を予測
するのにも応用可能であることが明らかになった。(3) Screening of Human VEGF Gene Transcriptional Activation by Test Drugs It is confirmed that the plasmid obtained in (2) above can be widely used for screening the VEGF gene transcriptional activation ability of an insulin sensitizer. Therefore, the following experiment was conducted. The reporter plasmid phVEGF2.2LUC and the β-galactosidase expression plasmid pCMV-β prepared in (2) above were introduced into A-172 cells using the lipofection method (hereinafter, the cells obtained here are referred to as A-172VEGF
Cell). 6-8 hours after the introduction of the reporter plasmid and the β-galactosidase expression plasmid, the test agent (pioclitazone 20 μM) or troglitazone (20 μM)
M), and after an additional 24 hours the cells were washed with saline and then lysed in cell lysis buffer. The luciferase and β-galactosidase activities of the supernatant obtained by centrifuging the cell lysate (12,000 g × 10 seconds) were measured, and the luciferase / β-galactosidase activity ratio was calculated. The results are shown in FIG. As is clear from the results shown in FIG. 2, troglitazone had a strong ability to activate VEGF gene transcription, whereas pioglitazone had a slightly stronger ability to activate VEGF gene transcription than control but weaker than troglitazone. That is, pioglitazone was superior to troglitazone when these two agents were compared in terms of the possibility of exacerbating diabetic retinopathy and the low edema-inducing effect. From the above results, by screening candidate compounds using the screening method of the present invention, the ability to activate VEGF gene transcription is lower, furthermore, there is no possibility of exacerbating diabetic retinopathy when administered to patients. It has been revealed that not only can an insulin sensitizer having no edema-inducing action be obtained, but also it can be applied to predict the above-mentioned side effects of existing insulin sensitizers.
【図１】 トログリタゾン応答領域を含むVEGF遺伝子プ
ロモーター／ルシフェラーゼレポータープラスミドphVE
GF2.2LUCの遺伝子マップを示す。FIG. 1. VEGF gene promoter / luciferase reporter plasmid phVE containing troglitazone response region
3 shows a gene map of GF2.2LUC.
【図２】 A-172VEGF細胞を用いた、チアゾリジン系イ
ンスリン抵抗性改善薬のVEGF遺伝子転写活性化能に関す
るスクリーニング実験の結果を示すグラフである。トロ
グリタゾン存在下で培養したA-172VEGF細胞（レーン
４）では、トログリタゾン非存在下で培養したA-172VEG
F細胞（コントロール、レーン３）に比べて、その上清
でのルシフェラーゼ／βガラクトシダーゼ活性比が著し
く増大する。一方、ピオグリタゾン存在下で培養したA-
172VEGF細胞（レーン５）ではルシフェラーゼ／βガラ
クトシダーゼ活性比の増大はコントロールよりやや高い
が、トログリタゾン存在下より弱かった。pGL3（プロメ
ガ社）だけを導入したA-172細胞（レーン１）およびCMV
プロモーターをルシフェラーゼ遺伝子の上流に配置した
pGL3-CMVを導入したA-172細胞（レーン２）の上清での
ルシフェラーゼ／βガラクトシダーゼ活性比を、この細
胞でのVEGFプロモーターの基礎活性を示すためのコント
ロールとして同時に示した。FIG. 2 is a graph showing the results of a screening experiment on the ability of a thiazolidine-based insulin sensitizer to activate VEGF gene transcription using A-172 VEGF cells. In A-172VEGF cells cultured in the presence of troglitazone (lane 4), A-172VEGF cultured in the absence of troglitazone was used.
Compared to F cells (control, lane 3), the luciferase / β-galactosidase activity ratio in the supernatant is significantly increased. On the other hand, A-cultured in the presence of pioglitazone
In 172VEGF cells (lane 5), the increase in the luciferase / β-galactosidase activity ratio was slightly higher than in the control, but weaker than in the presence of troglitazone. A-172 cells (lane 1) transfected only with pGL3 (Promega) and CMV
Promoter located upstream of luciferase gene
The luciferase / β-galactosidase activity ratio in the supernatant of A-172 cells (lane 2) into which pGL3-CMV was introduced was simultaneously shown as a control for indicating the basal activity of the VEGF promoter in these cells.
フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ テーマコート゛(参考） Ｇ０１Ｎ 33/15 Ｇ０１Ｎ 33/50 Ｚ 33/50 33/68 33/68 Ｃ１２Ｎ 15/00 Ａ Continued on the front page (51) Int.Cl. 7 Identification symbol FI Theme coat II (reference) G01N 33/15 G01N 33/50 Z 33/50 33/68 33/68 C12N 15/00 A
Claims (14)
因子遺伝子にレポーター遺伝子を結合させた組換え遺伝
子を哺乳動物細胞に導入し、この細胞のレポーター遺伝
子の発現によりヒト血管内皮増殖因子遺伝子の発現を検
出することを特徴とする、糖尿病網膜症を増悪させる可
能性がなく、浮腫惹起作用を有しないインスリン抵抗性
改善薬のスクリーニング法。1. A recombinant gene in which a reporter gene is linked to a human vascular endothelial growth factor gene containing a promoter region is introduced into mammalian cells, and the expression of the human vascular endothelial growth factor gene is determined by the expression of the reporter gene in the cells. A method for screening for an insulin sensitizer having no possibility of exacerbating diabetic retinopathy and having no edema-inducing action, which is characterized by being detected.
である請求項１記載のスクリーニング法。2. The screening method according to claim 1, wherein the reporter gene is a luciferase gene.
因子遺伝子にレポーター遺伝子を結合させた組換え遺伝
子。3. A recombinant gene in which a reporter gene is linked to a human vascular endothelial growth factor gene containing a promoter region.
法に用いるための請求項３記載の組換え遺伝子。4. The recombinant gene according to claim 3, which is used for a method for screening an insulin sensitizer.
である請求項３記載の組換え遺伝子。5. The recombinant gene according to claim 3, wherein the reporter gene is a luciferase gene.
法に用いるための請求項５記載の組換え遺伝子。6. The recombinant gene according to claim 5, which is used for a method of screening for an insulin sensitizer.
法を用いることにより得られるインスリン抵抗性改善
薬。7. An insulin sensitizer obtained by using the screening method according to claim 1 or 2.
因子遺伝子を、ベクターに組み込まれたレポーター遺伝
子の上流にクローニングして得られる発現ベクターを哺
乳動物細胞に導入し、この細胞のレポーター遺伝子の発
現によりヒト血管内皮増殖因子遺伝子の発現を検出する
ことを特徴とする、糖尿病網膜症を増悪させる可能性が
なく、浮腫惹起作用を有しないインスリン抵抗性改善薬
のスクリーニング法。8. An expression vector obtained by cloning a human vascular endothelial growth factor gene containing a promoter region upstream of a reporter gene incorporated into a vector, is introduced into a mammalian cell, and the expression of the reporter gene in the cell is carried out. A screening method for an insulin sensitizer having no possibility of exacerbating diabetic retinopathy and having no edema-inducing action, comprising detecting the expression of a human vascular endothelial growth factor gene.
である請求項８記載のスクリーニング法。9. The screening method according to claim 8, wherein the reporter gene is a luciferase gene.
殖因子遺伝子を、ベクターに組み込まれたレポーター遺
伝子の上流にクローニングして得られる発現ベクター。10. An expression vector obtained by cloning a human vascular endothelial growth factor gene containing a promoter region upstream of a reporter gene incorporated in the vector.
グ法に用いるための請求項１０記載の発現ベクター。11. The expression vector according to claim 10, which is used for a screening method for an insulin sensitizer.
子である請求項１０記載の発現ベクター。12. The expression vector according to claim 10, wherein the reporter gene is a luciferase gene.
グ法に用いるための請求項１２記載の発現ベクター。13. The expression vector according to claim 12, which is used for a method for screening an insulin sensitizer.
グ法を用いることにより得られるインスリン抵抗性改善
薬。[14] An insulin sensitizer obtained by using the screening method according to [8] or [9].
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2001103391A JP2001340080A (en) | 2000-03-31 | 2001-04-02 | Searching system for insulin-resistant ameliorant without possibility of exacerbating diabetic retinopathy and without edema inducing action |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2000-97365 | 2000-03-31 | ||
| JP2000097365 | 2000-03-31 | ||
| JP2001103391A JP2001340080A (en) | 2000-03-31 | 2001-04-02 | Searching system for insulin-resistant ameliorant without possibility of exacerbating diabetic retinopathy and without edema inducing action |
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|---|---|
| JP2001340080A true JP2001340080A (en) | 2001-12-11 |
Family
ID=26589107
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|---|---|---|---|
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003062427A1 (en) * | 2002-01-23 | 2003-07-31 | Yamanouchi Pharmaceutical Co., Ltd. | Method of screening drug for improving insulin resistance |
| WO2003099331A1 (en) * | 2002-05-24 | 2003-12-04 | Takeda Pharmaceutical Company Limited | Insulin resistance improving agents |
| WO2006059691A1 (en) * | 2004-12-03 | 2006-06-08 | Astellas Pharma Inc. | Method of screening antiobesity drug |
| EP1761638A4 (en) * | 2004-05-24 | 2009-01-14 | Ptc Therapeutics Inc | Methods and agents for screening for compounds capable of modulating vegf expression |
-
2001
- 2001-04-02 JP JP2001103391A patent/JP2001340080A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003062427A1 (en) * | 2002-01-23 | 2003-07-31 | Yamanouchi Pharmaceutical Co., Ltd. | Method of screening drug for improving insulin resistance |
| WO2003099331A1 (en) * | 2002-05-24 | 2003-12-04 | Takeda Pharmaceutical Company Limited | Insulin resistance improving agents |
| US7556926B2 (en) | 2002-05-24 | 2009-07-07 | Takeda Pharmaceuticals Company Ltd. | Methods for screening insulin-sensitizing agents |
| EP1761638A4 (en) * | 2004-05-24 | 2009-01-14 | Ptc Therapeutics Inc | Methods and agents for screening for compounds capable of modulating vegf expression |
| WO2006059691A1 (en) * | 2004-12-03 | 2006-06-08 | Astellas Pharma Inc. | Method of screening antiobesity drug |
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