JP3931218B2 - Preventive diastolic heart failure - Google Patents

Description

【００１９】
このスコアと下式に基づき、一次刺激スコアを計算した。
一次刺激スコア＝1／2（Ａ₂₄＋Ａ₄₈）＋1／2（Ｘ₂₄＋Ｘ₄₈）＋1／2（Ｂ₂₄＋Ｂ₄₈）＋1／2（Ｙ₂₄＋Ｙ₄₈）
ここで、
Ａ₂₄：正常皮膚に絆創膏貼付24時間後の紅斑スコア
Ａ₄₈：正常皮膚から絆創膏剥離48時間後の紅斑スコア
Ｂ₂₄：損傷皮膚に絆創膏貼付24時間後の紅斑スコア
Ｂ₄₈：損傷皮膚から絆創膏剥離48時間後の紅斑スコア
Ｘ₂₄：正常皮膚に絆創膏貼付24時間後の浮腫スコア
Ｘ₄₈：正常皮膚から絆創膏剥離48時間後の浮腫スコア
Ｙ₂₄：損傷皮膚に絆創膏貼付24時間後の浮腫スコア
Ｙ₄₈：損傷皮膚から絆創膏剥離48時間後の浮腫スコア
次に、一次刺激スコアと下記の基準に基づき、試験化合物の刺激度を判定した。
軽度刺激 ：一次刺激スコア０〜２未満
中程度刺激：一次刺激スコア２〜５未満
強度刺激 ：一次刺激スコア５以上
その結果、ＭＥＡの一次刺激スコアは０．２５であり、刺激度は軽度であった。
【００２０】
実施例
試料と方法
対象：離乳中の雄Dahl-S（DIS／Eis、Eisai、東京、日本）を0.3％のNaClを含む研究室固形試料で飼育し、７週齢で8％のNaClを含む研究室固形試料に切り換えた。これらのラットを次の２つの群に分けた。第一群は8週齢から200mg／kg／日の割合でＭＥＡ（和光純薬工業（株）製）の塩酸塩を経口投与したグループ（ｎ＝７）であり、第二群は無処置グループ（ｎ＝６）である。さらに、0.3％のNaClを含む研究室固形試料を連続的に与えられた雄Dahl-Sラットを年齢対応対照とした（ｎ＝５）。高食塩飼料によるラットの8週齢における収縮期血圧の平均値は161mmHgであり、年齢対応対照ラットのそれは130mmHgであった。
【００２１】
13および19週齢で心エコー図的研究を行った。19週齢では心エコー図検討のあとで、血行動態調査と心臓の回収を行った。著者らの先の研究結果に従ってこれらのスケジュールを決定した。実験中、飼料と水道水を自由に摂取できるようにした。尾部カフシステム（BP-98A、Softron、東京、日本）を用いて7、13、および19週齢で収縮期血圧を測定した。
【００２２】
心エコー図的研究：13および19週齢で経胸部心エコー図を記録した（Doi R.ら、J. Hypertens, 2000;18:111-120）。すなわち、ケタミンHCl（50mg／kg）およびキシラジンHCl（10mg／kg）の腹腔内投与によってラットを麻酔し、自発呼吸を伴う半左側位置にラットを保持した。7.5MHz探触子（SONOS 2000、Hewlett-Packard、アンドーバ、マサチューセッツ）を装着した市販の心エコー図装置を用いて記録を行い、左心室（LV）の内径と壁厚を測定した。また、下記式に従って、LV重量およびLV内径短縮率を計算した。
LV重量（ｇ）＝ 1.04 ｘ [(LVDd ＋ PWd ＋ AWd)³ − LVDd³] ｘ 0.8 ＋0.14
LV内径短縮率（％）＝ (LVDd − LVDs)／LVDd ｘ 100
ここで、LVDd：左心室拡張末期径
PWd ：拡張前壁厚
AWd ：拡張後壁厚
LVDs：左心室収縮末期径
なお、統計解析値には、LV重量を体重に対して補正した「LV重量／体重」を用いた。肥大心における収縮機能の過大推定を回避するため、Shimizuモデルを用いてLV壁中央部での短縮率も計算した。
【００２３】
血行動態的研究：19週齢における心エコー図的研究の後で、大気圧に対して校正したチップ先端装着型1.5Fカテーテル（SPR-407、Millar Instruments、ヒューストン、テキサス）を右頚動脈を介して左心室に挿入した。LV圧のトレーシングと心電図をディジタル化してLV拡張末期圧を決定し、非−零漸近線手法（Yamamoto K.ら、Circulation, 1995;91:192-200）を用いて等容積LV圧降下の時定数（τ）を計算した。Douglas法（Douglas PSら、J Am Coll Cardiol. 1987;9:945−951）を用いてLV収縮末期壁応力を計算した。
【００２４】
組織採取：血行動態的研究のあと、直ちに心臓を回収した。乳頭筋下左心室心尖部を除去し、重量を測定し、直ちに液体窒素中に入れ、StegmannとStalderの方法に従ってヒドロキシプロリン含有量を測定するため、-80℃で保存した。結果は組織の湿潤重量当りのヒドロキシプロリン含有量として計算した。mRNA量測定用左心室試料の重量を測定し、直ちに液体窒素中に入れ、-80℃で保存した。他の部分の重量を測定したあと、それを4％の冷パラホルムアルデヒド溶液中に16〜24時間浸した。肺も回収し、重量を測定した。
【００２５】
病理学的検討：4％のパラホルムアルデヒド溶液中に浸した検体をパラフィンに埋め込んだ。Azan Mallory染色法を用いて乳頭筋レベルのLV自由壁の2μm厚横断面切片を100倍の倍率の顕微鏡で調べた。
【００２６】
統計値：平均値±標準偏差値として結果を表現した。一方向ANOVAとFisherの保護最小有意差検定を用いて特異段階における群間差異を評価した。p＜0.05の確率値を統計的に有意であると考えた。なお、図１〜６のグラフにおいて、＊はp＜0.05対年齢対応対照ラットであり、＃はp＜0.05対未処置ラットである。
【００２７】
結果
本モデルにおける拡張不全型心不全への移行：未処置ラットの収縮期圧は年齢対応対照ラットに比べて13および19週で有意に上昇した（図1）。13週では、未処置ラットの拡張末期および収縮末期LV径は対照ラットのそれに比べて有意に小さかった（図1）。それゆえ、未処置ラットのLV収縮末期壁応力は対照ラットのそれよりも有意に低かった（47.5±4.4対103.0±9.4 10³dynes／cm²、p＜0.05）。未処置ラットのLV重量／体重は対照ラットのそれよりも有意に大きかった（図６）。19週では、未処置ラットは頻呼吸、労作性呼吸、および活動度の喪失といった明白な心不全の兆候を示した。未処置ラットでは、13週から19週にかけてLV重量／体重は進行的に増加した。このとき、LV収縮末期径および拡張末期径（図２、３）または左室壁中央部で評価した短縮率（図５）は19週でのそれと変わらなかった。19週では、未処置ラットと年齢対応対照ラットとの間でLV収縮末期圧の差はなかった（62.3±9.4対80.0±7.5 10³dynes／cm²）。対照ラット、未処置ラットおよびＭＥＡ処置ラットの19週での各定量値を示す下記表２から分かるように、対照ラットに比べて、未処置ラットのLV拡張末期圧（図２）および肺重量の体重に対する比率（肺／体重）は高く、τは長く、左心室のヒドロキシプロリン量は高かった。
【００２８】
【表２】

各値は、平均値±標準偏差値で示した。＊ｐ＜０．０５対対照；＃ｐ＜０．０５対未処置
ＬＶＥＤＰ＝左心室拡張末期圧；Pro-ＯＨ＝ヒドロキシプロリン濃度；τ＝LV弛緩の時定数
【００２９】
ＭＥＡの効果：ＭＥＡの投与は13週または19週における収縮期圧を低下させなかった。にもかかわらず、図２〜６に示すように13週で形態的変化が生じた。すなわち、LV重量／体重は未処置ラットのそれと匹敵するレベルまで増加し、LV拡張末期径は対照ラットのレベルまで拡大し、これによってLV収縮末期壁応力の正常化（116.1±12.5 10³dynes／cm²）が導かれた。内径短縮率は対照ラットに比し有意差なく、未処置ラットのそれよりも低かったが、左室壁中央部で評価した短縮率はＭＥＡの影響を受けなかった。
【００３０】
19週では、ＭＥＡ処置ラットは明白な心不全の兆候を示さなかった。ＭＥＡで処置したラットのLV拡張末期圧と肺／体重は未処置ラットのそれよりも有意に低く、対照群のそれと違わなかった（表２）。病理学調査（図７）が示すように、未処置ラットでは進行性の間質線維化が観察されたが、ＭＥＡで処置したラットでは観察されず、ＭＥＡは左心室中のヒドロキシプロリン量を正常化した（表２）。ＭＥＡは、未処置ラットで観察される13週から19週にかけてのLV重量／体重のさらなる増加を阻止した（図６）。ＭＥＡは、対照ラットと未処置ラットに比べて19週におけるLV拡張末期径を特徴的に拡大させたが、LV収縮末期壁応力は上昇しなかった（98.6士8.4 10³dyne／cm²）。未処置ラットに比較して、τはＭＥＡによって変化しなかった（表２）。19週における左室壁中央部で評価した短縮率はＭＥＡ投与の影響を受けなかった（図５）。
【００３１】
高血圧発症後にＭＥＡを長期投与した場合、ＭＥＡは、高血圧心モデルにおいて、降圧効果なしで明白な拡張不全型心不全への移行を阻止した。これはLV拡張末期圧よび肺重量に対するＭＥＡの効果によって証明された。内径短縮率と左室壁中央部で評価した短縮率によって評価したように、このような阻止効果はＭＥＡのLV収縮機能に対する効果によってもたらされたものではない。ＭＥＡは13週でのLV肥大の代償性変化を阻止しなかったが、13週から19週にかけてのLV肥大のさらなる進行と心筋線維化を阻止した。ＭＥＡは13週でのLV径の減少を完全に阻止し、これがLV収縮末期壁応力の正常化を導いた。対照および未処置ラットに比べて、ＭＥＡは19週でのLV径を特徴的に拡張したが、このLV拡張はLV収縮末期壁応力を正常範囲以上には増大しなかった。
【００３２】
ＭＥＡは予想通りにLV線維化の抑制に効果的であった。また、予期しなかったことに、ＭＥＡは代償性肥大に続くさらなるLV肥大の進行をも妨げ、その結果、拡張不全型心不全への進行を阻止した。
【００３３】
ＭＥＡはLV形状に対する独特の効果を示した。代償性肥大段階（13週）で、未処置ラットのLV径は対照ラットのそれに比べて減少したが、ＭＥＡはこのような減少を阻止しLV径を対照ラットレベルに維持した。本研究では、未処置ラットのLV径が小さくなるほどLV収縮末期壁応力は亜正常となり、ＭＥＡはそれを正常化した。LV腔が比較的小さいことに起因する亜正常LV収縮末期壁応力を有する高血圧患者では、降圧治療はLV肥大の退行に有効でない。従って、この段階におけるLV径のＭＥＡ誘導による正常化は亜正常左室収縮末期壁応力を有する患者のLV肥大を退行させる上で降圧治療を補完できる。心不全段階（19週）において、ＭＥＡは、対照ラットや未処置ラットに比べてLV径を拡張した。LVの拡張は通常LV収縮末期壁応力とLV拡張末期圧の増大によって心不全を悪化させると考えられるが、ＭＥＡによって誘導されたLV径の拡張はLV収縮末期壁応力もLV拡張末期圧をも正常レベル以上に増大させなかった。LV拡張末期圧が同様であるならば、左心室が大きくなるほど左心室の硬さは小さくなる。従って、ＭＥＡによって処置したラットの左心室は対照ラットのそれよりも硬くないように思われた。LV硬度の増強は心不全進行の主要な原因である。ＭＥＡによって処置したラットのLV重量は対照ラットのそれよりも重く、LVヒドロキシプロリン量はこの二つの群間で差異はなかった。これらの結果から、ＭＥＡは、LV肥大とLV線維化を抑制するだけでなく、LV肥大とLV線維化に無関係なメカニズムで心室硬度を低下することでも、高血圧心における拡張不全型心不全の阻止に有益であることが示唆された。
【００３４】
以上より、ＭＥＡは降圧効果なしでLV線維化とLV肥大を抑制することによって高血圧心における拡張不全型心不全を有効に阻止することが実証された。拡張不全型心不全のＭＥＡ誘導型阻止に関するさらなる薬理学的調査は必要であるが、ＭＥＡは、臨床的頻度にもかかわらず治療戦略が未だ確立されていない拡張不全型心不全の治療方式としての可能性を有する。
【００３５】
【図面の簡単な説明】
【図１】年齢対応対照ラット、未処置ラットおよびＭＥＡ処置済みラットにおける収縮期血圧の変化を示すグラフ。
【図２】年齢対応対照ラット、未処置ラットおよびＭＥＡ処置済みラットにおけるLV拡張末期径の変化を示すグラフ。
【図３】年齢対応対照ラット、未処置ラットおよびＭＥＡ処置済みラットにおけるLV収縮末期径の変化を示すグラフ。
【図４】年齢対応対照ラット、未処置ラットおよびＭＥＡ処置済みラットにおけるLV内径短縮率の変化を示すグラフ。
【図５】年齢対応対照ラット、未処置ラットおよびＭＥＡ処置済みラットにおける左室壁中央部で評価した短縮率の変化を示すグラフ。
【図６】年齢対応対照ラット、未処置ラットおよびＭＥＡ処置済みラットにおけるLV重量／体重の変化を示すグラフ。
【図７】19週での年齢対応対照ラット、未処置ラットおよびＭＥＡ処置済みラットの左心室Azan Mallory染色の顕微鏡写真。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a prophylactic agent for diastolic heart failure. In particular, the present invention relates to a prophylactic agent for diastolic heart failure containing an effective amount of N-methylethanolamine or a pharmaceutically acceptable salt thereof.
[0002]
[Problems to be solved by the invention]
There are two types of heart failure: a “systolic dysfunction type” that shows a decrease in ejection fraction, and a “diastolic dysfunction type” that does not show a decrease in contractility. The latter accounts for 30 to 50% of heart failure. So far, the former has proposed a drug with clinically prognostic effect, but the latter has no established clinical utility (Circulation 1995; 92: 2764-2784, Eur. Heart J. 1998; 19: 990-1003), because of the poor prognosis, development of a therapeutic agent useful for the latter heart failure is desired. The present invention provides such a prophylactic agent for diastolic heart failure.
[0003]
[Means for Solving the Problems]
The present invention resides in a prophylactic agent for diastolic heart failure containing an effective amount of N-methylethanolamine or a pharmaceutically acceptable salt thereof.
[0004]
The most common cardiovascular disease in patients with diastolic heart failure is hypertension, and ventricular fibrosis and ventricular hypertrophy are likely to cause diastolic heart failure in this type of patient. In our study, Dahl-Iwai salt-sensitive (Dahl-S) rats bred with high-salt diet developed hypertension from 7 weeks and showed compensatory left ventricular (LV) hypertrophy around 13 weeks. Around the week it has been demonstrated to shift to diastolic heart failure. This model is used for tachycardia, exertional breathing, and apparent heart failure such as increased LV end-diastolic pressure and lack of activity due to increased lung weight based on pulmonary congestion due to increased LV end-diastolic diameter or reduced rate of internal diameter shortening. Showed signs. This symptom is associated with progressive LV hypertrophy and fibrosis, which can be the primary cause of hardened LV cavity and diastolic dysfunction. Therefore, the therapeutic goal for diastolic dysfunction must focus on the suppression and regression of LV hypertrophy and fibrosis.
[0005]
In our in vitro studies, N-methylethanolamine (MEA) promotes cellular responsiveness of human fibroblasts to growth factor cytokines such as basic fibroblast growth factor (bFGF) It has been proven. Thus, the present inventors have assumed the involvement of cytokines in heart failure, tried to apply MEA, and completed the present invention.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
According to the present invention, MEA or a pharmaceutically acceptable salt thereof is administered during the change from compensated cardiac hypertrophy associated with hypertension to heart failure.
[0007]
Pharmaceutically acceptable salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, phosphate, and acetate, fumarate, maleate, tartrate, citrate, Mention may be made of organic acid salts such as p-toluenesulfonate.
[0008]
The prophylactic agent of the present invention is administered to humans orally or parenterally (intravenous injection, infusion, etc.).
[0009]
Examples of the dosage form for oral administration include solid preparations such as tablets, granules, powders, fine granules, and hard capsules, and liquids such as syrups, elixirs, and soft capsules. Such a preparation is produced by a conventional method, and tablets, granules, powders, fine granules are prepared from MEA or a pharmaceutically acceptable salt thereof, such as lactose, starch, crystalline cellulose, magnesium stearate, hydroxypropyl cellulose, It is manufactured by mixing with normal pharmaceutical additives such as talc, and hard capsules are manufactured by appropriately filling the above-mentioned fine granules and powders into capsules.
[0010]
The syrup is prepared by dissolving or penetrating MEA or a pharmaceutically acceptable salt thereof together with a preservative such as methyl paraoxybenzoate and propyl paraoxybenzoate in an aqueous solution containing sucrose, D-sorbitol, carboxymethylcellulose and the like. The elixir is produced by mixing glycerin, orange oil, lemon oil, coriander oil, anise oil, talc and the like into an ethanol solution of MEA or a pharmaceutically acceptable salt thereof.
[0011]
Soft capsules are produced by dissolving or suspending MEA or a pharmaceutically acceptable salt thereof in a lipid excipient such as vegetable oil, oil emulsion, glycols, etc., and filling the capsules into soft capsules.
[0012]
An injection is prepared by dissolving or emulsifying MEA or a pharmaceutically acceptable salt thereof in physiological saline or a lipid excipient such as vegetable oil, oily emulsion, glycols, etc., and aseptically enclosing it in an ampoule or vial. Manufactured.
[0013]
The dose varies depending on the patient's age, weight, symptoms, administration method, and the like, but is generally 10 to 1000 mg / kg body weight / day.
[0014]
【The invention's effect】
Administration of MEA or a pharmaceutically acceptable salt thereof suppresses excessive hypertrophy and fibrosis of the left ventricle, increases the softness of the heart, and improves the expandability of the heart. The transition to is inhibited and the symptoms of diastolic heart failure are reduced.
[0015]
【Example】
The present invention will be further described with reference to the following test examples, examples and drawings, but the present invention is not limited to the following examples.
[0016]
Test Example 1: Acute toxicity test A solution of MEA (manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to pH 7 with hydrochloric acid at a rate of 0.2 ml / kg body weight (2 g / kg body weight as a sample) ICR male mice (5 weeks Age, body weight 24-28 g, 5 mice per group). Thereafter, the mice were observed for 7 days, but no deaths were observed in any of the sample administration groups.
[0017]
Test Example 2: Skin irritation test Japanese native male rabbit (weight approximately 3 kg) was used, and the Drates method (APPRAISAL OF THE SAFETY OF CHEMICALS IN FOODS, DRAGS AND COSMETOCS, p.46, 1959, Edited and Published by THE EDITORIAL COMMITTEE, ASSOCIATION OF FOOD & DRUG OFFICIALS OF USA). That is, a wound site (damaged skin) was created on the back of a rabbit with the hair cut, and 1 w / w of MEA (manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to pH 7 with hydrochloric acid on each damaged skin and normal skin. A 0.1% v% aqueous solution was infiltrated into a patch test adhesive bandage (1.2 × 1.6 cm, Ribbon Aid (trademark), manufactured by River Tape Pharmaceutical Co., Ltd.) and attached. After 24 hours, the adhesive bandage was peeled off, and the erythema and edema state of the skin were observed. The erythema score and edema score were assigned according to the following evaluation criteria.
[0018]
[Table 1]

[0019]
Based on this score and the following equation, a primary stimulation score was calculated.
Primary Irritation Score _{= 1/2 (A 24 +} A 48) +1/2 (X 24 + X 48) +1/2 (B 24 + B 48) +1/2 (Y 24 + Y 48)
here,
A _24: normal skin bandages sticking 24 hours after erythema score A _48: Normal plaster from the skin peeling 48 hours after erythema score B _24: injured skin bandages sticking 24 hours after erythema score B _48: plaster peeling from damage skin Erythema score X ₂₄ after 48 hours: edema score X ₄₈ 24 hours after application of adhesive bandage to normal skin Y ₄₈ : edema score Y ₂₄ 48 hours after application of adhesive bandage to normal skin Y ₄₈ score edema score 24 hours after application of adhesive bandage to damaged skin : Edema score 48 hours after removal of adhesive bandage from damaged skin Next, the degree of irritation of the test compound was determined based on the primary irritation score and the following criteria.
Mild stimulus: Primary stimulus score less than 0 to 2 Moderate stimulus: Primary stimulus score less than 2 to 5 Intensity stimulus: Primary stimulus score of 5 or more As a result, the MEA primary stimulus score is 0.25, and the stimulus degree is mild It was.
[0020]
Example
Samples and methods Subjects: Weaning male Dahl-S (DIS / Eis, Eisai, Tokyo, Japan) is raised in laboratory solid samples containing 0.3% NaCl, 7 weeks old and 8% NaCl Switched to a laboratory solid sample containing These rats were divided into two groups: The first group is a group (n = 7) in which hydrochloride of MEA (manufactured by Wako Pure Chemical Industries, Ltd.) is orally administered at a rate of 200 mg / kg / day from the age of 8 weeks, and the second group is an untreated group (N = 6). In addition, male Dahl-S rats fed continuously with laboratory solid samples containing 0.3% NaCl served as age-matched controls (n = 5). The mean systolic blood pressure at 8 weeks of age in rats fed a high salt diet was 161 mmHg and that in age-matched control rats was 130 mmHg.
[0021]
Echocardiographic studies were performed at 13 and 19 weeks of age. At 19 weeks of age, after examining the echocardiogram, a hemodynamic survey and cardiac recovery were performed. These schedules were determined according to our previous research results. During the experiment, feed and tap water were freely available. Systolic blood pressure was measured at 7, 13, and 19 weeks of age using a tail cuff system (BP-98A, Softron, Tokyo, Japan).
[0022]
Echocardiographic study: Transthoracic echocardiograms were recorded at 13 and 19 weeks of age (Doi R. et al., J. Hypertens, 2000; 18: 111-120). That is, the rat was anesthetized by intraperitoneal administration of ketamine HCl (50 mg / kg) and xylazine HCl (10 mg / kg), and the rat was held in a semi-left position with spontaneous breathing. Recording was performed using a commercially available echocardiograph equipped with a 7.5 MHz probe (SONOS 2000, Hewlett-Packard, Andover, Mass.), And the inner diameter and wall thickness of the left ventricle (LV) were measured. Moreover, LV weight and LV inner diameter shortening rate were calculated according to the following formula.
LV weight (g) = 1.04 x [(LVDd + PWd + AWd) ^3- LVDd ³ ] x 0.8 +0.14
LV inner diameter shortening rate (%) = (LVDd-LVDs) / LVDd x 100
Where LVDd: left ventricular end-diastolic diameter
PWd: Wall thickness before expansion
AWd: Wall thickness after expansion
LVDs: Left ventricular end systolic diameter In addition, “LV weight / body weight” obtained by correcting LV weight with respect to body weight was used as a statistical analysis value. To avoid overestimation of contractile function in the hypertrophied heart, the shortening rate at the center of the LV wall was also calculated using the Shimizu model.
[0023]
Hemodynamic study: After an echocardiographic study at 19 weeks of age, a tip-tip 1.5F catheter (SPR-407, Millar Instruments, Houston, Texas) calibrated to atmospheric pressure was passed through the right carotid artery Inserted into the left ventricle. LV pressure tracing and electrocardiogram are digitized to determine LV end-diastolic pressure, and non-zero asymptotic method (Yamamoto K. et al., Circulation, 1995; 91: 192-200) The time constant (τ) was calculated. LV end systolic wall stress was calculated using the Douglas method (Douglas PS et al., J Am Coll Cardiol. 1987; 9: 945-951).
[0024]
Tissue collection: After the blood line kinetic study, was immediately recovered the heart. The subventricular left ventricular apex was removed, weighed, immediately placed in liquid nitrogen, and stored at -80 ° C. to determine hydroxyproline content according to the method of Stegmann and Stalder. The results were calculated as hydroxyproline content per wet weight of tissue. The left ventricular sample for measuring the amount of mRNA was weighed, immediately placed in liquid nitrogen, and stored at −80 ° C. After weighing the other part, it was immersed in a 4% cold paraformaldehyde solution for 16-24 hours. Lungs were also collected and weighed.
[0025]
Pathological examination: Specimens soaked in 4% paraformaldehyde solution were embedded in paraffin. Azan Mallory staining method was used to examine a 2 μm thick cross section of the LV free wall at the papillary muscle level with a microscope at 100 × magnification.
[0026]
Statistical values: Results were expressed as mean values ± standard deviation values. One-way ANOVA and Fisher's protected least significant difference test was used to evaluate the difference between groups at specific stage. A probability value of p <0.05 was considered statistically significant. In the graphs of FIGS. 1-6, * is p <0.05 vs age-matched control rats, and # is p <0.05 vs untreated rats.
[0027]
Results Transition to diastolic heart failure in this model: systolic pressure in untreated rats was significantly increased at 13 and 19 weeks compared to age-matched control rats (Figure 1). At 13 weeks, the end-diastolic and end-systolic LV diameters of untreated rats were significantly smaller than those of control rats (FIG. 1). Therefore, LV end-systolic wall stress in untreated rats was significantly lower than that in control rats (47.5 ± 4.4 vs. 103.0 ± 9.4 10 ³ dynes / cm ² , p <0.05). The LV weight / body weight of untreated rats was significantly greater than that of control rats (FIG. 6). At 19 weeks, untreated rats showed obvious signs of heart failure such as tachypnea, exertional breathing, and loss of activity. In untreated rats, LV weight / body weight progressively increased from 13 to 19 weeks. At this time, the LV end-diastolic diameter and end-diastolic diameter (FIGS. 2 and 3) or the shortening rate (FIG. 5) evaluated at the center of the left ventricular wall was not different from that at 19 weeks. At 19 weeks, there was no difference in LV end systolic pressure between untreated and age-matched control rats (62.3 ± 9.4 vs. 80.0 ± 7.5 10 ³ dynes / cm ² ). As can be seen from Table 2 below showing the respective quantitative values at 19 weeks of control rats, untreated rats and MEA-treated rats, LV end-diastolic pressure (FIG. 2) and lung weight of untreated rats compared to control rats. The ratio to body weight (lung / body weight) was high, τ was long, and the left ventricular hydroxyproline level was high.
[0028]
[Table 2]

Each value is shown as an average value ± standard deviation value. * P <0.05 vs control; #p <0.05 vs untreated LVEDP = left ventricular end diastolic pressure; Pro-OH = hydroxyproline concentration; τ = LV relaxation time constant
Effect of MEA: Administration of MEA did not reduce systolic pressure at 13 or 19 weeks. Nevertheless, morphological changes occurred at 13 weeks as shown in FIGS. That is, LV weight / body weight increased to a level comparable to that of untreated rats, and LV end diastolic diameter expanded to the level of control rats, thereby normalizing LV end systolic wall stress (116.1 ± 12.5 10 ³ dynes / cm ²⁾ was derived. The shortening rate of the inner diameter was not significantly different from that of the control rats and was lower than that of the untreated rats, but the shortening rate evaluated at the center of the left ventricular wall was not affected by MEA.
[0030]
At 19 weeks, MEA-treated rats showed no obvious signs of heart failure. The LV end-diastolic pressure and lung / body weight of rats treated with MEA were significantly lower than that of untreated rats and did not differ from those of the control group (Table 2). As the pathological study (FIG. 7) shows, progressive interstitial fibrosis was observed in untreated rats but not in rats treated with MEA, and MEA normalizes the amount of hydroxyproline in the left ventricle (Table 2). MEA prevented the further increase in LV weight / body weight from weeks 13 to 19 observed in untreated rats (FIG. 6). MEA characteristically increased LV end-diastolic diameter at 19 weeks compared to control and untreated rats, but LV end-systolic wall stress did not increase (98.6 8.4 10 ³ dyne / cm ² ). Compared to untreated rats, τ was not changed by MEA (Table 2). The shortening rate evaluated at the center of the left ventricular wall at 19 weeks was not affected by MEA administration (FIG. 5).
[0031]
When MEA was administered for a long time after the onset of hypertension, MEA blocked the transition to overt diastolic heart failure without hypertensive effects in the hypertensive heart model. This was evidenced by the effect of MEA on LV end-diastolic pressure and lung weight. As evaluated by the inner diameter shortening rate and the shortening rate evaluated at the central part of the left ventricular wall, such a blocking effect is not caused by the effect of MEA on the LV contraction function. MEA did not prevent compensatory changes in LV hypertrophy at 13 weeks, but prevented further progression of LV hypertrophy and myocardial fibrosis from 13 to 19 weeks. MEA completely prevented the decrease in LV diameter at 13 weeks, which led to normalization of LV end systolic wall stress. Compared to control and untreated rats, MEA characteristically expanded LV diameter at 19 weeks, but this LV expansion did not increase LV end systolic wall stress beyond the normal range.
[0032]
MEA was effective in inhibiting LV fibrosis as expected. Unexpectedly, MEA also prevented the progression of further LV hypertrophy following compensatory hypertrophy, thus preventing progression to diastolic heart failure.
[0033]
The MEA showed a unique effect on the LV shape. At the compensatory hypertrophy stage (13 weeks), the LV diameter of untreated rats decreased compared to that of control rats, but MEA prevented such reduction and maintained the LV diameter at the control rat level. In this study, LV end-systolic wall stress became subnormal as the LV diameter of untreated rats decreased, and MEA normalized it. In hypertensive patients with subnormal LV end systolic wall stress due to the relatively small LV cavity, antihypertensive therapy is not effective in regressing LV hypertrophy. Therefore, normalization of LV diameter by MEA induction at this stage can complement antihypertensive treatment in regressing LV hypertrophy in patients with subnormal left ventricular end systolic wall stress. At the heart failure stage (19 weeks), MEA expanded the LV diameter compared to control and untreated rats. LV dilation is usually thought to exacerbate heart failure by increasing LV end-systolic wall stress and LV end-diastolic pressure, but MEA-induced LV dilatation normalizes both LV end-systolic wall stress and LV end-diastolic pressure It did not increase beyond the level. If the LV end-diastolic pressure is similar, the larger the left ventricle, the less rigid the left ventricle. Thus, the left ventricle of rats treated with MEA appeared to be less stiff than that of control rats. Increased LV hardness is a major cause of heart failure progression. The LV weight of rats treated with MEA was heavier than that of control rats, and the amount of LV hydroxyproline was not different between the two groups. From these results, MEA not only suppresses LV hypertrophy and LV fibrosis, but also reduces ventricular stiffness by a mechanism unrelated to LV hypertrophy and LV fibrosis, thereby preventing diastolic heart failure in hypertensive hearts. It was suggested that it is beneficial.
[0034]
From the above, it was demonstrated that MEA effectively inhibits diastolic heart failure in hypertensive heart by suppressing LV fibrosis and LV hypertrophy without hypotensive effect. Although further pharmacological investigations are needed on MEA-induced prevention of diastolic heart failure, MEA has potential as a treatment modality for diastolic heart failure for which clinical strategy has not yet been established despite clinical frequency Have
[0035]
[Brief description of the drawings]
FIG. 1 is a graph showing changes in systolic blood pressure in age-matched control rats, untreated rats and MEA-treated rats.
FIG. 2 is a graph showing changes in LV end-diastolic diameter in age-matched control rats, untreated rats and MEA-treated rats.
FIG. 3 is a graph showing changes in LV end-systolic diameter in age-matched control rats, untreated rats and MEA-treated rats.
FIG. 4 is a graph showing changes in LV inner diameter shortening rate in age-matched control rats, untreated rats and MEA-treated rats.
FIG. 5 is a graph showing the change in shortening rate evaluated at the center of the left ventricular wall in age-matched control rats, untreated rats and MEA-treated rats.
FIG. 6 is a graph showing changes in LV weight / body weight in age-matched control rats, untreated rats and MEA-treated rats.
FIG. 7: Photomicrographs of left ventricular Azan Mallory staining of age-matched control, untreated and MEA-treated rats at 19 weeks.

Claims (1)

A prophylactic agent for diastolic heart failure containing an effective amount of N-methylethanolamine or a pharmaceutically acceptable salt thereof.

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