WO2012011572A1 - Therapeutic agent for diastolic heart failure - Google Patents
Therapeutic agent for diastolic heart failure Download PDFInfo
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- WO2012011572A1 WO2012011572A1 PCT/JP2011/066707 JP2011066707W WO2012011572A1 WO 2012011572 A1 WO2012011572 A1 WO 2012011572A1 JP 2011066707 W JP2011066707 W JP 2011066707W WO 2012011572 A1 WO2012011572 A1 WO 2012011572A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/205—Amine addition salts of organic acids; Inner quaternary ammonium salts, e.g. betaine, carnitine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
- A61K31/221—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin with compounds having an amino group, e.g. acetylcholine, acetylcarnitine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
Definitions
- the present invention relates to a therapeutic agent for diastolic heart failure.
- Heart failure is a leading cause of death in industrialized countries, including Japan, and it reduces the quality of life for patients.
- Heart failure is categorized as systolic heart failure, in which the left ventricle expands and movement decreases (lower left ventricular ejection fraction), and diastolic heart failure, in which the left ventricle does not expand and left ventricular ejection fraction is maintained.
- the ratio of heart failure patients is about 3: 2, and both have a poor prognosis.
- Patent Document 1 describes a medicine containing acylcarnitine as an active ingredient for the prevention and / or treatment of a disease involving the production of inflammatory cytokines, and is one of the diseases involving the production of inflammatory cytokines. As heart failure is described. However, according to the results of a large-scale clinical trial for contractile heart failure conducted before the filing of Patent Document 1, it has been reported that suppressing inflammatory cytokines is not necessarily effective as a treatment for heart failure ( It is not scientifically supported that the invention described in Patent Document 1 is effective for heart failure.
- Patent Document 2 describes that propionyl L-carnitine suppresses left ventricular hypertrophy in end-stage renal disease dialysis patients.
- diastolic heart failure indicates a pathological condition in which the left ventricle does not dilate as described above, it cannot be inferred from the description in Patent Document 2 that propionyl L-carnitine is effective for diastolic heart failure. .
- Non-Patent Document 6 describes the use of L-carnitine for the treatment of diastolic heart failure. However, there is no “foundation for diagnosing heart failure” necessary to determine whether the target patient meets the criteria as a patient with diastolic dysfunction, and how blood pressure and pulse have changed as a result of treatment. Since there is no description about it, it is undeniable that the evaluation index has simply changed due to an antihypertensive action or the like, and that the expanded function has not been changed directly. Therefore, Non-Patent Document 6 cannot be said to scientifically demonstrate that L-carcinin is effective in the treatment of diastolic heart failure.
- JP 2006-347935 A JP-T 2009-511619
- an object of the present invention is to provide a medicament effective for the prevention or treatment of diastolic heart failure.
- the present invention includes the following inventions in order to solve the above problems.
- a preventive or therapeutic drug for diastolic heart failure comprising carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
- a myocardial sclerosis inhibitor comprising carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
- a myocardial fibrosis inhibitor comprising carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
- a prostacyclin increasing agent comprising carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
- Carnitine which is an active ingredient of the present invention, is a compound synthesized in vivo and is already commercially available as a pharmaceutical product for other purposes, so it is very useful in terms of low safety concerns.
- FIG. 3 shows OCTN2 mRNA and protein expression levels in the heart. It is a figure which shows the mRNA and protein expression level of OCTN2 in a kidney. It is a figure which shows the free carnitine excretion rate (FE) in the kidney of a control group and a non-treatment group. It is a figure which shows the measured value of [ ⁇ 3 > H] proline uptake
- FIG. 1 It is a figure which shows the measured value of 6-keto-PGF1 ⁇ concentration in the heart. It is a figure which shows the measured value of 6-keto-PGF1 ⁇ concentration in the culture supernatant of cultured cardiac fibroblasts. It is a figure which shows the mRNA expression level of FADS1 in the heart. It is a figure which shows the mRNA expression level of FADS2 in the heart. It is a figure which shows the mRNA expression level of FADS1 in a cultured cardiac fibroblast. It is a figure which shows the mRNA expression level of FADS2 in a cultured cardiac fibroblast. It is a figure which shows the arachidonic acid (AA) density
- FIG. 6 is a graph showing measured values of 6-keto-PGF1 ⁇ concentration in cultured cardiac fibroblasts knocked down by FADS1 or FADS2. It is a figure which shows the AA density
- the present invention provides a preventive or therapeutic agent for diastolic heart failure containing carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
- the present invention also provides a myocardial sclerosis inhibitor, a myocardial fibrosis inhibitor, and a prostacyclin-increasing agent containing carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
- the present inventors conducted comprehensive metabolomic analysis on blood and urine samples collected from diastolic heart failure model rats (Dahl salt-sensitive rats) and control rats. As a result, it was found that L-carnitine was contained in a substance in which various changes were observed, and in the heart tissue of a diastolic heart failure model rat, it was confirmed that the carnitine concentration decreased during the heart failure stage. Accordingly, the present inventors conducted an experiment in which L-carnitine was administered to a diastolic heart failure model rat. As shown in Example 1, L-carnitine was fibrotic to the myocardium accompanied by an increase in myocardial prostacyclin concentration. Based on the suppression, it was found that the myocardial sclerosis was suppressed, and as a result, the onset of diastolic heart failure could be suppressed.
- Carnitine or a derivative thereof is not particularly limited as long as it is carnitine, an isomer thereof, or a derivative thereof (for example, a derivative that is converted into carnitine in vivo).
- Examples thereof include L-carnitine, DL-carnitine, acyl-L-carnitine, acetyl-L-carnitine, propionyl-L-carnitine, carnitine chloride, carnitine orotate and the like.
- the pharmaceutically acceptable salt thereof is not particularly limited, and examples thereof include metal salts such as sodium salt and calcium salt, inorganic acid salts such as hydrochloride, carbonate and sulfate, acetate, malate and succinate.
- organic acid salts such as Specific examples include DL-carnitine hydrochloride, L-carnitine hydrochloride, L-carnitine fumarate, L-carnitine tartrate, L-carnitine magnesium citrate, N-acetyl-L-carnitine hydrochloride, and the like.
- Carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof used in the present invention is preferably L-carnitine, carnitine chloride, L-carnitine hydrochloride, L-carnitine fumarate, L-carnitine tartrate, acyl-L -Carnitine, acetyl-L-carnitine and propionyl-L-carnitine, more preferably L-carnitine, carnitine chloride, L-carnitine hydrochloride, L-carnitine fumarate and L-carnitine tartrate.
- the preventive or therapeutic agent for diastolic heart failure, myocardial sclerosis inhibitor, myocardial fibrosis inhibitor, and prostacyclin-increasing agent of the present invention comprises the above active ingredient, a pharmaceutically acceptable carrier, and an additive as appropriate. It can be formulated. Specifically, oral agents such as tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, emulsions, parenterals such as injections, infusions, suppositories, ointments, patches, etc. can do. What is necessary is just to set suitably about the mixture ratio of a carrier or an additive based on the range normally employ
- Carriers or additives that can be blended are not particularly limited.
- various carriers such as water, physiological saline, other aqueous solvents, aqueous or oily bases, excipients, binders, pH adjusters, disintegrants, absorption
- Various additives such as an accelerator, a lubricant, a colorant, a corrigent, and a fragrance are included.
- Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Leavening agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red mono oil or cherry.
- a liquid carrier such as fats and oils can be further contained in the above type of material.
- Sterile compositions for injection can be formulated according to conventional pharmaceutical practice, such as dissolving or suspending active substances in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like.
- aqueous liquid for injection for example, isotonic solutions containing physiological saline, glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) are used.
- alcohols eg, ethanol
- polyalcohols eg, propylene glycol, polyethylene glycol
- nonionic surfactants eg, polysorbate 80 TM , HCO-50
- oily liquid for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.
- Buffers eg, phosphate buffer, sodium acetate buffer
- soothing agents eg, benzalkonium chloride, procaine, etc.
- stabilizers eg, human serum albumin, polyethylene glycol, etc.
- storage You may mix
- the preparation thus obtained is safe and has low toxicity, for example, it is administered to humans and other mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) By doing so, diastolic heart failure can be effectively prevented or treated.
- prostacyclin can be increased to suppress myocardial fibrosis and to suppress myocardial sclerosis.
- the dose varies depending on the patient's condition, symptom, administration method and the like, but in the case of oral administration, for example, generally about 0.01 g to 40 g as an active ingredient per day for a human body weight of about 60 kg, preferably Is about 0.1 to 10 g, more preferably about 0.3 to 4 g.
- the single dose varies depending on the patient's condition, symptom, administration method, etc.
- it is usually about 0.01 to 100 g in a human body weight of 60 kg, for example.
- the total daily dose may be a single dose or divided doses.
- Example 1 Suppression of diastolic dysfunction by administration of carnitine
- 1-1 Experimental Method (1) Administration of L-carnitine to a diastolic dysfunction model (Dahl salt-sensitive rats) Male Dahl salt-sensitive rats were bred on a high salt diet (8% NaCl-containing diet) from day 1 of 6 weeks of age. This was used as a model for diastolic dysfunction (reference 1: Doi R, et al. J Hypertens 2000; 18: 111-120. Reference 11: Masuyama T, et al. J Am Coll Cardiol 2000; 36: 2333- 2338).
- Echocardiography and cardiac catheterization SONOS 5500 were applied to 20-week-old Dahl salt-sensitive rats under general anesthesia with Ketamine HCl (50 mg / kg, intraperitoneal injection). (Netherlands) was used for echocardiography (see Reference 1). After echocardiography, a 1.5-Fr high-fidelity, manometer tipped catheter (SPR-407; Millar Instruments, USA) was inserted into the left ventricle from the right carotid artery for cardiac catheterization. In cardiac catheterization, left ventricular end-diastolic pressure, left ventricular relaxation time constant (Tau), and myocardial stiffness constant were measured (Reference 2: Yoshida J, et al. Hypertension 2004; 43: 686-691.).
- Solute carrier family 22 organic cation transporter
- member 5 OCTN2
- fatty acid desaturase 1 FADS1
- FADS2 fatty acid desaturase 2
- TaqMan Gene Expression Assay TaqMan Gene Expression Assay (respective assay IDs are Rn00570533_m1, Rn01759794_g1, Rn00580220_m1; RT-PCR was performed using Applied Biosystems, USA. Each mRNA amount was corrected using the GAPDH mRNA amount as an internal control.
- 6-keto-prostaglandin F1 ⁇ (6-keto-PGF1 ⁇ ) concentration measurement 6-keto-PGF1 ⁇ (stable metabolite of prostacyclin) in the culture supernatant of the heart and cultured cardiac fibroblasts was enzyme immunoassay kit. (Cat. No. 900-004; Assay Design, USA).
- Systolic blood pressure, pulse and weight Table 1 shows the systolic blood pressure, pulse and weight at the age of 19 weeks.
- the systolic blood pressure and pulse were significantly increased compared with the control group by administration of the 8% high NaCl diet, but in the carnitine group, the systolic blood pressure and pulse were increased by L-carnitine administration. There was no significant effect. There was no difference in body weight among the three groups.
- FIG. 1 (a) Plasma Free Carnitine Concentration in Heart
- FIG. 1 (b) the free carnitine concentration in the heart
- FIGS. 1 (a) and 1 (b) the free carnitine concentrations in plasma and heart are both decreased in the untreated group (diastolic dysfunction model). Significantly increased. From these results, it was confirmed that the carnitine concentration decreased in the diastolic dysfunction model was improved by administration of L-carnitine.
- Echocardiographic examination and cardiac catheter examination Table 2 shows the results of the echocardiographic examination and cardiac catheter examination.
- the increase in relative wall thickness was reduced in the carnitine group compared with the untreated group.
- the other echocardiographic indices showed no change due to L-carnitine administration, and the left ventricular diameter shortening rate was maintained in both the untreated group and the carnitine group.
- Lung weight and left ventricular end-diastolic pressure increased significantly in the untreated group, but these increases were not observed in the carnitine group, indicating that the onset of diastolic dysfunction was prevented.
- FIG. 2 shows Azan Mall stained images of the heart of each group of rats. The left ventricular fibrosis area was significantly increased in the untreated group, but the increase was suppressed in the carnitine group (see Table 2). L-carnitine was thought to have improved myocardial stiffness constant by inhibiting left ventricular fibrosis.
- FIG. 3 (a) OCTN2 mRNA and protein expression levels in heart
- FIG. 3 (b) OCTN2 mRNA and protein expression levels in kidney
- the upper graph shows the mRNA expression level
- the lower graph shows the protein expression level (western blotting result).
- FE free carnitine excretion rate
- 6-keto-PGF1 ⁇ concentration The measured value of 6-keto-PGF1 ⁇ concentration in the heart is shown in FIG. 6 (a), and the measured value of 6-keto-PGF1 ⁇ concentration in the culture supernatant of cultured cardiac fibroblasts is shown in FIG. This is shown in 6 (b).
- the 6-keto-PGF1 ⁇ concentration in the heart was significantly increased in the carnitine group.
- the 6-keto-PGF1 ⁇ concentration in the culture supernatant of cultured cardiac fibroblasts was significantly increased by the addition of L-carnitine. From these results, it is considered that an increase in tissue prostacyclin concentration contributes to suppression of cardiac fibrosis, and it is clear that L-carnitine acts directly on fibroblasts to promote prostacyclin production. It was.
- FIG. 7 (a) shows the FADS1 mRNA expression level in the heart
- FIG. 7 (b) shows the FADS2 mRNA expression level.
- the FADS1 mRNA expression level in cultured cardiac fibroblasts is shown in FIG. 8 (a)
- the FADS2 mRNA expression level is shown in FIG. 8 (b).
- Prostacyclin is synthesized in vivo using AA as a precursor.
- FADS1 and FADS2 are AA synthases. As is apparent from FIGS. 7 (a) and (b) and FIGS.
- AA Concentration The measured value of AA concentration in the heart is shown in FIG. As is clear from FIG. 9, the AA concentration in the heart increased significantly in the carnitine group.
Abstract
It has been discovered that carnitine, a derivative thereof or a pharmaceutically acceptable salt thereof is useful as an active ingredient of a prophylactic or therapeutic agent for diastolic heart failure. The present invention is capable of providing a pharmaceutical agent that is effective for prophylaxis or treatment of diastolic heart failure, on which an effective therapeutic method has not been established yet.
Description
本発明は、拡張不全型心不全治療薬に関するものである。
The present invention relates to a therapeutic agent for diastolic heart failure.
心不全患者はわが国に２００万人以上存在すると推定される。心不全はわが国を含む先進国の主要な死因であり、また、患者の生活の質を低下させている。心不全は左心室が拡大し動きが低下する（左室駆出率が低下する）収縮不全型心不全と、左心室が拡大せず左室駆出率も保持されている拡張不全型心不全に分類される。心不全患者に占める比は３：２程度であり、ともに予後不良である。
It is estimated that there are more than 2 million heart failure patients in Japan. Heart failure is a leading cause of death in industrialized countries, including Japan, and it reduces the quality of life for patients. Heart failure is categorized as systolic heart failure, in which the left ventricle expands and movement decreases (lower left ventricular ejection fraction), and diastolic heart failure, in which the left ventricle does not expand and left ventricular ejection fraction is maintained. The The ratio of heart failure patients is about 3: 2, and both have a poor prognosis.
収縮不全型心不全では予後改善効果のある治療法が複数明らかとなっている。日米欧の循環器学会がそれぞれ作成している治療ガイドライン（非特許文献１～３参照）でもいくつかの標準的治療法が記載されるに至り、過去２０年間に生命予後は改善傾向にある。しかし、拡張不全型心不全では有効な治療方法が全く確立しておらず、過去２０年間に生命予後は改善していない。また、拡張不全型心不全は高齢者、女性に多いため、心不全全体に占める患者の割合は先進国で年々増加しており、この病態に対する治療方法の確立は急務である。
複数 Multiple treatments that have a prognostic improvement effect have been revealed for systolic heart failure. Treatment guidelines (see Non-Patent Documents 1 to 3) prepared by the Japanese, American and European Cardiovascular Society have also described some standard treatments, and the prognosis has been improving over the past 20 years. . However, no effective treatment has been established for diastolic heart failure, and life prognosis has not improved over the past 20 years. In addition, since diastolic heart failure is more common among the elderly and women, the proportion of patients in total heart failure is increasing year by year in developed countries, and establishment of a treatment for this condition is urgent.
特許文献１には、炎症性サイトカインの産生が関与する疾患の予防および／または治療用の、アシルカルニチンを有効成分として含む医薬が記載されており、炎症性サイトカインの産生が関与する疾患の１つとして心不全が記載されている。しかし、特許文献１の出願前に実施された収縮不全型心不全を対象とした大規模臨床試験結果によれば、炎症性サイトカインを抑制することが必ずしも心不全治療として有効でないことが報告されており（非特許文献４，５参照）、特許文献１に記載の発明が心不全に有効であることは科学的に支持されない。
Patent Document 1 describes a medicine containing acylcarnitine as an active ingredient for the prevention and / or treatment of a disease involving the production of inflammatory cytokines, and is one of the diseases involving the production of inflammatory cytokines. As heart failure is described. However, according to the results of a large-scale clinical trial for contractile heart failure conducted before the filing of Patent Document 1, it has been reported that suppressing inflammatory cytokines is not necessarily effective as a treatment for heart failure ( It is not scientifically supported that the invention described in Patent Document 1 is effective for heart failure.
特許文献２には、プロピオニルＬ－カルニチンが末期腎臓疾患透析患者の左室肥大を抑制することが記載されている。しかし、拡張不全型心不全は、上記のように左心室が拡張しない病態を示すものであることから、特許文献２の記載から、プロピオニルＬ－カルニチンが拡張不全型心不全に有効であることは推認できない。
Patent Document 2 describes that propionyl L-carnitine suppresses left ventricular hypertrophy in end-stage renal disease dialysis patients. However, since diastolic heart failure indicates a pathological condition in which the left ventricle does not dilate as described above, it cannot be inferred from the description in Patent Document 2 that propionyl L-carnitine is effective for diastolic heart failure. .
非特許文献６には、Ｌ－カルチニンを拡張不全型心不全の治療に用いることが記載されている。しかし、対象患者が拡張不全患者として基準を満たしているか否かを判断する上で必要な「心不全と診断した根拠」が記載されておらず、また治療により血圧や脈拍がどのように変化したかについて記載がないので単に降圧作用などで評価指標が変化しただけであり拡張機能を直接的に変化させたわけではない可能性が否定できない。したがって、非特許文献６は、Ｌ－カルチニンが拡張不全型心不全の治療に有効であることを科学的に実証しているとは言えない。
Non-Patent Document 6 describes the use of L-carnitine for the treatment of diastolic heart failure. However, there is no “foundation for diagnosing heart failure” necessary to determine whether the target patient meets the criteria as a patient with diastolic dysfunction, and how blood pressure and pulse have changed as a result of treatment. Since there is no description about it, it is undeniable that the evaluation index has simply changed due to an antihypertensive action or the like, and that the expanded function has not been changed directly. Therefore, Non-Patent Document 6 cannot be said to scientifically demonstrate that L-carcinin is effective in the treatment of diastolic heart failure.
上述のように、拡張不全型心不全の有効な治療方法は未だ確立されておらず、この病態に対する治療方法の早期確立が望まれている。そこで、本発明は、拡張不全型心不全の予防または治療に有効な医薬を提供することを目的とする。
As described above, an effective treatment method for diastolic heart failure has not yet been established, and early establishment of a treatment method for this disease state is desired. Therefore, an object of the present invention is to provide a medicament effective for the prevention or treatment of diastolic heart failure.
本発明は、上記課題を解決するために、以下の各発明を包含する。
［１］カルニチンもしくはその誘導体またはその薬学的に許容される塩を有効成分として含有することを特徴とする拡張不全型心不全の予防または治療薬。
［２］カルニチンもしくはその誘導体またはその薬学的に許容される塩を有効成分として含有することを特徴とする心筋硬化抑制剤。
［３］カルニチンもしくはその誘導体またはその薬学的に許容される塩を有効成分として含有することを特徴とする心筋線維化抑制剤。
［４］カルニチンもしくはその誘導体またはその薬学的に許容される塩を有効成分として含有することを特徴とするプロスタサイクリン増加剤。
［５］プロスタサイクリンが心筋プロスタサイクリンである前記［４］に記載のプロスタサイクリン増加剤。
［６］哺乳動物に対して、カルニチンもしくはその誘導体またはその薬学的に許容される塩の有効量を投与することを特徴とする拡張不全型心不全の予防または治療方法。
［７］拡張不全型心不全の予防または治療薬を製造するための、カルニチンもしくはその誘導体またはその薬学的に許容される塩の使用。
［８］拡張不全型心不全の予防または治療に使用するための、カルニチンもしくはその誘導体またはその薬学的に許容される塩。 The present invention includes the following inventions in order to solve the above problems.
[1] A preventive or therapeutic drug for diastolic heart failure, comprising carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
[2] A myocardial sclerosis inhibitor comprising carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
[3] A myocardial fibrosis inhibitor comprising carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
[4] A prostacyclin increasing agent comprising carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
[5] The prostacyclin increasing agent according to the above [4], wherein the prostacyclin is a myocardial prostacyclin.
[6] A method for preventing or treating diastolic heart failure, comprising administering an effective amount of carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof to a mammal.
[7] Use of carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof for the manufacture of a preventive or therapeutic agent for diastolic heart failure.
[8] Carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of diastolic heart failure.
［１］カルニチンもしくはその誘導体またはその薬学的に許容される塩を有効成分として含有することを特徴とする拡張不全型心不全の予防または治療薬。
［２］カルニチンもしくはその誘導体またはその薬学的に許容される塩を有効成分として含有することを特徴とする心筋硬化抑制剤。
［３］カルニチンもしくはその誘導体またはその薬学的に許容される塩を有効成分として含有することを特徴とする心筋線維化抑制剤。
［４］カルニチンもしくはその誘導体またはその薬学的に許容される塩を有効成分として含有することを特徴とするプロスタサイクリン増加剤。
［５］プロスタサイクリンが心筋プロスタサイクリンである前記［４］に記載のプロスタサイクリン増加剤。
［６］哺乳動物に対して、カルニチンもしくはその誘導体またはその薬学的に許容される塩の有効量を投与することを特徴とする拡張不全型心不全の予防または治療方法。
［７］拡張不全型心不全の予防または治療薬を製造するための、カルニチンもしくはその誘導体またはその薬学的に許容される塩の使用。
［８］拡張不全型心不全の予防または治療に使用するための、カルニチンもしくはその誘導体またはその薬学的に許容される塩。 The present invention includes the following inventions in order to solve the above problems.
[1] A preventive or therapeutic drug for diastolic heart failure, comprising carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
[2] A myocardial sclerosis inhibitor comprising carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
[3] A myocardial fibrosis inhibitor comprising carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
[4] A prostacyclin increasing agent comprising carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
[5] The prostacyclin increasing agent according to the above [4], wherein the prostacyclin is a myocardial prostacyclin.
[6] A method for preventing or treating diastolic heart failure, comprising administering an effective amount of carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof to a mammal.
[7] Use of carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof for the manufacture of a preventive or therapeutic agent for diastolic heart failure.
[8] Carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of diastolic heart failure.
本発明により、これまで有効な治療法が皆無であった拡張不全型心不全の予防または治療薬を提供することができる。本発明の有効成分であるカルニチンは、生体内で合成される化合物であり、すでに他の目的で医薬品として市販されていることから、安全性に対する懸念が低い点で非常に有用である。
According to the present invention, it is possible to provide a preventive or therapeutic agent for diastolic heart failure, for which there has been no effective treatment until now. Carnitine, which is an active ingredient of the present invention, is a compound synthesized in vivo and is already commercially available as a pharmaceutical product for other purposes, so it is very useful in terms of low safety concerns.
本発明は、カルニチンもしくはその誘導体またはその薬学的に許容される塩を有効成分として含有する拡張不全型心不全の予防または治療薬を提供する。また、本発明は、カルニチンもしくはその誘導体またはその薬学的に許容される塩を有効成分として含有する心筋硬化抑制剤、心筋線維化抑制剤およびプロスタサイクリン増加剤を提供する。
The present invention provides a preventive or therapeutic agent for diastolic heart failure containing carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient. The present invention also provides a myocardial sclerosis inhibitor, a myocardial fibrosis inhibitor, and a prostacyclin-increasing agent containing carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
本発明者らは、拡張不全型心不全モデルラット（Ｄａｈｌ食塩感受性ラット）およびコントロールラットから採取した血液および尿サンプルを対象として、網羅的なメタボローム解析を行った結果、拡張不全型心不全モデルラットにおいて有意な変化が認められた物質中にＬ－カルニチンが含まれていることを見出し、拡張不全型心不全モデルラットの心臓組織において、心不全期にはカルニチン濃度が低下していることを確認した。そこで、本発明者らは、拡張不全型心不全モデルラットにＬ－カルニチンを投与する実験を行ったところ、実施例１に示すように、Ｌ－カルニチンは心筋プロスタサイクリン濃度上昇を伴う心筋の線維化抑制に基づいて心筋の硬化を抑制し、その結果拡張不全型心不全の発症を抑制できることを見出した。
The present inventors conducted comprehensive metabolomic analysis on blood and urine samples collected from diastolic heart failure model rats (Dahl salt-sensitive rats) and control rats. As a result, it was found that L-carnitine was contained in a substance in which various changes were observed, and in the heart tissue of a diastolic heart failure model rat, it was confirmed that the carnitine concentration decreased during the heart failure stage. Accordingly, the present inventors conducted an experiment in which L-carnitine was administered to a diastolic heart failure model rat. As shown in Example 1, L-carnitine was fibrotic to the myocardium accompanied by an increase in myocardial prostacyclin concentration. Based on the suppression, it was found that the myocardial sclerosis was suppressed, and as a result, the onset of diastolic heart failure could be suppressed.
カルニチンもしくはその誘導体は、カルニチン、その異性体、その誘導体（例えば生体内でカルニチンに変換される誘導体）であれば特に限定されない。例えば、Ｌ－カルニチン、ＤＬ－カルニチン、アシル－Ｌ－カルニチン、アセチル－Ｌ－カルニチン、プロピオニル－Ｌ－カルニチン、塩化カルニチン、オロチン酸カルニチンなどが挙げられる。
その薬学的に許容される塩は特に限定されず、例えば、ナトリウム塩、カルシウム塩などの金属塩、塩酸塩、炭酸塩、硫酸塩などの無機酸塩、酢酸塩、リンゴ酸塩、コハク酸塩などの有機酸塩などが挙げられる。具体的には、ＤＬ－カルニチン塩酸塩、Ｌ－カルニチン塩酸塩、Ｌ－カルニチンフマル酸塩、Ｌ－カルニチン酒石酸塩、Ｌ－カルニチンマグネシウムクエン酸塩、Ｎ－アセチル－Ｌ－カルニチン塩酸塩などが挙げられる。 Carnitine or a derivative thereof is not particularly limited as long as it is carnitine, an isomer thereof, or a derivative thereof (for example, a derivative that is converted into carnitine in vivo). Examples thereof include L-carnitine, DL-carnitine, acyl-L-carnitine, acetyl-L-carnitine, propionyl-L-carnitine, carnitine chloride, carnitine orotate and the like.
The pharmaceutically acceptable salt thereof is not particularly limited, and examples thereof include metal salts such as sodium salt and calcium salt, inorganic acid salts such as hydrochloride, carbonate and sulfate, acetate, malate and succinate. And organic acid salts such as Specific examples include DL-carnitine hydrochloride, L-carnitine hydrochloride, L-carnitine fumarate, L-carnitine tartrate, L-carnitine magnesium citrate, N-acetyl-L-carnitine hydrochloride, and the like. .
その薬学的に許容される塩は特に限定されず、例えば、ナトリウム塩、カルシウム塩などの金属塩、塩酸塩、炭酸塩、硫酸塩などの無機酸塩、酢酸塩、リンゴ酸塩、コハク酸塩などの有機酸塩などが挙げられる。具体的には、ＤＬ－カルニチン塩酸塩、Ｌ－カルニチン塩酸塩、Ｌ－カルニチンフマル酸塩、Ｌ－カルニチン酒石酸塩、Ｌ－カルニチンマグネシウムクエン酸塩、Ｎ－アセチル－Ｌ－カルニチン塩酸塩などが挙げられる。 Carnitine or a derivative thereof is not particularly limited as long as it is carnitine, an isomer thereof, or a derivative thereof (for example, a derivative that is converted into carnitine in vivo). Examples thereof include L-carnitine, DL-carnitine, acyl-L-carnitine, acetyl-L-carnitine, propionyl-L-carnitine, carnitine chloride, carnitine orotate and the like.
The pharmaceutically acceptable salt thereof is not particularly limited, and examples thereof include metal salts such as sodium salt and calcium salt, inorganic acid salts such as hydrochloride, carbonate and sulfate, acetate, malate and succinate. And organic acid salts such as Specific examples include DL-carnitine hydrochloride, L-carnitine hydrochloride, L-carnitine fumarate, L-carnitine tartrate, L-carnitine magnesium citrate, N-acetyl-L-carnitine hydrochloride, and the like. .
本発明に用いるカルニチンもしくはその誘導体またはその薬学的に許容される塩として、好ましくは、Ｌ－カルニチン、塩化カルニチン、Ｌ－カルニチン塩酸塩、Ｌ－カルニチンフマル酸塩、Ｌ－カルニチン酒石酸塩、アシル－Ｌ－カルニチン、アセチル－Ｌ－カルニチンおよびプロピオニル－Ｌ－カルニチンであり、より好ましくは、Ｌ－カルニチン、塩化カルニチン、Ｌ－カルニチン塩酸塩、Ｌ－カルニチンフマル酸塩およびＬ－カルニチン酒石酸塩である。
Carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof used in the present invention is preferably L-carnitine, carnitine chloride, L-carnitine hydrochloride, L-carnitine fumarate, L-carnitine tartrate, acyl-L -Carnitine, acetyl-L-carnitine and propionyl-L-carnitine, more preferably L-carnitine, carnitine chloride, L-carnitine hydrochloride, L-carnitine fumarate and L-carnitine tartrate.
本発明の拡張不全型心不全の予防または治療薬、心筋硬化抑制剤、心筋線維化抑制剤、プロスタサイクリン増加剤は、上記有効成分と薬学的に許容される担体、さらに添加剤を適宜配合して製剤化することができる。具体的には錠剤、被覆錠剤、丸剤、散剤、顆粒剤、カプセル剤、液剤、懸濁剤、乳剤等の経口剤、注射剤、輸液、坐剤、軟膏、パッチ剤等の非経口剤とすることができる。担体または添加剤の配合割合については、医薬品分野において通常採用されている範囲に基づいて適宜設定すればよい。配合できる担体または添加剤は特に制限されないが、例えば、水、生理食塩水、その他の水性溶媒、水性または油性基剤等の各種担体、賦形剤、結合剤、ｐＨ調整剤、崩壊剤、吸収促進剤、滑沢剤、着色剤、矯味剤、香料等の各種添加剤が挙げられる。
The preventive or therapeutic agent for diastolic heart failure, myocardial sclerosis inhibitor, myocardial fibrosis inhibitor, and prostacyclin-increasing agent of the present invention comprises the above active ingredient, a pharmaceutically acceptable carrier, and an additive as appropriate. It can be formulated. Specifically, oral agents such as tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, emulsions, parenterals such as injections, infusions, suppositories, ointments, patches, etc. can do. What is necessary is just to set suitably about the mixture ratio of a carrier or an additive based on the range normally employ | adopted in the pharmaceutical field | area. Carriers or additives that can be blended are not particularly limited. For example, various carriers such as water, physiological saline, other aqueous solvents, aqueous or oily bases, excipients, binders, pH adjusters, disintegrants, absorption Various additives such as an accelerator, a lubricant, a colorant, a corrigent, and a fragrance are included.
錠剤、カプセル剤などに混和することができる添加剤としては、例えば、ゼラチン、コーンスターチ、トラガント、アラビアゴムのような結合剤、結晶性セルロースのような賦形剤、コーンスターチ、ゼラチン、アルギン酸などのような膨化剤、ステアリン酸マグネシウムのような潤滑剤、ショ糖、乳糖またはサッカリンのような甘味剤、ペパーミント、アカモノ油またはチェリーのような香味剤などが用いられる。調剤単位形態がカプセルである場合には、上記タイプの材料にさらに油脂のような液状担体を含有することができる。注射のための無菌組成物は注射用水のようなベヒクル中の活性物質、胡麻油、椰子油などのような天然産出植物油などを溶解または懸濁させるなどの通常の製剤実施に従って処方することができる。注射用の水性液としては、例えば、生理食塩水、ブドウ糖やその他の補助薬を含む等張液（例えば、Ｄ－ソルビトール、Ｄ－マンニトール、塩化ナトリウムなど）などが用いられ、適当な溶解補助剤、例えば、アルコール（例、エタノール）、ポリアルコール（例、プロピレングリコール、ポリエチレングリコール）、非イオン性界面活性剤（例、ポリソルベート８０ＴＭ、ＨＣＯ－５０）などと併用してもよい。油性液としては、例えば、ゴマ油、大豆油などが用いられ、溶解補助剤である安息香酸ベンジル、ベンジルアルコールなどと併用してもよい。また、緩衝剤（例えば、リン酸塩緩衝液、酢酸ナトリウム緩衝液）、無痛化剤（例えば、塩化ベンザルコニウム、塩酸プロカインなど）、安定剤（例えば、ヒト血清アルブミン、ポリエチレングリコールなど）、保存剤（例えば、ベンジルアルコール、フェノールなど）、酸化防止剤などと配合してもよい。
Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Leavening agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red mono oil or cherry. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above type of material. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice, such as dissolving or suspending active substances in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As an aqueous liquid for injection, for example, isotonic solutions containing physiological saline, glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) are used. For example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80 ™ , HCO-50) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. Buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), storage You may mix | blend with an agent (for example, benzyl alcohol, phenol, etc.), antioxidant, etc.
このようにして得られる製剤は安全で低毒性であるので、例えば、ヒトや他の哺乳動物（例えば、ラット、マウス、ウサギ、ヒツジ、ブタ、ウシ、ネコ、イヌ、サルなど）に対して投与することにより、拡張不全型心不全を有効に予防または治療することができる。
また、プロスタサイクリンを増加させ、心筋の線維化を抑制し、心筋の硬化を抑制することができる。 Since the preparation thus obtained is safe and has low toxicity, for example, it is administered to humans and other mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) By doing so, diastolic heart failure can be effectively prevented or treated.
In addition, prostacyclin can be increased to suppress myocardial fibrosis and to suppress myocardial sclerosis.
また、プロスタサイクリンを増加させ、心筋の線維化を抑制し、心筋の硬化を抑制することができる。 Since the preparation thus obtained is safe and has low toxicity, for example, it is administered to humans and other mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) By doing so, diastolic heart failure can be effectively prevented or treated.
In addition, prostacyclin can be increased to suppress myocardial fibrosis and to suppress myocardial sclerosis.
投与量は、患者の状態、症状、投与方法などにより差異はあるが、経口投与の場合、一般的に例えば、体重約６０ｋｇのヒトにおいては、１日当たり有効成分として約０．０１ｇ～４０ｇ、好ましくは約０．１ｇ～１０ｇ、より好ましくは約０．３ｇ～４ｇである。非経口的に投与する場合は、その１回投与量は患者の状態、症状、投与方法などによっても異なるが、例えば注射剤では、通常例えば体重６０ｋｇのヒトにおいては、約０．０１ｇ～１００ｇ程度、好ましくは約０．１ｇ～２０ｇ程度、より好ましくは約０．５ｇ～１０ｇ程度を静脈注射により投与するのが好都合である。１日当たりの総投与量は、単一投与量であっても分割投与量であってもよい。
The dose varies depending on the patient's condition, symptom, administration method and the like, but in the case of oral administration, for example, generally about 0.01 g to 40 g as an active ingredient per day for a human body weight of about 60 kg, preferably Is about 0.1 to 10 g, more preferably about 0.3 to 4 g. When administered parenterally, the single dose varies depending on the patient's condition, symptom, administration method, etc. For example, in the case of an injection, it is usually about 0.01 to 100 g in a human body weight of 60 kg, for example. It is convenient to administer about 0.1 to 20 g, more preferably about 0.5 to 10 g by intravenous injection. The total daily dose may be a single dose or divided doses.
以下、実施例により本発明を詳細に説明するが、本発明はこれらに限定されるものではない。
Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.
〔実施例１：カルニチン投与による拡張不全の抑制〕
１－１ 実験方法
（１）拡張不全モデル（Ｄａｈｌ食塩感受性ラット）へのＬ－カルニチン投与
雄のＤａｈｌ食塩感受性ラットを６週齢１日目より高食塩食（８％ＮａＣｌ含有食）で飼育し、これを拡張不全モデルとして使用した（参考文献１：Doi R, et al. J Hypertens 2000; 18:111-120. 参考文献１１：Masuyama T, et al. J Am Coll Cardiol 2000;36:2333-2338）。コントロール群（通常食（０．３％ＮａＣｌ含有食）を継続した雄のＤａｈｌ食塩感受性ラット）、拡張不全モデルの無治療群（高食塩食＝８％ＮａＣｌ含有食を６週齢１日目より開始)、拡張不全モデルにカルニチン投与を行った群（拡張不全モデルにおいて、１７週齢から食餌を１％Ｌ－カルニチン（和光純薬工業、日本）混合高食塩食（食塩含有量は８％ＮａＣｌのまま）に変更）の３群に分けた。 [Example 1: Suppression of diastolic dysfunction by administration of carnitine]
1-1 Experimental Method (1) Administration of L-carnitine to a diastolic dysfunction model (Dahl salt-sensitive rats) Male Dahl salt-sensitive rats were bred on a high salt diet (8% NaCl-containing diet) fromday 1 of 6 weeks of age. This was used as a model for diastolic dysfunction (reference 1: Doi R, et al. J Hypertens 2000; 18: 111-120. Reference 11: Masuyama T, et al. J Am Coll Cardiol 2000; 36: 2333- 2338). Control group (male Dahl salt-sensitive rats with normal diet (0.3% NaCl-containing diet) continued), no treatment group of diastolic dysfunction model (high salt diet = 8% NaCl-containing diet from day 1 of 6 weeks old) Start), group in which carnitine was administered to the diastolic dysfunction model (in the diastolic dysfunction model, 1% L-carnitine (Wako Pure Chemical Industries, Japan) mixed high-saline diet from 17 weeks of age) (salt content is 8% NaCl It was divided into 3 groups.
１－１ 実験方法
（１）拡張不全モデル（Ｄａｈｌ食塩感受性ラット）へのＬ－カルニチン投与
雄のＤａｈｌ食塩感受性ラットを６週齢１日目より高食塩食（８％ＮａＣｌ含有食）で飼育し、これを拡張不全モデルとして使用した（参考文献１：Doi R, et al. J Hypertens 2000; 18:111-120. 参考文献１１：Masuyama T, et al. J Am Coll Cardiol 2000;36:2333-2338）。コントロール群（通常食（０．３％ＮａＣｌ含有食）を継続した雄のＤａｈｌ食塩感受性ラット）、拡張不全モデルの無治療群（高食塩食＝８％ＮａＣｌ含有食を６週齢１日目より開始)、拡張不全モデルにカルニチン投与を行った群（拡張不全モデルにおいて、１７週齢から食餌を１％Ｌ－カルニチン（和光純薬工業、日本）混合高食塩食（食塩含有量は８％ＮａＣｌのまま）に変更）の３群に分けた。 [Example 1: Suppression of diastolic dysfunction by administration of carnitine]
1-1 Experimental Method (1) Administration of L-carnitine to a diastolic dysfunction model (Dahl salt-sensitive rats) Male Dahl salt-sensitive rats were bred on a high salt diet (8% NaCl-containing diet) from
（２）心エコー図検査および心臓カテーテル検査
Ketamine HCl（50 mg/kg、腹腔内注射）にて全身麻酔下にある２０週齢のＤａｈｌ食塩感受性ラットに対して、SONOS 5500（Philips Medical System社、オランダ）を用いて心エコー図検査を施行した（参考文献１参照）。
心エコー図検査後に、右頸動脈より左室へ1.5-Frのhigh-fidelity，manometertipped catheter（SPR-407; Millar Instruments社、米国）を挿入して心臓カテーテル検査を施行した。心臓カテーテル検査では、左室拡張末期圧、左室弛緩時定数（Tau）、心筋スティッフネス定数を測定した（参考文献２：Yoshida J, et al. Hypertension 2004; 43:686-691.）。 (2) Echocardiography and cardiac catheterization SONOS 5500 (Philips Medical System, Inc.) was applied to 20-week-old Dahl salt-sensitive rats under general anesthesia with Ketamine HCl (50 mg / kg, intraperitoneal injection). (Netherlands) was used for echocardiography (see Reference 1).
After echocardiography, a 1.5-Fr high-fidelity, manometer tipped catheter (SPR-407; Millar Instruments, USA) was inserted into the left ventricle from the right carotid artery for cardiac catheterization. In cardiac catheterization, left ventricular end-diastolic pressure, left ventricular relaxation time constant (Tau), and myocardial stiffness constant were measured (Reference 2: Yoshida J, et al. Hypertension 2004; 43: 686-691.).
Ketamine HCl（50 mg/kg、腹腔内注射）にて全身麻酔下にある２０週齢のＤａｈｌ食塩感受性ラットに対して、SONOS 5500（Philips Medical System社、オランダ）を用いて心エコー図検査を施行した（参考文献１参照）。
心エコー図検査後に、右頸動脈より左室へ1.5-Frのhigh-fidelity，manometertipped catheter（SPR-407; Millar Instruments社、米国）を挿入して心臓カテーテル検査を施行した。心臓カテーテル検査では、左室拡張末期圧、左室弛緩時定数（Tau）、心筋スティッフネス定数を測定した（参考文献２：Yoshida J, et al. Hypertension 2004; 43:686-691.）。 (2) Echocardiography and cardiac catheterization SONOS 5500 (Philips Medical System, Inc.) was applied to 20-week-old Dahl salt-sensitive rats under general anesthesia with Ketamine HCl (50 mg / kg, intraperitoneal injection). (Netherlands) was used for echocardiography (see Reference 1).
After echocardiography, a 1.5-Fr high-fidelity, manometer tipped catheter (SPR-407; Millar Instruments, USA) was inserted into the left ventricle from the right carotid artery for cardiac catheterization. In cardiac catheterization, left ventricular end-diastolic pressure, left ventricular relaxation time constant (Tau), and myocardial stiffness constant were measured (Reference 2: Yoshida J, et al. Hypertension 2004; 43: 686-691.).
（３）血液・尿・臓器の回収と保存
心臓カテーテル検査後、速やかに膀胱穿刺による採尿、下大静脈穿刺による採血を施行し、遠心後の尿上清と血漿を－８０℃で保存した。
心臓は、大動脈より逆行性に４℃生理食塩水による灌流処理後に液体窒素で凍結し、－８０℃で保存した。腎臓は、腎動脈の近位部と遠位部をクリッピングで遮断した腹部大動脈から４℃生理食塩水にて灌流処理を行った後、液体窒素で凍結し、－８０℃で保存した。また、心臓の一部を１０％ホルマリンで固定後、パラフィンに包埋した。 (3) Collection and storage of blood, urine and organs After cardiac catheterization, urine collection by bladder puncture and blood collection by inferior vena cava puncture were performed, and the urine supernatant and plasma after centrifugation were stored at -80 ° C.
The heart was retroperfused from the aorta, frozen with liquid nitrogen after perfusion with 4 ° C. saline, and stored at −80 ° C. The kidney was perfused with 4 ° C. physiological saline from the abdominal aorta where the proximal and distal portions of the renal artery were blocked by clipping, frozen with liquid nitrogen, and stored at −80 ° C. A part of the heart was fixed with 10% formalin and then embedded in paraffin.
心臓カテーテル検査後、速やかに膀胱穿刺による採尿、下大静脈穿刺による採血を施行し、遠心後の尿上清と血漿を－８０℃で保存した。
心臓は、大動脈より逆行性に４℃生理食塩水による灌流処理後に液体窒素で凍結し、－８０℃で保存した。腎臓は、腎動脈の近位部と遠位部をクリッピングで遮断した腹部大動脈から４℃生理食塩水にて灌流処理を行った後、液体窒素で凍結し、－８０℃で保存した。また、心臓の一部を１０％ホルマリンで固定後、パラフィンに包埋した。 (3) Collection and storage of blood, urine and organs After cardiac catheterization, urine collection by bladder puncture and blood collection by inferior vena cava puncture were performed, and the urine supernatant and plasma after centrifugation were stored at -80 ° C.
The heart was retroperfused from the aorta, frozen with liquid nitrogen after perfusion with 4 ° C. saline, and stored at −80 ° C. The kidney was perfused with 4 ° C. physiological saline from the abdominal aorta where the proximal and distal portions of the renal artery were blocked by clipping, frozen with liquid nitrogen, and stored at −80 ° C. A part of the heart was fixed with 10% formalin and then embedded in paraffin.
（４）左室線維化面積
パラフィン包埋心臓サンプルから左室乳頭筋レベルのＡｚａｎ Ｍａｌｌｏｒｙ染色標本を作成し、左室線維化面積測定を行った（参考文献１参照）。 (4) Left ventricular fibrosis area An Azan Mall-stained specimen of the left ventricular papillary muscle level was prepared from a paraffin-embedded heart sample, and the left ventricular fibrosis area was measured (see Reference 1).
パラフィン包埋心臓サンプルから左室乳頭筋レベルのＡｚａｎ Ｍａｌｌｏｒｙ染色標本を作成し、左室線維化面積測定を行った（参考文献１参照）。 (4) Left ventricular fibrosis area An Azan Mall-stained specimen of the left ventricular papillary muscle level was prepared from a paraffin-embedded heart sample, and the left ventricular fibrosis area was measured (see Reference 1).
（５）心臓線維芽細胞の採取・培養
生後１日目の新生児Ｗｉｓｔａｒラットより、心臓線維芽細胞の回収を行った（参考文献３：Kinugawa KI, et al. Am J Physiol. 1997; 272: H35-47.）。回収後の心臓線維芽細胞は、５％ＣＯ２インキュベーター内で、１０％ウシ胎児血清（FCS; Invitrogen社、米国）入りDulbecco’s modified Eagle’s medium（DMEM; Invitrogen社、米国）を用いて培養を行った。以後４８時間毎に継代培養を行い、２回目の継代後の心臓線維芽細胞を実験に使用した。
薬剤あるいは［３Ｈ］プロリン導入の２４時間前に、培養液をＦＣＳ無しのＤＭＥＭへ交換した。また、薬剤導入無しのＤＭＥＭを各実験のコントロール群とした。回収した心臓線維芽細胞と培養液上清は、－８０℃で保存した。 (5) Collection and culture of cardiac fibroblasts Cardiac fibroblasts were collected from neonatal Wistar rats on the first day of life (Reference 3: Kinugawa KI, et al. Am J Physiol. 1997; 272: H35). -47.) The collected cardiac fibroblasts were cultured in a 5% CO 2 incubator using Dulbecco's modified Eagle's medium (DMEM; Invitrogen, USA) containing 10% fetal calf serum (FCS; Invitrogen, USA). . Thereafter, subculture was performed every 48 hours, and cardiac fibroblasts after the second passage were used for the experiment.
The culture medium was replaced with DMEM without FCS 24 hours before introduction of the drug or [ 3 H] proline. Moreover, DMEM without drug introduction was used as a control group for each experiment. The collected cardiac fibroblasts and culture supernatant were stored at −80 ° C.
生後１日目の新生児Ｗｉｓｔａｒラットより、心臓線維芽細胞の回収を行った（参考文献３：Kinugawa KI, et al. Am J Physiol. 1997; 272: H35-47.）。回収後の心臓線維芽細胞は、５％ＣＯ２インキュベーター内で、１０％ウシ胎児血清（FCS; Invitrogen社、米国）入りDulbecco’s modified Eagle’s medium（DMEM; Invitrogen社、米国）を用いて培養を行った。以後４８時間毎に継代培養を行い、２回目の継代後の心臓線維芽細胞を実験に使用した。
薬剤あるいは［３Ｈ］プロリン導入の２４時間前に、培養液をＦＣＳ無しのＤＭＥＭへ交換した。また、薬剤導入無しのＤＭＥＭを各実験のコントロール群とした。回収した心臓線維芽細胞と培養液上清は、－８０℃で保存した。 (5) Collection and culture of cardiac fibroblasts Cardiac fibroblasts were collected from neonatal Wistar rats on the first day of life (Reference 3: Kinugawa KI, et al. Am J Physiol. 1997; 272: H35). -47.) The collected cardiac fibroblasts were cultured in a 5% CO 2 incubator using Dulbecco's modified Eagle's medium (DMEM; Invitrogen, USA) containing 10% fetal calf serum (FCS; Invitrogen, USA). . Thereafter, subculture was performed every 48 hours, and cardiac fibroblasts after the second passage were used for the experiment.
The culture medium was replaced with DMEM without FCS 24 hours before introduction of the drug or [ 3 H] proline. Moreover, DMEM without drug introduction was used as a control group for each experiment. The collected cardiac fibroblasts and culture supernatant were stored at −80 ° C.
（６）フリーカルニチン濃度測定
酵素サイクリング法を用いて血漿中、尿中、心臓中のフリーカルニチン濃度を測定した（参考文献４：McGarry JD, Foster DW. Free and esterified carnitine. Radiometric method. In: Bergmeyer J, editors. Methods of enzymatic analysis. New York, Academic Press; 1981. p. 474-88.）。
腎臓フリーカルニチン***率（ＦＥ）は、以下の式を用いて求めた（参考文献５：Luci’a Go’ mez-Amores, et al. Pharmaceutical Research. 2003; 20(8): 1133-1140.）。
(6) Measurement of free carnitine concentration Free carnitine concentration in plasma, urine and heart was measured using enzyme cycling method (Reference 4: McGarry JD, Foster DW. Free and esterified carnitine. Radiometric method. In: Bergmeyer J, editors. Methods of analytical analysis. New York, Academic Press; 1981. p. 474-88.).
Renal free carnitine excretion rate (FE) was determined using the following formula (Reference 5: Luci'a Go 'mez-Amores, et al. Pharmaceutical Research. 2003; 20 (8): 1133-1140.) .
酵素サイクリング法を用いて血漿中、尿中、心臓中のフリーカルニチン濃度を測定した（参考文献４：McGarry JD, Foster DW. Free and esterified carnitine. Radiometric method. In: Bergmeyer J, editors. Methods of enzymatic analysis. New York, Academic Press; 1981. p. 474-88.）。
腎臓フリーカルニチン***率（ＦＥ）は、以下の式を用いて求めた（参考文献５：Luci’a Go’ mez-Amores, et al. Pharmaceutical Research. 2003; 20(8): 1133-1140.）。
Renal free carnitine excretion rate (FE) was determined using the following formula (Reference 5: Luci'a Go 'mez-Amores, et al. Pharmaceutical Research. 2003; 20 (8): 1133-1140.) .
（７）定量的リアルタイムＲＴ－ＰＣＲ法
－８０℃保存の心臓、腎臓あるいは心臓線維芽細胞からＲＮＡ抽出を行い、ｏｌｉｇｏ ｄ（Ｔ）１６をリバースプライマーとして逆転写反応を行い、ｃＤＮＡを得た（参考文献６：Yamamoto K, et al. Cardiovasc Res. 2000; 46: 421-432.）。ｃＤＮＡをABI PRISM 7900 HT Sequence Detection System and Software（Applied Biosystems社、米国）を用いてＲＴ－ＰＣＲ施行し、ｍＲＮＡ量を測定した。ＧＡＰＤＨのプライマーとＴａｑＭａｎ（登録商標）プローブは以前に述べられている方法で使用した（参考文献７：Nishikawa N, et al. Cardiovasc Res. 2003; 57: 766-774.）。Solute carrier family 22（organic cation transporter）, member 5（OCTN2）、fatty acid desaturase 1（FADS1）、fatty acid desaturase 2（FADS2）に関してはTaqMan Gene Expression Assay（それぞれのassay IDは、Rn00570533_m1、Rn01759794_g1、Rn00580220_m1; Applied Biosystems社、米国）を用いてＲＴ－ＰＣＲを行った。それぞれのｍＲＮＡ量は、ＧＡＰＤＨ ｍＲＮＡ量を内部コントロールとして補正した。 (7) Quantitative real-time RT-PCR method RNA was extracted from heart, kidney or cardiac fibroblasts stored at -80 ° C., and reverse transcription reaction was performed using oligo d (T) 16 as a reverse primer to obtain cDNA ( Reference 6: Yamamoto K, et al. Cardiovasc Res. 2000; 46: 421-432.). The cDNA was subjected to RT-PCR using ABI PRISM 7900 HT Sequence Detection System and Software (Applied Biosystems, USA), and the amount of mRNA was measured. GAPDH primers and TaqMan® probes were used as previously described (Ref. 7: Nishikawa N, et al. Cardiovasc Res. 2003; 57: 766-774.). Solute carrier family 22 (organic cation transporter), member 5 (OCTN2), fatty acid desaturase 1 (FADS1), fatty acid desaturase 2 (FADS2), TaqMan Gene Expression Assay (respective assay IDs are Rn00570533_m1, Rn01759794_g1, Rn00580220_m1; RT-PCR was performed using Applied Biosystems, USA. Each mRNA amount was corrected using the GAPDH mRNA amount as an internal control.
－８０℃保存の心臓、腎臓あるいは心臓線維芽細胞からＲＮＡ抽出を行い、ｏｌｉｇｏ ｄ（Ｔ）１６をリバースプライマーとして逆転写反応を行い、ｃＤＮＡを得た（参考文献６：Yamamoto K, et al. Cardiovasc Res. 2000; 46: 421-432.）。ｃＤＮＡをABI PRISM 7900 HT Sequence Detection System and Software（Applied Biosystems社、米国）を用いてＲＴ－ＰＣＲ施行し、ｍＲＮＡ量を測定した。ＧＡＰＤＨのプライマーとＴａｑＭａｎ（登録商標）プローブは以前に述べられている方法で使用した（参考文献７：Nishikawa N, et al. Cardiovasc Res. 2003; 57: 766-774.）。Solute carrier family 22（organic cation transporter）, member 5（OCTN2）、fatty acid desaturase 1（FADS1）、fatty acid desaturase 2（FADS2）に関してはTaqMan Gene Expression Assay（それぞれのassay IDは、Rn00570533_m1、Rn01759794_g1、Rn00580220_m1; Applied Biosystems社、米国）を用いてＲＴ－ＰＣＲを行った。それぞれのｍＲＮＡ量は、ＧＡＰＤＨ ｍＲＮＡ量を内部コントロールとして補正した。 (7) Quantitative real-time RT-PCR method RNA was extracted from heart, kidney or cardiac fibroblasts stored at -80 ° C., and reverse transcription reaction was performed using oligo d (T) 16 as a reverse primer to obtain cDNA ( Reference 6: Yamamoto K, et al. Cardiovasc Res. 2000; 46: 421-432.). The cDNA was subjected to RT-PCR using ABI PRISM 7900 HT Sequence Detection System and Software (Applied Biosystems, USA), and the amount of mRNA was measured. GAPDH primers and TaqMan® probes were used as previously described (Ref. 7: Nishikawa N, et al. Cardiovasc Res. 2003; 57: 766-774.). Solute carrier family 22 (organic cation transporter), member 5 (OCTN2), fatty acid desaturase 1 (FADS1), fatty acid desaturase 2 (FADS2), TaqMan Gene Expression Assay (respective assay IDs are Rn00570533_m1, Rn01759794_g1, Rn00580220_m1; RT-PCR was performed using Applied Biosystems, USA. Each mRNA amount was corrected using the GAPDH mRNA amount as an internal control.
（８）ウエスタンブロッティング
ＯＣＴＮ２抗体（Cat. No. ab79964; Abcam社、米国）を用いて、ウエスタンブロット解析を行い、タンパク質発現量評価を行った（参考文献８：Sakata Y, et al. Cardiovasc Res 2003; 57:757-765.）。 (8) Western blotting Using the OCTN2 antibody (Cat. No. ab79964; Abcam, USA), Western blot analysis was performed to evaluate the protein expression level (Reference 8: Sakata Y, et al. Cardiovasc Res 2003). ; 57: 757-765.).
ＯＣＴＮ２抗体（Cat. No. ab79964; Abcam社、米国）を用いて、ウエスタンブロット解析を行い、タンパク質発現量評価を行った（参考文献８：Sakata Y, et al. Cardiovasc Res 2003; 57:757-765.）。 (8) Western blotting Using the OCTN2 antibody (Cat. No. ab79964; Abcam, USA), Western blot analysis was performed to evaluate the protein expression level (Reference 8: Sakata Y, et al. Cardiovasc Res 2003). ; 57: 757-765.).
（９）培養心臓線維芽細胞への［３Ｈ］プロリン取り込み測定
培養心臓線維芽細胞におけるコラーゲン産生評価目的にて、［３Ｈ］プロリン取り込み測定を行った。アンジオテンシンII（5μmol/L; ペプチド研究所、日本）、Ｌ－カルニチン（30mmol/L）と同時に［３Ｈ］プロリンを導入し、導入４８時間後に液体シンチレーションカウンター（LS 6500; Beckman Coulter社、米国）を用いて［３Ｈ］プロリン取り込み量を測定した（参考文献９：Villarreal F, et al. Am J Physiol Cell Physiol. 2009; 296(5):C1178-C1184.）。 (9) Measurement of [ 3 H] proline incorporation into cultured cardiac fibroblasts [ 3 H] proline incorporation was measured for the purpose of evaluating collagen production in cultured cardiac fibroblasts. Angiotensin II (5 μmol / L; Peptide Institute, Japan), [ 3 H] proline was introduced simultaneously with L-carnitine (30 mmol / L), and liquid scintillation counter (LS 6500; Beckman Coulter, USA) 48 hours after introduction [ 3 H] proline uptake was measured using (Reference 9: Villarreal F, et al. Am J Physiol Cell Physiol. 2009; 296 (5): C1178-C1184.).
培養心臓線維芽細胞におけるコラーゲン産生評価目的にて、［３Ｈ］プロリン取り込み測定を行った。アンジオテンシンII（5μmol/L; ペプチド研究所、日本）、Ｌ－カルニチン（30mmol/L）と同時に［３Ｈ］プロリンを導入し、導入４８時間後に液体シンチレーションカウンター（LS 6500; Beckman Coulter社、米国）を用いて［３Ｈ］プロリン取り込み量を測定した（参考文献９：Villarreal F, et al. Am J Physiol Cell Physiol. 2009; 296(5):C1178-C1184.）。 (9) Measurement of [ 3 H] proline incorporation into cultured cardiac fibroblasts [ 3 H] proline incorporation was measured for the purpose of evaluating collagen production in cultured cardiac fibroblasts. Angiotensin II (5 μmol / L; Peptide Institute, Japan), [ 3 H] proline was introduced simultaneously with L-carnitine (30 mmol / L), and liquid scintillation counter (LS 6500; Beckman Coulter, USA) 48 hours after introduction [ 3 H] proline uptake was measured using (Reference 9: Villarreal F, et al. Am J Physiol Cell Physiol. 2009; 296 (5): C1178-C1184.).
（１０）6-keto-prostaglandin F1α（6-keto-PGF1α）濃度測定
心臓中および培養心臓線維芽細胞の培養液上清中の6-keto-PGF1α（prostacyclinの安定代謝物）を、enzyme immunoassay kit（Cat. No. 900-004; Assay Design社、米国）を用いて測定した。 (10) 6-keto-prostaglandin F1α (6-keto-PGF1α) concentration measurement 6-keto-PGF1α (stable metabolite of prostacyclin) in the culture supernatant of the heart and cultured cardiac fibroblasts was enzyme immunoassay kit. (Cat. No. 900-004; Assay Design, USA).
心臓中および培養心臓線維芽細胞の培養液上清中の6-keto-PGF1α（prostacyclinの安定代謝物）を、enzyme immunoassay kit（Cat. No. 900-004; Assay Design社、米国）を用いて測定した。 (10) 6-keto-prostaglandin F1α (6-keto-PGF1α) concentration measurement 6-keto-PGF1α (stable metabolite of prostacyclin) in the culture supernatant of the heart and cultured cardiac fibroblasts was enzyme immunoassay kit. (Cat. No. 900-004; Assay Design, USA).
（１１）ＡＡ濃度測定
心臓中および培養心臓線維芽細胞中のＡＡ濃度を、ガスクロマトグラフィーにより測定した（参考文献１０：Metcalfe LD, et al. Anal Chem. 1961; 33: 363-64.）。 (11) AA concentration measurement The AA concentration in the heart and in cultured cardiac fibroblasts was measured by gas chromatography (Reference 10: Metcalfe LD, et al. Anal Chem. 1961; 33: 363-64.).
心臓中および培養心臓線維芽細胞中のＡＡ濃度を、ガスクロマトグラフィーにより測定した（参考文献１０：Metcalfe LD, et al. Anal Chem. 1961; 33: 363-64.）。 (11) AA concentration measurement The AA concentration in the heart and in cultured cardiac fibroblasts was measured by gas chromatography (Reference 10: Metcalfe LD, et al. Anal Chem. 1961; 33: 363-64.).
（１２）培養心臓線維芽細胞におけるFADS1またはFADS2のノックダウン
培養心筋線維芽細胞に関して、FADS1またはFADS2に対するsiRNA（それぞれのCat. No. SRF27A-2134 と SYD21-325; いずれもコスモ・バイオ社、日本）を導入することによりFADS1またはFADS2のノックダウンを行った。 (12) Knockdown of FADS1 or FADS2 in cultured cardiac fibroblasts For cultured cardiac fibroblasts, siRNAs against FADS1 or FADS2 (Cat. No. SRF27A-2134 and SYD21-325, respectively; both Cosmo Bio, Japan) ) Knocked down FADS1 or FADS2.
培養心筋線維芽細胞に関して、FADS1またはFADS2に対するsiRNA（それぞれのCat. No. SRF27A-2134 と SYD21-325; いずれもコスモ・バイオ社、日本）を導入することによりFADS1またはFADS2のノックダウンを行った。 (12) Knockdown of FADS1 or FADS2 in cultured cardiac fibroblasts For cultured cardiac fibroblasts, siRNAs against FADS1 or FADS2 (Cat. No. SRF27A-2134 and SYD21-325, respectively; both Cosmo Bio, Japan) ) Knocked down FADS1 or FADS2.
（１３）統計処理
測定値は、平均値±標準偏差で表示した。多群間の検定には、one-factor analysis of varianceを使用し、有意差を認めた場合にpost hoc検定としてFisher protected least significant differenceを用いて解析を行った。Ｐ値＜０．０５を統計的に有意とした。
図、表においては、コントロール群に対してＰ＜０．０５である場合＊で示し、無治療群またはAngiotensin II群に対してＰ＜０．０５である場合†で示した。 (13) Statistical processing The measured values were displayed as an average value ± standard deviation. For testing between multiple groups, one-factor analysis of variance was used, and when a significant difference was observed, analysis was performed using Fisher protected least significant difference as a post hoc test. A P value <0.05 was considered statistically significant.
In the figures and tables, the case where P <0.05 was indicated for the control group, and the case where P <0.05 was indicated for the untreated group or the Angiotensin II group.
測定値は、平均値±標準偏差で表示した。多群間の検定には、one-factor analysis of varianceを使用し、有意差を認めた場合にpost hoc検定としてFisher protected least significant differenceを用いて解析を行った。Ｐ値＜０．０５を統計的に有意とした。
図、表においては、コントロール群に対してＰ＜０．０５である場合＊で示し、無治療群またはAngiotensin II群に対してＰ＜０．０５である場合†で示した。 (13) Statistical processing The measured values were displayed as an average value ± standard deviation. For testing between multiple groups, one-factor analysis of variance was used, and when a significant difference was observed, analysis was performed using Fisher protected least significant difference as a post hoc test. A P value <0.05 was considered statistically significant.
In the figures and tables, the case where P <0.05 was indicated for the control group, and the case where P <0.05 was indicated for the untreated group or the Angiotensin II group.
１－２ 実験結果
（１）収縮期血圧、脈拍および体重
１９週齢時における収縮期血圧、脈拍および体重を表１に示した。８％高ＮａＣｌ食投与により無治療群（拡張不全モデル）ではコントロール群に比して有意に収縮期血圧と脈拍の上昇を認めたが、カルニチン群ではＬ－カルニチン投与による収縮期血圧と脈拍への有意な影響は認めなかった。体重は３群間で差を認めなかった。 1-2 Experimental Results (1) Systolic blood pressure, pulse and weight Table 1 shows the systolic blood pressure, pulse and weight at the age of 19 weeks. In the untreated group (diastolic dysfunction model), the systolic blood pressure and pulse were significantly increased compared with the control group by administration of the 8% high NaCl diet, but in the carnitine group, the systolic blood pressure and pulse were increased by L-carnitine administration. There was no significant effect. There was no difference in body weight among the three groups.
（１）収縮期血圧、脈拍および体重
１９週齢時における収縮期血圧、脈拍および体重を表１に示した。８％高ＮａＣｌ食投与により無治療群（拡張不全モデル）ではコントロール群に比して有意に収縮期血圧と脈拍の上昇を認めたが、カルニチン群ではＬ－カルニチン投与による収縮期血圧と脈拍への有意な影響は認めなかった。体重は３群間で差を認めなかった。 1-2 Experimental Results (1) Systolic blood pressure, pulse and weight Table 1 shows the systolic blood pressure, pulse and weight at the age of 19 weeks. In the untreated group (diastolic dysfunction model), the systolic blood pressure and pulse were significantly increased compared with the control group by administration of the 8% high NaCl diet, but in the carnitine group, the systolic blood pressure and pulse were increased by L-carnitine administration. There was no significant effect. There was no difference in body weight among the three groups.
（２）血漿中、心臓中フリーカルニチン濃度
血漿中のフリーカルニチン濃度を図１（ａ）に、心臓中のフリーカルニチン濃度を図１（ｂ）にそれぞれ示した。図１（ａ）および（ｂ）から明らかなように、血漿中および心臓中のフリーカルニチン濃度は、ともに無治療群（拡張不全モデル）で低下しており、カルニチン群は、無治療群に対して有意に増加した。この結果から、拡張不全モデルにおいて低下したカルニチン濃度は、Ｌ－カルニチン投与により改善することが認められた。 (2) Plasma Free Carnitine Concentration in Heart The free carnitine concentration in plasma is shown in FIG. 1 (a), and the free carnitine concentration in the heart is shown in FIG. 1 (b). As is clear from FIGS. 1 (a) and 1 (b), the free carnitine concentrations in plasma and heart are both decreased in the untreated group (diastolic dysfunction model). Significantly increased. From these results, it was confirmed that the carnitine concentration decreased in the diastolic dysfunction model was improved by administration of L-carnitine.
血漿中のフリーカルニチン濃度を図１（ａ）に、心臓中のフリーカルニチン濃度を図１（ｂ）にそれぞれ示した。図１（ａ）および（ｂ）から明らかなように、血漿中および心臓中のフリーカルニチン濃度は、ともに無治療群（拡張不全モデル）で低下しており、カルニチン群は、無治療群に対して有意に増加した。この結果から、拡張不全モデルにおいて低下したカルニチン濃度は、Ｌ－カルニチン投与により改善することが認められた。 (2) Plasma Free Carnitine Concentration in Heart The free carnitine concentration in plasma is shown in FIG. 1 (a), and the free carnitine concentration in the heart is shown in FIG. 1 (b). As is clear from FIGS. 1 (a) and 1 (b), the free carnitine concentrations in plasma and heart are both decreased in the untreated group (diastolic dysfunction model). Significantly increased. From these results, it was confirmed that the carnitine concentration decreased in the diastolic dysfunction model was improved by administration of L-carnitine.
（３）心エコー図検査および心臓カテーテル検査
心エコー図検査および心臓カテーテル検査の結果を表２に示した。心エコー図検査の結果、カルニチン群において無治療群と比べRelative wall thicknessの増加は軽減されていた。その他の心エコー図指標には、Ｌ－カルニチン投与による変化を認めず、無治療群とカルニチン群のいずれにおいても、左室内径短縮率は保たれていた。
無治療群では肺重量と左室拡張末期圧が有意に増加したが、カルニチン群ではこれらの増加を認めず、拡張不全発症が阻止されたことが示された。
無治療群と比してカルニチン群においては、Ｔａｕに変化を認めなかったが、心筋スティッフネス定数の上昇が軽減されており、Ｌ－カルニチンは心筋スティッフネスを改善することにより拡張不全を抑制したものと考えられた。また、無治療群と比してカルニチン群において、左室重量の増加抑制も認めた。
図２に、各群のラットの心臓のＡｚａｎ Ｍａｌｌｏｒｙ染色像を示した。左室線維化面積は無治療群で有意に増加していたが、カルニチン群では増加が抑制されていた（表２参照）。Ｌ－カルニチンは左室線維化を抑制することにより、心筋スティッフネス定数を改善したと考えられた。 (3) Echocardiographic examination and cardiac catheter examination Table 2 shows the results of the echocardiographic examination and cardiac catheter examination. As a result of echocardiography, the increase in relative wall thickness was reduced in the carnitine group compared with the untreated group. The other echocardiographic indices showed no change due to L-carnitine administration, and the left ventricular diameter shortening rate was maintained in both the untreated group and the carnitine group.
Lung weight and left ventricular end-diastolic pressure increased significantly in the untreated group, but these increases were not observed in the carnitine group, indicating that the onset of diastolic dysfunction was prevented.
In the carnitine group, there was no change in Tau compared to the untreated group, but the increase in myocardial stiffness constant was reduced, and L-carnitine suppressed diastolic dysfunction by improving myocardial stiffness. It was considered a thing. In addition, an increase in left ventricular weight was also suppressed in the carnitine group compared to the untreated group.
FIG. 2 shows Azan Mall stained images of the heart of each group of rats. The left ventricular fibrosis area was significantly increased in the untreated group, but the increase was suppressed in the carnitine group (see Table 2). L-carnitine was thought to have improved myocardial stiffness constant by inhibiting left ventricular fibrosis.
心エコー図検査および心臓カテーテル検査の結果を表２に示した。心エコー図検査の結果、カルニチン群において無治療群と比べRelative wall thicknessの増加は軽減されていた。その他の心エコー図指標には、Ｌ－カルニチン投与による変化を認めず、無治療群とカルニチン群のいずれにおいても、左室内径短縮率は保たれていた。
無治療群では肺重量と左室拡張末期圧が有意に増加したが、カルニチン群ではこれらの増加を認めず、拡張不全発症が阻止されたことが示された。
無治療群と比してカルニチン群においては、Ｔａｕに変化を認めなかったが、心筋スティッフネス定数の上昇が軽減されており、Ｌ－カルニチンは心筋スティッフネスを改善することにより拡張不全を抑制したものと考えられた。また、無治療群と比してカルニチン群において、左室重量の増加抑制も認めた。
図２に、各群のラットの心臓のＡｚａｎ Ｍａｌｌｏｒｙ染色像を示した。左室線維化面積は無治療群で有意に増加していたが、カルニチン群では増加が抑制されていた（表２参照）。Ｌ－カルニチンは左室線維化を抑制することにより、心筋スティッフネス定数を改善したと考えられた。 (3) Echocardiographic examination and cardiac catheter examination Table 2 shows the results of the echocardiographic examination and cardiac catheter examination. As a result of echocardiography, the increase in relative wall thickness was reduced in the carnitine group compared with the untreated group. The other echocardiographic indices showed no change due to L-carnitine administration, and the left ventricular diameter shortening rate was maintained in both the untreated group and the carnitine group.
Lung weight and left ventricular end-diastolic pressure increased significantly in the untreated group, but these increases were not observed in the carnitine group, indicating that the onset of diastolic dysfunction was prevented.
In the carnitine group, there was no change in Tau compared to the untreated group, but the increase in myocardial stiffness constant was reduced, and L-carnitine suppressed diastolic dysfunction by improving myocardial stiffness. It was considered a thing. In addition, an increase in left ventricular weight was also suppressed in the carnitine group compared to the untreated group.
FIG. 2 shows Azan Mall stained images of the heart of each group of rats. The left ventricular fibrosis area was significantly increased in the untreated group, but the increase was suppressed in the carnitine group (see Table 2). L-carnitine was thought to have improved myocardial stiffness constant by inhibiting left ventricular fibrosis.
（４）心臓および腎臓におけるＯＣＴＮ２発現レベル
心臓におけるＯＣＴＮ２のｍＲＮＡおよびタンパク質発現レベルを図３（ａ）に、腎臓におけるＯＣＴＮ２のｍＲＮＡおよびタンパク質発現レベルを図３（ｂ）にそれぞれ示した。いずれも上段のグラフがｍＲＮＡ発現レベルであり、下段がタンパク質発現レベル（ウエスタンブロッティング結果）である。
また、コントロール群および無治療群の腎臓におけるフリーカルニチン***率（ＦＥ）を図４に示した。
図３（ａ）および（ｂ）から明らかなように、心臓および腎臓では、無治療群において、フリーカルニチンの特異的な輸送体であるＯＣＴＮ２発現が、ｍＲＮＡレベルおよびタンパク質レベルのいずれにおいても低下していた。
また、腎臓でのＯＣＴＮ２発現低下に伴い、フリーカルニチンの***率（ＦＥ）は無治療群で増加しており（図４参照）、血中フリーカルニチン濃度が低下した原因と考えられた。無治療群において心臓組織中のＯＣＴＮ２発現低下と血中フリーカルニチン濃度低下が、心臓組織中フリーカルニチン濃度低下に寄与していると考えられた。 (4) OCTN2 expression level in heart and kidney OCTN2 mRNA and protein expression levels in heart are shown in FIG. 3 (a), and OCTN2 mRNA and protein expression levels in kidney are shown in FIG. 3 (b). In either case, the upper graph shows the mRNA expression level, and the lower graph shows the protein expression level (western blotting result).
Moreover, the free carnitine excretion rate (FE) in the kidneys of the control group and the untreated group is shown in FIG.
As is apparent from FIGS. 3 (a) and 3 (b), in the untreated group, the expression of OCTN2, which is a specific transporter of free carnitine, decreased at both the mRNA level and the protein level in the untreated group. It was.
Moreover, with the decrease in OCTN2 expression in the kidney, the free carnitine excretion rate (FE) increased in the untreated group (see FIG. 4), which was considered to be the cause of the decrease in blood free carnitine concentration. It was considered that the decrease in OCTN2 expression in the heart tissue and the decrease in blood free carnitine concentration in the untreated group contributed to the decrease in free carnitine concentration in the heart tissue.
心臓におけるＯＣＴＮ２のｍＲＮＡおよびタンパク質発現レベルを図３（ａ）に、腎臓におけるＯＣＴＮ２のｍＲＮＡおよびタンパク質発現レベルを図３（ｂ）にそれぞれ示した。いずれも上段のグラフがｍＲＮＡ発現レベルであり、下段がタンパク質発現レベル（ウエスタンブロッティング結果）である。
また、コントロール群および無治療群の腎臓におけるフリーカルニチン***率（ＦＥ）を図４に示した。
図３（ａ）および（ｂ）から明らかなように、心臓および腎臓では、無治療群において、フリーカルニチンの特異的な輸送体であるＯＣＴＮ２発現が、ｍＲＮＡレベルおよびタンパク質レベルのいずれにおいても低下していた。
また、腎臓でのＯＣＴＮ２発現低下に伴い、フリーカルニチンの***率（ＦＥ）は無治療群で増加しており（図４参照）、血中フリーカルニチン濃度が低下した原因と考えられた。無治療群において心臓組織中のＯＣＴＮ２発現低下と血中フリーカルニチン濃度低下が、心臓組織中フリーカルニチン濃度低下に寄与していると考えられた。 (4) OCTN2 expression level in heart and kidney OCTN2 mRNA and protein expression levels in heart are shown in FIG. 3 (a), and OCTN2 mRNA and protein expression levels in kidney are shown in FIG. 3 (b). In either case, the upper graph shows the mRNA expression level, and the lower graph shows the protein expression level (western blotting result).
Moreover, the free carnitine excretion rate (FE) in the kidneys of the control group and the untreated group is shown in FIG.
As is apparent from FIGS. 3 (a) and 3 (b), in the untreated group, the expression of OCTN2, which is a specific transporter of free carnitine, decreased at both the mRNA level and the protein level in the untreated group. It was.
Moreover, with the decrease in OCTN2 expression in the kidney, the free carnitine excretion rate (FE) increased in the untreated group (see FIG. 4), which was considered to be the cause of the decrease in blood free carnitine concentration. It was considered that the decrease in OCTN2 expression in the heart tissue and the decrease in blood free carnitine concentration in the untreated group contributed to the decrease in free carnitine concentration in the heart tissue.
（５）培養心臓線維芽細胞への［３Ｈ］プロリン取り込み
培養心臓線維芽細胞への［３Ｈ］プロリン取り込み測定値を図５に示した。図５から明らかなように、アンジオテンシンII投与によって増加した［３Ｈ］プロリンの取り込みは、Ｌ－カルニチンの添加により抑制された。一方、無刺激の線維芽細胞に対しては、Ｌ－カルニチンはコラーゲン産生に影響を与えなかった。この結果から、Ｌ－カルニチンがコラーゲン産生を抑制したと考えられた。 (5) the [3 H] proline incorporation measurements to [3 H] proline incorporation cultured cardiac fibroblasts to the culture cardiac fibroblasts shown in FIG. As is apparent from FIG. 5, the uptake of [ 3 H] proline increased by administration of angiotensin II was suppressed by the addition of L-carnitine. On the other hand, for unstimulated fibroblasts, L-carnitine did not affect collagen production. From this result, it was considered that L-carnitine suppressed collagen production.
培養心臓線維芽細胞への［３Ｈ］プロリン取り込み測定値を図５に示した。図５から明らかなように、アンジオテンシンII投与によって増加した［３Ｈ］プロリンの取り込みは、Ｌ－カルニチンの添加により抑制された。一方、無刺激の線維芽細胞に対しては、Ｌ－カルニチンはコラーゲン産生に影響を与えなかった。この結果から、Ｌ－カルニチンがコラーゲン産生を抑制したと考えられた。 (5) the [3 H] proline incorporation measurements to [3 H] proline incorporation cultured cardiac fibroblasts to the culture cardiac fibroblasts shown in FIG. As is apparent from FIG. 5, the uptake of [ 3 H] proline increased by administration of angiotensin II was suppressed by the addition of L-carnitine. On the other hand, for unstimulated fibroblasts, L-carnitine did not affect collagen production. From this result, it was considered that L-carnitine suppressed collagen production.
（６）６－ｋｅｔｏ－ＰＧＦ１α濃度
心臓中の６－ｋｅｔｏ－ＰＧＦ１α濃度測定値を図６（ａ）に、培養心臓線維芽細胞の培養液上清中の６－ｋｅｔｏ－ＰＧＦ１α濃度測定値を図６（ｂ）にそれぞれ示した。図６（ａ）から明らかなように、心臓中の６－ｋｅｔｏ－ＰＧＦ１α濃度は、カルニチン群において有意に増加した。また、図６（ｂ）から明らかなように、培養心臓線維芽細胞の培養液上清中の６－ｋｅｔｏ－ＰＧＦ１α濃度は、Ｌ－カルニチン添加により有意に増加した。これらの結果から、組織プロスタサイクリン濃度の増加が心臓線維化抑制の一因であると考えられ、Ｌ－カルニチンは線維芽細胞に直接的に作用してプロスタサイクリン産生を促進することが明らかとなった。 (6) 6-keto-PGF1α concentration The measured value of 6-keto-PGF1α concentration in the heart is shown in FIG. 6 (a), and the measured value of 6-keto-PGF1α concentration in the culture supernatant of cultured cardiac fibroblasts is shown in FIG. This is shown in 6 (b). As is clear from FIG. 6 (a), the 6-keto-PGF1α concentration in the heart was significantly increased in the carnitine group. As is clear from FIG. 6 (b), the 6-keto-PGF1α concentration in the culture supernatant of cultured cardiac fibroblasts was significantly increased by the addition of L-carnitine. From these results, it is considered that an increase in tissue prostacyclin concentration contributes to suppression of cardiac fibrosis, and it is clear that L-carnitine acts directly on fibroblasts to promote prostacyclin production. It was.
心臓中の６－ｋｅｔｏ－ＰＧＦ１α濃度測定値を図６（ａ）に、培養心臓線維芽細胞の培養液上清中の６－ｋｅｔｏ－ＰＧＦ１α濃度測定値を図６（ｂ）にそれぞれ示した。図６（ａ）から明らかなように、心臓中の６－ｋｅｔｏ－ＰＧＦ１α濃度は、カルニチン群において有意に増加した。また、図６（ｂ）から明らかなように、培養心臓線維芽細胞の培養液上清中の６－ｋｅｔｏ－ＰＧＦ１α濃度は、Ｌ－カルニチン添加により有意に増加した。これらの結果から、組織プロスタサイクリン濃度の増加が心臓線維化抑制の一因であると考えられ、Ｌ－カルニチンは線維芽細胞に直接的に作用してプロスタサイクリン産生を促進することが明らかとなった。 (6) 6-keto-PGF1α concentration The measured value of 6-keto-PGF1α concentration in the heart is shown in FIG. 6 (a), and the measured value of 6-keto-PGF1α concentration in the culture supernatant of cultured cardiac fibroblasts is shown in FIG. This is shown in 6 (b). As is clear from FIG. 6 (a), the 6-keto-PGF1α concentration in the heart was significantly increased in the carnitine group. As is clear from FIG. 6 (b), the 6-keto-PGF1α concentration in the culture supernatant of cultured cardiac fibroblasts was significantly increased by the addition of L-carnitine. From these results, it is considered that an increase in tissue prostacyclin concentration contributes to suppression of cardiac fibrosis, and it is clear that L-carnitine acts directly on fibroblasts to promote prostacyclin production. It was.
（７）心臓および培養心臓線維芽細胞におけるＦＡＤＳ１、ＦＡＤＳ２発現レベル
心臓におけるＦＡＤＳ１のｍＲＮＡ発現レベルを図７（ａ）に、ＦＡＤＳ２のｍＲＮＡ発現レベルを図７（ｂ）にそれぞれ示した。また、培養心臓線維芽細胞におけるＦＡＤＳ１のｍＲＮＡ発現レベルを図８（ａ）に、ＦＡＤＳ２のｍＲＮＡ発現レベルを図８（ｂ）にそれぞれ示した。
プロスタサイクリンは、生体内においてＡＡを前駆物質として合成される。ＦＡＤＳ１およびＦＡＤＳ２はＡＡ合成酵素である。
図７（ａ）および（ｂ）、ならびに図８（ａ）および（ｂ）から明らかなように、心臓および培養心臓線維芽細胞のいずれも、Ｌ－カルニチンの投与または添加によりＦＡＤＳ１およびＦＡＤＳ２の発現が増加した。この結果は、Ｌ－カルニチンによるプロスタサイクリン産生増加の一因であると考えられた。
（８）ＡＡ濃度
心臓中のＡＡ濃度測定値を図９に示した。図９から明らかなように、心臓中のＡＡ濃度は、カルニチン群において有意に増加した。 (7) FADS1 and FADS2 expression levels in heart and cultured cardiac fibroblasts FIG. 7 (a) shows the FADS1 mRNA expression level in the heart, and FIG. 7 (b) shows the FADS2 mRNA expression level. The FADS1 mRNA expression level in cultured cardiac fibroblasts is shown in FIG. 8 (a), and the FADS2 mRNA expression level is shown in FIG. 8 (b).
Prostacyclin is synthesized in vivo using AA as a precursor. FADS1 and FADS2 are AA synthases.
As is apparent from FIGS. 7 (a) and (b) and FIGS. 8 (a) and (b), both cardiac and cultured cardiac fibroblasts expressed FADS1 and FADS2 upon administration or addition of L-carnitine. increased. This result was thought to contribute to increased prostacyclin production by L-carnitine.
(8) AA Concentration The measured value of AA concentration in the heart is shown in FIG. As is clear from FIG. 9, the AA concentration in the heart increased significantly in the carnitine group.
心臓におけるＦＡＤＳ１のｍＲＮＡ発現レベルを図７（ａ）に、ＦＡＤＳ２のｍＲＮＡ発現レベルを図７（ｂ）にそれぞれ示した。また、培養心臓線維芽細胞におけるＦＡＤＳ１のｍＲＮＡ発現レベルを図８（ａ）に、ＦＡＤＳ２のｍＲＮＡ発現レベルを図８（ｂ）にそれぞれ示した。
プロスタサイクリンは、生体内においてＡＡを前駆物質として合成される。ＦＡＤＳ１およびＦＡＤＳ２はＡＡ合成酵素である。
図７（ａ）および（ｂ）、ならびに図８（ａ）および（ｂ）から明らかなように、心臓および培養心臓線維芽細胞のいずれも、Ｌ－カルニチンの投与または添加によりＦＡＤＳ１およびＦＡＤＳ２の発現が増加した。この結果は、Ｌ－カルニチンによるプロスタサイクリン産生増加の一因であると考えられた。
（８）ＡＡ濃度
心臓中のＡＡ濃度測定値を図９に示した。図９から明らかなように、心臓中のＡＡ濃度は、カルニチン群において有意に増加した。 (7) FADS1 and FADS2 expression levels in heart and cultured cardiac fibroblasts FIG. 7 (a) shows the FADS1 mRNA expression level in the heart, and FIG. 7 (b) shows the FADS2 mRNA expression level. The FADS1 mRNA expression level in cultured cardiac fibroblasts is shown in FIG. 8 (a), and the FADS2 mRNA expression level is shown in FIG. 8 (b).
Prostacyclin is synthesized in vivo using AA as a precursor. FADS1 and FADS2 are AA synthases.
As is apparent from FIGS. 7 (a) and (b) and FIGS. 8 (a) and (b), both cardiac and cultured cardiac fibroblasts expressed FADS1 and FADS2 upon administration or addition of L-carnitine. increased. This result was thought to contribute to increased prostacyclin production by L-carnitine.
(8) AA Concentration The measured value of AA concentration in the heart is shown in FIG. As is clear from FIG. 9, the AA concentration in the heart increased significantly in the carnitine group.
（９）ＦＡＤＳ１またはＦＡＤＳ２ノックダウン細胞における６－ｋｅｔｏ－ＰＧＦ１α濃度およびＡＡ濃度
培養心臓線維芽細胞、ＦＡＤＳ１をノックダウンした培養心臓線維芽細胞、またはＦＡＤＳ２をノックダウンした培養心臓線維芽細胞を用いて６－ｋｅｔｏ－ＰＧＦ１α濃度を測定した結果を図１０（ａ）に、ＡＡ濃度を測定した結果を図１０（ｂ）にそれぞれ示した。
図１０（ａ）および（ｂ）から明らかなように、ＦＡＤＳ１およびＦＡＤＳ２の一方をノックダウンすれば、Ｌ－カルニチンの添加による６－ｋｅｔｏ－ＰＧＦ１α濃度増加およびＡＡ濃度増加がいずれも抑制された。これらの結果から、Ｌ－カルニチンによるプロスタサイクリン産生増加は、ＦＡＤＳ１およびＦＡＤＳ２の発現増加によるＡＡ合成亢進に起因するものと考えられた。 (9) 6-keto-PGF1α concentration and AA concentration in FADS1 or FADS2 knockdown cells Using cultured cardiac fibroblasts, cultured cardiac fibroblasts knocked down by FADS1, or cultured cardiac fibroblasts knocked down by FADS2 The results of measuring the 6-keto-PGF1α concentration are shown in FIG. 10 (a), and the results of measuring the AA concentration are shown in FIG. 10 (b).
As is clear from FIGS. 10 (a) and (b), when one of FADS1 and FADS2 was knocked down, the increase in 6-keto-PGF1α concentration and the increase in AA concentration due to the addition of L-carnitine were both suppressed. From these results, it was considered that the increase in prostacyclin production by L-carnitine was caused by the enhanced AA synthesis due to the increased expression of FADS1 and FADS2.
培養心臓線維芽細胞、ＦＡＤＳ１をノックダウンした培養心臓線維芽細胞、またはＦＡＤＳ２をノックダウンした培養心臓線維芽細胞を用いて６－ｋｅｔｏ－ＰＧＦ１α濃度を測定した結果を図１０（ａ）に、ＡＡ濃度を測定した結果を図１０（ｂ）にそれぞれ示した。
図１０（ａ）および（ｂ）から明らかなように、ＦＡＤＳ１およびＦＡＤＳ２の一方をノックダウンすれば、Ｌ－カルニチンの添加による６－ｋｅｔｏ－ＰＧＦ１α濃度増加およびＡＡ濃度増加がいずれも抑制された。これらの結果から、Ｌ－カルニチンによるプロスタサイクリン産生増加は、ＦＡＤＳ１およびＦＡＤＳ２の発現増加によるＡＡ合成亢進に起因するものと考えられた。 (9) 6-keto-PGF1α concentration and AA concentration in FADS1 or FADS2 knockdown cells Using cultured cardiac fibroblasts, cultured cardiac fibroblasts knocked down by FADS1, or cultured cardiac fibroblasts knocked down by FADS2 The results of measuring the 6-keto-PGF1α concentration are shown in FIG. 10 (a), and the results of measuring the AA concentration are shown in FIG. 10 (b).
As is clear from FIGS. 10 (a) and (b), when one of FADS1 and FADS2 was knocked down, the increase in 6-keto-PGF1α concentration and the increase in AA concentration due to the addition of L-carnitine were both suppressed. From these results, it was considered that the increase in prostacyclin production by L-carnitine was caused by the enhanced AA synthesis due to the increased expression of FADS1 and FADS2.
なお本発明は上述した各実施形態および実施例に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、異なる実施形態にそれぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。また、本明細書中に記載された学術文献および特許文献の全てが、本明細書中において参考として援用される。
The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope shown in the claims, and technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.
Claims (8)
- カルニチンもしくはその誘導体またはその薬学的に許容される塩を有効成分として含有することを特徴とする拡張不全型心不全の予防または治療薬。 A preventive or therapeutic agent for diastolic heart failure, comprising carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
- カルニチンもしくはその誘導体またはその薬学的に許容される塩を有効成分として含有することを特徴とする心筋硬化抑制剤。 A myocardial sclerosis inhibitor comprising carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
- カルニチンもしくはその誘導体またはその薬学的に許容される塩を有効成分として含有することを特徴とする心筋線維化抑制剤。 An inhibitor of myocardial fibrosis, comprising carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
- カルニチンもしくはその誘導体またはその薬学的に許容される塩を有効成分として含有することを特徴とするプロスタサイクリン増加剤。 A prostacyclin increasing agent characterized by containing carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
- プロスタサイクリンが心筋プロスタサイクリンである請求項４に記載のプロスタサイクリン増加剤。 The prostacyclin increasing agent according to claim 4, wherein the prostacyclin is a myocardial prostacyclin.
- 哺乳動物に対して、カルニチンもしくはその誘導体またはその薬学的に許容される塩の有効量を投与することを特徴とする拡張不全型心不全の予防または治療方法。 A method for preventing or treating diastolic heart failure, comprising administering an effective amount of carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof to a mammal.
- 拡張不全型心不全の予防または治療薬を製造するための、カルニチンもしくはその誘導体またはその薬学的に許容される塩の使用。 Use of carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof for the manufacture of a preventive or therapeutic agent for diastolic heart failure.
- 拡張不全型心不全の予防または治療に使用するための、カルニチンもしくはその誘導体またはその薬学的に許容される塩。 Carnitine or a derivative thereof or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of diastolic heart failure.
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| JPS59222412A (en) * | 1983-06-01 | 1984-12-14 | Earth Chem Corp Ltd | Remedy for heart disease |
| JPS62190190A (en) * | 1986-01-13 | 1987-08-20 | シグマ−タウ・インダストリエ・フアルマシウテイシエ・リウニテ・ソシエタ・ペル・アチオ−ニ | Phosphorylalkanolamide derivatives of l-carnitine and medicinal composition |
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