JPWO2005018316A1 - Hypertrophic cardiomyopathy model animal - Google Patents

Abstract

The present invention is an experimental model animal that develops human hypertrophic cardiomyopathy, which is produced by continuously administering a potassium channel inhibitor or a potassium channel inhibitor and an antibody to the whole body or heart of the animal. A model animal is provided. This animal can be used for elucidating the pathophysiology of hypertrophic cardiomyopathy and screening for prophylactic / therapeutic drugs, and is extremely useful as an experimental animal.

Description

  The present invention relates to a hypertrophic cardiomyopathy model animal, a production method thereof, and a drug evaluation method using the model animal.

Idiopathic hypertrophic cardiomyopathy may be due to a genetic abnormality or an unknown cause. From left ventricular morphology, (1) large ventricular septum, (2) centripetal hypertrophy, (3) apical hypertrophy, ventricular septal hyperplasia is left ventricular outflow tract obstruction type and non-occlusion type It is divided into capital ^{(the Heart (5 th edition)} , 1309, Hurst JW, et al, McGraw-Hill Book Co, NY). As it progresses, cardiomyocyte damage, fibrosis, and edema occur at the end of the stage, resulting in contraction of the left ventricle and congestive heart failure. That is, (4) dilated phase hypertrophic cardiomyopathy. The disease often results in sudden death, and no curative treatment has yet been found other than partial ventricular septal resection in an obstructive form.
In general, in a drug efficacy screening test for the purpose of drug development, a test method using a disease model animal that is not a healthy animal but in a certain disease state is closer to an actual clinical symptom, and is also preferable for drug efficacy evaluation.
In hypertrophic cardiomyopathy, attempts to produce animal models have been made conventionally. For example, a model by continuous injection of catecholamine (Brafuss AH, et al: Clin Res 23: 77A, 1975), a model by glycoprotein administration (Witzke DJ). et al: Circulation 53/54 (Suppl): 88, 1976), a model by triac administration (Teare D: Lancet 2: 221, 1977) and the like have been reported.
However, such an animal model is centripetal hypertrophy and can only produce one of the aforementioned four types of pathological conditions in the clinic, and a model that develops four pathologies in human hypertrophic cardiomyopathy has been obtained. There is no current situation.

The present invention provides a novel hypertrophic cardiomyopathy model animal, a production method thereof, and a drug evaluation method using the disease state model animal.
As a result of intensive studies in view of such circumstances, the present inventors have continuously administered a potassium channel inhibitor or an antibody against an animal used therewith, thereby causing thickening of the heart wall, resulting in human hypertrophic cardiomyopathy. It was found that a pathological condition very similar to each disease type is induced.
That is, the present invention is an experimental model animal that develops human hypertrophic cardiomyopathy, which is produced by continuously administering a potassium channel inhibitor or a potassium channel inhibitor and an antibody to the whole body or heart of the animal. A disease model animal is provided.
The present invention also relates to a method for producing an experimental model animal that develops human hypertrophic cardiomyopathy, characterized in that a potassium channel inhibitor or a potassium channel inhibitor and a systemic or heart antibody of the animal are continuously administered. A method for producing a hypertrophic cardiomyopathy model animal is provided.
The present invention also provides a drug evaluation method using the model animal.
The present invention also provides a prophylactic or therapeutic agent for hypertrophic cardiomyopathy comprising a potassium channel opener, particularly nicorandil as an active ingredient.
The present invention also provides use of a potassium channel opener for producing a prophylactic or therapeutic agent for hypertrophic cardiomyopathy.
Furthermore, the present invention provides a method for treating hypertrophic cardiomyopathy characterized by administering a potassium channel opener.

FIG. 1 is a photograph showing the morphological findings of the heart after drug administration.
A: control example, B: 5 mg / Kg administration example (arrow: thickened ventricular septum), C: 40 mg / Kg administration example, D: 40 mg / Kg and anti-rat systemic antibody 2 unit administration example (arrow: edema), E: Example of administration of 40 mg / Kg and nicorandil 20 mg / Kg. Scale: 1 mm.
FIG. 2 is a photograph showing myocardial tissue findings after drug administration.
A: Control example, B: 40 mg / Kg administration example, C: 40 mg / Kg and anti-rat systemic antibody 2 unit administration example (arrow: edema and fibrosis), D: 40 mg / Kg and nicorandil 20 mg / Kg administration example. Scale: 10 μm.

The hypertrophic cardiomyopathy model animal of the present invention is produced by continuously administering a potassium channel inhibitor or a potassium channel inhibitor and an antibody to the whole body or heart of the animal.
Here, “hypertrophic cardiomyopathy” is one of idiopathic cardiomyopathy, a disease in which the inner wall of the left ventricle becomes narrower due to the thickening of the heart wall, impeding blood flow from the atria to the ventricles It is. It is classified into ventricular septal hypertrophy, afferent hypertrophy, and apical hypertrophy according to the region where the myocardial wall becomes thick. The large ventricular septal manure is divided into a left ventricular outflow tract occlusion type and a non-occlusion type (see Non-Patent Document 1). As the thickening progresses, cardiomyocyte damage, fibrosis and edema occur at the end stage, resulting in dilated phase hypertrophic cardiomyopathy.
As the animal used as the model animal of the present invention, animals commonly used as experimental animals can be used, and examples include rats, mice, guinea pigs, hamsters, rabbits, dogs, monkeys, etc. Rats, mice, and guinea pigs are preferable from the viewpoint that they can be produced.
Any potassium channel inhibitor may be used as long as it has a function of reducing the movement of potassium ions through the cell membrane and, as a result, prolonging the action potential in the cell or depolarizing the membrane potential. For example, mono- or diaminopyridine such as 3,4-diaminopyridine, 2,3-diaminopyridine, 2,5-diaminopyridine, 2,6-diaminopyridine, 2-aminopyridine, 3-aminopyridine, 4-aminopyridine, etc. Derivatives (Uchida Y, et al: J Cardiovas Pharmacol 8:49, 1987), tetraethylammonium, glibenclamide, iberiotoxin, thiaribdotoxin and the like, among which 3,4-diaminopyridine is particularly preferable.
The antibody against the whole body or heart of the animal (hereinafter also referred to as “anti-animal antibody”) refers to an antibody obtained by crushing the whole body or heart of the animal to be used and administering it to an animal other than the animal. Administration of these in combination with potassium channel inhibitors promotes the onset of myocardial hypertrophy and increases the incidence of fibrosis and edema, resulting in changes similar to the diastolic phase seen at the end of hypertrophic cardiomyopathy Can do.
As the animal to be sensitized, mammals other than the animal to be modeled are preferable. For example, when a model rat is prepared, a rabbit is preferable.
For immunization, for example, when preparing a model rat, the whole body or heart of the rat is crushed with a homogenizer or the like and suspended in a normal buffer or physiological saline or mixed with Freund's complete or incomplete adjuvant. The solution may be administered to animals other than rats, for example, rabbits by an appropriate route such as intraperitoneal or subcutaneous, and after the primary stimulation, the same operation may be repeated as necessary. The dose of the immunizing antigen is appropriately determined according to the administration route, the type of animal and the like. After completion of immunization, anti-rat systemic antibody or anti-rat heart antibody can be obtained by collecting serum of the sensitized animal.
Administration of a potassium channel inhibitor or a potassium channel inhibitor to an animal can be performed by a commonly used administration route such as intravenous administration, intraperitoneal administration, or oral administration, and oral administration is particularly preferable. The dosage varies depending on the animal species, strain difference, sex difference, age, fasting state, etc., and a preferable amount may be set as appropriate. For example, when preparing a model rat, it is preferable to administer 5 to 1000 mg / kg, preferably 5 to 400 mg / kg daily for 30 to 60 days in a single administration of a potassium channel inhibitor alone. When a potassium channel inhibitor and an anti-animal antibody are used in combination, in addition to the above-mentioned potassium channel inhibitor, the anti-animal antibody is 0.5-100 units / kg, preferably 10-50 units / kg per week. It is preferable to administer subcutaneously once a week. In this case, the potassium channel inhibitor and the anti-animal antibody may be administered simultaneously or with a time difference.
In this way, animals that cause pathologies very similar to each type of human hypertrophic cardiomyopathy can be produced. That is, by adjusting the dose of a potassium channel inhibitor, the degree of ventricular septal thickening and lumen loss, and the increase in left ventricular myocardial area can be controlled. Ventricular septal hypertrophic cardiomyopathy or afferent hypertrophy Cardiomyopathy-like changes can be selectively developed. In addition, administration of high doses can cause interstitial fibrosis and edema, which can cause changes similar to the transition process of hypertrophic cardiomyopathy to the expansion phase.
In addition, when combined with anti-animal antibodies, the left ventricular myocardial area increased significantly, myocardial cell diameter increased, the degree of fibrosis increased, and the degree of edema increased compared to administration of a potassium channel inhibitor alone. The incidence is also significantly increased and easily causes a diastole-like change seen at the end of so-called hypertrophic cardiomyopathy.
As described above, the production of a hypertrophic cardiomyopathy model animal of the present invention is simple and easy in terms of the method, as it is merely a continuous administration of a potassium channel inhibitor or a potassium channel inhibitor and an anti-animal antibody.
This hypertrophic cardiomyopathy model animal is a pathologic model animal for elucidating the pathophysiology of hypertrophic cardiomyopathy, evaluating the efficacy of drugs for the prevention and treatment of hypertrophic cardiomyopathy, such as screening tests for the development of new drugs, etc. Can be used.
That is, an appropriate amount of a test substance is administered to the model animal of the present invention by an administration route such as injection or oral, and its action and effect over time is observed and evaluated by general means, or together with a potassium channel inhibitor or the like. By continuously administering a test substance to an animal and evaluating the degree of suppression of myocardial thickening, screening and drug efficacy tests can be performed.
Nicorandil (Uchida Y: Jpn Heart J 19: 904, 1978), which is known as an angina treatment, is a drug having a potassium channel opening action and a cyclic GMP stimulating action, together with a potassium channel inhibitor. When continuously administered to rats, myocardial thickening was suppressed, and fibrosis and edema were also suppressed. Therefore, potassium channel openers such as nicorandil, pinacidil, diazoxide, cromakalim, lebucromakalim, remacalim, minoxidil, particularly nicorandil are useful as preventive or therapeutic agents for hypertrophic cardiomyopathy. When such a potassium channel opener is used as a prophylactic or therapeutic agent for hypertrophic cardiomyopathy, the dosage is preferably 0.1 to 100 mg / kg per day, and the dosage form is tablets, powders, Any formulation such as pills, granules, capsules, suppositories, liquids, injections and the like may be used.

1. Preparation of rat hypertrophic cardiomyopathy model Using 4-week-old Wistar rats. Seven animals were divided into the following seven groups.
(1): Control group,
(2) to (5): 3,4-diaminopyridine aqueous solution 5 mg, 10 mg, 20 mg, 40 mg / Kg administration group (daily, oral administration),
(6): 3,4-diaminopyridine aqueous solution 40 mg / Kg daily oral administration and anti-rat whole body antibody 20 units / Kg (once a week, subcutaneous injection) combination group,
(7): 3,4-diaminopyridine aqueous solution 40 mg / Kg and nicorandil 20 mg / Kg combined use group (daily oral administration).
The dose was corrected by measuring the body weight once a week. The administration period was 8 weeks, and the heart was removed at 8 weeks, and the following morphological and histological examinations were performed.
1) (Myocardial wet weight / body weight) × 10 ³
2) Average value of the myocardial cross-sectional area in a circular slice of the region where the left ventricle is divided into three equal parts in the long axis direction (mm ² )
3) Ventricular nucleus thickness / left ventricular posterior wall thickness (1.3 or more was considered a septal thickening type)
4) The average value of the left ventricular empty area in a sliced section of the left ventricle divided into three equal parts in the long axis direction (mm ² )
5) Myocardial cell diameter (μm)
6) Presence or absence of left ventricular myocardial fibrosis 7) Presence or absence of left ventricular myocardial stromal edema Results 1) Administration of 3,4-diaminopyridine FIG. 1 shows a sample of the left ventricular short axis. Compared with the control example (FIG. 1, A), the administration of 3,4-diaminopyridine 5 mg / Kg (FIG. 1, B) shows a marked thickening of the ventricular septum (arrow) and a decrease in lumen. In the administration example of 3,4-diaminopyridine 40 mg / Kg (FIG. 1, C), thickening over the entire circumference and a decrease in lumen, that is, afferent thickening are observed.
Although cardiomyocytes were shown in FIG. 2, marked hypertrophy was seen in the 3,4-diaminopyridine 40 mg / Kg administration example (FIG. 2, B) compared to the control example (FIG. 2, A).
Table 1 shows the results of statistical processing. The myocardial weight / body weight ratio was significantly increased in the administration group of 3,4-diaminopyridine of 20 mg / Kg or more. The left ventricular myocardial area, which is an index of myocardial weight, significantly increased in the administration group of 3,4-diaminopyridine 10 mg / Kg or more. That is, myocardial hypertrophy occurred. Septal hypertrophic cardiomyopathy refers to cases where the ventricular septal thickness / left ventricular posterior wall thickness is 1.3 or more, but exceeds 1.3 at low doses of 3,4-diaminopyridine 5 mg / Kg and 10 mg / Kg. It was. That is, a change similar to ventricular septal thickening hypertrophic cardiomyopathy occurred. In the administration group of 3,4-diaminopyridine of 20 mg / Kg or more, it became thickened to the whole circumference, the left ventricular lumen decreased, and a change similar to afferent hypertrophic cardiomyopathy occurred. The cardiomyocyte diameter appeared in the administration group of 3,4-diaminopyridine 5 mg / Kg or more, and increased in a dose-dependent manner. That is, cardiomyocyte hypertrophy was determined to be the cause of left ventricular hypertrophy. Interstitial fibrosis and edema occurred at high doses. In the 3,4-diaminopyridine 40 mg / Kg administration group, the left ventricular lumen showed an increasing tendency, which was similar to the transition process of hypertrophic cardiomyopathy seen in humans to the expansion phase.
2) Administration of anti-animal antibody in combination FIG. 1D shows an anti-rat systemic antibody combination example. The left ventricular wall was markedly thickened and marked edema (arrow) was observed (FIG. 1, D). Moreover, edema and fibrosis (arrow) were observed (FIG. 2, C). As shown in Table 1, the left ventricular myocardial area is significantly increased, the myocardial cell diameter is increased, the degree of fibrosis is higher, and the incidence of edema as compared with 3,4-diaminopyridine 40 mg / Kg alone. Also increased significantly. That is, the anti-rat systemic antibody promoted myocardial hypertrophy due to 3,4-diaminopyridine, while causing myocardial damage, causing a so-called diastole-like change seen at the end of hypertrophic cardiomyopathy.
3) Effects of nicorandil administration By using nicorandil (manufactured by Chugai Pharmaceutical Co., Ltd.) 20 mg / Kg, as shown in E of FIG. 1, D of FIG. 2 and Table 1, myocardial thickening is suppressed, and fibrosis and edema are also suppressed. It was done. That is, the appearance of changes similar to hypertrophic cardiomyopathy was prevented. Therefore, nicorandil has been found to be useful as a preventive or therapeutic agent for hypertrophic cardiomyopathy.

  According to the present invention, a model animal that develops cardiomyopathy very similar to human hypertrophic cardiomyopathy can be produced very easily and in a short period of time. This animal can be used for elucidating the pathophysiology of hypertrophic cardiomyopathy and screening for prophylactic / therapeutic drugs, and is extremely useful as an experimental animal.

Claims (9)

An experimental model animal that develops human hypertrophic cardiomyopathy, which is produced by continuously administering a potassium channel inhibitor or a potassium channel inhibitor and an antibody to the whole body or heart of the animal. The model animal according to claim 1, wherein the potassium channel inhibitor is a pyridine derivative having an amino group, tetraethylammonium, or glibenclamide. The model animal according to claim 2, wherein the pyridine derivative having an amino group is 3,4-diaminopyridine. A method for producing an experimental model animal that develops hypertrophic cardiomyopathy in humans, comprising continuously administering a potassium channel inhibitor or a potassium channel inhibitor and an antibody to the whole body or heart of the animal. Of producing a disease model animal. The drug evaluation method using the model animal of any one of Claims 1-3. A prophylactic or therapeutic agent for hypertrophic cardiomyopathy comprising a potassium channel opener as an active ingredient. The preventive or therapeutic agent for hypertrophic cardiomyopathy according to claim 6, wherein the potassium channel opener is nicorandil or pinacidil. Use of a potassium channel opener for the manufacture of a prophylactic or therapeutic agent for hypertrophic cardiomyopathy. A method for treating hypertrophic cardiomyopathy, comprising administering a potassium channel opener.

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