JPH111439A - Therapeutic agent for dilated cardiomyopathy - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject therapeutic agent, effective for treating/ preventing dilated cardiopathy in humans and other mammals, slight in side effects through only acting on affected tissues, by including hepatocyte growth factor as active ingredient. SOLUTION: This therapeutic agent contains hepatocyte growth factor(HGF) as active ingredient. The HGF is prepared, for example, through the following process: a rat is intraperitoneally administered with carbon tetrachloride and brought to hepatitis; the liver of the resultant rat is extracted and ground, and then purified by ordinary protein purification process such as gel column chromatography with, e.g. S-sepharose. The daily dose of this agent is normally 1-100 mg in terms of HGF in one to several portions in intravenous, intraartery, subcutaneous, intramuscular way in the form of injection 0.001-0.1 w/v% in HGF content.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a drug effective for treating and preventing dilated cardiomyopathy. More specifically, the present invention relates to a therapeutic agent for dilated cardiomyopathy containing hepatocyte growth factor (HGF) as an active ingredient.

[0002]

BACKGROUND OF THE INVENTION The major condition of dilated cardiomyopathy is a decrease in myocardial contractility, which results in an enlarged left ventricular lumen. It is easy to cause decrease in left ventricular ejection blood volume and increase in left ventricular diastolic pressure,
The patient is susceptible to congestive heart failure. But,
Not all will result in congestive heart failure, and in this sense dilated cardiomyopathy is more rational than conventional congestive cardiomyopathy. Onset can be acute or insidious, but often presents with refractory heart failure at the end. Histopathologically, diffuse or local degeneration, fibrosis, and atrophy of myocardial tissue are observed. In many cases, the residual cardiomyocytes are enlarged. Possible causes include alcohol overdose, viral sequelae, microvascular spasm, and immune mechanism, but it is unknown. Some have familial outbreaks, suggesting recessive inheritance. In addition to heart failure, severe arrhythmias and thromboembolism occur, and the prognosis is extremely poor. What should be differentiated include ischemic heart disease, secondary myocardial disease causing heart failure, and hypertensive heart disease. But,
In the treatment of dilated cardiomyopathy, it is not enough to suppress each factor alone, and it is necessary to suppress multiple factors at the same time. In the future, treatment should be considered in such a direction.

[0003]

Dilated cardiomyopathy is an intractable disease that accounts for about one-quarter of heart failure patients in the United States and about half of patients undergoing heart transplantation. In recent years, several possible causes of dilated cardiomyopathy have been revealed. The definition of dilated cardiomyopathy by the WHO / ISFC committee in 1995 includes a statement suggesting that the cause is genetic predisposition, viral infection, alcohol, and the like. Current,
Dilated cardiomyopathy tends to be classified according to its etiology and pathological condition. When chronic inflammation persists due to viral infections or immune abnormalities, many transcription factors are activated at the same time, producing autoantibodies, increasing the expression of MHC and adhesion molecules, producing cytokines, activating oncogenes, etc. It is likely to last. It is also conceivable that cardiomyocytes are continuously damaged due to genetic predisposition due to mutated gene products or MHC-mediated immune abnormalities. These persistent damage to cardiomyocytes may lead to dilated cardiomyopathy-like lesions, but the mechanism is still unknown. However, when treating dilated cardiomyopathy, it is not enough to suppress each factor alone, and it is necessary to suppress multiple factors at the same time, and we must consider treatment in this direction in the future. It is the current situation.

[0004] The basic conditions of dilated cardiomyopathy are pulmonary congestion and limitation of cardiac output due to left heart failure, and daily life is often restricted due to dyspnea and effortlessness during exertion.
Peripheral edema is also observed in patients with right heart failure. Reducing symptoms by improving hemodynamics is an important treatment goal for the time being to gain patient confidence. However, since myocardial insufficiency of this disease is progressive and has a poor prognosis, long-term survival is also an important therapeutic goal. Although treatment for the cause of this disease is not possible at present,
In recent years, understanding of factors promoting and suppressing progression of myocardial insufficiency has been deepened, and drugs that suppress progression and have a life-prolonging effect have appeared. It is considered that enhancement of the sympathetic nerve and renin-angiotensin-aldosterone system is important as a myocardial failure promoting factor. Suppression of these factors not only suppresses the progression of heart failure, but can also be expected to relieve some degree of myocardial failure. Naturally, in that case, the heart failure symptoms are also reduced secondarily. Appropriate lifestyle guidance along with drug treatment is indispensable for the treatment goals of both reducing heart failure symptoms and suppressing progression of heart failure. Infection, anemia, thyroid dysfunction, etc. are factors that exacerbate heart failure, and thus preventive and early treatment are necessary. Diabetes, alcohol overdose, and long-lasting tachycardia can also result in myocardial insufficiency similar to dilated cardiomyopathy, but in these cases myocardial insufficiency usually improves significantly with correction of the underlying disease. It is. Although there is no specific complication of dilated cardiomyopathy, as in general heart failure, there are arrhythmias and embolism due to thrombosis lining the heart chamber, which are also important as direct causes of death. In any case, dilated cardiomyopathy has a 5-year survival rate of 50-60% and is a disease with a poor prognosis, and elucidation of its cause and development of an effective treatment are urgently desired. .

[0005] From the above viewpoint, the present inventors have found that HGF has an inhibitory, ameliorating or preventive effect on dilated cardiomyopathy, and that HGF is useful for the prevention and treatment of dilated cardiomyopathy. There was found. The above-mentioned HGF is a protein found as a factor for growing hepatocytes in vitro (Biochem. Biophys. Res. Commun., 122 ,
1450, 1984, Proc. Natl. Acad. Sci. USA, 83 , 6489,
1986, FEBS Letter, 22 , 311, 1987, Nature, 342 , 44
0, 1989, Proc. Natl. Acad. Sci. USA, 87 , 3200, 199
0). HGF, which was discovered as a factor that specifically promotes liver parenchymal cells, has shown various activities such as tissue injury healing in vivo by recent research results by many researchers including the present inventors. It becomes clear that
Expectations are high for its application not only as a research target but also as a therapeutic drug for humans and animals. It has been elucidated that HGF is mainly produced by mesenchymal cells, and it has been clarified that a so-called paracrine mechanism in which HGF is supplied from neighboring cells as necessary is established. However, when the liver or kidney is injured, the production of HGF is also increased in uninjured organs such as the lungs.
Is considered to have been supplied. Recent studies on the receptor for HGF have confirmed that the c-Met proto-oncogene encodes the HGF receptor (Bottaro et al., Science 251 , 802-804, 1991). ; Nald
ini et al., Oncogene 6 , 501-504, 1991). As described above, many findings have been obtained regarding HGF.
It is a new finding that F is capable of suppressing, improving or preventing dilated cardiomyopathy. The present invention has been made based on such findings, and an object of the present invention is to provide a novel therapeutic agent for dilated cardiomyopathy.

[0006]

Means for Solving the Problems The present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide hepatocyte growth factor (HGF).
As an active ingredient.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION As HGF used in the present invention, those prepared by various methods can be used as long as they are purified to the extent that they can be used as pharmaceuticals. Various methods are known for preparing HGF. For example, it can be extracted and purified from organs such as liver, spleen, lung, bone marrow, brain, kidney, and placenta of mammals such as rats, cows, horses, and sheep, blood cells such as platelets and leukocytes, plasma, and serum. (FEBS Letters, 224 ,
312, 1987, Proc. Natl. Acad. Sci. USA, 86 , 5844, 1
989). In addition, primary culture cells or cell lines that produce HGF are cultured and cultured (culture supernatant, cultured cells, etc.).
HGF can also be obtained by separation and purification from HGF. Alternatively, a gene encoding HGF is inserted into an appropriate vector by a genetic engineering technique, inserted into an appropriate host, and transformed, and the desired recombinant HGF can be obtained from the culture supernatant of the transformant. Yes (for example, Nature, 342 ,
440, 1989, JP-A-5-111383, Biochem. Biophy
s. Res. Commun., 163 , 967, 1989). The host cell is not particularly limited, and various host cells conventionally used in genetic engineering techniques, for example, Escherichia coli, Bacillus subtilis, yeast, filamentous fungi, plant or animal cells, and the like can be used.

More specifically, as a method for extracting and purifying HGF from a living tissue, for example, carbon tetrachloride is intraperitoneally administered to a rat, and the liver of a rat in a hepatitis state is extracted, excised and pulverized. -It can be purified by a conventional protein purification method such as gel column chromatography such as sepharose and heparin sepharose, and HPLC. In addition, animal cells, such as Chinese hamster ovary (CHO) cells, mouse C127 cells, monkeys, and the like, can be expressed by an expression vector in which a gene encoding an amino acid of human HGF has been incorporated into a vector such as bovine papilloma virus DNA by genetic recombination. COS cells can be transformed and obtained from the culture supernatant.

The HGF thus obtained may have a part of its amino acid sequence deleted or substituted by another amino acid, or may have another amino acid sequence partially inserted, as long as it is substantially equivalent to HGF. , N-terminal and / or C-terminal may have one or more amino acids bound thereto, or the sugar chain may be similarly deleted or substituted.

[0010] The therapeutic agent of the present invention contains the above-mentioned HGF as an active ingredient. As shown in the test examples described below, HGF is effective against dilated cardiomyopathy caused by hamster Bio53.58, which is a spontaneous model of dilated cardiomyopathy. It has the effect of suppression, improvement or prevention. Furthermore, since HGF does not act on unaffected tissues but acts only on impaired tissues, it has the advantage that it is less likely to cause side effects. Therefore, the therapeutic agent of the present invention is useful for treating dilated cardiomyopathy.
Effective for prevention. The therapeutic agent of the present invention is applied to the treatment and prevention of dilated cardiomyopathy in mammals (eg, cows, horses, pigs, sheep, dogs, cats, etc.) in addition to humans.

[0011] The therapeutic agent of the present invention can be in various preparation forms (eg, liquid, solid, capsule, etc.). In general, the active ingredient is HGF alone or an injection or inhalation thereof together with a conventional carrier. Preparations, suppositories or oral preparations. The injection can be prepared by a conventional method.
F can be prepared by dissolving F in a suitable solvent (eg, sterilized water, buffer, physiological saline, etc.), filtering with a filter or the like, sterilizing, and then filling an aseptic container. . The HGF content in the injection is usually 0.1.
It is adjusted to about 0002-0.2 (w / v%), preferably about 0.001-0.1 (w / v%). In addition, as oral drugs, for example, tablets,
It is formulated into dosage forms such as granules, powders, soft or hard capsules, liquids, emulsions, suspensions, syrups and the like, and these formulations can be prepared according to the usual methods of formulation. Suppositories can also be prepared by a conventional method using a conventional base (for example, cacao butter, laurin butter, glycerogelatin, macrogol, witepsol, etc.). Also, an inhalant can be prepared according to conventional means for preparation. The HGF content in the preparation can be appropriately adjusted according to the dosage form, applicable disease and the like.

At the time of formulation, a stabilizer is preferably added. Examples of the stabilizer include albumin, globulin, gelatin, glycine, mannitol, glucose, dextran, sorbitol, ethylene glycol and the like. Further, the preparation of the present invention may contain additives necessary for preparation, for example, excipients, solubilizing agents, antioxidants, soothing agents, isotonic agents and the like. In the case of a liquid preparation, it is desirable to preserve by removing the water by cryopreservation or freeze-drying. The lyophilized preparation is used after reconstitution by adding distilled water for injection or the like at the time of use.
The therapeutic agent of the present invention can be administered by an appropriate route depending on the form of the preparation. For example, it can be administered in the form of an injection to vein, artery, subcutaneous, intramuscular, and the like.
The dose is appropriately adjusted depending on the condition, age, body weight, etc. of the patient, but is usually 0.05 mg to 500 mg, preferably 1 mg to 100 mg as HGF, which is administered once or several times a day. It is appropriate to do.

[0013]

According to the present invention, the active ingredient HGF
Has an inhibitory, ameliorating, or preventing effect on dilated cardiomyopathy. Therefore, the therapeutic agent of the present invention is effective for the treatment and prevention of the aforementioned dilated cardiomyopathy. In addition, HGF
Since it acts only on the affected tissue, it is possible to obtain a drug with few side effects.

[0014]

EXAMPLES Hereinafter, the present invention will be described in more detail based on test examples and examples, but the present invention is not limited to these examples. Test Example 1 Hamster Bio53.
Hamster Bio53, a spontaneous model of dilated cardiomyopathy tested at 58 .
Using 58 (3 months old), HGF was subcutaneously administered at 450 μg / kg once a day for 30 days. HGF administration group is 10
The test was performed on 8 groups of the group administered with the saline as a control. The results are as follows. Ventricular diameter (Cardiac Dimensio)
n), the heart rate (HR), left ventricular volume [end diastolic diameter (E
DD), end-systolic diameter (ESD)], wall thickness [ventricular septum Th
(IVS), posterior wall Th (PW)] had no effect (see FIG. 1). For systolic function, left ventricular ejection fraction (LVEF), left ventricular diameter shortening rate (FS), wall thickening rate [ventricular septum Thickening (IVS), posterior wall Thickening (PW)] significantly improved (See FIG. 2). Regarding diastolic function, atrial inflow blood flow tends to be high, but early diastolic mitral flow inflow velocity (Early Diastolic Mitral Flow Velocit)
y), atrial systolic mitral orifice blood flow inflow rate (Peak Mitral Flo
w Velocity at Atrial Contraction), A / E, and decay time (Deceleration time) were not significantly different (see FIG. 3). There was no significant change in myocardial weight or left ventricular weight. In histopathological examination, HGF administration decreased collagen fibers [fibrosis (% Area Fibrosis in
the Ventricles), that is, the area of fibrous tissue stained with aniline blue, and the hydroxyproline content (Hydroxypr
oline Content), myocytes (Myocyte Diameter) were relatively large, and vacuolation and suppression of nuclear dysplasia were observed (see FIG. 4). In the HGF-administered group, calcium deposition and inflammatory cell infiltration were also small. From the above, it was found that HGF was effective for cardiomyopathy of hamster Bio53.58, which is a model of dilated cardiomyopathy.

Example 1 A solution containing 1 mg of HGF, 1 g of mannitol and 10 mg of polysorbate 80 in 100 ml of physiological saline is aseptically prepared, dispensed into vials in 1 ml portions, freeze-dried and sealed to freeze-dry the preparation. I got

Example 2 0.02 M phosphate buffer (containing 0.15 M NaCl and 0.01% polysorbate 80, pH 7.4) 10
HGF 1 mg and human serum albumin 100 in 0 ml
An aqueous solution containing mg was aseptically prepared, dispensed in 1 ml portions into vials, lyophilized and sealed to obtain a lyophilized preparation.

[Brief description of the drawings]

FIG. 1. Heart rate (HR), left ventricular volume [end-diastolic diameter (EDD), end-systolic diameter (ESD)], wall thickness [ventricular septum Th (IVS), after Wall Th (PW)].

FIG. 2: Left ventricular ejection fraction (LVEF), left ventricular diameter shortening rate (FS), wall thickening rate [ventricular septum Thickening (IVS), posterior wall Thickening (PW)] in the HGF administration group and the saline administration group FIG.

FIG. 3 is a diagram comparing the diastolic early mitral orifice blood flow inflow velocity, atrial systolic mitral orifice blood flow inflow velocity, A / E, and decay time between the HGF administration group and the saline administration group.

FIG. 4 is a diagram comparing% fibrosis area, hydroxyproline content, and myocyte diameter between the HGF administration group and the saline administration group.

Claims (1)

[Claims] 1. A therapeutic agent for dilated cardiomyopathy, comprising hepatocyte growth factor as an active ingredient.