HK1036019B - Treatment of cardiac hypertrophy - Google Patents

Description

Method for treating cardiac hypertrophy

Technical Field

The invention mainly relates to the function of gamma interferon on cardiac hypertrophy. More particularly, the invention relates to the prevention or treatment of cardiac hypertrophy and related disorders with interferon gamma.

Background

Gamma interferon (IFN-gamma)

Interferons are small single-chain glycoproteins secreted by cells invaded by viruses or some other substance. Currently, interferons are mainly classified into three types: leukocyte interferon (interferon- α, IFN- α 1), fibroblast interferon (interferon α 0, interferon α 3, IFN- β) and immune interferon (interferon- γ, interferon γ, IFN- γ). In response to viral infection, lymphocytes synthesize primarily interferon-alpha 2 (and a small amount of a different interferon, commonly referred to as interferon-omega), whereas infection of fibroblasts usually induces interferon-beta. Interferon-alpha has about 20-30% amino acid sequence homology with interferon-beta. The human IFN- β gene has no intron and the encoded protein has 29% amino acid sequence homology with human IFN- α I, indicating that IFN- α and IFN- β are from the same ancestor (Taniguchi et al,Nature 285,547-549(1980)). In contrast, IFN- γ is not induced by viral infection but is synthesized by lymphocytes in response to mitogens and has little relationship in amino acid sequence to the other two classes of interferons. Alpha interferon and beta interferon are known to induce MHC class I antigen, and IFN-gamma induces MHC class II antigen expression and enhances targetThe cells present the effect of the viral peptide bound to MHC class I molecules for recognition by cytotoxic T cells.

IFN-gamma is a member of the interferon family, with alpha and beta interferon (IFN-alpha and beta) antiviral and antiproliferative properties, but unlike them is unstable to PH 2.IFN- γ is originally produced by mitogen-induced lymphocytes. Gray, Goeddel and colleagues have first reported recombinant production of human IFN- γ (Gray et al,Nature 295，503-508[1982]) This is also the subject of U.S. patents 4,762,791, 4,929,544, 4,727,138, 4,925,793, 4,855,238, 5,582,824, 5,096,705, 5,574,137 and 5,595,888. Recombinant human IFN-. gamma.produced by Gray and Goeddel in E.coli has 146 amino acids, starting at the N-terminus of the molecule as CysTyrCys. It was later found that the native human IFN-gamma (i.e., a mitogen-induced production and purification of human peripheral blood lymphocytes) polypeptide did not have the CysTyrCys N-terminus as described by Gray et al, supra. Recently, the crystal structure of recombinant human IFN-. gamma.produced by E.coli (rhIFN-. gamma.) was determined (Ealick et al,Science 252，698-702[1991]) The protein is shown to be a non-covalent dimer with tight entanglement, in which the two polypeptide chains are antiparallel to each other.

IFN-gamma is known to have a number of biological activities, including anti-tumor, anti-bacterial and immunomodulatory activities. One form of recombinant human IFN- γ (rhIFN- γ -1b, actimune *, Genentech, inc. south San Francisco, california) is marketed as an immunomodulatory drug for the treatment of chronic granulomatosis, characterized by severe and recurrent infections of skin, lymph nodes, liver, lung and bone caused by dysfunction of phagocytes (Baehner, r.l.,Pediatric Pathol.10,143-153(1990)). Also proposed is the use of IFN-gamma for the treatment of allergic dermatitis, a common skin inflammatory condition characterized by severe itching, prolonged and recurrent disease progression, intermittent worsening, unique clinical morphology and distribution of skin lesions (see PCT application WO9I/07984, published 6/13/1991), vascular stenosis, including the treatment of restenosis following angioplasty and/or vascular surgery (PCT application WO90/03189, published 4/5/1990), various pulmonary disorders, including dyspnea syndrome (RDS)For example, Adult Respiratory Distress Syndrome (ARDS) and neonatal type, also known as idiopathic RDS or pulmonary hyaline membrane disease (PCT application WO89/01341, published on 23/2.1989). In addition, IFN-gamma has been proposed for the treatment of various allergies, such as asthma and conditions associated with HIV infection, such as opportunistic infections, e.g., Pneumocystis carinii pneumonia and traumatic sepsis. It has been found that IFN- γ production is impaired in patients with multiple sclerosis, and it has been reported that IFN- γ production is severely inhibited in mitogen-stimulated monocyte suspensions of AIDS patients. For discussion see, e.g., chapter 16, "pathogenic roles interferons may have in disease",Interferons and Regulation Cytokines，Edward de Maeyer(1988，John Wilet and SonsPublishers)。

it is now believed that IFN- γ, as well as other cytokines, are inducers of Inducible Nitric Oxide (iNOS) and are therefore described as a potential inflammatory mechanism responsible for heart failure, the response of the heart to sepsis or transplant rejection, and an important mediator in the development of dilated cardiomyopathy of various etiologies. Unkureanu-Longrois, etc.,Circ.Res. 77494-; the number of the molecules of Pinsky et al,J.Clin.Invest.95677-685 (1995); singh, etc., and the like,J.Biol. Chem.27028471-8 (1995); birks and yacobb, respectively,Coronary Arterry Disease 8389-402, (1997); hattori et al,J.Mol.Cell.Cardiol.29,1585-92(1997). In fact, it has been reported that IFN- γ is the most potent cytokine for myocyte iNOS induction (Watkins et al,J.Mol. & Cell Cardiol 27，2015-29(1995))。

cardiac hypertrophy

Hypertrophy generally refers to an abnormal increase in development of an organ or structure, but is not associated with tumor formation. Hypertrophy of organs or tissues is caused either by an increase in the volume of individual cells (true hypertrophy), by an increase in the number of cells constituting a tissue (hyperplasia), or by a combination of both.

Cardiac hypertrophy refers to enlargement of the heart by mechanical and hormonal stimulation to adapt the heart to increased cardiac output or injury. The Morgan and Baker are known to be,Circulation 83,13-52(1991). This response is often accompanied by a number of different pathological conditions, such as hypertension, aortic stenosis, myocardial infarction, cardiomyopathy, valve regurgitation, cardiac bypass, congestive heart failure, etc.

At the cellular level, the heart works as a syncytium consisting of myocytes and surrounding supporting cells called non-myocytes. Muscle cells are primarily fibroblasts/mesenchymal cells, but also include endothelial cells and smooth muscle cells. In fact, although adult cardiac muscle is composed primarily of myocytes, they account for only about 30% of the total cell count in the heart.

The fetal heart is enlarged primarily by an increase in the number of myocytes, which continues shortly after birth until the time when the myocytes lose proliferative capacity. Later progression is the enlargement of a single cell. Adult ventricular myocyte hypertrophy is a response to a variety of chronic hemodynamic overload conditions. Thus, in response to hormonal, physiological, hemodynamic, and pathological stimuli, adult ventricular myocytes can be altered to accommodate increased workload by activating a hypertrophic program. This response is characterized by an increase in myocytes and contractile protein content within individual cardiomyocytes, but is not accompanied by cellular differentiation and activation of embryonic genes, including the Atrial Natriuretic Peptide (ANP) gene. Chien et al, in the art,FASEB J.53037-3046 (1991); chien et al, in the art,Annu. Rev.Physiol.55,77-95(1993). It has been described that in left ventricular hypertrophy in humans caused by excessive stress, myocyte enlargement causes enlargement of myocardial tissue, and myocyte enlargement is associated with accumulation of interstitial collagen in the extracellular matrix and around the coronary artery in the myocardium (Caspari et al,Cardiovasc.Res.11554-8 (1977); schwarz et al, supra,Am.J.Cardiol.42895-903 (1978); hess et al, in the name of,Circulation 63360-371 (1981); pearlman, etc. in a mobile communication system,Lab Invest 46，158-164(1982))。most cardiac outcomes result in cardiac hypertrophy from chronic hemodynamic overload, a common feature of heart failure.

It has been suggested that paracrine factors produced by non-myocyte supporting cells may also be involved in the development of cardiac hypertrophy, and that a number of non-myocyte-produced hypertrophy factors, such as Leukocyte Inhibitory Factor (LIF) and endothelin, have been identified. The amount of the solvent in the solvent is Metcalf,Growth Factors 7169- > 173 (1992); the general name of Kurzrock et al,Endocrine Reviews 12208-217 (1991); the number of Inoue and the like,Proc.Natl.Acad.Sci.USA(published 11/12 in 1996). Other factors have been identified such as cardiac hypertrophy potential mediators, including cardiotrophin-1 (CT-1) (Pennica et al,Proc.Natl.Acad.Sci.USA 921142-46(1995)), catecholamines, adrenocorticosteroids, angiotensin and prostaglandins.

Adult myocyte hypertrophy is initially beneficial as a short-term response to impairment of cardiac function, reducing the load on individual muscle fibers. However, due to severe long-term overload, mast cells begin to degenerate and die. Katz, "Heart failure," Physiology of Heart (New York, Raven Press, 1992) pp.638-668, compiled by Kate A.M. In clinical heart failure, cardiac hypertrophy is a prominent lethal and pathogenic factor. The reaction solution of Katz is mixed with water,Trends Cardiovasc Med.5，37-44(1995)。

for more details on the cause and pathology of cardiac hypertrophy, see, e.g., Heart Disease, a textbook of cardiovacular Medicine, Braunwald, ed., w.b. saunders co., 1988, chapter 4, pathology of Heart failure.

Treatment of cardiac hypertrophy

Currently, the treatment of cardiac hypertrophy varies depending on the heart disease. Catecholamines, adrenocorticosteroids, angiotensin, prostaglandins, Leukemia Inhibitory Factor (LIF), endothelins (including endothelin-1, -2, -3, and macroendothelins), heartTrophic factor-1 (CT-1) and Cardiac Hypertrophy Factor (CHF), both of which are thought to be strong mediators of cardiac hypertrophy. For example, β -adrenergic receptor inhibitors (β -inhibitors such as propranolol, timolol, terbutalol, carteolol, nadolol, betaxolol, penbutolol, acetobourolol, atenolol, metoprolol, carvedilol, etc.) and verapamil have been widely used in the treatment of hypertrophic cardiomyopathy. The improvement in symptoms (e.g., chest pain) and exercise tolerance of β -inhibitors is primarily due to a slowing of heart rate, thereby prolonging diastole and increasing passive infusion into the ventricles. Thompson et al, in a similar manner,Br.Heart J.44, 488-98 (1980); the result of the work was Harrison et al,Circulation29, 84-89(1964). Verapamil is said to increase ventricular input and possibly reduce myocardial ischemia. The result of Bonow et al,Circulation27, 853-64(1985). Nifedipine and diltiazem * are also sometimes used to treat hypertrophic cardiomyopathy. The method of the present invention is described by Lorell et al,Circulation65, 499 and 507 (1982); betocchi et al, in the name of Betocchi,Am.J.Cardiol.78,451-7(1996). However, nifedipine may be harmful because of its strong vasodilatory properties, especially in patients with impaired output. The negative inotropic effect of propiram has been used to alleviate symptoms. Pollick.N.Eng.J.Med.307.997-9(1982). However, in many patients, the initial effects decline over time. A Wigle, etc., and a method for extracting,Circulation 92，1680-92(1995)。

treatment with antihypertensive agents has also been reported to be effective in hypertension-related cardiac hypertrophy. For example, drugs used alone or in combination for antihypertensive therapy are: calcium antagonists, such as nitrendipine; the aforementioned β -adrenergic receptor inhibitors; angiotensin converting enzyme (AIE) inhibitors such as quinapril, captopril, enalapril, ramipril, benazepril, fosinopril, lisinopril; diuretics, such as chlorothiazide, hydrochlorothiazide, hydroflumethiazide, methylchlorothiazide, benzylthiazide, dichlorofinamide, acetazolamide, indapamide; calcium channel inhibitors, such as diltiazem *, nifedipine, verapamil, nicardipine. For example, by DierTreatment of hypertension with sulfur * and captopril showed a reduction in left ventricular myocardium, but the Doppler index of diastolic action was not normalized. Szlachcic and the like,Am.J.Cardiol.63198-201 (1989); the results of Shahi et al,Lancet 336,458-61(1990). The above findings are interpreted as hints: excess interstitial collagen may be retained after the left ventricular hypertrophy is relieved. (ii) a Rossi, etc.,Am.Heart J.124,700-709(1992). Rossi et al (supra) studied the effect of captopril on preventing and reversing cellular hypertrophy, and on interstitial fibrosis in high pressure-loaded cardiac hypertrophy in experimental rats.

Because there is no general method of treating cardiac hypertrophy, the identification of factors capable of preventing or reducing cardiomyocyte hypertrophy is particularly important in developing new treatments to inhibit pathological cardiac growth.

Brief summary of the invention

We have unexpectedly found that IFN-gamma inhibits prostaglandin F in isolated cells from rats_2α(PGF_2α) And phenylephrine-induced cardiomyocyte expansion. We have also found that IFN-gamma inhibits PGF in vivo in a live rat model_2αThe antagonistic analogs fluprostenol-induced hypertrophy and stress-load-induced hypertrophy.

The present invention therefore relates to the treatment of cardiac hypertrophy of various underlying etiologies by administering a therapeutically effective amount of IFN-gamma. If human patients are treated, recombinant human IFN-gamma (rh IFN-gamma) is preferred, with rh IFN-gamma-1 b being most preferred, as described below. Treatment herein is used in its broadest concept and specifically includes prevention, alleviation, mitigation and cure of cardiac hypertrophy at various stages.

It is preferable to administer IFN-. gamma.in the form of a liquid preparation which is preserved to have long-term stability. Preserving the liquid medicament may contain multiple doses of IFN- γ and may therefore be reused.

IFN-gamma may be used in combination with one or more other agents for treating cardiac hypertrophy or for promoting the development of a pathological condition of cardiac hypertrophy such as hypertension, aortic stenosis or myocardial infarction.

The present invention also includes a method for manufacturing a pharmaceutical composition for treating cardiac hypertrophy comprising IFN- γ as an active ingredient.

The invention also relates to a pharmaceutical product comprising:

(a) a pharmaceutical composition comprising at least one therapeutically effective amount of IFN- γ;

(b) a container containing the pharmaceutical composition; and

(c) a label carried by the container or a package insert within the pharmaceutical product, indicating use of the IFN- γ for treating cardiac hypertrophy.

Brief Description of Drawings

In the figures and examples, "IFN" or "IFN- γ" refers to recombinant murine IFN- γ (Genentech, Inc., Southsan Francisco, CA or Genzyme, Cambridge, MA).

FIG. 1: IFN-gamma vs_2α(PGF_2α) Inhibition of induced enlargement. On the day of isolation, myocytes were pre-cultured with saline vehicle or IFN- γ (500U/ml). 24 hours after separation, a second addition of vehicle or IFN-gamma, together with vehicle or PGF_2α(10^-7M). After further culturing for 72 hours, the cells were fixed with glutaraldehyde, stained with yellow eosin, and observed with a fluorescence microscope. A. B, C reference and PGF_2αAnd PGF_2α+ IFN-. gamma.cardiomyocytes after 4 days of culture. Histograms showing the maximum width of the rod cardiomyocytes and the percentage of respiratory frequency. The maximum width of the rod cells was determined using fluorescence microscopy and image analysis software. Each group examined at least 200 rod cells of the same experiment. No observed effect of IFN- γ alone on cell morphology. P < 0.001 for all comparisons between groups.

FIG. 2: IFN-gamma (500-25U/ml) vs PGF_2αDose-responsive inhibition of the induced response. On the day of isolation, myocytes were pre-cultured with saline vehicle or IFN- γ. 24 hours after separation, a second addition of vehicle or IFN-gamma, together with vehicle or PGF_2α(10^-7M). After further culturing for 72 hours, the cells were fixed with glutaraldehyde, stained with yellow eosin, and observed with a fluorescence microscope. And (3) cell morphology quantification: control A, B PGF_2α，C PGF_2α+IFN-γ(25U/ml)，D PGF_2α+IFN-γ(100U/ml)，E PGF_2α+ IFN-. gamma.s (500U/ml). Histograms showing the maximum width of the rod cardiomyocytes and the percentage of respiratory frequency. The maximum width of the rod cells was determined using fluorescence microscopy and image analysis software. Each group examined at least 200 rod cells of the same experiment. No observed effect of IFN- γ alone on cell morphology. P < 0.001 for all comparisons between groups.

FIG. 3: inhibition of Phenylephrine (PE) induced swelling by IFN- γ. On the day of isolation, myocytes were pre-cultured with saline vehicle or IFN- γ (500U/ml). 24 hours after separation, a second addition of vehicle or IFN-. gamma.is carried out, together with the addition of vehicle or PE (10)^-5M). After further culturing for 72 hours, the cells were fixed with glutaraldehyde, stained with yellow eosin, and observed with a fluorescence microscope. A. B, C were cardiomyocytes cultured for 4 days with control, PE and PE + IFN-. gamma.respectively. Histograms showing the maximum width of the rod cardiomyocytes and the percentage of respiratory frequency. The maximum width of the rod cells was determined using fluorescence microscopy and image analysis software. Each group examined at least 200 rod cells of the same experiment. No observed effect of IFN- γ alone on cell morphology. All comparisons between groups P < 0.001.

FIG. 4: effect of IFN- γ on Fluprostenol-induced cardiac hypertrophy in rats. Data shown are mean ± SEM. In brackets are the number of animals per group. Compared with the group of the carriers,^*P＜0.05，^*p is less than 0.0 l. Compared with the Flup group, # P < 0.05, # P < 0.01. And (3) Flup: fluprostenol; IFN- γ; HW: heart weight; BW: body weight; VW: ventricular weight; LVW left ventricle weight.

FIG. 5: the effects of flu and/or IFN on MAP and HR. Data shown are mean ± SEM. In brackets are the number of animals per group. Compared with the group of the carriers,^*p is less than 0.05. Compared with the Flup group, # P < 0.05. Compared with the group of Flup + IFN, + P < 0.05. And (3) Flup: fluprostenol; IFN- γ; MAP: mean arterial pressure; HR: heart rate.

FIG. 6: histograms showing the effect of Fluprostenol (FLUP) and IFN- γ on the expression of: skeletal actin (SKA); b sarcoplasmic reticulum calcium atpase (srca); collagen i (coi); d Atrial Natriuretic Factor (ANF). Expression levels were normalized to glutaraldehyde-3-phosphate dehydrogenase (GAPDH) messenger. VEH is a vector. There were 7 animals per group and data are presented as mean ± SEM. P < 0.05 for VEH group.

FIG. 7: in high pressure loaded rats, IFN- γ effects on cardiac weight, ventricular weight and left ventricular weight. Data are presented as mean ± SEM. In brackets are the number of animals per group. In contrast to the dummy treatment group,^**p is less than 0.01. Compared to ligation + vehicle group, # # P < 0.01. Ligation group: rats with their aorta ligated; IFN: IFN-gamma; WH: heart weight; VW: ventricular weight; LVW left ventricle weight.

FIG. 8: effect of IFN- γ on heart weight, ventricular weight and left ventricular weight to body weight in high stress loaded rats. Data are presented as mean ± SEM. In brackets are the number of animals per group. In contrast to the dummy treatment group,^**p is less than 0.01. Compared to ligation + vehicle group, # # P < 0.01. Ligation group: rats with their aorta ligated; IFN: IFN-gamma; WH: heart weight; VW: ventricular weight; LVW left ventricle weight.

FIG. 9: effects of IFN- γ on systolic arterial pressure, mean arterial pressure and diastolic arterial pressure in pressure-loaded rats. In brackets are the number of animals per group. In contrast to the dummy treatment group,^**p is less than 0.01. Ligation group: rats with their aorta ligated; IFN: IFN-gamma; MAP: mean arterial pressure; DAP: arterial diastolic pressure.

Detailed description of the invention

A. Definition of

"Gamma interferon", "interferon gamma" or "IFN-gamma" refers to any form of gamma interferon (human and other animal) that exhibits biological activity in any cardiac hypertrophy assay, such as the hypertrophy assays described herein, including but not limited to mature, pre, altered and/or de (1-3) (also known as des CysTyrCys IFN-gamma) forms produced by natural, chemical synthetic or recombinant DNA techniques. For a comprehensive description of the preparation of recombinant human IFN- γ (rhu IFN- γ, including its cDNA and amino acid sequence) see, for example, U.S. Pat. Nos. 4,727,138, 4,762,791, 4,925,554, 5,582,824, 5,096,705, 4,855,238, 5,574,137 and 5,595,888 for CysTyrCys-free recombinant human IFN- γ, including differentially truncated derivatives, see European patent publication 146,354 for non-human animal interferons, including IFN- γ, see European patent publication 88,622, including differentially glycosylated forms of natural (wild-type) interferons and other variants (e.g., amino acid sequence variants) and derivatives, which are or will be known or will be obtained, such variants as alleles, which produce residue deletions, insertions and/or substituted site-directed mutagenesis products (see, e.g., European patent publication 146,354), it is known that the host range of IFN- γ is narrow, and therefore, human therapy homologous to the treated animal, preferably, the des-CysTyrCys variant of the sequence shown in U.S. Pat. No. 4,717,138 and its cognate EP77,670 is used, and optionally a C-terminal variant in which the last four residues are deleted post-translationally. For human therapy, the IFN- γ of the invention is preferably recombinant human IFN- γ (rhuIFN- γ), with or without the N-terminal amino acid CysTyrCys. More preferably, IFN-. gamma.is recombinant human IFN-. gamma. (recombinant human interferons-. gamma. -1b, rhu IFN-. gamma. -1b, containing 140 amino acids) as an active ingredient in a commercially available formulation, Actimmune * (Genentech, Inc., South San Francisco, Califonia). Since IFN- γ is known to have a high degree of species specificity, it is preferred to use the same species of IFN- γ as the animal being treated in animal experiments or for veterinary use. Therefore, in vivo experiments using rat animal models, murine (mouse) recombinant IFN-. gamma.was used (Genentech, Inc.). The association of rats with mice is sufficiently close that mouse IFN- γ can be used in rat models.

Pharmacologically, in the present invention, a "therapeutically effective amount" of IFN- γ refers to an amount that is effective in treating hypertrophy, particularly cardiac hypertrophy.

"hypertrophy" as used herein refers to an enlargement of an organ or structure that is not dependent on natural development and is not associated with tumor formation. Hypertrophy of an organ or tissue may be due to an increase in single cells (true hypertrophy), an increase in the number of cells constituting the tissue or organ (hyperplasia), or a combination thereof. Some organs, such as the heart, lose their ability to divide shortly after birth. Thus, "cardiac hypertrophy" is an increase in heart volume, manifested in myocyte enlargement and increased contractile protein content in adults, without accompanying cell division. The characteristics of stress that can induce hypertrophy (e.g., increased preload, increased afterload, decreased myocytes as in myocardial infarction, or suppression of contraction) appear to have a significant effect on the characteristics of the response. Early stages of cardiac hypertrophy are often manifested by morphologic myofibrillar and mitochondrial enlargement, and mitochondrial and nuclear enlargement. At this time, although the muscle cells are larger than normal, the cell tissue is substantially retained. In the further phase of cardiac hypertrophy, specific organelles, such as mitochondria, grow or increase and new contractile elements increase in an irregular manner to local areas. Long-term hypertrophied cells exhibit more pronounced histological abnormalities, including marked nuclear enlargement and high degrees of lobulation of the membrane, which displace nearby myofibrils, resulting in disruption of normal Z-band recordings. "cardiac hypertrophy" comprises the stages of disease progression characterized by different degrees of structural damage to the heart muscle, independent of the heart disease being suffered.

"Heart failure" refers to a cardiac dysfunction in which the heart pumps blood at a rate that is insufficient for tissue metabolism. Heart failure may be caused by a number of causes, including ischemia, congestion, rheumatism, or idiopathic.

"congestive heart failure" is a progressive pathological condition in which the heart is increasingly unable to provide sufficient cardiac output (i.e., the amount of blood pumped by the heart per unit time) to provide oxygenated blood to the surrounding tissues. Structural and hemodynamic damage occurs with the progression of congestive heart failure. Although these lesions appear in different forms, one of the characteristic symptoms is ventricular hypertrophy. Many different heart diseases ultimately lead to congestive heart failure.

The cause of "cardiac hypertrophy" is usually atherosclerosis of the coronary arteries, often accompanied by coronary thrombosis. It can be divided into two important classes: transmural infarctions, i.e. myocardial necrosis, involve the entire thickness of the ventricle; subendocardial infarction (non-transmural infarction), i.e. necrosis, involves only the subintimal membrane or the intraventricular myocardium or both, but does not reach the epicardium through the entire ventricular wall. Myocardial infarction is known to cause both hemodynamic changes and structural changes in the damaged and healthy areas of the heart. Thus, myocardial infarction reduces the maximum cardiac output and the amount of cardiac ejection. Also associated with myocardial infarction are stimulation of DNA synthesis occurring in the interstitium, and increased collagen formation in unaffected parts of the heart.

In chronic hypertension, for example, caused by an increase in total peripheral resistance, cardiac hypertrophy has been associated with hypertension as a result of increased stress or stress. One of the features of ventricular hypertrophy from chronic overpressure is diastolic dysfunction. Fouaad et al, in a laboratory,J.Am.Coll.Cardiol.41500-6 (1984); the number of the particles in the surface layer is Smith and the like,J.Am. Coll.Cardiol.5,869-74(1985). Prolonged left ventricular relaxation has been detected in early essential hypertension, but systolic function is normal or supernormal. Hartford et al, in a similar manner,Hypertension 6,329-338(1984). However, there is a lack of close parallelism between blood pressure levels and cardiac hypertrophy. Although, it has been reported that antihypertensive treatment in humans improves left ventricular function, it has been found that patients treated with diuretics (hydrochlorothiazide), beta inhibitors (propranolol) or calcium channel inhibitors (diltiazem *), respectively, shrink the left ventricle and do not improve diastolic function. Inouye, etc., and the like,Am.J.Cardiol.53，1583-7(1984)。

another complex heart disease associated with cardiac hypertrophy is "hypertrophic cardiomyopathy". The disease is characterized by a great diversity of morphologic, functional and clinical symptoms (Maron et al, 780-9 (1987); Spirito et al,N.Eng.J.Med.320749-55 (1989); the combination of Louie and Edwards,Prog.Cardiovasc.Dis.36275- "308 (1994); a Wigle, etc., and a method for extracting,Circulation 921680-92(1995)), which can occur in patients of various ages, which further increases its diversity (Sprito et al,N.Eng.J.Med.336,775-785(1997)). The causes of hypertrophic cardiomyopathy are also numerous and rarely known. Recent information suggests that β -myosin heavy chain mutations may be the cause of 30-40% of familial hypertrophic cardiomyopathies (Watkins et al,N.Eng.J.Med.3261108-14 (1992); the processes of Schwartz et al,Circulation 91532-40 (1995); the results of Marian and Roberts,Circulation 921336-47 (1995); thierfelder et al,Cell 77701-12 (1994); the Watkins et al, in the name of,Nat.Gen.11，434-7(1995))。

supravalvular "aortic stenosis" is a hereditary vascular disease characterized by ascending aortic stenosis, but other arteries, including the pulmonary artery, may also be affected. Aortic stenosis, if left untreated, can cause elevated intracardiac pressure, leading to myocardial hypertrophy and ultimately heart failure and death. The etiology of the disease is not fully understood, but hypertrophy and possible hyperplasia of the inner smooth muscle are prominent features of the disease. Variant molecules of elastin have been reported to be involved in the development and etiology of aortic stenosis. (U.S. patent 5,650,282 on 7/22 of 1997).

"valvular regurgitation" is the result of a heart disease that causes abnormalities in the heart valves. Many diseases, such as high rheumatic fever, cause the valve orifice to contract or pull open, others may cause endocarditis, i.e., inflammation of the endocardium or atrioventricular orifice, and require cardiac surgery. Defects such as valve stenosis or incomplete valve closure cause blood to accumulate in the heart chamber or to flow back through the valve. If left uncorrected, long-term valvular stenosis or dysfunction can cause cardiac hypertrophy-associated myocardial damage, eventually necessitating replacement of the valve.

The subject of the present invention is to treat all these and other heart diseases with accompanying cardiac hypertrophy.

"treatment" includes both treatment and prevention with the aim of avoiding or slowing (alleviating) hypertrophy. Those in need of treatment include those already diseased, susceptible to disease, or those in which the disease is to be avoided. Hypertrophy can be of various causes, including spontaneous, cardiac, ischemic, or ischemic attack, such as myocardial infarction.

By "chronic" administration is meant continuous administration of the drug as opposed to acute mode, whereby the initial anti-hypertrophic effect is maintained over a longer period of time.

"mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, captive or free-ranging animals, and ornamental, sports or pet animals, such as dogs, cats, cows, horses, sheep, pigs, and the like. Preferably a human.

"in combination with" one or more other drugs includes simultaneous administration and sequential administration in any order.

B. Best mode for carrying out the invention

1. Cardiac hypertrophy test

In vitro assay

a. Adult rat myocardial cell expansion induction

In this experiment, ventricular myocytes from one rat (male, Sprague-Dawley) were isolated, essentially according to Piper et al, "adult rat ventricular myocytes"Cell Culture Techniques in Heart and Vessel ResearchPip eds, Berlin: a modification of the method described by Springer-Verlag, 1990, pp.36-60. The method is capable of isolating adult ventricular myocytes and culturing these cells for long term rod-shaped phenotypes. Phenylephrine and prostate are knownGlandin F_2α(PGF_2α) Capable of inducing an exaggerated response in these cells. Piper et al, supra; lai, and the like,Am.J.Physiol.1996(ii) a 217(Heart Circ, physiol.40): h2197-2208. The PGF's were then measured for different potential inhibitors of cardiac hypertrophy_2αOr PGF_2αInhibition of analog (e.g., fluprostenol) and phenylephrine-induced myocyte expansion.

In vivo assay

a. Inhibition of fluprostenol-induced cardiac hypertrophy in vivo

The pharmaceutical model tests IFN- γ inhibits subcutaneous injection of Fluprostenol (PGF) in rats (e.g., male, Wistar or Sprague-Dawley)_2αOne of the analogs) ability to induce cardiac hypertrophy. It is known that rats with myocardial infarction-induced pathologic cardiac hypertrophy have a gradual accumulation of high levels of extractable PGF in their myocardium_2α. Lai, and the like,Am.J.Physiol.(Heart Circ.Physiol.)271H2197-H2208 (1996). Therefore, factors that inhibit the effect of fluprostenol on myocardial growth in vivo may be useful in treating cardiac hypertrophy. The effect of IFN- γ on cardiac hypertrophy was determined by measuring the weight of the heart, ventricles and left ventricle (normalized against body weight) and comparing to rats that did not receive IFN- γ. Detailed descriptions of this assay are provided in the examples.

b. Pressure-overload cardiac hypertrophy test

For in vivo testing, pressure-overload cardiac hypertrophy is generally induced by the contraction of the abdominal aorta of the test animal. In a typical protocol, rats (male, Wistar or Sprague-Dawley) are anesthetized and the abdominal aortic gap under the diaphragm is narrowed. The method of making a Benznak M.Can.J.Biochem.Physiol.33,985-94(1955). The aorta was surgically opened and a blunt needle was placed alongside the blood vessel. The blunt needle is wound with a wool ligature and immediately removed, whereupon the aortic lumen is narrowed to the needle diameter. See Rossi et al for a method of this,Am.Heart J.124700-.P.S.E.M.B.，200，95-100(1992)。For a detailed description of the process used in the present invention, reference is made to the following examples.

b. Effect of Experimental Induction of Myocardial Infarction (MI) on cardiac hypertrophy

Acute MI was induced in rats by left coronary artery ligation and confirmed by electrocardiographic examination. A sham-treated group of animals was also prepared as a control. Early data showed that cardiac hypertrophy was present in the MI group as an 18% increase in heart weight to body weight. Lai et al, supra. Treatment of these animals with a candidate inhibitor of cardiac hypertrophy, such as IFN-gamma, provides valuable information regarding the efficacy of the test candidate inhibitor.

Use, therapeutic compositions and administration of IFN-gamma

According to the invention, IFN-gamma can be used to treat cardiac hypertrophy, i.e., enlargement of the heart of various etiologies and pathologies. When the heart (ventricle) is subjected to excessive pressure or volume loading, cardiac hypertrophy occurs as a fundamental compensatory mechanism that allows the ventricle to withstand its load. Krayenbuehl et al,Eur.Heart J.4(supplement A), 29 (1983). The characteristics of stress that induce hypertrophy (e.g., increased preload, increased afterload, decreased myocytes as in myocardial infarction, or major inhibition of contractility) play an important role in the characteristics of the hypertrophic response. The Scheuer and the Buttrick,Circulation 75(supplement 1), 63 (1987). The present invention relates to the treatment of cardiac hypertrophy associated with any pathological condition including, but not limited to, post myocardial infarction, hypertension, aortic stenosis, cardiomyopathy, valve regurgitation, cardiac bypass, and congestive heart failure. The main features of these conditions have been discussed previously.

Of particular importance is the prevention of heart failure with IFN-gamma following myocardial infarction. In the united states, about 750,000 people suffer Acute Myocardial Infarction (AMI) annually, with about one-fourth of them dying from AMI. In recent years, thrombolytic agents, such as streptokinase, urokinase, and especially tissue plasminogen (t-PA) have greatly improved survival in patients with myocardial infarction. Continuous intravenous infusion for 1.5 to 4 hours at 69-90%In the treated patients, t-PA caused coronary artery patency at 90 minutes. Topol and the like,Am.J.Cardiol.61723-8 (1988); neuhaus et al,J. Am.Coll.Cardiol.12581-7 (1988); neuhaus et al,J.Am.Coll.Cardiol.14,1566-9(1989). The fastest rate is reported to be achieved with high doses or with faster dosing. Topol and the like,Am.J.Cardiol.15,922-4(1990). t-PA can also be administered as a pill, but because of its shorter half-life, infusion therapy is more suitable. The amount of Tebbe and the like,Am.J.Cardiol.64,448-53(1989). A more prolonged half-life and highly fibrin-specific T-PA variant, TNK T-PA (T103N, N117Q, KHRR (296-299) AAAA T-PA variant, Keyt et al,Proc.Natl.Acad.Sci.USA.91,3670-3674(1994)). However, despite the above advances, long-term prognosis of surviving patients depends largely on post-infarction monitoring and treatment of the patient, which should include monitoring and treatment of cardiac hypertrophy.

Another important therapeutic context is the treatment of cardiac hypertrophy associated with hypertension. As mentioned previously, long-term hypertension is known to cause cardiac hypertrophy. While some hypertensives are known to reduce left ventricular volume, treatment does not necessarily improve diastolic function. Thus, IFN- γ can be combined with a β adrenergic receptor inhibitor, such as propranolol, timolol, terbutalol, carteolol, nadolol, betaxolol, penbutolol, acetobourolol, atenolol, metoprolol, carvedilol; angiotensin converting enzyme (AIE) inhibitors such as quinapril, captopril, enalapril, ramipril, benazepril, fosinopril, lisinopril; diuretics, such as chlorothiazide, hydrochlorothiazide, hydroflumethiazide, methylchlorothiazide, benzylthiazide, dichlorofinamide, acetazolamide, indapamide; calcium channel inhibitors, such as diltiazem *, nifedipine, verapamil, nicardipine. Pharmaceutical compositions containing the therapeutic agents indicated by the above generic names are commercially available and can be administered according to the manufacturer's instructions for dosage, dosage form, side effects, contraindications, and the like (see, physicians' Desk Reference, medical economics Data Production co. montvale n.j., 51 th edition, 1997).

IFN-gamma may be administered prophylactically to patients with cardiac hypertrophy for arresting the development of disease conditions and avoiding sudden death, including asymptomatic sudden death. Such prophylactic treatment is particularly necessary for patients diagnosed with giant left ventricular hypertrophy (maximum wall thickness of an adult up to 35mm or more, or equivalent wall thickness of a child), or when the hemodynamic burden on the heart is particularly heavy.

IFN- γ may also be used to treat atrial fibrillation that occurs in most patients diagnosed with hypertrophic cardiomyopathy.

IFN-gamma is administered in the form of a pharmaceutical composition comprising IFN-gamma as an active ingredient and a pharmaceutically acceptable carrier. Therapeutic IFN- γ formulations for the treatment of cardiac hypertrophy were prepared for preservation as follows: IFN- γ of desired purity is mixed with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's pharmaceutical Science, supra) and prepared in the form of a lyophilized cake or an aqueous solution. Acceptable carriers, excipients, and stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphoric acid, citric acid, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than 10 residues) peptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other sugars including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; automatic generation of counter ions, such as sodium; and/or non-ionic surfactants such as Tween, Pluronic or polyethylene glycol (PEG).

IFN- γ must be sterile for in vivo administration. This can be done by filtration through sterile filtration membranes before or after freeze-drying and regeneration. IFN- γ is typically stored in lyophilized or solution form.

IFN- γ may be used in lyophilized form and mixed with the other ingredients for reconstitution with a suitable solvent at the time of use. Since IFN- γ is known to be acid labile, it is generally treated at neutral or slightly alkaline pH. See, for example, U.S. patent 4,499,014, which describes the reactivation of lyophilized acidic IFN- γ solution upon reaching a pH of 6-9. Neutral or weakly alkaline solutions of IFN-gamma at high concentrations are generally not suitable as infusion preparations because a visible precipitate forms rapidly. Such precipitation may cause embolism or decrease in drug efficacy upon administration. European patent publication 0196,203 discloses the reconstitution of lyophilized IFN- γ at pH 4-6.0.

U.S. patent 5,151,265, 9/29 1992, discloses a stable liquid pharmaceutical composition comprising an effective amount of non-lyophilized IFN- γ, a buffer capable of maintaining a pH of 4.0-6.0, a stabilizer, and a non-ionic detergent. The stabiliser is typically a polyhydric sugar alcohol, for example mannitol, and the non-ionic detergent may be a surfactant, for example polysorbate 80 or polysorbate 20. Preferably, the nonionic detergent is present in an amount of about 0.07 to 0.2mg/ml, more preferably 0.1 mg/ml. Suitable buffers are conventional buffers of organic acids and salts thereof, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, trisodium citrate mixture, monosodium citrate-sodium citrate mixture, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodium gluconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium gluconate mixture, etc.), oxalate buffers (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.), and acetate mixtures (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.).

A known commercial liquid formulation of IFN- γ (Actimmune * rhu IFN- γ -1b, Genentech, Inc.) is a sterile, clear, colorless, non-stock solution contained in single dose vials for subcutaneous injection. 100. mu.g (3X 10) per 0.5ml of Actimmune *⁶Unit, specific activity: 30 x 10⁶Unit/mg) IFN- γ -1b, formulated with 20mg mannitol, 0.36mg sodium succinate, 0.05mg polysorbate 20 and sterile water for injection.

Stock pharmaceutical compositions suitable for repeated use, for use in accordance with the present invention, preferably comprise:

a) non-lyophilized IFN- γ;

b) acetate buffers capable of maintaining a pH of about 4-6 (i.e., the pH range at which protein stability in solution is highest);

c) nonionic detergents, primarily used to stabilize proteins against aggregation caused by agitation;

d) an isotonicity adjusting agent;

e) a preservative selected from: phenol, benzyl alcohol and benzethonium halides, such as benzethonium chloride; and

f) and (3) water.

The non-ionic detergent (surfactant) may be, for example, a polysorbate (e.g. polysorbate (Tween)20 or 80) or a poloxamer (e.g. poloxamer 188). The use of nonionic surfactants enables the formulation to be free of protein denaturation due to surface shear forces. In addition, formulations containing such surfactants can be used in aerosol devices, such as those used for pulmonary administration, and needleless injection guns (see, EP257,956).

The isotonicity adjusting agent is included to ensure isotonicity of the liquid compositions of the present invention and includes polyhydric sugar alcohols, such as ternary or higher sugar alcohols, e.g., glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol. These sugar alcohols may be used alone or in combination. Alternatively, sodium chloride or other suitable inorganic salts may be used to maintain isotonicity of the solution.

The acetate buffer may be, for example, an acetic acid-sodium acetate mixture, an acetic acid-sodium hydroxide mixture, or the like. The pH of the liquid formulation of the present invention is between 4.0 and 6.0, more preferably between 4.5 and 5.5, and most preferably at pH5.

Preservatives, such as phenol, benzyl alcohol and benzethonium halides, such as benzethonium chloride, are known antimicrobial agents.

In a preferred embodiment, the IFN- γ is administered in the form of a liquid pharmaceutical composition comprising:

IFN-γ 0.1-2.0mg/ml

sodium acetate (pH5.0) 5-100mM

Tween 20 0.1-0.01wt％

Phenol 0.05-0.4 wt%

Mannitol 5 wt%

Water for injection, USP make up to 100%

Wherein the percentage amounts are based on the total weight of the composition. Phenol can be replaced by 0.5-1.0 wt% benzyl alcohol, and mannitol can be replaced by 0.9 wt% sodium chloride.

Preferably, the composition comprises:

IFN-γ 0.1-1.0mg/ml

sodium acetate (pH5.0) 10mM

Tween 20 0.01wt％

0.2 wt% of phenol

Mannitol 5 wt%

Phenol can be replaced by 0.75 wt% benzyl alcohol and mannitol can be replaced by 0.9 wt% sodium chloride.

The stock solution formulation preferably contains multiple doses of IFN- γ in a therapeutically effective amount. Because of the narrow host range of the polypeptide, it is desirable to treat human patients with liquid formulations containing human IFN- γ, preferably with human IFN- γ having its native sequence. As biological response modifiers, IFN-gamma has a variety of activities on various cell types in human and non-human mammals. Of course, a therapeutically effective amount will depend on a variety of factors, such as the pathological condition being treated (including prevention), the age, weight, general health, medical history of the patient, etc., which the practitioner is able to determine. The effective dose is generally about 0.001-1.0mg/kg, preferably about 0.01-1mg/kg, and most preferably about 0.01-0.1 mg/kg. In such formulations, hu IFN- γ preferably exhibits greater than or equal to about 2X 10 when tested against encephalomyocarditis virus using A549 cells⁷Specific activity of U/mg protein. It will be appreciated that endotoxin contamination must be maintained below safe levels, for example below 0.5ng/mg protein. Moreover, if administered to humans, liquid formulations must meet sterility, pyrogenicity, overall safety and purity requirements set forth by FDA and biological standards.

The administration route of IFN-. gamma.is by a known method such as intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial or intralesional injection or infusion, or by a sustained-release system as described later. The therapeutic IFN- γ composition is typically contained in a container having a sterile access port, such as an intravenous bag or vial having a stopper through which a hypodermic needle may be inserted. The formulations are preferably administered by multiple intravenous (i.v.), subcutaneous (s.c.) or intramuscular (i.m.) injections, or as an aerosol suitable for intranasal or intrapulmonary administration (for intrapulmonary administration, see EP257,956).

The stabilized IFN- γ aqueous composition is preferably contained in a vial containing up to about 30 doses of a therapeutically effective amount of IFN- γ. Preferably, the biological activity of IFN- γ is maintained at about 80% of that exhibited by the first administration for at least 14 days, more preferably at least 200 days, after the first administration.

IFN- γ may also be administered in the form of a sustained release formulation. Suitable ions for the sustained release agent include semipermeable matrices of solid hydrophobic polymers containing the protein, which matrices are in the form of films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate)) (Langer et al,J.Biomed.Mater. Res.，15167- "Alkanka 277(1981) and Langer,Chem.Tech.，1298-105(1982)), or poly (vinyl alcohol), polylactide (U.S. Pat. No. 3,773,919, EP58,481), copolymers of L-glutamic acid and gamma ethyl L-glutamate (Sidman et al,Biopolymers 22: 547-556(1983)), non-degradable ethylene ethyl acetate (Langer et al, supra), degradable lactic acid-glycolic acid copolymers, e.g., Lupron Depot^TM(injectable microspheres of lactic acid-glycolic acid copolymer and leuprolide acetate), poly-D- (-) -3-hydroxybutyric acid (EP133,988).

Polymers such as ethylene ethyl acetate and lactic acid-glycolic acid release molecules for up to 100 days, while some hydrogels release proteins for shorter periods. When the encapsulated proteins are retained in the body for a prolonged period of time, they denature or aggregate by exposure to moisture at 37 ℃, resulting in a loss of biological activity and possibly a change in immunogenicity. Rational dosing regimens may be designed to ensure protein stability, depending on the mechanism involved. For example, if the aggregation mechanism is found to be disulfide bond formation through sulfur-disulfide interconversion, stabilization may be achieved by modifying sulfhydryl residues, freeze drying acidic solutions, controlling humidity, using appropriate additives, and inventing particular polymer matrix compositions.

The slow release IFN-gamma composition also comprises liposomeEncapsulated IFN-gamma. The IFN-gamma containing liposomes can be prepared by known methods: DE3,218,121; an acid addition salt of Epstein and the like,Proc.Natl.Acad.Sci.USA，82: 3688-3692 (1985); the method of the Hwang and the like,Proc.Natl.Acad.Sci.USA，77: 4030-4034 (1980); EP52,322; EP36,676; EP88,046; EP143,949; EP142,641; japanese patent publication No. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP102,324. Typically, small (about 200-800 angstroms) monolithic lamellar liposomes are used, in which the lipid content is greater than 30 mol% cholesterol, the ratio being selected to optimize therapy.

The effective amount of IFN- γ for treatment depends, for example, on the purpose of the treatment, the route of administration, and the condition of the patient. Therefore, in order to obtain the best therapeutic effect, it is necessary for the physician to determine the dosage and modify the route of administration. Body surface area 0.5m when IFN-gamma is administered for treating chronic granulomatous patients²The recommended dose for the above patients is 50mcg/m²(1.5×10⁶U/m²) Body surface area less than or equal to 0.5m²The recommended dose for patients is 1.5 mcg/kg/dose, injected subcutaneously 3 times a week. This is instructive for the physician to determine the optimal effective dose for cardiac hypertrophy. The physician will administer IFN- γ until the desired dose is obtained for the effect of treating cardiac dysfunction. For example, if the goal is to treat congestive heart failure, it should be an amount that inhibits the progressive cardiac hypertrophy associated with such a condition. The progress of the above therapy can be conveniently monitored by echocardiography. Similarly, in patients with hypertrophic cardiomyopathy IFN- γ may be administered empirically based on the patient's subjective perception of therapeutic efficacy.

IFN- γ may be administered in combination with other therapeutic agents useful in the treatment of cardiac hypertrophy, including the treatment of cardiac hypertrophy. For example, IFN- γ therapy may be administered in combination with known inhibitors of cardiac myocyte hypertrophy factors (e.g., inhibitors of alpha adrenergic activators such as phenylephrine; endothelin-1; CT-1; LIF; vasodilator peptide convertase and vasodilator peptide II). For combination therapy, combination with a cardiac hypertrophy factor inhibitor (CHF, cardiotrophin or cardiotrophin-1, see U.S. Pat. No. 5,679,545) is particularly preferred.

Preferred drug candidates in combination therapy for cardiac hypertrophy are beta adrenergic receptor inhibitors (e.g. propranolol, timolol, terbutalol, carteolol, nadolol, betaxolol, penbutolol, acetobourdol, atenolol, metoprolol, carvedilol), verapamil, difedipine, diltiazem *. Treatment of cardiac hypertrophy associated with hypertension may require the use of antihypertensive therapy with calcium channel inhibitors such as diltiazem, nifedipine, verapamil, nicardipine; a beta adrenergic blocker; diuretics, such as chlorothiazide, hydrochlorothiazide, hydroflumethiazide, methylchlorothiazide, benzylthiazide, dichlorofinamide, acetazolamide, indapamide; and/or ACE inhibitors, such as quinapril, captopril, enalapril, ramipril, benazepril, fosinopril, lisinopril.

The effective amount of the therapeutic agent to be administered in combination with IFN- γ is determined by a physician or veterinarian. Dosage control and adjustment can be performed to achieve optimal effect, and the use of diuretics or digitalis and factors such as hypertension, hypotension, renal insufficiency, etc. are preferably considered. The dosage will also depend on factors such as the type of therapeutic agent used and the particular condition of the patient being treated. Generally, the same dose is used as if it were not combined with IFN- γ.

Examples

Example 1

IFN-gamma vs PGF_2αInhibition of induced adult myocyte expansion

Materials and methods

Adult muscle cell culture

The procedure described in modified Piper et al, supra, is used to isolate ventricular myocytes in adult rats, as described in Lai et al, supra. For preparing each muscle cell preparation, useSodium pentobarbital Male Sprague-Dawley rats weighing approximately 250g were anesthetized and hearts removed. Peripheral tissues were removed and fixed to a Langendorff system controlled at 37 ℃.40 ml of Krebs buffer (110mM NaCl, 2.6mM KCl, 1.2mM MgSO 2. sup. m)₄·7H₂O，25mM NaHCO₃And 11mM glucose) perfused the heart. Then 30mg collagenase and 12.5. mu.l 100mM CaCl were added₂The Krebs buffer was circulating in the heart for 30 minutes. The heart was removed from the Langendorff apparatus and the atria and connective tissue were removed. The ventricles were cut into 2mm pieces with a dissecting scissors and then treated with fresh collagenase solution (30mg collagenase and 400mg BSA in 100mM CaCl 12.5. mu.l₂Krebs buffer (r) was digested at 37 ℃ for 5 minutes. During digestion, the tissue suspension was gently shaken manually every minute. After digestion, the supernatant was separated and retained, and the remaining tissue was re-digested with fresh collagenase solution for 5 minutes.

The isolated adult rat myocytes were plated on a plate coated with laminin at a density of 3X 10³Cells/ml. After 72 hours of appropriate stimulation, cells were fixed with glyceraldehyde and stained with eosin yellow. The rod cells were looked up under a fluorescent microscope and the maximum width was determined with image analysis software (Simple 32, compax Imaging, Mars, PA).

Results

IFN-gamma inhibiting hypertrophy factor PGF_2αAnd phenylephrine-induced expansion of adult cardiomyocytes

As is known, PGF_2αAnd the alpha adrenergic activator phenylephrine induces enlargement of new myogenic cells in cultured adult rats (Adams et al,J.Biol.Chem.271: 1179-1186 (1996); lai, and the like,Am.J.Physiol.(Heart Circ.Physiol.)271: h2197-2208 (1996); meidell and the like,Am.J.Physiol.251：H1076-H1084(1986)；Simpson， J.Clin.Invest.72：732-738(1983)；Simpson， Circ.Res.56: 884-894(1985)). Adult rat ventricular myocytes in culture expand upon exposure to the above factors (Lai et al, supra; Piper et al)The "adult rat ventricular myocyte",Cell Culture Techniques in Heart and Vessel Researchpip eds, 1990, Springer-Verlag: berlin, p.36-60). Adult muscle cells are rod-shaped. When these cells were at 0.1. mu.M PGF_2αIn the middle, the rod cells expanded in their flattening (FIG. 1). Quantitative amplification reaction: the maximum cell width of at least 200 rods was determined and the values were plotted against their frequency of appearance in the cell population. PGF_2αThe maximum cell width was significantly altered, as indicated by a shift in the distribution of values within the cell population compared to control cells (p < 0.001). Treatment of cells with IFN-gamma significantly inhibited their PGF_2αResponse (PGF)_2αIFN-. gamma.and PGF_2αIn contrast, p < 0.001). IFN-gamma vs PGF in a certain concentration range_2αThe inhibitory effect of the induced myocyte expansion is dose-dependent, which corresponds to the biological response to IFN- γ in cardiomyocytes and other cellular systems (Singh et al,Biol.Chem.271: 1111-; the number of the molecules of Pinsky et al,J.Clin. Invest.95: 766 685 (1995); Unkureanu-Longrois, etc.,Circ.Res.77: 494-; Soderberg-Naucler et al,J.Clin.Invest.100: 3154 and 3163 (1997); gou et al, and the like,J.Clin.Invest. 100: 829-838 (1997); marra, etc. are used for the treatment of the diseases,Can.J.Cardiol.12: 1259-1267(1996)). Inhibition of PGF_2αThe ability to induce myocyte expansion appears to be unique to IFN- γ, as no other cytokines including IL-1 α, IL-1 β, IL-2, IL-6, TNF- α, INF- α and IFN- β inhibit the expansion response. The inhibitory effect of IFN-gamma is not limited to PGF_2α. IFN- γ also inhibited phenylephrine-induced enlargement (FIG. 3).

Example 2

Inhibiting cardiac hypertrophy in vivo

Materials and methods

Animal(s) production

The entire experimental procedure was in compliance with the guidelines of the american physiological association and was approved by the Genentech research animal care and use committee. Sprague/Dawley (SD) rats (8 weeks old, Charles river Breeding Laboratories, Inc.) were used for the experiments. Before the experiment, the animals are adapted in the laboratory for at least 1 week, fed with the pill-shaped mice for eating, freely drink water and are caged in a room with light control and temperature control.

Administration of Fluprostenol and/or IFN-gamma

Rats were injected subcutaneously with 0.15mg/kg of fluprostenol (Cayman Chemical, Ann Arbor, M), 0.008mg/kg of murine IFN- γ (Genentech inc., South San Francisco, CA), fluprostenol and murine IFN- γ, or saline vehicle, 2 times daily for 14 days. In the IFN- γ group and the fluprostenol + IFN- γ group, animals were pretreated with IFN- γ for 1 day. Body weights before and after treatment were measured. Previous studies have shown that the dose of fluprostenol used herein is the lowest dose that causes significant cardiac hypertrophy in rats. Lai et al, supra. One trial showed that IFN-. gamma.at the above doses inhibited fluprostenol-induced cardiac hypertrophy with little effect on rat body weight.

Hemodynamic evaluation

On day 13 post-treatment, rats were anesthetized with an intraperitoneal injection of ketamine 80mg/kg (Aveco Co. Inc., Fort Dodge, Iowa) and xylazine 10mg/kg (Rugby Laboratories, Inc., Rockville Center, NY). A catheter (PE-10 to PE-50) containing heparin-salt solution (50U/ml) was implanted in the abdominal aorta and passed through the femoral artery for measurement of Mean Arterial Pressure (MAP) and Heart Rate (HR). The catheter is fixed behind the neck after penetrating out.

1 day after catheterization, the arterial catheter was connected to a CP10 type pressure transducer (Century technology company, Inglewood, Calif., USA) connected to a Grass 7 type variety of wave scanners (Grass instruments. Quincy, MA, USA). MAP and HR were also determined in conscious, non-restrained mice.

Determination of organ weight

Under ketamine/xylazine anesthesia, the heart, kidneys and spleen were removed, dissected and weighed. The left ventricle was stored at 80 ℃ for evaluation of gene expression.

Pressure overload animal model

Inducing pressure overboost by partial ligation of rat abdominal aorta is described in the prior art. The Kimura et al, the fact,Am. J.Physiol.1989：256( Heart Circ.Physiol.25): H1006-H1011; batra, etc., in the form of a suspension,J.Cardiovasc. Pharmacol.17(supplement 2), S151-S153 (1991). Briefly, rats were anesthetized with ketamine/xylazine as previously described. A 3mm incision was made in the abdominal wall. The abdominal aorta between the septum and the renal arteries was exposed and ligated with 5-0 silk suture. The suture is tied tightly around a 23 gauge needle and the needle is withdrawn. Sham-treated animals also received surgery, but were not ligated with sutures.

Protocol for testing in pressure-overloaded rats

Aorta-ligated rats were randomly injected with 0.08mg/kg IFN-. gamma.s 2 times subcutaneously 1 day before surgery and 14 days after surgery. Sham-treated animals received no treatment. On day 13 post-treatment, a catheter was implanted in the right subclavian carotid artery as described previously. Arterial pressure and HR were measured in conscious rats 1 day after implantation. Hearts and other organs such as liver, kidney and spleen were removed, weighed and fixed with 10% buffered formalin for pathology. The left ventricles of some animals were excised quickly, frozen with liquid nitrogen, and stored at-80 ℃ for gene expression studies.

Statistical analysis

Results are expressed as mean ± SEM. One-way analysis of variance (ANOVA) was performed to determine the difference between the parameters. Then using Newman-Keuls method to carry out post-hoc analysis on the obvious difference; p < 0.05 was considered significant.

RNA preparation

Total RNA was isolated using RNeasy Maxi columns (Qiagen) according to the manufacturer's instructions.

RT-PCR

Real-time RT-PCR (TaqMan) technology was used to compare gene expression differences between different treatment groups. An oligonucleotide probe containing the fluorescent dye 6-carboxytetramethyl-rhodamine (TAMRA) at its 3' end is designed and used to hybridize with the amplicon defined by the two PCR primers. 3' end-capped phosphates avoid elongation of the probe. During the extension phase of the PCR reaction, the reporter dye is released from the probe due to the 5' exo-activity of Taq polymerase. The fluorescence produced is monitored in the reaction tube with a sequencer without further processing and quantified, hence the term "real-time". The threshold cycle number (Ct), i.e., the time at which the reporter fluorescence exceeds the baseline standard deviation by more than 10-fold, is proportional to the amount of replicon produced by the sample. Since fluorescence is measured in the log phase of the amplification reaction, all reaction components are not limiting. In each experiment, one control without RNA template was analyzed to monitor contamination and another control, omitting the RT step, was included to eliminate contaminating DNA amplification that could be the signal source. The reaction is optimized by adjusting the concentration of magnesium ions and primers so as to obtain the strongest fluorescence signal and the smallest Ct. The product was run on an agarose gel and only one band at the expected molecular weight was confirmed. In addition, the sequence of the amplicons is screened in the gene bank, eliminating the possibility of overlapping with closely related genes.

For each sample, the mRNA for each target gene was determined using a standard curve as described below, and normalized with respect to the glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) content in the sample (for specific calculations, see below). The relative abundance of each target gene relative GAPDH can then be compared between treatment groups.

RT-PCR

1ng of total RNA was taken for each reaction and subjected to RT-PCR using a TaqMan 7700 sequencer (ABI-Perkin Elmer) (Gibson et al,Genome Res.6,995-1001(1996)). Conditions for the amplification reaction (50. mu.l) were: 1 XTaqMan buffer A, 200. mu.M dATP, dCTP, dGTP and 400. mu.M dUTP, 10% glycerol, 6.5mM MgCl₂50U MuLV reverse transcriptase, 20U RNase inhibitor, 1.25U AmpliTaq Gold, 100nM forward and reverse primers, 100nM fluorescent probe. RT-PCR reagents and glycerol were purchased from Perkin Elmer and Sigma, respectively. The reaction was performed in a MicoAmp optical tube and lid (ABI-Perkin Elmer). TaqMan primers were designed according to the guidelines established by Perkin Elmer, synthesized in Genentech Inc., and the probe for murine GAPDH was given by Perkin Elmer. Reverse transcription was performed at 48 ℃ for 30 minutes, followed by activation of AmpliTaq gold at 95 ℃ for 10 minutes. The thermal cycle was 95 ℃ for 30 seconds and 60 ℃ for 1.5 minutes for 40 cycles.

Such as the Heid and the like,Genome Res.6: 986-. Briefly, a standard curve (1: 5 serial dilutions) of each gene of interest was assayed 2 times. The equation describing the curve was determined with Ct as the Y-axis and Log values for total RNA concentration as the X-axis. According to the corresponding standard curve, Ct (Y value) is substituted to obtain mRNA (X), and the mRNA of each target gene is measured. Then, the values of each target gene were normalized to GAPDH using the following equation: 10^X1/10^X2Wherein X1 is the target gene and X2 is GAPDH.

Results

IFN-gamma inhibition of cardiac hypertrophy in vivo

It is known that PGF is administered for a long period of time_2αThe activator analog of fluprostenol induces cardiac hypertrophy in vivo, and PGF can be extracted from myocardium of rats suffering from pathologic myocardial hypertrophy caused by myocardial infarction_2αLevels have been gradually increased (Lai et al, supra). Therefore, PGF can be inhibited in vivo_2αFactors that act on myocardial growth may be useful in treating myocardial hypertrophy. Fluprostenol was administered with or without IFN- γ for 2 weeks and the effect on cardiac hypertrophy was determined. The absolute weights of heart, ventricle and left ventricle tended to increase in the fluprostenol-treated rats compared to the vehicle control, but the weights were significantly reduced in the fluprostenol + IFN- γ -treated rats compared to the fluprostenol-treated rats (table 1). Fluprostenol treatment caused a significant increase in the ratio of heart, ventricular and left ventricular weight to Body Weight (BW), indicating that fluprostenol induced cardiac hypertrophy (fig. 4). IFN-gamma inhibition of fluoride precursorThe hypertrophy induced by the alcohol. Rats receiving fluprostenol + IFN- γ had significantly reduced relative weights of heart, ventricle and left ventricle ratio BW compared to the fluprostenol group (FIG. 4). Comparison of the IFN- γ group with the vehicle group showed no significant change in absolute weight or BW relative weight of heart, ventricle or left ventricle with IFN- γ alone (Table 1, FIG. 4).

Chronic administration of fluprostenol correlated with a significant decrease in Mean Arterial Pressure (MAP) compared to vehicle treated groups (fig. 5). Compared to vehicle, IFN- γ had no effect on MAP, nor did it have an effect on MAP in fluprostenol-treated animals. There was a significant change in heart rate in the 4 treatment groups (fig. 5). The above results indicate that IFN- γ does not inhibit cardiac hypertrophy induced by Fluprostenol by counteracting its hemodynamic effects.

IFN- γ not only inhibited cardiac mass associated with the use of fluprostenol, but also altered intracardiac gene expression associated with fluprostenol-induced hypertrophy (FIG. 6). Fluprostenol-treated rat hearts have increased mRNA abundance of alpha-skeletal actin, collagen I, and natriuretic factor compared to vehicle. Sarcoplasmic reticulum calcium ATPase was significantly reduced in these rats. IFN- γ suppresses all but the natriuretic factor response.

IFN- γ experiments were also performed in a mouse model in which one abdominal aorta was ligated causing pressure overload leading to cardiac hypertrophy. Hypertrophy of the heart from aortic stenosis is manifested by a significant increase in the absolute weight of the heart, atria, ventricles and left ventricle, as well as the relative weight to BW. IFN- γ treatment significantly reduced cardiac hypertrophy in this model (table 2, fig. 7 and 8).

The effects of IFN- γ on other organs were also examined (Table 2). Neither aortic ligation nor IFN- γ treatment altered the weight of the kidney and its ratio to BW. Rats treated with vehicle and ligated aortas tended to decrease in liver weight and its ratio to BW, and not with IFN- γ, compared to sham operated animals. Aortic ligation caused a significant increase in both the absolute spleen weight and BW normalized weight, which was exacerbated by IFN- γ treatment. Thus, the effects of IFN- γ on cardiac hypertrophy are not due to a generalized effect on organ weight.

Mean arterial, systolic and diastolic blood pressure were significantly higher in aorta-ligated rats than in sham-operated controls, and there was no difference in the increase in arterial pressure between IFN- γ -treated and vehicle-treated ligated rats (fig. 9). The results demonstrate that the alleviation of cardiac hypertrophy in ligated rats receiving IFN- γ is independent of afterload changes.

Aortic ligation caused changes in gene expression in part of the heart. In ligated rats, the relative abundance of mRNA for β -myosin heavy chain, α -smooth muscle actin, α -skeletal actin, atrial natriuretic factor, collagen 1 and III, and fibronectin was increased compared to sham controls. 2 of 3 genes: alpha-smooth muscle actin and collagen 1 were inhibited by IFN-gamma (Table 3).

In summary, the results of examples 1 and 2 show that IFN-. gamma.inhibits cardiac hypertrophy. IFN-gamma acts not only to inhibit hypertrophy stimulus-induced cardiac augmentation, but it also inhibits certain molecular changes occurring in hypertrophic heart at the gene expression level. It is particularly noteworthy that IFN-gamma inhibits collagen 1 gene expression in vivo, both in response to long-term stimulation with fluprostenol, and in a model of stress overload induced hypertrophy. About 75% of myocardial collagen is collagen 1(Ju et al,Can.J.Caridol.12: 1259-1267(1996)). Increased extracellular matrix deposition and interstitial fibrosis with cardiac hypertrophy may be due to the pathophysiology of heart failure. By inhibiting collagen 1 production, IFN- γ can reduce interstitial fibrosis in heart failure.

TABLE 1 body and organ weights of Flup and/or IFN-treated rats

	Body weight	Flup	Flup+IFN	IFN
					BWO(g)	292.4±1.7	292.3±2.2	292.8±2.1	292.5±3.2
BW(g)	391.6±6.3	381.1±4.4	377.6±4.5	380.8±6.1
					ΔBW(g)	99.2±5.5	91.9±4.0	84.8±3.9	88.3±5.0
HW(g)	0.966±0.22	1.000±0.018	0.9279±0.016#	0.956±0.029
					VW(g)	0.922±0.22	0.957±0.017	0.889±0.015#	0.914±0.029
LVW(g)	0.706±0.018	0.740±0.013	0.678±0.011#	0.696±0.023
					KW(g)	1.440±0.035	1.397±0.038	1.377±0.031	1.327±0.035
KW/BW(g/kg)	3.678±0.075	3.632±0.076	3.648±0.070	3.483±0.069
					SW(g)	0.799±0.050	0.880±0.048	1.009±0.042*	0.924±0.068
SW/BW(g/kg)	2.065±0.149	2.309±0.130	2.676±0.082**	2.415±0.149

Data given are mean ± SEM, and the number of animals in the vehicle, flu + IFN and IFN groups were 14, 14 and 9, respectively. Carrier: brine; and (3) Flup: fluprostenol; IFN: an interferon gamma; BWO: basal body weight; BW: the treated body weight; Δ BW: BW-BWO; HW: heart weight; VW: ventricular weight; LVW: left ventricular weight; KW: kidney weight; SW: spleen weight.^*p＜0.05，^**p is less than 0.01, compared with the carrier group; # p < 0.05, # p < 0.01, compared to the Flup group.

TABLE 2 weight, organ weight and HR of pressure overloaded rats

	False operation	PO + carrier	PO+IFN
				BWO(g)	278.8±1.9	279.4±1.3	279.0±1.3
BW(g)	367.9±6.8	347.5±5.7*	355.5±5.2
				ABW(g)	89.1±5.8	68.1±5.6*	76.5±4.7
AW(g)	0.038±0.02	0.056±0.002**	0.046±0.003*##

AW/BW(g)	0.104±0.04	0.162±0.006**	0.129±0.007*##
				KW(g)	1.438±0.51	1.334±0.033	1.349±0.071
KW/BW(g/kg)	3.894±0.078	3.841±0.070	3.776±0.071
				LW(g)	13.84±0.55	12.53±0.36	13.96±0.47#
LW/BW(g/kg)	37.46±0.96	36.01±0.71	38.94±0.92#
				SW(g)	0.724±0.030	0.839±0.026*	1.170±0.053**##
SW/BW(g/kg)	1.959±0.051	2.418±0.069**	3.261±0.121**##
				HR(bpm)	371±12	415±12*	418±19*

The data given are mean ± SEM, the number of animals in sham, PO + vehicle and PO + IFN groups was 16, 22 and 21, respectively, when all parameters were determined, and the number of animals in HR was 7, 8 and 7. PO: pressure overload; IFN: an interferon gamma; BWO: basal body weight; BW: the treated body weight;_ΔBW: BW-BWO; AW: atrial weight; KW: kidney weight; LW: liver weight; SW: spleen weight; HR: efficiency.^*p＜0.05，^**p is less than 0.01, compared with a sham operation group; # p < 0.05, # p < 0.01, compared to the PO + support group.

TABLE 3 IFN vs genesEffect of expression

Treatment of	Pseudo surgery + vector	PO + carrier	PO+IFN
				ANF	0.98±0.37	5.29±1.21*	3.61±1.32
βMHC	0.89±0.24	1.91±0.15*	1.71±0.14*
				SKA	0.95±0.13	3.35±0.46*	2.47+0.51*
SMA	0.71±0.06	0.89±0.04*	0.77±0.06
				COLI	0.55±0.05	0.91±0.09*	0.77±0.10
COLIII	0.44±0.05	0.66±0.08*	0.72±0.09*
				FIB	0.66±0.17	1.03±0.11*	0.97±0.12

PO: pressure overload; IFN: an interferon gamma; and (3) ANF: atrial natriuretic factor; β MHC: beta myosin heavy chain; SKA: alpha skeletal actin; SMA: alpha smooth muscle actin; COLI: collagen I; COLIII: collagen III; FIB: fibronectin. The expression level was calculated as the ratio to glyceraldehyde-3-phosphate dehydrogenase. N of each group is 6. Data are mean ± SEM.^*P < 0.05 compared to sham + vehicle group.

Claims (14)

1. Use of IFN- γ for the manufacture of a medicament for the treatment of cardiac hypertrophy associated with non-viral infection.
2. 2. The use according to claim 1, wherein the medicament is a human medicament.
3. 3. The use of claim 2, wherein the IFN- γ is recombinant human IFN- γ.
4. 4. The use of claim 2, wherein the IFN- γ is recombinant human IFN- γ -1 b.
5. 5. The use according to claim 2, wherein said cardiac hypertrophy is characterized by PGF_2αThe level increased.
6. 6. The use according to claim 2, wherein the cardiac hypertrophy is myocardial infarction induced.
7. 7. The use according to claim 2, wherein IFN- γ is combined with other drugs selected from the group consisting of: beta-adrenergic blockers, verapamil, difeidipine and diltiazem *.
8. 8. The use according to claim 7, wherein the beta adrenergic blocker is carvedilol, propranolol, metoprolol, timolol, oxprenolol, or terbutalol.
9. 9. The use of claim 2, wherein the IFN- γ is administered in combination with an antihypertensive agent.
10. 10. The use according to claim 2, wherein the IFN- γ is in combination with an ACE inhibitor.
11. 11. The use according to claim 2, wherein the IFN- γ is administered in combination with an endothelin receptor antagonist.
12. 12. The use according to claim 1, wherein said pharmaceutical composition is a liquid.
13. 13. The use according to claim 1, wherein the pharmaceutical composition contains a preservative.
14. 14. The use according to claim 1, wherein the pharmaceutical composition is an injection.