KR100540537B1 - Treatment of hepatic cirrhosis - Google Patents

Abstract

The present invention relates to compositions for the treatment of liver fibrosis and cirrhosis, and to methods of using and preparing the compositions. The composition comprises a quinazolinone derivative, preferably halofuginone.

Description

Treatment of cirrhosis {TREATMENT OF HEPATIC CIRRHOSIS}             

The present invention relates to a method for treating cirrhosis (hepatic cirrhosis), specifically a method for treating cirrhosis using a quinazolinone derivative (eg halofuginone).

Cirrhosis is observed by copper in chronic alcoholism, malnutrition, hemochromatosis, passive congestion, hypercholesterolemia, exposure to toxins or toxins (such as lead), exposure to drugs, immune responses, and Wilson's disease. Genetically determined susceptibility to certain substances such as, and by various parasitic infections, including but not limited to Schistosomiasis mansoni and S. japonica , viral hepatitis and syphilis It has a number of causes including hepatic fibrosis that results. For the reasons presented in more detail below, the disease is currently incurable and often fatal.

The development of cirrhosis proceeds in a number of stages. First, hepatomegaly is identified by various fat changes. Next, apparent fibrosis is identified as a concomitant decrease in liver function. Finally, atrophy of the liver begins with a corresponding decrease in the size and function of the liver. Necrosis of the liver can be observed at any stage but is especially pronounced by cirrhosis of the late stages. Observation under a microscope confirms that the normal structure of the liver has completely collapsed in the cirrhosis of the late stage.

As cirrhosis progresses, other pathological changes become apparent outside the liver. The formation of fibrous tissue in the liver reduces portal circulation, further reducing liver function. This reduced circulation increases lateral venous circulation, especially intravenous venous circulation. These esophageal vessels can be destroyed, resulting in fatal thrombosis. Thus, cirrhosis is a holistic pathological process with a non-limiting effect on the liver, but its root cause can be found in certain pathological changes to the liver itself.

One essential step in the development of cirrhosis is the formation of fibrous tissue in the liver. Liver fibrosis is not characteristic of cirrhosis alone, but of most chronic liver disease (S.L. Friedman, "New Eng. J. Med., 328: 1828-35, 1993"). In hepatic fibrosis, connective tissue accumulates in the liver and is replaced with normal hepatic glandular tissue and reduces liver function. Fibrous tissue is replaced with more complex normal liver tissue during the pathological process, reducing the amount of liver tissue available for normal function, such as removing toxic substances from the blood, and gradually blocking intrahepatic blood flow. The formation of fibrous tissue in the liver is characterized by abnormally large deposition of extracellular matrix components, including five or more collagens, especially type I, III and IV collagens, as well as other substrate proteins [L. Ala. -Kokko, Biochem. J. 244: 75-9, 1987.

Synthesis of collagen is also involved in many other pathological diseases. For example, clinical diseases and diseases associated with first or second fibrosis (eg, systemic sclerosis, graft-versus-host disease (GVHD), pulmonary fibrosis and various autoimmune diseases) It is characterized by excessive production of connective tissue, which eventually destroys the structure and function of normal tissue. These diseases can be interpreted as disturbances of cellular function, the main evidence of which is excessive collagen synthesis and deposition. Because of the decisive role of collagen in fibrosis, attempts have been made to develop drugs that inhibit its accumulation (KI Kivirikko's "Annals of Medicine, Vol. 25, pp. 113-126 (1993)"). .

Such drugs can act by regulating the synthesis of procollagen polypeptide chains or by inhibiting specific post-translational processes, which reduce the formation of extracellular collagen fibers or have fibers with modified properties. Will cause an accumulation. Unfortunately, very few collagen synthesis inhibitors are available, although these proteins are important in maintaining tissue integrity and are involved in a variety of diseases.

For example, cytotoxic drugs have been used to delay the proliferation of collagen-producing fibroblasts (JA Casas et al. "Ann. Rhem. Dis., 46: 763, 1987"), such as extracellular matrix. Not only colchicine (D. Kershenobich et al., "N. Engl. J. Med., 318: 1709, 1988") that delays collagen secretion into the body, but also inhibitors of key collagen metabolizing enzymes [Car D. Karvonen et al., J. Biol Chem., 265: 8414, 1990; C. J. Cunliffe et al., &Quot; J. Med. Chem., 35: 2652, 1992 "

Unfortunately, none of these inhibitors are specific for the collagen-type. There is also serious concern about the toxic consequences that interfere with the biosynthesis of other bioactive collagen molecules such as Clq in classical supplemental pathways, acetylcholine esterases in neuromuscular junctions, conglutinin and hepatic apoptotin.

Other drugs capable of inhibiting collagen synthesis, such as nifedipine and phenytoin, also inhibit the synthesis of other proteins, thereby non-specifically blocking the collagen biosynthetic pathway [T. Salo et al., J. Oral Pathol. Med. ., 19: 404, 1990 ".

Collagen crosslinking inhibitors (eg β-amino-propionitrile) can act as useful antifibrotic agents, but they are also nonspecific. Their long-term use leads to lathyritic syndrome, and elastin (ie other fibrous connective tissue proteins) also interferes with elastogenesis because it is crosslinked. In addition, collagen cross-linking inhibitory effect is secondary, if collagen is excessively produced, collagen decomposition should proceed by collagenase. Thus, type-specific inhibitors are clearly required as antifibrotic agents for the synthesis of collagen itself.

Such type-specific collagen synthesis inhibitors for treating fibrotic diseases are disclosed in US Pat. No. 5,449,678. The specific inhibitor is a composition having a pharmaceutically effective amount of a pharmaceutically active compound of formula (I):

Where
n is 1 or 2;

R ₁ is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy;

R ₂ is a member of the group consisting of hydroxy, acetoxy and lower alkoxy;

R ₃ is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl.

Pharmaceutically acceptable salts thereof are also included. In the crowd of compounds, halofuginone has been found to be particularly effective in the treatment.

U.S. Pat. No. 5,449,678 discloses that these compounds are effective in the treatment of fibrotic diseases such as scleroderma and GVHD. PCT Publication No. WO 96/06616 further discloses that these compounds are effective in treating restenosis. The first two diseases are associated with excessive collagen deposition, which can be inhibited by halofuginone. Restenosis is characterized by the accumulation of extracellular matrix in the lumen of invaded blood vessels in response to smooth muscle cell proliferation and vascular injury (Choi et al., Arch. Surg., 130: 257-261, 1995). "]. One evidence of such smooth muscle cell proliferation is a phenotypic transformation from a normal contractile phenotype to a synthetic phenotype. Collagen type I has been found to support this phenotypic modification, which can be blocked by halofuginone (C. et al., Arch. Surg., 130: 257-261, 1995); US Patent No. 5,449,678].

However, the ex vivo activity of halofuginone does not always predict in vivo effects. For example, halofuginone inhibits the synthesis of type I collagen in bone chondrocytes in vitro as demonstrated in US Pat. No. 5,449,678. However, chickens treated with halofuginone were not reported to have increased bone breakage, indicating that the effect was not observed in vivo. Thus, in vitro studies cannot always accurately predict the behavior of halofuginone in vivo.

In addition, the ability of halofuginone or other quinazolinones associated with it to block or inhibit pathological processes associated with cirrhosis has not been demonstrated. Other inhibitors of collagen synthesis, crosslinking and deposition (eg corticosteroids, penicylamine, methotrexate and colchicine) have been tested for their therapeutic effects on liver fibrosis, but there is no evidence that they are effective [Fredman's literature "New Eng. J. Med., 328: 1828-35, 1993 ". Although halofuginone has been shown to have a specific inhibitory effect on the synthesis of type I collagen, this inhibition has not been found to be otherwise effective in the treatment of cirrhosis. In fact, cirrhosis has a high mortality rate as currently available treatment options have significant side effects and generally do not have the effect of delaying or stopping the progression of fibrosis. In addition, many other types of extracellular matrix components, including five or more types of collagen, especially type I, III and IV collagens as well as other substrate proteins, are deposited during the development of liver fibrosis [Alla-Coco, Biochem. J., 244: 75-9, 1987 ". Thus, only inhibiting the synthesis of collagen type I will not necessarily delay or stop the growth of liver fibrosis.

Thus, simple administration of compounds found to inhibit collagen synthesis, deposition and crosslinking only in vitro in an attempt to treat cirrhosis is not effective. Clearly, there is a need for new therapies for such incurable diseases that specifically delay or stop the development of liver fibrosis in vivo without nonspecific effects or toxic side effects.

Therefore, the need for the treatment of cirrhosis and fibrosis that inhibits fiber production in vivo without substantially exhibiting undesirable nonspecific effects or toxic side effects is widely recognized and it would be very advantageous to provide such a therapy.

Summary of the Invention

As described in the Examples below, halofuginone can also inhibit the pathophysiological processes of liver fibrosis in vivo, possibly by inhibiting type I collagen synthesis (although other mechanism (s) may be involved). This turned out to be unexpected. Inhibition of type I collagen synthesis is proposed as a plausible mechanism, but it is not intended to be limited to the only mechanism, and the only mechanism because the in vivo data presented below clearly demonstrates the efficacy of halofuginone as an inhibitor of liver fibrosis in vivo. There is no need to limit.

According to the teachings of the present invention there is provided a composition for treating cirrhosis, comprising a compound which is a member of the group having the formula (1) in a pharmaceutically effective amount with a pharmaceutically acceptable carrier:

Formula 1

Where
n is 1 or 2;

R ₁ is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy;

R ₂ is a member of the group consisting of hydroxy, acetoxy and lower alkoxy;

R ₃ is a member of the group consisting of hydrogen and lower alkenoxy.

Pharmaceutically acceptable salts thereof are also included.

According to a further preferred embodiment of the invention, the preferred compound is halofuginone. Hereafter, the term "halofuginone" is defined as a compound having the formula (2) and pharmaceutically acceptable salts thereof:

The composition preferably comprises a pharmaceutically acceptable carrier for the compound.

According to another aspect of the present invention, there is provided a method of preparing a medicament for treating cirrhosis, comprising the step of including a pharmaceutically effective amount of a compound which is a member of the group having formula (I) in a pharmaceutically acceptable carrier Is provided:

Formula 1

Where
n is 1 or 2;

R ₁ is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy;

R ₂ is a member of the group consisting of hydroxy, acetoxy and lower alkoxy;

R ₃ is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl.

Pharmaceutically acceptable salts thereof are also included.

According to another aspect according to the invention, there is provided a method of treating cirrhosis in a subject comprising administering a pharmaceutical effective amount of a compound having Formula 1:

Formula 1

Where
n is 1 or 2;

R ₁ is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy;

R ₂ is a member of the group consisting of hydroxy, acetoxy and lower alkoxy;

R ₃ is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl.

Pharmaceutically acceptable salts thereof are also included.

According to another aspect according to the present invention, there is provided a composition for substantially preventing the occurrence of cirrhosis, comprising a compound which is a member of the group having formula (I) in a pharmaceutically effective amount with a pharmaceutically acceptable carrier:

Formula 1

Where
n is 1 or 2;

R ₁ is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy;

R ₂ is a member of the group consisting of hydroxy, acetoxy and lower alkoxy;

R ₃ is a member of the group consisting of hydrogen and lower alkenoxy.

Pharmaceutically acceptable salts thereof are also included.

According to another aspect of the present invention, there is provided a medicament for substantially preventing the occurrence of cirrhosis, comprising the step of incorporating a compound which is a member of the group having the formula Methods of making are provided:

Formula 1

Where
n is 1 or 2;

R ₁ is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy;

R ₂ is a member of the group consisting of hydroxy, acetoxy and lower alkoxy;

R ₃ is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl.

Pharmaceutically acceptable salts thereof are also included.

According to another aspect according to the invention, there is provided a method of substantially preventing the occurrence of cirrhosis in a subject comprising administering a pharmaceutical effective amount of a compound having Formula 1:

Formula 1

Where
n is 1 or 2;

R ₁ is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy;

R ₂ is a member of the group consisting of hydroxy, acetoxy and lower alkoxy;

R ₃ is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl.

Pharmaceutically acceptable salts thereof are also included.

According to another embodiment according to the present invention there is provided a composition for treating liver fibrosis, comprising a compound which is a member of the group having the formula 1 in a pharmaceutically effective amount with a pharmaceutically acceptable carrier:

Formula 1

Where
n is 1 or 2;

R ₁ is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy;

R ₂ is a member of the group consisting of hydroxy, acetoxy and lower alkoxy;

R ₃ is a member of the group consisting of hydrogen and lower alkenoxy.

Pharmaceutically acceptable salts thereof are also included.

According to the present invention, there is also provided a method of preparing a medicament for treating liver fibrosis, comprising the step of including a pharmaceutically effective amount of a compound that is a member of the group having formula (I) in a pharmaceutically acceptable carrier: :

Formula 1

Where
n is 1 or 2;

R ₁ is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy;

R ₂ is a member of the group consisting of hydroxy, acetoxy and lower alkoxy;

R ₃ is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl.

Pharmaceutically acceptable salts thereof are also included.

Also provided is a method of treating liver fibrosis in a subject comprising administering a pharmaceutical effective amount of a compound having Formula 1:

Formula 1

Where
n is 1 or 2;

R ₁ is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy;

R ₂ is a member of the group consisting of hydroxy, acetoxy and lower alkoxy;

R ₃ is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl.

Pharmaceutically acceptable salts thereof are also included.

According to another aspect according to the present invention, there is provided a composition for substantially preventing the occurrence of liver fibrosis, comprising a compound which is a member of the group having formula (I) in a pharmaceutically effective amount with a pharmaceutically acceptable carrier do:

Formula 1

Where
n is 1 or 2;

R ₁ is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy;

R ₂ is a member of the group consisting of hydroxy, acetoxy and lower alkoxy;

R ₃ is a member of the group consisting of hydrogen and lower alkenoxy.

Pharmaceutically acceptable salts thereof are also included.

Also provided is a method of preparing a medicament for substantially preventing the development of liver fibrosis, comprising the step of including a compound that is a member of the group having Formula 1 in a pharmaceutically acceptable amount in a pharmaceutically acceptable carrier: :

Formula 1

Where
n is 1 or 2;

R ₁ is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy;

R ₂ is a member of the group consisting of hydroxy, acetoxy and lower alkoxy;

R ₃ is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl.

Pharmaceutically acceptable salts thereof are also included.

According to another aspect according to the invention there is provided a method of substantially preventing the development of liver fibrosis in a subject comprising administering a pharmaceutical effective amount of a compound having Formula 1

Formula 1

Where
n is 1 or 2;

R ₁ is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy;

R ₂ is a member of the group consisting of hydroxy, acetoxy and lower alkoxy;

R ₃ is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl.

Pharmaceutically acceptable salts thereof are also included.

Preferably, all of the compounds mentioned above may be the compound itself as described by the formula and / or may be a pharmaceutically acceptable salt thereof.

Hereafter, the term “subject” refers to a human or lower animal to which halofuginone is administered. The term "patient" refers to a human subject. The term “treatment” includes not only delaying or stopping the progression of cirrhosis or liver fibrosis once developed, but also substantially preventing the occurrence of cirrhosis or liver fibrosis. The phrase “substantially prevents the development of cirrhosis or liver fibrosis” includes clinical or preclinical treatment of these diseases, including the prevention of their syndromes, such as thrombosis from blood vessels in the esophagus, which are indirectly involved in the fibrosis and cirrhosis process itself. It is understood to refer to preventing the manifestation of the syndrome.

Although certain quinazolinone derivatives "halofuginone" are mentioned throughout the specification, it is understood that other quinazolinone derivatives may also be used in place, and these derivatives have the following formula:

Formula 1

Where
n is 1 or 2;

R ₁ is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy;

R ₂ is a member of the group consisting of hydroxy, acetoxy and lower alkoxy;

R ₃ is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl.

Pharmaceutically acceptable salts thereof are also included.

The present invention will now be described with reference to certain preferred embodiments in the following figures and examples so that the embodiments thereof may be more fully understood, but are not intended to limit the invention to these specific embodiments. On the contrary, the present invention is intended to include all alternative embodiments, modified embodiments, and equivalent embodiments as long as it can be included within the scope of the present invention as defined by the appended claims. Accordingly, the following drawings and examples, which include preferred embodiments, will illustrate the practice of the invention, and the details presented are, by way of example only, to illustrate, by way of example, preferred embodiments of the invention, the principles of the invention And conceptual aspects as well as what is presented as what is believed to be the most useful and easily understood description of the formulation process of the present invention.

The invention is described herein by way of example only with reference to the accompanying drawings.

1A-1D show the effect of halofuginone on collagen α1 (I) gene expression in rat liver.

2 shows the effect of halofuginone on hydroxyproline concentration in the liver of rats.

3A-3D show the effect of halofuginone on mild fibrosis in the liver of rats.

As described in the Examples below, it is unexpectedly possible that halofuginone can inhibit the pathological process of cirrhosis in vivo, possibly by inhibiting type I collagen synthesis (although other mechanism (s) may be involved). Turned out. Indeed, regardless of the specific mechanism, the data presented below clearly demonstrate the efficacy of halofuginone in vivo for the inhibition of pathological progression of liver fibrosis.

This discovery is unexpected for many reasons. First, the behavior of halofuginone in vitro does not exactly correspond to that in vivo. This can be demonstrated by the different effects of halofuginone observed by bone chondrocytes in vivo and ex vivo. Halofuginone inhibits the synthesis of type I collagen in chondrocytes in vitro, as demonstrated in US Pat. No. 5,449,678. However, chickens treated with halofuginone were not reported to have increased bone breakage, indicating that the effect was not observed in vivo. Thus, the exact behavior of halofuginone in vivo may not always be accurately predicted from in vitro studies.

Second, other inhibitors of collagen synthesis, deposition and crosslinking have not been shown to be effective in the treatment of cirrhosis, demonstrating that inhibiting collagen production alone is not sufficient to determine the success or failure of liver fibrosis treatment. . Thus, the discovery that halofuginone can successfully inhibit liver fibrosis in vivo in a suitable animal model is new and unclear.

Third, halofuginone has only been found to be a type I collagen inhibitor. However, the formation of fibrous tissue in the liver is characterized by a large amount of abnormal deposition of extracellular matrix components, including five or more types of collagen, especially type I, III and IV collagens as well as other substrate proteins. Biochem. J., 244: 75-9, 1987. Thus, halofuginone's ability to inhibit type I collagen synthesis and deposition cannot predict the halofuginone's ability to delay, reduce or otherwise improve the development of liver fibrosis.

Fourth, halofuginone has not been taught as a prophylactic agent suitable for preventing complex pathophysiological processes such as liver fibrosis and cirrhosis in mammals (eg humans). For example, US Pat. No. 3,320,124 teaches the use of compounds associated with halofuginone only for the prevention of coccidiosis, an infectious disease in chickens. Chickens are very physiologically different from any mammal, including humans. Indeed, chickens are generally not considered acceptable as experimental models for mammals and are certainly not used as experimental models for liver diseases and diseases such as liver fibrosis and cirrhosis. Thus, prophylactic treatment of humans or other mammals using the compounds of the present invention for the prevention of liver fibrosis or cirrhosis is clearly not taught in the above references, but in fact is also taught in other references mentioned in the background. not.

Fifth, the complexity of the pathological process of liver fibrosis is suggested by the important differences between liver fibrosis and cirrhosis. Cirrhosis is not simply a disease associated with liver fibrosis. The development of cirrhosis progresses in many stages, which can potentially lead to late stage liver failure and death. All these stages, from the initial appearance of fat changes in the liver to the final stage of liver necrosis, are important for the development of cirrhosis. Outside the liver, other pathological changes manifest as the cirrhosis progresses. As fibrous tissue forms in the liver, portal circulation is reduced, further reducing liver function. This reduced circulation results in an increase in the lateral venous circulation, especially in the esophagus. These esophageal vessels are destroyed, resulting in fatal thrombosis. Thus, cirrhosis is a holistic pathological process that has an unlimited effect on the liver, but its root cause can be found in certain pathological changes to the liver itself. In order to inhibit this pathological process or to prevent the occurrence of such a process, a successful therapeutic agent must be able to intervene to clearly delay or prevent the development of the overall process. The present invention has found that halofuginone may be such a successful therapeutic as compared to many of the materials that are theoretically appropriate but fail in in vivo testing. Thus, the discovery that halofuginone is a successful therapeutic agent for delaying and / or preventing the development of a group of syndromes occurring during the pathological process of cirrhosis is clearly not only a new and non-obvious finding but also a clear advance.

Finally, all other prior art references only teach the efficacy of halofuginone on cells such as fibroblasts and smooth muscle cells. In the liver, Ito cells have been shown to be a source of extracellular matrix components produced during liver fibrosis, and thus the cell type plays a critical role in the development of liver fibrosis [Friedman's "New Eng. J. Med. , 328: 128-35, 1993 ". However, Ito cells are a completely different cell type than fibroblasts. Although the behavior of halofuginone on a particular type of cell could be predicted, this prediction is certainly unreliable for cells other than that type. Thus, the effects of halofuginone on Ito cells cannot be predicted from the effects on fibroblasts.

Thus, there is no teaching in the prior art that halofuginone is useful for the treatment of liver fibrosis in vivo. In addition, the ability to delay or stop the progression of fibrosis in the liver of halofuginone and related compounds is not only a new and non-obvious discovery but also a clear advance. If the response observed in vitro and in vivo for halofuginone differs, the demonstration of such capacity for in vivo treatment of mammals is not particularly predicted.

The invention may be more readily understood by reference to the following illustrative examples and figures. Although only halofuginone is mentioned, it should be noted that other quinazolinone derivatives described and claimed in US Pat. No. 3,320,124, the teachings of which are incorporated herein by reference, are believed to have similar properties.

The present invention relates to the treatment of cirrhosis using a quinazolinone-containing compound such as halofuginone. Compositions having specific pharmaceutical formulations and methods of using and preparing these compounds are described below.

Although the pathogenesis of cirrhosis is not fully understood, animal models suitable for the disease have been successfully developed. Liver fibrosis was induced with a relatively short onset of action by intraperitoneal injection of dimethylnitrosamine in rats. That is, liver fibrosis has already developed within 3 weeks of the administration of dimethylnitrosamine to rats (A. M. Jezequel et al., J. Hepatol., 5: 174-81, 1987).

Dimethylnitrosamine-induced liver fibrosis is characterized by increased deposition of extracellular matrix components, including various types of collagen, such as type I collagen. Thus, inhibition of fibrosis, such as in dimethylnitrosamine-induced and other types of liver fibrosis, depends on the delay or interruption of the pathological process that produces fibrous tissue.

Thus, compounds for the inhibition of cirrhosis are dimethylnitrosamines of hepatic fibrosis in experimental animal models in vivo, such as rats, for their ability to delay or stop pathological processes that result in the deposition of fibrous tissue. Should be tested in the model. This test was performed on the type I collagen synthesis inhibitor halofuginone as described in more detail in Examples 1 and 2 below.

In addition, once a clearly effective compound is found, the specific formulation and route of administration should be identified for maximum therapeutic efficacy. Such formulations and routes of administration should be able to effectively absorb and deliver the compound to the desired therapeutic site while at the same time minimizing nonspecific side effects caused by systemic distribution of the compound. Examples of these formulations and routes of administration for quinazolinone-containing compounds such as halofuginone are shown in Examples 3-5.

Example 1

Effect of Halofuginone on Tissue and Morphology of Rat Liver

Histological examination of liver samples from control rats and dimethylnitrosamine-treated rats revealed that dimethylnitrosamine induced specific morphological changes, including increased collagen fiber content in rat livers. Halofuginone substantially inhibited the occurrence of this morphological change, giving the rat liver a more normal appearance. The experimental method was as follows. Male Sprague-Dawley rats were divided into four groups. Two groups were injected with 1% dimethylnitrosamine in saline intraperitoneally at a dose of 1 mL / kg body weight for three consecutive days per week for three weeks. When administered at this dosage, severe liver fibrosis will be induced. The other two groups of rats (control rats) were injected with saline. One group of dimethylnitrosamine-treated rats and one control group received halofuginone at a dose of 5 mg / kg weight in the diet from 3 days prior to administration of the dimethylnitrosamine injection. At the end of the experiment, rats were killed and livers were weighed out.

Liver samples were taken for histology. In summary, tissue samples were collected in phosphate-buffered saline (PBS) and fixed overnight in 4% paraformaldehyde in PBS at 4 ° C. Samples were dehydrated in graded ethanol solution, cleared in chloroform and enclosed in Paraplast, to prepare a series of 5 μm sections. Collagen and non-collagen proteins were differentially stained with 0.1% fast green as 0.1% Sirius red and opposite dyes in picric acid. With this procedure collagen is stained red (Gascon-Barre, M. et al., "J. Histochem. Cytochem., 37: 377-381, 1989").

Liver samples were then hybridized using a probe for the expression of collagen α1 (I) in rats. For hybridization using the gene probe, sections were waxed in xylene, hydrated again through a graded series of ethanol solutions, rinsed in distilled water for 5 minutes, and then in 2X SSC at 70 ° C. for 30 minutes. Incubated at. The sections were then rinsed in distilled water and treated with 0.125 mg / mL of pronase in 50 mM Tris-HCl, 5 mM EDTA at pH 7.5 for 10 minutes. After digestion, the slides were rinsed with distilled water, post-fixed in 10% formalin in PBS and blocked in 0.2% glycine. After blocking, the slats were rinsed in distilled water, quickly dehydrated through a graded ethanol solution and air-dried for several hours. Before hybridization, 1600 bp of collagen α1 (I) inserts from rats were cut from the original plasmid (pUC18) and inserted into the pSafyre plasmid. Sections were then hybridized with the probe after digoxigenin-labeling (M. Pines et al., "Matrix Biology, 14: 765-71, 1996").

1 shows in situ hybridization of rat liver tissue sections using rat collagen α1 (I) probes. Low expression of the collagen α1 (I) gene is observed in the livers of control rats (FIG. 1A) or rats provided with halofuginone alone (FIG. 1B). Significant increases in collagen α1 (I) gene expression were observed in the livers of rats provided with dimethylnitrosamine alone (FIG. 1C). The gene expression occurred mainly in the septum surrounding the lobule at the site of scattered collagen tissue. Rats provided with both halofuginone and dimethylnitrosamine showed a marked reduction in collagen α1 (I) gene expression compared to rats provided with dimethylnitrosamine alone (FIG. 1D). Although this dose of halofuginone substantially reduces the increase in collagen α1 (I) gene expression in rats caused by dimethylnitrosamine, it does not completely inhibit the expression as evidenced (see arrow in FIG. 1D). It was. However, substantially reduced collagen α1 (I) gene expression in rats indicates that halofuginone is effective against the pathological induction of expression by dimethylnitrosamine.

Although the results are not shown graphically because the tissue samples must be examined in color to observe the effect, sections of rat liver tissues were stained with Sirius red to confirm the collagen content of the tissues. Collagen fibers were rarely observed in control rats or liver tissue harvested from rats provided with halofuginone alone. The livers of dimethylnitrosamine-treated rats showed an increase in collagen content, which gave the collagen bundles surrounding the lobules, resulting in a large fibrous septum. The thickening of these collagen bundles was markedly reduced in rats provided with both dimethylnitrosamine and halofuginone, which also indicates the ability of halofuginone to substantially inhibit the pathophysiological process of fibrosis induced by dimethylnitrasamine. .

Interestingly, relatively high doses of dimethylnitrosamine resulted in severe liver fibrosis, resulting in 4 of 6 dimethylnitrosamine-treated rats not given halofuginone killed at the end of the 3 week period. On the other hand, only one out of six mice given both dimethylnitrosamine and halofuginone died. Six rats survived in each of the two groups not given dimethylnitrosamine. Thus, halofuginone was not toxic by itself, but was able to almost completely prevent dimethylnitrosamine-induced death.

Dimethylnitrosamine-induced changes in gross morphology levels were also inhibited by halofuginone. Rats treated with dimethylnitrosamine alone were significantly lower liver weight (4.5 g, especially when compared to control rats and rats given halofuginone alone (liver weight 12 ± 1 g and 11 ± 1.5 g, respectively). And 5.0 g). Rats provided with both halofuginone and dimethylnitrosamine had a liver weight (8.5 ± 1.7 g) that was nearly two times higher than the liver weight of rats given dimethylnitrosamine alone, but was somewhat reduced compared to the control rats.

Thus, halofuginone was able to prevent the effects of dimethylnitrosamine-induced fibrosis at all levels. That is, it almost eliminates dimethylnitrosamine-induced lethality and significantly reduces the total and micromorphological changes caused by dimethylnitrosamine-induced fibrosis. Clearly, the effects of halofuginone are potent and specific for the prevention of morphological changes produced during the pathological process of liver fibrosis.

Example 2

Effect of Halofuginone on Mild Fibrosis in Rat Liver

Halofuginone substantially completely prevented dimethylnitrosamine-induced mild fibrosis, as confirmed by measuring collagen α1 (I) gene expression and hydroxyproline content. The specific experimental method used was similar to the method of Example 1 except that dimethylnitrosamine-treated rats provided only a very low dose, i.e. 0.25% of dimethylnitrosamine in saline, compared to the dose presented in Example 1 above. . In addition, the treatment period before killing the rats was further extended: 4 weeks in contrast to 3 weeks in Example 1.

Expression of the collagen α1 (I) gene was measured as described in Example 1 above. For hydroxyproline analysis, liver samples were hydrolyzed at 110 ° C. for 22 hours with 6N of HCl. Nitrogen was measured after Kjeldahl digestion by a spectrophotometric procedure using an autoanalyzer as described by Krom (MD Krom, Analyst, 105: 305). -16, 1980 "]. Hydroxyproline, an amino acid unique to collagen from the same hydrolysate, was subjected to amino acid analysis (Biotronic LC 5000, Germany) after post-column derivatization on a cation exchange column (BTC 2710, Biotronik). Was measured. Results are expressed as percentage of collagen in total liver protein.

Since hydroxyproline is an amino acid present in relatively large amounts in collagen, it serves as a measure of the total concentration of collagen in specific tissues. Thus, as shown in Figure 2, apparently dimethylnitrosamine significantly increases the hydroxyproline concentration, thus increasing the collagen concentration in the liver of the rat. This increase was completely suppressed by treatment with halofuginone. However, the administration of halofuginone to rats not provided with dimethylnitrosamine did not result in any change in hydroxyproline concentration. Thus, the effect of halofuginone was simply to inhibit dimethylnitrosamine-induced increase in hydroxyproline concentration.

3C demonstrates that such low doses of dimethylnitrosamine still result in an increase in collagen α1 (I) gene expression, particularly by cells surrounding blood vessels. 3D shows that the increased gene expression is lost by halofuginone. In addition, as in Example 1, halofuginone alone had no effect on the expression of collagen α1 (I) gene (FIG. 3B), but the control rat did not have any collagen α1 (I) gene expression. (FIG. 3A).

Thus, halofuginone apparently completely inhibited the increase in the level of collagen synthesis induced by dimethylnitrosamine in rat liver. However, halofuginone alone has not demonstrated this effect in rats, indicating that the effect of halofuginone is specific for the inhibition of pathophysiological processes (eg collagen synthesis) caused by dimethylnitrosamine-induced fibrosis. Indicates. In addition, apparently halofuginone was able to substantially completely eliminate the biochemical and physiological changes caused by dimethylnitrosamine, as demonstrated by both Examples 1 and 2.

Example 3

Inhibition of fibrosis induced by bile duct ligation

A second model of liver fibrosis in rats with dimethylnitrosamine-induced liver fibrosis is available. The model relies on surgical ligation of bile ducts rather than requiring the administration of exogenous or toxic chemicals to induce liver fibrosis, which has been suggested to be a suitable model for studying cirrhosis in humans [Conta Kotaras, J. et al., Br. J. Exp. Pathol, 65: 305-311, 1984; Muriel, P. et al., J. Hepatol., 21: 95-102, 1994; Muriel et al., J. Appl. Tox., 15: 449-453, 1995]. Thus, a particular advantage of the bile duct ligation model is that any prophylactic treatment does not indirectly modify the effects of the exogenous materials used to cause liver fibrosis in animal models, but rather directly affects the liver from pathological changes induced by fibrosis. It must be protected. The experimental method was as follows.

Male Wistar rats weighing 200-250 g were divided into four experimental groups with three rats in each group. Group 1 did not provide bile duct ligation and provided halofuginone. Group 2 provided halofuginone without biliary duct ligation. It should be noted that all animals in the first two groups had a sham operation including all stages of the actual surgical procedure except bile duct ligation itself. Group 3 underwent bile duct ligation and did not provide halofuginone. Group 4 underwent bile duct ligation and provided halofuginone. The actual surgical procedure was essentially similar to that recorded in the literature (Contaras et al., Br. J. Exp. Pathol., 65: 305-311, 1984).

All animals were given unlimited drinks. Rats given halofuginone were given halofuginone at a concentration of 5 mg / kg weight in normal rats during the experimental period of 1 week before surgery and 3 or 7 days after surgery. Rats were killed at the end of the experimental period. Both collagen content (via Sirius red staining) and collagen α1 (I) gene expression were measured as described in Example 1 above. In addition, serum alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase concentrations were measured colorimetrically by the Boerringer-Mannheim Hitachi Auto-analyzer System.

No collagen synthesis was observed in the sham-operated rats. In addition, these rats did not show any increase in weight or liver weight, or any modified liver tissue. Finally, these rats showed no change in the concentration of enzymes such as alkaline phosphatase, alanine aminotransferase or aspartate aminotransferase, 3 or 7 days after surgery, regardless of the halofuginone administration.

In contrast, elevated concentrations of all three enzymes were observed in rats ligated with bile ducts in both the halofuginone-treated and untreated groups. These elevated concentrations are a characteristic indication of the pathological process of liver fibrosis and cirrhosis. However, rats fed halofuginone had lower concentrations of these enzymes than rats that did not. Specifically, rats not provided with halofuginone had 56% higher alanine aminotransferase, 257% alkaline phosphatase and 15% higher aspartate aminotransferase than rats fed halofuginone. Thus, halofuginone apparently reduced the degree of elevation of enzyme concentration in rats ligated with bile ducts.

In addition, halofuginone can be used to determine bile duct ligation-induced collagen synthesis and collagen α1 (I) gene expression when compared to rats that are ligated with bile ducts and fed halofuginone only. The increase was significantly reduced. Thus, halofuginone apparently was able to inhibit the process of liver fibrosis in a model of bile duct ligation-induced fibrosis in rats.

Example 4

Formulations Suitable for Administration of Halofuginone

Halofuginone and related compounds of the present invention, as well as pharmaceutically acceptable salts thereof, can be administered to a subject in a number of ways well known in the art. Hereafter, the term “subject” refers to a human or lower animal to which halofuginone is administered. For example, it can be administered orally or parenterally, such as by intravenous drip or intraperitoneal, subcutaneous or intramuscular injection.

Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, sachets, capsules or tablets. Thickeners, diluents, flavors, dispersion aids, emulsifiers or binders may be preferred.

Formulations for parenteral administration may include, but are not limited to, sterile aqueous solutions that may also contain buffers, diluents and other suitable additives.

Dosages vary depending on the neutrality of the syndrome and the subject's response to halofuginone or one of the other compounds of the invention and pharmaceutically acceptable salts thereof. One skilled in the art can readily determine the optimal dosage, method of administration and repetition rate.

Example 5

Treatment of liver fibrosis and cirrhosis

As noted above, halofuginone has been shown to be an effective inhibitor of liver fibrosis, a preliminary stage of cirrhosis. The following examples merely illustrate, but are not intended to limit, methods for treating liver fibrosis and cirrhosis using halofuginone or one of the other compounds of the present invention and pharmaceutically acceptable salts thereof.

The method comprises administering to a subject to be treated a halofuginone or one of the other compounds of the invention and a pharmaceutically acceptable salt thereof in a pharmaceutically acceptable carrier as described in Example 4 above. . Halofuginone, depending on the effective method of administration, preferably until it reaches a predefined endpoint, such as until there is no further progression of liver fibrosis or cirrhosis in the subject, or until it inhibits liver fibrosis or cirrhosis, or It is administered until it prevents the formation of liver fibrosis or cirrhosis.

Examples of types of liver fibrosis in which the therapy is effective include chronic alcoholism, malnutrition, hemochromatosis, passive congestion, hypercholesterolemia, exposure to toxins or toxins (such as lead), exposure to drugs, immune responses, Wilson's disease. Genetically determined susceptibility to certain substances as observed by copper in and various parasitic infections, including, but not limited to, hemoptysis and Japanese schistosomiasis, and caused by infections such as viral hepatitis and syphilis. Liver fibrosis includes but is not limited to. In addition, these therapies will also be effective against liver fibrosis diseases of unknown or insufficiently defined etiology.

Specifically, the evidence described in the above examples clearly shows that halofuginone and other compounds of the present invention are suitable for the treatment of hepatic diseases caused by the ingestion of hepatotoxic substances. Substances that are not normally hepatotoxic can also lead to liver damage, such as when present in excessive concentrations as drugs. Since the liver is a major organ for metabolic detoxification of many different chemicals, hepatic disease caused by the ingestion of hepatotoxic substances is not uncommon. The efficacy of the present invention for the treatment of such hepatic diseases is clearly shown by experiments using a dimethylnitrosamine-induced model of hepatic fibrosis as described in Example 1 above.

All these methods are also hepatic alone, as not limited to a single mechanism for the action of the compounds of the present invention, and because liver fibrosis is an essential underlying factor for the development of cirrhosis, which is substantially prevented or improved by the compounds of the present invention. In addition to treating diseases characterized by fibrosis, it can also be used to treat cirrhosis.

Example 6

Method for producing a drug containing halofuginone

The following examples are examples of methods for preparing halofuginone or one of the other compounds of the present invention and their pharmaceutically acceptable salts. Although an example of preparation of halofuginone has been described as an example, it is understood that the above description includes not only the pharmaceutically acceptable salts of halofuginone itself but also other compounds of the present invention and methods for preparing the pharmaceutically acceptable salts thereof. First, halofuginone is synthesized according to appropriate pharmaceutical preparation practices. Examples of methods for synthesizing halofuginone and related quinazolinone derivatives are given in US Pat. No. 3,338,909. Next, the halofuginone is then added back into a suitable pharmaceutical carrier as described in Example 4 above according to appropriate pharmaceutical preparation practices.

The above description is provided by way of example only, and it will be understood that many other aspects are possible within the spirit and scope of the invention.

Claims (14)

A composition for treating cirrhosis in a subject comprising a pharmaceutically effective amount of a compound having Formula 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier: Formula 1

Where n is 2, R ₁ is Br or Cl, R ₂ is hydroxy; R ₃ is hydrogen. The method of claim 1, The compound is

Phosphorus composition. delete The method of claim 1, Liver cirrhosis is caused by a hepatotoxic substance. A composition for treating hepatic fibrosis in a subject, comprising a pharmaceutically effective amount of a compound having Formula 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier: Formula 1

Where n is 2, R ₁ is Br or Cl; R ₂ is hydroxy; R ₃ is hydrogen. The method of claim 5, Compound

Phosphorus composition. delete The method of claim 5, A composition in which liver fibrosis is caused by a hepatotoxic substance. A method according to claim 1 for the treatment of cirrhosis in a subject, comprising the step of including a pharmaceutically effective amount of a compound or a pharmaceutically acceptable salt thereof, which is a member of the group having the formula Method of Preparation of the Composition: Formula 1

Where n is 2, R ₁ is Br or Cl; R ₂ is hydroxy; R ₃ is hydrogen. The method of claim 9, Compound

How to be. The method of claim 9, How cirrhosis is caused by hepatotoxic substances. A method of treating liver fibrosis in a subject comprising the step of incorporating a compound or pharmaceutically acceptable salt thereof in a pharmaceutically acceptable carrier in a pharmaceutically acceptable carrier. Process for the preparation of the composition according to: Formula 1

Where n is 2, R ₁ is Br or Cl; R ₂ is hydroxy; R ₃ is hydrogen. The method of claim 12, Compound

How to be. The method of claim 12, How liver fibrosis is caused by hepatotoxic substances.

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