KR20010012838A - 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}

Cirrhosis is observed by copper in chronic alcoholism, malnutrition, hemochromatosis, passive hyperemia, hypercholesterolemia, exposure to toxins or toxins (such as lead), exposure to drugs, immune responses, and Wilson's disease. Genetically determined susceptibility to certain substances, and infections such as many parasitic infections, viral hepatitis and syphilis, including but not limited to Schistosomiasis mansoni and S. japonica. It has a number of causes, including hepatic fibrosis caused by it. 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, the swollen liver is known by various fat changes. Next, obvious 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 certain stages, but is particularly 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.

Other pathological changes have been identified outside the liver as evidence of cirrhosis progression. The formation of fibrous tissue in the liver reduces portal circulation, and further reduces 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, is replaced by 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 interstitial components, including at least five collagens, specifically type I, III and IV collagens as well as other cytoplasmic proteins [Alla-Coco (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 in 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 their accumulation (KI Kivirikko's "Annals of Medicine, Vol. 25, pp. 113-126 (1993)"). .

Such agents can act by regulating the synthesis of procollagen polypeptide chains or by inhibiting specific post-translational processes, which inhibit the formation of extracellular collagen fibers or have fibers with modified properties. Will inhibit accumulation. Unfortunately, despite the importance of these proteins, only a few inhibitors of collagen synthesis are available for maintaining tissue integrity and its contents in various diseases.

For example, cytotoxic agents have been used to delay the proliferation of collagen-producing fibroblasts (JA Casas et al., "Ann. Rhem. Dis., 46: 763, 1987"). In addition to delaying collagen secretion into extracellular epilepsy (D. Kershenobich et al., "N. Engl. J. Med., 318: 1709, 1988"), it is also an inhibitor of key collagen metabolizing enzymes. Used in D. Karvonen et al., J. Biol Chem., 265: 8414, 1990; C. J. Cunliffe et al., J. Med. Chem., 35: 2652, 1992].

Unfortunately, none of these inhibitors are specific for the collagen-type. In addition, serious consideration should be given to the toxic consequences involved in the biosynthesis of other bioactive collagen molecules such as Clq in existing supplementary pathways, acetylcholine esterases in neuromuscular junctions, conglutinin and hepatic surfactant apoprotein.

Other agents capable of inhibiting collagen synthesis, such as nifedipine and phenytoin, can inhibit the synthesis of other proteins as well as thereby non-specifically block the collagen biosynthetic pathway (T. Salo et al., J. Oral Pathol). Med., 19: 404, 1990 "].

Collagen crosslinking inhibitors (eg β-amino-propionitrile) can be provided as useful antifibrotic agents, but they are also nonspecific. Their prolonged use leads to lathyritic syndrome and intervenes in elastogenesis because elastin (ie other fibrous connective tissue proteins) is also crosslinked. In addition, collagen cross-linking inhibitory effect is secondary, and collagen is inevitably degraded by collagenase when collagen is excessively produced. Thus, type-specific inhibitors of the synthesis of collagen are clearly required as antifibrotic agents per se.

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 1 below:

Where

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 was found to have a particular effect on the treatment.

U.S. Patent 5,449,678 discloses that these compounds are effective in the treatment of fibrotic diseases such as scleroderma and GVHD. PCT patent WO 96/06616 further discloses that these compounds are effective in treating restenosis. The two diseases are associated with excessive collagen deposition, which can be inhibited by halofuginone. Restenosis is characterized by smooth muscle cell proliferation and the accumulation of extracellular epilepsy in the lumen of invasive vessels in response to vascular damage (Choi et al., Arch. Surg., 130: 257-261). , 1995 "]. One evidence of this smooth muscle cell proliferation is the transformation of a normal contractile trait into a synthetic trait. Collagen type I has been shown to support this transformation, 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 are not reported to have increased bone breakage, indicating that the effect is not observed in raw vegetables. Thus, the exact behavior of halofuginone in vivo cannot always be accurately predicted from in vitro studies.

In addition, the ability of halofuginone or other quinazolinones 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 does not appear to be otherwise effective in the treatment of cirrhosis. In fact, cirrhosis has a high mortality rate because the treatment options currently in use 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 interstitial components are deposited during the development of liver fibrosis, which includes five or more types of collagen, specifically type I, III and IV collagens as well as other interstitial proteins [alka-cocos]. See, Biochem. J., 244: 75-9, 1987. Thus, only inhibiting the synthesis of type I collagen does not necessarily delay or stop the growth of liver fibrosis.

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

Thus, the need for the treatment of cirrhosis and fibrosis that inhibits fiber production in vivo without substantially exhibiting unwanted nonspecific 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 other mechanism (s) also), possibly by inhibiting collagen I synthesis. It turned out unexpectedly. Although the inhibitory action of type I collagen synthesis is proposed as a plausible mechanism, it is not intended to be limited to the only mechanism, and the only mechanism is 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 to.

In accordance with 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 formula (I) in a pharmaceutically effective amount with a pharmaceutically acceptable carrier:

Formula 1

Where

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 of 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

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 method of treating cirrhosis in any subject comprising administering a pharmaceutical effective amount of a compound having Formula 1:

Formula 1

Where

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

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

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 any subject, comprising administering a pharmaceutical effective amount of a compound having Formula 1:

Formula 1

Where

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

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

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 are methods of treating liver fibrosis in any subject, comprising administering a compound of formula 1 in a pharmaceutically effective amount:

Formula 1

Where

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

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 carrier in a pharmaceutically acceptable carrier:

Formula 1

Where

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 method of substantially preventing the development of liver fibrosis in any subject, comprising administering a pharmaceutical effective amount of a compound having Formula 1:

Formula 1

Where

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 previously mentioned herein may be the compound itself and / or a pharmaceutically acceptable salt thereof, as described by the formula.

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 that occurred once but substantially preventing the occurrence of cirrhosis or liver fibrosis. The phrase “substantially prevents the development of cirrhosis or liver fibrosis” includes clinical or preclinical clinical trials of these diseases, including the prevention of their syndromes, such as thrombosis from blood vessels in the esophagus, which are indirectly involved in fibrosis and cirrhosis process itself. It is understood to refer to preventing the appearance of red 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

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 cover all alternative embodiments, modified embodiments, and equivalent embodiments as may 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 performance of the invention, and the details set forth are merely illustrative of the preferred embodiments of the invention through examples, and the principles of the invention. And conceptual aspects as well as descriptions which are presented to provide a description that is believed to be the most useful and easy to understand of the formulation process of the present invention.

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

The invention is described herein by merely illustrating 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, halofuginone can also inhibit the pathological process of cirrhosis in vivo, possibly by inhibiting collagen I synthesis (although other mechanism (s) may also be involved). This turned out to be unexpected. 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 no effect was 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 inhibition of collagen production alone is not sufficient to determine the success or failure of treatment of liver fibrosis. . Thus, the discovery that halofuginone can successfully inhibit liver fibrosis in vivo in a suitable animal model is new and evident.

Third, halofuginone was shown to be the only type I collagen inhibitor. However, the formation of fibrous tissue in the liver is characterized by a large amount of abnormal deposition of extracellular interstitial components, including five or more types of collagen, specifically type I, III and IV collagens as well as other interstitial proteins. Coco, "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 this complex pathophysiological process as liver fibrosis and cirrhosis in mammals (eg humans). For example, US Pat. No. 3,320,124 teaches the use of halofuginone related compounds for the prevention of infectious coccidiosis in chickens only. Chickens are physiologically very different from any mammal, including humans. Indeed, chickens are generally not considered acceptable as experimental models for mammals and are not particularly used as experimental models for liver disease (eg liver fibrosis and cirrhosis). Thus, prophylactic treatment of humans or other animals using the compounds of the present invention for the prevention of liver fibrosis or cirrhosis is clearly not taught herein by reference or in fact in the background other references.

Fifth, the complexity of the pathological process of liver fibrosis is suggested by the important differences between liver fibrosis and cirrhosis. Cirrhosis is not just a disease associated with liver fibrosis. The development of cirrhosis develops in a number of stages, which can potentially result in end-stage liver failure and death. All these steps, from the first appearance of fat change in the liver to the final-stage of hepatic necrosis, are important steps in the development of cirrhosis. External, ie, other pathological changes in the liver are evident as the cirrhosis progresses. Portal circulation decreases as fibrous tissue is formed in the liver, further reducing liver function. This reduced circulation results in an increase in the lateral venous circulation, especially in the esophagus. Blood vessels in these esophagus can be destroyed, resulting in fatal thrombosis. Thus, cirrhosis is a holistic pathological process that has an unlimited effect on the liver, but the root cause can be found in specific pathological changes to the liver itself. In order to inhibit this pathological process or to prevent the occurrence of such a process, successful treatment must be able to intervene to clearly delay or prevent the development of the overall process. The present invention only suggests that halofuginone can represent such a successful treatment in comparison with a number of theoretically appropriate materials, but still fails in in vivo testing. Thus, the discovery that halofuginone is a successful treatment to delay and / or prevent the development of a group of syndromes occurring during the pathological process of cirrhosis is not only a new and 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 appear to be the source of extracellular interstitial components produced during liver fibrosis, so this 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 against a particular type of cell is foreseen, this prediction is not particularly reliable 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 of delaying or stopping the progression of fibrosis in the liver of halofuginone and related compounds is not only a new and obvious finding but also a clear advance. Since different responses observed in vitro and in vivo are provided for halofuginone, the demonstration of such capacity for in vivo treatment of mammals is not particularly predicted.

The invention may be more readily understood with reference to the following illustrative examples and figures. Although only halofuginones are exclusively referenced, it should be noted that other quinazolinone derivatives described and claimed in US Pat. No. 3,320,124, which is incorporated herein by reference and taught, are known to have similar properties. do.

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, mammalian models suitable for the disease have been successfully developed. Hepatic fibrosis with a relatively short onset of action was induced in rats by intraperitoneal injection of dimethylnitrosamine: Liver fibrosis was already developed within 3 weeks of dimethylnitrosamine administration in rats (AM Jezequel et al. "J. Hepatol., 5: 174-81, 1987".

Dimethylnitrosamine-induced liver fibrosis is characterized by increased deposition of extracellular interstitial components, which include various types of collagen such as type I collagen. Thus, the inhibitory action of fibrosis, such as in dimethylnitrosamine-induced and other types of liver fibrosis, depends on delaying or stopping the pathological process of producing fibrous tissue.

Thus, compounds for the inhibition of cirrhosis are dimethylnitrosamines of hepatic fibrosis in experimental animal models, such as rats, as described above, 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 for the type I collagen synthesis inhibitor halofuginone as described in more detail in Examples 1 and 2 below.

In addition, once a verifiable effective compound is found, the specific formulation and route of administration should be identified for maximum efficacy of the treatment. 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 the 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 liver. Halofuginone substantially inhibited the occurrence of morphological changes, which eventually gave the rat liver a more normal appearance. The experimental method was as follows. Male Sprague-Dawley rats were divided into four groups. In two groups, 1% dimethylnitrosamine in saline was injected 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 of food from 3 days before 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 Paralast, followed by 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 et al. "J. Histochem. Cytochem., 37: 377-1989").

Liver samples were then hybridized using a probe for the expression of collagen α1 (I) in rats. For hybridization using the gene probe, the compartments are dewaxed in xylene, hydrated again through a graded series of ethanol solutions, rinsed in distilled water for 5 minutes, and then 2X SSC at 70 ° C. for 30 minutes. Incubated in-house. The compartments 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, 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. The sections were then digoxigenin-labeled and hybridized with the probe (M. Pines et al., "Matrix Biology, 14: 765-71, 1996").

1 shows hybridization of compartments of rat liver tissue using rat collagen α1 (I) probes in situ. Low expression of the collagen α1 (I) gene is observed in the liver of control rats (FIG. 1A) or in the liver of rats provided with halofuginone alone (FIG. 1B). Significant increases in expression of the collagen α1 (I) gene were observed in the liver of rats given dimethylnitrosamine alone (FIG. 1C). The gene expression occurs mainly in the diaphragm surrounding the lobule at the site of scattered collagen tissue. Rats provided with both halofuginone and dimethylnitrosamine showed a significant decrease in expression of the collagen α1 (I) gene as compared to rats provided with dimethylnitrosamine alone (FIG. 1D). Although this dose of halofuginone substantially reduced the increase in collagen α1 (I) gene expression in rats caused by dimethylnitrosamine, it did not completely inhibit the expression, which can be observed as a tracer (indicated by arrow). 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 vivid because the tissue samples must be examined in color to observe the effect, the 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 tissues or liver tissue harvested from rats provided with halofuginone alone. The livers of dimethylnitrosamine-treated rats show an increase in collagen content, which provides a bundle of collagen that envelops the lobules, resulting in a large fibrous septum. The thickening of these collagen bundles is markedly reduced in rats provided with both dimethylnitrosamine and halofuginone, indicating 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, one in six mice that received both dimethylnitrosamine and halofuginone died. Six rats each survived in two groups that were not given dimethylnitrosamine. Thus, halofuginone is not toxic by itself and still almost completely prevents dimethylnitrosamine-induced death.

Dimethylnitrosamine-induced changes in gross morphology levels were also inhibited by halofuginone. Rats treated with dimethylnitrosamine alone had significantly lower liver weights (4.5 g and 5.0 g), especially when compared to control rats and rats given halofuginone alone. Rats provided with both halofuginone and dimethylnitrosamine had a liver weight (8.5 ± 1.7 g) that was almost twice as high as the liver weight of rats provided with dimethylnitrosamine alone, although somewhat reduced compared to the control rats.

Thus, halofuginone was able to prevent the emergence of the effects of dimethylnitrosamine-induced fibrosis: the total elimination of most of the dimethylnitrosamine-induced high mortality, and the change in the total and fine morphology caused by dimethylnitrosamine-induced fibrosis Marked reduction in. 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 was substantially completely prevented dimethylnitrosamine-induced mild fibrosis as determined by measuring collagen α1 (I) gene expression and hydroxyproline content. The specific experimental method used was similar to that of Example 1 except that the dimethylnitrosamine-treated rats provided only a very low dose, i.e. 0.25% dimethylnitrosamine in saline, relative to the doses 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 "]. Collagen-only amino acid hydroxyproline from the same hydrolysate was measured by amino acid analysis (Biotronic LC 5000, Germany) after post-column derivatization on a cation exchange column (BTC2710, Biotronik) It was. Results are expressed as percentage of collagen in total liver protein.

Hydroxyproline is an amino acid present in relatively large amounts in collagen and therefore serves as a measure of all levels of collagen in a particular tissue. Thus, as shown in FIG. 2, dimethylnitrosamine apparently results in a significant increase in hydroxyproline concentrations, resulting in an increase in collagen levels in the rat liver. This increase was completely inhibited by treatment with halofuginone. However, administration of halofuginone to rats not provided with dimethylnitrosamine did not result in any change in hydroxyproline concentration. Thus, the effect of halofuginone simply inhibited the 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 the blood vessels. 3D shows that the increased gene expression is lost by halofuginone. In addition, as in Example 1, halofuginone alone does not have any effect on the expression of collagen α1 (I) gene (FIG. 3B), but the control rat does not have any collagen α1 (I) gene expression. (FIG. 3A).

Thus, halofuginone apparently completely inhibited the increased levels of collagen synthesis induced by dimethylnitrosamine in the rat's liver. However, with halofuginone alone, it demonstrates the above effects in rats, indicating that the effect of halofuginone is specific for the inhibition of pathophysiological processes (eg collagen synthesis) caused by dimethylnitrosamine-induced fibrosis. I did not. In addition, halofuginone was able to substantially completely eliminate the biochemical and physiological changes caused by dimethylnitrosamine, as clearly 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, Pl et al., J. Hepatol., 2195-102, 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 directly modifies the liver from pathological changes induced by fibrosis, rather than indirectly modifying the effect of the exogenous material used to cause liver fibrosis in the animal model. It must be prevented. The experimental model 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 that included all steps of the actual surgical procedure except the bile duct 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 for 1 week before surgery and during the experimental period of 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 levels were measured colorimetrically by the Boerringer-Mannheim Hitachi Auto-analyzer System.

No collagen synthesis was observed in apparently 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 that gall bile ducts in both the halofuginone-treated and untreated groups. These elevated concentrations are characteristic markers of pathological processes of liver fibrosis and cirrhosis. However, rats fed halofuginone had lower concentrations of these enzymes than rats that did not. In particular, 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 elevated enzyme concentration in rats that ligated bile ducts.

In addition, halofuginone was associated with an increase in bile duct ligation-induced collagen synthesis and collagen α1 (I) gene expression when compared to rats ligated with bile ducts and fed with halofuginone. Significantly reduced. Thus, halofuginone could clearly 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 its related compounds, 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 in non-aqueous media, sachets, capsules or tablets. Thickeners, diluents, flavors, dispersion aids, emulsifiers or binders may be required.

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

Dosage depends on the neutrality of the syndrome and the subject's response to one of the halofuginone or 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 precursors of liver fibrosis and cirrhosis. The following examples only illustrate methods of treating liver fibrosis and cirrhosis using halofuginone or one of the other compounds of the present invention and pharmaceutically acceptable salts thereof, but are not intended to be limiting.

The method comprises administering to a subject to be treated one of the halofuginone or 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 any subject, until it inhibits liver fibrosis or cirrhosis, Or 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 hyperemia, hypercholesterolemia, exposure to toxins or toxins (such as lead), exposure to drugs, immune responses, and Wilson's disease. Genetically determined susceptibility to certain substances as observed by copper in and caused by a number of parasitic infections, including but not limited to pancreatic nephropathy and Japanese schistosomiasis, and infections such as viral hepatitis and syphilis. Liver fibrosis, including but not limited to. In addition, these therapies are also effective against liver fibrosis diseases of unknown or poorly defined etiology.

Specifically, the evidence described in the preceding examples indicates that the halofuginone and other compounds of the invention are suitable for the treatment of hepatic diseases caused by the ingestion of hepatotoxic substances. In the case of substances that are not normally hepatotoxic, for example, even when the drug is present in excessive concentrations, it can cause liver damage. Since the liver is a major organ for detoxification by the metabolism 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. It can be used to treat cirrhosis as well as to treat diseases characterized by fibrosis.

Example 6

Method for producing a drug containing halofuginone

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

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 the treatment of cirrhosis in any subject, comprising a pharmaceutically effective amount of a compound having Formula 1 and a pharmaceutically acceptable salt thereof: Formula 1 Where 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. The method of claim 1, The composition is a halofuginone. The method of claim 1, A composition further comprising a pharmaceutically acceptable carrier. The method of claim 1, Liver cirrhosis is caused by a hepatotoxic substance. A composition for treating liver fibrosis in any subject, comprising a pharmaceutically effective amount of a compound having Formula 1 and a pharmaceutically acceptable salt thereof: Formula 1 Where 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. The method of claim 5, The composition is a halofuginone. The method of claim 6, A composition further comprising a pharmaceutically acceptable carrier. The method of claim 7, wherein A composition in which liver fibrosis is caused by a hepatotoxic substance. Preparation of a medicament for treating cirrhosis in any subject, comprising the step of including a compound that is a member of the group having Formula 1 and a pharmaceutically acceptable salt thereof in a pharmaceutically acceptable carrier in a pharmaceutically effective amount Way: Formula 1 Where 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. The method of claim 9, The compound is halofuginone. The method of claim 9, How cirrhosis is caused by hepatotoxic substances. A medicament for treating liver fibrosis in any subject, comprising the step of including a compound that is a member of the group having Formula 1 and a pharmaceutically acceptable salt thereof in a pharmaceutically acceptable carrier Manufacturing Method: Formula 1 Where 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. The method of claim 12, The compound is halofuginone. The method of claim 12, How liver fibrosis is caused by hepatotoxic substances.