CN1265034A - Treatment of hepatic cirrhosis - Google Patents

Abstract

A composition for treating hepatic fibrosis and hepatic cirrhosis, and methods of using and manufacturing the composition are disclosed. The composition includes a quinazolinone derivative, preferably Halofuginone.

Description

The treatment of liver cirrhosis

The present invention relates to the treatment of liver cirrhosis, particularly treat liver cirrhosis with Quinazol derivative such as halofuginone.

The reason of liver cirrhosis has many, comprises by chronic alcoholism nutritional disorder, hemochromatosis, passive hyperemia, hypercholesteremia has contacted poison or toxin such as lead, the contact medicine, immunoreation, the sensitivity to some material of heredity decision, as can in hepatolenticular degeneration, see to the copper sensitivity, and infectious disease such as viral hepatitis, syphilis and various parasitic infection, include but not limited to the hepatic fibrosis that schistosomiasis mansoni and schistosomiasis japanica cause.For the more detailed reason that provides hereinafter, this disease is incurable at present, and usually causes death.

Pathogenesis of cirrhosis will be passed through many stages.At first, see that the liver that has various fat to change enlarges.Secondly, significantly fibre modification follows liver function obviously to reduce.At last, liver begins atrophy, simultaneously reduce corresponding with function of size of liver.The necrosis of liver all can be seen in any stage, but particularly remarkable in hardening late.By microscopic examination, late period, the normal configuration of liver was obviously broken fully when hardening.

When liver cirrhosis further develops, except liver, other tangible pathology also occur and change.When the formation fibre modification was organized in the liver, Portal circulation reduced, and has further reduced the function of liver.This circulation minimizing causes side to prop up venous circulation to be increased, particularly in esophagus.These esophagus blood vessels can break, and cause severe haemorrhage.Therefore, although basic reason can find it is the specificity pathology variation of liver self, liver cirrhosis is the whole pathological process that an influence is not only limited to liver.

An inevitable step in the pathogenesis of cirrhosis is to form the fibre modification tissue in liver.Hepatic fibrosis is the feature of most chronic hepatopathys, and not merely is the feature (S.L.Friedan, " New England Journal of Medicine " 328:1828-35,1993) of liver cirrhosis.In hepatic fibrosis, connective tissue gathers in liver, has replaced normal liver parenchyma, and has reduced liver function.In pathological process, this fibre modification tissue has replaced more complicated normal liver tissue, makes to can be used for normal function, reduce as the hepatic tissue quantity of removing the toxicant in the blood, has interrupted blood flow in the liver gradually.Form the deposition that the fibre modification tissue is characterised in that a large amount of unusually extracellular matrix compositions in the liver, comprise at least five types collagen, particularly I type, III type and IV Collagen Type VI, and other stromatins (L.Ala-Kokko, " journal of biological chemistry " 244:75-9,1987).

Synthesizing of collagen also relates to many other pathology situations.For example, clinical scenarios and the disease relevant with constitutional fibre modification or Secondary cases fibre modification, such as Sjogren's syndrome disease, graft versus host disease (GVHD), pnemnofibrosis and a large amount of autoimmune disease, their distinguishing characteristics is that connective tissue excessively produces, thereby has caused the destruction of normal structure 26S Proteasome Structure and Function.These diseases can be interpreted as having upset cell function rightly, and it mainly shows is the synthetic and deposition of excessive collagen.The decisive role of collagen in fibre modification encouraged exploitation to suppress the trial (K.T.Kivirikko, " medical science record event " the 25th volume, 113-126 page or leaf (1993)) of its medicine of accumulating.

These medicines can be by regulating the synthetic of precollagen polypeptide chain or working by suppressing specificity translation back incident, the accumulating of fiber of reducing the formation of the outer collagen fiber of born of the same parents thus or causing performance change.Unfortunately, though this protein is very important in keeping tissue integrity, and closely related with multiple disease, it is available having only minority collagen synthetic inhibitor.

For example, cytotoxic drug has attempted being used to slow down collagenic fibroblasts proliferation (J.A.Casas etc., Ann.Rhem.Dis., 46:763,1987), such as colchicine, it has slowed down the secretion (people such as D.Kershenobich of collagen in extracellular matrix, " New England Journal of Medicine " 35:2652,1992).

Unfortunately, not having a kind of in these inhibitor is that collagen type is specific.And, exist and disturb other important tropocollagen molecules, as the relevant serious worry of biosynthetic toxicity consequence of the acetylcholinesterase of the Clq in the CCP, neuromuscular junction soleplate, conglutinin regulating liver-QI surfactant apoprotein etc.

Can suppress the synthetic other drug of collagen,, also suppress other proteinic synthesizing as nifedipine and phenytoin, thus the non-specific biosynthesis pathway of blocking collagen (T.Salo etc., " oral pathology medical journal " 19:404,1990).

The cross-linked inhibitor of collagen as beta-amino-propionitrile, although can be used as the fibrosis medicine, also is nonspecific.Their life-time service can cause the lathyrism syndrome and disturb elasticity that (elastogenesis) takes place, because another kind of fibrous connective tissue protein-elastin laminin also is crosslinked.In addition, the crosslinked inhibitory action of collagen is insecondary, and the excessive generation of collagen has to take place before it is by degraded by collagenase.Therefore, significant need is as the synthetic Idiotype inhibitor of the collagen self of fibrosis medicine.

United States Patent (USP) 5,449 discloses this Idiotype collagen synthetic inhibitor that is used for the treatment of fibrotic conditions in 678.This specific inhibitor is the compositions that a kind of pharmaceutically active compound of representing with pharmacy effective dose, following formula forms:

Wherein:

R ₁Be hydrogen, halogen, nitro, benzo, low alkyl group, phenyl or lower alkoxy; R ₂Be hydroxyl, acetoxyl group or lower alkoxy; And R ₃Be hydrogen or rudimentary alkenyloxy-carbonyl, also comprise its pharmaceutically acceptable salt.In this group chemical compound, found that halofuginone is particularly effective for this treatment.

United States Patent (USP) 5,449, disclosing these chemical compounds in 678 is effective in the treatment of fibrotic conditions such as scleroderma and GVHD.PCT application WO 96/06616 further discloses these chemical compounds and can treat restenosis effectively.Preceding two kinds of diseases are relevant with the over-deposit of collagen, and they can suppress with halofuginone.After restenosis was characterised in that blood vessel injury, smooth muscle cell proliferation, extracellular matrix were accumulated (Choi etc., " surgery's document " 130:257-261,1995 in affected lumen of vessels; United States Patent (USP) 5,449,678).

But the interaction in vitro of halofuginone does not always indicate effect in its body.For example, as United States Patent (USP) 5,449, in 678 prove that halofuginone is vitro inhibition type i collagen synthetic in the bone chondrocyte.Yet, do not report that this shows does not see this effect in vivo with the bone fracture rate increase of the chicken of halofuginone treatment.Therefore, halofuginone definite behavior in vivo can not always obtain correct prophesy from vitro study.

In addition, do not prove that also halofuginone or other relevant quinazolinones have the ability of blocking-up or the inhibition pathological process relevant with liver cirrhosis.Tested synthetic, the crosslinked and deposition inhibitor of other collagens, as corticosteroid, penicillamine, methotrexate and colchicine,, but do not prove that they are effective (S.L.Friedman to the therapeutical effect of hepatic fibrosis, " New England Journal of Medicine " 328:1828-35,1993).Have specific inhibitory effect although shown halofuginone to type i collagen is synthetic, it is effective that this inhibition is not presented in the treatment of liver cirrhosis as yet.Really, the mortality rate of liver cirrhosis is very high, and present available Therapeutic Method has pronounced side effects, and generally fails to slow down effectively or stop hardened further developing.In addition, in the pathogenic process of hepatic fibrosis, deposited the extracellular matrix composition of many other types, comprise at least five types collagen, particularly I, III and IV Collagen Type VI, and other stromatins (L.Ala-Kokko, " journal of biological chemistry " 244:75-9,1987).Therefore, only suppress the synthetic development that differs and slow down surely or stop hepatic fibrosis of type i collagen.

Therefore, giving simply to have shown can be synthetic at vitro inhibition collagen, to attempt to treat liver cirrhosis be invalid for deposition and crosslinked chemical compound.Clearly, for this incurable disease, need be can specificity slow down or cessative aspect in hepatic fibrosis morbidity and do not have the new treatment of non-specific or toxic side effects.

Therefore, now extensively have recognized the need to treat liver cirrhosis and fibrotic method, and this will be very useful, this method should suppress in fact that fiber takes place in the body, and the non-specific or toxic side effects of not expecting.

Unexpectedly, as described in embodiment hereinafter, have now found that halofuginone also can suppress the pathophysiological processes of hepatic fibrosis in the body, may type i collagen is synthetic to be realized by suppressing, but also may be another kind of or several mechanism work.When imagination type i collagen synthetic inhibition when seeming rational mechanism, do not expect to only limit to this single mechanism, and be unnecessary, because the interior data of hereinafter listed body have clearly proved the usefulness of halofuginone as hepatic fibrosis inhibitor in the body.

A kind of compositions for the treatment of liver cirrhosis is provided in accordance with the teachings of the present invention, has comprised that the chemical compound of pharmacy effective dose combines with pharmaceutically acceptable carrier, this chemical compound is a member in the chemical compound represented of following formula: Wherein:

R ₁Be hydrogen, halogen, nitro, benzo, low alkyl group, phenyl or lower alkoxy; R ₂Be hydroxyl, acetoxyl group or lower alkoxy; R ₃Be hydrogen or rudimentary alkenyloxy.Also comprise its pharmaceutically acceptable salt.

According to another preferred embodiment of the present invention, this chemical compound is preferably halofuginone.Hereinafter, term " halofuginone " is defined as the chemical compound that following formula is represented:

And pharmaceutically acceptable salt.Said composition preferably includes the pharmaceutically acceptable carrier of this chemical compound.

According to another embodiment of the invention, it provides the medicaments preparation that is used for the treatment of liver cirrhosis method, comprises that chemical compound with pharmacy effective dose places the step of pharmaceutically acceptable carrier, and this chemical compound is a member in the chemical compound represented of following formula: Wherein:

R ₁Be hydrogen, halogen, nitro, benzo, low alkyl group, phenyl or lower alkoxy; R ₂Be hydroxyl, acetoxyl group or lower alkoxy; R ₃Be hydrogen or rudimentary alkenyloxy-carbonyl.Also comprise its pharmaceutically acceptable salt.

According to another embodiment of the invention, it provides the method for treatment main body liver cirrhosis, comprises the step of the chemical compound that the following formula that gives pharmacy effective dose is represented: Wherein:

R ₁Be hydrogen, halogen, nitro, benzo, low alkyl group, phenyl or lower alkoxy; R ₂Be hydroxyl, acetoxyl group or lower alkoxy; R ₃Be hydrogen or rudimentary alkenyloxy-carbonyl.Also comprise its pharmaceutically acceptable salt.

According to another embodiment of the invention, it provides the compositions that is used for preventing in fact liver cirrhosis to take place, and comprises that the chemical compound of pharmacy effective dose combines with pharmaceutically acceptable carrier, and this chemical compound is a member in the chemical compound represented of following formula:

Wherein:

R ₁Be hydrogen, halogen, nitro, benzo, low alkyl group, phenyl or lower alkoxy; R ₂Be hydroxyl, acetoxyl group or lower alkoxy; R ₃Be hydrogen or rudimentary alkenyloxy.Also comprise its pharmaceutically acceptable salt.

According to another embodiment of the invention, it provides the medicaments preparation method that is used for preventing in fact the liver cirrhosis generation, comprise that chemical compound with pharmacy effective dose places the step of pharmaceutically acceptable carrier, this chemical compound is a member in the chemical compound represented of following formula:

Wherein:

R ₁Be hydrogen, halogen, nitro, benzo, low alkyl group, phenyl or lower alkoxy; R ₂Be hydroxyl, acetoxyl group or lower alkoxy; R ₃Be hydrogen or rudimentary alkenyloxy-carbonyl.Also comprise its pharmaceutically acceptable salt.

According to another embodiment of the invention, it provides the method for preventing main body generation liver cirrhosis in fact, comprises the step of the chemical compound that the following formula that gives pharmacy effective dose is represented:

Wherein:

R ₁Be hydrogen, halogen, nitro, benzo, low alkyl group, phenyl or lower alkoxy; R ₂Be hydroxyl, acetoxyl group or lower alkoxy; R ₃Be hydrogen or rudimentary alkenyloxy-carbonyl.Also comprise its pharmaceutically acceptable salt.

According to another embodiment of the invention, a kind of compositions for the treatment of hepatic fibrosis is provided, comprise that the chemical compound of pharmacy effective dose combines with pharmaceutically acceptable carrier, this chemical compound is a member in the chemical compound represented of following formula:

Wherein:

R ₁Be hydrogen, halogen, nitro, benzo, low alkyl group, phenyl or lower alkoxy; R ₂Be hydroxyl, acetoxyl group or lower alkoxy; R ₃Be hydrogen or rudimentary alkenyloxy.Also comprise its pharmaceutically acceptable salt.

Also provide the medicaments preparation that is used for the treatment of hepatic fibrosis method according to the present invention, comprised that chemical compound with pharmacy effective dose places the step of pharmaceutically acceptable carrier, this chemical compound is a member in the chemical compound represented of following formula:

Wherein:

R ₁Be hydrogen, halogen, nitro, benzo, low alkyl group, phenyl or lower alkoxy; R ₂Be hydroxyl, acetoxyl group or lower alkoxy; R ₃Be hydrogen or rudimentary alkenyloxy-carbonyl.Also comprise its pharmaceutically acceptable salt.

The method of treatment main body hepatic fibrosis also is provided, has comprised the step of the chemical compound that the following formula that gives pharmacy effective dose is represented:

Wherein:

R ₁Be hydrogen, halogen, nitro, benzo, low alkyl group, phenyl or lower alkoxy; R ₂Be hydroxyl, acetoxyl group or lower alkoxy; R ₃Be hydrogen or rudimentary alkenyloxy-carbonyl.Also comprise its pharmaceutically acceptable salt.

According to another embodiment of the invention, it provides the compositions that is used for preventing in fact hepatic fibrosis to take place, and comprises that the chemical compound of pharmacy effective dose combines with pharmaceutically acceptable carrier, and this chemical compound is a member in the chemical compound represented of following formula: Wherein:

R ₁Be hydrogen, halogen, nitro, benzo, low alkyl group, phenyl or lower alkoxy; R ₂Be hydroxyl, acetoxyl group or lower alkoxy; R ₃Be hydrogen or rudimentary alkenyloxy.Also comprise its pharmaceutically acceptable salt.

The medicaments preparation method that is used for preventing in fact hepatic fibrosis to take place also is provided, has comprised that chemical compound with pharmacy effective dose places the step of pharmaceutically acceptable carrier, this chemical compound is a member in the chemical compound represented of following formula:

Wherein:

R ₁Be hydrogen, halogen, nitro, benzo, low alkyl group, phenyl or lower alkoxy; R ₂Be hydroxyl, acetoxyl group or lower alkoxy; R ₃Be hydrogen or rudimentary alkenyloxy-carbonyl.Also comprise its pharmaceutically acceptable salt.

According to another embodiment of the invention, it provides the method for preventing main body generation hepatic fibrosis in fact, comprises the step of the chemical compound that the following formula that gives pharmacy effective dose is represented:

Wherein:

R ₁Be hydrogen, halogen, nitro, benzo, low alkyl group, phenyl or lower alkoxy; R ₂Be hydroxyl, acetoxyl group or lower alkoxy; R ₃Be hydrogen or rudimentary alkenyloxy-carbonyl.Also comprise its pharmaceutically acceptable salt.

Preferably, all above alleged chemical compounds can be the chemical compounds of being described by structural formula itself, and/or its pharmaceutically acceptable salt.

Hereinafter, term " main body " is meant people or the rudimentary animal that gives halofuginone.Term " patient " is meant the human agent.Term " treatment " comprises and prevents liver cirrhosis or fibrotic generation in fact, and in case after liver cirrhosis or fibre modification occurring, slows down or stops its development.Phrase " prevents liver cirrhosis or fibrotic generation in fact " and is interpreted as to be meant the appearance of symptom before these diseases of prevention clinical or clinical, comprises those symptoms that prevention is directly related with fibre modification and hardening process self, as the esophagus angiorrbagia.

Although in whole description, should be mentioned that specificity Quinazol derivative " halofuginone ", it is to be understood that other Quinazol derivatives also can be used for the place of its usefulness, these derivants have following structural:

Wherein:

R ₁Be hydrogen, halogen, nitro, benzo, low alkyl group, phenyl or lower alkoxy; R ₂Be hydroxyl, acetoxyl group or lower alkoxy; R ₃Be hydrogen or rudimentary alkenyloxy-carbonyl.Also comprise its pharmaceutically acceptable salt.

To get in touch some preferred embodiment now, describe the present invention with following drawings and Examples, so that various aspects of the present invention can obtain understanding more completely and understanding, but intention is not to limit the present invention in these particular.Antithesis, intention is to comprise by appended claims all selections, improvement and equivalent definition, that can be included in the scope of the present invention.Therefore, the following drawings and Examples that comprise preferred embodiment will be used to illustrate enforcement of the present invention, everybody should understand, these shown special cases are the modes via embodiment, purpose only is in order to set forth the discussion of the preferred embodiment of the invention, be the explanation that is trusted as the compound method that is the most useful and easy understanding in order to provide in addition, and the explanation of principle of the present invention and notion aspect.

Brief description of drawings

Only utilize embodiment herein, and with reference to the accompanying drawings to describe the present invention, wherein:

Figure 1A-1D has illustrated halofuginone effect to collagen α 1 (I) gene expression in rats'liver;

Fig. 2 has illustrated halofuginone effect to hydroxyproline concentration in rats'liver;

Fig. 3 A-3D illustrated halofuginone in rats'liver to the fibrotic effect of moderate.

Unexpectedly, as described in embodiment hereinafter, have now found that Halofuginone can suppress body The pathological process of interior cirrhosis may synthesize realization by suppressing type i collagen, but Also may be that another kind or several mechanism work. Really, no matter this specificity mechanism is how, Lower column data has proved that clearly Halofuginone suppresses the pathology development of hepatic fibrosis in vivo Usefulness.

There are many reasons to prove that this discovery is surprisingly. At first, Halofuginone External behavior is not exclusively corresponding with the interior behavior of its body. The halogen husband who observes with the bone cartilage cell Ketone in vivo with the external provable this point of not same-action. Such as United States Patent (USP) 5,449,678 In institute prove that Halofuginone is in external inhibition type i collagen synthesizing in the cartilage cell. Yet, Do not report that this shows does not see this in vivo with the bone fracture rate increase of the chicken of Halofuginone treatment Effect. Therefore, Halofuginone definite behavior in vivo can not always just obtain from vitro study True prophesy.

Secondly, synthetic, the deposition of other collagens and cross-linked inhibitor also do not prove and can effectively control The treatment cirrhosis, this explanation, the generation that suppresses separately collagen are not enough to determine the liver fiber is become The treatment of property is success or failure. Therefore, find that Halofuginone can be successfully in suitable moving It is novel and non-obvious suppressing hepatic fibrosis in the object model body.

The 3rd, Halofuginone only shows it is the type i collagen inhibitor. Yet, form fiber in the liver The sex change tissue is characterised in that the deposition of unusually a large amount of extracellular matrix compositions, comprises at least five Type collagen, particularly I, III and IV Collagen Type VI, and other stromatins (L.Ala-Kokko, " journal of biological chemistry " 244:75-9,1987). Therefore, halogen The type i collagen ability synthetic and deposition that suppresses husband's ketone can not indicate that Halofuginone has and slow down, subtracts Less or other improve the ability of hepatic fibrosis morbidity.

The 4th, also there is not instruction to point out that Halofuginone can be used as suitable prophylactic treatment, is used for Prevent mammal as the people resemble hepatic fibrosis and the sclerosis complicated Pathological Physiology mistake Journey. For example, United States Patent (USP) 3,320, the compound relevant with Halofuginone used in 124 only instructions The infectious disease global-worm illness of prevention chicken. It is very big that chicken and any mammal comprise that people's physiology has Difference. Really, do not think that generally chicken is acceptable mammiferous experimental model, and Certainly be not used as the experimental model of hepatopathy such as hepatic fibrosis and cirrhosis. Therefore, this reference Document does not point out The compounds of this invention to have the prevention people or other mammiferous liver fibers become Property and this clearly shortsightedness therapeutic action of cirrhosis, other in the background technology are with reference to literary composition Offer and also really do not make this prompting.

The 5th, the important difference between hepatic fibrosis and the cirrhosis has shown hepatic fibrosis The complexity of the pathological process that takes place. Cirrhosis not merely is a kind of and hepatic fibrosis Relevant disease. The morbidity of cirrhosis divides many stages to carry out, and this can cause potentially at last Stage hepatic failure and necrosis. From at first fatty change owning to last hepatonecrosis occurring the liver The generation of stage for cirrhosis all is important. When cirrhosis further develops, except Beyond the liver, other obvious pathology also occur and change. In liver, form the fibre modification tissue The time, portal circulation reduces, and has further reduced the function of liver. This circulation minimizing causes side Propping up venous circulation increases, particularly in oesophagus. These oesophagus blood vessels can break, and cause tight The blood of reppearing. Therefore, although basic reason can find it is the specificity pathology variation of liver self, But cirrhosis is the whole pathological process that an impact is not only limited to liver. The present invention's demonstration, Yet with think in theory be suitable in vivo the experiment in the failure many materials Compare, only have Halofuginone can successfully finish this treatment. Therefore, Halofuginone can be successfully Various the influencing each other that is used for slowing down and/or prevents the pathological process at cirrhosis to produce The appearance of symptom, this discovery are novel and non-obvious, and have demonstrated significantly Creative.

At last, every other formerly technical literature all just instructed Halofuginone to cell as becoming The effect of fibrocyte and smooth muscle cell. In liver, Ito cell has shown it is the liver fiber The source of the extracellular matrix composition that produces in the degenerative process, therefore this cellular type is for the liver fibre The morbidity of dimension sex change is conclusive (S.L.Friedman, " New England Journal of Medicine " 328:1828-35,1993). But Ito cell is a kind of fiber that is different from into fully The cellular type of cell. Even can predict Halofuginone to the behavior of certain type cell, this Planting prediction also is insecure for the cell except this type certainly. Therefore, halogen Husband's ketone can not be predicted out to fibroblastic effect from it to the effect of Ito cell.

Therefore, in the prior art without any being used for the treatment of in vivo liver about Halofuginone Fibrotic instruction. In addition, Halofuginone and related compound slow down or stop in the liver fine The ability of dimension sex change development is novel and non-obvious, and has shown obvious creation The property. In view of the Halofuginone shown difference of replying in vitro and in vivo, this moving for lactation Proving of the interior therapeutic ability of thing is especially unexpected.

Can understand the present invention more easily with reference to following illustrative embodiment and accompanying drawing.Although should be noted that, should believe that at United States Patent (USP) 3,320 describe in 124 and also have similar performance with other Quinazol derivatives of asking for protection, this article content is incorporated herein by reference with reference to just carrying out at halofuginone.

The present invention treats liver cirrhosis with the chemical compound such as the halofuginone that contain quinazolinone.Compositions with certain drug preparation is described below, and the use of these chemical compounds and preparation method.

Although the pathogenic process of liver cirrhosis imperfectly understands, successfully develop the suitable animal model of this disease.By inducing the generation hepatic fibrosis to injection N-nitrosodimethylamine in the rat peritoneum, has the short relatively onset time: giving in three weeks of rat N-nitrosodimethylamine, hepatic fibrosis is (A.M.Jezequel etc., " hepatology magazine " 5:174-81,1987) clearly.

The feature of the inductive hepatic fibrosis of N-nitrosodimethylamine is that the deposition of extracellular matrix composition increases, and comprises various types of collagens such as type i collagen.Therefore, no matter be the inductive hepatic fibrosis of N-nitrosodimethylamine or the hepatic fibrosis of other types, fibrotic inhibition is depended on the pathological process that slows down or stop causing the fibre modification tissue to produce.

Therefore, being intended to be used to suppress the chemical compound of liver cirrhosis must be with experimental animal model in the body, test the ability that it slows down or stops causing the pathological process of fibre modification tissue deposition such as the fibrotic N-nitrosodimethylamine model of above-mentioned rats'liver.With type i collagen synthetic inhibitor halofuginone carry out these the experiment, among the embodiment 1 and 2 below with more detailed description.

In addition, in case found to prove compounds effective, mandatory declaration produces the particular formulations and the route of administration of maximum therapy usefulness.These preparations and route of administration must be able to make this chemical compound absorb effectively and be discharged into the expection therapentic part, and the non-specific side effect that causes that will be distributed by the system of this chemical compound simultaneously reduces to minimum.The chemical compound such as these preparations of halofuginone and the illustrative embodiment of route of administration that contain quinazolinone have been provided in the following examples 3 to 5.

Embodiment 1

Halofuginone is to the histology and the morphology effect of rats'liver

The Microscopic examination showed of the liver sample of the rat that control rats and N-nitrosodimethylamine are handled, N-nitrosodimethylamine has been induced the specificity morphological change in rats'liver, comprise that collagen contents increases.Halofuginone has suppressed the generation of these morphological changes in fact, makes that the outward appearance of rats'liver is more normal.This experimental technique is as follows.Male Sprague-Dawley rat is divided into four groups.The saline solution of two groups of peritoneal injection 1% N-nitrosodimethylamines was injected weekly three days continuously, continued for three weeks, and dosage is the 1ml/kg body weight.This dosage will be induced serious hepatic fibrosis.Two groups of control rats pump pickles in addition.From first three day with the N-nitrosodimethylamine injection, the rat group of a N-nitrosodimethylamine processing and a matched group add halofuginone with the heavy dosage of 5mg/kg meal in diet.When experiment finishes, these rats to be put to death, the taking-up liver is also weighed.

These liver samples are carried out histological examination.In brief, these tissue samples are focused in the phosphate buffered solution (PBS), the PBS solution with 4% polyformaldehyde under 4 ℃ fixedly spends the night.After sample is with the dehydration of gradient alcoholic solution, prepare successive 5 μ m section, clean and implant among the Paraplast with chloroform.As the after stain in the picric acid collagen and noncollagen protein are carried out coloured differently with 0.1% sunset profit red (Sirius red) and 0.1% fast green.The method makes collagen dye redness (Gascon-Barre, M. etc., " histochemistry and cytochemistry magazine " 37:377-381,1989).

These liver samples and rat collagen α 1 (I) probe hybridization of expressing then.For with the hybridization of genetic probe, these sections with the dimethylbenzene dewaxing, by serial gradient alcoholic solution rehydration, with distilled water flushing 5 minutes, were cultivated 30 minutes down at 70 ℃ in 2X SSC then.These section reuse distilled water flushings are also used pronase, and 0.125mg/ml is at 50mMTris-HCl, and 5mM EDTA among the pH7.5, handled 10 minutes.After the digestion, the microscope slide distilled water flushing with fixing behind the PBS solution of 10% formalin, and seals with 0.2% glycine.After the sealing, the microscope slide distilled water flushing is by gradient alcoholic solution rapidly dehydration and air drying a few hours.Before hybridization, downcut 1600bp rat collagen α 1 (I) from original plasmid and insert fragment pUC18, and be inserted in the pSafyre plasmid.Behind the digoxigenin labelling, these probe hybridizations (M.Pines etc., " substrate biology " 14:765-71,1996) therewith of cutting into slices.

Fig. 1 has shown the on the spot hybridization of liver tissues of rats section with rat collagen α 1 (I) probe.In the liver of control rats (Figure 1A) or in the liver of the rat that gives halofuginone separately (Figure 1B) see the weak expression of collagen α 1 (I) gene.In the liver of the rat that gives N-nitrosodimethylamine separately, see collagen α 1 (I) expression of gene and significantly increase (Fig. 1 C).Gene expression mainly around the lobule in every in, at sparse collagen sex organization position.Compare with the rat that gives N-nitrosodimethylamine separately, the rat that gives halofuginone and N-nitrosodimethylamine demonstrates collagen α 1 (I) gene expression (Fig. 1 D) of remarkable minimizing.Although the halofuginone of this dosage has reduced the increase of rat collagen α 1 (I) gene expression that is caused by N-nitrosodimethylamine in fact, it does not suppress this expression fully, still can be observed a small amount of expression (seeing the arrow labelling).Yet rat collagen α 1 (I) gene expression has reduced in fact, shows that halofuginone has resisted N-nitrosodimethylamine effectively to inducing of expressing.

Liver tissues of rats section is with sharp red colouring of sunset, and proving the collagen content of this tissue, the result shows with figure, because histology's sample must check that color just knows the effect situation.Control rats or give separately almost not observe collagen fiber in the hepatic tissue of rat of halofuginone.The liver of the rat of handling with N-nitrosodimethylamine shows that collagen content increases, and shows multi beam collagen around the lobule, produced in the big fibroid every.In the rat that gives N-nitrosodimethylamine and halofuginone, these collagen bundles thicken remarkable minimizing, shown that once more halofuginone suppresses the ability by the inductive fibrotic pathophysiological processes of N-nitrosodimethylamine in fact.

Interesting is, when the N-nitrosodimethylamine with suitable high dose causes this serious hepatic fibrosis, with N-nitrosodimethylamine handle and six rats not giving halofuginone when three weeks, experiment finished death four.On the contrary, give to have only a death in six rats of N-nitrosodimethylamine and halofuginone.Six rats that do not give in those two groups of N-nitrosodimethylamine all survive.Therefore, independent halofuginone does not have toxicity, can also almost completely stop the inductive death of N-nitrosodimethylamine.

Halofuginone has also suppressed the variation of the inductive total morphology level of N-nitrosodimethylamine.Separately the liver weight of the rat of handling with N-nitrosodimethylamine significantly reduces (4.5g and 5.0g), particularly when with control rats or the rat that gives halofuginone separately (be respectively 12 ± 1g and 11 ± 1.5g) when contrasting.The liver of rat that gives halofuginone and N-nitrosodimethylamine is heavy, and (8.5 ± 1.7g) almost is the twice that gives the rat of N-nitrosodimethylamine separately, though compare with control rats reduction is arranged slightly.

Therefore, halofuginone can prevent the appearance of the inductive fibre modification effect of N-nitrosodimethylamine on all levels: almost eliminated the inductive death of N-nitrosodimethylamine, and significantly reduced by N-nitrosodimethylamine inductive fibrotic total and tiny morphological change.Obviously, halofuginone prevents that the effect of the morphological change that produces from being strong in the pathological process of hepatic fibrosis, and is specific.

Embodiment 2

Halofuginone in rats'liver to the fibrotic effect of moderate

By measuring collagen α 1 (I) gene expression and hydroxyproline content, prove that halofuginone has stoped the inductive fibre modification of a small amount of N-nitrosodimethylamine in fact fully.Used concrete experimental technique is similar to Example 1, and just the rat of handling with N-nitrosodimethylamine only gives the saline solution of 0.25% N-nitrosodimethylamine, and is more much lower than the dosage of being given in the foregoing description 1.And the processing persistent period before rat is put to death is also longer: around with respect to three weeks among the embodiment 1 being.

Described in above-mentioned embodiment 1, measure collagen α 1 (I) gene expression.For the hydroxyproline analysis, the liver sample is used 6N HCl hydrolysis 22 hours down at 110 ℃.As (M.D.Krom, " chemist " 105:305-16,1980) as described in the Krom, after Kjeldahl digestion, utilize autoanalyzer to determine nitrogen by spectrophotography.(BTC2710 Biotronik) goes up through behind the post-column derivation, and (Biotronik LC5000 Germany) determines single collagen amino acid hydroxyproline in the same hydrolysis product by amino acid analysis at cation exchange column.The result is expressed as the collagen percent in the liver gross protein.

Hydroxyproline is quite a large amount of aminoacid that exist in a kind of collagen, so it can be used as the indicator of collagen total concentration in the particular organization.Therefore, as shown in Figure 2, N-nitrosodimethylamine causes clearly that hydroxyproline concentration significantly increases in the rats'liver, so collagen concentration also significantly increases.This increase is subjected to suppressing fully by handling with halofuginone.But, give the rat halofuginone and do not give N-nitrosodimethylamine and then do not make hydroxyproline concentration produce any variation.Therefore, the effect of halofuginone only is the increase that suppresses the inductive hydroxyproline concentration of N-nitrosodimethylamine.

Fig. 3 C shows that the N-nitrosodimethylamine of low dosage still makes collagen α 1 (I) gene expression increase like this, particularly to circumvascular cell.Fig. 3 D shows that halofuginone has been eliminated the gene expression of this increase.With the same in the foregoing description 1, proved independent halofuginone once more to not effect (Fig. 3 B) of collagen α 1 (I) gene expression, control rats does not have collagen α 1 (I) gene expression (Fig. 3 A) yet.

Therefore, halofuginone has clearly suppressed the rising of the synthetic level of the inductive collagen of N-nitrosodimethylamine in the rats'liver fully.But independent halofuginone does not show any such effect in rat, shows that halofuginone is specific for those pathophysiological processeses, as the synthetic inhibitory action of collagen that caused by the inductive fibre modification of N-nitrosodimethylamine.In addition, such as in embodiment 1 and 2 proof, halofuginone obviously can eliminate the biochemistry that caused by N-nitrosodimethylamine in fact fully and the physiology changes.

Embodiment 3

Utilize the inductive fibrotic inhibition of bile duct ligation

Except the inductive hepatic fibrosis of N-nitrosodimethylamine, also can obtain second kind of rats'liver fibre modification model.This model is to rely on the surgical ligation of bile duct to induce hepatic fibrosis, and do not need to give exogenous material or poisonous chemical substance, shown that this is a kind of model (Kountaras, J. etc. of suitable research people liver cirrhosis, " Britain's experimental pathology magazine " 65:305-311,1984; Muriel, P. etc., " hepatology magazine " 21:95-102,1994; Muriel, P. etc., " applied toxicology magazine " 15:449-453,1995).Therefore; the special usefulness of bile duct ligation model is that the treatment of any protectiveness must directly protect liver the inductive pathology variation of fibre modification not occur, rather than change is used to cause animal model the effect of the exogenous material of hepatic fibrosis to occur indirectly.Experimental technique is as follows.

The male Wistar rat of heavy 200-250g is divided into four experimental grouies, every group of three rats.First group is not carried out the bile duct ligation operation, does not give halofuginone yet.Second group is not carried out the bile duct ligation operation, but gives halofuginone.All animals in it should be noted that two groups all will experience false operation, the institute that comprises the actual operation process in steps, not ligation bile duct just.The 3rd group is carried out the bile duct ligation operation, but does not give halofuginone.The 4th group is carried out the bile duct ligation operation, and gives halofuginone.Similar substantially (Kountaras, J. etc., " Britain's experimental pathology magazine " 65:305-311,1984) reported in actual operation process and the document.

All animals arbitrarily give drinking water.The rat that gives halofuginone was promptly performed the operation back 3 days or 7 days during operation the last week and Therapy lasted, and adding concentration in the normal rat diet is the heavy halofuginone of 5mg/kg meal.When experiment finishes these rats are put to death.Described in above-mentioned embodiment 1, measure collagen content (by sharp red colouring of sunset) and collagen α 1 (I) gene expression.In addition, utilize the autoanalyzer system of Hitachi of Boerringher-Mannheim, colorimetric measurement serum alkaline phosphatase, alanine aminotransferase and aspartate transaminase concentration.The result is as follows.

It is synthetic not observe collagen in the rat of the false operation of experience.In addition, these rats do not show that body weight or liver heavily have any increase, or any liver histological changes.At last, no matter whether give halofuginone, these rats did not show all that enzyme concentrations such as alkali phosphatase, alanine aminotransferase and aspartate transaminase had any variation in 3 or 7 days after operation.

On the contrary, in the group of handling with halofuginone and not handling with halofuginone, the rat of experience bile duct ligation all observes these three kinds of enzyme concentrations and raises.The concentration of these risings is characteristic signs of the hardened pathological process of hepatic fibrosis regulating liver-QI.But these enzyme concentrations that give the rat of halofuginone are lower than the rat that does not give halofuginone.Specifically, the alanine aminotransferase concentration ratio that does not give the rat of halofuginone has given that the rat of halofuginone is high by 56%, and alkaline phosphatase concentration is high by 257%, and aspartate transaminase concentration is high by 15%.Therefore, obvious halofuginone has reduced the rising degree of enzyme concentration in the rat that experiences the bile duct ligation.

In addition, when experience bile duct ligation and when giving the rat of halofuginone and comparing with the rat that only experiences the bile duct ligation, halofuginone has significantly reduced the increase of synthetic and collagen α 1 (I) gene expression of the inductive collagen of bile duct ligation.Therefore, halofuginone obviously can suppress the process of hepatic fibrosis in the inductive rats'liver fibre modification of the bile duct ligation model.

Embodiment 4

The appropriate formulation that is used for the halofuginone administration

Halofuginone of the present invention and related compound, with and pharmaceutically acceptable salt, can give main body with many modes of knowing in this area.Hereinafter, term " main body " is meant people or the rudimentary animal that gives halofuginone.For example, can pass through oral administration, or parenteral, for example by intravenous drip or intraperitoneal, subcutaneous or intramuscular injection.

Be used for liquid preparations for oral administration and comprise powder agent or granule, in water or suspension in the non-hydrated matrix or solution, cachet, capsule or tablet.Thickening agent, diluent, flavoring agent, dispersing aid, emulsifying agent or binding agent also are desirable.

The preparation that is used for parenteral includes but not limited to aseptic aqueous solution, and it also can contain buffer, diluent and other suitable additives.

Dosage depends on the order of severity of symptom and the main body responding ability to one of halofuginone or other chemical compounds of the present invention and pharmaceutically acceptable salt thereof.Those of ordinary skills can easily determine optimal dose, medication and repetitive rate.

Embodiment 5

The hardened method of treatment hepatic fibrosis regulating liver-QI

As mentioned above, halofuginone has shown it is hepatic fibrosis, effective inhibitor of liver cirrhosis precursor.Following examples only are in order to illustrate that intention does not lie in restriction with one of halofuginone or other chemical compounds of the present invention and the hardened method of pharmaceutically acceptable salt treatment hepatic fibrosis regulating liver-QI thereof.

This method may further comprise the steps: give main body with one of described in the embodiment 4, in pharmaceutically acceptable carrier, halofuginone or other chemical compounds of the present invention and pharmaceutically acceptable salt thereof and treat.Halofuginone preferably up to reaching predesigned end point, no longer further develops as hepatic fibrosis in the main body or liver cirrhosis according to the administration of effective dose methodology, and hepatic fibrosis or liver cirrhosis are suppressed, or has prevented the formation of hepatic fibrosis or liver cirrhosis.

This treatment at the example of hepatic fibrosis type can include but not limited to effectively: by chronic alcoholism, nutritional disorder, hemochromatosis, passive hyperemia, hypercholesteremia, poison or toxin such as lead have been contacted, the contact medicine, immunoreation, the sensitivity to some material of heredity decision, as can in hepatolenticular degeneration, see to the copper sensitivity, and infectious disease such as viral hepatitis, syphilis and various parasitic infection include but not limited to the hepatic fibrosis that schistosomiasis mansoni and schistosomiasis japanica cause.In addition, such treatment hepatic fibrosis disease unknown for etiology or that imperfectly understand also is effective.

Particularly, the evidence of describing among the embodiment in front clearly illustrates, halofuginone and other chemical compounds of the present invention are suitable for treating the hepatopathy that the picked-up by the hepatotoxicity material causes.Even generally do not have when the material of hepatotoxicity exists with excessive concentrations and can cause hepatic injury, for example medicine yet.Because liver is the antidotal major organs by the metabolism of many different chemical materials, so the hepatopathy that the picked-up of hepatotoxicity material causes is not a rare phenomenon.The experiment of the inductive hepatic fibrosis model of N-nitrosodimethylamine has as described in example 1 above clearly illustrated the therapeutic efficacy of the present invention to this hepatopathy.

We do not want to limit with single mechanism the effect of The compounds of this invention, because hepatic fibrosis is to have prevented in fact with The compounds of this invention or the essential Fundamentals of the pathogenesis of cirrhosis improved, except treating with independent hepatic fibrosis is that all these methods also can be used for treating liver cirrhosis those diseases of feature.

Embodiment 6

The medicaments preparation method that contains halofuginone

It below is the example of the preparation method of one of halofuginone or other chemical compounds of the present invention and pharmaceutically acceptable salt thereof.What describe as an example is the preparation of halofuginone, comprises other chemical compounds of the present invention and pharmaceutically acceptable salt thereof but should understand this description, and the preparation method of the pharmaceutically acceptable salt of halofuginone self.At first, prepare the synthetic halofuginone of convention according to excellent drug.United States Patent (USP) 3,338 has provided the example of the method for synthetic halofuginone and relevant Quinazol derivative in 909.Secondly, as described in example 4 above, prepare convention according to excellent drug once more, halofuginone is placed suitable pharmaceutical carrier.

The intention that should be understood that above-mentioned description only is as embodiment also have many other embodiments within the spirit and scope of the present invention.

Claims (14)

1, a kind of compositions that is used for the treatment of the main body liver cirrhosis, said composition comprise chemical compound and the pharmaceutically acceptable salt thereof that the following formula of pharmacy effective dose is represented:

Wherein:

R ₁Be hydrogen, halogen, nitro, benzo, low alkyl group, phenyl or lower alkoxy;

R ₂Be hydroxyl, acetoxyl group or lower alkoxy; With

R ₃Be hydrogen or rudimentary alkenyloxy-carbonyl.

2, compositions as claimed in claim 1, wherein said chemical compound is a halofuginone.

3, compositions as claimed in claim 1, it further comprises pharmaceutically acceptable carrier.

4, compositions as claimed in claim 1, wherein liver cirrhosis is caused by the hepatotoxicity material.

5, a kind of compositions that is used for the treatment of the main body hepatic fibrosis, it comprises chemical compound and the pharmaceutically acceptable salt thereof that the following formula of pharmacy effective dose is represented:

Wherein:

R ₁Be hydrogen, halogen, nitro, benzo, low alkyl group, phenyl or lower alkoxy;

R ₂Be hydroxyl, acetoxyl group or lower alkoxy; With

R ₃Be hydrogen or rudimentary alkenyloxy-carbonyl.

6, compositions as claimed in claim 5, wherein said chemical compound is a halofuginone.

7, compositions as claimed in claim 6, it further comprises pharmaceutically acceptable carrier.

8, compositions as claimed in claim 7, wherein hepatic fibrosis is caused by the hepatotoxicity material.

9, be used for the treatment of the medicaments preparation method of main body liver cirrhosis, it comprises the step that the chemical compound of pharmacy effective dose is placed pharmaceutically acceptable carrier, and described chemical compound is a member and the pharmaceutically acceptable salt thereof in the chemical compound represented of following formula:

Wherein: R ₁Be hydrogen, halogen, nitro, benzo, low alkyl group, phenyl or lower alkoxy; R ₂Be hydroxyl, acetoxyl group or lower alkoxy; And R ₃Be hydrogen or rudimentary alkenyloxy-carbonyl.

10, method as claimed in claim 9, wherein said chemical compound is a halofuginone.

11, method as claimed in claim 9, wherein liver cirrhosis is caused by the hepatotoxicity material.

12, be used for the treatment of the medicaments preparation method of main body hepatic fibrosis, this method comprises the step that the chemical compound of pharmacy effective dose is placed pharmaceutically acceptable carrier, and described chemical compound is a member in the chemical compound represented of following formula:

Wherein:

R ₁Be hydrogen, halogen, nitro, benzo, low alkyl group, phenyl or lower alkoxy;

R ₂Be hydroxyl, acetoxyl group or lower alkoxy; With

R ₃Be hydrogen or rudimentary alkenyloxy-carbonyl; And pharmaceutically acceptable salt.

13, method as claimed in claim 12, wherein said chemical compound is a halofuginone.

14, method as claimed in claim 12, wherein hepatic fibrosis is caused by the hepatotoxicity material.

Images