Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to the treatment of hepatic cirrhosis and, in particular, to the treatment of hepatic cirrhosis with quinazolinone derivatives such as Halofuginone.
• Hepatic cirrhosis has a number of causes, including hepatic fibrosis caused by chronic alcoholism, malnutrition, hemochromatosis, passive congestion, hypercholesterolemia, exposure to poisons or toxins such as lead, exposure to drugs, immune reactions, genetically determined sensitivities to certain substances as seen with copper in Wilson's disease and infections such as viral hepatitis, syphilis and various parasitic infections including, but not limited to,
• liver function The pathogenesis of hepatic cirrhosis progresses in a number of stages. First, an enlarged liver is seen with various fatty changes. Next, overt fibrosis is evident with a concomitant decrease in liver function. Finally, atrophy of the liver begins, with a corresponding reduction in the size and functionality of the liver. Necrosis of the liver can be seen at any stage, but is particularly pronounced by late stage cirrhosis. Microscopically, by late stage cirrhosis a complete disruption of the normal architecture of the liver is evident.
• cirrhosis is an entire pathological process with effects that are not limited to the liver, although the root causes can be found in specific pathological changes to the liver itself.
• Hepatic fibrosis is a feature of most chronic liver diseases, not just cirrhosis [S.L. Friedman, New Eng. J. Med., 328:1828-35, 1993].
• connective tissue accumulates in the liver, replacing normal hepatic parenchymal tissue, and reducing liver functionality.
• the fibrotic tissue replaces more complex normal liver tissue in a pathological process which reduces the amount of liver tissue available for normal functions, such as the removal of toxic substances from the blood, and which progressively disrupts intrahepatic blood flow.
• fibrotic tissue in the liver is characterized by the deposition of abnormally large amounts of extracellular matrix components, including at least five types of collagen, in particular collagen types I, III, and IV, as well as other matrix proteins [L. Ala-Kokko, Biochem. J, 244:75-9, 1987].
• Such drugs can act by modulating the synthesis of the procollagen polypeptide chains, or by inhibiting specific post-translational events, which will lead either to reduced formation of extra-cellular collagen fibers or to an accumulation of fibers with altered properties.
• cytotoxic drugs have been used in an attempt to slow the proliferation of collagen-producing fibroblasts [J.A. Casas, et al, Ann. Rhem. Dis., 46: 763, 1987], such as colchicine, which slows collagen secretion into the extracellular matrix [D. Kershenobich, et al., N. Engl. J. Med., 318: 1709, 1988], as well as inhibitors of key collagen metabolism enzymes [K. Karvonen, et al., J. Biol Chem., 265: 8414, 1990; C.J. Cunliffe, et al., J. Med. Chem., 35:2652, 1992].
• Collagen cross-linking inhibitors such as ⁇ -amino- propionitrile, are also non-specific, although they can serve as useful anti-fibrotic agents. Their prolonged use causes lathritic syndrome and interferes with elastogenesis, since elastin, another fibrous connective tissue protein, is also cross-linked. In addition, the collagen cross-linking inhibitory effect is secondary, and collagen overproduction has to precede its degradation by collagenase. Thus, a type-specific inhibitor of the synthesis of collagen itself is clearly required as an anti-fibrotic agent.
• Such a type-specific collagen synthesis inhibitor is disclosed in U.S. Patent No. 5,449,678 for the treatment of a fibrotic condition.
• This specific inhibitor is a composition with a pharmaceutically effective amount of a pharmaceutically active compound of a formula:
• Ri is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy;
• R2 is a member of the group consisting of hydroxy, acetoxy and lower alkoxy;
• R- is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl.
• Pharmaceutically acceptable salts thereof are also included.
• Halofuginone has been found to be particularly effective for such treatment.
• U.S. Patent No. 5,449,678 discloses that these compounds are effective in the treatment of fibrotic conditions such as scleroderma and GVHD.
• PCT Application No. WO 96/06616 further discloses that these compounds are effective in treating restenosis.
• the two former conditions are associated with excessive collagen deposition, which can be inhibited by Halofuginone.
• Restenosis is characterized by smooth muscle cell proliferation and extracellular matrix accumulation within the lumen of affected blood vessels in response to a vascular injury [Choi et al, Arch. Surg., 130:257-261, 1995].
• smooth muscle cell proliferation is a phenotypic alteration, from the normal contractile phenotype to a synthetic one.
• Type I collagen has been shown to support such a phenotypic alteration, which can be blocked by Halofuginone [Choi et al, Arch. Surg., 130: 257-261, 1995; U.S. Patent No. 5,449,678].
• Halofuginone inhibits the synthesis of collagen type I in bone chrondrocytes in vitro, as demonstrated in U.S. Patent No. 5,449,678.
• chickens treated with Halofuginone were not reported to have an increased rate of bone breakage, indicating that the effect is not seen in vivo.
• the exact behavior of Halofuginone in vivo cannot always be accurately predicted from in vitro studies.
• Halofuginone or other related quinazolinone to block or inhibit pathological processes related to hepatic cirrhosis.
• Other inhibitors of collagen synthesis, cross-linking and deposition such as corticosteroids, penicillamine, methotrexate and colchicine, have been tested for their therapeutic effect on hepatic fibrosis, but have not proved effective [S.L. Friedman, New Eng. J. Med., 328:1828-35, 1993].
• Halofuginone has been shown to have a specific inhibitory effect on the synthesis of type I collagen, such inhibition has not been otherwise shown to be efficacious in the treatment of hepatic cirrhosis.
• hepatic cirrhosis has a high mortality rate, as currently available therapeutic options have significant side effects and are not generally efficacious in slowing or halting the progression of the fibrosis.
• many other types of extracellular matrix components are deposited during the pathogenesis of hepatic fibrosis, including at least five types of collagen, in particular collagen types I, III, and IV, as well as other matrix proteins [L. Ala-Kokko, Biochem. J., 244:75-9, 1987].
• collagen types I, III, and IV as well as other matrix proteins
• liver cirrhosis and fibrosis which inhibits fibrogenesis in vivo substantially without undesirable non-specific or toxic side effects.
• Halofuginone can also inhibit the pathophysiological process of hepatic fibrosis in vivo, possibly by inhibiting collagen type I synthesis, although another mechanism or mechanisms could also be responsible. While inhibition of collagen type I synthesis is proposed as a plausible mechanism, it is not desired to be limited to a single mechanism, nor is it necessary since the in vivo data presented below clearly demonstrate the efficacy of Halofuginone as an inhibitor of hepatic fibrosis in vivo.
• composition for treating hepatic cirrhosis including a pharmaceutically effective amount of a compound in combination with a pharmaceutically acceptable carrier, the compound being a member of a group having a formula:
• Ri is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy; R2 is a member of the group consisting of hydroxy, acetoxy, and lower alkoxy; and R is a member of the group consisting of hydrogen and lower alkenoxy.
• Pharmaceutically acceptable salts thereof are also included.
• the compound is preferably Halofuginone.
• Halofuginone is defined as a compound having a formula:
• composition preferably includes a pharmaceutically acceptable carrier for the compound.
• a method of manufacturing a medicament for treating hepatic cirrhosis including the step of placing a pharmaceutically effective amount of a compound in a pharmaceutically acceptable carrier, the compound being a member of a group having a formula:
• Ri is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy;
• R2 is a member of the group consisting of hydroxy, acetoxy, and lower alkoxy, and RT is a member of the group consisting of hydrogen and lower alkenoxy- carbonyl.
• Pharmaceutically acceptable salts thereof are also included.
• a method for the treatment of hepatic cirrhosis in a subject including the step of administering a pharmaceutically effective amount of a compound having a formula:
• Ri is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy
• R2 is a member of the group consisting of hydroxy, acetoxy and lower alkoxy
• RT is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl.
• composition for substantially preventing the genesis of hepatic cirrhosis including a pharmaceutically effective amount of a compound in combination with a pharmaceutically acceptable carrier, the compound being a member of a group having a formula:
• Ri is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy;
• R2 is a member of the group consisting of hydroxy, acetoxy, and lower alkoxy; and
• R- is a member of the group consisting of hydrogen and lower alkenoxy.
• Pharmaceutically acceptable salts thereof are also included.
• a method of manufacturing a medicament for substantially preventing the genesis of hepatic cirrhosis including the step of placing a pharmaceutically effective amount of a compound in a pharmaceutically acceptable carrier, the compound being a member of a group having a formula:
• Ri is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy;
• R2 is a member of the group consisting of hydroxy, acetoxy, and lower alkoxy; and
• R3 is a member of the group consisting of hydrogen and lower alkenoxy- carbonyl.
• Pharmaceutically acceptable salts thereof are also included. According to still other embodiments of the present invention, there is provided a method for substantially preventing the genesis of hepatic cirrhosis in a subject, including the step of administering a pharmaceutically effective amount of a compound having a formula:
• Ri is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy;
• R2 is a member of the group consisting of hydroxy, acetoxy and lower alkoxy;
• R- is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl. Pharmaceutically acceptable salts thereof are also included.
• composition for treating hepatic fibrosis including a pharmaceutically effective amount of a compound in combination with a pharmaceutically acceptable carrier, the compound being a member of a group having a formula:
• Ri is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy
• R2 is a member of the group consisting of hydroxy, acetoxy, and lower alkoxy
• Ro is a member of the group consisting of hydrogen and lower alkenoxy.
• Pharmaceutically acceptable salts thereof are also included.
• a method of manufacturing a medicament for treating hepatic fibrosis including the step of placing a pharmaceutically effective amount of a compound in a pharmaceutically acceptable carrier, the compound being a member of a group having a formula:
• R is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy;
• R2 is a member of the group consisting of hydroxy, acetoxy, and lower alkoxy; and
• R-> is a member of the group consisting of hydrogen and lower alkenoxy- carbonyl.
• Pharmaceutically acceptable salts thereof are also included.
• hepatic fibrosis in a subject, including the step of administering a pharmaceutically effective amount of a compound having a formula:
• Ri is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy;
• R2 is a member of the group consisting of hydroxy, acetoxy and lower alkoxy, and R3 is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl.
• Pharmaceutically acceptable salts thereof are also included.
• composition for substantially preventing the genesis of hepatic fibrosis including a pharmaceutically effective amount of a compound in combination with a pharmaceutically acceptable carrier, the compound being a member of a group having a formula:
• Ri is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy;
• R2 is a member of the group consisting of hydroxy, acetoxy, and lower alkoxy, and R-, is a member of the group consisting of hydrogen and lower alkenoxy.
• Pharmaceutically acceptable salts thereof are also included.
• a method of manufacturing a medicament for substantially preventing the genesis of hepatic fibrosis including the step of placing a pharmaceutically effective amount of a compound in a pharmaceutically acceptable carrier, the compound being a member of a group having a formula:
• Ri is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy
• R2 is a member of the group consisting of hydroxy, acetoxy, and lower alkoxy
• R- is a member of the group consisting of hydrogen and lower alkenoxy- carbonyl.
• Pharmaceutically acceptable salts thereof are also included.
• a method for substantially preventing the genesis of hepatic fibrosis in a subject including the step of administering a pharmaceutically effective amount of a compound having a formula:
• Ri is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy;
• R2 is a member of the group consisting of hydroxy, acetoxy and lower alkoxy, and RT is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl.
• Pharmaceutically acceptable salts thereof are also included.
• all of the compounds referred to hereinabove can be either the compound itself as described by the formula, and/or pharmaceutically acceptable salts thereof.
• the term "subject” refers to the human or lower animal to whom Halofuginone was administered.
• the term "patient” refers to human subjects.
• treatment includes both substantially preventing the genesis of hepatic cirrhosis or fibrosis, as well as slowing or halting the progression of hepatic cirrhosis or fibrosis once it has arisen.
• substantially preventing the genesis" of hepatic cirrhosis or fibrosis is understood to refer to the prevention of the appearance of clinical or preclinical symptoms of these conditions, including the prevention of those symptoms which are indirectly related to the fibrotic and cirrhotic processes themselves, such as hemorrhage from esophageal blood vessels.
• Ri is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy;
• R2 is a member of the group consisting of hydroxy, acetoxy and lower alkoxy, and R is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl.
• Pharmaceutically acceptable salts thereof are also included.
• FIGS. 1A-1D illustrate the effect of Halofuginone on collagen ⁇ l(I) gene expression in rat liver
• FIG. 2 illustrates the effect of Halofuginone on hydroxyproline concentration in rat liver
• FIGS. 3A-3D illustrate the effect of Halofuginone on moderate fibrosis in rat liver.
• Halofuginone can inhibit the pathological process of hepatic cirrhosis in vivo, possibly by inhibiting collagen type I synthesis, although another mechanism or mechanisms could also be responsible. Indeed, irrespective of the specific mechanism, the data presented below clearly demonstrate the efficacy of Halofuginone in vivo for inhibition of the pathological progression of hepatic fibrosis. Such a finding is unexpected for a number of reasons. First, the behavior of Halofuginone in vitro does not exactly correspond to its behavior in vivo. This can be demonstrated by the differential effect of Halofuginone observed with bone chondrocytes in vivo and in vitro.
• Halofuginone inhibits the synthesis of collagen type I in chrondrocytes in vitro, as demonstrated in U.S. Patent No. 5,449,678.
• chickens treated with Halofuginone were not reported to have an increased rate of bone breakage, indicating that the effect is not seen in vivo.
• the exact behavior of Halofuginone in vivo cannot always be accurately predicted from in vitro studies.
• Halofuginone has only been shown to be a collagen type I inhibitor.
• the formation of fibrotic tissue in the liver is characterized by the deposition of abnormally large amounts of extracellular matrix components, including at least five types of collagen, in particular collagen types I, III, and IV, as well as other matrix proteins [L. Ala-Kokko, Biochem. J, 244:75-9, 1987].
• the ability of Halofuginone to inhibit collagen type I synthesis and deposition cannot predict the ability of Halofuginone to slow, reduce or other ameliorate the pathogenesis of hepatic fibrosis.
• Halofuginone has not been taught as a suitable prophylactic treatment to prevent such complex pathophysiological processes as hepatic fibrosis and cirrhosis in mammals such as humans.
• U.S. Patent No. 3,320,124 only teaches the use of compounds related to Halofuginone for the prevention of the infectious disease coccidiosis in chickens.
• Chickens are physiologically very different from any mammal including humans. Indeed, chickens are not generally considered to be acceptable experimental models for mammals, and are certainly not used as experimental models for hepatic diseases and conditions such as hepatic fibrosis and cirrhosis.
• the prophylactic treatment of a human or other mammal with the compounds of the present invention for the prevention of hepatic fibrosis of cirrhosis is not taught by this reference, or indeed by other references in the background art.
• Hepatic cirrhosis is not merely a condition which is related to hepatic fibrosis.
• the pathogenesis of hepatic cirrhosis progresses in a number of stages, which can potentially lead to end-stage hepatic failure and death. All of these stages, from the first presentation of fatty changes in the liver to late-stage liver necrosis, are important for the genesis of hepatic cirrhosis. Outside of the liver, other pathological changes become evident as cirrhosis progresses.
• cirrhosis is an entire pathological process with effects that are not limited to the liver, although the root causes can be found in specific pathological changes to the liver itself. In order to inhibit such a pathological process, or to prevent the genesis of the process, clearly a successful treatment must be able to intervene to slow or prevent the occurrence of the entire process.
• Halofuginone can be such a successful treatment, as compared to the many substances which should, theoretically, have been appropriate, yet which failed in an in vivo test.
• the finding that Halofuginone is a successful treatment for slowing and/or preventing the occurrence of the constellation of symptoms which arise during the pathological process of hepatic cirrhosis is clearly both novel and non-obvious, as well as showing a clear inventive step.
• all other prior art references have only taught the efficacy of Halofuginone on cells such as fibroblasts and smooth muscle cells.
• Halofuginone would be useful in the treatment of hepatic fibrosis in vivo.
• the ability of Halofuginone, and related compounds, to slow or halt progression of fibrosis in the liver is both novel and non-obvious, as well as showing a clear inventive step.
• the demonstration of such an ability for in vivo treatment of a mammal is particularly unexpected, given the differential responses seen in vitro and in vivo to Halofuginone.
• the present invention may be more readily understood with reference to the following illustrative examples and figures. It should be noted that although reference is made exclusively to Halofuginone, it is believed that the other quinazolinone derivatives described and claimed in U.S. Patent 3,320,124, the teachings of which are incorporated herein by reference, have similar properties.
• the present invention is of a treatment for hepatic cirrhosis with quinazolinone- containing compounds such as Halofuginone. Compositions with specific pharmaceutical formulations, and methods of using and manufacturing these compounds are described below. Although the pathogenesis of hepatic cirrhosis is not fully understood, suitable animal models for the disease have been successfully developed.
• Hepatic fibrosis has been induced in rats by the intraperitoneal injection of dimethylnitrosamine, with a relatively short onset of action: within three weeks of administration of dimethylnitrosamine to rats, hepatic fibrosis was already evident [A. M. Jezequel et al, J. Hepatol, 5:174-81, 1987].
• Dimethylnitrosamine-induced hepatic fibrosis is characterized by increased deposition of extracellular matrix components, including various types of collagen such as collagen type I.
• inhibition of fibrosis depends upon the slowing or halting of the pathological process leading to the production of fibrotic tissue.
• Liver samples were then hybridized with a probe for rat collagen ⁇ l(I) expression.
• the sections were deparaffinized in xylene, rehydrated through a graded series of ethanol solutions, rinsed in distilled water for 5 minutes and then incubated in 2X SSC at 70 °C for 30 minutes.
• the sections were then rinsed in distilled water and treated with pronase, 0.125 mg/ml in 50 mM Tris-HCl, 5 mM EDTA, pH 7.5, for 10 minutes. After digestion, the slides were rinsed with distilled water, post-fixed in 10% formalin in PBS and blocked in 0.2% glycine.
• the slides were rinsed in distilled water, rapidly dehydrated through graded ethanol solutions and air-dried for several hours.
• the 1600 bp rat collagen ⁇ l(I) insert was cut out from the original plasmid, pUC18, and inserted into the pSafyre plasmid. The sections were then hybridized with this probe after digoxigenin-labeling [M. Pines et al, Matrix Biology, 14:765-71, 1996].
• Figure 1 shows in situ hybridization of a section of rat liver tissue with rat collagen l(I) probe.
• a low expression of collagen l(I) gene is seen in liver of control rats ( Figure 1A) or rats given Halofuginone alone ( Figure IB).
• Figure IB A marked increase in the expression of collagen ⁇ l(I) gene was seen in the liver of rats given dimethylnitrosamine alone ( Figure 1C).
• the gene expression was mainly in the septa surrounding the lobules at the site of sparse collagenous tissue.
• Rats given both Halofuginone and dimethylnitrosamine show a marked reduction in the expression of collagen ⁇ l(I) gene ( Figure ID), as compared to rats given dimethylnitrosamine alone.
• Halofuginone is effective against the pathological induction of expression by dimethylnitrosamine.
• Sections of rat liver tissue were stained with Sirius red to demonstrate collagen content of the tissue, although results are not shown pictorially since the histological samples must be viewed in color in order to see the effects. Almost no collagen fibers were observed in liver tissue taken from control rats or rats given Halofuginone alone.
• the livers of the dimethylnitrosamine-treated rats exhibited an increase in collagen content, displaying bundles of collagen surrounding the lobules, resulting in large fibrous septa. The thickening of these collagen bundles was markedly reduced in rats given both dimethylnitrosamine and Halofuginone, again indicating the ability of Halofuginone to substantially inhibit the pathophysiological process of fibrosis induced by dimethylnitrosamine.
• Halofuginone was able to prevent the appearance of the effects of dimethylnitrosamine-induced fibrosis on all levels: near-elimination of dimethylnitrosamine- induced fatalities, and marked reduction of gross and fine morphological changes caused by dimethylnitrosamine-induced fibrosis.
• the effects of Halofuginone are both potent and specific for the prevention of the morphological changes produced during the pathological process of hepatic fibrosis.
• Example 2 Effect of Halofuginone on Mild Fibrosis in Rat Liver Halofuginone substantially completely prevented mild dimethylnitrosamine-induced fibrosis, as demonstrated by the measurement of collagen ⁇ l(I) gene expression and hydroxyproline content.
• Example 1 The specific experimental method used was similar to that of Example 1, except that the dimethylnitrosamine-treated rats were only given 0.25% dimethylnitrosamine in saline, a much lower dose than that given in Example 1 above. Also, the duration of treatment was longer before the rats were sacrificed: 4 weeks as opposed to 3 weeks in Example 1.
• the expression of the collagen ⁇ l(I) gene was measured as described in Example 1 above.
• liver samples were hydrolyzed for 22 hours at 110 °C with 6 N HC1. Nitrogen was determined after Kjeldahl digestion by the spectrophotometric procedure using an autoanalyzer as described by Krom [M.D. Krom, Analyst, 105:305-16, 1980].
• the collagen-unique amino acid hydroxyproline from the same hydro lysate was determined by amino acid analysis (Biotronik LC 5000, Germany) after post-column derivatization on a cation exchange column (BTC 2710, Biotronik). The results are expressed as the percentage of collagen in total liver proteins.
• Hydroxyproline is an amino acid which is present in relatively large amounts in collagen, and therefore serves as an indicator for the overall level of collagen in a particular tissue.
• dimethylnitrosamine clearly caused a significant increase in hydroxyproline concentration, and therefore of collagen levels, in the livers of rats. This increase was completely inhibited by treatment with Halofuginone.
• administration of Halofuginone to rats which were not given dimethylnitrosamine did not cause any change in hydroxyproline concentration. Therefore, the effect of Halofuginone was simply to inhibit the dimethylnitrosamine-induced increase in hydroxyproline concentration.
• Figure 3C demonstrates that such a low dose of dimethylnitrosamine still caused an increase in collagen ⁇ l(I) gene expression, especially by cells surrounding the blood vessels.
• Figure 3D shows that this increased gene expression was abolished by Halofuginone. Again, as in Example 1 above, Halofuginone alone had no effect on collagen ⁇ l(I) gene expression (Figure 3B), while control rats also had no collagen ⁇ l(I) gene expression ( Figure 3 A).
• Halofuginone completely inhibited the increased levels of collagen synthesis induced by dimethylnitrosamine in the livers of rats.
• Halofuginone alone did not demonstrate any such effect in rats, indicating that the effect of Halofuginone is specific for inhibition of those pathophysiological processes, such as collagen synthesis, which are caused by dimethylnitrosamine-induced fibrosis.
• Halofuginone was clearly able to substantially completely abrogate the biochemical and physiological changes caused by dimethylnitrosamine, as demonstrated by both Examples 1 and 2.
• liver fibrosis In addition to dimethylnitrosamine-induced liver fibrosis, a second model of liver fibrosis in rats is available. This model relies upon surgical ligation of the bile duct to induce liver fibrosis, rather than requiring the administration of exogenous substances or toxic chemicals, and has been shown to be a suitable model for studying human liver cirrhosis [Kountaras, J. et al, Br. J. Exp. Pathol, 65:305-311, 1984; Muriel, P. et al, J. Hepatol, 21:95-102, 1994; Muriel P. et al, J. Appl. Tox., 15:449-453, 1995].
• the particular advantage of the bile duct ligation model is that any protective treatments must directly protect the liver from the pathological changes induced by fibrosis, rather than indirectly altering the effects of the exogenous substance which is used to cause liver fibrosis in the animal model.
• the experimental method was as follows. Male Wistar rats, weighing 200-250 g, were divided into four experimental groups with 3 rats in each group. The first group did not have bile duct ligation surgery and was not given Halofuginone. The second group did not have bile duct ligation surgery and was given Halofuginone. It should be noted that all animals in the first two groups underwent sham operations which included all steps of the actual surgical procedure, with the exception of the bile duct ligation itself. The third group had bile duct ligation surgery and was not given
• Halofuginone The fourth group had bile duct ligation surgery and was given Halofuginone. The actual surgical procedure was essentially similar to that reported in the literature [Kountaras, J. et al, Br. J. Exp. Pathol, 65:305-311, 1984].
• Halofuginone significantly reduced the bile duct ligation-induced increased in collagen synthesis and collagen ⁇ l(I) gene expression, when rats which underwent bile duct ligation and which were fed Halofuginone were compared to rats which only underwent bile duct ligation.
• Halofuginone clearly was able to inhibit the process of liver fibrosis in the model of bile duct ligation-induced fibrosis in rats.
• Halofuginone and related compounds of the present invention can be administered to a subject in a number of ways, which are well known in the art.
• the term "subject" refers to the human or lower animal to whom Halofuginone was administered.
• administration may be done orally, or parenterally, for example by intravenous drip or intraperitoneal, subcutaneous, or intramuscular injection.
• Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, sachets, capsules or tablets. Thickeners, diluents, flavorings, dispersing aids, emulsifiers or binders may be desirable.
• Formulations for parenteral administration may include but are not limited to sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
• Dosing is dependent on the severity of the symptoms and on the responsiveness of the subject to Halofuginone or one of the other compounds of the present invention and pharmaceutically acceptable salts thereof. Persons of ordinary skill in the art can easily determine optimum dosages, dosing methodologies and repetition rates.
• Halofuginone has been shown to be an effective inhibitor of hepatic fibrosis, a precursor of hepatic cirrhosis.
• the following example is an illustration only of a method of treating hepatic fibrosis and cirrhosis with Halofuginone or one of the other compounds of the present invention and pharmaceutically acceptable salts thereof, and is not intended to be limiting.
• the method includes the step of administering Halofuginone or one of the other compounds of the present invention and pharmaceutically acceptable salts thereof, in a pharmaceutically acceptable carrier as described in Example 4 above, to a subject to be treated.
• Halofuginone is administered according to an effective dosing methodology, preferably until a predefined endpoint is reached, such as the absence of further progression of hepatic fibrosis or cirrhosis in the subject, the inhibition of hepatic fibrosis or cirrhosis or the prevention of the formation of hepatic fibrosis or cirrhosis.
• hepatic fibrosis examples include, but are not limited to, hepatic fibrosis caused by chronic alcoholism, malnutrition, hemochromatosis, passive congestion, hypercholesterolemia, exposure to poisons or toxins such as lead, exposure to drugs, immune reactions, genetically determined sensitivities to certain substances as seen with copper in Wilson's disease and infections such as viral hepatitis, syphilis and various parasitic infections including, but not limited to, Schistosomiasis mansoni and S. japonica.
• such a treatment would also be effective for hepatic fibrotic conditions of unknown or poorly defined etiology.
• Halofuginone and other compounds of the present invention are suitable for the treatment of hepatic disease which is caused by the ingestion of hepatotoxic substances. Even substances which are not normally hepatotoxic may cause liver damage when present in excessive concentrations, as for example drugs. Since the liver is the main organ for detoxification by metabolism of many different chemicals, hepatic disease caused by ingestion of a hepatotoxic substance is not a rare phenomenon.
• the efficacy of the present invention for the treatment of such hepatic disease is clearly shown by experiments with the dimethylnitrosamine-induced model of hepatic fibrosis, as described in Example 1 above.
• liver cirrhosis is a necessary underlying factor for the pathogenesis of liver cirrhosis which is substantially prevented or ameliorated by the compounds of the present invention
• all of these methods can also be used to treat liver cirrhosis, in addition to treating those conditions characterized by liver fibrosis alone.
• Example 6 Method of Manufacture of a Medicament Containing Halofuginone
• a method of manufacturing Halofuginone or one of the other compounds of the present invention and pharmaceutically acceptable salts thereof As an example, manufacture of Halofuginone is described, it being understood that this description encompasses methods of manufacture of the other compounds of the present invention and pharmaceutically acceptable salts thereof, as well as of pharmaceutically acceptable salts of Halofuginone itself.
• Halofuginone is synthesized in accordance with good pharmaceutical manufacturing practice. Examples of methods of synthesizing Halofuginone, and related quinazolinone derivatives, are given in U.S. Patent No. 3,338,909.

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• 1998
  • 1998-05-22 JP JP55068298A patent/JP2002515905A/ja not_active Ceased
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  • 1998-05-22 AU AU76922/98A patent/AU748754B2/en not_active Ceased
  • 1998-05-22 CN CNB988053896A patent/CN1160073C/zh not_active Expired - Fee Related
  • 1998-05-22 EP EP98924847A patent/EP1014988A2/de not_active Withdrawn
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  • 1998-05-22 KR KR1019997010803A patent/KR100540537B1/ko not_active IP Right Cessation
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