AU737094B2 - Treatment for pulmonary fibrosis - Google Patents

Description

WO 98/43642 PCT/IL97/00115 TREATMENT FOR PULMONARY FIBROSIS FIELD AND BACKGROUND OF THE INVENTION The present invention relates to the treatment of pulmonary fibrosis and, in particular, to the treatment of pulmonary fibrosis with quinazolinone derivatives such as Halofuginone.

Pulmonary fibrosis is a chronic and incurable disease in which interstitial connective tissue accumulates in the lungs, reducing lung functionality and efficiency of gas exchange Phan, New Strategiesfor Treatment of Pulmonary Fibrosis, 50:415-421, 1995]. The fibrotic tissue replaces more complex pulmonary tissue in a pathological process which progressively reduces the surface area for gas exchange in the lungs.

Pulmonary fibrosis often follows such therapeutic interventions as bone marrow transplantation, radiotherapy and chemotherapy [Nagler, A. et al., Am. J. Respir. Crit.

Care Med., 154:1082-1086, 1996]. For reasons given in greater detail below, the disease is frequently fatal.

The pathogenesis of pulmonary fibrosis includes the formation of fibrotic tissue in the lungs. The formation of fibrotic tissue is characterized by the deposition of abnormally large amounts of collagen. The synthesis of collagen is also involved in a number of other pathological conditions. For example, clinical conditions and disorders associated with primary or secondary fibrosis, such as systemic sclerosis, graft-versushost disease (GVHD), pulmonary and hepatic fibrosis and a large variety of autoimmune disorders, are distinguished by excessive production of connective tissue, which results in the destruction of normal tissue architecture and function. These diseases can best be interpreted in terms of perturbations in cellular functions, a major manifestation of which is excessive collagen synthesis and deposition. The crucial role of collagen in fibrosis has prompted attempts to develop drugs that inhibit its accumulation Kivirikko, Annals ofMedicine, Vol. 25, pp. 113-126 (1993)].

WO 98/43642 PCT/IL97/00115 2 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. Unfortunately, only a few inhibitors of collagen synthesis are available, despite the importance of this protein in sustaining tissue integrity and its involvement in various disorders.

For example, cytotoxic drugs have been used in an attempt to slow the proliferation of collagen-producing fibroblasts Casas, et al., Ann. Rhem. Dis., 46: 763, 1987], such as colchicine, which slows collagen secretion into the extracellular matrix Kershenobich, et al., N. Engl. J Med., 318:1709, 1988], as well as inhibitors of key collagen metabolism enzymes 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 collagen-type specific. Also. there are serious concerns about the toxic consequences of interfering with biosynthesis of other vital collagenous molecules, such as Clq in the classical complement pathway, acetylcholine esterase of the neuro-muscular junction endplate, conglutinin and pulmonary surfactant apoprotein.

Other drugs which can inhibit collagen synthesis, such as nifedipine and phenytoin, inhibit synthesis of other proteins as well, thereby non-specifically blocking the collagen biosynthetic pathway Salo, et al.. J. Oral Pathol. Med., 19: 404 ,1990].

Collagen cross-linking inhibitors, such as P-amino- propionitrile, are also nonspecific, 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 3 composition with a pharmaceutically effective amount of a pharmaceutically active compound of a formula:

I

aC u o 83 wherein: n 1 or 2; R, is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy; R, is a member of the group consisting of hydroxy, acetoxy and lower alkoxy, and

R

3 is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl; as well as pharmaceutically acceptable salts thereof. Of this group of compounds, Halofuginone 8 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. WO Application No.

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 .:26 i 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].

One hallmark of such smooth muscle cell proliferation is a phenorypic alteration, from the normal contractile phenorype 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].

However, the in vitro action of Halofuginone does not always predict its in vivo effects. For example, Halofuginone inhibits the synthesis of collagen type I in bone chrondrocytes in vitro, as demonstrated in U.S. Patent No. 5,449,678. However, chickens eef with Halofuinone were not reported to have an increased rate of bone breakage, 7Zi OFi' WO 98/43642 PCT/IL97/00115 4 indicating that the effect is not seen in vivo. Thus, the exact behavior of Halofuginone in vivo cannot always be accurately predicted from in vitro studies.

Furthermore, the ability of Halofuginone or other related quinazolinone to block or inhibit pathological processes related to pulmonary fibrosis has only been shown in one reference [Nagler, A. et al., Am. J Respir. Crit. Care Med., 154:1082-1086, 1996], but is not otherwise known in the prior art. Although 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 pulmonary fibrosis. Indeed, pulmonary fibrosis 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 [Nagler, A. et al., Am. J. Respir. Crit. Care Med., 154:1082- 1086, 1996]. For example, steroids, penicillamine, colchicine, alkylating agents, nonsteroidal anti-inflammatory preparations, anti-oxidants and immunosuppressants have all been shown to be largely ineffective for slowing or halting progression of the disease.

Other medications, such as protease inhibitors and cell adhesion factors, were largely non-specific in their effects, with potentially dangerous side-effects. As described above, various inhibitors of the process of collagen production have been tried against pulmonary fibrosis, but have had non-specific effects H. Phan in Lung Cell Biology, D. Massaro, ed., Marcel Dekker, New York, 1989, p. 907-979].

Thus, simply administering known in vitro inhibitors of collagen synthesis, deposition and cross-linking in an attempt to treat pulmonary fibrosis is ineffective.

Clearly, new treatments for this incurable disease are required which specifically slow or halt the pathogenesis of fibrosis, without non-specific or toxic side effects.

There is thus a widely recognized need for, and it would be highly advantageous to have, a treatment for pulmonary which inhibits fibrogenesis substantially without undesirable non-specific or toxic side effects.

SUMMARY OF THE INVENTION Unexpectedly, it has been found, as -described in the examples below, that Halofuginone can also inhibit the pathophysiological process of pulmonary 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 pulmonary fibrosis in vivo.

According to the teachings. of the present invention, there is provided a composition for treating pulmonary 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:

N

R' II 1 :wherein: 3 n= or2; SR is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy; R 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; and pharmaceutically acceptable salts thereof.

According to further preferred embodiments of the present invention, the compound is preferably Halofuginone. The composition preferably includes a pharmaceutically acceptable carrier for the compound. Most preferably, the composition is administered to lung tissue, for example as an aerosol.

According to another embodiment of the present invention, there is provided a method of manufacturing a medicament for treating pulmonary 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: <4,c o R 1 IEzocaC z I N3 wherein: o n 1or 2; R is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy; 1 2 is a member of the group consisting of hydroxy, acetoxy, and lower alkoxy, and R 3 is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl; and pharmaceutically acceptable salts thereof.

According to yet another embodiment of the present invention, there is provided a method for the treatment of pulmonary fibrosis in a subject, including the step of administering a pharmaceutically effective amount of a compound having a formula: po o wherein: tr n I or 2; RI is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy; 2 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; and pharmaceutically acceptable salts thereof.

Hereinafter, the term "subject" refers to the human or lower animal to whom Halofuginone was administered. The term "patient" refers to human subjects. The term "pulmonary fibrosis" refers to any fibrotic condition in the lungs or respiratory tract of \~tfesubiect. The term "treatment" includes both substantially preventing the process of 7 pulmonary fibrosis from starting and slowing or halting the progression of pulmonary fibrosis once it has arisen.

Hereinafter, the term "oral administration" includes, but is not limited to, administration by mouth for absorption through the gastrointestinal tract, buccal administration and sublingual administration.

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.

The term "parenteral administration" includes, but is not limited to, administration by intravenous drip or intraperitoneal, subcutaneous, or intramuscular injection.

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

Although the specific quinazolinone derivative "Halofuginone" is referred to 15: throughout the specification, it is understood that other quinazoinone derivatives may be used in its place, these derivatives having the formula: 3

P::

wherein: n 1 or 2; R 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, and R 3 is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl; and pharmaceutically acceptable salts thereof.

While the invention will now be described in connection with certain preferred Tf~ bodiments in the following figures and examples so that aspects thereof may be more WO 98/43642 PCT/IL97/00115 8 fully understood and appreciated, it is not intended to limit the invention to these particular embodiments. On the contrary, it is intended to cover all alternatives, modifications and equivalents as may be included within the scope of the invention as defined by the appended claims. Thus, the following figures and examples which include preferred embodiments will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of preferred embodiments of the present invention only, and arepresented in the cause of providing what is believed to be the most useful and readily understood description of formulation procedures as well as of the principles and conceptual aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is herein described, by way of example only, with reference to the accompanying drawings, wherein: FIG. 1 is a graph of the effect of Halofuginone on hydroxyproline concentration in rat lung; FIG. 2 is a graph of the effect of Halofuginone on protein concentration in rat lung; FIG. 3 is a graph of the effect of Halofuginone on hydroxyproline\protein ratios in rat lung; FIG. 4 is a graph of the effect of Halofuginone on DNA levels in rat lung; and FIGS. 5A and 5B show histologically-stained sections of lung from bleomycintreated rats.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Unexpectedly, it has been found, as described in the examples below, that Halofuginone can also inhibit the pathological process of pulmonary fibrosis in vivo, possibly by inhibiting collagen type I synthesis, although another mechanism or mechanisms could also be responsible. Indeed, irrespective of the specific mechanism, SUBSTITUTE SHEET (RULE 26) WO 98/43642 PCT/IL97/00115 9 the data presented below clearly demonstrate the efficacy of Halofuginone in vivo for inhibition of the pathological progression of pulmonary fibrosis.

Such a finding is unexpected for three 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. However, chickens treated with Halofuginone were not reported to have an increased rate of bone breakage, indicating that the effect is not seen in vivo. Thus, the exact behavior of Halofuginone in vivo cannot always be accurately predicted from in vitro studies.

Second, other inhibitors of collagen synthesis, deposition and cross-linking have not proved effective for the treatment of pulmonary fibrosis, demonstrating that inhibition of collagen production alone is not sufficient for determining the success or failure of a treatment for pulmonary fibrosis. Thus, the finding that Halofuginone can successfully inhibit pulmonary fibrosis in vivo is both novel and non-obvious.

Thus, apart from the Nagler reference [Nagler, A. et al., Am. J. Respir. Crit. Care Med., 154:1082-1086, 1996], nothing in the prior art taught that Halofuginone would be useful in the treatment of pulmonary fibrosis in vivo. Furthermore, the ability of Halofuginone. and related compounds, to slow or halt progression of fibrosis in the lungs is both novel and non-obvious. The demonstration of such an ability in vivo 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 pulmonary fibrosis with quinazolinonecontaining compounds such as Halofuginone. Both compositions with specific pharmaceutical formulations and methods of using these compounds are described below.

WO 98/43642 PCT/IL97/00115 Although the pathogenesis of pulmonary fibrosis is not fully understood, animal models for the disease have been successfully developed. Pulmonary fibrosis has been induced in rats by the parenteral administration of bleomycin. Interestingly, bleomycin is used as a chemotherapeutic agent [Nagler, A. et al., Am. J. Respir. Crit. Care Med., 154:1082-1086, 1996]. As noted above, chemotherapy is one of the causes of pulmonary fibrosis in patients. Thus, the bleomycin model is directly and specifically applicable to idiopathic, medication-induced pulmonary fibrosis, as well as being more generally exemplary of the pathogenesis of pulmonary fibrosis.

Bleomycin-induced pulmonary fibrosis is characterized by inflammation of the lower respiratory tract, interstitial edema and alveolar capillary damage. This capillary damage in turn results in the infiltration of macrophages, mast cells and inflammatory cells into the alveolar space. Subsequently, enhanced fibroblast proliferation and activation result in increased interstitial deposition of collagen type I and fibrosis [Nagler, A. et al., Am. J. Respir. Crit. Care Med., 154:1082-1086, 1996]. Thus, inhibition of fibrosis, as in both bleomycin-induced and other types of pulmonary fibrosis, depends upon the slowing or halting of the pathological process leading to the production of fibrotic tissue.

Therefore, compounds which are intended for the inhibition of pulmonary fibrosis must be tested in an in vivo model, such as the bleomycin model described above, for their ability to slow or halt the pathological process leading to deposition of fibrotic tissue. Such experiments were conducted for the collagen type I synthesis inhibitor Halofuginone, as described in greater detail in Examples 1-3 below.

Furthermore. once demonstrably effective compounds have been discovered, specific formulations and routes of administration must be elucidated for maximum efficacy of the treatment. Such formulations and routes of administration must enable the compound to be effectively absorbed and delivered to the desired site of treatment, while minimizing non-specific side effects caused by systemic distribution of the compound.

Illustrative examples of these formulations and routes of administration for quinazolinone-containing compounds such as Halofuginone are given in Examples 4-6 below.

WO 98/43642 PCT/IL97/00115 11 Example 1 Effect of Halofuginone on Biochemical Markers in Rat Lung The experimental protocol for the in vivo studies and the specific results for the effect of Halofuginone on specific biochemical markers in lung tissue are given below.

Essentially, Halofuginone inhibited bleomycin-induced increased collagen levels, as demonstrated by a variety of biochemical markers. The specific experimental method used was as follows.

Eighty male Sabra rats weighing from 350-400 g were divided into four groups of rats each. The first group was a control group which did not receive any injections.

The second group received intraperitoneal injections of bleomycin, 5 mg/kg body weight, for seven consecutive days. The third group received intraperitoneal injections of Halofuginone, 5 mg per rat, every other day for the entire experimental period. The fourth group received separate intraperitoneal injections of bleomycin and Halofuginone at the above dosages in separate syringes following a two hour interval.

All rats were given a regular diet and received drinking water ad libitum. The weight and food intake of the rats were monitored during the entire experimental period.

Ten rats from each group were killed after 4 and 6 weeks with an overdose of pentobarbital. The lungs were removed from the killed rats and washed with phosphatebuffered saline. One lung was immediately frozen in liquid nitrogen and lyophilized for the analysis of hydroxyproline concentration. The entire dried right lower lobe was weighed and finely minced and homogenized in distilled water. The tissue was subsequently boiled in distilled water for 30 minutes. After cooling and centrifugation, the tissue residue was subjected to a second cycle of the extraction, boiling and centrifugation process. Both supernatants were then pooled, and aliquots were taken for the analysis of hydroxyproline concentration and protein levels.

Hydroxyproline was determined by subjecting the aliquots to acid hydrolysis with 6 N HC1 at 138 C for 3 hours, followed by the assay of Stegemann and Stalder [H.

Stegemann and K. Stalder, Clin. Chim. Acta, 18:267-273, 1967]. The results are WO 98/43642 PCT/IL97/00115 12 expressed in Figure 1 as micrograms of hydroxyproline per mg dry tissue weight. Protein levels were determined by the Bradford assay Bradford, Analyt. Biochem., 72:248- 254, 1976]. Hydroxyproline\protein ratios were determined from samples taken from the same aliquot.

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. Thus, as shown in Figure 1, bleomycin clearly caused a significant increase in hydroxyproline concentration, and therefore of collagen levels, in the lungs of rats sacrificed after 4 or 6 weeks. This increase was completely inhibited by treatment with Halofuginone. However, administration of Halofuginone to rats which were not given bleomycin did not cause any change in hydroxyproline concentration. Therefore, the effect of Halofuginone was simply to inhibit the bleomycin-induced increase in hydroxyproline concentration.

Figure 2 is a graph of the effect of Halofuginone on protein concentration in rat lung. Again, bleomycin caused a significant increase in protein levels in the lungs of animals sacrificed after 4 or 6 weeks. Halofuginone again completely inhibited this bleomycin-induced increase in protein levels in the lungs of animals sacrificed after 4 weeks, although no such inhibition was seen in tissue taken from animals sacrificed after 6 weeks.

Figure 3 is a graph of the effect of Halofuginone on hydroxyproline\protein ratios in rat lung. Again. bleomycin treatment caused a significant increase in the hydroxyproline\protein ratio in rat lung, indicating that the increased protein synthesis was largely due to increased collagen synthesis. Halofuginone reduced this ratio to the level seen in tissue taken from control rats. Thus, clearly Halofuginone completely inhibited the increased levels of collagen synthesis induced by bleomycin in the lungs of rats. However, Halofuginone alone did not demonstrate an anorectic effect in rats, indicating that the effect of Halofuginone is specific for inhibition of collagen synthesis.

WO 98/43642 PCT/IL97/00115 13 Example 2 Effect of Halofuginone on DNA Levels The experimental protocol and results are given for the effect of Halofuginone on DNA levels in rat lung. Essentially, a significant decrease in levels of DNA was induced by bleomycin. However, this decrease was abolished by Halofuginone. The experimental method was as follows.

Lung tissue from four groups of rats were prepared as described in Example 1, except that the preparation stopped at the lyophilization of the tissue. The quantity of DNA in the dried lung tissue was determined by the method of Burton Burton, Meth.

Enzymol., XII-B:163-166, 1968]. Results are given in Figure 4 as micrograms of DNA per mg dry tissue weight.

As shown in Figure 4, bleomycin caused a significant reduction in levels of DNA in lung tissue taken from rats which were sacrificed at 4 or 6 weeks. Halofuginone abolished this reduction of DNA levels in bleomycin-treated rats, yet had no effect on the level of DNA in non-bleomycin-treated rats.

Previous in vitro studies with cultured fibroblasts have shown that bleomycin reduces levels of DNA in these cells as well [Nagler, A. et al., Am. J. Respir. Crit. Care Med., 154:1082-1086, 1996]. Thus, this effect of bleomycin correlates well with previous results. The ability of Halofuginone to abolish this bleomycin-induced effect may be related to both the inhibition of the overall fibrotic process and the corresponding inhibition of specific effects of bleomycin on DNA levels. Regardless of the mechanism, Halofuginone specifically abolishes this bleomycin-induced effect without altering DNA levels in non-bleomycin-treated rats.

Example 3 Effect of Halofuginone on Histology and Morphology of Rat Lung Rats were treated as described in Example 1 and then sacrificed. One lung was taken for biochemical analysis and the other for histological examination, as described in further detail in this Example. The histological examination revealed that bleomycin- WO 98/43642 PCT/IL97/00115 14 induced specific morphological changes in rat lung, including diffuse pneumonitis and thickened alveolar walls. Halofuginone prevented these changes from occurring, resulting in airspaces of normal appearance in the rat lung. The experimental method was as follows.

The lung which was taken for histological examination was placed in Caroy's fixative after removal from the rat. The Carnoy-fixed lung was then embedded in paraffin and histologic sections, 6 mm thick, were then cut from the apex, central portion and lower end of the left lung. These sections were then incubated with monoclonal antibodies to rat mast cell chymase, and were stained with alkaline phosphatase to detect the antibodies, and with hematoxylin-eosin for determination of morphological features.

Figures 5A and 5B show the resulting stained sections of lung from bleomycintreated rats. The tissue shown in Figure 5A was from a rat treated with bleomycin alone and sacrificed after 6 weeks. Diffuse pneumonitis and thickened alveolar walls can clearly be seen, showing the extensive morphological changes caused by the bleomycininduced process of fibrosis. No such changes can be seen in Figure 5B, which shows lung tissue taken from a rat treated with both Halofuginone and bleomycin. Instead, airspaces with substantially normal morphology can be seen. Clearly, the effects of Halofuginone are specific for the prevention of the morphological changes produced during the pathological process of fibrosis.

Example 4 Suitable Formulations for Administration of Halofuginone Halofuginone can be administered to a subject in a number of ways, which are well known in the art. Although a number of routes of administration are possible, such as oral or parenteral administration, the most preferred route of administration for the treatment of pulmonary fibrosis is by inhalation, either through the nose, mouth or both.

There are a number of reasons for this preference. First, inhalation would permit direct exposure of the affected tissue to the pharmaceutical composition containing Halofuginone or another quinazolinone derivative. Second, such direct exposure would minimize systemic absorption, thus minimizing any potential side effects. Inhalation for SUBSTITUTE SHEET (RULE 26) WO 98/43642 PCT/IL97/00115 drug delivery to the lungs is thus more comparable to topical application on the skin in terms of systemic exposure. Third, direct exposure would minimize the amount of Halofuginone which would be required for treatment, since substantially all of the Halofuginone would be delivered to the tissue to be treated. Indeed, if the dose given to the rats (5 mg per kg) is extrapolated to an average human subject, about half a gram would be required, which is a large amount for administration and could potentially reduce patient compliance. Fourth, direct exposure could be particularly important for subjects with significant fibrotic tissue already present in the lungs, as this tissue does not contain the normal structure of the capillary network, potentially reducing the ability of the blood vessels to deliver Halofuginone to the tissue to be treated. Fifth, inhalation of pharmaceutical compositions allows for a rapid onset of therapeutic effect. Finally, other routes of administration could prove inconvenient for various reasons. For example, intraperitoneal delivery by injection, used in the above-described experiments, would be inconvenient for regular administration of Halofuginone. The efficacy of oral administration of Halofuginone has not been established in rats and lower animals, and has not been examined at all in humans. Furthermore, for some human subjects, such as those undergoing chemotherapy, oral administration is highly problematic because many chemotherapeutic agents cause extensive nausea and vomiting. Thus, administration of Halofuginone by inhalation bypasses many of these problems with other routes of administration.

For administration by inhalation, Halofuginone could be given in aerosolized form from a pneumatic or ultrasonic nebulizer, for example. Alternatively and preferably. Halofuginone could be suspended in micronized form in a fluorocarbon propellant solvent and delivered in metered doses from a pressurized canister. Following aerosolization, most of the propellant solvent is lost through flash evaporation and replaced by moisture in the respiratory tract, leading to the deposition of hydrated micronized particles. However, both of these methods rely upon deposition of the free form of Halofuginone to the respiratory tract.

An alternative and preferable method would use liposome-based aerosols for drug delivery. Examples of such aerosols are given in PCT Application No. WO 86/01714.

WO 98/43642 PCT/IL97/00115 16 Halofuginone would be trapped in the liposomes, which would then be suspended in an aqueous solution, and delivered by a nebulizer or other metered-dose system. Further examples of drug delivery by inhalation are described in U.S. Patent No. 5,340,587.

Whatever the route of administration, dosing is dependent on the severity of the symptoms and on the responsiveness of the subject to Halofuginone. Persons of ordinary skill in the art can easily determine optimum dosages, dosing methodologies and repetition rates.

Example Method of Treatment of Pulmonary Fibrosis As noted above, Halofuginone has been shown to be an effective inhibitor of pulmonary fibrosis. The following example is an illustration only of a method of treating pulmonary fibrosis with Halofuginone, and is not intended to be limiting.

The method includes the step of administering Halofuginone, 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 pulmonary fibrosis in the subject.

Examples of types of pulmonary fibrosis for which such a treatment would be effective include, but are not limited to, pulmonary fibrosis following such therapeutic interventions as bone marrow transplantation. radiotherapy and chemotherapy. Other examples include pulmonary fibrosis caused by contact with injurious chemicals, inflammation, neoplasms, toxic substances, auto-immune diseases, vasculitis, trauma.

post-surgical effects, genetic disorders, scleroderma, viral diseases such as cytomegalovirus, lung transplants, bums, congenital malformations and chemical compounds like monocrotaline.

WO 98/43642 PCT/IL97/00115 17 Example 6 Method of Manufacture of a Medicament Containing Halofuginone The following is an example of a method of manufacturing Halofuginone. First, 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. Next, Halofuginone is placed in-a suitable pharmaceutical carrier, as described in Example 4 above, again in accordance with good pharmaceutical manufacturing practice.

It will be appreciated that the above descriptions are intended only to serve as examples, and that many other embodiments are possible within the spirit and the scope of the present invention.

Claims (8)

1. Use of a compound being a member of a group having a formula: RI C M, COCB H, wherein: 3 n=l or 2; R is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower S* 15 alkoxy; 9*9* R 2 is a member of the group consisting of hydroxy, acetoxy, and lower alkoxy, and R 3 is a member of the group consisting of hydrogen and lower alkenoxy; and pharmaceutically acceptable salts 20 thereof, together with a pharmaceutically acceptable carrier, in the manufacture of a medicament for treating pulmonary fibrosis. e

2. The use of claim 1, wherein said compound is 25 Halofuginone.

3. The use of claim 1, further comprising a pharmaceutically acceptable carrier for said compound.

4. The use of claim 3, wherein said pharmaceutically acceptable carrier enables administration of the composition as an aerosol.

A method for the treatment of pulmonary fibrosis in a subject, comprising the step of administering to a subject suffering from pulmonary fibrosis a j harmaceutically effective amount of a compound having a >r H:\suzanneg\Keep\20420-97 SPEC DATED 22 JUNE 2001.doc 22/06/01 FF\Cl 19 formula: a 2 CBCOCN 0* 0 wherein: n=l or 2; Ri 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, and 15 R 3 is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl; and pharmaceutically acceptable salts thereof.

6. The method of claim 5, wherein said compound is 20 Halofuginone.

7. The method of claim 5 or claim 6, wherein said compound further includes a pharmaceutically acceptable carrier.

8. The method of claim 7, wherein said pharmaceutically acceptable carrier enables administration of the composition as an aerosol. 0*00*0 0 S S. Dated this 25th day of June 2001. AGRICULTURAL RESEARCH ORGANIZATION MINISTRY OF AGRICULTURE and HADASIT MEDICAL RESEARCH SERVICES DEVELOPMENT COMPANY LTD By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and !PFUS Trade Mark Attorneys of Australia H:\suzanneg\Keep\20420-97 SPEC DATED 22 JUNE 2001.doc 25/06/01