CA2669123A1 - Acat inhibitors and their use in the prevention or treatment of fibrosis - Google Patents

Abstract

The present invention relates to the use of ACAT inhibitors, {e.g. F-1394, Avasimibe (CI-1011), Pactimibe (CS505), Efluimibe (F12511), Eldacimibe, NTE 122, AS-183, KW-3033, E5324, FY087, FCE27677, CI-976, K-604, TEI6522, Octimibate, FR179254, and S 58-035, or mixtures thereof), compositions comprising the same, and methods for preventing or treating fibrosis, for preventing or reducing collagen deposition in a tissue, and in the prevention and reduction of excessive fibrous connective tissue

Description

ACAT INHIBITORS AND THEIR USE
IN THE PREVENTION OR TREATMENT OF FIBROSIS
FIELD OF THE INVENTION

The present invention provides novel methods and compositions for preventing and reducing fibrosis associated with fibrotic disorders using ACAT
inhibitors.
More particularly, the present invention relates to the use of ACAT inhibitors in compositions and methods for preventing or treating fibrosis, for modulating collagen deposition in a tissue, and in the prevention and reduction of excessive fibrous connective tissue in an organ.

BACKGROUND OF THE INVENTION

The process of tissue repair as part of wound healing involves two phases. The first phase is the regenerative phase, in which injured cells are replaced by cells of the same type. The second phase is the formation of fibrous tissues, also called fibroplasias or fibrosis, in which connective tissue replaces parenchyma tissues. The tissue repair process can become pathogenic if the fibrosis phase continues unrestrained, leading to extensive tissue remodelling and the formation of permanent scar tissue (Wynn 2004).

Fibrotic disorders affect almost every tissues and organ systems. Major organ fibrosis diseases include interstitial lung disease (ILD) which leads to pulmonary inflammation and fibrosis. ILD is known to have a number of causes such as sarcoidosis, silicosis, collagen vascular diseases. The most common type of ILD
is idiopathic pulmonary fibrosis (IPF), which still has no known cause (idiopathic).
Other fibrotic disorders include liver cirrhosis and fibrosis originating from viral hepatitis, kidney disorders associated with unregulated TGF-R activity and excessive fibrosis such as glomerulonephritis (GN), renal interstitial fibrosis, renal fibrosis in transplant patients, and eye diseases such as macular degeneration and retinal and vitreal retinopathy. In addition, fibroproliferative disorders include systemic and local scleroderma, keloids and hypertrophic scars, and collagen disorders associated with the occurrence of Raynaud's syndrome. Excessive scarring resulting from surgery, chemotherapeutic drug-induced fibrosis, radiation-induced fibrosis, and burns are also part of fibroproliferative diseases.
Acyl-CoA: Cholesterol acyltransferase (ACAT) catalyzes the formation of cholesteryl esters using both cholesterol and long chain fatty acyl co-enzyme A
as substrate. ACAT is present in a variety of tissue including intestinal mucosa, liver, adrenal, testes and macrophages. ACAT inhibitors are known to reduce or prevent the appearance of atheromatous lesions in animal models.

Current treatments for fibrotic disorders involve general immunosuppressive drugs such as corticosteroids and other anti-inflammatory drugs. These therapies are not always effective in reducing or preventing fibrosis probably because the mechanisms involved in regulation of fibrosis appear to be distinct from those of inflammation, and anti-inflammatory therapies. Few investigational therapies have been proven to alter or reverse the inflammatory process that is associated with fibrosis.

Moreover, a wide range of likely molecular targets for antifibrotic therapy have been identified, for example those directly involved in fibrogenesis, including matrix metalloproteinases and their inhibitors, and pro-fibrogenic cytokines, such as tumor growth factor R(TGF-P). TGF-R plays a key role initiating the cascade of events that culminates in the production of cytokines, and in excessive and faulty expression of collagen, and of other extracellular matrix components.

Several animal models are useful to identify and characterize new anti-fibrotic agents. Antifibrotic effects of pirfenidone were recently demonstrated in the bleomycin-hamster model of lung fibrosis (lyer, Margolin et al. 1998) and in a unilateral ureteral obstruction model of kidney fibrosis (Shimizu, Kuroda et al.
1998). An inhibitor of ALK5 (TGF-P receptor II) was shown to be protective in the dimethyinitrosamine-induced liver fibrosis (DMN) rat model (de Gouville and Huet 2006).

The present inventors have now found that compounds having ACAT inhibitory capability are molecules of choice for the treatment and prevention of fibrosis.

SUMMARY OF THE INVENTION

The present invention provides novel methods and compositions for preventing and reducing fibrosis and/or fibrotic disorders using ACAT inhibitors.

In one aspect, the present invention provides an antifibrotic composition comprising an effective amount of at least one antifibrotic agent and an acceptable excipient.

In another aspect, the present invention concerns the use of an ACAT inhibitor for preventing or reducing collagen deposition in a tissue.

Further, another aspect of the present invention concerns the use of an ACAT
inhibitor for preventing the formation or development of excess fibrous connective tissue in an organ.

The present invention also encompasses a method for reducing the level of collagen in a tissue, the method comprising providing a tissue, contacting the tissue with at least one ACAT inhibitor and measuring a reduced level of collagen in the tissue.

According to another aspect, the present invention relates to a method for preventing the formation or development of excess of fibrous connective tissue in an organ of a subject, the method comprising administering to the subject an effective amount of at least one ACAT inhibitor.

In another aspect, the invention provides a method for preventing or treating fribrosis or a fibrotic disorder in a subject, the method comprising:

-identifying a subject suffering from or being at risk of developing fibrosis;
-administering to the subject an ACAT inhibitor in an amount sufficient to decrease the level of collagen; and -measuring a reduced level of collagen in the subject.

DESCRIPTION OF THE FIGURES

Figure 1 shows a schematic representation of the TGF-P pathway in human with worm homologs.

Figure 2 shows that ACAT inhibitors decrease TGF-P signalling. Dauer formation was measured in the daf-14(m77) mutant. The following compounds were tested, Avasimibe (n=651), F-1394 (n=935), Pactimibe (n=1166), FR179254 (n =1056), and S 58-035 (n=765). DMSO was used as a control solvent (n=941). All the compounds tested enhance dauer formation, relative to the control treatment.
Figure 3 shows that ACAT inhibitors decrease TGF-R signalling in more than one TGF-P constitutive mutant. Dauer formation induced by Avasimibe (a), and F-1394 (b) was measured in dauer mutants as described in the Material and Method section of Example 1. (a) Avasimibe was tested in daf-7(e1372) (n=560, vs. 539 for control); in daf-1(m40) (n=870, vs. 636 for control); and in daf-8(e1393) (n=852, vs. 502 for control). (b) F-1394 was tested in daf-7(e1372) (n=99, vs. 134 for control); in daf-1(m40) (n=145, vs. 108 for control); and in daf-8(e1393) (n=47, vs. 140 for control). The compounds tested enhance dauer formation in all the mutants, relative to the control treatment.

Figure 4 shows the relative quantification of al (I) collagen mRNA expression levels in A549 cells unstimulated (control) or stimulated with transforming growth factor (TGF; 5 ng ml-')-(3i in the absence or presence of F-1394 (0.3, 0.6 and g ml"1, as indicated). Exposure time was 72 h for TGFR,. F-1394 was present from 1 h before TGFP, to the end of the experiment. TGFP,-induced increase of al (I) collagen mRNA expression was concentration-dependently inhibited by F-1394. a1 (I) collagen mRNA expression was determined by using real-time RT-PCR by the OOCt method; columns show the fold increase in expression of a1 (I) collagen mRNA relative to GAPDH values as mean SEM of the 2-AOCt values of three independent experiments. *: p<0.05 versus control; #: p<0.05 versus TGFP1.

Figure 5 shows the relative expression of a1 (I) collagen mRNA in lung tissue of mice receiving intratracheal bleomycin (BLM) or saline (control). Columns are mean s.e.m. of 3-5 animals per group. *P<0.05 vs. control, #P<0.05 vs. BLEO by ANOVA.

5 Figure 6 shows representative photomicrographs of lung histology in mice treated with drug vehicle + saline (a), F-1394 + saline (b), N-acetylcysteine + saline (c), drug vehicle + bleomycin (d,e,f), F-1394+bleomycin (g,h) and N-acetylcyteine+bleomycin (i). All lung sections were stained with haematoxylin-eosin. Magnification x40. Saline-treated animals show normal architecture.
Bleomycin-exposed mice showed marked peribronchial interstitial infiltration with inflammatory cells, thickened alveolar septa, oedema and foci of dense fibrosis.
These pulmonary lesions were reduced in animals orally treated with F-1394 and with N-acetylcysteine.

Figure 7 shows the evaluation of fibrotic changes in the lung by numerical fibrotic score. Bars denote Ascroft scores (mean SEM) of each experimental group as indicated. *P<0.05 vs. vehicle+BLEO by Mann-Whitney test. Pulmonary lesions were reduced in animals orally treated with F-1394 as well as with N-acetylcysteine.

Figure 8 shows the fibrosis percentage measured after Sirius red coloration on ten non-overlapping fields in cortical arere expressed as the mean percentage of the interstitial area stained; ***p<0.01 compared to Vehicle group; n=10 per group; data were analyzed with ANOVA followed by a Newman-Keuls test.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel methods and compositions for preventing and reducing fibrosis associated with fibrotic disorders using ACAT
inhibitors.
Definitions As used herein, the expression ACAT inhibitor means any compound that inhibits the enzyme Acyl-CoA:Cholesterol acyltransferase (ACAT). Examples of ACAT inhibitors include small organic compounds, i.e., having a molecular weight of more than 50 yet less than about 2500. The ACAT inhibitors include functional chemical groups necessary for structural interactions with proteins and/or nucleic acid molecules, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical. The ACAT
inhibitors can include cyclic carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups.

The ACAT inhibitors according to the present invention are for instance the following compounds: F-1394, Avasimibe, Pactimibe (CS-505), Eflucimibe (F
12511), Eldacimibe, NTE 122, AS-183, KW-3033, E5324, FY 087, FCE 27677, Cl 976, TEI 6522, K-604, Octimibate, FR17924 and S 58-035 as identified in Table 1.

As used herein the term fibrosis refers to the formation of fibrous tissue as a reparative or reactive process. Fibrosis is characterized by fibroblast accumulation and collagen deposition in excess of normal deposition in a particular tissue.

As used herein the expressions fibrotic disorder, fibroproliferative disease or fibrotic disease refer to conditions involving fibrosis in one or more tissues.

Fibrotic disorder include, but is not limited to scleroderma, keloids and hypertrophic scars, collagen disorders associated with the occurrence of Raynaud's syndrome, pulmonary inflammation and fibrosis, interstitial lung diseases, idiopathic pulmonary fibrosis, sarcoidosis, liver cirrhosis and liver fibrosis resulting from viral or from parasitical infection, kidney disorders associated with unregulated TGF-P activity and excessive fibrosis such as glomerulonephritis (GN), renal interstitial fibrosis, renal fibrosis in transplant patients, focal glomerulosclerosis, fibrosis caused by Marfan's disease, cardiac fibrosis, radiation-induced fibrosis, and fibrosis arising from wound healing, eye diseases such as macular degeneration and retinal and vitreal retinopathy. It also includes peritoneal fibrosis, intestinal fibrosis, chemotherapeutic drug-induced fibrosis and burns.

As used herein, the term scleroderma means a rare, chronic disease characterized by excessive deposits of collagen, which affects the skin, and in more serious cases it can affect the blood vessels and internal organs.
Typically, the most evident symptom is the hardening of the skin and associated scarring, and blood vessels may also be more visible.

Idiopathic pulmonary fibrosis is an inflammatory lung disorder of unknown origin (idiopathic) characterized by abnormal formation of fibrous tissue (fibrosis) between the tiny air sacs (alveoli) or ducts of the lungs.

Liver fibrosis refers to the accumulation of tough, fibrous scar tissue in the liver of a subject.

As used herein the term subject refers to animals and humans. This term thus includes, without being limited to, primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, mice, rats, rabbits, guinea pigs, captive animals such zoo animals, and wild animals.

As used herein the term tissue refers to an organ or set of specialized cells such as skin tissue, lung tissue, kidney tissue, and other types of cells.

As used herein, the term treatment / treating refer to a process by which the symptoms of fibrotic disorders, fibrotic diseases, fibrosis and related disorders as exemplified hereinabove, are alleviated or completely eliminated.

As used herein, the term prevention / preventing refer to a process by which symptoms of fibrotic disorders, fibrotic diseases, fibrosis and related disorders as exemplified hereinabove are obstructed, delayed or averted.

As used herein the expression effective amount indicates an amount that produces the desired effect as judged by clinical trial results and/or animal models. This amount can be routinely determined by one skilled in the art. An effective amount of a composition to be employed will depend, for example, upon the treatment context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the type of ACAT inhibitor delivered, the indication for which the ACAT inhibitor is being used, the route of administration, and the size (body weight, body surface or organ size) and condition (the age and general health) of the patient.
Accordingly, the clinician may titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.

For any compound, the effective dose can be estimated initially either in cell culture assays or in animal models such as mice, rats, rabbits, dogs, pigs, or monkeys. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

As used herein, the expression acceptable excipient means an ingredient used in a composition which does not interfere with the effectiveness of the biological activity of the active ingredient(s) of the composition, and which is not toxic to the host, tissue or organ intended to be treated.

The expression acceptable excipient as used herein means an ingredient used in a composition which does not interfere with the effectiveness of the biological activity of the active ingredients of the composition, and which is not toxic to the host or patient. Furthermore, the excipient is advantageously a compound with minimum probability of being rejected by the immune system of the subject being treated.

Such acceptable and/or pharmaceutically acceptable excipients are used for various purposes, such as stabilizers, buffers, suspending agents, carriers and the like and are listed and described in a number of texts including for example, the British Pharmacopeia, the Japanese Pharmacopeia and the United States Pharmacopeia XXII and National Formulary XVII and supplements thereto.

Use of ACAT inhibitors in the prevention and treatment of fibrosis or fibortic disorders The inventors of the present invention found that a series of compounds having ACAT inhibitory capability are useful to reduce or prevent fibrosis and related disorders in well accepted animal models of fibrotic disorder.

In one aspect, the present invention provides antifibrotic compositions comprising an effective amount of at least one antifibrotic agent and an acceptable excipient.
More particularly, the antifibrotic agent is an ACAT inhibitor. For instance, the ACAT inhibitor may be one of the ACAT inhibitor defined in Table 1 or a mixture thereof.

The CAS registry numbers or/and the publication/patent that describe the preferred ACAT inhibitors contemplated by the present invention are found in Table 1. They include the following compounds: F-1394, Avasimibe, Pactimibe (CS-505), Eflucimibe (F12511), Eldacimibe, NTE 122, AS-183, KW-3033, E5324, FY 087, FCE 27677, Cl 976, TEI 6522, K-604, Octimibate, FR 179254 and S 58-035.

In a related aspect, the invention concerns a method for preventing or treating fibrosis or a fibrotic disorder in a subject. The method comprises a first step of identifying a subject suffering from or being at risk of developing fibrosis, followed by a step of administering to the subject an ACAT inhibitor in an amount sufficient to decrease the level of collagen. The method also includes a further step of measuring a reduced level of collagen in the subject. The ACAT inhibitor is preferably one of the ACAT inhibitors defined in Table 1 or a mixture thereof.

The fibrotic disorder to be treated or averted is for instance scleroderma, keloids and hypertrophic scars, collagen disorders associated with the occurrence of Raynaud's syndrome, pulmonary inflammation and fibrosis, interstitial lung diseases, idiopathic pulmonary fibrosis, sarcoidosis, liver cirrhosis and liver fibrosis resulting from viral, or from parasitical infection, kidney disorders associated with unregulated TGF-f3 activity, excessive fibrosis, renal interstitial fibrosis, renal fibrosis in transplant patients, focal glomerulosclerosis, fibrosis caused by Marfan's disease, cardiac fibrosis, radiation-induced fibrosis, fibrosis arising from wound healing, eye diseases, peritoneal fibrosis, intestinal fibrosis, and chemotherapeutic drug-induced fibrosis or burns.

5 Use of ACAT inhibitors for the prevention or reduction of coliagen deposition in a tissue It is well known that some types of fibrosis are characterized by abnormal collagen deposition resulting from increased collagen synthesis and/or decreased collagen degradation.

10 Accordingly, the present invention concerns the use of at least one ACAT
inhibitors for preventing or reducing collagen deposition in a tissue.

By the expression "preventing or reducing collagen deposition in a tissue" it is meant that collagen deposition in excess of normal deposition is obstructed, delayed or averted in a particular tissue, or that the excess of collagen deposited in the tissue, compared to a normal deposition, is alleviated or eliminated.

For instance, the ACAT inhibitor used for preventing or reducing collagen deposition in the tissue may be one of the ACAT defined in Table 1 or a mixture thereof.

The present invention also provides a method for reducing the level of collagen in a tissue which comprises a step of providing a tissue followed by a step of contacting the tissue with at least one ACAT inhibitor. The next step consists of measuring a reduced level of collagen in the tissue. According to a preferred embodiment, the ACAT inhibitor used in this method is one of the compounds defined in Table 1 or a mixture thereof. The tissue is preferably a fibrotic tissue. It may also be a tissue of heart, lung, brain, eye, stomach, spleen, bone, pancreas, kidney, liver, intestine, skin, uterus or biadder.

In a related aspect, the present invention also concerns a composition useful in the prevention or reduction of collagen deposition in a tissue, the composition comprising an effective amount of at least one ACAT inhibitor as defined in Table 1 or a mixture thereof, and an acceptable excipient.

One will appreciate that the tissue is for instance an organ tissue of a subject, such as a human. The organ tissue is for instance a tissue of an organ selected from heart, lungs, brain, eye, stomach, spleen, bones, pancreas, kidneys, liver, intestines, skin, uterus and bladder.

Use of ACAT inhibitors in the prevention of formation or development of excess of fibrous tissue in an organ In the process of tissue repair, the fibrosis phase consists of the formation of fibrous tissues in which connective tissue replaces parenchyma tissues. If the fibrosis phase continues unrestrained, the tissue repair process can become pathogenic, leading to extensive tissue remodeling.

Accordingly, the present invention proposes to use an ACAT inhibitor to prevent the formation or development of excess of fibrous connective tissue in an organ.
More particularly, the ACAT inhibitor may be one of the compounds defined in Table 1 or a mixture thereof.

By the expression "to prevent the formation or development of excess of fibrous connective tissue in an organ" it is meant that the formation of fibrous connective tissue, in excess of normal formation is obstructed, delayed or averted in a particular organ.

One will appreciate that the organ is for instance an organ of a subject, such as a human. The organ is for instance a heart, a lung, a brain, an eye, a stomach, a spleen, a bone, a pancreas, a kidney, a liver, an intestine, a skin, an uterus or a bladder.

In a related aspect, the present invention provides a method to prevent the formation or development of excess of fibrous connective tissue in an organ of a subject, which comprises administering to the subject an effective amount of at least one ACAT inhibitor. More particularly, the ACAT inhibitor may one of the ACAT inhibitors defined in Table 1 or a mixture thereof. In a preferred embodiment, the organ is selected from heart, lungs, brain, eye, stomach, spleen, bones, pancreas, kidneys, liver, intestines, skin, uterus and bladder and the subject is a human.

Table 1:

ACAT Structure CAS registry Reference inhibitor number F-1394 135392-43-7 Kusunoki, (Fujirebio 0XO Aragane et Inc.) o p N a1.1995) ~q~
~
US patent (1 S,2S)-2-[3-(2,2-dimethylpropyl)-2- 5,120,738 nonylureido]cyclohexane-1 -yl-3-[(4R)-N-(2,2,5,5-tetram ethyl-1, 3-d ioxane-4-carbon I amino ro ionate Avasimibe CH3 CH3 166518-60-1 (Delsing, (CI-1011) / CH3 H3C "' Offerman et (Pfizer) I o s \ ~0 0 CH3 al. 2001) ~
~ H US patent H3~ CH3 5,491,172 N-((2,6-bis(1-methylethyl)phenoxy) sulfonyl)-2,4,6-tris(1-methylethyl)-benz lacetamide Pactimibe HO 0 CH3 189198-30-9 US patent (CS505) 5,990,150 iiIIiiiiiI:jiiiiIi> O

NH

1 H-Indole-5-acetic acid, 7-((2,2-dimethyl-1 -oxopropyl)amino)-2,3-dihdro-4,6-dimeth I-1-oct I-Eflucimibe 202340-45-2 (Junquero, (F12511) ~ Oms et al.
õ 2001) \ N
I / O ==y5 HO
S)-2', 3',5'-Trimethyl-4'-hydroxy-alpha-dodec Ithioacetanilide Eldacimibe H,C "' 141993-70-6 US patent (Wyeth- a>~o 5,179, 216 Ayerst/
AHP) H3C CH3 O I O
H3 C - 1) \ NH N CH3 HO

CH, [5-(((3, 5-bis(1,1-d i m ethylethyl)-4-hydroxylphenyl)amino)(((4-(2,2-dimethyl-propyl)phenyl)methyl)hexylamino)methyl ene-2,2-dimeth I -1,3-dioxane-4,6-dione NTE 122 (Azuma, (N Kawasaki et (Nissin õ,C ;"
~ ~
iN ~ GC i`"' a1.1998;
Food Azuma, Seto Products õ'` N0 O", Co., Ltd.) "N et al. 1999) Trans-l,4-bis[[1-cyclohexyl-3-(4-dimethylaminophenyl)ureido]methyl] US patent c clohexane 5,733,931 AS-183 CH3 CH3 CH3 CH3 CH3 (Kuroda, o CH3 Yoshida et al. 1993) OH O

KW-3033 (Kumazawa, ~ Yanase et al.
1996) NH
0 Br US patent 5,340,807 Enantiomer (-)-1 of the above molecule:
(-)-2-Bromo-N-(2,6-diisopropylphenyl)-6,11-dihydrodibenz[b,e]oxepin-11-carboxamide - -1 E5324 141799-76-0 US patent 5,668,136 N" NH N
H
H3C / O`
\ I

(n-butyl-N'-[2-[3-(5-ethyl-4-phenyl-1 H-imidazol-l-yl)propoxy]-6-meth I hen I urea FY 087 (Nagata, "'C CH 3 Yonemoto et p / c", a1.1995) S (~ "~ N
~~ US patent C"3 5,405,873 (N-[2-[N'-pentyl-(6,6-dimethyl-2,4-heptadiynyl)amino]ethyl]- (2-methyl-1-na hth Ithio acetamide FCE 27677 (Chiari, /r - (\ Lovisolo et 0 o al. 1996) N~(N

N I
I
(-)N-[2,6-bis(1-methylethyl)phenyl]-N'-[(4R,5R)-2-(4-dimethylaminoph eny I)-4,5-dimeth I-dioxolan-2- I meth lurea C1976 114289-47-3 (Bocan, õ,C Mueller et al.
1991) H,G~ O
(Sinz, Black et al. 1997) (2,2-dimethyl-N-(2,4,6-trimetho hen I dodecanamide K-604 / (Ikenoya, Yoshinaka, et al. 2007) N \ /
H N
`~ ^ ~N \

SJ V

2-[4-[2-(benzimidazol-2-ylthio)ethyl]
piperazin-I yl]-N-[2,4-bis(methylthio)-6-meth I-3- rid I acetamide.

TEI 6522 C"3 (Kataoka, Shiota et al.
1995) o o H,c H3C' 0 H CH3 N-(7-methoxy-4-oxoch roman-8-yl)-2, 2-dimeth Idodecanamide Octimibate 89838-96-0 (Jackson, õ Gee et al.
N 1990) ( / \

8-((1,4,5-Triphenylimidazol-2-I ox octanoic acid FR179254 õ' _ (Tanaka, A., "H Cõ et al. 1998) H,C

S 58-035 H H3C\ 1-1 CH3 78934-83-5 (Ross, A.C., (Sandoz) "S'"I (CH2)yCH3 et al. 1984) 3-[Decyldimethylsilyl]-N-[2-(4-methyl hen I-1- heneth I ro anamide ACAT inhibitors compositions of the invention The compositions according to the invention contain at least one excipient or formulation material, including for example a carrier or vehicle useful for 5 delivering the ACAT inhibitor, while maintaining and preserving the antifibrotic agent. The formulations can be adapted to the condition to be treated. For example, treatment of fibrotic disorders may be delivered topically, orally or by injection. Alternatively, the compositions may be delivered by inhalation therapy.
Other suitable means for the introduction of the therapeutic molecule include 10 implantable drug delivery devices.

The optimal composition will be determined by one skilled in the art depending upon, for example, the desired route of administration, its mode of delivery and intended dosage.

A suitable vehicle for parenteral injection preferably contemplated by the present invention, is sterile distilled water in which the desired ACAT inhibitor is formulated as a sterile, isotonic solution, properly preserved.

When the compositions of the invention consist of aqueous injection suspensions, they preferably contain substances increasing the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the desired ACAT inhibitor may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic amino polymers may also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or agents for increasing the solubility of the ACAT inhibitor and allowing for the preparation of highly concentrated solutions.

The composition of the invention may be formulated for inhalation. For example, an ACAT inhibitor may be formulated as a dry powder for inhalation. ACAT
inhibitor inhalation solutions may also be formulated with a propellant for aerosol delivery. Alternatively, solutions may be nebulized.

The compositions comprising the ACAT inhibitors may be formulated for topical administration. An example of topical compositions may take the form of a cream, ointment or lotion.

It is also contemplated that compositions of the invention may be administered orally. Compositions for oral administration can be formulated using acceptable carriers well known in the art in appropriate dosages for oral administration.
Such carriers enable the compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

Moreover, a capsule may be also designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to facilitate absorption of the ACAT inhibitor. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.

One will appreciate that an example of an oral formulation may consist of a capsule which can be prepared by granulating the ACAT inhibitor contemplated by the present invention with lactose and corn starch as the excipient and hydroxypropylcellulose as the binder. Such a capsule may consist of a white hard gelatin capsule containing the desired amount of the ACAT inhibitor.

Another example of a capsule formulation for the ACAT inhibitor F-1394 is provided in Table 2. It consists of a gelatin capsule provided with an enteric coat.
Table 2:

In redient m Capsule content F-1394 25.0 Triethyl citrate 35.0 Propyleneglycol fatty acid ester 110.0 Polyoxyethylene castor oil-60 170.0 Sub total 340.0 Capsule body Hard capsule (gelatine) 70.0 Caspule coat Methacrylic acid copolymer LD 29.84 Triethyl citrate 3.0 Talc 9.16 Sub total 42.0 Total 452.0 Formulations for oral use can also be obtained through combining the desired ACAT inhibitor with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores.
Suitable excipients for such formulations include for example, carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol;
starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, or sodium carboxymethylcellulose; and proteins, such as gelatin and collagen.

It is within the scope of the present invention to provide sustained- or controlled-delivery formulations of the ACAT inhibitors of the invention. One skilled in the art will know how to prepare such formulations.

The dosage levels for treatment will vary depending, in part, upon the type of ACAT inhibitor delivered, the specific indication for which the ACAT inhibitor is being used, the route of administration, and the size (body weight, body surface or organ size) and condition (the age and general health) of the patient. For instance, the clinician may titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.

For any compound, the effective dose can be estimated initially either in cell culture assays or in animal models such as mice, rats, rabbits, dogs, pigs, or monkeys. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

The frequency of dosing will depend upon the pharmacokinetic parameters of the ACAT inhibitor in the specific formulation that is used. Typically, a composition is administered until a dosage is reached that achieves the desired effect. The composition may therefore be administered as a single dose or as multiple doses (at the same or different concentrations/dosages) over time, or as a continuous infusion. Further refinement of the appropriate dosage is routinely made.
Appropriate dosages may be ascertained through use of appropriate dose-response data.

As an example, a capsule as described in Table 2, comprising 25 mg of the ACAT inhibitor F-1394 could be administered at least once a day, preferably at least twice a day. However, the daily dosage as well as the F-1394 capsule content could be adapted in order to obtain the optimal therapeutic effect.

The following examples illustrate the invention, with reference to the accompanying figures. These examples are illustrative of the wide range of applicability of the present invention and are not intended to limit its scope.
Modifications and variations can be made therein without departing from the spirit and scope of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred methods and materials are described.
EXAMPLES

Example 1: ACAT inhibitors inhibit the TGF-0 signalling pathway TGF-P belongs to a large family of secreted peptide growth factors that play critical roles in animal development. Upon ligand binding, the type I and type II
receptors are recruited into a complex and the type II receptor phosphorylates, and thereby activates, the type I receptor. In turn, the type I receptor phosphorylates Smads of the R-Smad (receptor-regulated) subfamily. R-Smads form complexes with Co-Smads and accumulate in the nucleus to regulate gene transcription via other transcription factors (Figure 1). This pathway regulates many cellular processes and plays crucial roles in normal development.
Disruption of the pathway can lead to a range of diseases including cancer, cardiovascular, inflammatory and fibrotic diseases, as well as Marfan's Syndrome.

In worms, TGF-P pathway regulates entry into the dauer larval stage, an alternative larval stage that is specialized for survival under adverse conditions.
Disruption of the TGF-R pathway leads to constitutive dauer formation, that is, the formation of dauer larvae even under favourable conditions. Thus, an increase in dauer formation indicates a decrease in signalling through the pathway.
However, there are other signalling pathways, such as the insulin-like signalling pathway, which affect dauer formation in worms. Constitutive dauer formation via the TGF-P pathway requires the activities of DAF-3 and DAF-5 whereas constitutive dauer formation via the insulin-like signalling pathway does not.

The goal of this study was to establish that ACAT inhibitors have the capacity to interfere with the TGF-P signalling pathway.

Materials and methods General Methods 5 All assays were carried out in 24-well plates. Wells contained 1.5 mL of nematode growth media (NGM). On day one, the appropriate volume of the compound stock solutions was added to the surface of the NGM wells and left to dry in the dark for at least 2 hours. The wells were then seeded with 3 to 5 pL of OP50 bacterial culture, which had been concentrated 15 times. Worms were 10 transferred to the wells as eggs on day 1 and then left to grow for 3 days at 20 C
(for daf-8, daf-14, and daf-4;daf-5) or at 22 C (for daf-1 and daf-7). The percentage of dauer-stage worms was determined on day 4 and/or day 5.

Worms Worm strains were obtained from the Caenorhabditis Genetics Center (CGC) and 15 maintained at 20 C using standard culture methods. The wild-type strain used was the Caenorhabditis elegans Bristol strain, N2. The mutants used in this study are: daf-7(e1372)III, daf-1(m40)IV, daf-4(m63)III, daf-8(e1393)I, daf-14(m77)IV, and daf-5(e1386)11.

Compound 20 Avasimibe, Pactimibe, F-1394, FR19254, S58035 were kept as 10 mM stock solutions at -20 C. All compounds were dissolved in DMSO. Compounds were assayed at a final concentration of 82.5 NM, except avasimibe which was assayed at 10.3 pM in the daf-14 mutant, and at 55 pM in the other mutants.
Results and discussion ACAT inhibitors affect dauer formation by reducing signalling through the TGF-R
atp hway All the ACAT inhibitors tested enhance dauer formation in the C. elegans daf-mutant, which is a dauer constitutive mutant in the TGF-R pathway (Figure 2).

This effect requires the activity of DAF-5, as all the ACAT inhibitors tested have no effect in the daf-4; daf-5 double mutant (Table 3). Indeed daf-5 is necessary for dauer formation due to TGF-P signalling inhibition, and the absence of induction of dauer formation by ACAT inhibitors in a daf-5 mutant background indicate that their effect on dauer is through TGF-R signalling pathway inhibition.
Moreover, the inventors specifically tested F-1394 and avasimibe in several other dauer constitutive mutants in the TGF-R pathway that are daf-7, daf-1, and daf-8.
The inventors found that F-1394 as well as avasimibe enhance dauer formation in these mutants (Figure 3), consistent with the results described in Figure 2.
Altogether, this indicates that ACAT inhibitors affects dauer formation specifically by reducing signalling through the TGF-R pathway.

Table 3:

Treatment % dauer Sample size Avasimibe 0 321 Pactimibe 0 50 Table 3: ACAT inhibitors do not induce dauer formation in the daf-4(m63);daf-5(e1386) double mutant. The following compounds were tested, as described in the Material and Methods section: Avasimibe (n=651), F1394 (n=935), Pactimibe (n=1166), FR179254 (n =1056), and S 58-035 (n=765). The DMSO solvent was used as a control (n=941). The tested compounds have no dauer inducing activity in the tested worm background.

Example 2: Effects of F-1394 on TGFRI-induced matrix protein expression in human lung epithelial cell line A549, an in vitro animal model relevant to human pulmonary fibrosis and COPD

Transforming growth factor (TGF)-Ri, a multifunctional or pleiotropic cytokine, participates in numerous biological processes, including cell proliferation, differentiation, apoptosis, fibrosis, wound repair and inflammation (Ning W.
et al.
2002; Martin G.E.M. et al. 2006). In particular, TGFPI is a growth factor with a role as critical mediator of the lung tissue response to injury; therefore, it is involved in the mechanisms of lung repair and fibrosis that follow inflammatory processes (Bellocq A. et al. 1999). Reactive oxygen and nitrogen intermediates increase TGFR, release from human epithelial alveolar cells in what may well constitute a molecular link between inflammatory and fibrotic processes (Bellocq A. et a/.
1999).
Fibrilar collagen type I is characteristically synthesised by fibroblastic-type cells (Kasai H. et al. 2005).

Previous work has demonstrated that TGFP, but not other inflammatory cytokines induced A549 cells with an alveolar epithelial type II cell phenotype to undergo an epithelial mesenchymal transition which includes the expression of the fibroblast phenotypic markers (Kasai H. et al. 2005).

Materials and methods Experimental model The human alveolar epithelial cell carcinoma line A549 (American Type Culture Collection; Rockville, MD) was grown as outlined in Mata M. et al. 2005.
Recombinant human TGFP, was used at a concentration of 5ng/mL. Medium was changed every 24 h with re-addition of TGFP, and F-1394 as appropriate. F-1394 was present from 1 h before first addition of TGFP, until the end of the experiment.

mRNA analysis For analysis of mRNA, cultured A549 cells were prepared as outlined in Mata M.
et al. 2005. Total RNA was extracted by using TriZol ReagentTM (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions. Integrity of the extracted RNA was confirmed by using the BioanalizerTM (Agilent, Palo Alto, CA, USA). a1 (I) collagen mRNA was determined by quantitative RT-PCR as outlined (Manoury B. et al. 2006). Time of determination: Time point at 72 h as previously outlined (Kasai H. et al. 2005).

Experimental groups The in vitro experiments were carried out with the appropriate negative and positive controls as well as treated groups with F-1394 and reference compound and their respective controls as follows:
1) control vehicle (DMSO) 2) F-1394 (in DMSO) dose level 3 (3 pg/mL) 3) TGFO, + DMSO

4) TGF(3l + F-1394 (in DMSO) dose level 1(0.3 pg/mL) 5 TGF(3, + F-1394 (in DMSO) dose level 2 (0.6 pg/mL) 6) TGFR, + F-1394 (in DMSO) dose level 3 (3 pg/mL).
Results and discussion Data are presented as mean SEM. Statistical analysis of results was carried out by analysis of variance (ANOVA) followed by Bonferroni test (GraphPad Software Inc, San Diego, CA, USA). Significance was accepted when P<0.05.

TGF(ii produced a marked increase in the a1 (I) collagen mRNA expression as shown in Figure 4.

This TGFR,-induced augmented expression was significantly reduced in a concentration-dependent fashion in the presence of F-1394 (Fig. 4).

F-1394 as a prototypic ACAT inhibitor is effective to reduce markedly the augmented a1 (I) collagen mRNA expression evoked by TGF(3, in cultured A549 cells, a human airway epithelial cell line.

Example 3: Effects of F-1394 on bleomycin-induced pulmonary damage, an in vivo animal model relevant to human pulmonary fibrosis Bleomycin-induced pulmonary damage is an established model considered relevant in the investigation of anti-inflammatory and anti-fibrotic (remodeling) activities of new compounds.

Materials and methods A total number of 66 mice entered the study. The number of animals per group was apporximately10-12 (as required). Each mouse had a body weight at delivery (6 weeks) of approximately 20 g.

F-1394 was orally administered at 300 mg/kg as a suspension with 0.5%
carboxymethylcellulose (CMC) for 14 days. N-acetyl-L-cysteine (NAC) was used as reference article. Both F-1394 and NAC were administered orally by gavage at the volume of 10 mi/kg.

Experimental model Male adult C57BL/6 male mice, each weighing about 20 g, received endotracheally, by the transoral route, a single dose of 0.075 U bleomycin sulphate (Sigma Catalogue B 5507, (conservation at 2-8 C, 1.2-1.5 units per mg solid) dissolved in 50 pl saline (0.9% NaCI). Control animals were subjected to the same protocol but received intratracheal saline instead of bleomycin.
Tracheal instillation was carried out under halothane anaesthesia. Fourteen days after endotracheal bleomycin/saline administration, the animals were killed by anaesthesia (sodium pentobarbital) followed by exsanguination from abdominal aorta. Lungs were obtained and weighed. Then the right and left lungs were processed separately for histological and biochemical studies as indicated below.

The 14th-day after bleomycin was selected as peak time for collagen synthesis (Lazemby et al 1990).

Histological evaluation For histological examination, the lung was first perfused through its main 5 bronchus with a fixative solution (10% neutral-buffered formalin) maintained at 25 cm of hydrostatic pressure for 15 min, immersed in the fixative for 24 h, and blocks were taken. Only lungs that were well inflated by the fixative were analyzed. Tissue blocks were placed in formalin, dehydrated in a graded series of ethanol, embedded in paraffin, cut into 4-pm-thick serial sections, and stained 10 with haematoxylin - eosin to identify inflammatory cells and fibrotic areas.
Histologic grading of lesions was performed by using a blinded semiquantitative scoring system for extent and severity of inflammation and fibrosis in lung parenchyma based on previous studies from this laboratory (Cortijo et al., 2001;
Mata et al., 2003; Serrano-Mollar et al., 2002; Serrano-Mollar et al., 2003).
The 15 severity of fibrosis was scored according to Ashcroft and co-workers.
Briefly, the grade of lung fibrosis was scored on a scale from 0 to 8 by the following criteria:
grade 0, normal lung; grade 1, minimal fibrous thickening of alveolar or bronchiolar walls; grade 3, moderate thickening of walls without obvious damage to lung architecture; grade 5, increased fibrosis with definite damage to lung 20 structure and formation of fibrous bands or small fibrous masses; grade 7, severe distortion of structure and large fibrous areas; grade 8, total fibrous obliteration of fields. Grades 2, 4 and 6 were used as intermediate pictures. The severity of fibrotic changes in each lung section was assessed as the mean score of severity from the observed microscopic fields. Ten random fields in each section were 25 analysed. Grading was performed in a blinded fashion by two observers, and the mean value was taken as the fibrosis score).

Experimental groups The in vivo experiment was carried out with the appropriate negative and positive controls as well as groups receiving the different drug treatments and their respective controls (drug vehicle; treated-negative controls) as necessary.

= Negative control: F-1394-vehicle (oral) + saline (intratracheal) = Positive control: F-1394-vehicle (oral) + BLEOMYCIN (intratracheal) = F-1394 300 mg/kg (oral) + BLEOMYCIN (intratracheal) = NAC 500mg/kg (oral) + BLEOMYCIN (intratracheal) Results and discussion Data are presented as mean SEM. Statistical analysis of results was carried out by analysis of variance (ANOVA) followed by Bonferroni test or by non-parametric tests as appropriate (GraphPad Software Inc, San Diego, CA, USA). Survival curves were analysed by logrank test by using GraphPad Software Inc (Prism 4.03). Significance was accepted when P<0.05.

a1 (I) collagen mRNA in lung tissue F-1394 as well as NAC showed a marked inhibitory effect on the expression of a1 (I) collagen transcripts (Figure 5) which indicates an anti-fibrotic effect with decrease of collagen deposition.

F-1394 as a prototypic ACAT inhibitor is a valuable compound endowed with anti-fibrotic properties at the level of a1 (I) collagen mRNA expression in lung tissue.
Histological evaluation Lungs from control animals were histologically normal. The administration of bleomycin resulted in characteristic histological lesions including areas of marked peribronchiolar and interstitial infilltration with inflammatory cells (predominantly mononuclear cells including macrophages and lymphocytes with fewer numbers of neutrophils and scattered eosinophils), extensive cellular thickening of interalveolar septa, interstitial oedema, and increases in interstitial cells with a fibroblastic appearance. The pattern of distribution of lesions was multifocal (i.e.
patchy areas of pulmonary fibrosis). Although multifocal parenchymal lesions were also present, treatment with F-1394 significantly reduced the changes in lung morphology; there were fewer inflammatory infiltrates, less collagen deposition and less septal widening (Fig. 6). A significant reduction in the Ashcroft score was noted (Fig. 7). Similar results were obtained for N-acetylcysteine (NAC).

Example 4: Effect of F-1394 in Unilateral Ureteral Obstruction in mice Unilateral Ureteral Obstruction (UUO) is a well-established accelerated experimental model that mimics the different features of obstructive nephropathy leading to tubulointerstitial fibrosis. Because of its fastness and high reproducibility, this model was used for providing a rapid proof of concept for the use of F-1394 as a prototypic ACAT inhibitor in the prevention interstitial collagen accumulation.

The aim of the present study was to evaluate the effects of a preventive treatment with F-1394 on whole interstitial fibrillar collagen deposition by Sirius red histochemistry. The effects of F-1394 were evaluated by quantification of the specific stained areas relatively to the whole obstructed kidney section surface.

Materials and methods Animals Male C57BL/6J mice, weighing 25-30 g at the beginning of the experiments, were used. Mice were housed per group of five (5) in polypropylene cages on wood litter with free access to food (TekladTM 5018; HARLAN, Gannat, France) and water. The animal house was maintained under artificial lighting (12 hours) between 7:00 and 19:00 in a controlled ambient temperature of 20 1 C, and relative humidity maintained at 60%. Mice were sacrificed by cervical dislocation.
Animals were not submitted to autopsy.

Study materials F-1394 dosage formulation was prepared with two different ways; 200 mg/kg solution using 1%Tween80 and 0.5% CMC, and 300mg/kg solution using 0.5%
CMC alone. F-1394 was administered orally at a dose of 200 mg/kg/d for the first 10 days and 300 mg/kg/d for the 5 remaining days of the protocol. Captopril (Sigma-Aldrich, St Louis, USA) was dissolved in 1% Tween80 and 0.5% CMC
and administered orally at a dose of 32 mg/kg/d for 15 days.

Principal equipment Morphometric measurement was based on computerized image analysis of Sirius red-stained sections, observed through a microscope (Eclipse 600TM , Nikon Instruments; Champigny sur Marne, France) and numeric camera (MicroFireTM
Optronics; Avanex, Nozay, France) with a 20X objective lens (final calibration 0.366 pm/pixel). Quantitative analysis of the pictures was performed using the ExploraNova MorphoLiteTM and ExpertT"" softwares (La Rochelle, France).

Experimental protocol Four experimental groups with 10 mice each were used:
1) Sham operated mice 2) Obstructed mice treated with vehicle (0.1 % Tween80 / 0.5% CMC) 3) Obstructed mice treated with F-1394 4) Obstructed mice treated with captopril Oral treatments lasted fifteen (15) days and obstruction fourteen (14) days.
Experimental unilateral ureteral obstruction Unilateral ureteral obstruction was performed as described by Schanstra et al., 2002. Briefly, mice were anesthetized with a mix of oxygen-isoflurane (0.2 L/min and 2% isoflurane; Centravet, Lapalisse, France). A 5 mm cutaneous incision was performed at 1 cm from the last left costal edge of the mouse, followed by a 5mm muscular incision. The left ureter was exposed, and ligated (6/0 cardionyl thread; Peters Surgical, Bobigny, France) at the uretero-pelvic junction.
Then, the muscle wall was closed (5/0 Ethicrin thread; Ethicon, Auneau, France), and the cutaneous wall was closed by a suture clip. All along the surgery, animals were set on a 35 C hotplate.

Food and water were given ad libitum. The oral treatments began one day before UUO, obstruction lasted 14 days. At the end of the protocol, mice were killed by cervical dislocation; obstructed kidneys were removed, and divided for preparation of histology and further gene expression analyses.

Histological analysis After the obstructed kidneys were removed, the central fragment used for histology was fixed in Carnoy's solution for 24 hours and embedded in paraffin according to standard procedures. Transverse 3-4-pm-thick histological sections were performed and stained with Sirius red for whole interstitial fibrillar collagen deposition evaluation.

Sections were deparaffinized, rehydrated, and then stained with 0.1% Sirius red F3BTM (BDH Laboratory; VWR, Fontenay sous Bois, France) in saturated aqueous picric acid for 1 hour, differentiated in 0.01 N HCI for 1 minute, and rapidly dehydrated and mounted.
The tubulointerstitial fibrillar collagen surface was determined by measuring the area of stained tissue within either the whole cortical area or a given field.
Glomeruli and vasculature were excluded from evaluation in the present study.
Recording analysis Analyses were performed by an operator unaware of the origin of each kidney section. Double blind quantifications of the selected objects within a given image were recorded into a Microsoft Excel file combined to the ExploraNovaTM
software. Removal of the histological codes was performed at the end of the recordings for results classification.
Results were expressed as percentage (%) of fibrosis in relation to the whole area studied.

Analysis and expression of results The results were given as mean values standard error of the mean (SEM). A
one-way ANOVA was used for comparison of within-group difference followed by a Newman-Keuls test for comparison of all pairs of columns (GraphPad Prism, San Diego, USA). A p<0.05 was accepted for statistical significance.

Results and discussion Effects of F-1394 and captopril on UUO-induced tubulointerstitial fibrosis Interstitial fibrosis was significantly higher in the UUO group treated with vehicle group as compared with sham operated animals (Figure 8), with an interstitial 5 collagen expression that represented 13.36 0.60% and 0.26 0.03% of the kidney sections, respectively (n=10, p<0.001, ANOVA with Newman-Keuls test). This effect was in accordance with the previous publication (Schanstra et al, 2003 and 2002).

F-1394 decreased UUO-induced tubulointerstitial fibrosis comparing with vehicle 10 group (5.43 0.29% vs. 13.36 0.60% in F-1394 and vehicle groups, respectively, n=10, p<0.001, ANOVA with Newman-Keuls test). Captopril treatment led to an expected decrease in the UUO-induced tubulointerstitial fibrosis (2.89 0.12%
vs.
13.36 0.60% in captopril and vehicle groups, respectively, n=10, p<0.001, ANOVA with Newman-Keuls test).
15 These results show that the administration of F-1394 as a prototypic ACAT
inhibitor significantly reduces tubulointerstitial fibrosis in the UUO-model with comparable efficacy to captopril.

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Claims (27)

1- A composition useful for the prevention or reduction of collagen deposition in a tissue, the composition comprising an effective amount of at least one ACAT
inhibitor comprising F-1394, Avasimibe, Pactimibe (CS505), Eflucimibe (F12511), Eldacimibe, NTE 122, AS183, KW3033, E5324, FY087, FCE27677, C1976, TEI6522, K604, Octimibate, FR179254, S58-035, or a mixture thereof and an acceptable excipient.

2- The composition of claim 1, wherein the ACAT inhibitor is F-1394.

3- The composition of claim 1, wherein the tissue is a tissue of a heart, a lung, a brain, an eye, a stomach, a spleen, a bone, a pancreas, a kidney, a liver, an intestine, a skin, an uterus or a bladder.

4- An antifibrotic composition comprising an effective amount of at least one antifibrotic agent and an acceptable excipient.

5- The antifibrotic composition of claim 4, wherein the antifibrotic agent is an ACAT inhibitor.

6- The antifibrotic composition according to claim 5, wherein the ACAT
inhibitor is F-1394, Avasimibe, Pactimibe (CS505), Eflucimibe (F12511), Eldacimibe, NTE 122, AS183, KW 3033, E5324, FY 087, FCE 27677, Cl 976, TEI
6522, K604, Octimibate, FR179254, S 58-035 or a mixture thereof.

7- The antifibrotic composition of claim 6, wherein the ACAT inhibitor is F-1394, Avasimibe, Pactimibe, FR179254 or S 58-035.

8- The antifibrotic composition of claim 7, wherein the ACAT inhibitor is F-1394.

9- Use of an ACAT inhibitor for preventing or reducing collagen deposition in a tissue.

10- Use according to claim 9, wherein the ACAT inhibitor is F-1394, Avasimibe, Pactimibe (CS505), Eflucimibe (F12511), Eldacimibe, NTE 122, AS183, KW 3033, E5324, FY 087, FCE 27677, Cl 976, TEI 6522, K604, Octimibate, FR179254, S 58-035 or a mixture thereof.

11- The use according to claim 9, wherein the tissue is a tissue of a heart, a lung, a brain, an eye, a stomach, a spleen, a bone, a pancreas, a kidney, a liver, an intestine, a skin, an uterus or a bladder.

12- Use of an ACAT inhibitor for preventing the formation or development of excess fibrous connective tissue in an organ.

13- The use according to claim 12, wherein the ACAT inhibitor is F-1394, Avasimibe, Pactimibe (CS505), Eflucimibe (F12511), Eldacimibe, NTE 122, AS183, KW 3033, E5324, FY 087, FCE 27677, Cl 976, TEI 6522, K604, Octimibate, FR179254, S 58-035 or a mixture thereof.

14- The use according to claim 12, wherein the tissue is a tissue of a heart, a lung, a brain, an eye, a stomach, a spleen, a bone, a pancreas, a kidney, a liver, an intestine, a skin, a uterus or a bladder.

15- A method for reducing the level of collagen in a tissue, the method comprising providing a tissue, contacting the tissue with at least one ACAT
inhibitor and measuring a reduced level of collagen in the tissue.

16- The method of claim 15, wherein the ACAT inhibitor is F-1394, Avasimibe, Pactimibe (CS505), Eflucimibe (F12511), Eldacimibe, NTE 122, AS183, KW
3033, E5324, FY 087, FCE 27677, Cl 976, TEI 6522, K604, Octimibate, FR179254, S 58-035 or a mixture thereof.

17- The method of claim 15, wherein the tissue is a fibrotic tissue.

18- The method of claim 15, wherein the tissue is a tissue of a heart, a lung, a brain, an eye, a stomach, a spleen, a bone, a pancreas, a kidney, a liver, an intestine, a skin, an uterus or a bladder.

19- A method for preventing the formation or development of excess of fibrous connective tissue in an organ of a subject, the method comprising administering to said subject an effective amount of at least one ACAT inhibitor.

20- The method of claim 19, wherein the at least on ACAT inhibitor is F-1394, Avasimibe, Pactimibe (CS505), Eflucimibe (F12511), Eldacimibe, NTE 122, AS183, KW 3033, E5324, FY 087, FCE 27677, CI 976, TEI 6522, K604, Octimibate, FR179254, S 58-035 or a mixture thereof.

21- The method of claim 19, wherein the organ is a heart, a lung, a brain, an eye, a stomach, a spleen, a bone, a pancreas, a kidney, a liver, an intestine, a skin, a uterus or a bladder.

22- The method of claim 21, wherein the subject is a human.

23- A method for preventing or treating fribrosis or a fibrotic disorder in a subject, the method comprising:

-identifying a subject suffering from or being at risk of developing fibrosis;

-administering to said subject an ACAT inhibitor in an amount sufficient to decrease the level of collagen; and -measuring a reduced level of collagen in the subject.

24- The method of claim 23, wherein the ACAT inhibitor is F-1394, Avasimibe, Pactimibe (CS505), Eflucimibe (F12511), Eldacimibe, NTE 122, AS183, KW
3033, E5324, FY 087, FCE 27677, CI 976, TEI 6522, K604, Octimibate, FR179254, S 58-035 or a mixture thereof.

25- The method of claim 24, wherein the ACAT inhibitor is F-1394.

26- The method of claim 23, wherein the subject is a human.

27- The method of claim 23, wherein the fibrotic disorder is scleroderma, keloids and hypertrophic scars, collagen disorders associated with the occurrence of Raynaud's syndrome, pulmonary inflammation and fibrosis, interstitial lung diseases, idiopathic pulmonary fibrosis, sarcoidosis, liver cirrhosis and liver fibrosis resulting from viral, or from parasitical infection, kidney disorders associated with unregulated TGF-.beta. activity, excessive fibrosis, renal interstitial fibrosis, renal fibrosis in transplant patients, focal glomerulosclerosis, fibrosis caused by Marfan's disease, cardiac fibrosis, radiation-induced fibrosis, fibrosis arising from wound healing, eye diseases, peritoneal fibrosis, intestinal fibrosis, and chemotherapeutic drug-induced fibrosis or burns.