BRPI0718630A2 - ACAT INHIBITORS AND THEIR USE IN FIBROSIS PREVENTION OR TREATMENT. - Google Patents

Description

ACAT INHIBITORS AND THEIR USE IN FIBROSIS PREVENTION OR TREATMENT

FIELD OF INVENTION

The present invention provides novel methods and compositions for the prevention and reduction of 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 for preventing and reducing excess fibrous connective tissue in an organ. .

BACKGROUND OF THE INVENTION

The process of tissue repair as part of healing involves two phases. The first phase is regenerative phase 15, in which the injured cells are replaced by cells of the same type. The second phase is the formation of fibrous tissues, also called fibroplasia or fibrosis, in which connective tissue replaces the parenchyma. Tissue repair can become pathogenic if the fibrotic phase remains unrefragable, leading to extensive tissue remodeling and permanent scarring (Wynn 2004).

Fibrotic disorders affect almost all tissue and organ systems. Major fibrotic disorders include interstitial lung disease (PID), which leads to pulmonary inflammation and fibrosis. PID is known to have a number of causes such as sarcoidosis, silicosis, collagen vascular diseases. The most common type of PID is idiopathic pulmonary fibrosis (IPF), which has no known cause yet. Other fibrotic disorders include liver cirrhosis and fibrosis originating from viral hepatitis, renal diseases associated with unregulated TGF-β activity and excessive fibrosis such as glomerulonephritis (NG), renal interstitial fibrosis, renal fibrosis in 10 transplanted patients, and eye diseases such as macular degeneration and retinal and vitreous retinopathy. In addition, fibroproliferative disorders include systemic and local scleroderma, keloids, 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 disorders.

Acyl CoA: Cholesterol Acyltransferase (ACAT)

It catalyzes the formation of cholesterol esters using both cholesterol and coenzyme A long chain fatty acid as a substrate. ACAT is present in a variety of tissues 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 generally involve immunosuppressive drugs such as corticosteroids and other anti-inflammatory drugs. These treatments are not always effective in reducing or preventing fibrosis probably because the mechanisms involved in regulating fibrosis appear to be distinct from these inflammation and anti-inflammatory therapies. Few research therapies 10 have been shown to alter or nullify the inflammatory process that is associated with fibrosis.

In addition, a wide range of similar molecular targets for antifibrotic therapy has been identified, for example, in those directly involved with fibrogenesis, including matrix metalloproteinases and their inhibitors, pro-fibrogenic cytokines, such as tumor growth factor P (TGF-P). P (TGF-P) plays a key role in initiating the cascade of events that culminates in cytokine production and in the excess and failure of collagen expression and other extracellular matrix components.

Several animal models are useful for identifying and characterizing new antifibrotic agents. The antifibrotic effects of pirfenidone have recently been demonstrated in the pulmonary fibrosis hamster bleomycin model (Iyer, Margolin et al. 1998) and in a unilateral ureteral renal fibrosis obstruction model (Shimizu, Kuroda et al. 1998). An ALK5 inhibitor (TGF-Preceptor II) has been shown to be protective against dimethylnitrosamine-induced liver fibrosis (DMN) in the rat model (de Gouville Huet and 2006).

The present inventors have realized that ACAT inhibitory compounds are molecules of choice for the treatment and prevention of fibrosis.

SUMMARY OF THE INVENTION The present invention provides novel methods and

compositions for the prevention and reduction of fibrosis and / or fibrotic disorders using ACAT inhibitors.

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

In another aspect, the present invention relates to the use of an ACAT inhibitor to prevent or reduce collagen tissue deposition.

In addition, another aspect of the present invention relates to

regarding the use of an ACAT inhibitor to prevent the formation or development of excess fibrous connective tissue in an organ.

The present invention also includes 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 the 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 fibrous connective tissue in an organ of an individual. The method comprises administering to the individual an effective amount of at least one ACAT inhibitor. .

In another aspect, the invention provides a method for

prevent or treat fibrosis or a fibrotic disorder in an individual, the method comprising:

- identify an individual suffering from fibrosis or at risk of developing it;

administering to said individual an ACAT inhibitor in

an amount sufficient to decrease the level of collagen; and

- measure a reduced level of collagen in the individual.

DESCRIPTION OF THE FIGURES

Figure 1 shows a schematic representation of the

via TGF-β in humans with homologous larvae.

Figure 2 shows that ACAT inhibitor decreases TGF-β signaling. The dauer formation was measured in the daf-14 (thousand) mutant. The following compounds were

tested, Avasimiba (n = 651), F-1394 (n = 935), Pactimiba (η = 1166), FR179254 (η = 1056), and S 58-035 (η = 765). DMSO was used as a control solvent (n = 941). All tested compounds reinforced dauer formation in relation to treatment control.

Figure 3 shows that the ACAT inhibitor decreases the

TGF-β signaling in more than one constitutive TGF-β mutant. Dauer formation induced by Avasimiba (a), and F-1394 (b) was measured in Dauer mutants as described in the Materials and Methods section of Example I. (a) Avasimiba was tested on daf-7 (el372) (n = 560, versus 539 for control); in daf-1 (M40) (n = 870, versus 636 for control), and in daf-8 (el393) (n = 852, versus 502 for control). (b) F-1394 was tested on daf-7 (el372) (n = 99, versus 134 for control); in daf-1 (M40) (n = 145, 15 versus 108 for control), and in daf-8 (el393) (n = 47, versus 140 for control). The tested compounds reinforce dauer formation in all mutants with respect to treatment control.

Figure 4 shows the quantitation relationship of the 20 levels of al (I) collagen mRNA expression in unstimulated (control) or growth factor stimulated (TGF; 5 ng ml ^ 1) -β A cells if transformed in the absence or presence of F-139 (0.3, 0.6 and 1 μς ml-1 as indicated). Exposure time was 72 h for TGFPi. F-1394 was present from 1h before TGFPx until the end of the experiment. Induced TGFβ1 plus mRNA expression of collagen al (I) was inhibited depending on the concentration by F-1394. Collagen mRNA expression al (I) was calculated using real-time RT-PCR by the AACt method; the 5 columns show the increased fold in mRNA expression of collagen al (I) in relation to GAPDH values as mean and pm of 2-AACt values from three independent experiments. *: p <0.05 versus control; #: p <0.05 versus TGFPx.

Figure 5 shows the relationship of mRNA expression of collagen al (I) in lung tissue of endotracheal bleomycin (BLM) or saline (control) mice. The columns are average of 3 to 5 animals per group. * P <0.05 versus control, # P <0.05 versus BLEO by ANOVA.

Figure 6 shows representative photomicrographs of the

pulmonary 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 20 N-acetyl cysteine + bleomycin (i). All pulmonary sections were stained with hematoxylin-eosin. The x40 magnification. Animals treated with saline solution showed a normal architecture. Mice exposed to bleomycin showed peribronchial interstitial infiltration marked with inflammatory cells, thickening of the alveolar septa, edema, and dense foci of fibrosis. These lung lesions were reduced in animals treated with oral F-1394 and N-acetylcysteine.

Figure 7 shows the evaluation of fibrotic changes in the lung by numerical fibrotic score. The Ascroft score denotes Bars (mean ± epm) of each experimental group as indicated. * P <0.05 versus vehicle + BLEO by Mann-Whitney test. Lung lesions were reduced in animals treated with oral F-1394 as well as N-acetylcysteine.

Figure 8 shows the percentage of fibrosis measured after red Sirius staining in ten non-overlapping cortical fields are expressed as the average percentage of stained interstitial area; *** 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 the prevention and reduction of fibrosis associated with fibrotic disorders using ACAT inhibitors. Definitions

As used herein, the term ACAT inhibitor means any compound that inhibits the enzyme acyl-CoA: cholesterol acyltransferase (ACAT). Examples of ACAT inhibitors include small organic compounds, that is, having a molecular weight of more than 50 yet and less than about 2500. ACAT inhibitors include functional chemical groups required for interactions with proteins and / or molecules. nucleic acids and usually include at least one amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemicals. ACAT inhibitors may include carbons of cyclic or heterocyclic structures and / or aromatic or polycyclic structures substituted with one or more of the functional groups identified above.

ACAT inhibitors according to the present invention are, for example, the following compounds: F-1394, Avasimiba, Pactimiba (CS-505), Eflucimiba (F 12511), Eldacimiba, NTE 122, AS-183, KW- 3033, E5324, FY 087, FCE 20 27677, CI 976, TEI 6522, K-604, Octimibate, FR17924 and S 58-035, 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 the accumulation of fibroblasts and excessive collagen deposition above the normal deposition of a given tissue.

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

Fibrotic disorder includes, but is not limited to scleroderma, keloids and hypertrophic scars, collagen disorders associated with Raynaud's syndrome, pulmonary inflammation and fibrosis, pulmonary interstitial diseases, idiopathic pulmonary fibrosis, sarcoidosis, liver cirrhosis, and hepatic fibrosis. resulting from viral or parasitic infection, kidney disorders associated with unregulated TGF-β activity, excess fibrosis such as glomerulonephritis (GN), renal interstitial fibrosis 15, renal fibrosis in transplant patients, focal glomerulosclerosis, fibrosis caused by Marfan disease, cardiac fibrosis, radiation-induced fibrosis, fibrosis due to wound healing, ophthalmic diseases such as macular degeneration and vitreous and retinal retinopathy. It also includes peritoneal fibrosis, intestinal fibrosis, burn-induced fibrosis, and chemotherapeutic drugs.

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

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

Liver fibrosis refers to the accumulation of hard scarring fibrosis in an individual's liver.

As used herein, the term individual refers to animals and humans. This term includes, but is not limited to, primates, domestic animals such as dogs, cats, sheep, cattle, goats, pigs, mice, rats, rabbits, guinea pigs, captive animals such as zoo animals, and animals. wild.

As used herein, the term refers to a specialized tissue or organ or cell set, such as skin tissues, lung tissue, renal tissue, and other cell types.

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

As used herein, the term prevention / prevention refers to a process whereby the symptoms of fibrotic disorders, fibrosis diseases, fibrosis and related diseases as exemplified above are obstructed, retarded or prevented.

As used herein the term effective amount indicates an amount that produces the desired effect as appreciated by the results of clinical trials and / or animal models. This amount may be routinely determined by a person skilled in the art. The effective amount of a composition to be employed will depend, for example, on the context and objectives of the treatment. One skilled in the art will appreciate that appropriate dose levels for treatment will vary, in part, by the type of ACAT inhibitor delivered, by indicating that the ACAT inhibitor is being used, by the route of administration, and by by size (body weight, body surface 20 or organ size) and conditions (age and overall health) of the patient. Thus, the clinician can titrate the dose and modify the route of administration for better therapeutic effects.

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 and monkeys. An animal model can also be used to determine the appropriate concentration range and route of administration. This information can then be used to determine doses and routes useful for administration to humans.

As used herein, the term acceptable excipient means an ingredient used in a composition that does not interfere with the effectiveness of the biological activity of the active ingredient (s) of the composition, and is non-toxic to the host, tissues or organs intended for use. be treated.

The term used herein as an acceptable excipient means an ingredient used in a composition that does not interfere with the effectiveness of the biological activity of the active ingredients of the composition and is non-toxic to the host or patient. In addition, the excipient is advantageously a compound with a minimal probability of being rejected by the immune system of the subject to be treated.

Such acceptable and / or pharmaceutically acceptable excipients are used for various purposes, such as stabilizers, buffers, suspending agents, vehicles and the like, and are listed and described in various articles including, for example, British Pharmacopoeia XXII and National Pharmacopoeia XVII. and its supplements.

Use of ACAT inhibitors in the prevention and treatment of fibrosis or fibrotic disorders.

5th

The inventors of the present invention have found that a series of ACAT inhibitory compounds are useful for reducing or preventing fibrosis and related diseases and are well accepted in animal models of fibrotic disorder.

In one aspect, the present invention provides compositions

antifibrotic agent comprising an effective amount of at least one agent and an acceptable antifibrotic excipient. More particularly, the antifibrotic agent is an ACAT inhibitor. For example, the ACAT inhibitor may be an ACAT inhibitor as defined in Table 1 or a mixture thereof.

CAS registration number and / or publication / patent describing the preferred ACAT inhibitor is contemplated by the present invention and are found in Table 1. Including the following compounds: F-1394, Avasimiba, Pactimiba (CS-505), Eflucimiba (F12511), Eldacimiba, NTE 122, AS-183, KW-3033, E5324, AF 087, FCE 27677, CI 976, TEI 6522, K-604, Octimibate, FR 179254 and S 58-035.

In a related aspect, the invention relates to a method for preventing or treating fibrosis or fibrotic disease in an individual. The method includes a first step of identifying an individual suffering from at risk of developing fibrosis, followed by a step of administering to the individual an ACAT inhibitor in an amount sufficient to decrease the collagen level. The method also includes a 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 prevented is by

example: scleroderma, scleroderma, keloids and hypertrophic scars, collagen disorders associated with Raynaud's syndrome, pulmonary inflammation and fibrosis, pulmonary interstitial diseases, idiopathic pulmonary fibrosis, sarcoidosis, liver cirrhosis, and liver fibrosis resulting from viral or parasitic infection , kidney disorders associated with unregulated TGF-β activity, excess fibrosis, renal interstitial fibrosis, renal fibrosis in transplant patients, focal glomerulosclerosis, fibrosis caused by Marfan's disease, cardiac fibrosis, radiation-induced fibrosis, fibrosis due to wound healing. wounds, eye diseases, peritoneal fibrosis, intestinal fibrosis, chemotherapy drug-induced fibrosis or burns.

25 Using ACAT Inhibitor to Prevent or Reduce Collagen 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.

Thus, the present invention relates to the use of at least one ACAT inhibitor to prevent or reduce collagen tissue deposition.

By the term "preventing or reducing collagen deposition of a tissue" is meant that excess collagen deposition relative to normal deposition is obstructed, retarded or prevented in a given tissue, or that excess collagen deposited in tissue in comparison with a normal deposition is attenuated or eliminated.

For example, ACAT inhibitors may be used to prevent or reduce collagen deposition, tissues may be one of the ACATs defined in Table 1 or a mixture thereof.

The present invention also provides a method for reducing the level of collagen in a tissue comprising a step of providing a tissue followed by a step of making contact with the tissue with at least one ACAT inhibitor. The next step is to measure 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 can also be a tissue of the heart, lung, brain, eyes, stomach, spleen, bones, pancreas, kidneys, liver, intestines, skin, uterus and bladder.

In a related aspect, the present invention also relates to a composition useful in preventing or reducing collagen deposition in a tissue, composition 10 comprising an effective amount of at least one ACAT inhibitor as defined in Table 1 or a mixture thereof, and an acceptable excipient.

It will be understood that the tissue, for example, is a tissue of an organ of an individual, such as a human being. Organ tissue, for example, is a tissue selected from an organ such as the heart, lungs, brain, eyes, stomach, spleen, bones, pancreas, kidneys, liver, intestines, skin, uterus and bladder.

Use of ACAT inhibitors to prevent formation or development of excess fibrous tissue in an organ

In the tissue repair process, the fibrosis phase consists of the formation of fibrous tissue in the connective tissue that replaces the tissue parenchyma. If the fibrosis phase remains uncontracted, the tissue repair process can become pathogenic, leading to extensive tissue remodeling.

Thus, the present invention proposes to use an ACAT inhibitor to prevent excess formation or development 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 fibrous connective tissue in an organ "means that formation of fibrous connective tissue in addition to normal formation is obstructed, retarded or prevented in a particular organ.

It will be appreciated that the organ is, for example, a

of an individual, such as a human. The organ is, for example, heart, lung, brain, eye, stomach, spleen, bone, pancreas, kidney, liver, intestine, skin, uterus or bladder.

In a related aspect, the present invention

provides a method for preventing the formation or development of excess fibrous connective tissue in an organ of an individual comprising administering to an individual effective amount of at least one inhibitor of

ACAT More particularly, the ACAT inhibitor may be an ACAT inhibitor as defined in Table 1 or a mixture thereof. In a preferred embodiment, the organ is selected from the heart, lungs, brain, eyes, stomach, spleen, bones, pancreas, kidneys, liver, intestines, skin, uterus and bladder and the individual is a human being.

Table 1:

INBIDOR Structure CAT Reference Number CAS registration F-1394 X ^ 135392-43-7 Patent (Fujirebio (1S, 2S) -2- [3- (2,2-dimethylpropyl) -2-American Inc.) nonylureido] cyclohexane -1-yl-3 - [(4R) -N- (2,2,5,5,5,120,738 tetramethyl-1,3-dioxane-4-Kusunoki, carbonyl) amino] propionate Aragane et al. 1995) Avasimiba CH3 166518-60-1 Patent (CI-111) I Zch = American CH3 (Pfizer) ^ #; s \ / ^ '' '-ch3 H3C - ^ / / 5,491,172 Yvo ^ 0AJJ CHl (Delsing, H = C3 -CH3 T Offerman et al H3C CH3 2001) N - ((2,6-bis (1-methylethyl) phenoxy) sulfonyl) -2,4,6-tris (1-methylethyl) benzylacetamide Pactimiba OH CH3 189198-30-9 US Patent (CS505) °% NH / Γ-CH3 / 5,990,150 H3C [CH3] H3C 1H-Indole-5-acetic acid, 7 - ((2,2-dimethyl-1-oxopropyl) amino) -2,3-dihydro-4,6-dimethyl-1-octyl-Eflucimib S) -2 ', 3', 5'-Trimethyl-4'-hydroxy-alpha-202340-45-2 (Junquero, Oms (F12511) dodecylthioacetanilide et al. 2001) Eldacimiba CH3 141993-70-6 Patent (Wyeth- "X0 American Ayerst / H3C --- vy 5,179,216 ΑΗΡ) H3C CH" (CH3 [5 - (((3,5-bis (1,1-dimethylethyl) -4-hydroxyphenyl) amino) ((((4- (2,2-dimethylpropyl) phenyl) methyl) hexylamino) methylene-2,2-dimethyl] -1,3-dioxane-4,6-dione ΝΤΕ 122 - ----- '--- Patent (Nissin Food Trans-1,4-bis [[1-cyclohexyl-3- (4-American Products Co., dimethylaminophenyl) ureido] methyl] 5,733,931 Ltd.) cyclohexane) (Azuma, Kawasaki et al. 1998; Azuma, Seto et al. 1999) AS-183 CH3 CH3 CH3 CH3 CH3 CH3 (Kuroda, 0γ4νΑ ^ ΛΑ / ΟΗ3 Yoshida et al. OH ^ V0 1993) CH3 KW-3033 Br Patent --- The American Enantiomer (-) - 1 of the above molecule: 5,340,807 ( -) - 2-Bromo-N- (2,6-diisopropylphenyl) -6,11 - (Kumazawa, dihydrodibenz [6, e] oxepin-11-carboxamide Γ (-) - 1] Yanase et al. 1996) Ε5324 CH3141799-76-0 US Patent IX 5,668,136 H 3 C (n-butyl-N '- [2- [3- (5-ethyl-4-phenyl-1 H -imidazol-1-yl) propoxy] -6-methylphenyl ] urea) FY 087 H, C US Patent CH 3 O 5,405,873 LkA 4 (Nagata, II H Yonemoto et al. 1995) (N- [2- [N'-pentyl- (6,6-dimethyl-2,4-heptadinyl) amino] ethyl] - (2-methyl-1-naphthylthio) acetamide) FCE 27677 KY ( Chiari, (-) N- [2,6-bis (1-methylethyl) phenyl] -N '- [(4R, 5R) -2- (4-Lovisolo et al. Dimethylaminophenyl) -4,5-dimethyl dioxolan (1996) iljmethylurea Cl 976 wS, 114289-47-3 (Bocan, I Mueller et al. CH3 1991) (2,2-dim ethyl-N- (2,4,6- (Sinz, Black et trimethoxyphenyl) dodecanamide) al. 1997) 60-604 / (Ikenoya, Rnr Yoshinaka, et 2- [4- [2- (benzimidazol-2-ylthio) ethyl] al. 2007) piperazin-1-yl] -7V- [2,4-bis (methylthio) -6-methyl-3-pyridylacetamide. TEI6522 / CH3 (Kataoka, (FJJ) Sqj Shiota et al. J "1995) H3C N- (7-methoxy-4-oxochroman-8-yl) -2,2-dimethyldodecanamide Octimibate GO 89838-96-0 (Jackson, Gee 8 - ((1,4,5-Triphenylimidazol-2-yl) oxy) acid et al. 1990) octanoic FR179254 (Tanaka, A., et al. 1998) S 58-035 H 78934- 83-5 (Ross, AC, et (Sandoz) x (CH 2) 9 CH 3 al. 1984) 3 - [Decyldimethylsilyl] -N- [2- (4-methylphenyl) -1-phenyl] propanamide ACAT inhibitor 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 delivery of the ACAT inhibitor, maintaining and preserving the antifibrotic agent. The formulations may be adapted to the condition to be treated. For example, the treatment of fibrotic disorders may be delivered topically, orally or by injection. Alternatively, the compositions may be delivered by therapeutic inhalation. Other suitable means for introducing the therapeutic molecule include implantable drug delivery devices.

The ideal composition will be determined by one of ordinary skill in the art, depending for example on the desired route of administration, mode of delivery and desired dosage.

A carrier suitable for parenteral administration preferably provided by the present invention is sterile distilled water in which the desired ACAT inhibitor is formulated as a properly preserved sterile isotonic solution.

When the composition of the invention consists of aqueous injection suspensions, preferably containing substances to increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. In addition, suspensions of the desired ACAT inhibitor may be prepared as appropriate or appropriate oily injection suspensions. Lipophilic solvents or carriers 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 stabilizers or agents suitable for enhancing the solubility of the ACAT inhibitor and permitting the elaboration 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 inhalation powder. ACAT inhibitor delivery solutions may also be formulated with an aerosol propellant. Alternatively, the solutions may be nebulized.

Compositions comprising ACAT inhibitors may be formulated for topical administration. Examples of topical compositions may be in the form of cream, ointment or lotion.

It is also contemplated that the compositions of the invention may be administered orally. Compositions for oral administration may be formulated using acceptable carriers well known in the art at 20 doses suitable for oral administration. These carriers allow the compositions to be formulated as tablets, pills, pills, capsules, liquids, gels, syrups, suspensions, and the like for patient ingestion.

In addition, a capsule may also be designed to release the active part of the formulation at a point in the gastrointestinal tract when bioavailability is maximized and presystemic degradation is minimized. Additional agents may be included to facilitate absorption of the ACAT inhibitor. Diluents, flavors, low melting waxes, vegetable oils, lubricants, suspending agents, tablet dissolving agents and binders may also be employed.

It will be appreciated that an example of an oral formulation 10 may consist of a capsule which may be prepared by granulating the ACAT inhibitor contemplated in the present invention, with lactose and cornstarch as excipient and hydroxypropylcellulose as the binder. Such a capsule may consist of a hard white gelatin capsule containing the desired amount of the ACAT inhibitor.

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

Table 2:

Ingredient (mg) Capsule content F-1394 25.0 Triethyl citrate 35.0 Propylene glycol fatty acid ester 110.0 Polyoxyethylene castor oil 60 170.0 Sub-total 340.0 Capsule body Hard capsule (gelatin) 70.0 Capsule Coating LD 29.84 Methacrylic Acid Copolymer Triethyl Citrate 3.0 Talc 9.16 Sub Total 42.0 Total 452.0 Oral formulations may also be obtained by combining the desired ACAT inhibitor with the solid excipient and the resulting processing of mixing the granule mixture (optionally after milling) p For 5 get tablets or pill cores. Suitable excipients for such formulations include, for example, protein or carbohydrate excipients, such as sugars, including lactose, sucrose, mannitol, sorbitol, and cornstarch, wheat, rice, potato, or other vegetables; 10 cellulose, such as methylcellulose, or sodium carboxymethylcellulose, and proteins such as gelatin and collagen.

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

Dosage levels for treatment will vary, in part, depending on the type of ACAT inhibitor delivered, the specific indication for which the ACAT inhibitor will be used, the route of administration, and the size 20 (weight, body surface area). or organ size) and condition (age and overall health) of the patient. For example, the clinician may titrate the dose and modify the route of administration to obtain the best therapeutic effects.

For any compound, the effective dose can be estimated initially, either in cell culture or animal model assays such as mice, rats, rabbits, dogs, pigs and monkeys. An animal model may also be used to determine the appropriate concentration range and route of administration. This information can then be used to determine doses and routes useful for administration to humans. 10 The frequency of administration will depend on the

pharmacokinetic parameters of the ACAT inhibitor in the specific formulation that is used. Typically, a composition is administered to be achieved until the desired effect is achieved. The composition may therefore be administered as a single dose or in multiple doses (at the same or different concentrations / doses) over time, or as a continuous infusion. In addition, refinement of the proper dosage is routinely done. Proper dosage can be verified by appropriate use of dosage response data.

As an example, a capsule as described in Table

2, with 25 mg of ACAT F-1394 inhibitor can be administered at least once a day, preferably at least twice a day. However, the daily dose as well as the capsule content of F-1394 may be adapted to obtain better therapeutic effects.

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

EXAMPLES Example 1: ACAT_ inhibitors inhibit the TGF-β signaling pathway

TGF-β belongs to a large peptide family of

secreted growth factors that play critical roles in animal development. Following binding, type I and type II receptors are recruited by the type II phosphorylate complex and receptor, and thus activates type I receptor 20. In turn, the type I phosphorylate receptor of the subfamily Smads R Smad (receptor-regulated) ). R-Smads form complexes with Co-Smads and accumulate in the nucleus to regulate gene transcription through other transcription factors (Figure 1). This pathway regulates many cellular processes and plays a crucial role in normal development. Rupture of the pathway can lead to a variety of diseases, including cancer, cardiovascular disease, inflammatory disease and fibrosis, as well as Marfan's syndrome.

In larvae, the TGF-β pathway regulates entry into the

larval, an alternative larval phase that specializes in survival under adverse conditions. The disruption of the TGF-β pathway leads to constitutive Dauer formation, ie the formation of Dauer larvae even under favorable conditions. Thus, an increase in Dauer formation indicates a decrease in signaling across the pathway. However, there are other signaling pathways, such as the insulin-like signaling pathway, that affect the formation of the dauer larva. Dauer constitutive formation via the pathway requires the activities of daf-3 and daf-5 while Dauer constitutive formation via the insulin-like signaling pathways does not require them.

The aim of this study was to establish that ACAT inhibitors have the ability to interfere with the TGF-β signaling pathway.

Materials and methods

General Methods

All assays were performed in 24-well plate. The wells contained 1.5 mL nematode growth average (NGM). On one day, the appropriate volume of compound solutions provided was added to the surface of the NGM wells and allowed to dry in the dark for at least 2 hours. The wells were then seeded with 3 to 5 μΐι of OP50 bacterial culture, which had been concentrated 15 5 times. Larvae were transferred to egg wells on Day 1 and then grown for 3 days at 20 ° C (for daf-8, then-14 and then-4; hence-5) or at 22 ° C (for thereafter-1). and hence-7). The percentage of larvae in the dauer phase was determined on the 4th or 5th day.

Larvae

Larval strains were obtained from the Caenorhabditis Genetics Center (CGC) and maintained at 20 ° C using standard culture methods. The wild-type strain used was the Caenorhabditis elegans Bristol strain, 15 N2. The mutants used in this study are: daf-7 (e! 372) III, daf-1 (m40) IV, daf-4 (m63) III, daf-8 (e! 393) I, daf-14 (zn77) IV , and daf-5 (el386) II.

Compound

Avasimiba, Pactimiba, F-1394, FR19254, S58035 were maintained as 10 mM solutions supplied at -20 ° C. All compounds were dissolved in DMSO. The compounds were tested at a final concentration of 82.5 μΜ, except Avasimiba which was tested at 10.3 μΜ in the mutant thence-14, and 55 μΜ in the other mutants.

25 Result and Discussion ACAT inhibitors that affect dauer formation by TGF-β reduction

All ACAT inhibitors tested increased formation in the daf-lAC elegans mutant, which is a constitutive dauer mutant in the TGF-β pathway (Figure 2). This effect requires daf-5 activity, as all ACAT inhibitors tested had no effect on daf-4; daf-5 double mutant (Table 3). In fact daf-5 is required for Dauer formation due to inhibition of TGF-β signaling, and the absence of induction of dauer formation by ACAT inhibitors in a daf-5 mutant the history indicated that its effects on dauer are through inhibition of the TGF-β signaling pathway. In addition, the inventors specifically tested F-1394 and Avasimiba on several other Dauer constitutive mutants in the TGF-β pathway which are daf-I, daf-1, and daf-8. The inventors found that F-1394 as well as Avasimiba increased Dauer formation in these mutants (Figure 3), consistent with the results described in Figure 2.

In total, this indicates that ACAT inhibitors affect dauer formation specifically by signaling by reducing the TGF-β pathway. Table 3:

Treatment% dauer Sample Size DMSO 0 50 Avasimiba 0 321 F-1394 0 83 Pactimiba 0 50 FRl79254 0 50 S 58-035 0 50 Table 3: ACAT inhibitors do not induce dauer formation in the daf-4 double mutant (M63); daf-5 (el386). The following 5 compounds were tested as described in the Material and Methods section: Avasimiba (n = 651), F1394 (n = 935), Pactimiba (n = 1166), FR179254 (n = 1056), and S 58-035 (n = 765). DMSO solvent was used as a control (n = 941). The tested compounds did not have dauer 10 activity induced in previously tested larvae.

Example 2: Effects of F-1394 on TGF-β induced protein matrix expression in A 549 epithelial cell line in human lungs, an in vitro animal model relevant for human pulmonary fibrosis and COPD Transforming Growth Factor (TGF) -βι , an

multifunctional or pleiotropic cytokine participates in numerous biological processes, including cell proliferation, differentiation, apoptosis, fibrosis, inflammation, and lesion repair (Ning W. et al. 2002; Martin G.E.M. et al. 2006). In particular, TGFPi is a growth factor that plays a crucial role in mediating the response to lung tissue injury and is therefore involved in the repair mechanisms and pulmonary fibrosis that follow inflammatory processes (Bellocq A. et al. 1999). Reactive oxygen and nitrogen mediate the TGFPi increase by releasing it from human alveolar epithelial cells which may well constitute a molecular link between inflammatory and fibrotic processes (Bellocq A. et al. 1999).

Type I fibrillar collagen is characteristically

synthesized by fibroblast cells type I (Kasai H. et al. 2005).

Previous work has shown that TGFP1 but not other inflammatory cytokines induced in A549 cell with a type II alveolar epithelial cell phenotype undergoing a mesenchymal epithelial transition, which includes expression of fibroblast phenotypic markers (Kasai H. et al. 2005) .

Materials and methods Experimental model

A549 human alveolar epithelial cell carcinoma line (American Type Culture Collection, Rockville, MD) was cultured as described in Mata M. et al. 2005. The human recombinant TGFPi was used at a concentration of 5ng / mL. Medium was changed every 24 h with the addition of TGFP1 and F-1394 when appropriate. F-1394 was present from 1 h before the first addition of TGFPi until the end of the experiment.

MRNA analysis

For mRNA analysis, A549 cells were

grown and prepared as described in Mata M. et al. 2005. Total RNA was extracted using Trizol Reagent ™ (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions. The integrity of the extracted RNA was confirmed by the use of Bioanalizer ™ (Agilent, Palo Alto, CA, USA). Collagen al (I) mRNA was determined by quantitative RT-PCR as indicated (Manoury B. et al. 2006). Determination time: 0 time point in 72 hours as previously described (Kasai H. et al. 2005).

Experimental Groups

In vitro experiments were performed with the appropriate negative and positive controls, as well as groups treated with F-1394 and reference compounds and their respective controls, as follows:

1) Vehicle Control (DMSO)

2) F-1394 (in DMSO) dose level 3 (3 pg / mL)

3) TGFpi + DMSO

4) TGFpi + F-1394 (in DMSO) dose level 1 (0.3 pg / mL)

5) TGFPi + F-1394 (in DMSO) dose level 2 (0.6 pg / ml) 6) TGFpi + F-1394 (in DMSO) dose level 3 (3 pg / ml).

Result and Discussion

Data are presented as mean + SEM. Statistical analysis of the results was performed by analysis of variance (ANOVA) followed by Bonferroni test (GraphPad Software Inc, San Diego, CA, USA). Significance was accepted when P <0.05.

TGFPi produced a marked increase in mRNA expression of collagen al (I) as shown in Figure 4.

This increased expression of induced TGFβ1 was significantly reduced at a concentration dependent form in the presence of F-1394 (Fig. 4).

F-1394 as a prototype ACAT inhibitor is effective for reducing TGFP1-evoked collagen al (I) mRNA expression in A549 cells cultured in a human respiratory epithelial cell line.

Example 3: Effects of F-1394 on bleomycin-induced lung damage in vivo in an animal model relevant for pulmonary fibrosis.

Bleomycin-induced lung damage is a model considered to be considered relevant in the investigation of the activities of new anti-inflammatory and antifibrotic compounds (remodeling).

Materials and methods

A total of 66 mice entered the study. The number of animals per group was approximately 10 to 12 (as required). Each rat had a body weight delivered in (6 weeks) of about 20 g.

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

Experimental model

Adult male C57BL / 6 mice, each weighing about 20 g, transorally received a single dose of 0.075 U bleomycin sulphate (Sigma Catalog B 5507 (conserved at 2 to 8 ° C, 1.2 1.5 units / mg solid) dissolved in 50 μΐ saline (0.9% NaCl) Control animals underwent the same protocol but received intratracheal serum instead of bleomycin. Tracheal instillation was performed under anesthesia. Fourteen days after administration of bleomycin saline / endotracheal, the animals were sacrificed by anesthesia (pentobarbital sodium), followed by exsanguination of the abdominal aorta. The obtained lungs were weighed. Then, the right and left lungs were processed separately. for histological and biochemical studies as indicated below.

14 days after bleomycin was chosen as the peak time for collagen synthesis (Lazemby et al 1990).

Histological Evolution

By histological examination, the lungs were first perfused through their main bronchus with a fixative solution (10% neutral buffered formalin) maintained at 10 25 cm of hydrostatic pressure for 15 min, immersed in the fixative for 24 h, and blocks were taken. . Only the lungs well inflated by the fixative were analyzed. Tissue blocks were placed in formaldehyde, dehydrated in a series of ethanol grades, embedded in paraffin, 15 cut into 4pm series sections, and stained with hematoxylin-eosin to identify inflammatory cells and fibrotic areas.

Histological grading of the lesions was performed using a semi-quantitative scoring system for the extent and severity of inflammation and fibrosis in the lung parenchyma based on previous studies from this laboratory (Cortijo et al., 2001; Mata et al., 2003; Serrano et al. -Mollar al., 2002; Mollar-Serrano et al., 2003). The severity of fibrosis was classified according to Ashcroft and co-authors. Briefly, the degree of pulmonary fibrosis was rated on a scale of 0 to 8 by the following criteria: grade 0, normal lung, grade 1, minimal alveolar fibrous thickening or bronchiolar walls; grade 3, moderate wall thickening without obvious damage to the pulmonary architecture; grade 5, increased fibrosis with definitive damage to the lung structure and formation of fibrous bands or small fibrous masses; grade 7, severe structure distortion and large fibrous areas; grade 8, total fibrous obliteration of the fields. Grades 2, 4 and 6 were 10 used as intermediate images. The severity of fibrotic changes in each pulmonary section was assessed according to the mean score observed from the severity of the microscopic fields. Ten random fields in each section were analyzed. The classification was performed 15 masked by two observers, and the mean value was considered according to the fibrosis score.

Experimental group

The in vivo experiment was performed with appropriate negative and positive controls, as well as groups that received the different drug treatments and their respective controls (drug vehicle; negative treated controls) when necessary.

• Negative control: F-1394-vehicle (oral) + saline (endotracheal)

· Positive control: F-1394-vehicle (oral) + bleomycin (endotracheal)

• F-1394 300 mg / kg (oral) + bleomycin (endotracheal)

• NAC 500mg / kg (oral) + bleomycin (intratracheal)

Result and Discussion

Data are presented as mean ± SEM. Statistical analysis of the results was performed by analysis of variance (ANOVA) followed by the Bonferroni test or by nonparametric tests, as appropriate (GraphPad Software 10 Inc, San Diego, CA, USA). Survival curves were analyzed by logrank testing using GraphPad Software Inc (Prism 4.03). Significance was accepted when P <0.05.

al (!) collagen mRNA in lung tissue

F-1394 as well as NAC had a marked inhibitory effect on the expression of al (I) collagen transcripts (Figure 5), which indicates an antifibrotic effect with decreased collagen deposition.

F-1394 as a prototype ACAT inhibitor is a valuable compound endowed with antifibrotic properties at the level of al (I) collagen mRNA expression in lung tissue.

Histological Evolution

The lungs of the control animals were histologically normal. The characteristic administration of bleomycin resulted in histological lesions, including areas of inflammatory cells with marked interstitial and peribronchiolar infiltration (predominantly mononuclear cells including macrophages and lymphocytes with fewer scattered neutrophils and eosinophils), 5 extensive interalveolar septal cell thickening, interstitial edema , and increases in interstitial cells with a fibroblastic appearance. The pattern of lesion distribution was multifocal (ie irregular areas of pulmonary fibrosis). Although multifocal parenchymal lesions were also present, treatment with F-1394 significantly reduced changes in lung morphology; There were few infiltrations of inflammation, less collagen deposition and less septal enlargement (Fig. 6). A significant reduction in the 15 Ashcroft score was observed (Fig. 7). Similar results were obtained for N-acetylcysteine (NAC).

Example 4: Effect of F-1394 on unilateral ureteral obstruction in rats

Unilateral ureteral obstruction (UUO) is a well-established accelerated experimental model 20 that reproduces the different characteristics of obstructive nephropathy leading to tubulointerstitial fibrosis. Given its robustness and high reproducibility, this model has been used to provide rapid proof of concept for the use of F-1394 as a prototype ACAT inhibitor in preventing interstitial collagen accumulation.

The aim of the present study was to evaluate the effects of preventive treatment with F-1394 on all fibrillary interstitial collagen deposition by 5 Sirius red histochemical analysis. The effects of F-1394 were assessed by quantifying specific stained areas relative to the entire surface section of the clogged kidney.

Materials and methods Animals

Male C57BL / 6J mice weighing 25 to 30 g in the

beginning of the experiments were used. The mice were housed by each group of five (5) in opaque polypropylene cages with free access to food and water (TekladTM 5018; HARLAN, Gannat, France). The animal was kept under artificial illumination (12 hours) between 7:00 and 19:00, at a controlled room temperature of 20 ± 1 ° C, and the relative humidity maintained at 60%. The mice were sacrificed by cervical dislocation. The animals were not submitted to autopsy.

Study Materials

The F-1394 dosage formulation was prepared in two different ways, 200 mg / kg solution with 1% Tween80 and 0.5% CMC, and 300 mg / kg solution with 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 remaining 5 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.

Main equipment

Morphometric measurement was based on computerized image analysis of red Sirius stained sections, observed through a microscope (Eclipse 600TM, Nikon Instruments; Champigny sur Marne, France) and 10 numerical camera (MicroFireTM, Optronics; Avanex, Nozay, France) , with a 20X objective lens (final calibration 0.366 pm / pixel). Quantitative analysis of the images was performed using ExploraNova MorphoLite ™ and Expert ™ software (La Rochelle, France).

15

Experimental Protocol

Four experimental groups with 10 mice each were used:

1) Sham operated mice 2) Obstructed vehicle treated mice (0.1%

Tween80 / 0.5% CMC)

3) Blocked mice treated with F-1394

4) Captopril-treated clogged mice

Oral treatments lasted 15 (fifteen) 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 an oxygen-isoflurane mixture (0.2 L / min and 2% isoflurane; Centravet, Lapalisse, France). A 5 mm skin incision was made from 1 cm from the last left costal edge of the mouse, followed by a 5 mm muscle incision. The left ureter was exposed and ligated (6/0 cardionyl thread; Peters Surgical, Bobigny, France) at the ureteropelvic junction. Then the muscle wall was closed (5/0 Ethicrin thread; Ethicon, Auneau, France), and the skin walls were closed by a suture clip. Throughout the surgery, the animals were kept on a plate heated to 35 ° C.

Food and water were provided ad libitum. Oral treatments began one day before UUO, the obstruction lasted 14 days. At the end of the protocol, the mice were sacrificed for cervical dislocation; obstructed kidneys 20 were removed and divided for histology preparation and gene expression analysis.

Histological analysis

After the clogged kidneys were removed, the central fragment used for histology was fixed in Carnoy's solution for 24 hours and embedded in paraffin according to normal procedures. Histological cross sections of 3 to 4-μπι thickness were performed and stained with red Sirius throughout the evaluation of fibrillar interstitial collagen deposition.

The cuts were deparaffinized, rehydrated, and then

stained with 0.1% Sirius red F3B ™ (BDH Laboratory; VWR, Fontenay sous Bois, France) in the saturated aqueous picric acid solution for 1 hour, differentiated by Ο, ΟΙΝ HCl by

1 minute, and quickly dehydrated and assembled.

The surface of the tubulointerstitial fibrillar collagen

was determined by measuring the area of stained tissues within the entire cortical area or within a given field. Glomeruli and vasculature were excluded from the evaluation of the present study.

15

Registry Analysis

Analyzes were performed by an operator of unknown origin from each section of the kidney. Double blind quantifications of selected objects within a given image were recorded in a Microsoft Excel file combined with ExploraNovaTM software. The removal of histological codes was performed at the end of the recordings for the classification results.

Results were expressed as a percentage (%) of fibrosis in relation to the whole area studied. Analysis and expression of results

Results were presented as mean values ± standard error of the mean (SEM). One way ANOVA was used to compare the difference within the group followed by a Newman-Keuls test to compare all column pairs (GraphPad Prism, San Diego, USA). A p <0.05 was accepted for statistical significance.

Result and Discussion

Effects of F-1394 and captopril on induced UUO tubulointerstitial fibrosis

Interstitial fibrosis was significantly higher in the vehicle-treated UUO group compared to sham operated animals (Figure 8), with an interstitial collagen expression representing 13.36 ± 0.60% and 0.26 15 ± 0.03 % of kidney sections, respectively (n = 10, p <0.001, ANOVA with Newman-Keuls). This effect was, according to the previous publication (Schanstra et al, 2003 and 2002).

F-1394 decreased the induced UUO tubulointerstitial fibrosis compared with the vehicle group (5.43 ± 0.29% 20 versus 13.36 ± 0.60% in the F-1394 and vehicle group, respectively, n = 10, p <0.001, ANOVA with Newman-Keuls). Captopril treatment led to an expected decrease in induced UUO tubulointerstitial fibrosis (2.89 ± 0.12% vs 13.36 ± 0.60% in vehicle and captopril, 25 respectively, n = 10, p <0.001 , ANOVA with Newman-Keuls). These results show that administration of F-1394 as a prototype ACAT inhibitor significantly reduces tubulointerstitial fibrosis in the UUO model, with efficacy comparable to captopril.

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Claims (27)

1. Composition useful for the prevention or reduction of collagen deposition in a tissue, the composition being characterized by the fact that it comprises an effective amount of at least one ACAT inhibitor comprising F-1394, Avasimiba, Pactimiba (CS505), Eflucimiba (F12511). ), Eldacimiba, NTE 122, AS183, KW3033, E5324, FY087, FCE27677, CI97 6, TEI6522, K604, Octimibate, FR179254, S58-035, or a mixture of these and an acceptable excipient. Composition according to Claim 1, characterized in that the ACAT inhibitor is F-1394. Composition according to Claim 1, characterized in that the tissue is a tissue of a heart, lung, brain, eye, stomach, spleen, bone, pancreas, kidney, liver, intestine, skin, uterus or bladder. . 4. Antifibrotic composition, characterized in that it comprises an effective amount of at least one antifibrotic agent and an acceptable excipient. Antifibrotic composition according to claim 4, characterized in that the antifibrotic agent is an ACAT inhibitor. Antifibrotic composition according to claim 5, characterized in that the ACAT inhibitor is F-1394, Avasimiba, Pactimiba (CS505), Eflucimiba (F12511), Eldacimiba, NTE 122, AS183, KW 3033, E5324, FY087, FCE 27677, CI 976, TEI 6522, K604, Octimibate, FR179254, S 58-035 or a mixture thereof. Antifibrotic composition according to claim 6, characterized in that the ACAT inhibitor is F-1394, Avasimiba, Pactimiba, FR179254 or S 58-035. Antifibrotic composition according to claim 7, characterized in that the ACAT inhibitor is F-1394. 9. Use of an ACAT inhibitor, characterized in that it serves to prevent or reduce collagen tissue deposition. Use according to claim 9, characterized in that the ACAT inhibitor is F-1394, Avasimiba, Pactimiba (CS505), Eflucimiba (F12511), Eldacimiba, NTE 122, AS183, KW 3033, E5324, FY. 087, FCE27677, CI 976, TEI 6522, K604, Octimibate, FR179254, S 58-035 or a mixture thereof. Use according to claim 9, characterized in that the tissue is a tissue of a heart, lung, brain, eye, stomach, spleen, bone, pancreas, kidney, liver, intestine, skin, uterus or bladder. . 12. Use of an ACAT inhibitor, characterized in that it prevents the formation or development of excess fibrous connective tissue in an organ. Use according to claim 12, characterized in that the ACAT inhibitor is F-1394, Avasimiba, Pactimiba (CS505), Eflucimiba (F12511), Eldacimiba, ΝΤΕ 122, AS183, KW 3033, E5324, AF. 087, FCE27677, Cl 976, TEI 6522, 604, Octimibate, FR179254, S 58-035 or a mixture thereof. Use according to claim 12, characterized in that the tissue is a tissue of the heart, lung, brain, eye, stomach, spleen, bone, pancreas, kidney, liver, intestine, skin, uterus or bladder. 15. Method for reducing the level of collagen in a tissue, the method being characterized by understanding providing a tissue, contacting the tissue, with at least one ACAT inhibitor and measuring a reduced level of collagen in the tissue. Method according to Claim 15, characterized in that the ACAT inhibitor is F-1394, Avasimiba, Pactimiba (CS505), Eflucimiba (F12511), Eldacimiba, NTE 122, AS183, KW 3033, E5324, FY. 087, FCE27677, CI 976, TEI6522, K604, Octimibate, FR179254, S 58-035 or a mixture thereof. Method according to claim 15, characterized in that the tissue is a fibrotic tissue. Method according to claim 15, characterized in that the tissue is a tissue of the heart, lung, brain, eye, stomach, spleen, bone, pancreas, kidney, liver, intestine, skin, uterus or bladder. A method for preventing the formation or development of excess fibrous connective tissue in an organ of an individual, the method being characterized by administering to said individual an effective amount of at least one ACAT inhibitor. The method according to claim 19, characterized in that at least one ACAT inhibitor is F-1394, Avasimiba, Pactimiba (CS505), Eflucimiba (F12511), Eldacimiba, NTE 122, AS183, KW 3033, E5324. , FY087, FCE 27677, CI 976, TEI 6522, K604, Octimibate, FR179254, S 58-035 or a mixture thereof. Method according to claim 19, characterized in that the organ is a heart, lung, brain, eye, stomach, spleen, bone, pancreas, kidney, liver, intestine, skin, uterus or bladder. Method according to claim 21, characterized by the fact that the individual is a human being. A method for preventing or treating fibrosis or a fibrotic disorder in an individual, the method being characterized by: identifying an individual suffering from fibrosis or at risk of developing it; administering to said individual an ACAT inhibitor in an amount sufficient to decrease the level of collagen; and - measure a reduced level of collagen in the individual. A method according to claim 23, characterized in that the ACAT inhibitor is F-1394, Avasimiba, Pactimiba (CS505), Eflucimiba (F12511), Eldacimiba, 122, AS183, KW 3033, E5324, FY. 087, FCE 27677, Cl 976, TEI 6522, K604, Octimibate, FR179254, S 58-035 or a mixture thereof. Method according to claim 24, characterized in that the ACAT inhibitor is F-1394. A method according to claim 23, characterized by the fact that the individual is a human being. Method according to claim 23, characterized in that the fibrotic disorder is scleroderma, keloids and hypertrophic scars, collagen disorders associated with the occurrence of Raynaud's syndrome, pulmonary inflammation and fibrosis, pulmonary interstitial diseases, pulmonary fibrosis. idiopathic, sarcoidosis, liver cirrhosis and liver fibrosis resulting from viral or parasitic infection, kidney disorders associated with unregulated TGF-β activity, excess fibrosis, renal interstitial fibrosis, renal fibrosis in transplant patients, focal glomerulosclerosis, fibrosis caused by Marfan disease , cardiac fibrosis, radiation-induced fibrosis, wound healing fibrosis, eye diseases, peritoneal fibrosis, intestinal fibrosis, chemotherapy drug-induced fibrosis or burns.