KR20100014267A - 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

ACT inhibitors and their use in the prevention or treatment of fibrosis {ACAT INHIBITORS AND THEIR USE IN THE PREVENTION OR TREATMENT OF FIBROSIS}

The present invention provides novel methods and compositions for preventing and reducing fibrosis associated with fibrotic diseases using ACAT inhibitors. More specifically, the present invention relates to the use of ACAT inhibitors in compositions and methods for the prevention or treatment of fibrosis, for controlling collagen deposition in tissues, and in the prevention and reduction of excessive fibrous connective tissue in organs.

The process of tissue repair as part of wound healing involves two phases. The first phase is the regenerator, in which damaged cells are replaced with cells of the same type. The second phase is the formation of fibrous tissue, also called fibrous formation or fibrosis, in which connective tissue replaces milk tissue. The breakfast recovery process can become pathogenic if fibrosis is not suppressed continuously, leading to extensive tissue remodeling and the formation of permanent reactive tissue (Wynn 2004).

Fibrotic diseases affect almost all tissue and organ systems. Major fibrosis diseases include interstitial lung disease (ILD), which causes pulmonary inflammation and fibrosis. ILD is known to have many causes, such as sarcoidosis, silicosis, collagen vascular disease. The most common type of ILD is idiopathic pulmonary fibrosis (IPE), which is still unknown (idiopathic). Other fibrotic diseases include cirrhosis and fibrosis derived from viral hepatitis, kidney disorders associated with unregulated TGF-β activity and excessive fibrosis such as glomerulonephritis (GN), renal interstitial fibrosis, renal fibrosis in transplant patients , Eye diseases such as macular degeneration and retinal and vitreoretinopathy. Fibrotic disorders also include collagen disorders associated with the development of systemic and local scleroderma, keloid and hypertrophic scars and Raynaud's syndrome. Excessive scars resulting from surgery, chemotherapy drug induced fibrosis, radiation induced fibrosis and burns are also part of the fibrotic disease.

Acyl-CoA: cholesterol acyltransferase (ACAT) promotes the formation of cholesteryl esters using both cholesterol and long chain fatty acyl promoter A as substrates. ACAT is present in a variety of tissues including intestinal medulla, liver, adrenal glands, testes and macrophages. ACAT inhibitors are known to reduce or prevent the development of atherosclerotic lesions in animal models.

Current treatments for fibrotic diseases include common immunosuppressive drugs such as corticosteroids and other anti-inflammatory drugs. Such therapies are not always effective in reducing or preventing fibrosis, and in anti-inflammatory therapies because the mechanisms involved in fibrosis control usually appear to be separate from the mechanism of inflammation. Very few review therapies have been found to alter or reverse inflammatory processes associated with fibrosis.

Moreover, a wide range of similar molecular targets of antifibrotic therapy, such as those directly related to fibrosis, such as matrix metalloproteinases and inhibitors thereof and pro-fibrogenic cytokines such as tumor growth factor β (TGF-β) Confirmed. TGF-β plays an important role in initiating multistage events that peak in cytokine production and excessive and defective expression of collagen and other extracellular matrix components.

Some animal models are useful for identifying and characterizing new antifibrotic agents. The antifibrotic effect of pirfenidone has recently been confirmed in the bleomycin-hamster model of pulmonary fibrosis (Iyer, Margolin et al. 1998) and in the unilateral urinary obstruction model of kidney fibrosis (Shimizu, Kuroda et al. 1998). Inhibitors of ALK5 (TGF-β receptor II) have been shown to be protective in dimethylnitrosamine induced liver fibrosis (DMN) rat model (de Gouville and Huet 2006).

The inventors have found that compounds having an ACAT inhibitory ability are the selection molecules 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 disease using ACAT inhibitors.

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

In another aspect, the invention relates to the use of an ACAT inhibitor for preventing or reducing collagen deposition in tissues.

Another aspect of the invention also relates to the use of ACAT inhibitors to prevent the formation and development of excessive fibrous connective tissue in organs.

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

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

In another aspect, the invention provides a method of preventing or treating fibrosis or fibrotic disease in a subject,

Identifying a subject suffering from or at risk of developing fibrosis;

Administering to the subject an ACAT inhibitor in an amount sufficient to reduce the level of collagen; And

Measuring the reduced level of collagen in the subject

It provides a method comprising a.

Brief description of the drawings

1 shows a schematic representation of the TGF-β pathway in humans by worm homologues.

2 shows that ACAT inhibitors reduce TGF-β signal transduction. Dower formation was measured in daf-14 (m77) mutant. The following compounds, 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 compounds tested increased Dow formation compared to the control treatment.

3 shows that ACAT inhibitors reduce TGF-β signaling in one or more TGF-β structural mutants. Dower formation induced by Abashimaib (a) and F-1394 (b) was measured in Dow mutants as described in the Materials and Methods section of Example 1. (a) Abashimaib was treated with daf-7 (e1372) (n = 560 versus 539 as a control); daf-1 (m40) (n = 870 vs 636 as control); And daf-8 (e1393) (n = 852 vs 502 as a control). (b) F-1394 is daf-7 (e1372) (n = 99 versus 134 as a control); daf-1 (m40) (n = 145 versus 108 as control); And daf-8 (e1393) (n = 47 vs 140 as control). Compounds tested increased Dow formation compared to control treatment in all mutants.

4 shows 4579 cells stimulated with unstimulated converting growth factor (TGF; 5 ng ml ⁻¹ ) -β ₁ in the absence or presence of F-1394 (0.3, 0.6 and 1 μg ml ⁻¹ , as specified) or Relative quantification of α1 (I) collagen mRAN expression levels in unstimulated A579 cells (control). The exposure time was 72 hours for TGFβ ₁ . F-1394 was present from 1 hour before TGFβ ₁ to the end of the experiment. TGFβ ₁ induced increase in α1 (I) collagen mRAN expression was concentration dependently inhibited by F-1394. α1 (I) collagen mRNA expression was measured using real-time RT-PCR by the ΔΔC _t method; The column shows a several-fold increase in the expression of α1 (I) collagen mRNA compared to the GAPDH value as the mean ± SEM of the 2-ΔΔC _t values of three independent experiments. *: p <0.05 vs control; #: p <0.05 vs. TGFβ ₁ .

5 shows the relative expression of α1 (I) collagen mRNA in lung tissue of mice receiving intratracheal bleomycin (BLM) or saline (control). The column is the mean ± s.e.m. Of 3-5 animals per group. * P <0.05 vs. Control, BLEO by #P <0.05 vs ANOVA.

6 shows drug vehicle + saline (a), F-1394 + saline (b), N-acetylcysteine + saline (c), drug vehicle + bleomycin (d, e, f), 1394 + bleomycin (g, h) and representative micrographs of lung tissue structure in mice treated with N-acetylcysteine + bleomycin (i). All lung sections were stained with hematoxylin-eosin. Magnification x40. Saline treated animals show normal structure. Bleomycin exposed mice exhibited significant peribronchial interstitial infiltration, thickened cell septum, edema and dense fibrosis by inflammatory cells. This lung injury was reduced in animals treated orally with F-1394 and N-acetylcysteine.

Figure 7 shows the evaluation of fibrous changes in the lung by a number of fibrous scores. Bars represent Ascroft scores (mean ± SEM) for each experimental group as specified. * P <0.05 vs. Beagle + BLEO by Mann-Whitney test. Lung damage was reduced in animals treated orally with N-acetylcysteine as well as F-1394.

FIG. 8 shows the percentage of fibrosis measured after Sirius red staining of 10 non-overlapping zones in the cortical area, expressed as the average percentage of stained interstitial areas. *** p <0.01 compared by vehicle group; N = 10 per group; Data was analyzed by ANOVA followed by Newman-Keuls test.

The present invention provides novel methods and compositions for using ACAT inhibitors to prevent and reduce fibrosis associated with fibrotic disease.

Justice

As used herein, the term ACAT inhibitor refers to any compound that inhibits the enzyme Acyl-CoA: cholesterol acyltransferase (ACAT). Examples of ACAT inhibitors include small organic compounds, ie compounds having a molecular weight greater than 50 and less than about 2500. ACAT inhibitors contain chemical functional groups necessary for structural interaction with protein and / or nucleic acid molecules, and typically include at least an amine, carbonyl, hydroxyl or carbonyl group, preferably two or more of the chemical functional groups. The ACAT inhibitor may comprise a cyclic carbon or heterocyclic structure and / or an aromatic or polyaromatic structure substituted by one or more of the above identified functional groups.

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

As used herein, the term fibrosis refers to the formation of fibrous tissue as a repair or reactive process. Fibrosis is characterized by fibroblast accumulation and deposition of collagen above normal deposition of certain tissues.

As used herein, fibrotic diseases, fibrosis, proliferative disease or fibrotic diseases; refers to conditions including a fibrosis in one or more tissues.

Fibrotic diseases include, but are not limited to, scleroderma, keloid and hypertrophic scars, collagen disorders associated with the development of Raynaud's syndrome, pulmonary inflammation and fibrosis, interstitial lung disease, idiopathic pulmonary fibrosis, sarcoidosis, viral or parasitic infections And hepatic fibrosis, renal disorders associated with unregulated TGF-β activity and excessive fibrosis such as glomerulonephritis (GN), renal interstitial fibrosis, renal fibrosis in transplant patients, focal glomerulosclerosis, Marfan's disease Fibrosis resulting from fibrosis, cardiac fibrosis, radiation induced fibrosis and wound healing, eye diseases such as macular degeneration and retinal and vitreous retinopathy. It also includes peritoneal fibrosis, intestinal fibrosis, chemotherapy drug-induced fibrosis and burns.

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

Idiopathic pulmonary fibrosis is an unknown (idiopathic) inflammatory lung disorder characterized by a small air pocket (alveoli) or abnormal formation of fibrous tissue (fibrosis) between the catheter of the lung.

Liver fibrosis refers to the accumulation of tough fibrous scar tissue between subjects.

As used herein, the term subject means animal or human. Thus, the term means, but is not limited to, primates, domestic animals such as dogs, cats, sheep, cattle, goats, pigs, mice, rats, rabbits, guinea pigs, capture animals such as zoo animals, and wild animals.

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

As used herein, the term treating / treating means a method in which fibrotic disease, fibrotic disease, fibrosis and related disorders as exemplified above are ameliorated or completely eliminated.

As used herein, the term preventing / preventing means how fibrotic disease, fibrotic disease, fibrosis and related disorders as exemplified above are blocked, delayed or avoided.

As used herein, an expression effective amount refers to an amount that produces a predetermined effect as judged by clinical trial results and / or animal models. Such amounts can be routinely determined by one skilled in the art. The effective amount of the composition to be used will depend, for example, on the content and purpose of treatment. Those skilled in the art will therefore appreciate that the appropriate dosage level for treatment is, in part, the type of ACAT inhibitor administered, the indication that the ACAT inhibitor is used, the route of administration and the size (weight, body area or organ size) and condition (age and overall health) of the patient. Will be different depending on Thus, the clinician can titrate the dosage and change the route of administration to obtain the optimal therapeutic effect.

For any compound, the effective dosage can be assessed for the first time in a cell culture assay or animal model such as a mouse, rat, rabbit, dog, pig or monkey. Animal models can also be used to determine appropriate concentration ranges and routes of administration. This information can then be used to determine dosages and routes useful for administration to humans.

As used herein, the term acceptable excipient means a component used in the composition that does not interfere with the biologically active effects of the active ingredient (s) of the composition and is not toxic to the host, tissue or organ being treated.

As used herein, the term acceptable excipient means an ingredient used in the composition that does not interfere with the biologically active effects of the active ingredient of the composition and is not toxic to the host or patient. Moreover, excipients are advantageously compounds which are least likely to be rejected by the immune system of the subject being treated.

Such acceptable and / or pharmaceutically acceptable excipients such as stabilizers, buffers, suspending agents, carriers and the like are used for a variety of purposes and many literatures, such as the British Positron, the Japanese Pharmacopoeia and the American Pharmacopoeia XXII and the National Prescription XIII and its appendices It is described and recorded in.

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

The inventors of the present invention have found that a series of compounds that possess ACAT inhibitory activity is useful for reducing or preventing fibrosis and related disorders in well-accepted animal models of fibrotic disease.

In one aspect, the invention provides an antifibrotic composition comprising an effective amount of at least one antifibrotic agent and an acceptable excipient. More specifically, the antifibrotic agent is an ACAT inhibitor. For example, the ACAT inhibitor may be one of the ACAT inhibitors or mixtures thereof as defined in Table 1.

CAS registry numbers and / or publications / patents describing preferred ACAT inhibitors contemplated by this invention are identified in Table 1. These include the following compounds: F-1394, Avasimive, Factmive (CS-505), Eflusimive (F12511), Eldasimive, NTE 122, AS-183, KW-3033, E5324, FY 087, FCE 27677, CI 976, TEI 6522, K-604, Octimibate, FR 179254 and S 58-035.

In a related aspect, the present invention relates to a method of preventing or treating fibrosis or fibrotic disease in a subject. The method includes after the first step of identifying a subject suffering from or at risk of developing fibrosis, administering an ACAT inhibitor to the subject in an amount sufficient to reduce the level of collagen. The method also includes the additional step of measuring reduced collagen levels in the subject. The ACAT inhibitor is preferably one of the ACAT inhibitors or mixtures thereof as defined in Table 1.

Fibrotic diseases to be treated or ameliorated include, for example, scleroderma, keloid and hypertrophic scars, collagen disorders associated with the development of Raynaud's syndrome, pulmonary inflammation and fibrosis, interstitial lung disease, idiopathic pulmonary fibrosis, sarcoidosis, viral or parasites Liver cirrhosis and liver fibrosis resulting from infection, renal disorders associated with unregulated TGF-β activity and excessive fibrosis, renal interstitial fibrosis, renal fibrosis in transplant patients, focal glomerulosclerosis, fibrosis caused by marfan disease, cardiac fibrosis, Fibrosis, eye disease, peritoneal fibrosis, intestinal fibrosis and chemotherapy drug induced fibrosis and burns resulting from radiation induced fibrosis and wound healing.

Use of ACAT inhibitors to prevent or reduce collagen deposition in tissues

It is well known that some types of fibrosis are characterized by abnormal collagen deposition resulting from increased collagen synthesis and / or reduced collagen degradation.

Accordingly, the present invention relates to the use of one or more ACAT inhibitors for the prevention or reduction of collagen deposition in tissues.

The expression 'prevention or reduction of collagen deposition in tissue' means that collagen deposition in excess of normal deposition is blocked, delayed or avoided in certain tissues, or that excess collagen deposited on the tissue is improved or eliminated compared to normal deposition. do.

For example, the ACAT inhibitor used for the prevention or reduction of collagen deposition in tissues may be one of ACAT or a mixture thereof as defined in Table 1.

The present invention also provides a method of reducing collagen levels in a tissue, the method comprising contacting the tissue with at least one ACAT inhibitor after providing the tissue. The next step consists in measuring the reduced collagen levels in the tissues. According to a preferred embodiment, the ACAT inhibitor used in the method is one of the compounds or mixtures thereof defined in Table 1. Preferably, the tissue is a fibrous tissue. It may also be tissues of the heart, lungs, brain, eye, stomach, spleen, bone, pancreas, kidney, liver, intestine, skin, uterus or bladder.

In a related aspect, the present invention also relates to a composition useful for preventing or reducing collagen deposition in tissue, comprising a composition comprising an effective amount of one or more ACAT inhibitors as defined in Table 1 and acceptable excipients.

It will be understood that the tissue is organ tissue of a subject, such as a human. The tissue is, for example, tissue of an organ selected from the heart, lungs, brain, eye, stomach, spleen, bone, pancreas, kidney, liver, intestine, skin, uterus and bladder.

Use of ACAT inhibitors in the prevention of excessive fibrous tissue formation or development in organs

In the process of tissue repair, the fibrosis phase consists of the formation of fibrous tissue in which connective tissue replaces milk tissue. If the fibrosis phase remains unrestricted, the tissue repair process can become pathogenic, leading to proliferative tissue remodeling.

Accordingly, the present invention proposes the use of ACAT inhibitors to prevent the formation or development of excess fibrous connective tissue in organs. More specifically, the ACAT inhibitor can be one of the compounds or mixtures thereof defined in Table 1.

The term 'preventing the formation or development of excess fibrous connective tissue in organs' refers to blocking, delaying or avoiding, in certain organs, the formation of fibrous connective tissue in excess of normal cells.

It is to be understood that the organ is, for example, the organ of a subject, such as a human. The organ is, for example, the heart, lungs, brain, eye, stomach, spleen, bone, pancreas, kidney, liver, intestine, skin, uterus or bladder.

In a related aspect, the invention provides a method of preventing the formation or development of excess fibrous connective tissue in an organ of a subject, comprising administering to the subject an effective amount of at least one ACAT inhibitor. More specifically, the ACAT inhibitor may be one of the ACAT inhibitors or mixtures thereof as defined in Table 1. In a preferred embodiment, the organ is selected from heart, lung, brain, eye, stomach, spleen, bone, pancreas, kidney, liver, intestine, skin, uterus and bladder, and the subject is a human.

ACAT Inhibitor Compositions of the Invention

The composition according to the invention contains a carrier or vehicle useful for the delivery of one or more excipients or formulation materials, such as ACAT inhibitors, while at the same time maintaining and preserving antibiotics. The formulation can be applied to the condition to be treated. For example, treatment of fibrotic disease can be delivered locally, orally or by injection. Alternatively, the composition can be delivered by inhalation therapy. Other suitable means for introducing therapeutic molecules include implantable drug delivery devices.

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

Suitable vehicles for parenteral injection contemplated by the present invention are sterile distilled water in which the desired ACAT inhibitor is formulated as a sterile isotonic solution and properly preserved.

When the compositions of the present invention consist of aqueous injection suspensions, they preferably contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. In addition, suspensions of certain ACAT inhibitors may be prepared as appropriate oil 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 can also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or agents for increasing the solubility of the ACAT inhibitor and for preparing highly concentrated solutions.

The composition of the present invention may be formulated for inhalation. For example, ACAT inhibitors can be formulated as dry powders for inhalation. ACAT inhibitor inhalation solutions can also be formulated with propellants for aerosol delivery. Alternatively, the solution may be nebulized.

Compositions comprising ACAT inhibitors may be formulated for topical administration. Examples of topical compositions may take the form of creams, ointments or lotions.

It is also contemplated that the compositions of the present invention may be administered orally. Compositions for oral administration may be formulated using an acceptable carrier known to those skilled in the art in a suitable dosage for oral administration. With such carriers, the compositions can be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like for ingestion by a patient.

Moreover, the capsule may be designed to release the active portion of the formulation at the gastrointestinal tract point where bioavailability is maximized and total systemic degradation is minimized. Additional agents can be included to facilitate the absorption of ACAT inhibitors. Diluents, flavors, low melting waxes, vegetable oils, lubricants, emulsifiers, tablet disintegrants and binders may also be used.

Examples of oral formulations may consist of capsules that can be prepared by granulating the ACAT inhibitor contemplated herein by lactose and corn starch as excipients and hydroxypropylcellulose as a binder. Such capsules may consist of white hard gelatin capsules containing a predetermined amount of ACAT inhibitor.

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

Ingredient (mg) Capsule Contents F-1394 25.0 Triethyl citrate 35.0 Propylene Glycol Fatty Acid Ester 110.0 Polyoxyethylene Caster Oil-60 170.0 Subtotal 340.0 Capsule Hard Capsule (Gelatin) 70.0 Capsule coating Methacrylic acid copolymer LD 29.84 Triethyl citrate 3.0 Talc 9.16 Subtotal 42.0 Grand total 452.0

Formulations for oral use can be obtained by combining certain ACAT inhibitors with solid excipients and processing the resulting granule mixture (optionally after grinding) to obtain a tablet or dragee core. Suitable excipients of the formulations include, for example, sugars, including carbohydrate or protein fillers such as lactose, sucrose, mannitol and sorbitol; Starch from corn, wheat, rice, potatoes or other plants; Celluloses such as methyl cellulose or sodium carboxymethylcellulose; And proteins such as gelatin and collagen.

Within the scope of the present invention, sustained or controlled delivery formulations of the ACAT inhibitors of the invention are provided. Those skilled in the art will know how to prepare such formulations.

Dosage levels for treatment will depend, in part, on the type of ACAT inhibitor delivered, the specific indications on which the ACAT inhibitor is used, the route of administration and the size of the patient (weight, body surface or organ size) and the condition (age and overall health). do. For example, the clinician can titrate the dosage and change the route of administration to achieve the optimal therapeutic effect.

For any compound, the effective dosage can be assessed for the first time in a cell culture assay or animal model such as a mouse, rat, rabbit, dog, pig or monkey. Animal models can also be used to determine appropriate concentration ranges and routes of administration. This information can then be used to determine dosages and routes useful for administration to humans.

The frequency of administration will vary depending on the pharmacokinetic parameters of the ACAT inhibitor of the particular formulation employed. Typically the composition is administered until a dosage is achieved that achieves the desired effect. Thus, the composition may be administered as a single dose or multiple doses (same or different concentrations / dosages) over a period of time, or as a continuous infusion. Additional consideration of appropriate dosages is made regularly. Appropriate dosages can be ascertained using appropriate dosage response data.

By way of example, a capsule as described in Table 2 comprising 25 mg of ACAT inhibitor F-1394 may be administered at least once a day, preferably at least twice a day. However, the optimal therapeutic effect can be obtained by adjusting the F-1394 capsule content as well as the daily dosage.

The following examples illustrate the invention with reference to the accompanying drawings. The above examples illustrate the broad applicability of the present invention and are not intended to suggest the scope thereof. Modifications and variations can be presented herein 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 experimental runs of the present invention, the preferred methods and materials are described.

Example 1 ACAT Inhibitors Inhibit the TGF-β Signaling Pathway

TGF-β belongs to a large family of secretory peptide growth factors that play an important role in animal development. Upon ligand binding, type I and II receptors are supplemented to the complex, and type II receptors phosphorylate and thus activate the type I receptor. As a result, type I receptors phosphorylate Smad in the R-Smad (receptor regulatory) subgroup. R-Smad forms complexes with Co-Smad and accumulates in the nucleus to regulate gene transcription through other transcription factors (FIG. 1). These pathways regulate many cellular processes and play an important role in normal development. Disruption of this pathway can lead to a wide range of diseases such as cancer, cardiovascular, inflammatory and fibrotic diseases as well as Marfan's syndrome.

In worms, the TGF-β pathway regulates entry into the Dower larval stage, an alternative larval stage that differentiates for survival under negative conditions. Blocking the TGF-β pathway leads to structural Doyer formation, ie the formation of Dower larvae even under favorable conditions. Thus, an increase in dower formation indicates a decrease in signaling through this pathway. However, other signaling pathways exist, such as analogous insulin signaling pathways, which affect Doer formation in worms. Structural Doyer formation via the TGF-β pathway requires the activity of DAF-3 and DAF-5, while structural Doyer formation via the similar insulin signaling pathway is not.

The purpose of this study was to establish that ACAT inhibitors possess interference of the TGF-β signaling pathway.

Substances and Methods

Common way

All assays were performed in 24 well plates. Wells contained 1.5 mL of nematode growth medium (NGM). On day 1, an appropriate volume of compound mother liquor was added to the NGM well surface and dried in the dark for at least 2 hours. The wells were then seeded with 3-5 μL of OP50 bacterial medium and concentrated 15-fold. The worms were transferred to the wells as eggs per day, then at 20 ° C. (for daf-8, daf-14 and daf-4; daf-5 ) or at 22 ° C. (for daf-1 and daf-7 ). It was allowed to grow for 3 days. The percentage of Dower stage worms was measured at 4 days and / or 5 days.

Worm

Nematode strains were obtained from Caenorhabditis gene center (CGC) and maintained at 20 ° C. using standard culture methods. The wild type strain used was Caenorhabditis elegans Britol strain, N2. Mutants used 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) There is II .

compound

Avashimave, Factive, F-1394, FR19254, S58035 were stored at −20 ° C. as a 10 mM mother liquor. All compounds were dissolved in DMSO. Compounds were analyzed at a final concentration of 82.5 μM with the exception of Avashimaib, which was analyzed at 10.3 μM for the daf-14 mutant, and at a final concentration of 55 μM for other mutations.

Results and discussion

ACAT inhibitors affect Doyer formation by reducing signaling through the TGF-β pathway

All ACAT inhibitors tested increased Doer formation in the Elfin daf-14 mutant, which was a Dow structural mutant in the TGF-β pathway (FIG. 2). All ACAT inhibitors tested were daf-4; Since there is no effect in the daf-5 double mutant, this effect requires the activity of DAF-5 (Table 3). Indeed , daf-5 is required for Doyer formation due to inhibition of TGF-β signaling, and the absence of induction of Doyer formation by ACAT inhibitors in the background of daf-5 mutants has the effect of TGF-β on Doyer. Indicating that the signaling pathway is through inhibition. Moreover, we tested F-1394 and Avasimive in several other Dow structural mutants in the TGF-β pathway, in particular daf-7, daf-1 and daf-8 . We have found that Avashimave as well as F-1394 enhances Doer formation in this mutant (FIG. 3), which is consistent with the results described in FIG. 2.

It has also been shown that ACAT inhibitors affect Doer formation, in particular by reducing signaling through the TGF-β pathway.

process % Dow Sample size DMSO 0 50 Abashima Live 0 321 F-1394 0 83 Factive 0 50 FR179254 0 50 S 58-035 0 50

Table 3: ACAT inhibitors do not induce DAU formation in daf-4 (m63); daf-5 (e1386) double mutants. The following compounds were tested as described in the Materials and Methods section: Abashimaib (n = 651), F1394 (n = 935), Fantimive (n = 1166), FR179254 (n = 1056) and S 58- 035 (n = 765). DMSO solvent was used as a control (n = 941). The compounds tested were free from Dow inducing activity in the tested worm background.

Example 2: TGFβ in an In Vitro Animal Model Associated with Human Lung Epithelial Cell Line A549, Human Lung Fibrosis and COPD _One  Effect of F-1394 on Induced Substrate Protein Expression

Converting growth factor (TGF) -β ₁ , multifunctional or pleiotropic cytokines are involved in numerous biological processes such as cell proliferation, differentiation, apoptosis, fibrosis, wound repair and inflammation (Ning W. et al. 2002; Martin GEM et al. 2006). In particular, TGFβ ₁ is a growth factor that has a role as an important mediator of lung tissue response to the wound; Thus, it is involved in lung repair, and the mechanism of fibrosis after the inflammatory process (Bellocq A. et al. 1999). Reactive oxygen and nitrogen intermediates increase the release of TGFβ ₁ from human epithelial alveolar cells that may well constitute molecular binding between inflammatory and fibrous processes (Bellocq A. et al. 1999).

Fibrous collagen type I is characterized by being synthesized by fibroblast-like cells (Kasai H. et al. 2005).

Previous work has demonstrated that TGFβ ₁ , but not other inflammatory cytokines, induces A549 cells with alveolar epithelial type II cell phenotype to undergo epithelial mesenchymal conversion involving expression of fibroblast phenotypic markers (Kasai H. et al. 2005).

Substances and Methods

Experimental model

Human alveolar epithelial cell carcinoma line A549 (American Microbial Conservation Center; Rockville, Maryland) is described in Mata M. et al. 2005, as outlined above. Recombinant human TGFβ ₁ was used at a concentration of 5 ng / mL. The medium was changed every 24 hours with the appropriate addition of TGFβ ₁ and F-1394. F-1394 was present before the first addition of TGFβ ₁ from 1 hour until the end of the experiment.

mRNA analysis

For mRNA analysis, cultured A549 cells were described in Mata M. et al. 2005, as outlined above. Total RNA was extracted using TriZol Reagent ^™ (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The integrity of the extracted RNA was confirmed using Bioanalizer ^™ (Agilent, Cala Alto, CA, USA). α1 (I) collagen mRNA was measured by quantitative RT-PCR as outlined (Manoury B. et al. 2006). Measurement time: 72 hour time point as outlined above (Kasai H. et al. 2005).

Experimental group

In vivo experiments were conducted with treatment groups with F-1394 and reference compounds as well as their respective controls as follows, as well as appropriate negative and positive controls:

(1) control vehicle (DMSO)

(2) F-1394 (in DMSO) Dose Level 3 (3 μg / mL)

(3) TGFβ ₁ + DMSO

(4) TGFβ ₁ + F-1394 (in DMSO) Dose Level 1 (0.3 μg / mL)

(5) TGFβ ₁ + F-1394 (in DMSO) Dose Level 2 (0.6 μg / mL)

(6) TGFβ ₁ + F-1394 (in DMSO) Dose Level 3 (3 μg / mL).

Results and discussion

Data are shown as mean ± SEM. Statistical analysis of the results was performed by an analysis of variance followed by Bonferroni's test (GraphPad Software Inc, San Diego, CA, USA). An effective value was obtained when P <0.05.

TGFβ ₁ markedly increased α1 (I) collagen mRNA expression as shown in FIG. 4.

The TGFβ ₁ induced augmented expression was significantly reduced in a concentration dependent manner in the presence of F-1394 (FIG. 4).

F-1394 as a prototypical ACAT inhibitor is effective at significantly reducing the enhanced α1 (I) collagen mRNA expression caused by TGFβ ₁ in cultured A549 cells, the human airway epithelial cell line.

Example 3: Effect of F-1394 on an In Vivo Animal Model Associated with Bleomycin-Induced Lung Injury, Human Lung Fibrosis

Bleomycin-induced lung injury is an established model considered to be related to the study of anti-inflammatory and antifibrotic (remodeling) activity of new compounds.

Substances and Methods

A total of 66 mice were fed to the study. The number of animals per group was approximately 10-12 (as needed). Each mouse weighed approximately 20 g at the time of dispensing (6 weeks).

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

Experimental model

Male mature C57BL / 6 male mice (weight of about 20 g each) stored at 0.075 U bleomycin sulfate (Sigma Catalog B 5507, 2-8 ° C.) dissolved in 50 μl saline (0.9% NaCl), 1.2-mg / mg solids Organ administration by oral route in a single dose of 1.5 units). Control animals were subjected to the same protocol and infused saline in time instead of bleomycin. Institutional instillation was performed under halosane anesthesia. At 14 days after intratracheal bleomycin / saline administration, the animals were killed by anesthesia (sodium pentobarbital) followed by bleeding from the abdominal artery. Lungs were obtained and metered. The right and left lungs were then treated separately for histological and biochemical studies as indicated below. 14 days after bleomycin was selected as the peak time of collagen synthesis (Lazemby et al 1990).

Histological evaluation

For histological examination, the lungs were first sprayed through their main bronchus with fixative (10% neutral buffered formalin), kept at hydrostatic pressure 25 cm for 15 minutes, impregnated with the fixative for 24 hours and taken a block. . Only lungs that were well inflated with fixative solution were analyzed. Tissue blocks are placed in formalin, dehydrated in a graded series of ethanol, immersed in paraffin, cut into a series of 4 μm thick, and stained with hematoxylin-eosin to stain inflammatory cells and fibrous regions. Confirmed.

Histological grading of the injury was performed using a blinded semiquantitative scoring system for the severity and severity of inflammation and fibrosis in pulmonary milk tissues based on previous studies from the laboratory (Cortijo et al. ., 2001; Mata et al, 2003;. Serrano-Mollar et al, 2002;.. Serrano-Mollar et al, 2003). The severity of fibrosis was scored by Ashcroft and coworkers. Briefly, grades of pulmonary fibrosis were scored in stages 0-8 according to the following criteria: grade 0, normal lung; Grade 1, minimal fibrosis thickening in the alveoli or bronchial walls; Grade 3, thickening of the wall usually without obvious damage to the lung structure; Grade 5, increased fibrosis with obvious damage to the lung structure and formation of fibrous bands or small fibrous masses; Grade 7, severe distortion of the structure and large fibrous areas; Grade 8, erasure of the whole field of fibrous. Grades 2, 4 and 6 apply as intermediate photographs. The severity of fibrous changes in each lung section was assessed as the mean score of severity from the observed microscopic fields. Random fields of each region were analyzed. The grading was performed in a blind manner by two observers and the mean value was taken as the fibrosis score.

Experimental group

In vivo experiments were conducted with appropriate negative and positive controls as well as with groups receiving different drug treatments as needed and their respective controls (drug vehicle, treated negative controls).

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

Results and discussion

Data are shown as mean ± SEM. Statistical analysis of the results was performed after variance analysis by Bonferroni test or an appropriate non-parametric test (GraphPad Software Inc., San Diego, CA, USA). Survival curves were analyzed by logrank using Graphpad Software Inc. (Prism 4.03). An effective value was obtained when P <0.05.

Α1 (I) Collagen mRNA in Lung Tissues

NAC as well as F-1394 show a significant inhibitory effect on the expression of α1 (I) collagen transcription, which exhibits an antifibrotic effect of reduced collagen deposition (FIG. 5).

F-1394 as a prototypical ACAT inhibitor is a meaningful compound endowed with antifibrotic properties at the level of α1 (I) collagen mRNA expression in lung tissue.

Histological evaluation

Lungs from control animals were histologically normal. Administration of bleomycin may be characterized by prominent peribronchial and interstitial areas of interstitial infiltration by inflammatory cells (mainly mononuclear cells, including macrophages and lymphocytes with little neutrophils and dispersible eosinophils), proliferative cell thickening in the alveolar septum. And characteristic histological damage, including an increase in interstitial cells with interstitial edema and fibroblast development. The damage distribution pattern was multiple (ie, patchy area of pulmonary fibrosis). Multiple oily tissue damage is also present, but treatment with F-1394 significantly reduced lung form deformation; There was little inflammatory penetration, less collagen deposition, and less septal expansion (FIG. 6). It was noted that the Ashcroft score was significantly reduced (FIG. 7). Similar results were obtained for N-acetylcysteine (NAC).

Example 4: Effect of F-1394 on Unilateral Urinary Occlusion in Mice

Unilateral urinary tract obstruction (UUO) is a stable palpable experimental model that mimics the different characteristics of obstructive nephropathy causing tubular interstitial fibrosis. Due to its rapidness and high productivity, this model is used to provide rapid evidence for using F-1394 as a prototype ACAT inhibitor to prevent interstitial collagen accumulation.

The purpose of this study was to evaluate the effect of prophylactic treatment with F-1394 on total interstitial fiber collagen deposition by serious red histochemistry. The effect of F-1394 was assessed by quantification of specific stained areas on the entire closed kidney area surface.

Substances and Methods

animal

Male C57BL / 6J mice (body weight 25-30 g at the start of the experiment) were used. Mice were housed in groups of five (5) in polypropylene gauges on a wood litter with free access to food (Teklad ^™ 5018; HARLAN, Cannat, France). The animal house was kept under artificial light at 7: 00-19: 00 (12 hours) at a controlled room temperature of 20 ± 1 ° C., and the relative humidity was kept at 60%. Mice were sacrificed by cervical dislocation. Animals were not dissected.

Research material

F-1394 dosage formulations were prepared in two different ways; 200 mg / kg of solution using 1% Tween80 and 0.5% CMC, and 300 mg / kg of solution using 0.5% CMC only. F-1394 was administered orally at a dose of 20 mg / kg / day for the first 10 days and 300 mg / kg / day for the remaining 5 days of the protocol. Captopril (Sigma-Aldrich, St. Louis, USA) was dissolved in 1% Tween 80 and 0.5% CMC and administered orally at a dose of 32 mg / kg / day for 15 days.

Main device

Morphometric metrology measurements are based on computerized image analysis of serious red stained sections, microscope (Eclipse 600 ^TM , Nikon Instruments; Champignon-Schschein, France) and objective lens at 20 magnification (final calibration 0.366). (μm / pixel) with a numerical camera (MicroFire ^™ , Optronics, Novei Avanex, France). Quantitative analysis of the photographs was performed using ExploraNova MorphoLite ^™ and Expert ^™ software (La Rochelle, France).

Experimental protocol

Four experimental groups with 10 mice each were used:

(1) simulated mice

(2) closed mice treated with vehicle (0.1% Tween80 / 0.5% CMC)

(3) closed mice treated with F-1394

(4) closed mice treated with captopril

Oral treatment continued for fifteen (15) days and closure for fourteen (14) days.

Experimental Unilateral Urinary Tract Obstruction

Schanstra et al. , 2002] Unilateral urinary obstruction was performed. Briefly, mice were anesthetized with a mix of oxygen-isoprulane (0.2 L / min and 2% isoflurane, La Palis Centrabet, France). A 5 mm skin incision was made at 1 cm from the last left rib corner of the mouse, followed by a 5 mm muscle incision. The left urinary tract was exposed and ligated in the ureteric right marginal region (6/0 cardionyl thread; Peters Surgical, Bobbini, France). The muscle wall was then closed (5/0 eticlean yarn, Onu Eticon, France) and the skin wall was closed by suture clips. Throughout the surgery, animals were placed on a 35 ° C. hot rate.

Food and water were provided arbitrarily. Oral treatment started one day before UUO and closure was continued for 14 days. At the end of the protocol, the mice are killed by cervical dislocations; Obstructed kidneys were removed and divided for histology and preparation for further gene expression analysis.

Histological analysis

After the obstructed kidneys were removed, the central fragments used for histology were fixed in carnoy solution for 24 hours and immersed in paraffin according to standard procedures. Histological sections 3-4 μm thick and stained with serious red for overall interstitial fibrous collagen deposition evaluation.

Sections were deparaffinized, rehydrated, stained with 0.1% Serious Red F3B ^™ (BDH Laboratories, Fontenay Subois VWR, France) in saturated aqueous picric acid, differentiated for 1 minute in 0.01 N HCl. , Dewatered and mounted quickly.

The tubular interstitial fiber collagen surface was calculated by measuring the total cortical area or the stained tissue area within a given filter. Glomeruli and vasculature were excluded from the evaluation of this study.

Record analysis

Analysis was performed by an operator who did not know the origin of each kidney region. Double blind quantification of selected targets in a given image was recorded in a Microsoft Excel file linked with ExploraNova ^™ software. Removal of histological code was performed at the end of the results classification record.

Results are expressed as percentage of fibrosis with respect to the total area studied.

Result analysis and presentation

Results are presented as mean values ± mean standard error (SEM). One-way ANOVA was used to compare intragroup differences, followed by a Newman-Cools test for comparison of all column pairs (GraphPad Prism, San Diego, USA). P <0.05 was taken as statistical significance.

Results and discussion

Effect of F-1394 and Captopril on UUO Induced Vascular Interstitial Fibrosis

Interstitial fibrosis was significantly higher in the UUO group treated by the vehicle group compared to mimicked animals (FIG. 8), and interstitial collagen expression was 13.36 ± 0.60% and 0.26 ± 0.03% in the kidney region, respectively (n = 10, p <0.001, ANOVA with Newman Coulse test. This effect is in accordance with previous publications (Schanstra et al , 2003 and 2002).

F-1394 reduces UUO-induced tubular interstitial fibrosis compared to vehicle group (5.43 ± 0.29% vs 13.36 ± 0.60%, n = 10, p <0.001, respectively, in the F-1394 and vehicle groups, with Neumann Cools test) ANOVA). Captopril treatment led to a desirable reduction in UUO induced tubular interstitial fibrosis (2.89 ± 0.12% vs 13.36 ± 0.60%, n = 10, p <0.001, ANOVA with Newman Cools test, respectively in captopril and vehicle groups) .

The results indicate that administration of F-1394 as a prototypical ACAT inhibitor significantly reduces tubular interstitial fibrosis in the UUO model, equivalent to captopril efficacy.

Reference

Claims (27)

Compositions useful for the prevention or reduction of collagen deposition in tissues include, but are not limited to, F-1394, Avasimive, Factive (CS505), Eflushimaive (F12511), Eldasimive, NTE 122, AS183, KW3033, E5324, FY087 A composition comprising an effective amount of at least one ACAT inhibitor and an acceptable excipient, including FCE27677, CI976, TEI6522, K604, Octimibate, FR179254, S58-035, or mixtures thereof. The composition of claim 1, wherein the ACAT inhibitor is F-1394. The composition of claim 1, wherein the tissue is heart, lung, brain, eye, stomach, spleen, bone, pancreas, kidney, liver, intestine, skin, uterus or bladder. An antifibrotic composition comprising an effective amount of at least one antifibrotic agent and an acceptable excipient. The composition of claim 4, wherein the antifibrotic agent is an ACAT inhibitor. The method according to claim 5, wherein the ACAT inhibitor is F-1394, Avasimive, Factive (CS505), Eflushimaive (F12511), Eldasimive, NTE 122, AS183, KW 3033, E5324, FY 087, The antifibrotic composition of FCE 27677, CI 976, TEI 6522, K604, Octimibate, FR179254, S 58-035 or mixtures thereof. 7. The antifibrotic composition according to claim 6, wherein the ACAT inhibitor is F-1394, abashimaib, factive, FR179254 or S 58-035. The antifibrotic composition of claim 7 wherein the ACAT inhibitor is F-1394. Use of an ACAT inhibitor for the prevention or reduction of collagen deposition in tissues. 10. The method of claim 9, wherein the ACAT inhibitor is F-1394, Avasimive, Factive (CS505), Eflushimaive (F12511), Eldasimive, NTE 122, AS183, KW 3033, E5324, FY 087, FCE 27677, CI 976, TEI 6522, K604, Octimibate, FR179254, S 58-035 or mixtures thereof. 10. The use according to claim 9, wherein said tissue is heart, lung, brain, eye, stomach, spleen, bone, pancreas, kidney, liver, intestine, skin, uterus or bladder. Use of an ACAT inhibitor to prevent the formation or development of excessive fibrous connective tissue in an organ. The method according to claim 12, wherein the ACAT inhibitor is F-1394, Avasimive, Factive (CS505), Eflushimaive (F12511), Eldasimive, NTE 122, AS183, KW 3033, E5324, FY 087, FCE 27677, CI 976, TEI 6522, K604, Octimibate, FR179254, S 58-035 or mixtures thereof. 13. The use according to claim 12, wherein the tissue is heart, lung, brain, eye, stomach, spleen, bone, pancreas, kidney, liver, intestine, skin, uterus or bladder. A method of 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. The method according to claim 15, wherein the ACAT inhibitor is F-1394, Avasimive, Factive (CS505), Eflushimaive (F12511), Eldasimive, NTE 122, AS183, KW 3033, E5324, FY 087, FCE 27677, CI 976, TEI 6522, K604, Octimibate, FR179254, S 58-035 or mixtures thereof. The method of claim 15, wherein the tissue is fibrous tissue. The method of claim 15, wherein the tissue is heart, lung, brain, eye, stomach, spleen, bone, pancreas, kidney, liver, intestine, skin, uterus or bladder. A method of preventing the formation or development of excess fibrous connective tissue in an organ of a subject, comprising administering to the subject an effective amount of at least one ACAT inhibitor. The method of claim 19, wherein the one or more ACAT inhibitors are F-1394, Avasimive, Factive (CS505), Eflushimaive (F12511), Eldasimive, NTE 122, AS183, KW 3033, E5324, FY. 087, FCE 27677, CI 976, TEI 6522, K604, Octimibate, FR179254, S 58-035 or mixtures thereof. The method of claim 19, wherein the organ is a heart, lung, brain, eye, stomach, spleen, bone, pancreas, kidney, liver, intestine, skin, uterus or bladder. The method of claim 21, wherein the subject is a human. A method of preventing or treating fibrosis or fibrotic disease in a subject, Identifying a subject suffering from or at risk of developing fibrosis; Administering to the subject an ACAT inhibitor in an amount sufficient to reduce the level of collagen; And Measuring the reduced level of collagen in the subject How to include. The method of claim 23, wherein the ACAT inhibitor is selected from F-1394, Avasimive, Factive (CS505), Eflushimaive (F12511), Eldasimive, NTE 122, AS183, KW 3033, E5324, FY 087, FCE 27677, CI 976, TEI 6522, K604, Octimibate, FR179254, S 58-035 or mixtures thereof. The method of claim 24, wherein the ACAT inhibitor is F-1394. The method of claim 23, wherein the subject is a human. 24. The method of claim 23, wherein the fibrotic disease is from scleroderma, keloid and hypertrophic scars, collagen disorders associated with the development of Raynaud's syndrome, pulmonary inflammation and fibrosis, interstitial lung disease, idiopathic pulmonary fibrosis, sarcoidosis, viral or parasitic infections. Induced cirrhosis and hepatic fibrosis, kidney disorders associated with unregulated TGF-β activity, excessive fibrosis, renal interstitial fibrosis, renal fibrosis in transplant patients, focal glomerulosclerosis, fibrosis caused by marfan disease, cardiac fibrosis, radiation induction Sex fibrosis, fibrosis resulting from wound healing, eye disease, peritoneal fibrosis, intestinal fibrosis and chemotherapy drug-induced fibrosis or burns.

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