EP1981493A1 - Inhibiteurs d'acat et leur utilisation dans la prévention ou le traitement de la fibrose - Google Patents

Inhibiteurs d'acat et leur utilisation dans la prévention ou le traitement de la fibrose

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Publication number
EP1981493A1
EP1981493A1 EP07845502A EP07845502A EP1981493A1 EP 1981493 A1 EP1981493 A1 EP 1981493A1 EP 07845502 A EP07845502 A EP 07845502A EP 07845502 A EP07845502 A EP 07845502A EP 1981493 A1 EP1981493 A1 EP 1981493A1
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EP
European Patent Office
Prior art keywords
fibrosis
tissue
acat inhibitor
collagen
acat
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07845502A
Other languages
German (de)
English (en)
Other versions
EP1981493A4 (fr
Inventor
Siegfried Hekimi
Abdelmadjid K. Hihi
Robyn S. Branicky
Xihua Jia
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sosei Co Ltd
Original Assignee
Chronogen Inc
Sosei Co Ltd
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Publication date
Application filed by Chronogen Inc, Sosei Co Ltd filed Critical Chronogen Inc
Publication of EP1981493A1 publication Critical patent/EP1981493A1/fr
Publication of EP1981493A4 publication Critical patent/EP1981493A4/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/08—Vasodilators for multiple indications

Definitions

  • the present invention provides novel methods and compositions for preventing and reducing fibrosis associated with fibrotic disorders using ACAT inhibitors. More particularly, the present invention relates to the use of ACAT inhibitors in compositions and methods for preventing or treating fibrosis, for modulating collagen deposition in a tissue, and in the prevention and reduction of excessive fibrous connective tissue in an organ.
  • the process of tissue repair as part of wound healing involves two phases.
  • the first phase is the regenerative phase, in which injured cells are replaced by cells of the same type.
  • the second phase is the formation of fibrous tissues, also called fibroplasias or fibrosis, in which connective tissue replaces parenchyma tissues.
  • fibrous tissues also called fibroplasias or fibrosis, in which connective tissue replaces parenchyma tissues.
  • the tissue repair process can become pathogenic if the fibrosis phase continues unrestrained, leading to extensive tissue remodelling and the formation of permanent scar tissue (Wynn 2004).
  • ILD interstitial lung disease
  • ILD interstitial lung disease
  • silicosis pulmonary inflammation
  • collagen vascular diseases The most common type of ILD is idiopathic pulmonary fibrosis (IPF), which still has no known cause (idiopathic).
  • fibrotic disorders include liver cirrhosis and fibrosis originating from viral hepatitis, kidney disorders associated with unregulated TGF- ⁇ activity and excessive fibrosis such as glomerulonephritis (GN), renal interstitial fibrosis, renal fibrosis in transplant patients, and eye diseases such as macular degeneration and retinal and vitreal retinopathy.
  • fibroproliferative disorders include systemic and local scleroderma, keloids and hypertrophic scars, and collagen disorders associated with the occurrence of Raynaud's syndrome. Excessive scarring resulting from surgery, chemotherapeutic drug-induced fibrosis, radiation-induced fibrosis, and burns are also part of fibroproliferative diseases.
  • Acyl-CoA:Cholesterol acyltransferase catalyzes the formation of cholesteryl esters using both cholesterol and long chain fatty acyl co-enzyme A as substrate.
  • ACAT is present in a variety of tissue including intestinal mucosa, liver, adrenal, testes and macrophages.
  • ACAT inhibitors are known to reduce or prevent the appearance of atheromatous lesions in animal models.
  • TGF- ⁇ tumor growth factor ⁇
  • the present invention provides novel methods and compositions for preventing and reducing fibrosis and/or fibrotic disorders using ACAT inhibitors.
  • the present invention provides an antifibrotic composition comprising an effective amount of at least one antifibrotic agent and an acceptable excipient.
  • the present invention concerns the use of an ACAT inhibitor for preventing or reducing collagen deposition in a tissue.
  • Another aspect of the present invention concerns the use of an ACAT inhibitor for preventing the formation or development of excess fibrous connective tissue in an organ.
  • the present invention also encompasses a method for reducing the level of collagen in a tissue, the method comprising providing a tissue, contacting the tissue with at least one ACAT inhibitor and measuring a reduced level of collagen in the tissue.
  • the present invention relates to a method for preventing the formation or development of excess of fibrous connective tissue in an organ of a subject, the method comprising administering to the subject an effective amount of at least one ACAT inhibitor.
  • the invention provides a method for preventing or treating fribrosis or a fibrotic disorder in a subject, the method comprising:
  • Figure 1 shows a schematic representation of the TGF- ⁇ pathway in human with worm homologs.
  • FIG. 2 shows that ACAT inhibitors decrease TGF- ⁇ signalling.
  • FIG. 3 shows that ACAT inhibitors decrease TGF- ⁇ signalling in more than one TGF- ⁇ constitutive mutant.
  • Figure 4 shows the relative quantification of ⁇ 1 (I) collagen mRNA expression levels in A549 cells unstimulated (control) or stimulated with transforming growth factor (TGF; 5 ng ml "1 )- ⁇ i in the absence or presence of F-1394 (0.3, 0.6 and 1 ⁇ g ml "1 , as indicated). Exposure time was 72 h for TGF ⁇ -i. F-1394 was present from 1 h before TGF ⁇ i to the end of the experiment. TGF ⁇ -Hnduced increase of ⁇ 1 (I) collagen mRNA expression was concentration-dependently inhibited by F- 1394.
  • TGF transforming growth factor
  • ⁇ 1 (I) collagen mRNA expression was determined by using real-time RT- PCR by the ⁇ C t method; columns show the fold increase in expression of ⁇ 1 (I) collagen mRNA relative to GAPDH values as mean ⁇ SEM of the 2- ⁇ C t values of three independent experiments.
  • Figure 5 shows the relative expression of ⁇ 1 (I) collagen mRNA in lung tissue of mice receiving intratracheal bleomycin (BLM) or saline (control). Columns are mean ⁇ s.e.m. of 3-5 animals per group.
  • Figure 6 shows representative photomicrographs of lung histology in mice treated with drug vehicle + saline (a), F-1394 + saline (b), N-acetylcysteine + saline (c), drug vehicle + bleomycin (d,e,f), F-1394+bleomycin (g,h) and N- acetylcyteine+bleomycin (i). All lung sections were stained with haematoxylin- eosin. Magnification x40. Saline-treated animals show normal architecture. Bleomycin-exposed mice showed marked peribronchial interstitial infiltration with inflammatory cells, thickened alveolar septa, oedema and foci of dense fibrosis. These pulmonary lesions were reduced in animals orally treated with F-1394 and with N-acetylcysteine.
  • Figure 7 shows the evaluation of fibrotic changes in the lung by numerical fibrotic score. Bars denote Ascroft scores (mean ⁇ SEM) of each experimental group as indicated. * P ⁇ 0.05 vs. vehicle+BLEO by Mann-Whitney test. Pulmonary lesions were reduced in animals orally treated with F-1394 as well as with N- acetylcysteine.
  • the present invention provides novel methods and compositions for preventing and reducing fibrosis associated with fibrotic disorders using ACAT inhibitors.
  • ACAT inhibitor means any compound that inhibits the enzyme Acyl-CoA:Cholesterol acyltransferase (ACAT).
  • ACAT inhibitors include small organic compounds, i.e., having a molecular weight of more than 50 yet less than about 2500.
  • the ACAT inhibitors include functional chemical groups necessary for structural interactions with proteins and/or nucleic acid molecules, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical.
  • the ACAT inhibitors can include cyclic carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups.
  • the ACAT inhibitors according to the present invention are for instance the following compounds: F-1394, Avasimibe, Pactimibe (CS-505), Eflucimibe (F
  • fibrosis refers to the formation of fibrous tissue as a reparative or reactive process. Fibrosis is characterized by fibroblast accumulation and collagen deposition in excess of normal deposition in a particular tissue.
  • fibrotic disorder As used herein the expressions fibrotic disorder, fibroproliferative disease or fibrotic disease refer to conditions involving fibrosis in one or more tissues.
  • Fibrotic disorder include, but is not limited to scleroderma, keloids and hypertrophic scars, collagen disorders associated with the occurrence of Raynaud's syndrome, pulmonary inflammation and fibrosis, interstitial lung diseases, idiopathic pulmonary fibrosis, sarcoidosis, liver cirrhosis and liver fibrosis resulting from viral or from parasitical infection, kidney disorders associated with unregulated TGF- ⁇ activity and excessive fibrosis such as glomerulonephritis (GN), renal interstitial fibrosis, renal fibrosis in transplant patients, focal glomerulosclerosis, fibrosis caused by Marfan's disease, cardiac fibrosis, radiation-induced fibrosis, and fibrosis arising from wound healing, eye diseases such as macular degeneration and retinal and vitreal retinopathy. It also includes peritoneal fibrosis, intestinal fibrosis, chemotherapeutic drug-induced fibrosis and burns.
  • scleroderma means a rare, chronic disease characterized by excessive deposits of collagen, which affects the skin, and in more serious cases it can affect the blood vessels and internal organs. Typically, the most evident symptom is the hardening of the skin and associated scarring, and blood vessels may also be more visible.
  • Idiopathic pulmonary fibrosis is an inflammatory lung disorder of unknown origin (idiopathic) characterized by abnormal formation of fibrous tissue (fibrosis) between the tiny air sacs (alveoli) or ducts of the lungs.
  • Liver fibrosis refers to the accumulation of tough, fibrous scar tissue in the liver of a subject.
  • the term subject refers to animals and humans. This term thus includes, without being limited to, primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, mice, rats, rabbits, guinea pigs, captive animals such zoo animals, and wild animals.
  • tissue refers to an organ or set of specialized cells such as skin tissue, lung tissue, kidney tissue, and other types of cells.
  • treatment I treating refer to a process by which the symptoms of fibrotic disorders, fibrotic diseases, fibrosis and related disorders as exemplified hereinabove, are alleviated or completely eliminated.
  • prevention / preventing refer to a process by which symptoms of fibrotic disorders, fibrotic diseases, fibrosis and related disorders as exemplified hereinabove are obstructed, delayed or averted.
  • the expression effective amount indicates an amount that produces the desired effect as judged by clinical trial results and/or animal models. This amount can be routinely determined by one skilled in the art.
  • An effective amount of a composition to be employed will depend, for example, upon the treatment context and objectives.
  • One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the type of ACAT inhibitor delivered, the indication for which the ACAT inhibitor is being used, the route of administration, and the size (body weight, body surface or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician may titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
  • the effective dose can be estimated initially either in cell culture assays or in animal models such as mice, rats, rabbits, dogs, pigs, or monkeys.
  • animal models such as mice, rats, rabbits, dogs, pigs, or monkeys.
  • An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • the expression acceptable excipient means an ingredient used in a composition which does not interfere with the effectiveness of the biological activity of the active ingredient(s) of the composition, and which is not toxic to the host, tissue or organ intended to be treated.
  • the expression acceptable excipient as used herein means an ingredient used in a composition which does not interfere with the effectiveness of the biological activity of the active ingredients of the composition, and which is not toxic to the host or patient. Furthermore, the excipient is advantageously a compound with minimum probability of being rejected by the immune system of the subject being treated.
  • Such acceptable and/or pharmaceutically acceptable excipients are used for various purposes, such as stabilizers, buffers, suspending agents, carriers and the like and are listed and described in a number of texts including for example, the British Pharmacopeia, the Japanese Pharmacopeia and the United States Pharmacopeia XXII and National Formulary XVII and supplements thereto.
  • the inventors of the present invention found that a series of compounds having ACAT inhibitory capability are useful to reduce or prevent fibrosis and related disorders in well accepted animal models of fibrotic disorder.
  • the present invention provides antifibrotic compositions comprising an effective amount of at least one antifibrotic agent and an acceptable excipient.
  • the antifibrotic agent is an ACAT inhibitor.
  • the ACAT inhibitor may be one of the ACAT inhibitor defined in Table 1 or a mixture thereof.
  • the CAS registry numbers or/and the publication/patent that describe the preferred ACAT inhibitors contemplated by the present invention are found in Table 1. They include the following compounds: F-1394, Avasimibe, Pactimibe (CS-505), Eflucimibe (F12511), Eldacimibe, NTE 122, AS-183, KW-3033, E5324, FY 087, FCE 27677, Cl 976, TEI 6522, K-604, Octimibate, FR 179254 and S 58- 035.
  • the invention concerns a method for preventing or treating fibrosis or a fibrotic disorder in a subject.
  • the method comprises a first step of identifying a subject suffering from or being at risk of developing fibrosis, followed by a step of administering to the subject an ACAT inhibitor in an amount sufficient to decrease the level of collagen.
  • the method also includes a further step of measuring a reduced level of collagen in the subject.
  • the ACAT inhibitor is preferably one of the ACAT inhibitors defined in Table 1 or a mixture thereof.
  • the fibrotic disorder to be treated or averted is for instance scleroderma, keloids and hypertrophic scars, collagen disorders associated with the occurrence of Raynaud's syndrome, pulmonary inflammation and fibrosis, interstitial lung diseases, idiopathic pulmonary fibrosis, sarcoidosis, liver cirrhosis and liver fibrosis resulting from viral, or from parasitical infection, kidney disorders associated with unregulated TGF- ⁇ activity, excessive fibrosis, renal interstitial fibrosis, renal fibrosis in transplant patients, focal glomerulosclerosis, fibrosis caused by Marfan's disease, cardiac fibrosis, radiation-induced fibrosis, fibrosis arising from wound healing, eye diseases, peritoneal fibrosis, intestinal fibrosis, and chemotherapeutic drug-induced fibrosis or burns.
  • ACAT inhibitors for the prevention or reduction of collagen deposition in a tissue
  • the present invention concerns the use of at least one ACAT inhibitors for preventing or reducing collagen deposition in a tissue.
  • preventing or reducing collagen deposition in a tissue it is meant that collagen deposition in excess of normal deposition is obstructed, delayed or averted in a particular tissue, or that the excess of collagen deposited in the tissue, compared to a normal deposition, is alleviated or eliminated.
  • the ACAT inhibitor used for preventing or reducing collagen deposition in the tissue may be one of the ACAT defined in Table 1 or a mixture thereof.
  • the present invention also provides a method for reducing the level of collagen in a tissue which comprises a step of providing a tissue followed by a step of contacting the tissue with at least one ACAT inhibitor.
  • the next step consists of measuring a reduced level of collagen in the tissue.
  • the ACAT inhibitor used in this method is one of the compounds defined in Table 1 or a mixture thereof.
  • the tissue is preferably a fibrotic tissue. It may also be a tissue of heart, lung, brain, eye, stomach, spleen, bone, pancreas, kidney, liver, intestine, skin, uterus or bladder.
  • the present invention also concerns a composition useful in the prevention or reduction of collagen deposition in a tissue, the composition comprising an effective amount of at least one ACAT inhibitor as defined in Table 1 or a mixture thereof, and an acceptable excipient.
  • the tissue is for instance an organ tissue of a subject, such as a human.
  • the organ tissue is for instance a tissue of an organ selected from heart, lungs, brain, eye, stomach, spleen, bones, pancreas, kidneys, liver, intestines, skin, uterus and bladder.
  • ACAT inhibitors in the prevention of formation or development of excess of fibrous tissue in an organ
  • the fibrosis phase consists of the formation of fibrous tissues in which connective tissue replaces parenchyma tissues. If the fibrosis phase continues unrestrained, the tissue repair process can become pathogenic, leading to extensive tissue remodeling.
  • the present invention proposes to use an ACAT inhibitor to prevent the formation or development of excess of fibrous connective tissue in an organ.
  • the ACAT inhibitor may be one of the compounds defined in Table 1 or a mixture thereof.
  • the organ is for instance an organ of a subject, such as a human.
  • the organ is for instance a heart, a lung, a brain, an eye, a stomach, a spleen, a bone, a pancreas, a kidney, a liver, an intestine, a skin, an uterus or a bladder.
  • the present invention provides a method to prevent the formation or development of excess of fibrous connective tissue in an organ of a subject, which comprises administering to the subject an effective amount of at least one ACAT inhibitor.
  • the ACAT inhibitor may one of the ACAT inhibitors defined in Table 1 or a mixture thereof.
  • the organ is selected from heart, lungs, brain, eye, stomach, spleen, bones, pancreas, kidneys, liver, intestines, skin, uterus and bladder and the subject is a human.
  • compositions according to the invention contain at least one excipient or formulation material, including for example a carrier or vehicle useful for delivering the ACAT inhibitor, while maintaining and preserving the antifibrotic agent.
  • the formulations can be adapted to the condition to be treated. For example, treatment of fibrotic disorders may be delivered topically, orally or by injection. Alternatively, the compositions may be delivered by inhalation therapy. Other suitable means for the introduction of the therapeutic molecule include implantable drug delivery devices.
  • the optimal composition will be determined by one skilled in the art depending upon, for example, the desired route of administration, its mode of delivery and intended dosage.
  • a suitable vehicle for parenteral injection preferably contemplated by the present invention is sterile distilled water in which the desired ACAT inhibitor is formulated as a sterile, isotonic solution, properly preserved.
  • compositions of the invention consist of aqueous injection suspensions
  • they preferably contain substances increasing the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the desired ACAT inhibitor may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers may also be used for delivery.
  • the suspension may also contain suitable stabilizers or agents for increasing the solubility of the ACAT inhibitor and allowing for the preparation of highly concentrated solutions.
  • composition of the invention may be formulated for inhalation.
  • an ACAT inhibitor may be formulated as a dry powder for inhalation.
  • ACAT inhibitor inhalation solutions may also be formulated with a propellant for aerosol delivery.
  • solutions may be nebulized.
  • compositions comprising the ACAT inhibitors may be formulated for topical administration.
  • An example of topical compositions may take the form of a cream, ointment or lotion.
  • compositions of the invention may be administered orally.
  • Compositions for oral administration can be formulated using acceptable carriers well known in the art in appropriate dosages for oral administration. Such carriers enable the compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • a capsule may be also designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized.
  • Additional agents can be included to facilitate absorption of the ACAT inhibitor. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.
  • an example of an oral formulation may consist of a capsule which can be prepared by granulating the ACAT inhibitor contemplated by the present invention with lactose and corn starch as the excipient and hydroxypropylcellulose as the binder.
  • a capsule may consist of a white hard gelatin capsule containing the desired amount of the ACAT inhibitor.
  • ACAT inhibitor F-1394 Another example of a capsule formulation for the ACAT inhibitor F-1394 is provided in Table 2. It consists of a gelatin capsule provided with an enteric coat.
  • Formulations for oral use can also be obtained through combining the desired ACAT inhibitor with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores.
  • Suitable excipients for such formulations include for example, carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, or sodium carboxymethylcellulose; and proteins, such as gelatin and collagen.
  • the dosage levels for treatment will vary depending, in part, upon the type of ACAT inhibitor delivered, the specific indication for which the ACAT inhibitor is being used, the route of administration, and the size (body weight, body surface or organ size) and condition (the age and general health) of the patient. For instance, the clinician may titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
  • the effective dose can be estimated initially either in cell culture assays or in animal models such as mice, rats, rabbits, dogs, pigs, or monkeys.
  • animal models such as mice, rats, rabbits, dogs, pigs, or monkeys.
  • An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • the frequency of dosing will depend upon the pharmacokinetic parameters of the ACAT inhibitor in the specific formulation that is used.
  • a composition is administered until a dosage is reached that achieves the desired effect.
  • the composition may therefore be administered as a single dose or as multiple doses (at the same or different concentrations/dosages) over time, or as a continuous infusion. Further refinement of the appropriate dosage is routinely made. Appropriate dosages may be ascertained through use of appropriate dose- response data.
  • a capsule as described in Table 2, comprising 25 mg of the ACAT inhibitor F-1394 could be administered at least once a day, preferably at least twice a day.
  • the daily dosage as well as the F-1394 capsule content could be adapted in order to obtain the optimal therapeutic effect.
  • Example 1 ACAT inhibitors inhibit the TGF- ⁇ signalling pathway
  • TGF- ⁇ belongs to a large family of secreted peptide growth factors that play critical roles in animal development.
  • the type I and type Il receptors Upon ligand binding, the type I and type Il receptors are recruited into a complex and the type Il receptor phosphorylates, and thereby activates, the type I receptor.
  • the type I receptor phosphorylates Smads of the R-Smad (receptor-regulated) subfamily.
  • R-Smads form complexes with Co-Smads and accumulate in the nucleus to regulate gene transcription via other transcription factors ( Figure 1). This pathway regulates many cellular processes and plays crucial roles in normal development. Disruption of the pathway can lead to a range of diseases including cancer, cardiovascular, inflammatory and fibrotic diseases, as well as Marfan's Syndrome.
  • TGF- ⁇ pathway regulates entry into the dauer larval stage, an alternative larval stage that is specialized for survival under adverse conditions. Disruption of the TGF- ⁇ pathway leads to constitutive dauer formation, that is, the formation of dauer larvae even under favourable conditions. Thus, an increase in dauer formation indicates a decrease in signalling through the pathway.
  • signalling pathways such as the insulin-like signalling pathway, which affect dauer formation in worms.
  • Constitutive dauer formation via the TGF- ⁇ pathway requires the activities of DAF-3 and DAF-5 whereas constitutive dauer formation via the insulin-like signalling pathway does not. The goal of this study was to establish that ACAT inhibitors have the capacity to interfere with the TGF- ⁇ signalling pathway.
  • Worm strains were obtained from the Caenorhabditis Genetics Center (CGC) and maintained at 2O 0 C using standard culture methods.
  • the wild-type strain used was the Caenorhabditis elegans Bristol strain, N2.
  • the mutants used in this study are: daf-7(e1372) ⁇ , daf-1 (m40) ⁇ / , daf-4(m63) ⁇ , daf-8(e1393) ⁇ , daf-14(m77) ⁇ /, and daf-5(e1386)11.
  • Compound Avasimibe, Pactimibe, F-1394, FR19254, S58035 were kept as 10 mM stock solutions at -2O 0 C. All compounds were dissolved in DMSO. Compounds were assayed at a final concentration of 82.5 ⁇ M, except avasimibe which was assayed at 10.3 ⁇ M in the daf-14 mutant, and at 55 ⁇ M in the other mutants.
  • ACAT inhibitors affect dauer formation by reducing signalling through the TGF- ⁇ pathway
  • Table 3 ACAT inhibitors do not induce dauer formation in the da/-4(m63);daf- 5(e1386) double mutant.
  • the tested compounds have no dauer inducing activity in the tested worm background.
  • Example 2 Effects of F-1394 on TGF ⁇ i-induced matrix protein expression in human lung epithelial cell line A549, an in vitro animal model relevant to human pulmonary fibrosis and COPD
  • TGF- ⁇ - ⁇ Transforming growth factor- ⁇ - ⁇ , a multifunctional or pleiotropic cytokine, participates in numerous biological processes, including cell proliferation, differentiation, apoptosis, fibrosis, wound repair and inflammation (Ning W. et al.
  • TGFp 1 is a growth factor with a role as critical mediator of the lung tissue response to injury; therefore, it is involved in the mechanisms of lung repair and fibrosis that follow inflammatory processes (Bellocq A. et al. 1999). Reactive oxygen and nitrogen intermediates increase
  • Fibrilar collagen type I is characteristically synthesised by fibroblastic-type cells (Kasai H. ef a/. 2005).
  • TGFp 1 but not other inflammatory cytokines induced A549 cells with an alveolar epithelial type Il cell phenotype to undergo an epithelial mesenchymal transition which includes the expression of the fibroblast phenotypic markers (Kasai H. et al. 2005).
  • the human alveolar epithelial cell carcinoma line A549 (American Type Culture Collection; Rockville, MD) was grown as outlined in Mata M. et al. 2005. Recombinant human TGFp 1 was used at a concentration of 5ng/mL Medium was changed every 24 h with re-addition Of TGFp 1 and F-1394 as appropriate. F-1394 was present from 1 h before first addition of TGFp 1 until the end of the experiment. mRNA analysis
  • TGFp 1 + F-1394 (in DMSO) dose level 1 (0.3 ⁇ g/mL) 5
  • TGFp 1 + F-1394 (in DMSO) dose level 2 (0.6 ⁇ g/mL) 6)
  • TGFp 1 + F-1394 (in DMSO) dose level 3 (3 ⁇ g/mL).
  • TGFp 1 produced a marked increase in the ⁇ 1 (I) collagen mRNA expression as shown in Figure 4.
  • F-1394 as a prototypic ACAT inhibitor is effective to reduce markedly the augmented ⁇ 1 (I) collagen mRNA expression evoked by TGFp 1 in cultured A549 cells, a human airway epithelial cell line.
  • Example 3 Effects of F-1394 on bleomvcin-induced pulmonary damage, an in vivo animal model relevant to human pulmonary fibrosis
  • Bleomycin-induced pulmonary damage is an established model considered relevant in the investigation of anti-inflammatory and anti-fibrotic (remodeling) activities of new compounds.
  • mice entered the study.
  • the number of animals per group was apporximately10-12 (as required).
  • Each mouse had a body weight at delivery (6 weeks) of approximately 20 g.
  • F-1394 was orally administered at 300 mg/kg as a suspension with 0.5% carboxymethylcellulose (CMC) for 14 days.
  • CMC carboxymethylcellulose
  • NAC N-acetyl-L-cysteine
  • mice Male adult C57BL/6 male mice, each weighing about 20 g, received endotracheal ⁇ , by the transoral route, a single dose of 0.075 U bleomycin sulphate (Sigma Catalogue B 5507, (conservation at 2-8 0 C, 1.2-1.5 units per mg solid) dissolved in 50 ⁇ l saline (0.9% NaCI). Control animals were subjected to the same protocol but received intratracheal saline instead of bleomycin. Tracheal instillation was carried out under halothane anaesthesia.
  • the lung was first perfused through its main bronchus with a fixative solution (10% neutral-buffered formalin) maintained at 25 cm of hydrostatic pressure for 15 min, immersed in the fixative for 24 h, and blocks were taken. Only lungs that were well inflated by the fixative were analyzed. Tissue blocks were placed in formalin, dehydrated in a graded series of ethanol, embedded in paraffin, cut into 4- ⁇ m-thick serial sections, and stained with haematoxylin - eosin to identify inflammatory cells and fibrotic areas.
  • a fixative solution (10% neutral-buffered formalin) maintained at 25 cm of hydrostatic pressure for 15 min, immersed in the fixative for 24 h, and blocks were taken. Only lungs that were well inflated by the fixative were analyzed. Tissue blocks were placed in formalin, dehydrated in a graded series of ethanol, embedded in paraffin, cut into 4- ⁇ m-thick serial sections, and stained with haematoxylin - eosin to identify
  • Histologic grading of lesions was performed by using a blinded semiquantitative scoring system for extent and severity of inflammation and fibrosis in lung parenchyma based on previous studies from this laboratory (Cortijo et al., 2001 ; Mata et al., 2003; Serrano-Mollar et al., 2002; Serrano-Mollar et al., 2003). The severity of fibrosis was scored according to Ashcroft and co-workers.
  • the grade of lung fibrosis was scored on a scale from 0 to 8 by the following criteria: grade 0, normal lung; grade 1 , minimal fibrous thickening of alveolar or bronchiolar walls; grade 3, moderate thickening of walls without obvious damage to lung architecture; grade 5, increased fibrosis with definite damage to lung structure and formation of fibrous bands or small fibrous masses; grade 7, severe distortion of structure and large fibrous areas; grade 8, total fibrous obliteration of fields. Grades 2, 4 and 6 were used as intermediate pictures. The severity of fibrotic changes in each lung section was assessed as the mean score of severity from the observed microscopic fields. Ten random fields in each section were analysed. Grading was performed in a blinded fashion by two observers, and the mean value was taken as the fibrosis score).
  • Negative control F-1394-vehicle (oral) + saline (intratracheal)
  • F-1394 as well as NAC showed a marked inhibitory effect on the expression of ⁇ 1 (I) collagen transcripts ( Figure 5) which indicates an anti-fibrotic effect with decrease of collagen deposition.
  • F-1394 as a prototypic ACAT inhibitor is a valuable compound endowed with anti- fibrotic properties at the level of ⁇ 1 (I) collagen mRNA expression in lung tissue.
  • Lungs from control animals were histologically normal.
  • the administration of bleomycin resulted in characteristic histological lesions including areas of marked peribronchiolar and interstitial infilltration with inflammatory cells (predominantly mononuclear cells including macrophages and lymphocytes with fewer numbers of neutrophils and scattered eosinophils), extensive cellular thickening of interalveolar septa, interstitial oedema, and increases in interstitial cells with a fibroblastic appearance.
  • the pattern of distribution of lesions was multifocal (i.e. patchy areas of pulmonary fibrosis).
  • UUO Unilateral Ureteral Obstruction
  • the aim of the present study was to evaluate the effects of a preventive treatment with F-1394 on whole interstitial fibrillar collagen deposition by Sirius red histochemistry.
  • the effects of F-1394 were evaluated by quantification of the specific stained areas relatively to the whole obstructed kidney section surface.
  • mice Male C57BL/6J mice, weighing 25-30 g at the beginning of the experiments, were used. Mice were housed per group of five (5) in polypropylene cages on wood litter with free access to food (TekladTM 5018; HARLAN, Gannat, France) and water. The animal house was maintained under artificial lighting (12 hours) between 7:00 and 19:00 in a controlled ambient temperature of 20 ⁇ 1 0 C, and relative humidity maintained at 60%. Mice were sacrificed by cervical dislocation. Animals were not submitted to autopsy.
  • F-1394 dosage formulation was prepared with two different ways; 200 mg/kg solution using 1 %Tween80 and 0.5% CMC, and 300mg/kg solution using 0.5% CMC alone. F-1394 was administered orally at a dose of 200 mg/kg/d for the first 10 days and 300 mg/kg/d for the 5 remaining days of the protocol. Captopril (Sigma-AIdrich, St Louis, USA) was dissolved in 1% Tween ⁇ O and 0.5% CMC and administered orally at a dose of 32 mg/kg/d for 15 days.
  • Morphometric measurement was based on computerized image analysis of Sirius red-stained sections, observed through a microscope (Eclipse 600TM, Nikon Instruments; Champigny sur Marne, France) and numeric camera (MicroFireTM, Optronics; Avanex, Nozay, France) with a 2OX objective lens (final calibration 0.366 ⁇ m/pixel). Quantitative analysis of the pictures was performed using the ExploraNova MorphoLiteTM and ExpertTM softwares (La Rochelle, France).
  • mice Four experimental groups with 10 mice each were used:
  • Oral treatments lasted fifteen (15) days and obstruction fourteen (14) days.
  • mice were anesthetized with a mix of oxygen-isoflurane (0.2 L/min and 2% isoflurane; Centravet, Lapalisse, France).
  • a 5 mm cutaneous incision was performed at 1 cm from the last left costal edge of the mouse, followed by a 5mm muscular incision.
  • the left ureter was exposed, and ligated (6/0 cardionyl thread; Peters Surgical, Bobigny, France) at the uretero-pelvic junction.
  • the muscle wall was closed (5/0 Ethicrin thread; Ethicon, Auneau, France), and the cutaneous wall was closed by a suture clip. All along the surgery, animals were set on a 35 0 C hotplate.
  • Sections were deparaffinized, rehydrated, and then stained with 0.1 % Sirius red F3BTM (BDH Laboratory; VWR, Fontenay sous Bois, France) in saturated aqueous picric acid for 1 hour, differentiated in 0.01 N HCI for 1 minute, and rapidly dehydrated and mounted.
  • Sirius red F3BTM BDH Laboratory; VWR, Fontenay sous Bois, France
  • tubulointerstitial fibrillar collagen surface was determined by measuring the area of stained tissue within either the whole cortical area or a given field. Glomeruli and vasculature were excluded from evaluation in the present study.
  • Results were expressed as percentage (%) of fibrosis in relation to the whole area studied.
  • TGF-beta1 induces human alveolar epithelial to mesenchymal cell transition (EMT)". Respir Res. 6:56.
  • TGF-b transforming growth factor-beta
  • TGF-beta1 stimulates HO-1 via the p38 mitogen-activated protein kinase in A549 pulmonary epithelial cells. Am J Physiol Lung Cell MoI Physiol.

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Abstract

La présente invention porte sur l'utilisation d'inhibiteurs de ACAT (par exemple, 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 et S 58-035, ou des mélanges de ceux-ci), sur des compositions les contenant et sur des procédés pour la prévention ou le traitement de la fibrose, pour empêcher ou réduire le dépôt de collagène dans un tissu, et dans la prévention et la réduction d'un tissu conjonctif fibreux excessif.
EP07845502A 2006-11-13 2007-11-13 Inhibiteurs d'acat et leur utilisation dans la prévention ou le traitement de la fibrose Withdrawn EP1981493A4 (fr)

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US7625890B2 (en) 2005-11-10 2009-12-01 Smithkline Beecham Corp. Substituted imidazo[4,5-c]pyridine compounds as Akt inhibitors
WO2008138350A1 (fr) * 2007-05-14 2008-11-20 Sygehuset Glostrup Prévention d'une fibrose intra-oculaire
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CA2798735A1 (fr) * 2010-05-19 2011-11-24 Haruki Shibata Agent prophylactique et/ou therapeutique pour une steatohepatite non alcoolique
WO2013116096A1 (fr) 2012-01-31 2013-08-08 Purdue Research Foundation Procédés de détermination de l'agressivité d'un cancer et de traitement associés
CN102670796A (zh) * 2012-05-22 2012-09-19 广西天天乐药业股份有限公司 一种治疗脾胃虚弱的中药及其制备方法
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WO2000040227A2 (fr) * 1999-01-05 2000-07-13 University Of Utah Procedes de traitement d'etats associes a l'accumulation d'un excedent de matrice extracellulaire
EP1666038A1 (fr) * 2003-08-29 2006-06-07 Kowa Co., Ltd. Procede pour stabiliser une plaque riche en lipides et procede pour prevenir la rupture de ladite plaque

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000040227A2 (fr) * 1999-01-05 2000-07-13 University Of Utah Procedes de traitement d'etats associes a l'accumulation d'un excedent de matrice extracellulaire
EP1666038A1 (fr) * 2003-08-29 2006-06-07 Kowa Co., Ltd. Procede pour stabiliser une plaque riche en lipides et procede pour prevenir la rupture de ladite plaque

Non-Patent Citations (3)

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Title
HORI M ET AL: "Up-regulation of acyl-coenzyme A:cholesterol acyltransferase-1 by transforming growth factor-beta1 during differentiation of human monocytes into macrophages" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 320, no. 2, 23 July 2004 (2004-07-23), pages 501-505, XP004516659 ISSN: 0006-291X *
MARK H. BEERS, M.D., ROBERT BERKOW, M.D.: "The Merck Manual of Diagnosis and Therapy" 1999, MERCK RESEARCH LABORATORIES , WHITEHOUSE STATION, N.J. , XP002546105 * page 1655 - page 1657 * *
See also references of WO2008058383A1 *

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IL198721A0 (en) 2010-02-17
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