WO1996025156A1 - Utilisation d'inhibiteurs de la metalloproteinase matricielle pour le traitement de maladies induites par tgf-alpha - Google Patents

Utilisation d'inhibiteurs de la metalloproteinase matricielle pour le traitement de maladies induites par tgf-alpha Download PDF

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Publication number
WO1996025156A1
WO1996025156A1 PCT/GB1996/000280 GB9600280W WO9625156A1 WO 1996025156 A1 WO1996025156 A1 WO 1996025156A1 GB 9600280 W GB9600280 W GB 9600280W WO 9625156 A1 WO9625156 A1 WO 9625156A1
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Prior art keywords
tgf
methyl
broad spectrum
matrix metalloproteinase
methylcarbamoyl
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PCT/GB1996/000280
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English (en)
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Edward Eliot Hodgkin
Karen Margrete Miller
Lindsey Ann Needham
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British Biotech Pharmaceuticals Limited
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Priority to EP96902330A priority Critical patent/EP0809491A1/fr
Publication of WO1996025156A1 publication Critical patent/WO1996025156A1/fr

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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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • 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
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • 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
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings

Definitions

  • This present invention relates to the novel use of certain hydroxamic acid and carboxylic acid derivatives, previously known in the art as inhibitors of matrix metalloproteinases such as collagenase, for inhibiting the production and processing of transforming growth factor alpha (TGF- ⁇ ) by cells, and thus is useful in the management of diseases or conditions mediated by overproduction of, or over- responsiveness to, TGF- ⁇ .
  • TGF- ⁇ matrix metalloproteinases
  • TGF- ⁇ Transforming growth factor alpha
  • EGF epidermal growth factor
  • TGF- ⁇ is known to stimulate epithelial cell migration, promote angiogenesis, induce bone resorption, stimulate hypercalcemia and inhibit gastric acid secretion (R Derynck (1992) Advances in Cancer Research. 58:27-52; R Derynck (1988) Cell. 54:593-595;
  • TGF- ⁇ which is also constitutively produced by human blood eosinophils, may participate in the inflammatory reaction by interacting with mesenchymal and epithelial cells, thus promoting fibrosis or neovascularization (Xalz et al. (1993) Leukemia. 7(10):1531 -1537).
  • TGF- ⁇ is expressed in embryonic tissues and also in a wide range of normal cells and tissues including: keratinocytes, macrophages, eosinophils, mammary epithelia, corneal epithelia. gastric mucosa, pancreas, pituitary and brain.
  • Overexpression of TGF- ⁇ promotes transformation and anchorage-independent growth of cells in vitro, and enhanced production of TGF- ⁇ is frequently observed in neoplasia (Massague
  • the TGF- ⁇ precursor is a 160 ammo acid, 20-22 kDa polypeptide with a hydrophobic transmembrane sequence of 23 ammo acids and is oriented with the N-termmus on the extracellular side of the membrane.
  • the soluble 50 ammo acid form of TGF- ⁇ is generated by cleavage of pro-TGF- ⁇ between residues Ala38-Val39 and Ala88-
  • pro-TGF- ⁇ is cleaved at the site proximal to the N- terminus, thereby shortening the membrane-anchored precursor to 17kDa
  • This cleavage step appears to proceed rapidly with a /2 of 15 minutes
  • the second step which is rate limiting, involves cleavage at the second site distal to the N-termmus, releasing the soluble, bioactive form into the medium along with the 5kDa glycosylated N-termmus This terminal portion of the molecule has no known function In non-stimulated cells this second stage of the cleavage process has a t 2 of about 4 hours, leading to the accumulation of pro-TGF- ⁇ on the cell surface.
  • Stimulation of secretion via protein kinase C-dependent or independent mechanisms dramatically decreases the t-
  • Some tumour-derived or retrovirally transformed cells release soluble forms that contain the N-terminal glycosylated region of the precursor still linked to the mature 6kDa TGF- ⁇ sequence, suggesting that regulation of cleavage differs between different cell types. These forms are referred to as meso-TGF- ⁇ . It is not known if the meso forms of TGF- ⁇ differ in function to the final 50 amino acid product. The enzyme or enzymes responsible for the cleavage of pro-TGF- ⁇ to the mature form have not been identified.
  • Neovascularisation is a critical component for normal cell growth, for injury repair and particularly for the development of the vascular system.
  • clinical hyperplastic conditions characterised by abnormal neovascularisation, for example psoriasis and diabetic retinopathy.
  • Abnormal neovascularisation is also associated with neoplastic cancer conditions.
  • the development of solid tumors and unrestricted tumour growth necessitates the induction of angiogenesis to provide a network of blood capillaries in order to provide nourishment for the tumour cells, and to remove waste metabolites from the tumour cells.
  • agents capable of inhibiting tumour cell mediated neovascularisation will prove useful in inhibiting the growth of tumours.
  • agents capable of inhibiting or regressing capillary formation in general may also be therapeutically useful in treating all diseases which are accompanied by angiogenesis.
  • WO 94/18967 discloses the use of a particular class of imidazoies useful in inhibiting angiogenesis
  • EP application 424,193 discloses the use of actinonin as an inhibitor of angiogenesis
  • WO 93/16716 teaches that various peptide fragments of thrombospondin are capable of inhibiting angiogenesis.
  • Inhibitors of matrix metalloproteinases have also been implicated in inhibiting angiogenesis (Tamargo et al. (1991 ) Cancer Res. 51 :672-675; Fischer et al. (1994) Dev. Biol. 162:499-510 and Galardy et ⁇ l (1994) Cancer Res.
  • TGF- ⁇ is a critical mediator in the pathology of several diseases.
  • TGF- ⁇ is elevated in skin lesions of psoriasis and of ultraviolet- induced skin hyperplasia. in wounds leading to scar and keloid formation, and in breast cancer, liver cancer, squamous carcinoma, ovarian carcinoma and keratoacanthoma.
  • Transgenic mice overexpressing TGF- ⁇ display hyperproliferative skin lesions similar to psoriasis, and are prone to develop liver and mammary tumours. Henry et al. (1987; Proceedings, 78th Ann. meeting Amer. Soc. Cancer Res.
  • Blocking production of TGF- ⁇ should be beneficial in malignant disorders where TGF- ⁇ is an autocrine or paracrine growth factor for the tumour, where TGF- ⁇ mediates angiogenesis associated with tumour growth, or where TGF- ⁇ mediates bone resorptio ⁇ and hypercalcemia. Blocking production of TGF- ⁇ should also be beneficial in conditions of hyperplasia (non-cancerous cell proliferation) such as fibrolytic conditions including skin disorders such as eczema. In inflammatory diseases blocking TGF- ⁇ production should be beneficial where TGF- ⁇ is acting as a growth factor for connective tissue cells, mediating hyperproliferation, or where excessive neo-vascularisation occurs.
  • TGF- ⁇ stimulates TGF- ⁇ in speeding wound healing, which leads to scar and keloid formation. It would therefore be desirable to be able to provide a therapeutic agent to inhibit TGF- ⁇ . leading to a reduction in the pathological actions of TGF- ⁇ .
  • TGF- ⁇ tumor necrosis fibroblasts
  • diseases for example where angiogenesis is important such as diabetic retinopathy, pterygia, psoriasis and atherosclerosis, or other hyperplastic diseases such as fibrinolytic diseases or ultraviolet-induced skin hyperplasia or in the control of wound healing.
  • Metalloproteinases are characterised by the presence in the structure of a zinc(ll) ionic site. It is now known that there exists a range of metalloproteinase enzymes that includes fibroblast collagenase (Type 1), PMN-collagenase, 72 kDa-gelatinase, 92 kDa-gelatinase, stromelysin, stromelysin-2 and PUMP-1 (J.F. Woessner, FASEB J, 5:2145-2154, 1991). Many known MMP inhibitors are peptide derivatives, based on naturally occurring amino acids, and are analogues of the cleavage site in the collagen molecule. A recent paper by Chapman et al. (J.
  • MMP inhibitors are less peptidic in structure, and may more properly be viewed as pseudopeptides or peptide mimetics.
  • Such compounds usually having a functional group capable of binding to the zinc (II) site in the MMP, and known classes include those in which the zinc binding group is a hydroxamic acid, carboxylic acid, sulphydril, and oxygenated phosphorus (eg phosphinic acid and phosphonamidate including aminophosphonic acid) groups.
  • MMP inhibitors Two known classes of pseudopeptide or peptide mimetic MMP inhibitors have a hydroxamic acid group and a carboxylic group respectively as their zinc binding groups. With a few exceptions, such known MMPs may be represented by the structural formula (I)
  • R ⁇ , R 2 and R 3 groups are believed to occupy respectively the P1 , P1 ' and P2' ammo acid side chain binding sites for the natural enzyme substrate.
  • a larger R T substituent can enhance activity against stromelysin, and that a (C ⁇ -Ce)alkyl group (sucn as iso-butyl) at R 2 may be preferred for activity against collagenase whilst an alkylphenyl group (such as phenylpropyl) at R 2 may provide selectivity for gelatmase over the other metalloproteinases
  • MMPIs matrix metalloproteinase inhibitors
  • MMPIs matrix metalloproteinase inhibitors
  • the art for example WO 93/13741 , also contains speculative claims suggesting that such inhibitors might be useful in inflammation and hyperplasia conditions such as psoriasis
  • the rationale for such claims is that matrix metalloproteinases cause the breakdown of the connective tissue thus enabling neovascularisation to develop.
  • TGF- ⁇ as the primary causative factor in the angiogenic and fibrotic disease states
  • the present invention has shown that broad spectrum MMPIs are capable of preventing the production of TGF- ⁇ and therefore justifiably demonstrates their utility in treating hyperplasia conditions.
  • the present invention has therefore opened up these disease end-points as targets treatable by broad spectrum hydroxamic acid- and carboxylic acid MMPIs.
  • the present invention therefore concerns a method of management of diseases mediated by TGF- ⁇ in mammals, in particular humans, the method comprising administering to the mammal an effective amount of a non-endogenous matrix metalloproteinase inhibitor or a pharmaceutically or veterinary acceptable salt thereof.
  • Matrix metalloproteinases have been shown to play an important role in the cancer- mediated degradation of extracellular matrix.
  • This matrix acts as a "host-defence" mechanism in xenograft models of cancer spread, forming an impenetrable barrier (DeVore et al., (1980) Expl. Cell Biol. 48:367-373).
  • Degradation enables the cancer cells to move through the extracellular matrix, and both to enter and exit the vasculature; these steps are integral to the process of metastasis, which may occur early or late in the growth of the primary tumour, depending on tumour type.
  • the present invention does not relate to the use of MMPIs to inhibit the cancer-mediated degradation of extracellular matrix, or in metastasis, but rather to the novel use of certain broad spectrum MMPIs in inhibiting TGF- ⁇ .
  • This invention therefore extends considerably the known clinical utility of matrix metalloproteinase inhibitors by enabling the treatment of patients with diseases mediated by TGF- ⁇ . Description of the Invention
  • TGF- ⁇ transforming growth factor alpha
  • MMPI broad spectrum matrix metalloproteinase inhibitor
  • Another aspect of this invention concerns:
  • a method of management by which is meant treatment or prophylaxis of diseases or conditions mediated by or primarily mediated by TGF- ⁇ in mammals, particularly in humans, which method comprises administration to the mammal an effective dose of a broad spectrum MMPI compound as defined herein, or a pharmaceutically acceptable salt thereof;
  • an exogenous broad spectrum MMPI compound as defined herein for use in human or veterinary medicine, particularly in the management (by which is meant treatment or prophylaxis) of diseases or conditions mediated by or primarily mediated by TGF- ⁇ ;
  • Salts of the compounds of the invention include physiologically acceptable acid addition salts for example hydrochlorides, hydrobromides, sulphates, methane sulphonates, p-toluenesulphonates, phosphates, acetates, citrates, succinates, lactates. tartrates, fumarates and maleates. Salts may also be formed with bases, for example sodium, potassium, magnesium, and calcium salts.
  • TGF- ⁇ Diseases or conditions mediated by TGF- ⁇ include but are not limited to inflammation, autoimmune disease, wound healing including scar and keloid formation, fibrinolytic disorders, angiogenic disorders and cancer.
  • TGF- ⁇ is predicted to be a potent therapeutic strategy for many disease states. These include, but are not restricted to, psohasis, scleroderma, hemangioma, diabetic retinopathy, neovascular glaucoma, retrolental fibroplasia, atherosclerosis, artehovenous malformations, vascular adhesions, arthritis, wound healing , fibrotic disorders, systemic sclerosis, systemic lupus erythematosus, vasculaities, vasculitides, hypercalcemia of malignancy, ovarian carcinoma, breast cancer, liver cancer, squamous carcinoma, ovarian carcinoma, keratoacanthoma, pancreatic carcinoma, colon carcinoma, erythroleukaemia and in any disease state where TGF- ⁇ is a mediator of host injury or where TGF- ⁇ production is an important feature of the disease pathology.
  • a broad spectrum matrix metalloproteinase inhibitor compound in the treatment of any of the following diseases or conditions mediated by overproduction of, or over- responsiveness to TGF- ⁇ : psoriasis, scleroderma, hemangioma. diabetic retinopathy, neovascular glaucoma, retrolental fibroplasia, atherosclerosis, artehovenous malformations, vascular adhesions, arthritis, wound healing , fibrotic disorders, systemic sclerosis, systemic lupus erythematosus.
  • MMP inhibitors have been proposed with hydroxamic acid or carboxylic acid zinc binding groups.
  • MMPI compounds may be identified amongst those disclosed in the above publications, by assaying such known MMPIs for their ability to inhibit each of collagenase, stromelysin and gelatmase Inhibition of these enzymes is assessed using the assays described infra.
  • the potency of compounds as inhibitors of collagenase is determined by the procedure of Cawston and Barrett, (Anal. Biochem. 99.340-345, 1979), hereby incorporated by reference, or the following adaptation, whereby a 1 mM solution of the compound being tested, or a dilution thereof, is incubated at 37°C for 16 hours with collagen and collagenase (buffered with 25mM Hepes, pH 7 5 containing 5mM CaCI 2 , 0 05% Brij 35 and 0.02% NaN 3 ).
  • the collagen is acetylated ⁇ C collagen prepared by the method of Cawston and Murphy, (Methods in Enzymology.
  • the samples are cent ⁇ fuged to sediment undigested collagen, and an aliquot of the radioactive supernatant is removed for assay on a scintillation counter as a measure of hydrolysis
  • the collagenase activity in the presence of 1 mM of the test compound, or a dilution thereof, is compared to activity in a control devoid of inhibitor and the inhibitor concentration effecting 50% inhibition of the collagenase activity (IC 50 ) is obtained
  • the potency of compounds as inhibitors of stromelysin is determined by the procedure of Cawston et al, (Biochem. J 195.159-165, 1981 ), hereby incorporated by reference, or the following adaptation, whereby a 1 mM solution of the compound being tested, or a dilution thereof, is incubated at 37oC for 16 hours with stromelysin and ⁇ *C acetylate casein (buffered with 25mM Hepes, pH 7 5 containing 5mM CaCI 2 , 0 05% Brij 35 and 0.02% NaN 3 )
  • the casein is acetylated ⁇ *C casein prepared by the method of Cawston et al (ibid)
  • the stromelysin activity in the presence of 1 mM of the test compound, or a dilution thereof, is compared to activity in a control devoid of inhibitor and the result of inhibitor concentration effecting 50% inhibition of the stromelysin activity (IC5
  • the potency of compounds as inhibitors of gelatmase is determined by the procedure of Sellers et al., (Biochem J 171 493-496,1979), hereby incorporated by reference, or the following adaptation, whereby a 1 mM solution of the compound being tested, or a dilution thereof, is incubated at 37°C for 16 hours with 50ng ⁇ C gelatin in 8mM acetic acid, and gelatmase (buffered with 100mM Tris, pH 7 5 containing 5mM CaCI , 0.05% Brij 35 and 0 02% NaN 3 )
  • the 1*C gelatin is freshly prepared by the thermal denaturation (57°C for 10 minutes) of 1 C-labeiled collagen
  • the collagen is acetylated 1 ⁇ *C collagen prepared by the method of Cawston and Murphy, (Methods in Enzymology 80:71 1 , 1981 ), hereby incorporated by reference
  • IC 50 values for each of collagenase, stromelysin and gelatmase of 300nM or less identifies broad spectrum MMPI compounds useful in the invention Any of these broad spectrum MMPI compounds can be used in the treatment of diseases mediated by TGF- ⁇ production but preferred compounds include those which contain a hydroxamic acid or carboxylic acid moiety including inter alia 3R-(1 S- methylcarbamoyl-2-phenylethylcarbamoyl)-5-methyl-2S-(th ⁇ en-2-ylsulphanylmethyl)- hexanohydroxamic acid.
  • compositions adapted for inhibition of TGF- ⁇ release which composition comprises at least one exogenous broad spectrum matrix metalloproteinase inhibitor compound as defined hereinbefore, in admixture with at least one pharmaceutically or veterinary acceptable carrier.
  • Suitable MMP inhibitors may be prepared for administration by any route.
  • the compositions may be in the form of tablets, capsules, powders, granules, lozenges, liquid or gel preparations, such as oral, topical, or sterile parenteral solutions or suspensions.
  • Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinyl-pyrollidone; fillers for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricant, for example magnesium stearate.
  • binding agents for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinyl-pyrollidone
  • fillers for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or gly
  • Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatin hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p- hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents.
  • the active ingredient may also be administered parenterally (e.g.
  • a sterile medium which is preferably isotonic with the blood of the recipient.
  • the drug can either be suspended or dissolved in the vehicle.
  • adjuvants such as a local anaesthetic, preservative and buffering agents can be dissolved in the vehicle.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • the dosage unit involved in oral administration may contain from about 1 to 250mg, preferably from about 25 to 250mg of a compound of general formula I supra.
  • a suitable daily dose for a mammal may vary widely depending on the condition of the patient. However, a dose of a compound of general formula I of about OJ to 300mg/kg body weight, particularly from about 1 to 100mg/kg body weight may be appropriate.
  • the drug may be made up into a cream, lotion or ointment.
  • Cream or ointment formulations which may be used for the drug are conventional formulations well known in the art, for example as described in standard textbooks of pharmaceutics such as the British Pharmacopoeia.
  • the drug may be made up into a solution or suspension in a suitable sterile aqueous or non aqueous vehicle.
  • Additives for instance buffers such as sodium metabisulphite or disodium edeate; preservatives including bactericidal and fungicidal agents such as phenyl mercuric acetate or nitrate, benzaikonium chloride or chlorhexidine, and thickening agents such as hypromellose may also be included.
  • each dose may typically be in the range from 10 to 100mg of the drug.
  • Compound 1 ( , 2S-(4-hvdroxy-Dhenylsulphanylmethyl)-3R-('3-methoxycarbonyl-1 S- methylcarbamoyl-propylcarbamoyl)-5-methyl-hexanohydroxamic acid), a broad spectrum matrix metalloproteinase inhibitor, can prevent the release of TGF- ⁇ from cells.
  • TGF- ⁇ convertase enzyme
  • Cells are seeded three days prior to experiment in 1 ml/well of a 24-well plate at a density of 2-5x105/ml of appropriate growth medium, in order to generate a confluent monolayer of cells on test day.
  • test cell-line The confluent monolayers of the test cell-line are washed to remove all traces of foetal calf serum. Cells are then allowed to condition serum free culture medium (Assay medium) MEM (with Earles salts, Gibco BRL 21090-022) containing 1 % Nutridoma- SR (Boehringer BCL Cat. No. 1271091 ) and Penicillin + Streptomycin antibiotics at 50U/ml final concentration and Glutamine at 2mM final concentration.
  • serum free culture medium Assay medium
  • MEM with Earles salts, Gibco BRL 21090-022
  • Nutridoma- SR Boehringer BCL Cat. No. 1271091
  • Penicillin + Streptomycin antibiotics 50U/ml final concentration
  • Glutamine 2mM final concentration
  • the conditioned cells are now ready for addition of the various stimuli and or inhibitor compound.
  • a suitable experimental format comprises:
  • Phorbol 12 - Myristate 13 - acetate (PMA, Sigma Cat. No. P8139).
  • Interferon- ⁇ IFN- ⁇ ,Boehringer Mannheim 100,000U/ml. Cat. No. 1040 596.
  • Interleukin-1 ⁇ Interleukin-1 ⁇ (IL-1 ⁇ ,Boehringer Mannheim 100,000U/ml. Cat. No. 1457 756).
  • LPS lipopolysaccharide
  • PMA was used at a final concentration of 5ng/ml.
  • IFN- ⁇ was used at a final concentration of 100U/ml.
  • IL-1 ⁇ was used at a final concentration of 10U/ml.
  • DMSO dimethyl sulphoxide
  • samples of the conditioned media were removed into a phosphate buffer saline protease inhibitor cocktail (Leupeptin (0.6 ⁇ g/ml), Pepstatin (0.4 ⁇ g/ml), Bestatin (5 ⁇ g/ml), PMSF(phenyl-methyl-sulfonyl fluoride)(10 ⁇ g/ml) and Phosphoramidon (10 ⁇ g/ml), Sigma, concentrations given are final) and centrifuged at 4°C for 5 minutes at 300g. Aliquots of the supernatant were then assayed for TGF- ⁇ using a commercially available sandwich enzyme immunoassay (Oncogene Science Inc.).
  • test cells were HS294T melanoma cells which were incubated with the test compound for 24 hours.
  • concentration of TGF- ⁇ (pg/ml) was plotted versus inhibitor concentration and the IC 50 value (50% inhibitory concentration) calculated (see table 2).
  • HT1080 human fibrosarcoma cells
  • DMEM + 10% foetal calf serum supplemented with non-essential amino acids (Gibco-BRL).
  • TGF- ⁇ release was stimulated with PMA at a final concentration of 1 ng/ml in the presence of the compound to be tested at concentrations ranging from 200 ⁇ M to 0.02 ⁇ M.
  • PMA a commercially available sandwich enzyme immunoassay
  • the concentration of TGF- ⁇ (pg/ml) was plotted versus inhibitor concentration and the IC 50 value calculated (see table 3).

Abstract

L'invention porte sur des dérivés d'acide hydroxamique et carboxylique à spectre large, connus jusqu'à présent en tant qu'inhibiteurs de métalloprotéinases matricielles comme la collagénase, et qui sont en mesure d'inhiber la production et la maturation moléculaire du facteur de croissance transformant alpha (TGF-α) par des cellules. Ces dérivés s'avèrent, donc, utiles dans le traitement de maladies ou d'états pathologiques induits par une surproduction du TGF-α ou une hypersensibilité à ce facteur.
PCT/GB1996/000280 1995-02-14 1996-02-13 Utilisation d'inhibiteurs de la metalloproteinase matricielle pour le traitement de maladies induites par tgf-alpha WO1996025156A1 (fr)

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GBGB9502858.5A GB9502858D0 (en) 1995-02-14 1995-02-14 Novel use of matrix metalloproteinase inhibitors
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EP0809491A1 (fr) 1997-12-03

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