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Title:
A COMBINATION OF A LONG-ACTING β2-AGONIST AND A GLUCOCORTICOSTEROID IN THE TREATMENT OF FIBROTIC DISEASES
Document Type and Number:
WIPO Patent Application WO/2004/028545
Kind Code:
A1
Abstract:
The invention relates to new uses of glucocorticosteroids and long-acting β2-agonists in the treatment of various fibrotic diseases, e.g. idiopathic pulmonary fibrosis, allergic alveolitis and cystic fibrosis. The preferred combination of active substances consists of budesonide and formoterol fumarate dihydrate.

Inventors:
Trofast, Jan (AstraZeneca R & D Lund, Lund, S-221 87, SE)
Westergren-thorsson, Gunilla (Rösträttsgatan 12, Lund, S-227 60, SE)
Application Number:
PCT/SE2003/001486
Publication Date:
April 08, 2004
Filing Date:
September 24, 2003
Export Citation:
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Assignee:
ASTRAZENECA AB (Södertälje, S-151 85, SE)
Trofast, Jan (AstraZeneca R & D Lund, Lund, S-221 87, SE)
Westergren-thorsson, Gunilla (Rösträttsgatan 12, Lund, S-227 60, SE)
International Classes:
A61K31/167; A61K31/58; A61K45/06; A61P11/00; A61P19/00; A61P43/00; (IPC1-7): A61K31/58; A61K31/167; A61P11/00; A61P19/00; A61P43/00
Domestic Patent References:
WO1993011773A1
WO1999000134A1
WO2001078746A1
WO2001078693A2
WO2000048587A1
WO1990006775A1
WO2002017894A2
WO2001078735A1
WO2002000879A2
WO1998041232A2
Foreign References:
US5376359A
US6013244A
Other References:
F. M. SPOELSTRA ET AL.: 'Budesonide and formoterol inhibit ICAM-1 and VCAM-1 expression of human lung fibroblasts' EUR RESPIR J vol. 15, 2000, pages 68 - 74
Attorney, Agent or Firm:
ASTRAZENECA AB (Global Intellectual Property, Södertälje, S-151 85, SE)
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Claims:
Claims
1. The use of a composition comprising, in admixture or separately: (a) a first active ingredient which is a longacting ß2agonist, a pharmaceutically acceptable salt or solvate thereof, or a solvate of such a salt; (b) a second active ingredient which is a glucocorticosteroid. in the manufacture of a medicament for use in prevention and/or treatment of fibrotic diseases.
2. Use according to claim 1 in which the ß2agonist is selected from salmeterol, formoterol, bambuterol, 2 (1H)Quinolone, 8hydroxy5 [1hydroxy2 [ [2 (4methoxy phenyl)lmethylethyl] amino] ethyl]monohydrochloride, [R (R*, R*)]. and formanilide derivatives e. g. 3 (4f [6 (1 (2R)2 [3 (formylamino)4hydroxyphenyl]2 hydroxyethyl} amino) hexyl] oxy}butyl) benzenesulfonamide, and benzenesulfonamide derivatives e. g. 3 (4{[6({(2R)2hydroxy2[4hydroxy3 (hydroxymethyl) phenyl] ethyl} amino) hexyl] oxy} butyl) benzenesulfonamide.
3. Use according to claim 1 in which the ß2agonist is formoterol, a pharmaceutically acceptable salt or solvate thereof, or a solvate of such a salt.
4. Use according to claim 1 in which the ß2agonist is formoterol fumarate dihydrate.
5. Use according to any one of claims 1 to 4 in which the glucocorticosteroid is selected from budesonide, fluticasone, mometasone, beclomethasone, ciclesonide, triamcinolone, flunisolide, zoticasone, flumoxonide, rofleponide, prednisolone, prednisone, tipredane, steroid esters e. g. 6α,9αdifluoro17α[(2furanylcarbonyl)oxy]11ßhydroxy16αmethyl 3oxoandrosta1, 4diene17ßcarbothioic acid Sfluoromethyl ester, 6a, 9adifluorollß hydroxy16amethyl3oxo17apropionyloxyandrosta1, 4diene17pcarbothioic acid S (2oxotetrahydrofuran3Syl) ester and 6a, 9adifluoro11 (3hydroxy16amethyl17a [ (4methyl1, 3thiazole5carbonyl) oxy]3oxoandrosta1, 4diene17pcarbothioic acid S fluoromethyl ester, steroid (II) and the long acting ß2agonists, without limitation, include: salmeterol, formoterol, bambuterol, 2 (1H)Quinolone, 8hydroxy5 [1hydroxy2 [ [2 (4methoxyphenyl)1methylethyl] amino] ethyl] monohydrochloride and formanilide derivatives e. g. 3(4{[6((2R)2[3(formylamino)4hydroxyphenyl]2 hydroxyethyl} amino) hexyl] oxy}butyl) benzenesulfonamide, and benzenesulfonamide derivatives (IV) e. g. 3 (4 { [6 ( { (2R)2hydroxy2 [4hydroxy3 (hydroxymethyl) phenyl] ethyl} amino) hexyl] oxy} butyl) benzenesulfonamide.
6. Use according to any one of claims 1 to 4 in which the glucocorticosteroid is budesonide.
7. Use according to claim 1 where the first and second active ingredients are selected from fluticasone propionate/salmeterol xinafoate, ciclesonide/formoterol fumarate dihydrate, mometasone furoate/formoterol fumarate dihydrate, fluticasone propionate/formoterol fumarate dihydrate, and budesonide/formoterol fumarate dihydrate.
8. Use according to claim 1 where the first and second active ingredients are selected from budesonide/formoterol fumarate dihydrate.
9. Use according to any one of claims 1 to 8 in which the molar ratio of the first active ingredient to the second active ingredient of from 1: 2500 to 12: 1.
10. Use according to any one of claims 1 to 9 in which the fibrotic disease is idiopathic pulmonary fibrosis.
11. Use according to any one of claims 1 to 9 in which the fibrotic disease is allergic alveolitis.
12. Use according to any one of claims 1 to 9 in which the fibrotic disease is cystic fibrosis.
13. A method of preventing and/or treating a fibrotic disease in patient suffering from, or at risk of, said disease, which comprises administering to the patient a therapeutically effective amount of a composition comprising, in admixture or separately: (a) a first active ingredient which is a longacting (32agonist, a pharmaceutically acceptable salt or solvate thereof, or a solvate of such a salt; (b) a second active ingredient which is a glucocorticosteroid.
Description:
A combination of a long-acting p2-agonist and a glucocorticosteroid in the treatment of fibrotic diseases

Background of the invention The connective tissue consists of extracellular matrix (ECM) molecules and forms the architectural framework of the vertebrate body. The extracellular matrix molecules are plentiful and play an important role in modulation of the connective tissue. During the formation of peribronchial fibrosis, remodelling of connective tissue is due to an increase deposition of ECM components like that of specific types of collagens and proteoglycans. The connective tissue is rich in fibrous proteins, and it is rather the ECM molecules than the cells that bear the mechanical supporty characteristics of the connective tissue. There is an enormous variation in composition and physiological properties of different connective tissues, varying from elasticity in fibrous connective tissue to a strength and rigidity in cartilage and bone. The fibrous connective tissue in the lung is crucial for its elasticity.

The extracellular matrix forms a complex network of proteins and proteoglycans that embed cells in an interactive environment. This environment provides a surrounding in which cells can migrate and interact with other cells and effector molecules. The ECM constituents are affected during several pathological conditions, and have in many cases been shown to be involved in, and even to directly influence these processes. Major components in the ECM are the collagens, proteoglycans, hyaluronan, fibronectin and laminin, which together with effecter molecules, such as cytokines and proteases, form a dynamic connective tissue.

Proteoglycans are a large family of molecules synthesized by a wide variety of cells, and are distributed in many types of tissues. Several deverse biological functions have been ascribed to proteoglycans, such as control of cell differentiation and migration, storage of cytokines, influence of tumor cell growth, regulation of ECM remodelling and tissue reorganization. Proteoglycans are proteins with one or more than a hundred glycosaminoglycan chains covalently attached to the protein core. They are often subdivided into groups according to size, structure and function. The large proteoglycans produced by lung fibroblasts are mainly versican and perlecan and the small proteoglycans are decorin and biglycan.

The main cell type found in fibrous connective tissue is the fibroblast. Several diseases are associated with the connective tissue, and there is often an altered composition of ECM

molecules over time. In several diseases the wound healing process is never turned off, resulting in an assembly of connective tissue molecules leading to the formation of fibrotic lesions. This pattern is observed in certain diseases in the lung, for example asthma, systemic sclerosis, alveolitis, sarcoidosis and idiopathic pulmonary fibrosis, but also in chronic obstructive pulmonary disease. Controversy exists as to whether the latter observation involves fibrotic lesions or just emphysema. There are also other diseases certain intestinal bowel diseases like Crohn's disease and ulcerative colitis; fibromyalgia, rheumatoid arthritis, atherosclerosis and osteoporosis where fibrotic lesions are formed.

The small proteoglycans biglycan and decorin, as well as other proteoglycans are involved in all the above mentioned diseases.

In asthma, which is considered to be a chronic inflammation. However, today it is general excepted that also an abnormal woundhealing process is running in parallell with the inflammatory process. It has been shown that the ECM molecules, such as collagen, hyaluronan and versican are highly infiltrated into the subepithelial area of the pulmonary tissue. The cytokines also play a central role in this phenomenon. There is an alteration in the observed proteoglycan pattern, with an increase in biglycan and perlecan with increasing severity of the disease.

The treatment of asthma generally consists of inhaled corticosteroids and inhaled ß2 agonists. Both beneficial and deleterious effects have been described when glucocorticosteroids and ß2-agonists are administered simultanously.

In patients with asthma increased levels of GM-CSF have been found, but could be blocked in an additive way by the combination of the long-acting (32-agonist formoterol and the glucocorticosteroid budesonide (Eur. Resp. J. 17 (2001), 1070). Bihl et al describe data on differences in the modulatory effect of long-acting 02-agonists for ECM deposition, the effect is depending on the type of 02-agonist used. Some agonists will mimic the effect of glucocorticosteroids while other decrease EMC deposition (AM. J.

Resp. Crit. Care Med. 165 (8), April 2002: A508).

Description of the invention It has been found that a combination of a glucocorticosteroid and a long-acting 02-agonist have very potent effects on the proteoglycan production of human embryonic lung fibroblasts. The combination seems to have effect on all major types of proteoglycans such

as versican, perlecan, biglycan and decorin effecting both the inflammatory type of matrix, which is dominated by verican, perlecan and biglycan and the fibrotic type of matrix, which is dominated by decorin. The steroid alone, however, only affects the inflammatory type of matrix, containing versican and biglycan. These results may further explain why the steroids have positive effect on peribronchial fibrosis if treatment starts early in the disease process as it repress formation of an inflammatory matrix and thereby effects these molecules that potentiate the activation of the fibroblast. However, when the patients have reached a more fibrotic state this treatment does not work, therefore it would be more beneficial to use the combination of a glucocorticosteroid and a long-acting (32-agonist.

In a first aspect the invention therefore provides the use of a glucocorticosteroid and a long-acting (32-agonist for the treatment of fibrotic diseases mediated by the proteoglycan production of human embryonic lung fibroblasts.

The combination has more than an additive effect on a parameter like the decorin, which dominates the fibrotic type of matrix.

These results show that the combination of a glucocorticosteroid and a long-acting ß2- agonist would have good effects in prevention and/or treatment of fibrotic diseases in the airways and the pulmonary tissue like nasal polyps and interstitial lung diseases like idiopathic pulmonary fibrosis (also called cryptogenic or idopathic fibrosing alveolitis) and pulmonary fibrosis in association with systemic sclerosis, systemic lupus erythernatosus, rheumatoid arthritis, dermatomyositis and polymyositis, Goodpasture's syndrome, and other immunologically mediated systemic diseases, and furthermore allergic alvelolitis (also called hypersensitivity pneumonitis), silicosis, asbestosis and other pulmonary granulomatoses, bronchiliotis, pulmonary vasculitis, drug-induced pneumonites, histiocytosis X, chronic eosinophilic pneumonia, pulmonary hemosiderosis and cystic fibrosis. There are also intestinal bowel diseases e. g. Crohn's disease and ulcerative colitis which may be associated with pulmonary fibrotic lesions. Asthma and COPD are excluded form the diseases that can be treated according to the present invention.

A preferred disease that can be treated is idiopathic pulmonary fibrosis.

A futher preferred disease that can be treated is allergic alveolitis.

A further preferred disease that can be treated is cystic fibrosis.

A further preferred disease that can be treated is rheumatoid arthritis.

The pharmacologically active agents in accordance with the present invention include glucocorticosteroids such as: budesonide, fluticasone (e. g. as propionate ester), mometasone (e. g. as furoate ester), beclomethasone (e. g. as 17-propionate or 17,21- dipropionate esters), ciclesonide, triamcinolone (e. g. as acetonide), flunisolide, zoticasone, flumoxonide, rofleponide, butixocort (e. g. as propionate ester), prednisolone, prednisone, tipredane, steroid esters according to WO 2002/12265, WO 2002/12266 and WO 2002/88167 (1) e. g. 6a, 9a-difluoro-17a-[(2-furanylcarbonyl) oxy]-11 ß-hydroxy-16a- methyl-3-oxo-androsta-1, 4-diene-17p-carbothioic acid S-fluoromethyl ester, 6a, 9a- difluoro-11 ß-hydroxy-16cc-methyl-3-oxo-17a-propionyloxy-androsta-1, 4-diene-17ß- carbothioic acid S- (2-oxo-tetrahydro-furan-3S-yl) ester and 6a, 9a-difluoro-1 lß-hydroxy- 16cc-methyl-17a-[(4-methyl-1, 3-thiazole-5-carbonyl) oxy]-3-oxo-androsta-1, 4-diene- 17p- carbothioic acid S-fluoromethyl ester, steroid esters according to DE 4129535 (II) and the like. Long-acting (32agonists, without limitation, include: salmeterol, formoterol, bambuterol, TA 2005 (chemically identified as 2 (1H)-Quinolone, 8-hydroxy-5- [1- hydroxy-2- [ [2- (4-methoxy-phenyl)-1-methylethyl] amino] ethyl] -monohydrochloride, [R- (R*, R*) ] also identified by Chemical Abstract Service Registry Number 137888-11-0 and disclosed in U. S. Patent No 4.579. 854, formanilide derivatives (E) e. g. 3-(4-{[6-({(2R)-2- [3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl} amino) hexyl] oxy}- butyl) benzenesulfonamide as disclosed in WO 2002/76933, benzenesulfonamide derivatives (IV) e. g. 3- (4- { [6- ( { (2R)-2-hydroxy-2- [4-hydroxy-3- (hydroxymethyl) phenyl] ethyl} amino) -hexyl] oxy} butyl) benzenesulfonamide as disclosed in WO 2002/88167 and the like. Several of these compounds could be administered in the form of pharmacologically acceptable esters, salts, solvates, such as hydrates, or solvates of such esters or salts, if any. Both racemic mixtures as well as one or more optical isomers of the above compounds are within the scope of the invention.

The preferred pharmacologically active glucocorticosteroid agents for use in accordance with the present invention include mometasone furoate, ciclesonide, zoticasone, flumoxonide, steroids from WO 2002/88167 e. g. 6a, 9a-difluoro-17a-[(2- <BR> <BR> <BR> furanylcarbonyl) oxy]-11 ß-hydroxy-16a-methyl-3-oxo-androsta-1, 4-diene-17ß-carbothioic acid S-fluoromethyl ester, 6a, 9a-difluoro-llß-hydroxy-16a-methyl-3-oxo-17a- propionyloxy-androsta-l, 4-diene-17p-carbothioic acid S- (2-oxo-tetrahydro-furan-3S-yl) ester and 6a, 9α-difluoro-11ß-hydroxy-16α-methyl-17α-[(4-methyl-1, 3-thiazole-5-

carbonyl) oxy]-3-oxo-androsta-1, 4-diene-17ß-carbothioic acid S-fluoromethyl ester, steroids from DE 4129535, fluticasone propionate and budesonide, and even more preferred is budesonide. The preferred pharmacologically active long-acting 02-agonist is salmeterol xinafoate, formanilide derivatives (III), benzenesulfonamide derivatives (IV) and formoterol (e. g. as fumarate dihydrate) and even more preferred is formoterol fumarate dihydrate.

The preferred combinations include fluticasone propionate/salmeterol xinafoate, ciclesonide/formoterol fumarate dihydrate, mometasone furoate/formoterol fumarate dihydrate, fluticasone propionate/formoterol fumarate dihydrate, and budesonide/formoterol fumarate dihydrate.

Other preferred combinations include steroids from WO 2002/88167/formanilide derivatives from WO 2002/76933, steroids from WO 2002/88167/benzenesulfonamide derivatives from WO 2002/88167, steroids from DE 4129535/formoterol fumarate dihydrate, zoticasone/benzenesulfonamide derivatives from WO 2002/88167 and zoticasone/formanilide derivative.

The most preferred combination is budesonide/formoterol fumarate dihydrate.

According to the invention there is provided the use of a composition comprising, in admixture or separately: (a) a first active ingredient which is a long-acting ß2-agonist, a pharmaceutically acceptable salt or solvate thereof, or a solvate of such a salt; (b) a second active ingredient which is a glucocorticosteroid. in the manufacture of a medicament for use in the prevention and/or treatment of fibrotic diseases.

The invention also provides a method of preventing and/or treating a fibrotic disease in patient suffering from, or at risk of, said disease, which comprises administering to the patient a therapeutically effective amount of a composition comprising, in admixture or separately: (a) a first active ingredient which is a long-acting 02-agonist, a pharmaceutically acceptable salt or solvate thereof, or a solvate of such a salt; (b) a second active ingredient which is a glucocorticosteroid.

Suitably the molar ratio of the first active ingredient to the second active ingredient is from 1: 2500 to 12: 1.

The molar ratio of the first active ingredient to the second active ingredient is preferably from 1: 555 to 2: 1 and more preferably from 1: 150 to 1: 1. The molar ratio of the first active ingredient to the second active ingredient is more preferably from 1 : 133 to 1: 6. The molar ratio of the first active ingredient to the second active ingredient is most preferably 1: 70 to 1: 4.

The components of the invention can be administered in admixture, i. e. together, or separately. When administered together the components can be administered as a single pharmaceutical composition such as a fixed combination. Alternatively the components can be administered separately, i. e. one after the other. The time interval for separate administration can be anything from sequential administration to administration several hours apart.

Examples of fibrotic diseases that can be prevented or treated according to the invention include diseases in the airways and the pulmonary tissue like nasal polyps and interstitial lung diseases like idiopathic pulmonary fibrosis (also called cryptogenic or idiopathic fibrosing alveolitis) and pulmonary fibrosis in association with systemic sclerosis, systemic lupus erythematosus, rheumatoid arthritis, dermatomyositis and polymyositis, Goodpasture's syndrome, and other immunologically mediated systemic diseases, and furthermore allergic alvelolitis (also called hypersensitivity pneumonitis), silicosis, asbestosis and other pulmonary granulomatoses, bronchiliotis, pulmonary vasculitis, drug- induced pneumonites, histiocytosis X, chronic eosinophilic pneumonia, pulmonary hemosiderosis and cystic fibrosis. There are also intestinal bowel diseases e. g. Crohn's disease and ulcerative colitis which may be associated with pulmonary fibrotic lesions.

For the above-mentioned therapeutic uses the dosage administered will, of course, vary with the compound employed, the mode of administration, the treatment desired and disorder indicated. The daily dosage of the first active ingredient may be in the range of from 0.001 mg to 30 mg, in the case of the preferred formoterol (as fumarate dihydrate) from 0. 001 mg to 0.100 mg. The daily dosage of the second active ingredient may be in the range of from 0.01 mg to 30 mg, in the case of the preferred budesonide from 0.01 mg to 10 mg.

The invention further provides a process for the preparation of a pharmaceutical composition of the invention which comprises mixing (a) a first active ingredient which is a long-acting 02-agonist, a pharmaceutically acceptable salt or solvate thereof, or a solvate of such a salt; (b) a second active ingredient which is a glucocorticosteroid ; with a pharmaceutically acceptable adjuvant, diluent or carrier.

The therapeutically active ingredients may be administered prophylactically as a preventive treatment or during the course of a medical condition as a treatment of cure.

The pharmaceutical compositions may be administered topically (e. g. to the lung and/or airways or to the skin) in the form of solutions, suspensions, fluoroalkane aerosols and dry powder formulations; or systemically, e. g. by oral administration in the form of tablets, capsules, syrups, powders or granules, or by parenteral administration in the form of solutions or suspensions, or by subcutaneous administration or by rectal administration in the form of suppositories or foams or transdermally.

The composition used in the invention optionally additionally comprises one or more pharmaceutically acceptable additives (e. g. pH or tonicity adjustment), diluents and/or carriers. The composition is preferably in the form of a dry powder for inhalation, wherein the particles of the pharmaceutically active ingredients have a mass median diameter of less than 10 um.

Suitable physiologically acceptable include acid addition salts derived from inorganic and organic acids, for example the chloride, bromide, sulphate, phosphate, maleate, fumarate, citrate, tartrate, benzoate, 4-methoxybenzoate, 2-or 4-hydroxybenzoate, 4-chlorobenzoate, p-toluenesulphonate, methane-sulphonate, ascorbate, acetate, succinate, lactate, glutarate, tricarballylate, hydroxynaphthalene-carboxylate (xinafoate) or oleate salts or solvates thereof. The first active ingredient is preferably formoterol fumarate dihydrate or salmeterol xinafoate.

The invention is illustrated by the following non-limiting examples:

Materials Concentration of the various drugs were selected according to Korn et al. (Eur. Respir. J. 17 (2001), 1070-1077). Budesonide (BUD) was dissolved in ethanol as a stock concentration of 10-4 M. Serial dilutions to reach 10-8 M was further made in Eagle's minimal essential medium (EMEM). Formoterol (FORM) was dissolved in the same way but in a stock concentration of 10-3 M followed by serial dilutions in EMEM to reach 10-'and 10-10 M.

As controls, ethanol in 0.01 and 0.001 % was used as well as EMEM containing 10 % fetal calf serum.

Proteoglycan determination At confluence, Human Embryonic Lung Fibroblast (HFL-1) cultures were incubated with BUD 10-8 M, FORM 10-8-10-1° M and combinations thereof like BUD 10-'M + FORM 10-8 M and BUD 10-8 M + FORM 10-1° M. Controls were used as described above. After 2h incubation the cells were labeled with 100, uCi/ml 35S-sulphate (ARC St. Louis, MO, USA) for 24 h in sulfate-low EMEM supplemented with 10 % fetal calf serum. The culture medium was then harvested and analyzed for amounts and type of secreted proteoglycans.

The total amount of proteoglycans was determined by ion exchange chromatography as previously described (Westergren Thorsson et al (Eur. J. Biochem. 205 (1992), 277-286)).

Briefly, the cell medium was applied to a DEAE-cellulose DE-52 column (0.7 x 4 cm: Whatman, Maidstone, UK), which had been equilibrated with 6 M urea, 50 mM acetic acid, pH 5.8, 5 mM N-ethylmaleimide, 1 mM EDTA and 5 ug/ml of ovalbumin. To remove unincorporated radioactive precursors, the columns were washed with 60 bed volumes of the same solution. Hyaluronan was eluted with 6 bed volumes of 6 M urea, 500 mM acetic acid, and 5 ug/ml of ovalbumin. Finally, proteoglycans were eluted twice with 3 bed volumes of 4 M guanidium chloride, 50 mM sodium acetate, pH 5.8, and 5, ug/ml of ovalbumin.

Different types of proteoglycans were then identified and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) before and after treatment with cohondroitinase ABC (5 U/ml). Electrophoresis was performed on 3-12 % polyacrylamide gradient gel (T/C=30/0.8) (Scot lab, Luton, UK), using a 3 % stacking gel and the buffer system of Laemmli (Nature, 227 (1970), 680-685). The intensity of the bands containing Versican, Perlecan, Biglycan or Decorin were further analyzed and visualized on an image plate which was scanned by a Fuji BAS 2000 image analyzer (Seikagaku Kogyo, Tokyo, Japan). These proteoglycans have earlier been identified in previous work by Western blot,

with specific antibodies against the various proteoglycans (Tufvesson and Westergren Thorsson, J. Cell Biochem. 77 (2000), 298-309) and also by MALDI-TOF (Tufvesson et al. , Eur. J. Biochem. 269 (2002), 3688-3696) Protein determination Production of proteoglycans were related to amount of protein, which was analyzed by the bicinchoninic acid (BCA) method.

Statistical Mean values +/-standard deviation was calculated. Students T-test was used to evaluate the differences between groups.

Results The most evident effects on total proteoglycan production was seen after incubating the cells for 24 h with the combination of BUD 10-8 + FORM 10-8 M, where the production decreased to 0.39 +/-0.21 (n=4, p=0.01) compared to control, which was calculated as 1. A smaller but still significant decrease 0.61 +/-0.25 (n=4, p= 0.05) of total proteoglycan production was seen after incubating the cultures with the combination of BUD and FORM, using a lower dose of FORM 10-1° M (Fig 1). Incubating the cultures with 0.01 and 0.001 % ethanol did not affect the total proteoglycan production at all (data not shown).

BUD itself in a dose of 10-8 M, inhibited the total proteoglycan production to 0.77 +/-0.12 (n=4, p=0.04), see Fig 1.

Furthermore, no significant changes could be seen on total proteoglycan production after incubating the cultures by FORM alone. The effect of FORM (10-8 M) on total proteoglycan production was 1.09 +/-0.32 (n=4, p=0.61) compared to control and by using FORM in a dose of 10-1° M the effect on the production was 0.63 +/-0.35 (n=4, p=0. 13) compared to control (Fig. 1).

The above changes could not be due to a changed level of total protein concentration in the various cultures as there was no changes after treatment with the various drugs (Fig. 2).

Treatment with BUD (10-8 M) alone selectively decreased the production of some proteoglycans such as versican and biglycan. Both versican and biglycan production decreased by 34-36 %. However no effect was seen on the production of either perlecan or decorin.

The combination of BUD (10'8 M) +FORM (10-1° M) decreased the production of all four types of proteoglycans like versican, perlecan, biglycan and decorin by 62-68% and the combination of BUD (10-8 M) + FORM (10-1° M) was even more potent, decreasing the production all four types of proteoglycans by 80-88%. This clearly shows that the combination also has effect on the more fibrotic type of matrix, where decorin is known to be a marker.

FORM alone in any doses tested, did not have any clear effect on the production of none of these proteoglycans.