MONO- AND DISULFO- SUBSTITUTED ANTHRAQUINONES AND THEIR USE FOR THE TREATMENT

OF BONE MATRIX DISORDERS.

The present invention relates to compounds useful for the treatment of the pathologies in which the erosion of the cartilaginous and bone matrix occurs in the most advanced steps of the disease, such as osteoarthrosis and rheumatoid arthritis, and to pharmaceutical compositions containing them. Furthermore, the invention relates to novel anthraquinone derivatives and to the process for the preparation thereof.

Osteoarthrosis are known to be treated mainly using substances acting on the pain and exerting their symptomatic effect thanks to their antiinflammatory activity. Said medicaments are usually referred to as non steroidal antiinflammatory drugs (NSAID) such as for example indo ethacin and steroidal ones, such as hydrocortisone and betha etasone.

Other used compounds comprise copper chelating agents, such as penicillamine, and those compounds which interfere with collagene synthesis, with DNA or with synovial membranes, such as cyclophosphamide.

Among the most recent substances used in the above mentioned pathologies there are diacetylrhein, a pro- drug of rhein, which exerts its therapeutical activity being a copper chelating agent, moreover it inhibits the formation of ceruloplasmin during the acute phase of arthritis inflammation and it is also a calcium

chelating agent, forming soluble complexes with calcium thanks to the solubilizing COOH group present in its structure. Such a characteristic of forming soluble complexes with calcium is likely to be of paramount importance, since it avoids the formation and precipitation of microcrystals at the articulation sites, thereby preventing inflammatory reactions from occurring or from going on (Friedman, U.S. Pat. n.

4,244,968 del 13/1/1981). Furthermore, rhein inhibits the formation and release of the superoxide anion from NADPH-dependent biological systems (Mian M. et al. J. Pharm. Pharmacol. 1987; 39: 845-847) and the activity of serine proteases, such as elastase and cathepsin G from man (Raimondi I. et al. Pharmacol. Res. Comm. 1982; 14 (2): 103-112); Zembower DE. et al. J. Med. Che . 1992; 35: 1597-1605 1992) .

Further pharmacological and clinical studies described in literature proved that, in addition to the above mentioned mechanisms, other pathogenetic mechanisms act in the diseases affecting articulations, causing the erosion of the cartilaginous and bone matrix.

Among such mechanisms, the increase in some enzyme activities or an unbalance among the latter and the inhibitors thereof should be stressed. As recently proved by some researchers, cysteine proteases, such as cathepsin B and L, are enzymes strictly connected with the degradation of the cartilago and therefore with the related pathologies. It has widely been reported in literature, that cysteine proteases, cathepsins B and L, are capable of inducing directly or indirectly (by

activation of proenzymes), the degradation of the main components of the cartilaginous and bone extracellular matrix (Roughley PJ. et al. Biochem. J. 1977; 167; 639-

637; Sakamoto S. et al. MO . Aspects Med. 1988; 10; 299- 428; Nguyen Pj . et al. Biochem. J. 1991 Aug. 15; 278 (pt

1): 143-7; Maciewiez RA. et al. Biomed. Biochim. Acta

1991; 50 (4-6): 561-4; Buttle DJ. Arthritis Rheum. 1993;

36 (12); 1709-17; Pelletier JP. et al. Osteoarthritis

1993; 19 (3); 545-568). Moreover, the interest in these enzyme activities, has been confirmed by studies carried out on laboratory animals (rats) in which rheumatoid arthritis had been induced. In said animals, the effect of the inhibitors of cysteine proteases, such as fluoromethylketones, on the development of the disease was evaluated particularly on the cartilaginous and bone articular lesions.

The results of said studies showed that the enzyme inhibitors can be clinically valuable in the treatment of arthritis (Ahmed NK. et al. Biochem. Pharmacol. 1992; 44 (6); 1201-7; Meijers M, Billingham M. et al. Agents actions 1993; 39 (1); 219-21; Esser RE. et al. J. Reu atol. 1993; 20 (7); 1176-83).

Other authors (Gabrijelcic D. et al. J. Clin. Chim. Biochem. 1990; 28 (3); 149-53) evidenced the presence of cathepsin B and H in the synovial fluid of patients with different diseases of the articulation whereas Martel- Pelletier J. et al., J. Orthop. (1990): 8 (3:336) proved the existence of an unbalance between the levels of cathepsin B and the inhibitors thereof in the cartilaginous tissue of osteoarthrosic patients.

Huet et al. Arthritis Rheum. (1993): 36 (3): 772 showed that I -1 and TNF stimulate the activity of cysteine proteases in synovial cells of explants from patients affected with osteoarthrosis and arthritis.

From what stated above, it is clear that cysteine proteases are actively involved in the osteoarthrosic and arthritic pathologies, therefore the use of a therapeutical agent markedly inhibiting the activity of said proteases is widely justified.

Now it has surprisingly been found that anthraquinone mono- and disulfonic acid derivatives have remarkable activity against serine proteases.

It is an object of the present invention the use for the preparation of a medicament useful for the treatment of pathologies in which the erosion of the cartilaginous and bone matrix occurs in the most advanced steps of the disease, in particular osteoarthrosis and rheumatoid arthritis, of the compounds of general formula (I)

wherein:

A is a group of formula -SO _β R, in which R is hydrogen or a cation capable of giving a water-soluble derivative; or A is a group of formula -SO2R ¹ , wherein R ¹ is a -NR ² R ³ group, in which R ² is hydrogen or C-^-Cg

straight or branched alkyl,

R ³ is -CH(COOH)-R ⁵ , wherein R ⁵ is ^-Cg alkyl or

^C 7~ ^c 12 arylalkyl; -(CH ₂ ) _n -COOH, wherein n is a integer from 1 to 6, C^-Cg straight or branched alkyl, -CgH ₄ -0-(CH ₂ ) _m -CH ₃ , wherein is a integer from 1 to 4; or

R- is a -O ^ group, in which R^ is a C^-Cg straight or branched alkyl group or an optionally substituted Cg-C ₁₀ aryl group; B is a hydrogen atom; or B has the same meanings as A; with the proviso that A and B are simultaneously -SO3R or -SO2 ¹ . With the expression "a cation capable of giving a water-soluble derivative", those skilled in the art can evaluate which cations are able to play the solubilizing function and, at the same time, to give rise to non- toxic derivatives which do not affect adversely the pharmacological activity of the compounds of formula (I). Examples of said type of cation are metal cations such as lithium, sodium, potassium.

Examples of C^-Cg straight or branched alkyl group are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl.

Examples of arylalkyl group are benzyl, 2- phenylethyl, 1-phenylethyl, 3-phenγlpropyl, 1- naphthylmethyl, 2-naphthylmethyl.

Examples of ^c 6~ ^c ιo ^{grc p are} Phenyl, naphthyl. Optional substituents groups can be for example amino, mono- or of-(C ₁ ~Cg)-alkylamino, such as diethylamino,

hydroxy, C^-Cg alkoxy, such as isopropoxy, thio.

The following novel compounds according to the present invention are included within the above general formula:

2,6-anthraquinonesulfonamido-N,N-caproic acid; N.N*-diethy1-2,6-anthraquinonedisulfonamide; N, '-(p-ethoxypheny1)-2,6-anthraquinonedisulfonamide; bis-2-(2,6-anthraquinonedisulfon)-N,N'-diamidopropionic acid; bis-2-(2,6-anthraquinonedisulfon)-N,N-diamido-3-phenyl- propionic acid.

Anthraquinone dibasic sulfonamides with antiviral activity have been described by M. Grisar et al. in Journal of Medicinal Chemistry, 1974, vol. 17, n. 8, p. 890-893.

Anthraquinone sulfonamides are described in GB 2025954 to be generally useful as intermediates for dyes, chemical products for agriculture and pharmaceuticals. Particularly, they are described as components of aqueous solutions for removing hydrogen sulfide from gases.

The compounds of the invention can be prepared from anthraquinone mono- and/or disulfonic acid by means of the intermediates:

in which X is a halogen, in particular chlorine

Such an intermediate is known to be prepared reacting mono or disulfonic acid with chlorosulfonic acid or with phosphorous pentachloride or with phosphorous oxychloride or with mixtures of the last two.

The resulting halogenated derivative is reacted with a compound of general formula HNR ² R ³ .

The technique which can be applied to the latter step varies with the type of R ² and R ³ , the purity and the obtainable yields. In some cases, the sulfonic acid chloride is reacted with a HNR ² R ³ strong excess, whereas in other cases it is preferred to carry out the reaction in a suitable solvent; as examples of appropriate solvents, ethyl ether and methylene chloride can be mentioned. When the presence of a base is suitable, preferred are those such as amines, for example triethylamine and paradimethylaminopyridine.

The reaction conditions depend on the nature of the reagents, the solvent and the presence or the absence of a base as well as its amount, if any. Temperatures range from -10°C and the solvent boiling point. The most suited operative conditions range from 5°C to 30°C. The reaction times vary depending on other parameters, but they usually range from 2 hours to 3 days.

Analogously, the halogenated derivative is reacted with a R---OH alcohol, wherein R ¹ is C^-Cg alkyl, thereby obtaining the sulfonic acid ester. The preferred method consists in reacting the sulfonic acid sodium salt with chlorosulfonic acid in excess at room temperature for about 24 hours.

After that, sulfonyl chloride is recovered by filtration, after hydrolysis of the chlorosulfonic acid excess with water and ice. Sulfonyl chloride is reacted with an amine HNR ¹ R ² or with an alcohol R ³ OH, in which R- , R ² , R ³ have the meanings defined above, preferably at room temperature and in the presence of tertiary amines, particularly in when operating in a solvent.

Anthraquinone mono- and disulfonic acids and substituted derivatives of the present invention are remarkably effective in various pharmacological tests.

By way of example, the experimental results of some enzyme tests carried out with mono- and disulfonic derivatives having SO3R groups at the positions 1,5 and 2,6 are reported (see Tables 1 and 2). Effects on human cathepsin B and enzymatic activity.

The anthraquinone onosulfonated and disulfonated compounds were dissolved in distilled water whereas the derivatives thereof were dissolved in di ethylsulfoxide. The concentration of the tested product was added to the reaction mixture in a volume of 100 μl.

Composition of the reaction mixture: 0.005 M acetate buffer pH 6.0 + 2 M cysteine + 1 mM EDTA; Enzyme 1 U/ml (cathepsin B or ) (Calbiochem); substrate 0.2 M Z-Phe-Arg-AMC (Novabioche ) final reaction volume 2 ml; temperature 25"C; observation time for cathepsin B : 2 minutes; for L: 8 minutes. This is a fluorimetric determination wherein the excitation wavelength was 380 nm, the emission one being instead 460 n . Fluorescence was read by means of a Perkin-Elmer spectrophotometer mod. LS-3B.

Studied concentrations:

Anthraquinone-2,6-disulfonic acid : 1 - 5 - 10 μM Anthraquinone-1,5-disulfonic acid : 1 - 5 - 10 μM N,N'-(p-Ethoxypheny1)-2,6-anthraquinone- disulfonamide : 1 - 10 μM.

The results reported in Tables 1 and 2 show that the tested compounds markedly inhibit the activity of cathepsins B and L from humans.

TABLE 1 EFFECT OF ANTHRAQUINONE MONO- AND DISULFONIC ACIDS ON HUMAN CATHEPSIN B ENZYMATIC ACTIVITY

TABLE 2

EFFECT OF ANTHRAQUINONE MONO- AND DISULFONIC ACIDS ON

HUMAN CATHEPSIN L ENZYMATIC ACTIVITY

The test evaluates the effect of the compounds anthraquinone-2,6-disulfonic acid and N,N'-diethy- lanthraquinone-2,6-disulfonamide on rheumatoid arthritis in the rat induced by administration of complete Freund's adjuvant. Diacetylrhein was used as control.

For the test, albino Sprague Dawley rats weighing 200 ± 10 g, were subdivided into four groups of six ani¬ mals each. All of the compounds were administered orally at a dosage of 20 mg/kg. Arthritis was induced admini- stering all the animals, in the left rear paw, with 5 mg of Freund's complete adjuvant suspended in 0.05 ml of

paraffin oil (prior to the treatment, the paw volume of both rear paws was measured). The treatment with the te¬ sted products started 5 days after the arthritis induc¬ tion and was carried out for ten days. Al the end of this time, the animals were killed to effect the analy¬ sis of the following evaluation parameters: measurement of the volume of both left and right rear paws; Rx in lateral and antero-posterior position of the tibio-tar- sal articulation of the left paw; histology evaluation of the articulation of both left and right rear paws.

The obtained results show that the compounds anthraquinone-2,6-disulfonic acid and N,N'-diethy- lanthraquinone-2,6-disulfonamide inhibit by 18.6 and 21.4% respectively the increase in the volume of the left rear paw, whereas diacetylrhein caused an 8.3% re¬ duction. The effect of the two disulfonated compounds on the increase in the volume of the right paw was much more marked. In fact, an inhibition of respectively 67.5 and 62.3% (acid anthraquinone-2,6-disulfonic acid and N,N'-diethylanthraquinone-2,6-disulfonamide) was obser¬ ved, versus a complete lack in activity by diacetylrhein (see Table 3). Furthermore, radiographies show that the two disulfonated compounds inhibit their loss in bone mass and significantly protect the tibio-tarsal articu- lation It is indeed evident from the histological exami¬ nation of the latter, that the disulfonated compounds markedly inhibit the formation of fibrous tissue in the articulation whereas this is remarkably present in the articulation of the control groups and in those treated with diacetylrhein.

TABLE 3

EFFECT OF ANTHRAQUINONE MONO- AND DISULFONIC ACIDS IN

THE OEDEMA INDUCED BY FREUND'S COMPLETE ADJUVANT

Percent increase in the oedema

Compound Dose after 15 days mg/kg/os Left paw Right paw

Control 252 ± 26 7.7 ± 29 Acid 2,6-an- 20 205 ± 41 25 ± 20 thraquinonedi- sulfonic acid

N,N'-diethyl- 20 198 ± 31 29 ± 12

2,6-anthra- quinonedi- sulfonamide

Diacetylrhein 20 231 ± 22 92 ± 30

Such a protecting activity is also evidenced by the histological examinations carried out on the rat tibio- tarsal articulation.

Contrary to rhein, said products are nor copper neither calcium chelating agents, they do not inhibit the production and the release of the superoxide anion from NADPH-dependent biological systems, do not inhibit the activity of superoxide dismutase, they are not serine proteases inhibitors, they are not mutagenic in the Ames test and cause no chromosomal aberrations as well. Test of induction of chromosomal aberrations

The clastogenetic effect of rhein and of 2,6-

anthraquinonedisulfonic acid, was evaluated using the test of the induction of chromosomal aberrations in cell cultures of Chinese hamster ovary (CHO). In this test, the cell line was treated with the tested substances and with suitable controls, in the presence of a metabolic activation system for four hours. After that, the cell in growth exponential phase were treated with colchicine (Colcemid ^R ) for 90 minutes, so as to obtain a signifi¬ cant number of cells in metaphase and to recover them for the preparation of the chromosome suspension. As the positive standard, cyclophosphamide was used (1-5-10- 12.5-25 μg/ml) .

The toxicity of the tested compounds was evaluated by means of the assay of the efficiency of plating, tre- ating the cells for four hours, then seeding them at a concentration of 200 cells/5 ml of fresh culture medium.

On the ground of the results obtained by means of this test, dosages were selected for the treatment in the test of induction of chromosomal aberrations, so as not to exceed, with the highest dosage, 50% toxicity levels.

The selected doses were: rhein: 25-50-100-200 μg/ml

Acid 2,6-anthraquinonedi- sulfonic acid: 50-100-150-200-250 μg/ml.

The chromosomal aberrations were recorded descri¬ bing the type of structural damage. The numeric aberra¬ tions and gaps were discarded. Metaphases showing a good chromosomal opening, a good staining and an acceptable chromosome number (20 ± 2) were selected. For statisti¬ cal evaluations of the results, the quadratic Chi test

was used .

The results prove that the compound 2,6-anthraqui- nonedisulfonic acid induces no chromosomal aberrations whereas rhein is positive at doses of 100 and 200 μg/ml (see Tables 4 and 5).

TABLE 4

CHROMOSOMAL ABERRATION ASSAY ON CHINESE HAMSTER OVARY

CELLS

Compound Dose Total meta- Metaphases with P phases aberrations

Control // 220 13

+ S - 9

10 32 12 P < 0.001 μg/ml CPS + S - 9 5 100 25 P < 0.001 μg/ml

1 100 10 N.S. μg/ml

N.S

N.S,

CPS = Cyclophosphamide S - 9 = metabolic activation system

TABLE 5 CHROMOSOMAL ABERRATION ASSAY ON CHINESE HAMSTER OVARY CELLS

25 87 87 P<0.001

CPS I- ¹ en 100 me- 12.5 44 44 P<0.001 taphases

200

Rhein 100 me- 100 taphases 25 1 1

Therefore, such characteristics markedly differentiate the compounds of the invention from rhein.

Action of disulfonated anthraquinone on copper

"scavenging" effect

The study involves the production of superoxide anion by means of the hypoxanthine/xanthine oxidase system and the target is the reduction in cytochrome C iron content. Copper is known to be capable of capturing the radical produced by said system. Copper chelants inhibit such a "scavenging" effect.

Table 6 shows that rhein inhibits said effect, whereas the compounds 1-anthraquinonesulfonic acid and 2-anthraquinonesulfonic acid are nearly inactive.

TABLE 6 EFFECT ON COPPER "SCAVENGING" ACTION

Compound % Reduction

1-AQ 3 2-AQ 5 1,5-AQ 4 2,6-AQ 6 rhein 45

1-AQ : anthraquinone-1-sulfonic

2-AQ : anthraquinone-2-sulfonic

1,5-AQ: anthraquinone-l,5-disulfonic acid

2,6-AQ: anthraquinone-2,6-disulfonic acid

The present invention also relates to pharmaceutical compositions containing a therapeutically effective amount of a compound of formula I in admixture

with conventional carriers and excipients.

The compositions according to the invention are prepared according to known methods, for example as described in "Remington's Pharmaceutical Sciences Handbook", XVII Ed., Mack Pub. Inc., N.Y., U.S.A..

Examples of compositions for the enteral, parenteral, topic administrations are tablets, capsules, granules, controlled-release formulations, liquid drinkable formulations, injectables, suppositories, creams, transdermal formulations.

In the oral formulations, the dosage of the active ingredient will range from 10 to 500 mg depending on the activity of the product and on the therapeutical uses. The systemic use will range from 1 to 100 mg. In the following, illustrative examples are reported. EXAMPLE 1, N.N'-Diethvl-2.6-anthraαuinonedisulfonamide

13.0 g of anthraquinone-2,6-disulfonylchloride are suspended in 300 ml of methylene chloride, under strong stirring. 300 ml of diethylamine are added thereto, by means of a dropping funnel at such a rate as not to exceed 25-30"C, cooling, if necessary, the solution. The resulting suspension is stirred for 6 hours, then left to stand overnight. The suspension is then added with 100 ml of IN NaOH under stirring, stirred for 2 hours and then the resulting solid is filtered, washing it thoroughly with water.

9,3 g of a product with a good purity (95-97% by HPLC) are obtained, which, if necessary, can be recrystallized from a 3/1 dimethylacetamide/water

solution .

IR and NMR analysis confirm the identity of the product.

EXAMPLE 2 N.N'-fp-Ethoxvphenvl ) -2.6-anthraσuinonedisulfonamide

15.5 ml of p-phenetidine are added to 150 ml of methylene chloride, under strong stirring. 6.1 g of anthraquinone-2,6-disulfonylchloride are gradually added, in small portions, so as not to exceed 15-20 ^β C, cooling, if necessary, the solution. The resulting suspension is stirred for 3 hours, then left to stand for 3 days; the obtained solid is filtered, washing thoroughly with IM hydrochloric acid and then with water. 9.8 g of a product are obtained, which is recrystallized twice from dimethylacetamide/water solutions. Finally 3,8 g of the pure product are obtained.

IR and NMR analysis confirm the identity of the product. EXAMPLE 3

2.6-Anthraαuinonedisulfonamido-N,N'-caproic acid

16.4 g of methyl-6-aminocaproate hydrochloride, 18 ml of triethylamine and 200 mg of p-dimethyl- aminopyridine are added to 150 ml of diethyl ether, stirring the whole thoroughly. 6.1 g of anthraquinone- 2,6-disulfonylchloride are gradually added, in sπvll portions, so as not to exceed 15-20°C, cooling, if necessary, the solution. The resulting suspension is stirred for 3 hours at room temperature, then 3 hours under reflux, then it is left to stand overnight; after that the solvent is evaporated off and the obtained

solid is taken up with 35 g of KOH dissolved in 500 ml of water/methanol 1/1. The mixture is stirred for 1 hour at room temperature, then is acidified with 20% hydrochloric acid to markedly acidic pH; after stirring for 5 minutes the resulting solid is filtered, washing it thoroughly with water. 5.0 g of a product are obtained, which is recrystallized twice from dimethylacetamide/water solutions. Finally 2.1 g of the pure product are obtained. IR and NMR analysis confirm the identity of the product. EXAMPLE 4 Soft gelatin capsules:

Each soft gelatin capsule contains: 1) 2)

Active ingredient 100 mg 300 mg

Soy oil 50 mg 150 mg

The dispersion of the active ingredient in soy oil is dosed in soft gelatin capsules using the suitable device.

The active ingredients used in this formulation are preferably:

1) - N,N'-diethyl-2,6-anthraquinonedisulfonamide

2) - N,N'-(p-ethoxyphenyl)-2,6-anthraquinonedisulfonami- de.

EXAMPLE 5

Hard gelatin capsules

Each capsule contains: Active ingredient 40 mg 200 mg Lactose 200 mg 285 mg

Magnesium stearate 10 mg 15 mg

The products are mixed and then dosed in capsules of suitable size.

The active ingredients used in this formulation are preferably selected from: - 2,6- or 1,5-anthraquinonedisulfonic acid sodium salts;

- 2,6-anthraquinonedisulfonamido-N,N'-caproic acid.

EXAMPLE 6

Tablets

Each tablet contains: Active ingredient

Microcrystalline cellulose

Anhydrous lactose

Sodium carboxymethyl starch

Magnesium stearate The active ingredient is mixed thoroughly with microcrystalline cellulose and the anhydrous lactose, sodium carboxymethyl starch and magnesium stearate are added mixing again, then the resulting mixture is sieved and tabletted to obtain tablets weighing 700 and 920 mg respectively.

The active ingredients used in this formulation are preferably:

1) - N,N-diethyl-2,6-anthraquinonedisulfonamide

2) - N,N-(p-ethoxyphenyl)-2,6-anthraquinonedisulfonamide 3) - anthraquinonesulfonic acid of formula (I) sodium salts. EXAMPLE 7 Cream

Active ingredient 2.000 g Mixture of cetyl and stearyl alcohols 15.000 g

Sodium lauryl sulfate 1.500 g

Decyl oleate 10.000 g

Vaselin oil 5.000 g

Depurated water 66.000 g Methyl-p-oxy-benzoate 0.160 g

Propyl para-oxy-benzoate 0.040 g

Rose essence 0.300 g

The mixture of cetyl and stearyl alcohols is melted with sodium laurylsulfate and decyl oleate, adding the active ingredient and mixing thoroughly. The obtained product is then added to water, brought to the same temperature at which methyl-p-oxybenzoate and propyl-p- oxybenzoate had been dissolved, emulsified carefully in a suitable device, cooled to about 60 ^β C under stirring, added with the rose essence and then cooled and distributed in suitable containers.

Particularly suitable active ingredients of the preparations are:

- N,N'-diethyl-2,6-anthraquinonedisulfonamide; - N,N'-(p-ethoxyphenyl) -2 ,6-anthraquinonedisulfonamide.

EXAMPLE 8

Ointment

With similar procedures, ointments can be prepared with active ingredients consisting of the salts of the compounds of formula (I) using compositions of the following type:

Active ingredient 3.000 g

Hamamelis water 27.000 g

Cetyl alcohol 16.500 g Free and esterified sterols 1.000 g

Methyl p-oxybenzoate 0.150 g

Propyl gallate 0.030 g

Propylene glycol 7.000 g

Rose essence 0.250 g

Disodium versenate 0.050 g

Vitamin F 80% 1.250 g

Polyglycol esters of saturated and unsaturated fatty amides

^C 13 ^~C 20 0.500 g

Depurated water q.ε. to 100 g. EXAMPLE 9 Intramuscular vials

Each vial contains: Active ingredient 25.00 g

Sodium chloride 3.80 g

Sodium citrate dihydrate 25.73 g

Citric acid monohydrate 7.87 g

Water for injectable preparations q.ε. to 2 ml.

The solution of the active ingredient and of the excipients is filtered sterilely and partitioned in vials which are subsequently sterilized in autoclave at 121"C for 15 minutes.

The active ingredients used in this formulation are preferably the anthraquinonesulfonic acid of formula (I) sodium salts. EXAMPLE 10 Intra-articular vials

Each vial of freeze-dried product contains: Active ingredient 1 mg

Mannitol 50 mg

Sodium hydroxide q.s. to pH 6.5

Each solvent vial contains:

Water for injectable preparations 2 ml.

The solution of the active ingredient and of the excipients is sterilely filtered and partitioned in vials, then freeze-dried in a suitable device.

The active ingredients used in this formulation are preferably the anthraquinonesulfonic acid of formula (I) sodium salts.