Abstract:
A process is described for the preparation of esters of diacerein with hyaluronic acid by means of which stable products are obtained, which are practically free of impurities and characterized by a prolonged anti-inflammatory activity. Also described are pharmaceutical compositions containing said esters having prolonged anti-inflammatory activity and are suitable for intra-articular administration.

Description:
STATE OF THE ART 
       [0001]    Hyaluronic acid is a disaccharide polymer composed of D-glucuronic acid and N-acetylglucosamine. It is distributed in the body as intracellular tissue and in the fluids, such as the vitreous humour and synovial liquid. In Europe hyaluronic acid sodium salt is used for intra-articular and intra-ocular administration. The wide interval in molecular weight and consequent intrinsic viscosity allow its use in the healing of wounds, in ocular operations, and in treatment of osteo-arthritis of the large joints (for example the knee). 
         [0002]    Hyaluronic acid, solution 20-30 mg/vial, has been used for over 15 years for intra-articular administration with the aim of replacing the hyaluronic acid normally present in the joint, which, with the progression of the illness undergoes a more rapid depolymerisation with consequent increase of severity of the pathology. 
         [0003]    The therapeutic value of its intra-articular administration is well documented (J. Rheumatol. 25:2203-2212, 1998; ibid 26:1216, 1999; ibid 38:602-607, 1999) and its efficacy is substantially attributed to the “lubrication” of the joint (Arch. Int. Med. 162:245-7, 2002). These results suggest that the intra-articular administration as a “supplementation” of high-viscosity acid and the regulating state of the product was changed in the last years from a medicinal drug product to a “medical device”. 
         [0004]    Diacerein is a drug authorised for oral use in various European countries for the treatment of osteoarthritis, because it is able to inhibit the synthesis of interleukin-1 (IL-1) and the production of nitrous oxides (NO), induced by the same IL-1, which are among the agents responsible for cartilaginous degeneration. The aetiological action of the diacerein was confirmed “in vivo” and “in vitro” (Presse Med. 2004 May 22; 33(9 Pt 2):S10-2; Biorheology 2002; 39(1-2):277-85; Arthritis Rheum. 2001 November; 44(11):2539-47; J. Rheumatol. 2001 April; 28(4):814-24; Arthritis Rheum. 2000 October; 43(10):2339-48; Osteoarthritis Cartilage 2000 May; 8(3): 186-96; Clin. Exp. Rheumatol. 2003 March-April; 21(2):171-7; Pharmacol. Toxicol., 2002 July; 91(1):22-8; Osteoarthritis Cartilage, 2001 April; 9(3):257-63). 
         [0005]    Diacerein has a limited bioavailability: its intestinal metabolism produces metabolites with a laxative action; it is a pro-drug of rhein, that penetrates rapidly into the synovial liquid at low concentrations (1-10 mmol/L) and it is rapidly eliminated. These characteristics—low absorption, hydrolysis in the stomach into rhein, anthraquinonic metabolites with a laxative effect, rapid elimination—suggest the reason for the poor tolerability of oral diacerein therapy and of the advantage of using local administration in the target area. 
         [0006]    Intra-articular administration of diacerein is difficult to realize due to the lack of solubility of the drug in a carrier that is compatible with the synovial liquid, and because of its permanence time in the joint, which has been shown to be too short for an effective block of the IL-1 synthesis. 
         [0007]    International Patent Application WO 2005/085293 describes esters of rhein with hyaluronic acid, prepared by warm reacting hyaluronic acid with rhein chloride and following purification by ultra filtration or dialysis. 
         [0008]    This method of synthesis, due to the stressed conditions of reaction, causes the formation of a number of red purple coloured by-products of rhein, which were found to be very difficult to remove, either by ultra filtration or by dialysis, for the occurrence of secondary reactions with hyaluronic acid itself. After ultra-filtration or dialysis, the final compound appears to be a red powder, having a negligible content of hydrolysable rhein (less that 1%), with a solubility in water or saline of less that 1 mg/ml, that is useless for the local administration. 
         [0009]    If the reaction of hyaluronic acid and rhein chloride is performed in controlled mild conditions (30-40° C., hydrophobic solvent with moisture content of less than 1%, absence of Lewis bases), rhein remains simply “trapped” in the hyaluronic acid structure, and it is washed out during dialysis or ultra-filtration. The final compound after drying appears almost white but it contains less than 0.1% of active drug. 
         [0010]    In said patent application WO 2005/085293 there are, generically mentioned, but not exemplified, other possible esters of hyaluronic acid with acyl derivatives of rhein, including the diacetyl derivative or diacerein. As to the preparation of the diacerein ester of the hyaluronic acid the method described in WO2005/085293 proved to be unsuccessful: at the process conditions, the diacerein is hydrolized into rhein and other derivatives, with subsequent secondary reactions and the formation of by-products. 
     
    
     DESCRIPTION OF THE INVENTION 
       [0011]    The object of the present invention is a process for the preparation of stable esters of diacerein with hyaluronic acid having a high purity level, that are endowed with an interesting anti-arthritis activity free from side effects and suitable for intra-articular administration. The hyaluronic acid used in these esters is preferably of a molecular weight in the range of about 100,000 to 1,500,000 Da. 
         [0012]    The esters of hyaluronic acid and diacerein, prepared according to the method of the invention, display the following characteristics:
       they do not contain detectable impurities of diacerein derivatives, such as non-linked diacerein, rhein and their derivatives;   they are white or straw-white in colour, the intensity of which depends on the diacerein content;   they can be administered in physiological solution or in aqueous organic solution, for example as saline-glycerol or saline-PEG solution;   when administered directly in the joint, they allow a residence time of diacerein that is inversely proportional to the depolymerization rate of the hyaluronic acid carrier;   they perform a double mechanism of action on the pathology: a local supplementation of hyaluronic acid and the diacerein inhibition of the collagenolytic activity induced by IL-1β; and   they do not cause local or systemic toxic risks because they do not contain free diacerein, and the total amount of diacerein is negligible in comparison to the dosage authorized in humans.       
 
         [0019]    The process of the object of the invention, enables obtaining a non-hydrolyzed diacerein ester with hyaluronic acid that is straw-white in colour, is without detectable impurities, and has the formula 
         [0000]    
       
                 
         
             
             
         
       
     
         [0020]    This process envisages protection of the carboxylic group of diacerein with N,N-carbodiimidazole (CDI) in accordance with a classic method used to esterify aminoacids (Synthesis 833, 1982). 
         [0021]    The synthesis of imidazolyl diacerinate (CDIDIAC) occurs by stoichiometric reaction in anhydrous organic solvent at a temperature of 30-40° C. The preferred solvent is dimethylformamide (DMF), but the synthesis can also occur in other aprotic, polar solvents, such as dimethyl sulfoxide (DMSO). 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    Said reaction does not cause the formation of by-products: the excess of CDI decarboxylates with the liberation of carbon dioxide and imidazole. 
         [0022]    The next step of the synthesis, the esterification of the protected diacerein with hyaluronic acid, requires the use of previously salified hyaluronic acid with low quaternary strong bases, such as, for example, the tetrabutylammonium hydroxide (TBAI). Salification makes hyaluronic acid soluble in the same material that is a solvent for CDIDIAC, and makes it available to esterify the DIAC by substitution of the imidazole amide. 
         [0023]    The salification of the hyaluronic acid with TBAI can be carried out by ionic exchange on a resin in accordance with traditional techniques (Butyric and Retinoic Mixed Ester of Hyaluronan, The Journal of Biological Chemistry, Vol. 279, No. 22, Issue of May 28, pp. 23574-23579, 2004; Hyaluronic-acid butyric esters as promising antineoplastic agents in human lung carcinoma: A pre-clinical study, Investigational New Drugs 22: 207-217, 2004.). For example, saturating a sulphonated resin, of the Amberlite IR-20 type, with a concentrated TBAI solution, washing with water to remove the excess of TBAI, then percolating a dilute solution of hyaluronic acid sodium salt. The tetrabutylammonium salt of the hyaluronic acid (HA-TBA) is separated in solid form by lyophilization and stored in a refrigerator in a container with dehydrating silica gel. 
         [0024]    Esterification occurs by the addition of HA-TBA to the CDIDIAC solution in the selected solvent, e.g. DMF, allowing it to react under mechanical stirring at a temperature lower than 40° C. for 4-48 hours in an anhydrous environment that has been made inert with nitrogen. 
         [0025]    For the recovery of the ester, the reaction mass can be dialysed, for example, with a pH 7 aqueous buffer, and/or ultra filtered, to remove the by-products of the reactions, imidazole and TBAI, and then lyophilized. Alternately, the ester can be isolated by precipitation with a suitable organic solvent, i.e. ethyl alcohol or acetone. In both cases a product that is obtained has a residual water content of less than 10%, is straw-white in colour and has a specific viscosity that is either the same or different with respect to that of the starting hyaluronic acid, and depending on the diacerein content. 
         [0026]    The diacerein content of the ester product can vary according to the molecular ratio of the reagents and, as a consequence, the colour of the final product can vary: it can reach approximately 5% substitution ratio using hyaluronic acid having a low molecular weight (LMW, approx. 0.1 10 6  Dalton) and decrease to 1.5% using hyaluronic acid with a higher molecular weight (HMW, 1.2 10 6  Dalton). The colour of the latter derivative is a lighter straw-yellow than the former. 
         [0027]    The level of esterification of the hyaluronate hydroxy groups depends on many factors, including molecular mass, viscosity, concentration of the HA-TBA solution, as well as the stoichiometric ratio in reaction with the CDIDIAC, and time and temperature used. 
         [0028]    The following examples illustrate this invention but are not to be considered as limiting the scope of this invention 
       Example 1 
       [0029]    Hyaluronic Acid Tetrabutylammonium Salt (HA-TBA) 
         [0030]    The resin (Amberlite IR-20, in acid form CAS 9002-23-7) has a declared capacity of 1.9 eq/L. To salify 1 litre of it, approximately 1,250 mL of tetrabutylammonium hydroxide solution at 40% (TBAI) are necessary. To obtain an efficient exchange using a ratio of 10:1 between the resin sulphonic acid groups, and the hyaluronic acid carboxylic acid groups, one litre of activated resin is sufficient for approximately 75-80 g of sodium hyaluronate. 
         [0031]    Preparation is carried out on a chromatographic column packed with approximately 0.1 L of resin medium, washed with 0.5 L of demineralized water. The quantity of TBAI, solution at 40% (1.25 L/litre of resin), is percolated and recycled with a pump having a flow of approximately 0.05-0.1 volumes of resin/hour for a time corresponding to 3-4 recycles (approximately 2-3 days). Once the cycle is completed, the resin is washed with demineralized water (equal to at least 5-6 volumes of resin) to obtain an eluate with a stable pH of about 9.5-10. To obtain a more efficient exchange it is appropriate to use a jacketed column, thermostatically controlled at 40° C. 8 Grams of sodium (LMW) HA are dissolved in approximately 2 L of demineralized water (suggested concentration 2-4 g/L of HA of M.W. 0.6 10 6  Dalton) and the solution is treated on a resin column (0.1 L) at a temperature lower than 35° C. at a flow rate of approximately 0.1-0.2 L/h. The column percolate and the washing waters (approximately 0.5 L) are collected and subjected to lyophilization. 7.5 Grams of the product are obtained having a water content that is less than 10%. 
       Example 2 
       [0032]    Hyaluronic Acid Tetrabutylammonim Salt (HA-TBA) 
         [0033]    8 Grams of (HMW) sodium hyaluronate are dissolved in approximately 5 L of demineralized water (suggested concentration 1-2 g/L per HA of M.W. 1.2 10 6  Dalton) and the solution is treated on a resin column (0.1 L) at a temperature lower than 35° C. at a flow rate of approximately 0.4 L/h. The percolate from the column and the washing waters (approximately 0.5 L) are collected and are subjected to lyophilization. 
         [0034]    7.2 Grams of the product are obtained having water content that is less than 10%. 
       Example 3 
       [0035]    (LMW) Hyaluronic Acid Diacerein Ester 
         [0036]    2.20 Grams of diacerein (˜6 mmol) are dissolved in 100 mL of anhydrous dimethylformamide to which 1.30 g (˜7 mmol) of N,N-carbonyldiimidazole are added with stirring. The mass is left to react at room temperature for 12 hours or overnight in a flask protected from humidity. Then 5 g of (HMW) HA-TBA dissolved in 250 mL of DMF are added and left under stirring for 24-48 hours until the mass of the reaction becomes a transparent and homogeneous red gel. To the residue after sedimentation, 100 mL of pH 7 phosphate buffer are added and transferred into a dialysis bag (PTFE membrane, size 200-400 nominal Dalton). Outer dialysis solution was monitored for colour and changed many times over 48 hours to form a colourless solution. The dialyzed solution is lyophilized and 5.8 g of product are obtained having water content of less that 10%. 
       Example 4 
       [0037]    (HMW) Hyaluronic Acid Diacerein Ester 
         [0038]    In a flask under nitrogen atmosphere, 2.2 g of diacerein (˜6 mmol) are dissolved in 100 mL of anhydrous DMF and 1.45 g of N,N-carbonyldiimidazole are added thereto under stirring. The mass is left to react at room temperature until complete solution or transparency is achieved. 5 Grams of HA-TBA (HMW), previously dissolved in 50 mL of dimethylformamide, are added thereto through a dropping funnel and the reaction mixture, protected from humidity, is kept under agitation for 24-48 hours. 
         [0039]    The reaction is stopped by adding, under stirring, 100 mL of a saturated solution of sodium chloride. The mass is precipitated by addition of about 2 volumes of 96% ethanol and the supernatant is discharged. The residue is washed several times with ethanol at different concentration and finally dried under vacuum. 
         [0040]    5.3 Grams of product are obtained having water content that is less than 10%. 
         [0041]    Pharmaceutical Formulations Containing the Hyaluronic Acid Diacerein Ester 
         [0042]    All the preparation must be performed in sterile area with previously sterilised equipment.
       a. 500 Milligrams of the (HMW) hyaluronic acid diacerein ester, as prepared in Example 4, are dissolved in 50 mL of saline and kept under stirring for 1 h. The final solution is sterilized by saturated steam at an appropriate time and temperature, validated by means of F 0 , to give an SAL of 10 −6  or better. Then 2 mL of the obtained solution are filled into a vial.   b. 500 Milligrams of the (HMW) hyaluronic acid diacerein ester, as prepared in Example 4, are dissolved in 50 mL of pH 7 phosphate buffer 0.01 M and kept under stirring for 1 h. The final solution is sterilized by saturated steam at an appropriate time and temperature, validated by means of F 0 , to give an SAL of 10 −6  or better. Then 2 mL of the obtained solution are filled into a vial.   c. 500 Milligrams of the (LMW) hyaluronic acid diacerein ester, as prepared in Example 3, are dissolved in 50 mL of pH 6.5 phosphate buffer 0.01 M/glicerol (6:4 v/v) and kept under stirring for 1 h. The final solution is sterilized by saturated steam at an appropriate time and temperature, validated by means of F 0 , to give an SAL of 10 −6  or better. Then 2 mL of the obtained solution are filled into a vial.       
 
         [0046]    Structural Identification of Hyaluronic Acid Diacerein Ester 
         [0047]    The derivatives of hyaluronic acid and diacerein, prepared according to the process of the invention, have been proved by  1 H and  13 C NMR performed on the TBA salt in DMSO. The final esters as sodium salts do not have enough solubility in DMSO to produce significant NMR signals, whereas the TBA salts have sufficient solubility. Due to the low concentration of diacerein in the DMSO solution, it was necessary to accumulate the signals in both  1 H and  13 C NMR overnight, in order to identify the specific signals for diacerein. 
         [0048]    The  1 H NMR spectrum showed a chemical shift of the aromatic signals of diacerein of about 0.5 ppm, from 7.5-8.5 ppm (pure diacerein) to 7.0-8.0 ppm (ester), due to the ester bond between diacerein and hyaluronic acid. 
         [0049]    The high noise of signals (due to the overnight accumulation) did not allow the signals of the acetyl groups to be seen so, in order to demonstrate that diacerein was not hydrolyzed by the reaction, the  13 C NMR was performed. As expected, the  13 C NMR spectrum demonstrated that the  13 C carbon&#39;s signal of diacerein&#39;s acetyl groups was shifted about 1 ppm, from 20 ppm (pure diacerein) to 21 ppm (ester). The above results combined together demonstrated that the diacerein was not hydrolyzed and it was structurally bonded to hyaluronic acid. 
       Analytical Properties 
       [0050]    Solubility: &gt;5 mg/mL in water
 
pH in water: 7.0-8.0
 
       Moisture (K.F.): &gt;5% 
       [0051]    Identification of diacerein by transacetylation of benzylamine: positive
 
Free diacerein (HPLC): &lt;0.01%
 
Free rhein (HPLC): &lt;0.01%
 
Substitution ratio: 2-5%