ITPD950089A1 - HYALURONIC ACID AND ITS FOREIGN DERIVATIVES FOR THE PREPARATION OF MATRICES FOR THE CONTROLLED RELEASE OF DRUGS - Google Patents

Abstract

The present invention relates to a process for the preparation of matrices for the controlled release of molecules with pharmacological activity, chemically linked to hyaluronic acid or its esters by means of a succinic group. The succinyl derivatives of hyaluronic acid or its esters conjugated with the anti-inflammatory drugs described in the present invention can be advantageously used directly or in pharmaceutical formulations for the treatment of various forms of arthrosis and joint inflammation.

Description

Description of an industrial invention entitled: "Hyaluronic acid and its ester derivatives for the preparation of matrices for the controlled release of drugs"

OBJECT OF THE INVENTION

The present invention relates to a process for the preparation of matrices for the controlled release of molecules with pharmacological activity, chemically linked to hyaluronic acid or its esters by means of a succinic group.

FIELD OF THE INVENTION

Hyaluronic acid is a linear polysaccharide whose structure is made up of alternating units of 1,4-P-D-glucuronic acid and 1,3-β-N-acetyl-D-glucosamine.

Hyaluronic acid is a fundamental component of animal connective tissue being present, for example, in the skin and cartilage. It is also found in greater quantities in the synovial fluid, in the vitreous humor of the eye and in the umbilical cord. Currently the main sources of commercially available hyaluronic acid are cockscomb and vitreous humor. The production of biotechnological hyaluronic acid from Streptococcus cultures is becoming increasingly important. Hyaluronic acid, being a fundamental constituent of connective tissue, is biocompatible, bioadsorbable and non-immunogenic and therefore plays a decisive role in many biological functions such as, for example, tissue hydration, the organization of proteoglycans in cartilage, tissue repair, embryonic development, as well as lubrication and protection of cartilage in the joints. This polysaccharide is currently used in the treatment of some diseases affecting the joints such as, for example, rheumatoid arthritis. It is also used in the so-called microvisosurgery, in particular in eye surgery. In this application, the rheological characteristics and biocompatibility of high molecular weight hyaluronic acid solutions are exploited. The biological functions of hyaluronic acid are closely related to the molecular weight of the polymer as they depend on the viscoelastic and rheological properties that the polymer imparts to the solutions (T.C. Laurent et al, The Faseb Journal, 6: 2397-2404, 1992).

Finally, considering its exceptional biocompatibility characteristics, hyaluronic acid represents the preferential carrier to which biologically active molecules are incorporated in controlled release systems. The properties of this material and environmental factors determine the release rate of the active molecule.

Drug controlled release systems are the subject of considerable interest from the scientific world given their enormous application potential. Among the possible sectors of use it is certainly worth mentioning the pharmaceutical one, which requires continuous improvements of these systems given the pressing need to optimize the diffusion of the drug in the desired application sites. Typically, most of these slow-release systems consist of a physical mixture of active compound and appropriate polymer matrix. However, different types of controlled release formulations are gaining attention, in which the drug is covalently bound to the matrix and the release rate is in this case modulated by the rate of hydrolysis of the chemical bond between polymer and drug.

Polymers such as starch used as binding matrices of drugs are known in the state of the art (Ferrati et al, Macromol. Chem., 180: 375-380, 1979). For example, the treatment of starch succinic esters with 4-aminobenzoic acid, or with L-DOPA (3,4-dihydroxyphenylalanine) in Ν, Ν-dimethylformamide or dimethyl sulfoxide in the presence of imidazole and N, N-dicyclohexylcarbodiimide, provides a polymeric derivative in which the drug is covalently bound to the starch matrix by means of a succinate group (P. Ferrati et al, Macromol. Chem., 180 (1979) 375-380). However, there are no known compounds of this type in which the polysaccharide is the hyaluronic addus.

On the other hand, systems consisting of hyaluronic acid derivatives to which the drug has been chemically bonded are known (US 4,851,521).

DESCRIPTION OF THE INVENTION

The present invention describes for the first time the preparation of derivatives in which the drug is not directly bound to the polysaccharide but is chemically reacted with a "spacer arm" before binding to the polysaccharide. If in the cases already reported in the literature the position on which the substitution reaction took place was the acid group of the glucuronic residue of the repeating unit, in the derivatives object of this invention a reaction has been developed with which the positions subject to substitution are consisting of the free hydroxyl functions of the two residues glucuronic acid and N-acetylglucosamine of the repeating unit. The innovative aspect of the present invention is therefore represented by the identified synthesis process, which allows to bind a greater quantity of drug to the hyaluronic acid thanks to the introduction of this "spacer arm".

Hyaluronic acid was chosen, among the various existing biocompatible polysaccharides, as its properties certainly make it very interesting. As mentioned, its presence in different compartments of the human body makes it much more suitable than other polysaccharides for the development of new drug matrices. Finally, it should be remembered that hyaluronic acid has shown therapeutic efficacy as a re-epithelizing, tissue repairer and in restoring the normal conditions of the joints involved in arthritic processes, therefore, the use of this biopolymer as a matrix for the release of drugs, in addition to ensuring the biocompatibility of the formulation, it allows to associate the intrinsic activity of hyaluronic acid with the therapeutic efficacy of the drug linked to it.

An example of a drug linked to hyaluronic acid by means of a spacer arm according to the present invention is prednisone. In this case the hyaluronic addus is not only interesting for its previously listed biocompatibility and bioabsorbable properties, but also for its therapeutic efficacy per se. In particular, the advantage of using this type of polysaccharide, replaced by means of a spacer arm with compounds with anti-inflammatory activity, is represented by the possibility of having a slow release of the drug and of obtaining a synergistic effect of the two components after hydrolysis of the bond. In fact, both hyaluronic acid and steroid-type anti-inflammatory drugs (for example, methylprednisolone) have been shown to be effective in reducing symptoms associated with knee osteoarthritis. (G. Leardini et al, Clinical and Experimental Rheumatology, 9 (1991) 375-381). Alkyl and aryl esters of hyaluronic acid have been used in preparations containing methylprednisolone, both as polymeric matrices in the form of microspheres in which the drug was physically incorporated, and as substrates on which methylprednisolone was chemically bonded (K. Kyyrònen, et al, Int.}. Pharmaceutics, 80, (1992), 161-169; L. Benedetti, et al, New Polymer Material, 3 (1991) 41-48).

In particular, with the present invention an alternative and convenient methodology is provided for covalently binding drugs to hyaluronic acid or its esters by means of a spacer arm to give compounds of general formula I, in which R represents an alkyl or aryl group, R ' it represents an alcoholic or amino function of the aliphatic, araliphatic, heterocyclic type of the molecule of pharmacological interest. This method allows the introduction of a greater quantity of active compound than the direct derivatization of the polysaccharide. This reaction makes it possible to prepare derivatives of the same type using the carboxyl esters of hyaluronic acid (US Patent 4,851,521) as polymeric substrate.

FORMULA I

Another example of a biologically interesting molecule that has been covalently linked to hyaluronic acid by means of a spacer arm is represented by an antioxidant compound that carries a phenolic group, namely Propofol (2,6-diisopropylphenol). Also in this case, the introduction of propofol in pharmaceutical preparations containing hyaluronic acid was interesting considering its activity. In fact, it should be remembered that the superoxide radical (02 "), generated by enzymatic processes, can depolymerize hyaluronic acid (J. M. McCord," Free radical and inflammation. Protection of synovial fluid by super oxide dismutase ", Science, 185, (1974 ) 529-531) consequently reducing the lubricating, protecting and "shock absorber" capacity of the synovial fluid. The phagocytosis by polymorphonuclear leukocytes produces superoxide radicals which can therefore constitute one of the degradation mechanisms of the synovial fluid in vivo in inflammatory situations It has been shown that many anti-inflammatory and free radical sequestering drugs are effective in protecting hyaluronic acid from radical depolymerization (P. Puig-Parellada et al, Biochem. Pharmacology, 27, (1978) 535-537; C. Parenti et al, Pharmaziè, 45 (1990) 680-681).

Propofol is an anesthetic used intravenously whose effectiveness, as a radical sequestering agent, is due to its ability to form stable radicals. In fact, its efficacy as a sequestering agent of hydroxy radicals generated by the xanthine oxidase / hypoxanthine system has been demonstrated by evaluating the depolymerization of hyaluronic acid in artificial synovial fluid in vitro (C. Kvam et al, Biochem. Biophys. Res. Commun., 193 ( 1993) 927-933). From these premises comes the interest in the use of Propofol to protect hyaluronic acid. The use of the succinic group as a spacer arm is particularly important in this case since the formation of a direct ester bond between the phenolic group and the carboxylic group of the polymer is particularly difficult for reasons of etheric bulk.

According to the present invention, a drug, such as prednisone, propofol, or cholesterol, or any other molecule with alcoholic functions, is first reacted with succinic anhydride in the presence of an organic base, such as pyridine, to obtain the corresponding hemiester; the hemiester is then transformed into the corresponding adlic chloride by reaction with oxalyl chloride in non-polar and aprotic solvents such as, for example, methylene chloride in the presence of catalytic quantities of N, N-dimethylformamide; the succinyl-drug halide is then reacted with the pyridinium salt of the hyaluronic acid or with the benzyl ester of the hyaluronic acid in Ν, Ν-dimethylformamide in the presence of pyridine and 4-dimethylamino pyridine as catalysts.

The dicarboxylic acid used as the "spacer arm" can be glutaric, adipic or any other bifunctional carboxylic addus. In this case it was considered appropriate to use the addon sucdnic.

As regards the catalyst for the esterification, which can be given from any base, pyridine and 4-dimethylamino pyridine were used while the solvent used, among those aprotid, was IV, N-dimethylformamide.

The chlorinating agent for transforming the carboxylic group of the hemiester into the corresponding acyl chloride can be any chlorinating reagent such as, for example, thionyl chloride or sulfuryl chloride. In this case oxalyl chloride was used.

In order to prepare the succimi derivatives conjugated with the anti-inflammatory drugs, samples of hyaluronic acid and its alkyl and ardic esters of any molecular weight can be used. The following examples were prepared using hyaluronic addo samples with molecular weights ranging from 30,000 to 760,000.

The succinyl derivatives of hyaluronic acid or of its esters conjugated with the anti-inflammatory drugs described in the present invention can therefore be advantageously used directly or in pharmaceutical formulations for the treatment of various forms of arthrosis and inflammation of the joints. The invention relates in particular to new derivatives of hyaluronic acid or esters of hyaluronic acid, in which a succinyl-drug group such as, for example, sucdnyl-prednisone, succinylpropofol is chemically linked to the polymer by means of an ester bond. and succinyl-cholesterol.

The compounds mentioned above constitute a probable substrate for the action of lipases in the human body and provide, in one case, hyaluronic acid, succinic acid and the drug and, in the case of benzyl derivatives of hyaluronic acid, hyaluronic acid, benzyl alcohol, addo sucdnico and the drug.

For purely illustrative purposes, we report some examples of preparation of sucdnyl derivatives of hyaluronic addus or of its esters covalently linked to biologically active molecules: Example 1

Preparation of propofol hemisuccinate

To a solution of Propofol (10 mL, 54 mmoles) in pyridine (25 mL), sucdnic anhydride (4.86 g, 48.6 mmoles) was added while the system was stirred at 70 ° C for 96 hours. The solution was then concentrated under reduced pressure conditions until a syrup was obtained. The residue was subjected to chromatography on a silica gel column, using diethyl ether and petroleum ether (low boiling fraction) (2: 3), after crystallization from diethyl ether and petroleum ether yielded the compound propofol hemisucdnate (7.0 g), fusion point:

101-102 ° C.

The spectrum <n m r> - of the product in DMSO-dg (50.3 MHz) shows the following signals: d 179.8 (COOH), 171 (C = 0), 145.63, 140.54, 126.79, 124.11 (aromatic carbons), 28.04, 28.83 ( CH2), 27.64 (CH), 23.38 (CH3).

The proton spectrum in DMSO-dg shows the following signals: d 1.21, 1.23 (CH3), 2.71-3.0 (CH2, CH), 7.12-7.27 (aromatic protons). Preparation of propofol-succinyl-hyaluronate

Hyaluronic acid (MW 30000, 100 mg) was dissolved in distilled water (25 mL) and the pH was brought to approximately 2.5, using an ion exchange resin (IRA 120 H <+>). The resin was then removed by filtration and the solution concentrated to approximately 10 mL. 50 mL of Ν, Ν-dimethylformamide (DMF) was then added and the solution was concentrated to approximately 20 mL. This procedure was repeated three times to replace most of the water with DMF. The solution was then neutralized with an excess of pyridine (5 mL) to give a gelatinous consistency solution which was then treated with a solution of propofol succimi chloride [prepared from propofol hemisuccinate (36 mg, 0.14 mmol), DMF (1 drop), oxalyl chloride (120 pL, 0.14 mmoles) in anhydrous methylene chloride (3 mL) for 1 hour] and stirred at room temperature for 24 hours. The reaction mixture was then dialyzed against distilled water (6 times 1 L). The resulting (opalescent) solution was lyophilized to provide propofolsuccinyl-hyaluronate as a flaky white lyophilic (72 mg). The product has limited solubility in water. Spectra n.m.r. del and del show the presence of signals due to the protons and carbons of the succinic group. The spectrum confirms the presence of aromatic protons. The degree of substitution based on results n.m.r. qualitative is 11-15%.

Hyaluronic acid (MW 30000, 250 mg) was dissolved in distilled water (50 mL) and the pH was brought to approximately 2.5, using an ion exchange resin (IRA 120 H +). The resin was then removed by filtration and the solution concentrated to approximately 10 mL. 50 mL of Ν, Ν-dimethylformamide (DMF) was then added and the solution was concentrated to approximately 20 mL. This procedure was repeated three times to replace most of the water with DMF. The solution was then neutralized with an excess of pyridine (5 mL) to give a gelatinous solution which was then treated with a solution of propofol succinyl chloride [prepared from Propofol hemisuccinate (90 mg,), DMF (1 drop) , oxalyl chloride (300 µL) in anhydrous methylene chloride (7.5 mL)] and stirred at room temperature for 24 hours. The product was then precipitated with ether, washed with methylene chloride to remove all reactants and dried under vacuum for 24 hours to yield propofolsuccinyl-hyaluronate (250mg).

In Table 1 the chemical displacements in DMSO of the sample are reported. In all the tables reporting data n.m.r. G indicates the glucuronic acid residue of the hyaluronic acid (for example, G-1 indicates the anomeric carbon of the glucuronic acid), N. represents the glucosamine residue (for example, N-6 indicates the 6 carbon of the glucosamine residue).

TABLE 1

The degree of substitution calculated on the basis of esterification in N-6 was estimated to be around 100%.

Example 2

Preparation of propofol-succinii derivative of the benzyl ester of hyaluronic acid (HABE)

A suspension of HABE (MW 140,000, 250 mg) in N, Ardirne tylformamide (DMF, 16 mL) was stirred at room temperature for 0.5 hours and then: pyridine (5 mL), 4-dimethylaminopyridine (20 mg) and a solution of propofol succimi chloride [prepared from Propofol hemisucdnate (410 mg,), DMF (2 drops), oxalyl chloride (130 μί) in ether (3 mL)]. The mixture was kept under stirring at room temperature for 24 hours. The product was then precipitated from ether, repeatedly washed with methylene chloride to remove all the reagents used and then dried under vacuum to provide propofol succinyl derivative of the benzyl ester of hyaluronic acid (250 mg).

Table 2 shows the chemical displacement values for the sample in DMSO.

TABLE 2

From the spectrum n.m.r. it was possible to estimate the degree of substitution based on the integration relative to carbon N-6 equal to 45%. Example 3

Synthesis of hemisuccinate cholesterol

Succinic anhydride (2.04 g, 20.4 mmoles) was added to a solution of cholesterol (7.5 g, 19.4 mmoles) in pyridine (20 ml), keeping the mixture at 60 ° C under stirring for 48 hours. The solution was concentrated under reduced pressure to provide, after crystallization from butan-2-one, the hemisuccinate cholesterol (6.62g).

Preparation of cholesterol-succinyl-hyaluronate

Hyaluronic acid (MW 30000, 500 mg) was dissolved in distilled water (125 mL) and the pH was brought to approximately 2.5, using an ion exchange resin (IRA 120 H +). The resin was then removed by filtration and the solution concentrated to approximately 45 mL. 100 mL of Ν, Ν-dimethylformamide (DMF) was then added and the solution was concentrated to approximately 50 mL. This procedure was repeated three times to replace most of the water with DMF. The solution was then neutralized with an excess of pyridine (17 mL) to give a gelatinous solution which was then treated with a solution of cholesterol succinyl chloride [prepared from hemisuccinate cholesterol (2.68 g, 5.5 mmol), DMF (1 drop), oxalyl chloride (0.51 mL, 5.8 mmoles) in anhydrous methylene chloride (8 mL)] and stirred at room temperature for 24 hours. The reaction mixture was then dialyzed against distilled water to provide a water soluble fraction which was lyophilized (230 mg) and a water insoluble fraction which was vacuum dried in the presence of phosphorus pentoxide to obtain cholesterol-succinylyaluronate. (1.5 g).

The water-soluble derivative is characterized by a lower degree of derivatization with cholesterol-succinate than the water-insoluble fraction.

Example 4

Preparation of cholesterol-succinyl derivative of the benzyl ester of hyaluronic acid (HABE)

A suspension of HABE (MW 140,000, 500 mg) in N, N-dimethylformamide (DMF, 16 mL) was stirred at room temperature for 0.5 hours and then: pyridine (4 mL), 4-dimethylaminopyridine ( 5 mg) and a solution of cholesterol succimi chloride [prepared from cholesterol hemisuccinate (1.04 g, 4.29 mmol), DMF (2 drops), oxalyl chloride (0.392 mL, 45 mmol) in ether (9 mL)]. The mixture was kept under stirring at room temperature for 24 hours. The reaction mixture was concentrated to about half volume and the product was then precipitated from ether, taken up with ethanol to give cholesterol-sucdnil-HABE in the form of solid product (550 mg).

Table 3 shows the assignment of signals 13C-n.m.r. cholesterol-succinyl-HABE.

TABLE 3

Example_5

Preparation of Prednisone hemisuccinate

To a solution of prednisone (750 mg, 2.1 mmoles) in anhydrous pyridine (10 ml), sucdnic anhydride (314 mg, 3.14 mmoles) was added by keeping the mixture at 60 ° C under stirring for 96 hours. The solution was concentrated under reduced pressure, the residue dissolved in methylene chloride and then passed on a silica gel column and then dried under vacuum, silica gel chromatography, using methylene-petroleum ether colide (2: 1 ), provides prednisone hemisucdnate (650 mg).

Preparation of prednisone succimi hyaluronate

Hyaluronic acid (MW 30000, 500 mg) was dissolved in distilled water (125 mL) and the pH was brought to approximately 2.5, using an ion exchange resin (IRA 120 H <+>). The resin was then removed by filtration and the solution concentrated to approximately 45 mL. 100 mL of Ν, Ν-dimethylformamide (DMF) was then added and the solution was concentrated to approximately 50 mL. This procedure was repeated three times to replace most of the water with DMF. The solution was then neutralized with an excess of pyridine (17 mL) to give a solution with a gelatinous consistency which was then treated with a solution of prednisone succinyl chloride [prepared from prednisone hemisucdnate (280 mg, 0.58 mmol), partially dissolving it in chloroform (3 mL) and dioxane (8 mL) and DMF (1 drop), and then adding oxalyl doride (0.51 mL, 5.8 mmol) and stirring the mixture for 1 hour] and stirring at room temperature for 18 hours. The solution was concentrated to total 20 mL taken up with hot water (50 ° C) and then dialyzed against distilled water (6 times, 1 L). The prednisone-succinyl-hyaluronate was then recovered by lyophilization (350 mg).

Table 4 shows the assignment of the 13C-n.m.r spectrum. in DMS0: D20 (1: 1.5) of prednisone sucdnii hyaluronate.

TABLE 4

From the data n.m.r. the degree of substitution of succinyl-prednisone on the polymer is about 5%.

Example 6

Preparation of succinyl prednisone derivative of the benzyl ester of hyaluronic acid (HABE)

A suspension of HABE (MW 140,000, 100 mg) in N, N-dimethylphonnamide (DMF, 16 mL) was stirred at room temperature for 0.5 hours and then: pyridine (4 mL), 4-dimethylaminopyridine ( 5 mg) and a solution of prednisone succinyl chloride [prepared from prednisone hemisuccinate (170 mg), DMF (2 drops), oxalyl chloride (34 μL) in ether (5 mL)]. The mixture was kept under stirring at room temperature for 24 hours. The reaction mixture was concentrated to about half volume and the product was then precipitated from ether, taken up with ethanol and kept under stirring for 0.5 hours to give prednisone-sucdnil-HABE (120 mg).

Table 5 shows the assignment of the 13C-n.m.r spectrum of prednisone-succinyl-HABE.

TABLE 5

From the data l ^ C-n.m.r. ^ degree of substitution of the polymer with succinyl prednisone is about 10%.

Since the invention is thus described, it is clear that these methods can be modified in various ways. These modifications are not to be considered as divergences from the spirit and perspectives of the invention and all those modifications that would appear evident to an expert in the field are included in the scope of the following claims:

Claims (1)

CLAIMS 1. Derivatives of hyaluronic acid of formula I in which one or more hydroxyl groups of the polysaccharide react with the non-esterified carboxylic group of the succinic hemiester of a molecule of pharmacological interest. FORMULA 1 2. Derivatives according to claim 1, wherein the molecule of pharmacological interest contains an alcoholic function of the aliphatic, araliphatic, aromatic, heterocyclic type, which contributes to the formation of the hemiester. 3. Process according to claims 1-2 for the preparation of succimi derivatives of hyaluronic acid or of its esters, in which the molecule of pharmacological interest contains an alcoholic function of the aliphatic, araliphatic, aromatic, heterocyclic type. 4. Process for the preparation of succimi derivatives of hyaluronic acid according to riv. 1-2 which includes the following steps: The reaction of aliphatic, araliphatic, aromatic, heterocyclic alcohol with succinic anhydride in the presence of a catalyst to provide the corresponding hemisuccinate (a). The reaction of hemisuccinate (a) with oxalyl chloride in an aprotic solvent, such as methylene chloride, in the presence of a catalytic amount of N, N-dimethylformamide to give the acyl chloride of hemisuccinate (b). The reaction of the acyl chloride of hemisuccinate (b) with the pyridinium salt of hyaluronic acid in N, N-dimethylformamide in the presence of a basic catalyst to provide the succinyl-hyaluronate compound of general formula I. Recovery of the product after dialysis and lyophilization or by precipitation from an organic solvent such as ether or methylene chloride or both. 5. Use of the derivatives according to claims 1-4 for the preparation of controlled release systems of biologically active molecules. 6. Use of the derivatives according to claim 5 wherein the biologically active molecule is represented by steroidal anti-inflammatory agents. 7. Controlled drug release systems characterized by a polymeric matrix consisting of succimi derivatives of hyaluronic acid or its esters in the treatment of joint diseases.