NO311621B1 - Crosslinked copolymers based on polymers containing carboxylic acid, process for their preparation, pharmaceutical compositions containing copolymers, and use of the pharmaceutical composition - Google Patents

Abstract

The invention concerns cross-linked copolymers with a base of non cross-linked polycarboxylic polymers, said copolymers containing at least one polycarboxylic polysaccharide. The invention also concerns a method for preparing these copolymers and their use in particular as support in pharmaceutical compositions.

Description

The invention relates to cross-linked copolymers based on non-cross-linked polycarboxylic polymers, the copolymers containing at least one polycarboxylic polysaccharide. The invention also relates to a method for the production of these copolymers and their use in particular as a carrier in pharmaceutical preparations.

Certain compounds with a polymer structure containing a polycarboxylic polysaccharide, possibly modified, are described in the literature. For example, patent application WO 89/02445 describes a gel based on hyaluronic acid; but this gel comprises only hyaluronic acid in its structure, and no other polycarboxylic polymer. Furthermore, no cross-linking agent is used in the production of this gel. The compound obtained in this way is mainly used in surgery. Patent application WO 91/16881 describes, among other things, the combination of an active ingredient with a matrix which consists of a modified polymer, i.e. to which saccharides are grafted. This modified polymer can be a natural polymer such as chondroitin sulfate. However, this matrix contains only one type of polymer.

The copolymers according to the invention based on polycarboxylic polymers contain at least one polycarboxylic polysaccharide and at least one polycarboxylic polymer which is not a polysaccharide. The combination of a polysaccharide and another type of polycarboxylic polymer enables modulation of the properties of the polysaccharides such as hydrophilicity. In this way, copolymers can be obtained with appropriate degradation properties according to their applications. Furthermore, the copolymers according to the invention are advantageously prepared in an aqueous medium. This is a real advantage since it is almost impossible to completely eliminate the solvents in a polymer structure; the presence of traces of aqueous residual solvents is generally easier to accept and accept than traces of organic residual solvents such as dimethylsulfoxide or dimethylformamide.

An object of the invention are cross-linked copolymers based on non-cross-linked polycarboxylic polymers and a cross-linking agent comprising at least two amine functions, the copolymers comprising at least one polycarboxylic polysaccharide and at least one other non-cross-linked polycarboxylic polymer which is not a polycarboxylic polysaccharide.

The non-crosslinked polycarboxylic polysaccharides can for example be chosen from glycosaminoglycans, pectinic acid, alginic acid, carboxylic derivatives of dextran such as carboxymethyldextrans, or the carboxylic derivatives of cellulose such as carboxymethyl cellulose types. Among the glycosaminoglycans, hyaluronic acid, chondroitin sulfate, heparin, dermatan sulfate, heparan sulfate, keratan sulfate or a mixture of these can be mentioned. Among the polycarboxylic polymers which are not polysaccharides, mention may be made of poly(glutamic acid), poly(aspartic acid), poly(maleic acid), poly(malic acid) and poly(fumaric acid), the polycarboxylic acrylic polymers such as poly(acrylic acid), poly(methacrylic acid) or the copolymers thereof such as Eudragits® L and S. The term polycarboxylic polymers includes polymers as defined above, but also the partially or totally substituted derivatives of these polymers such as for example their esters, their amides or their salts, copolymers containing the units found in these polycarboxylic polymers or in their derivatives as defined above, but also a mixture of these polymers and/or their derivatives and/or their copolymers as defined above.

A more particular subject of the invention are crosslinked copolymers as defined above, characterized in that the polysaccharide is selected from pectinic or alginic acid, glycosaminoglycans and preferably hyaluronic acid, chondroitin sulfate, heparin, dermatan sulfate, heparan sulfate, keratan sulfate or mixtures thereof.

A more particular object of the invention are cross-linked copolymers as defined above, characterized in that the non-cross-linked polycarboxylic polymer which is not a polycarboxylic polysaccharide is selected from polycarboxylic acrylic polymers, poly(glutamic acid), poly(aspartic acid), poly(maleic -acid), poly(malic acid) or poly(fumaric acid). The non-crosslinked polycarboxylic polymer which is not a polycarboxylic polysaccharide is preferably a polycarboxylic acrylic polymer, and more particularly poly(acrylic acid) or poly(methacrylic acid).

The polycarboxylic polymers according to the invention are bound together with a cross-linking agent. This crosslinking agent comprises at least two amine functions which can react with the free carboxylic functions in the non-crosslinked carboxylic polymers. It can be chosen, for example, from proteins, polyamines, triamines, diamines, natural or synthetic amino acids or the derivatives of compounds as defined above, such as for example their salts, esters or amides. Examples of amino acids include arginine, lysine, histidine and ornithine. Among the diamines can be mentioned ethylenediamine, butanediamine, hexanediamine, heptanediamine, octanediamine and dodecanediamine. Among the polyamines, mention may be made of chitosan, poly(amino acids) such as polylysine or polyornithine, as well as the copolymers of these polyamines. The cross-linking agent can also be selected from compounds such as spermine, spermidine, melamine, guanidine and diethylenetriamine. The cross-linking agent used is preferably an amino acid and advantageously lysine, ornithine or histidine.

A more particular object of the invention are also cross-linked copolymers as defined above, characterized in that the polycarboxylic polysaccharide is a polycarboxylic polysaccharide that can be broken down by the microbial flora in the colon, such as chondroitin sulfate, hyaluronic acid, pectin acid and heparin.

A more particular object of the invention are cross-linked copolymers as defined above, characterized in that the polycarboxylic polysaccharide is chondroitin sulfate and the other polycarboxylic polymer is selected from poly(acrylic acid) and poly(methacrylic acid), and the cross-linking agent is lysine or histidine.

An object of the invention is also a method for the production of cross-linked copolymers as defined above, the method being characterized in that the non-cross-linked polycarboxylic polymers that make up the cross-linked copolymer are reacted in the presence of an activator and a cross-linking agent comprising at least two amine functions, in a suitable reaction medium. The preparation of cross-linked copolymers as defined above is preferably carried out in an aqueous medium. The term aqueous medium denotes a medium containing only water or water mixed with one or more solvents which are miscible with water, such as for example acetone, or lower alcohols such as ethanol. The aqueous medium preferably comprises only water. The method according to the invention can be carried out in different ways. The method may actually consist of mixing cross-linked polycarboxylic polymers and the cross-linking agent, and then adding the activator. The cross-linking method according to the invention can also consist of mixing non-cross-linked polycarboxylic polymers and the activator, and then adding the cross-linking agent. The method can also consist of cross-linking one of the non-cross-linked polycarboxylic polymers that make up the copolymer, mixing the polymer with the cross-linking agent and then the activator, or with the activator and then the cross-linking agent, and then adding at least one other non-cross-linked polycarboxylic polymer to the reaction medium, for cross-linking it with the polymer present in the reaction mixture. During the execution of the method, the introduced reagents can be solubilized in advance in the selected reaction medium. The non-crosslinked polycarboxylic polymers and the crosslinking agent are preferably mixed together in an aqueous medium for solubilization, and then the activator is added. The method is carried out at a temperature of between -30 and 100°C, preferably between 0 and 40°C, and most preferably at ambient temperature. The output temperature for the cross-linking process is of course lower than the breakdown or decomposition temperatures for the introduced reagents.

The relative proportions of the reagents comprising the uncrosslinked polycarboxylic polymers, the crosslinking agent and the activator may vary according to the properties of the desired copolymers. The proportions of the non-crosslinked polycarboxylic polymers are defined with respect to the molar amounts of the carboxylic functions present per base unit. The non-crosslinked polycarboxylic polymers may vary within a molar ratio of between 0.01 and 100. The molar ratio of the crosslinking agent with respect to the total carboxylic functions may vary from 0.01 to 100. The molar ratio of the activator with respect to the total carboxylic functions may vary from 0.01 to 100.

The activator can be selected from coupling agents in standard use in peptide synthesis. The activator can thus, for example, be selected from among carbodiimides, quinoline derivatives or mixed anhydrides. Examples of carbodiimides include hydrohalides such as N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide (CMC). Examples of quinoline derivatives include 2-ethoxy-N-ethoxycarbonyl-1,2-dihydro-quinoline (EEDQ), N-isobutoxycarbonyl-2-isobutoxy-1,2-dihydroquinoline (IIDQ), N-isobutoxycarbonyl-2-methoxy- 1,2-dihydroquinoline (IMDQ) and N-isobutoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline (IEDQ). Examples of mixed anhydrides include chloroformates and more particularly isobutyl chloroformate (IBC). The activator used is preferably N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride.

The crosslinked copolymers according to the invention can, for example, be used in the pharmaceutical, cosmetic, biomedical, veterinary medicine, chemical, agrochemical or agro-nutrition areas.

A more particular object of the invention is a pharmaceutical preparation containing at least one active ingredient, and as an internal carrier or an excipient, at least one cross-linked copolymer according to the invention. The term active ingredient denotes any substance or mixture of substances with therapeutic activity.

Such a preparation can be prepared from these various components using any standard technique known to a person skilled in the art. It can, for example, be presented in the form of matrix tablets, tablets coated with the copolymers according to the present invention, multi-layer tablets, matrix pellets, pellets or microparticles coated with the copolymers according to the present invention. These microparticles and pellets can be enclosed in capsules, or not. It can also be presented in the form of microparticles or nanoparticles of which at least one component is a copolymer according to the present invention, or otherwise in any other form that enables oral administration. It may also be presented in any other form suitable for the chosen or appropriate method of administration such as suppositories or preparations for local application or injection. The amount of active ingredient that enables effective pharmacological action, especially therapeutic action, can vary according to the type of active ingredient, the patient's age and/or the disease to be treated.

An object of the present invention is also the use of a pharmaceutical preparation according to the invention for long-term release of the active ingredient(s) it contains.

Such preparations can also have other properties, which possibly depend on the properties of the initial polycarboxylic polymers such as bio-integration. A pharmaceutical preparation according to the invention can thus also be used as a bioadhesive pharmaceutical system. An object of the present invention is therefore also the use of a pharmaceutical preparation according to the invention as a biointegration system.

Preparations as defined above, where the polycarboxylic polysaccharide can be broken down by the flora in the colon, can also be used as a specific release system at colonic level by the influence of the microflora. The principle of specific release at the colonic level by the influence of microbial flora is based on the ability of the large intestine to have a very abundant microbial flora which further has the potential to metabolize substances which are broken down a little, or which are not broken down, by the upper part of the digestive tract. Such preparations are particularly suitable for transporting active ingredients intended for the treatment of diseases of the large intestine, which enables their effectiveness to be increased and their side effects to be reduced. These active ingredients include steroids such as dexamethasone and hydrocortisone, non-steroidal anti-inflammatory agents such as 5-aminosalicylic acid, antineoplastic agents such as methotrexate, tamoxifen, antispasmodics and chemotherapy agents. Such preparations are also particularly suitable for transporting active ingredients that are absorbed more efficiently at the level of the large intestine, such as steroids or xanthine. Direct administration of them at the level of the colon allows their effectiveness to be increased. Such preparations are also particularly suitable for transporting active ingredients that break down in the upper parts of the digestive tract. Among these active ingredients can be mentioned peptides and proteins such as oral vaccines, insulin, antifertility peptides, plasminogen activator peptides, growth peptides, LH/RH.

The following examples are shown to illustrate the above methods.

EXPERIMENTAL PART

Example 1

1.33 g of the sodium salt of chondroitin sulfate (70% A, 30% C) (CS), 0.29 g of the sodium salt of polymethacrylic acid (PMA) and 3.35 g of L-lysine monohydrochloride are mixed together in 9 ml of double distilled water until a clear solution is obtained, which is then degassed. 4.59 g of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) are then added. The pH is kept at between 6 and 7 by successive addition of 2.5 N hydrochloric acid. The reaction is carried out at ambient temperature for 6 hours. The reaction medium is transferred to a dialysis machine (Spectra/por, exclusion threshold 12-14 KD) and dialyzed 4 times against 4 liters of water each time. The precipitate thus obtained is washed with water and then dried. The desired chondroitin sulfate and polymethacrylic acid copolymer is obtained with an average mass of 1.53 ± 0.12 g. The use of sulfur as a marker for chondroitin sulfate enables definition by elemental analysis of the mass percentage of chondroitin sulfate in the copolymer, which is equal to 59 +2% .

Example 2

The procedure is carried out in the same way as in example 1, but with the use of 1.77 g of L-lysine monohydrochloride and 2.76 g of EDC. The mass of the copolymer obtained is 1.06 + 0.15 g; the mass percentage of CS in the precipitate is 55 +2.

Example 3

The method is carried out in the same way as in example 1, but with the use of 7.06 g of L-lysine monohydrochloride and 8.21 g of EDC. The mass of the obtained copolymer is 1.61 ± 0.12 g; the mass percentage of CS in the precipitate is 61 ±1.

Example 4

The procedure is carried out in the same way as in example 1, but with the use of 3 g of histidine instead of L-lysine. The mass of the obtained copolymer is 1.93 ± 0.01 g; the mass percentage of CS in the precipitate is 48 + 3.

Example 5

The method is carried out in the same way as in example 1, but with the use of 0.43 g of PMA, 4.5 g of L-lysine monohydrochloride and 5.82 g of EDC. The mass of the obtained copolymer is 1.86 ± 0.05 g; the mass percentage of CS in the precipitate is 58 ± 2.

Example 6

The method is carried out in the same way as in example 1, but with the use of 0.58 g of PMA, 5.45 g of L-lysine monohydrochloride and 7.05 g of EDC. The mass of the obtained copolymer is 2.07 ± 0.01 g; the mass percentage of CS in the precipitate is 54 ± 2.

Example 7

Tests regarding the solubilization of the copolymer according to example 1 are carried out in the following solvents and mixtures of solvents: water at pH 3 and 7, acetonitrile, ethanol, tetrahydrofuran, dichloromethane, dimethylsulfoxide, dimethylacetamide, acetone, dioxane, triethylamine, chloroform, petroleum ether, hexane, dimethylformamide, benzyl alcohol, heptane, isopropyl alcohol, 1,2-propanediol, water/acetone mixture (50%/50%), water/ethanol mixture (50%/50%).

The copolymer is insoluble in all these solvents, indicating its cross-linked nature.

Investigation of the enzymatic degradation of copolymers

1. Spectroscopic examination

We are investigating here the degradation of the copolymers according to the invention based on chondroitin sulfate with the help of chondroitinases, enzymes in the microbial flora of the large intestine.

Suspensions of the copolymers according to examples 1-6, in a buffer (acetate/tris/albumin) at pH 7.3, are prepared and agitated for a couple of hours to stabilize them. The suspensions contain 67 mg of CS per ml of buffer. A solution of chondroitinases is added in an amount of 3.103 EU (enzymatic unit) for each mg of CS present in the suspension. The mixture is incubated at 37°C. At predetermined time points, a suspension is centrifuged at 4°C and then filtered. An examination of the UV absorbance in the supernatant is carried out. The disaccharides resulting from the breakdown of CS have a maximum absorption at 230-240 nm (T. Ya-magata et al., J. Biol. Chem., 243(7): 1523-1535 (1968); A. Salyers et al., J. Bacteriol., 143(2): 772-780). The control is a solution of non-crosslinked CS prepared under the same process conditions as above.

The kinetics of the behavior in solution of disaccharides originating from the degradation of the non-crosslinked CS and the copolymer obtained in Example 1 is shown in Fig. 1 below.

These results show that the copolymer according to example 1 is broken down by the enzymes. Comparison of the degradation of the copolymer according to example 1 with the degradation of the control shows that the copolymer, although it is cross-linked, is rapidly degraded by the enzymes.

The same tests are carried out for the copolymers of Examples 2-6; the results show that these copolymers containing CS are broken down by the chondroitinases.

2. Rheological investigation

Enzymatic degradation of the copolymers leads to the appearance of molecular chains of smaller sizes and should therefore lead to a reduction in the viscosity of the medium in which they are suspended.

A suspension of the copolymer according to example 1 in the buffer mixture (tris/acetate/albumin) is prepared under the same process conditions as those used in the spectroscopic examination mentioned above. Then 4 ml of suspension are incubated in the cylinder of the viscometer (Haake RS100) which is kept at 37°C. Measurement of the initial viscosity (h) is carried out. Next, 0.8 EU of chondroitinase dissolved in 160 ml of water is added to the suspension. The control is a suspension of the copolymer according to example 1, prepared under the process conditions described earlier, without the addition of any enzyme and diluted in 160 ml of water. The development of the viscosity is monitored over time. The experiment is performed twice for each test.

Fig. 2 is a semi-logarithmic illustration of the development of the viscosity of the suspension of the copolymer according to example 1 in the presence of enzymes (continuous line) or in the absence of enzymes (dotted control line). The viscosity in the control, which is in the order of 17 ± 3 mPa.s, does not vary overtime. On the other hand, the viscosity drops progressively in the presence of enzymes, from 17 mPa.s to 3 mPa.s within 55 minutes and then becomes quasi-stable. This significant drop in viscosity is explained by the breakdown of the copolymer with the enzymes.

After the above-mentioned spectroscopic and rheological investigations carried out under the same process conditions, after incubation in the presence of enzymes for 55 minutes, a real total drop in viscosity can be seen, although only a part of the disaccharides originating from the breakdown of CS can be detected in solution. The degradation of a few places in the copolymer by enzymes is sufficient to bring about a collapse of the three-dimensional network in the copolymer.

Investigation of prolonged-release tablets

The cross-linked copolymers according to examples 1-6 are sieved and then mixed with aminosalicylic acid (5ASA) and magnesium stearate (mass ratio 79.5/20/0.5). Then 250 mg tablets with a hardness > 100 N are produced by direct compression.

Dissolution tests are carried out for the tablets prepared in this way, in a device with a rotating paddle (DISSULOTEST) at 37°C with agitation at 50 rpm. The dissolution media used is a buffer mixture of pH 1.2 and 7.5, respectively, corresponding to the artificial gastric and intestinal media (without enzymes). For each design and in each medium, the test is performed three times. At specific times, a sample of the dissolution medium is taken and filtered. Dosing of 5ASA is carried out using UV spectroscopy.

Table 1 below summarizes the time (in hours) required to release 50% of the initial dose of 5ASA (tso%) obtained in artificial gastric and intestinal media.

In a gastric medium, t50% varies from 1.2 to 8 hours, whereby release of the active ingredient to be modulated according to the copolymer type becomes possible. Among these copolymers, the copolymers according to examples 3, 5 and 6, which have t50o/o of 6.5, 7.9 and 8 hours respectively, significantly moderate the release of the active ingredient.

In an intestinal medium, the t50% values vary from 1.6 to 11 hours and also enable modulation of the release of the active ingredient according to the copolymer type. Furthermore, a significant moderation of the release of the active ingredient is obtained with the copolymers according to examples 1, 3, 5 and 6. In fact, the t50% values obtained with these copolymers are respectively 7.7, 8.3, 8.7 and 11 hours.

The synthesized copolymers therefore enable the production of pharmaceutical systems with prolonged release, according to the properties of the cross-linked copolymers. More particularly, it appears that those which have the property of significantly moderating the release of the active ingredient and are degradable with chondroitinases, are suitable candidates for the production of systems with prolonged release at the colonic level by the influence of the microflora.

Claims (23)

1. Cross-linked copolymers based on non-cross-linked polycarboxylic polymers and a cross-linking agent comprising at least two amine functions, characterized in that the copolymers comprise at least one polycarboxylic polysaccharide and at least one other non-cross-linked polycarboxylic polymer which is not a polycarboxylic polysaccharide. 2. Copolymers according to claim 1, characterized in that the polycarboxylic polysaccharide is selected from glycosaminoglycans, pectinic acid or alginic acid. 3. Copolymers according to one of claims 1 or 2, characterized in that the polycarboxylic polysaccharide is a glycosaminoglycan selected from hyaluronic acid, chondroitin sulfate, heparin, dermatan sulfate, heparan sulfate and keratan sulfate. 4. Copolymers according to one of claims 1-3, characterized in that the non-crosslinked polycarboxylic polymer which is not a polycarboxylic polysaccharide is selected from polycarboxylic acrylic polymers, poly(glutamic acid), poly(aspartic acid), poly(maleic acid), poly-(malic acid) and poly(fumaric acid). 5. Copolymers according to any one of claims 1-4, characterized in that the non-crosslinked polycarboxylic polymer which is not a polycarboxylic polysaccharide, is a polycarboxylic acrylic polymer. 6. Copolymers according to claim 5, characterized in that the polycarboxylic acrylic polymer is poly(acrylic acid) or poly(methacrylic acid). 7. Copolymers according to any one of claims 1-6, characterized in that the cross-linking agent is selected from diamines, neutral or synthetic amino acids or polyamines. 8. Copolymers according to claim 7, characterized in that the amino acid is chosen from lysine, histidine or ornithine. 9. Copolymers according to claim 7, characterized in that the diamine is selected from ethylenediamine, butanediamine, hexanediamine, heptanediamine, octanediamine and dodecanediamine. 10. Copolymers according to claim 7, characterized in that the polyamine is chosen from among chitosan, polyornithine and polylysine. 11. Copolymers according to any one of claims 1-10, characterized in that the polycarboxylic polysaccharide can be broken down by the flora in the large intestine. 12. Copolymers according to claim 11, characterized in that the polycarboxylic polysaccharide is selected from chondroitin sulfate, hyaluronic acid, pectin acid and heparin. 13. Copolymers according to any one of claims 1-8, 11 and 12, characterized in that the polycarboxylic polysaccharide is chondroitin sulfate, the other mentioned polycarboxylic polymer is poly(acrylic acid) or poly(methacrylic acid) and the cross-linking agent is lysine or histidine. 14. Process for producing copolymers according to any one of claims 1-13, characterized in that the non-crosslinked polycarboxylic polymers are reacted in an aqueous medium, in the presence of an activator and of said crosslinking agent. 15. Method according to claim 14, characterized in that the activator is selected from carbodiimides, quinoline derivatives and mixed anhydrides. 16. Pharmaceutical preparation, characterized in that it contains at least one active ingredient, and, as an internal carrier or excipient, at least one copolymer according to one of claims 1-10. 17. Pharmaceutical preparation, characterized in that it contains at least one active ingredient, and, as an internal carrier or excipient, at least one copolymer according to one of claims 11-13. 18. Pharmaceutical preparation according to one of claims 16-17, characterized in that it is in the form of a preparation with prolonged release. 19. Pharmaceutical preparation according to one of claims 16-17, characterized in that it is in the form of a bioadhesive pharmaceutical system. 20. Use of a pharmaceutical preparation according to claim 17 for the production of a preparation adapted for specific release of the active ingredient in the colon. 21. Use according to claim 20 to transport the active ingredient intended for the treatment of diseases of the large intestine. 22. Use according to claim 20 to transport the active ingredient which is absorbed at the level of the large intestine. 23. Use according to claim 20 to transport the active ingredient which breaks down in the upper part of the digestive tract.