Cross-linked copolymers based polycarboxylic polymers, method for their production and pharmaceutical compositions containing cross-linked copolymers

 

(57) Abstract:

Described cross-linked copolymers based unstitched polycarboxylic polymers and a crosslinking agent containing at least two AMINOPHENYL and named the unstitched copolymers contain at least one polycarboxylic polysaccharide and at least another unstitched polycarboxylic polymer non polycarboxylic polysaccharide, and polycarboxylic polymer selected from the group comprising polycarboxylic polyacid or their partially or completely substituted derivatives such as their esters, amides or salts, copolymers containing units of these polycarboxylic acids or their named derivatives, or mixtures of these polycarboxylic polymers, and/or named their derivatives, and/or their copolymers, as well as pharmaceutical compositions for the controlled selection of the active principle, in particular in the colon. 4 C. and 16 h.p. f-crystals, 1 tab., 2 Il.

The invention relates to cross-linked copolymers based unstitched polycarboxylic polymers, in which the copolymer contains at least one polycarboxylic polysaccharide. The invention also concerns the method of obtaining these copolymers and their PR some connection with the polymer structure, containing polycarboxylic polysaccharide, possibly modified. For example, in the application WO 89/02445 described a gel based on hyaluronic acid. However, this gel has its only hyaluronic acid and any other polycarboxylic polymers. On the other hand, in the preparation of this gel does not use any cross-linking agents. Thus obtained compound used mainly in surgery. In the application WO 91/16881 among other things describes the combination of the active principle with a matrix consisting of the modified polymer, i.e. a polymer to which is grafted sugars. This modified polymer may be a natural polymer, such as chondroitin sulfate. However, the above matrix contains only one type of polymer.

The copolymers of the present invention on the basis of polycarboxylic polymers contain at least one polycarboxylic polysaccharide and at least another polycarboxylic polymer, which is not a polysaccharide. The combination of the polysaccharide with polycarboxylic polymer of a different type allows you to modulate the properties such as hydrophilicity. The result can be obtained copolymers having degradability, zavidnoi environment. This is a real advantage, so as to completely remove the solvent from the polymer structure is almost impossible - the presence of residual traces of water solvent, usually more acceptable and allowed in more than residual traces of such organic solvents as dimethyl sulfoxide or dimethylformamide.

The object of the invention is citye copolymers based unstitched polycarboxylic polymers and a crosslinking agent containing at least two AMINOPHENYL and named the copolymers contain at least one polycarboxylic polysaccharide and at least another unstitched polycarboxylic polymer non polycarboxylic polysaccharide.

Unstitched polycarboxylate polysaccharides may be selected, for example, from the group of compounds comprising glycosaminoglycans, pectic acid, alginic acid, carboxyl derivatives of dextran such as carboxymethylcysteine or carboxyl derivatives of cellulose such as carboxymethylcellulose. Of glycosaminoglycans can be called hyaluronic acid, chondroitin sulfate, heparin, dermatan sulfate, heparan sulfate, keratan sulfate or SMEs, poliasparaginovaya acid, primulina acid, Polyana acid or polygonarea acid, polycarboxylate acrylic polymers such as polyacrylic acid, polymethacrylic acid or copolymers of these latter acids, such as EudragitsL and S. the Term "polycarboxylate polymers" means polymers defined above, as well as partially or fully substituted derivatives of these polymers, for example, their esters, amides or salts; copolymers containing units present in the named polycarboxylic polymers or above derivatives; and a mixture of polymers and/or their derivatives and/or their copolymers defined above.

More specifically, an object of the invention are as defined above, cross-linked copolymers, characterized in that the polysaccharide is selected from the group comprising pectin or alginic acid, glycosaminoglycans and, preferably, hyaluronic acid, chondroitin sulfate, heparin, dermatan sulfate, heparan sulfate, keratan sulfate or a mixture of the latter.

More specifically, an object of the invention are as defined above, cross-linked copolymers, characterized in that unstitched polikar roxannie acrylic polymers, polyglutamine acid, poliasparaginovaya acid, primulina acid, Polyana acid and polygonarea acid. Preferably, unstitched polycarboxylic polymer non polycarboxylic polysaccharide, is polycarboxylic acrylic polymer, more specifically, polyacrylic or polymethacrylic acid.

Proposed in the invention polycarboxylate polymers linked by using a cross-linking agent. This crosslinking agent contains at least two AMINOPHENYL, capable of reacting with free carboxyl functions of the above unstitched carboxylic polymers. The agent chosen, for example, from among proteins, polyamines, triamino, diamines, natural or synthetic amino acids or derivatives of these compounds such as their salts, esters or amides. Of the amino acids can be named, for example, arginine, lysine, histidine and ornithine. From diamines can be called Ethylenediamine, diaminobutane, diaminohexane, diaminoheptane, diaminooctane or diaminododecane. From polyamines can be called chitosan, polyaminoamide such as polylysine and polyalanine, as well as copolymers of these polyamines. Skyways streamin. Preferably, as a cross-linking agent used amino acid, mainly lysine, ornithine and histidine.

More specifically, an object of the invention are as defined above, cross-linked copolymers, characterized in that polycarboxylic the polysaccharide is polycarboxylic polysaccharide biodegradable microbial flora of the colon, namely chondroitin sulfate, hyaluronic acid, pectic acid or heparin.

More specifically, an object of the invention are as defined above, cross-linked copolymers, characterized in that polycarboxylic the polysaccharide is chondroitin sulfate, other polycarboxylic polymer is a polyacrylic or polymethacrylic acid, and a crosslinking agent is lysine or histidine.

The object of the invention is also a method of obtaining a certain higher cross-linked copolymers, characterized in that the above unstitched polycarboxylate polymers, forming a cross-linked copolymer, is subjected to the interaction in the presence of the activator with a crosslinking agent containing at least two AMINOPHENYL, in a suitable reaction medium. Obtaining defined above crosslinked copolymers carried the water with one or more miscible with water by solvents, for example, acetone or lower alcohols, such as ethanol. Preferably, the aqueous medium contains water only. The method of the invention can be realized according to different variants. Thus, the method may comprise mixing unstitched polycarboxylic polymers and cross-linking agent followed by the addition of activator. Joining mode in accordance with the invention may also consist in mixing unstitched polycarboxylic polymers with activator and then adding a crosslinking agent. The method may also consist in the stitching of one of the forming copolymer unstitched polycarboxylic polymers by mixing of the polymer with a crosslinking agent and then with an activator or first activator and then cross-linking agent, after which the reaction medium add at least one other unstitched polycarboxylic polymer for blending with the polymer already present in the reaction mixture. In the process of the invention introduce the reagents can be pre-dissolved in the chosen reaction medium. As a rule, unstitched polycarboxylate polymers and a crosslinking agent are mixed together in any aquatic environment prior to their dissolution, and then add activato mainly operate at room temperature. It is obvious that the temperature at which is way below the temperature of decomposition of the used reagents.

The relative proportions of the reagents, which are unstitched polycarboxylate polymers, crosslinking agent and the activator can vary depending on the desired characteristics of the copolymers. The proportions unstitched polycarboxylic polymers defined relative to the molar amount of carboxylic functions that reside in the source link. The molar proportion of unstitched polycarboxylic polymers may be in the range from 0.01 to 100. The molar proportion of the crosslinking agent relative to the total number of carboxylic functions can be in the range from 0.01 to 100. The molar proportion of the activator relative to the total number of carboxylic functions can be in the range from 0.01 to 100.

The activator can be selected from among the agents combinations commonly used in peptide synthesis. In particular, as an activator can be used carbodiimide, derivatives of quinoline or mixed anhydrides. As examples of carbodiimides can be called hydrogenogenic such as hydrochloride N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) is Ana 2 ethoxy-N-etoxycarbonyl-1,2-dihydroquinoline(EEDQ), N-isobutoxide-2-isobutoxy-1,2-dihydroquinoline(IIDQ), N-isobutoxide-2-methoxy-1,2-dihydroquinoline(IMDQ), N-isobutoxide-2-ethoxy-1,2-dihydroquinoline(IEDQ). As examples of mixed anhydrides can be called chloroformiate and more specifically isobutylparaben(IBC). As activator mostly use hydrochloride N-(3-dimethylaminopropyl) -N'-ethylcarbodiimide.

Proposed in the invention cross-linked copolymers can be used, for example, in the pharmaceutical industry, cosmetics, Biomedicine, veterinary medicine, chemistry, Agrochemistry and agri-food industry.

More specifically, an object of the invention is a pharmaceutical composition containing at least one active principle, and, as an inert filler or carrier, at least one proposed by the invention cross-linked copolymer. The term "active principle" means any substance or mixture of substances having therapeutic activity.

Such a composition can be made from various components using well-known conventional methods. The composition may be obtained in the form of pressed pellets consisting of a copolymer matrix; Tana matrix; pills or microparticles coated with the copolymers of the present invention. These microparticles and pills can be used in free form or can be enclosed in capsules. The composition can also be presented in the form of microparticles or nanoparticles, in which at least one component is a copolymer of the present invention, or in any other form, allowing for oral administration. The composition may be presented in any other form adapted to the selected method of administration, or be in the form of suppositories or topical preparations or injections. The number of active principle, ensure the effectiveness of pharmacological and, in particular therapeutic effect may vary depending on the nature of the active principle, from age and/or disease to be treated of the patient.

The object of the present invention is also the application of the proposed composition for the controlled selection it contained the active principle or active principles.

Such compositions may also have other characteristics, which in some cases depend on the characteristics of the original polycarboxylic polymers, for example, Imeretia as bioadhesive pharmaceutical system. Thus, an object of the present invention is also the application of the proposed invention the pharmaceutical composition as bioadhesive system.

The above-described composition, in which polycarboxylic polysaccharide decomposes flora of the colon, can also be used as a system with specific allocation in the large intestine under the action of microbial flora. The concept of a specific allocation in the large intestine under the action of the microbial flora is based on the fact that the colon contains very abundant microbial flora, which also has the ability to metabolize substances which decay weakly or not disintegrate in the upper digestive tract. Such compositions are particularly suitable for the implementation of transport of the active principles for the treatment of diseases of the colon that can improve the effectiveness of these compositions and to reduce their side effects. Among the active principles are steroids such as dexamethasone and hydrocortisone, non-steroidal anti-inflammatory agents such as 5-aminosalicylic acid, antitumor agent, such as methotrexate and tamoxifen, antispasmodic in the sport such active principles, which are absorbed in the colon more effective than steroids or xanthine. Direct introduction of these compositions at the level of the colon can improve their efficiency. Well suited to these songs and for the transport of active principles, which fall in the upper gastrointestinal tract. Of the active principles can be mentioned peptides and proteins such as oral injected vaccines, insulin, birth control peptides, plasminogen-activating peptides, peptide growth, LH/RH.

The following examples illustrate the above procedures and in any case should not be construed as limiting the scope of the invention.

EXPERIMENTAL PART

Example 1

of 1.33 g of sodium salt of chondroitin sulfate (70%-30%)(CS), 0.29 grams of sodium salt of polymethacrylic acid (PMA) and the 3.35 g of L-lysine-monohydrochloride stirred in 9 ml of double-distilled water to obtain a clear solution which is then Tegaserod. After that add 4.59 g of the hydrochloride of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC). pH maintained within the range of from 6 to 7, gradually adding 2,5 N. hydrochloric acid. The reaction is carried out at room temperature for 6 h, after which the reaction mixture is from 4 l of water. Resulting from this precipitate is washed with water and then dried. Get the desired copolymer chondroitin sulfate and polymetacrylate acid with an average output 1,530,12, the Use of sulfur as a marker of chondroitin sulfate helps to determine with the help of elemental analysis, the mass content of chondroitin sulfate copolymer, which is 592%.

Example 2

Repeating the procedure described in example 1, but using 1,77 g L-lysine-monohydrochloride and 2.76 g of the hydrochloride of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide, get 1,060,15 g of the copolymer; the content in the sediment chondroitin sulfate 552 wt.%.

Example 3

Repeating the procedure described in example 1, but using 7,06 g L-lysine-monohydrochloride and 8,21 g N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide, get 1,610,12 g of the copolymer; the content of chondroitin sulfate in the sediment is 611 wt.%.

Example 4

Repeating the procedure described in example 1, but using 3 g of histidine instead of L-lysine, get 1,940,01 g of the copolymer; the percentage of chondroitin sulfate is 483 wt.%.

Example 5

Repeating the procedure described in example 1, but using of 0.43 g of sodium salt polymetacrylate acid, 4,5 Gomera; the content of chondroitin sulfate in the sediment is 582 wt.%.

Example 6

Repeating the procedure described in example 1, but using of 0.58 g of sodium salt polymetacrylate acid, of 5.45 g of L-lysine-monohydrochloride and 7,05 g of the hydrochloride of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide, get 2,070,01 g of the copolymer; the content of chondroitin sulfate in the sediment is 542 wt.%.

Example 7

Test the solubility of the copolymer obtained in example 1 is produced in the following solvents or mixtures of solvents: water with a pH from 3 to 7, acetonitrile, ethanol, tetrahydrofuran, dichloromethane, dimethylsulfoxide, dimethylacetamide, acetone, dioxane, triethylamine, chloroform, petroleum ether, hexane, dimethylformamide, benzyl alcohol, heptane, isopropyl alcohol, propandiol-1,2, a mixture of water/acetone (50/50) and the mixture of water/ethanol (50/50). The copolymer was insoluble in these solvents, which proves its crosslinked structure.

The study of the enzymatic degradation of copolymers of

1-Spectroscopic study

Investigated the collapse of the proposed invention copolymers based on chondroitin sulfate using chondroitinase - enzymes microbial flora of the colon.

Preparation, is Itachi within a few hours. Suspension containing 67 mg of chondroitin sulfate in 1 ml of buffer. The solution chondroitinase added at the rate of 310-3units of enzyme activity per 1 mg contained in the suspension chondroitin sulfate. The mixture is incubated at 37oC. After a certain period of time, the suspension is centrifuged at 4oC and then filtered. The supernatant is subjected to investigation on UV absorption. Formed by the collapse of chondroitin sulfate disaccharides have a maximum absorption at 230-240 nm (Yamagata T., and others, J. Biol.Chem., 1968, 243(7), 1523-1535; Salyers, A. and others, J. Bacteriol., 143(2), 772-780). As a control, use a solution unstitched chondroitin sulfate prepared in the above-described conditions.

The kinetics of appearance in the solution of the disaccharides formed by the collapse of unstitched chondroitin sulfate, and the copolymer obtained in example 1 below and Fig.1.

The results show that the copolymer of example 1 is decomposed with the action of enzymes. Comparison of disintegration of the copolymer of example 1 with the control sample indicates that the copolymer, although the stitching is rapidly decomposed under the action of enzymes.

Similarly explore the copolymers of examples 2-6. The results show that speckletone

Enzymatic degradation of copolymers leads to the emergence of molecular targets with a smaller size and as a consequence would lead to lowering of the viscosity of the environment in which suspended copolymers.

The suspension of the copolymer from example 1 in Tris-acetate-albumen buffer is prepared in the same conditions as the slurry used in the above spectroscopic study. Then 4 ml of the suspension incubated in the cylinder rheometer (Haake RS100), thermostatted at 37oTo measure the initial viscosity (h). Then the suspension add 0.8 units of enzyme activity chondrons dissolved in 160 ml of water. The reference sample is a suspension of the copolymer of example 1 prepared in the above conditions without addition of the enzyme dissolved in 160 ml of water. We measure the change in viscosity over time. For each test is performed on two series of measurements.

In Fig. 2 are presented in semi-logarithmic form, the change in viscosity of the copolymer from example 1 in the presence of enzymes (continuous line) and in the absence of enzymes (curve control in the form of dashed lines). The viscosity of the control sample, with approximately 173 mpss not the giving 55 min and then remains almost constant. Such a significant drop in viscosity due to the collapse of the copolymer under the action of enzymes.

Along with this, based on the results above in the same conditions spectroscopic and rheological studies can draw attention to the fact that after 55 min of incubation in the presence of enzymes viscosity is almost entirely falls, while in the solution is detected only part of disaccharides resulting from the collapse of chondroitin sulfate. Thus, for the destruction of the three-dimensional grid copolymer enough enzymatic disintegration of the several parts of copolymer.

The study of the tablets on the adjustable selection

Cross-linked copolymers of examples 1-6 sift, mix with 5-aminosalicylic acid and magnesium stearate in a mass ratio of 79.5/20/0.5 and then by direct compression prepare 250 mg tablets having a hardness of more than 100 H.

Thus prepared tablets were tested for solubility in the apparatus with a rotary plate (Dissolutest) at 37oC and a stirring speed of 50 rpm was Used for dissolving medium: buffer solutions with a pH of 1.2 and 7.5, corresponding artificial gastric and intestinal is i.i.d. intervals take the sample test on the solubility of environment and filtered. The dosage of 5-aminosalicylic acid is carried out using UV spectroscopy.

The table below gives the time (in hours) the allocation of 50% of the initial dose 5-aminosalicylic acid (t50%), obtained for the artificial gastric and intestinal environments.

In the gastric environment, the value of t50%ranges from 1.2 to 8 hours, thus enabling you to modulate the selection of the active principle, which at the same time depends on the nature of the copolymer. All of the copolymers the copolymers obtained in examples 3, 5 and 6, for which value of t50%respectively 6,5; 7,9 and 8 h, materially delay the release of active beginning.

In the intestinal environment, the value of t50%varies between 1.6 and 11 h, also allowing you to modulate the selection of the active principle, which, in turn, depends on the nature of the copolymers. However, a significant delay of excretion of the active principle obtained with the copolymers of examples 1, 3, 5 and 6. In particular, the value of t50%for these copolymers, respectively 7,7; 8,3; 8.7 and 11 o'clock

Thus, the synthesized copolymers allow you to create a pharmaceutical system with adjustable selection, which depends on characteristic selection active beginning to disintegrate under the action of chondroitinase, are promising candidates for the development of systems with variable selection in the large intestine under the action of microbial flora.

1. Cross-linked copolymers based unstitched polycarboxylic polymers and a crosslinking agent containing at least two AMINOPHENYL and named the crosslinked copolymers contain at least one polycarboxylic polysaccharide and at least another unstitched polycarboxylic polymer non polycarboxylic polysaccharide, with polycarboxylic polymer selected from the group comprising polycarboxylic acid or partially or completely substituted derivatives such as their esters, amides or salts; copolymers containing units of these polycarboxylic acids, or their named derivatives, or mixtures of these polycarboxylic acids, and/or named their derivatives, and/or their copolymers.

2. The copolymers under item 1, characterized in that polycarboxylic the polysaccharide is a glycosaminoglycan, pectic acid or alginic acid.

3. The copolymers according to any one of paragraphs.1 and 2, characterized in that polycarboxylic the polysaccharide is glycosaminoglycan, selected from among the following compounds:"ptx2">

4. The copolymers according to any one of paragraphs.1-3, characterized in that the unstitched polycarboxylic polymer non polycarboxylic the polysaccharide is chosen from among the following compounds: polycarboxylate acrylic polymers, polyglutamine acid, poliasparaginovaya acid, primulina acid, Polyana acid and polygonarea acid.

5. The copolymers according to any one of paragraphs.1-4, characterized in that the unstitched polycarboxylic polymer non polycarboxylic the polysaccharide is polycarbosilanes acrylic polymer.

6. The copolymers under item 5, characterized in that polycarboxylic acrylic polymer is a polyacrylic or polymethacrylic acid.

7. The copolymers according to any one of paragraphs.1-6, characterized in that the crosslinking agent is selected from diamines, natural or synthetic amino acids and polyamines.

8. The copolymers according to p. 7, characterized in that the amino acid is lysine, histidine or ornithine.

9. The copolymers according to p. 7, characterized in that is selected from among the following compounds: Ethylenediamine, diaminobutane, diaminohexane, diaminoheptane, diaminooctane and diaminododecane.

10. The copolymers under item 1, characterized in that the floor of the WMD of PP.1-10, characterized in that polycarboxylic polysaccharide is degradable under the action of the colonic flora.

12. The copolymers according to p. 7, characterized in that polycarboxylic the polysaccharide is chondroitin sulfate, hyaluronic acid, pectic acid or heparin.

13. The copolymers according to any one of paragraphs.1-8, 11 and 12, characterized in that polycarboxylic the polysaccharide is chondroitin sulfate, other polycarboxylic polymer is a polyacrylic or polymethacrylic acid and cross-linking agent is lysine or histidine.

14. A method of obtaining a cross-linked copolymer according to any one of paragraphs.1-13, namely, that unstitched polycarboxylic polymer, including polycarboxylic polysaccharide described in paragraph 1, is subjected to the interaction in aqueous medium in the presence of an activator and a crosslinking agent, and a crosslinking agent selected from proteins, polyamines, triamino, diamines, natural or synthetic amino acids or their derivatives, such as esters, salts or amides.

15. The method according to p. 14, characterized in that the activator is selected from the group of compounds, which includes carbodiimide, derivatives of quinoline and mixed anhydrides.

16. Pharma is the one, at least one active principle and as an inert carrier or excipient, at least one copolymer according to any one of paragraphs.1-10.

17. Pharmaceutical composition for controlled specific allocation contained in the active start or started in the colon, comprising at least one active principle and as an inert carrier or excipient, at least one copolymer according to any one of paragraphs. 11-13.

18. The pharmaceutical composition according to p. 17 engaged in the transport of the active agent intended for the treatment of diseases of the colon.

19. The pharmaceutical composition according to p. 17 engaged in the transport of the active agent, visionage in the colon.

20. The pharmaceutical composition according to p. 17 engaged in the transport of the active agent that undergoes degradation in the upper gastrointestinal tract.

 

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