CA2963582C

CA2963582C - Oral preparation comprising mucoadhesive, biocompatible hydrophilic polymer for preventing and/or treating inflammatory conditions of the gastrointestinal tract

Info

Publication number: CA2963582C
Application number: CA2963582A
Authority: CA
Inventors: Zdenka Sedlakova; Lenka POLAKOVA
Original assignee: USTAV MAKROMOLEKULARNI CHEMIE AV CR VVI
Current assignee: USTAV MAKROMOLEKULARNI CHEMIE AV CR VVI
Priority date: 2014-11-28
Filing date: 2015-11-20
Publication date: 2017-12-05
Anticipated expiration: 2035-11-20
Also published as: CZ305391B6; CZ2014825A3; CA2963582A1; WO2016082808A1

Abstract

The present invention relates to a preparation for oral use destined for prevention and treatment of inflammatory affections of gastrointestinal tract, said preparation containing a mucoadhesive, biocompatible hydrophilic polymer containing: - at least one type of structural units A selected from a group comprising units derived from acrylic and methacrylic acids and their salts, hydroxyalkyl acrylates and methacrylates, hydroxyalkyl acrylamides and methacrylamides, N,N-dialkylaminoalkyl acrylates and methacrylates, N,N-dialkylaminoalkyl acrylamides and methacrylamides, N- vinyl-2-pyrrolidone and mixtures thereof; - at least one type of structural units B selected from a group comprising units derived from hydroxy terminated (polyoxyethylene) acrylates and methacrylates, alkyloxy terminated (polyoxyethylene) acrylates and methacrylates, hydroxy terminated (polyoxyethylene) acrylamides and methacrylamides, alkyloxy terminated (polyoxyethylene) acrylamides and methacrylamides and mixtures thereof; and - at least one type of structural units C derived from sterically hindered amines.

Description

ORAL PREPARATION COMPRISING MUCOADHESIVE, BIOCOMPATIBLE HYDROPHILIC POLYMER
FOR
PREVENTING AND/OR TREATING INFLAMMATORY CONDITIONS OF THE GASTROINTESTINAL
TRACT
Field of Art The present invention relates to a polymeric preparation, which adjusts the internal environment of gastrointestinal tract (GIT) by removal of harmful substances, introduced into the GIT together with food or originated from processes taking place in GIT, mainly from inflammatory processes.
The preparation has a beneficial influence on functioning of the mucose of the gastrointestinal tract and/or on inflammatory disease cure.
Background Art Inner surfaces of GIT mucose are exposed to a very aggresive environment of gastrointestinal juices, characterized by extremely low pH values, enzymatic activity of gastrointestinal juices and actions of substances introduced into the GIT together with food. The inner surface of stomach wall and of other parts of GIT are covered by a layer of mucus, which has, among others, a protective function for the mucous membrane. When such layer is disrupted from any reason, the mucous membrane of the organ wall is irritated by contact with gastrointestinal juices, which can lead to inflammation. Nowadays, stomach ulcers, Crohn disease and ulcerative colitis belong among the most frequent inflammatory diseases of GIT.
No reliable prophylactic or therapeutic method exists at present to prevent GIT mucous membrane from inflammatory changes. The most frequently used therapeutic methods use antibiotics and steroid based anti-inflammatory drugs. However, their excessive use might cause very harmful side effects, e.g. malaise, hypertension, diabetes, osteoporosis, glaucoma.
Patients, who cannot use antibiotics or steroid based anti-inflammatory drugs, are treated by immunosuppressants which weaken their immune system and therefore reduce the symptoms of the disease.
The effect of immunosuppressive treatment of Crohn disease and ulcerative colitis, based on weakening of the autoimmunity reaction, is disadvantageous in long-term usage because it makes the patient more predisposed towards other diseases.
Excessive production of reactive oxygen free radicals (reactive oxygene species, ROS) is a very important factor during tissue damage in the first (inflammatory) phase, caused by

2 activities of anti-inflammatory mediators. Oxygen free radicals are for example hydroxyl, peroxyl and superoxide anionradicals. If the ROS concentration exceeds antioxidation capacity of the surrounding cells, the so-called oxidative stress occurs. The oxidative stress caused by ROS might cause further damage of the tissue, therefore worsen the overall progression of the inflammation, and importantly prolongate the course of treatment.
Use of hindered amine stabilizers (HAS) and their oxidated derivatives such as nitroxides and hydroxylamines in inflammatory treatment is mentioned in patent documents.
CZ
293419 B6 relates to hydrophilic polymers in the form of a gel or a cover foil, which enhances the treatment process upon its application on a skin inflammation wound. The starting polymers contain a covalently bound sterically hindered amine or its oxidated derivative (nitroxide or hydroxylamine), which serve as very efficient free radical scavengers. Materials and preparations according to the above mentioned patent can be used on external injuries, wherein the localised effect is reached by applying the preparation directly on the injury. However, such preparations are not suitable for the complex and diverse GIT environment, where the effect of the preparation needs to be assured in a relatively large and changing volume of gastrointestinal juices, and changing properties and composition of the environment, eg. different pH values in different parts of the GIT. For oral use, the accessibility of functional groups to the noxas present, localization of the preparation effect on the GIT mucous membrane and minimal systemic effect on the organism, are most important. Such properties might be achieved by a combination of an antioxidant with a polymer.
JP 2012-111700 A relates to polymeric antioxidants for use in GIT. According to this document, the polymeric antioxidant is in the form of particles or micelles and contains derivatives of cyclic nitroxides in its structure. The described block copolymers contain chemically bound blocks of polyethylene glycol and polystyrene, whereas the polystyrene blocks contain styrene units functionalized in position 4 by functional groups capable of undergoing chemical modifications enabling introduction of cyclic nitroxides.
A
disadvantage of such solution is the binding of functional nitroxides on the polystyrene block of the copolymer, which forms the hydrophobic core of a micelle or a particle.
respectively, therefore it is separated from its surroundings by a layer of polyethylene glycol which forms the hydrophilic coating of the micelle or the particle, respectively. It results in a limited accessibility of hydrophilic ROS to the antioxidant placed in the core of the micelle.

3 Another factor influencing the progression of an inflammatory disease in GIT
is the extremely low pH value, e.g. in stomach juices. Compounds chemically binding hydrochloric acid, contained in stomach juices, can serve as a supporting agent to prevent the inflammation of the stomach mucous membrane and as a protection of the mucous membrane during the inflammatory stage (gastritis, irritated stomach, heartburn, reflux, gastroesophageal reflux disease, stomach ulcer and duodenum ulcer). It particularly concerns inorganic compounds based on bicarbonates, hydroxides, metal oxides (allurninium, magnesium), and optionally their combinations with omeprazole, which acts as a proton pump inhibitor. Low molecular weight compounds are usually dispersed in a polymeric carrier which swells in stomach juices and therefore ensures their controlled release into the surrounding medium. However, the presence of low molecular weight compounds can cause a whole range of side effects to sensitive patients, due to the absorption of the compounds into the bloodstream. Another disadvantage of the use of such metal containing preparations is their interaction with commonly used medicaments, especially with antibiotics -their absorption is significantly inhibited by those metal cations.
The above mentioned disadvantages of the present state of the art can be eliminated by using the oral preparation according to the present invention.
Disclosure of the Invention Object of the invention is a preparation for oral administration destined for prevention and treatment of inflammatory affections of the gastrointestinal tract. The preparation contains a biocompatible, hydrophilic polymer with affinity towards the glycoproteins present on the GIT
mucous membrane surface, said polymer containing functional groups in its structure, which are able to bind harmful substances from the GIT juice. Individual functional components are covalently bound within polymeric chains, therefore they are effectively prevented from being released and absorbed into the bloodstream, which would lead to adverse systemic effects.

3a According to a particular aspect, there is provided a preparation for oral administration for prevention and/or treatment of inflammatory conditions of gastrointestinal tract, characterized in that it contains a mucoadhesive, biocompatible hydrophilic polymer containing:
- at least one type of structural units A selected from the group consisting of units derived from acrylic and methacrylic acids and their salts, hydroxyalkyl acrylates and methacrylates, hydroxyalkyl acrylamides and methacrylamides, N,N-dialkylaminoalkyl acrylates and methacrylates, N,N-dialkylaminoalkyl acrylamides and methacrylamides, N-vinyl-2-pyrrolidone and mixtures thereof;
- at least one type of structural units B selected from the group consisting of units derived from hydroxy terminated (polyoxyethylene) acrylates and methacrylates, alkyloxy terminated (polyoxyethylene) acrylates and methacrylates, hydroxy terminated (polyoxyethylene) acrylamides and methacrylamides, alkyloxy terminated (polyoxyethylene) acrylamides and methacrylamides and mixtures thereof; and - at least one type of structural units C derived from monomers of general formula:
X
R3 ________________________________________ wherein RI is ¨H or ¨OH or oxygen radical, R2 to R5 is (CI - C4) alkyl, X is ¨CH(Y)¨ or ¨
CH(Y)CH2¨ and Y is a chemical group capable of undergoing radical polymerization:
H2C ______________________________________ _______________________________________________ 0 wherein R is ¨H or ¨CH3 and Z is ¨0¨ or ¨NH¨.
According to another particular aspect, there is provided the use of the preparation as defined herein, for the manufacture of a medicament useful for the prevention and/or treatment of inflammatory conditions of the gastrointestinal tract.
Said polymer contains three types of structural units which have different functions in the polymeric structure and in the functioning of the preparation. Those structural units are:

4 Structural units A, characterized by an ability to change their hydrophilic properties depending on the surrounding pH. These structural units ensure good solubility of the linear polymer, and, eventually, good swellability of branched and cross-linked polymeric structures in gastrointestinal tract juices in a wide range of pH (from 1.0 to 7.0). They are also able to adjust the pH of gastric fluid. These units are selected from acrylic and methacrylic acids and their salts, hydroxyalkyl acrylates and methacrylates, N-viny1-2-pyrrolidone, N,N-dialkylaminoalkyl acrylates and methacrylates, N,N-dialkylaminoalkyl acrylamides and methacrylamides.
Structural units B are selected from a group of compounds characterized by their affinity towards glycoproteins present on the gastrointestinal tract mucous membrane surface. The presence of such units in the polymeric structure enhances the adhesion of the polymer on the mucous membrane surface, and therefore keeps the active substance, incorporated in the polymeric chain, in contact with the afflicted site of the mucous membrane. Monomers suitable for providing affinity towards gastrointestinal tract mucous membrane surface are selected from hydroxy terminated (polyoxyethylene) acrylates and methacrylates, alkyloxy terminated (polyoxyethylene) acrylates and methacrylates, hydroxy terminated (polyoxyethylene) acrylamides and methacrylamides, alkyloxy terminated (polyoxyethylene) acrylamides and methacrylamides, preferably of an average molecular mass /1411 in the range of 200 to 1000.
Preferably, the term õalkyl" means C1-C6 alkyls, if it is not stated otherwise.
Salts include, in particular, salts of alkali metals and ammonium.
Structural units C contain chemical groups capable of binding or inactivating substances which are toxic to the gastrointestinal tract or which negatively influence inflammatory processes. Suitable hydrophilic monomers capable of binding and inactivating free radicals are selected from a group of sterically hindered amines and their oxidated derivatives of general formula:

X
R3 __________________________________ I
wherein Rl is ¨H or ¨OH or oxygen radical, R2 to R5 is (C1 - C4) alkyl, X is ¨CH(Y)¨ or ¨
CH(Y)CH2¨ and Y is a chemical group capable of undergoing radical polymerization:

5 H2C

wherein R is ¨H or ¨CH3 and Z is ¨0¨ or ¨NH¨, whereas the content of structural units C
in the polymer is at least 1 %, preferably from 3 to 30 mol.%.
Structural units C are preferably derived from N-(2,2,6,6-tetramethylazinan-4-y1) methacrylamide or N-(2.2,6,6-tetramethylazinan-4-y1) methacrylate.
Preferably, the polymer contains up to 98 % of units A, from 1 to 99 % of units B and at least 1 % of units C (in molar %).
Hydrophilic polymer according to the invention is prepared by radical polymerization of a mixture of precursors of structural units A, B and C.
Initiators are used to initiate the radical copolymerization. Suitable initiators of the radical polymerization, as well as suitable polymerization procedures and conditions, are generally known to a skilled person. For example, radical thermic initiators such as azoinitiators, diacylperoxides and other peroxocompounds, UV initiators generating free radicals by UV
irradiation or redox initiators, generating free radicals by oxidation-reduction reaction, can be suitable. Initiation does not have to be limited by the types of initiators mentioned above.
Polymers can be branched or crosslinked. Crosslinking reagents for the preparation of branched or crosslinked polymers can be, for example, ethylenedi(meth)acrylate, diglycol

6 and oligoglycol acrylates and methacrylates, ethylendiamindi(meth)acrylate, 1,1'-divinyl-3 ,3 '-(ethan-1,1'-diy1)bis(pyrrolidin-2-one), 2,3-dihydroxybutan-1,4-diy1 diacrylate or dimethacrylate, N,N'-methylenebisacrylamide or other crosslinkers commonly used in the field.
The hydrophilic polymer is, therefore, usually in the form of a high-molecular weight linear polymer, branched polymer or in the form of particles composed of covalently crosslinked gel. It is not absorbed by the gastrointestinal tract mucous membrane, neither does it enter the bloodstream, nor is it decomposed into low-molecular weight compounds which could be absorbed by the gastrointestinal tract. Non-absorbance of the polymer eliminates systemic contraindication. Moreover, the polymer is chemically neutral and does not undergo chain degradation in GIT juices.
The hydrophilic polymer is soluble or it has a high swelling ability in GIT
juices. The polymer, upon its contact with juices of gastrointestinal tract, gradually swells, which makes the active structural units C, incorporated in polymeric chains, accessible. Upon its gradual swelling, the polymer at the same time adheres to the gastrointestinal tract mucose because of the presence of structural units B, showing affinity towards mucose proteins.
The active substance gets therefore in contact with the afflicted site of the mucous membrane; therewithal, the time for which the polymer (together with its active substance) rests in the particular part of the gastrointestinal tract is prolongated due to the adhesion of the polymer to the mucose.
The preparation according to the present invention can preferably be in a form of a tablet (simple or multilayered, coated and non-coated, effervescent), capsule suitable for oral use.
syrup, solution, emulsion or suspension.
Besides the polymer according to the present invention, the preparation can further contain other pharmaceutically acceptable auxiliary substances, for example excipients for easier tablet making, commonly used in the field; acid and base components ensuring the effervescent way of administration; disintegration agents, e.g.
microcrystalline cellulose.
starches (corn, potato and modified starches). The content of disintegration agents in the preparation can reach up to 20 wt. %, preferably from 2 to 10 wt. %.

7 Sweeteners can be used as auxiliary substances in order to mask the bitter taste of active compounds or to enhance the sweet taste of the dosage form. Sweeteners, which can be used in the present invention, are selected from saccharides, such as monosaccharides or disaccharides (D-glucose, D-fructose, D-xylose, maltose, sucrose or sorbitol), polyols (glycerol, dulcitol, mannitol, sorbitol or xylitol), artificial sweeteners (saccharin and its sodium, potassium or calcium salts, cyclamate and its sodium or calcium salt, aspartame, acesulfame or their potassium salts). The content of sweeteners in the preparation can range from 2 to 60 wt. %, preferably from 5 to 10 wt. % (relative to the weight of the dosage form).
The auxiliary substances can include also aromatic compounds, which can be used in the present invention and which are not limited to the following examples: e.g., orange, lemon or grape flavour, peppermint flavour etc. The aromatic compounds can be used in the range of 0.01 to 5 wt. % (relative to the weight of the dosage form).
The polymer structure is designed to enable preferential concentration of the preparation close to the gastrointestinal tract mucose, e.g. utilising the adhesion of the preparation towards the mucose surface. The protective effect of the preparation towards the gastrointestinal tract mucose is enhanced by its adhesion to the mucose, the adjustment of the local pH in the mucose proximity or by protection of the mucose cells against harmful substances. The preparation includes chemical structures which specifically bind harmful substances of gastrointestinal tract juices, e.g. structures which bind and inactivate free radicals, structures adjusting pH of the gastric fluid by binding hydrochloric acid etc.
Functional structures specifically binding harmful substances are covalently incorporated into the material of the preparation. They are therefore prevented from releasing into the bloodstream, leading to potential adverse systemic effects. Preferably, such specific binding is via covalent bonding.
In comparison with JP 2012-111700 A, the present invention is a polymeric preparation composed of copolymers, in which different types of hydrophilic units (A, B, C) are statistically incorporated into the whole length of the polymeric chain. It ensures an even swelling of the polymer, which highly enhances the accessibility of the bound ROS

8 PCT/CZ2015/050011 antioxidant and enables molecules of the antioxidant to act in a close proximity of the mucose surface.
Another advantage of the present invention is the method of incorporating the molecules of antioxidants into the polymer by radical copolymerization, in which the antioxidant acts as comonomer. It simplifies the production of the preparation on industrial scale, which increases the industrial applicability of the invention.
Brief Description of Drawings Fig. 1: Time dependence of relative fluorescence intensity of the reaction mixture containing peroxyl free radicals and the polymer according to Example 4. The curve a corresponds to the reference sample without polymer, curves b, c, d, e correspond to samples containing 5 mg, 10 mg, 20 mg and 40 mg of the polymer.
Fig. 2: EPR spectrum of the polymer solution according to Example 5, after the reaction with in situ generated peroxyl free radicals.
Fig. 3: In vivo imaging of three mice 2 h, 5 h and 12 h after p.o. application of polymer solution labeled according to Example 8 (ca 50 MBq/mouse).
Fig. 4 Ex vivo biodistribution of the polymer according to Example 8 in DBA/2 mice; 2 h, 6 h and 24 h after the p.o. application, expressed as a percentage of the total dosage (%
ID).
Fig. 5 A part of a large intestine of a BALB/c mouse before (left) and after the exposure to the ABC polymer suspension (right) under UV light.
Fig. 6 Time dependence of the polymer swelling according to Example 14 in simulated GIT juices (stomach juice ¨ pH=1.6, small intestine juice ¨ pH=6.5, large intestine juice ¨
pH=7.0).
Fig. 7 Time dependence of the relative fluorescence intensity of the reaction mixture containing hydroxyl free radicals and the polymeric network according to Example 14. The continuous curve corresponds to a blank sample, other dependences belong to samples with polymer concentrations of 0.9 mg/mL (o). 4.5 mg/mL (A) and 9.0 mg/mL (0).
Fig. 8 Time dependence of the polymer swelling according to Example 17 in simulated GIT juices (stomach juice ¨ pH=1.6, small intestine juice ¨ pH=6.5, large intestine juice ¨
pH=7.0).

9 Fig. 9 Development of average weight of mice tested in individual groups in the course of the treatment described in Example 20.
Examples Example 1 Mixture of 0.2 g of N-(2,2,6,6-tetramethylazinan-4-y1) methacrylate and 6 g of N-viny1-2-pyrrolidone, 0.1 g of 1.1--diviny1-3,3--(ethan-1.1--diy1)bis(pyrrolidin-2-one) and 1.68 g of poly(ethyleneglycol) methyl ether methacrylate (average M. = 500) was dissolved in 120 ml of methanol. After an addition of 12 mg of 2,2'-azobis(2-methylpropionitrile), the mixture was heated under inert atmosphere to 55 C for 24 h. The resulting copolymer was extracted by ethanol and dried under vacuum. The powdered polymer has good swelling properties in water.
Example 2 Mixture of 0.35 g of N,N-(dimethylaminoethyl) methacrylate, 4.68 g of poly(ethyleneglycol) methyl ether methacrylate (average M. = 300). 1.0 g of N-(2,2,6,6-tetramethylazinan-4-y1) methacrylamide and 35 mg of 2.2 r-azobis(2-methylpropionitrile) was dissolved in 20 mL of tetrahydrofurane (THF). The solution was heated at 60 C for 24 h. After the polymerization was finished, the viscose solution was precipitated with diethylether and repeatedly precipitated with diethylether from a THF
solution. After drying, the resulting polymer has a good solubility in water.
Example 3 Mixture of 0.72 g of poly(ethyleneglycol) methyl ether methacrylate (average M. = 300).
1.8 g of N,N-(diethylaminopropyl) methacrylamide, 0.4 g of N-(2,2,6,6-tetramethylazinan-4-y1) methacrylamide and 100 mg of 2,2'-azobis(2-methylpropionitrile) was dissolved in 27 mL of toluene and heated at 70 C for 24 h. The resulting solution was partially evaporated, the polymer was precipitated with heptane and repeatedly precipitated with heptane from a THF solution. After drying, the resulting polymer was obtained in a powder form, having good solubility in water.
Example 4 Mixture of 2.00 g of 2-hydroxyethyl methacrylate, 1.84 g of poly(ethyleneglycol) methyl ether methacrylate (average Mn = 300), 2.1 g of N-(2,2,6,6-tetramethylazinan-4-y1) methacrylamide and 0.25 g of 2.2'-azobis(2-methylpropionitrile) was dissolved in 118 mL
of toluene and heated at 70 C for 24 h. The resulting polymer was filtered off and re-

10 precipitated with ether from methanol solution. The resulting polymer had a powder consistence and a good water solubility.
Example 5 Antioxidation ability of a solution of the linear polymer obtained in Example 4 was determined against in situ generated peroxyl radicals. The experiment was as follows:
Stock buffered solutions of the polymer at concentrations 8 mg/mL, 16 mg/mL, 32 mg/mL
and 64 mg/mL were prepared. In each vial, 650 tL of the polymer stock solution was placed together with 1.3 mL of fluorescein solution (2.4 x 10-5 M) and 1.95 mL
of AAPH
solution (0,16 M). Directly after addition of all components, a continuous decrease of fluorescence intensity was measured during 60 minutes at 37 C. The measurement was repeated twice for each polymer concentration. Fig. 1 shows that the polymer at the above mentioned conditions acts as a very efficient peroxyl radical scavenger.
Example 6 EPR spectrum of the polymer solution from Example 5 was measured after a reaction with peroxyl radicals (Fig. 2). EPR spectrum confirmed the presence of the oxidated form of a sterically hindered amine.
Example 7

11 0.17 g of N-(2,2,6,6-tetramethylazinan-4-y1) methacrylamide, 1.48 g of 2-hydroxyethyl methacrylate, 1.95 g of poly(ethyleneglycol) methyl ether methacrylate (average 1110 =
300), 36 mg of N-methacryloyl tyrosinamide (a precursor for radioisotope labeling) and 153 mg of 2,2'-azobis(2-methylpropionitrile) was dissolved in 20 mL of dioxane. After an addition of 70 mL of toluene the solution was heated at 70 C for 24 h. After a partial evaporation of solvents, the polymer was precipitated with diethylether. The precipitation was repeated several times from methanol solution to diethylether. The resulting polymer was dried under vacuum, and had a powder consistence and a good water solubility. The sample was purified using a desalting column.
Example 8 Polymer sample with incorporated tyrosinamide according to Example 7 was labeled with radioisotope 1311 according to the following procedure:
6 mg of the polymer was dissolved in 100 pL of phosphate buffer (PBS), followed by an addition of 50 pL, of Chloramine-T solution (20 mg chloramine / 1 mL PBS).
After 20 minutes of incubation, 260 pL of Na131I solution (corresponding to an activity of 420 MBq) and another 3 [t1_, of Chloramine-T solution were added. After 90 minutes of incubation, the reaction was ended by adding 20 pL of ascorbic acid solution (50 mg / 1 mL PBS), 50 pL of 2-amino-2-hydroxymethyl-propan-1,3-diole solution (100 mg /
1 mL
PBS) and 501..LL of freshly prepared NaBH4 solution (20 mg of hydride / 1 mL
PBS). High-molecular fractions expressing overall ca 89 % of the total radioactivity were lyophilised.
Example 9 5 mg of the lyophilised product from Example 8, having specific activity of 20 MBq/mg, was dissolved in 2.5 mL of saline. In order to image in vivo biodistribution, the solution of the labeled polymer was administered per orally (p.o.) to three Balb/c mice in a dose of cca 50 MBq/mouse. The imaging was performed on In vivo MS FX PRO for small animals. The experimental animals were under anesthesia during the whole time of the procedure. Static scanning was performed in time intervals of 2 h, 5 h and 12 h after polymer administration. Already 12 hours after polymer solution administration, almost all

12 activity was eliminated (Fig. 3). The experiment proved that the polymer is neither uptaken by a bloodstream, nor specifically absorbed and cumulated in a particular organ.
Example 10 5 mg of the lyophilised product from Example 8, having specific activity of 20 MBq/mg, was dissolved in 2.5 mL of saline. Ex vivo biodistribution of the polymer solution was monitored in three time intervals: 2 h, 6 h and 24 h after p.o. application.
The ex vivo biodistribution study of the radiolabeled polymer solution (dose ca 1 MBq/mouse) was performed p.o. to nine DBA/2 mice. The experimental animals were sacrified within the predetermined time limits (three animals per each time limit). The following organs were then taken away: blood, spleen, pancreas, stomach, intestines, kidneys, liver, heart, lungs, muscle, bone and thyroid. The radioactivity was measured in the organs using an automatic gamma counter. The results were expressed as percentages of the originally applied dose (% ID). The ex vivo biodistribution study confirmed a complete elimination of the polymer via the gastrointestinal tract (Fig. 4).
Example 11 Mixture of 3.0 g of N-(2,2,6,6-tetramethylazinan-4-y1) methacrylamide. 2.91 g of 2-hydroxyethylmethacrylate, 2.67 g of poly(ethyleneglycol) methyl ether methacrylate (average /14-11 = 300). 45 mg of 5-1[3-(methacryloylamino)propyl]thioureidyl }
fluoresceine, 18 mg of N,N--methylenebisacrylamide and 366 mg of 2.2'-azobis(2-methylpropionitrile) was dissolved in 30 mL of toluene and 3.5 mL of methanol. The polymerization was carried out for 24 h at the temperature of 70 C. The precipitated polymer (ABC) was filtered off, extracted by dioxane and dried into a constant weight.
Analogical procedure was used to prepare a polymeric sample (AC), which does not contain the structural unit based on poly(ethyleneglycol) methyl ether methacrylate.
Isolated parts of a large intestine of a male mouse BALB/c were exposed to suspensions of polymers ABC and AC (20 mg / 1 mL PBS; pH = 7.4) for 5 min. A strong fluorescence of the ABC polymer is observable under a UV lamp on the inner mucose of the exposed intestine. The fluorescence persists even after repeated washing with a buffer (Fig. 5). The

13 experiment proved markedly stronger mucoadhesive properties of the ABC polymer in comparison with the AC polymer, which does not contain the structural units B.
Example 12 Mixture of 2.6 g of poly(ethyleneglycol) methyl ether methacrylate (average Mn = 950).
0.8 g of 2-hydroxypropyl methacrylamide, 0.8 g of N-(2,2,6,6-tetramethylazinan-4-y1) methacrylate and 32 mg of 2,2'-azobis(2-methylpropionitrile) was dissolved in 28 mL of acetone and heated at 50 C for 24 h. The resulting polymer is water soluble.
Example 13 Mixture of 2.91 g of 2-hydroxyethyl methacrylate, 2.67 g of poly(ethyleneglycol) methacrylate (average Mt, = 300), 3.0 g of N-(2,2,6,6-tetramethylazinan-4-y1) methacrylamide, 18 mg of N,N--methylenebisacrylamide and 366 mg of 2,2'-azobis(2-methylpropionitrile) was dissolved in 90 mL toluene and heated at 70 C for 24 h. The polymer in a form of a powder precipitates during the reaction and has good swelling properties in water.
Example 14 Mixture of 0.78 g of N-(2,2,6,6-tetramethylazinan-4-ye methacrylate, 0.8 g of hydroxypropyl methacrylamide, 0.2 g of poly(ethyleneglycol) methacrylate (average M11 =
360) and 0.15 g of 2,2'-azobis(2-methylpropionitrile) was dissolved in 12 mL
of toluene and heated at 70 'V for 24 h. The resulting polymer was precipitated from methanol solution to diethylether. Dry polymer was in the form of a powder, soluble in water.
Example 15 2.54 g of N,N-(dimethylaminopropyl) methacrylamide, 2.0 g of N-(2,2,6,6-tetramethylazinan-4-ye methacrylamide, 1.78 g poly(ethyleneglycol) methyl ether methacrylate (average Mn = 300). 92 mg of N,N--methylenebisacrylamide and 0.24 g of 2,2'-azobis(2-methylpropionitrile) were heated in a mixture of 20 mL of dioxane and 40

14 mL of heptane at 70 C for 24 h. The resulting polymer network was filtered off, several times extracted with heptane and dried under vacuum.
Example 16 Swelling properties of the polymer network prepared in Example 15 were determined at the temperature of 37 C in a solution simulating gastrointestinal juices. 0.2 g of the polymer was placed into a centrifuge tube together with the above mentioned juice, giving the overall mass of 10 g (including the tube). The tube was placed into a bath temperated to 37 'V for the time listed in Fig. 6. The contents of the tube were centrifuged, the upper layer was taken off and the tube was weighted. The degree of swelling was calculated using the following formula:
swelling (%) = 100 x (mt ¨ 'no)/'no wherein mt is the mass of the swollen polymer in time t, and mo is the mass of the dry polymer before the experiment. The polymer network shows from 800 to 1300% of swelling in a wide range of pH of the simulated GIT juices (pH = 1.6 to 7.0;
Fig. 6).
Example 17 Antioxidation capacity against in situ generated hydroxyl free radicals in solution of the polymer network prepared in Example 15 was determined, according to the following procedure. Eight Eppendorf tubes containing appropriate amounts of the polymer were placed into a thermoblock and tempered to 37 'C. Then 900 [IL of a buffer, 200 litL of Na2W04.2 FI70 solution in a buffer (7.3 mM), 10 0_, of fluorescein solution (2.4 x 10 M) were placed into each tube and the polymer was kept to swell in this mixture for 5 minutes.
Hydroxyl radical generation was started by an addition of 10 L of hydrogen peroxide solution. The tubes were shaken and tempered to 37 C during the whole time of the reaction. Then Eppendorf tubes were opened in predetermined time intervals.
Their content was filtered using a microne filter, and the fluorescence values of the clear solutions were determined. Each sample was measured twice. Fig. 7 shows that even very low concentrations of the polymer network effectively scavenge the in situ generated hydroxyl radicals.
Example 18 4.0 g of N,N-(dipropylaminoethyl) methacrylate, 1.76 g of N-(2,2,6.6-tetramethylazinan-4-yl) methacrylate, 0.3 g of 2-hydroxyethyl acrylate, 0.28 g of poly(ethyleneglycol) methyl ether methacrylate (average Mll = 300), 15 mg of ethylenedimethacrylate and 0.22 g of 2,2'-azobis(2-methylpropionitrile) were heated in a mixture of dioxane and heptane at 70 10 'V for 24 h. The resulting polymer network was filtered off, several times extracted with heptane and dried under vacuum.
Example 19

15 Swelling properties of the polymer network prepared in Example 18 were determined using the same procedure as in Example 16. The results of this experiment are shown in Fig. 8. The polymer network swells in the range of 1000 % to 1800 % in simulated GIT
juices (pH = 1.6 to 7.0).
Example 20 The therapeutic effect of the preparation prepared in Example 15 in the GIT
was performed with the Balb/C induced chronic colitis experimental model.
20 male Balb/C mice were divided into three groups:
K ¨ control group of healthy mice without colitis (n=4) N ¨ non-treated group with induced colitis (n=8) L ¨ treated group with induced colitis (n=8).
Testing was started by chronic colitis induction in the N and L groups:
experimental animals drank 3.5% sodium dextran sulfate (DSS) solution in the following scheme: 7 days DSS solution, 7 days water, 7 days DSS solution, 7 days water, 7 days DSS
solution; then treatment was started in the L group. The tested preparation was administered to the animals of the L group twice per day by stomach probe (2x250 jiL 2% solution) for 7 days.

16 The mice were regularly weighted during the experiment (weight loss is a disease indicator, weight gain is an indicator of therapeutic effect). The day after the last therapeutic dose, dissection was performed, colons were taken away and their length was measured (shortening of the colon is an indicator of disease, its lengthening is an indicator of therapeutic effect).
Fig. 9 shows the comparison of average weight of mice in individual groups during the course of the treatment. There is a clearly visible difference between the N
group and the L
group which shows slowing down (or even reversing) of the pathological process in the L
group.
The results based on the colon length are in good agreement with the weight-based results, which demonstrate slowing down or even reversing of the pathological process in the large intestine. Healthy animals showed an average colon length of almost 10 cm, the non-treated (N) group animals showed an average colon length of about half of this value. The treated (L) group showed more than 20% extension of the colon length in comparison to the non-treated (N) group.

Claims

17

1. A preparation for oral administration for prevention and/or treatment of inflammatory conditions of gastrointestinal tract, characterized in that it contains a mucoadhesive, biocompatible hydrophilic polymer containing:
- at least one type of structural units A selected from the group consisting of units derived from acrylic and methacrylic acids and their salts, hydroxyalkyl acrylates and methacrylates, hydroxyalkyl acrylamides and methacrylamides, N,N-dialkylaminoalkyl acrylates and methacrylates, N,N-dialkylaminoalkyl acrylamides and methacrylamides, N-vinyl-2-pyrrolidone and mixtures thereof;
- at least one type of structural units B selected from the group consisting of units derived from hydroxy terminated (polyoxyethylene) acrylates and methacrylates, alkyloxy terminated (polyoxyethylene) acrylates and methacrylates, hydroxy terminated (polyoxyethylene) acrylamides and methacrylamides, alkyloxy terminated (polyoxyethylene) acrylamides and methacrylamides and mixtures thereof; and - at least one type of structural units C derived from monomers of general formula:
wherein R1 is ¨H or ¨OH or oxygen radical, R2 to R5 is (C1 - C4) alkyl, X is ¨CH(Y)¨ or ¨
CH(Y)CH2¨ and Y is a chemical group capable of undergoing radical polymerization:
wherein R is ¨H or ¨CH3 and Z is ¨O¨ or ¨NH¨.

2. The preparation according to claim 1, characterized in that the polymer contains up to 98 mol.
% of units A, from 1 to 99 mol. % of units B, and at least 1 mol. % of units C.

3. The preparation according to claim 1 or 2, characterized in that structural units C are derived from N-(2,2,6,6-tetramethylazinan-4-yl) methacrylamide or N-(2,2,6,6-tetramethylazinan-4-yl) methacrylate.

4. The preparation according to any one of claims 1 to 3, characterized in that the polymer is in the form of a linear polymer, a branched polymer or a covalently crosslinked polymer.

5. The preparation according to any one of claims 1 to 4, characterized in that it further contains pharmaceutically acceptable auxiliary substances selected from the group consisting of excipients for easier tablet making, effervescent acid and base components, disintegration agents, sweeteners and aromatic compounds.

6. The preparation according to any one of claims 1 to 5, characterized in that it is in the form of a tablet, a capsule, syrup, solution, emulsion or suspension.

7. Use of the preparation as defined in any one of claims 1 to 6, for the manufacture of a medicament useful for the prevention and/or treatment of inflammatory conditions of the gastrointestinal tract.