CZ349499A3 - Pharmaceutical preparation intended for oral administration, process of its preparation and use - Google Patents

Abstract

The proposed solution describes disintegrating preparations a gel skeleton containing the protein and its use for oral administration administering bioactive agents in the active form to the intestine vertebrate.

Description

ADMINISTRATION, METHOD

TECHNICAL FIELD

The present invention provides compositions comprising a disintegrating gel skeleton containing a protein and its use for oral administration of bioactive agents in active form to the intestine of a vertebrate.

BACKGROUND OF THE INVENTION

Two major problems remain in the development of compositions for the oral administration of insulin-like protein substances. The first problem is inactivation of most proteins by digestive enzymes in the gastrointestinal tract (GI), especially in the stomach. This can be prevented by means of a suitable carrier which will protect the protein from the adverse environment in the stomach before the substance is released in a more convenient environment of the gastrointestinal tract, particularly in the more distal regions of the intestine. In addition, protease inhibitors may be used to suppress the effect of enzymes present in the gastrointestinal tract that could inactivate the orally administered protein. Another problem is the very slow transport of large whole protein molecules through the intestinal walls into the bloodstream. Experts are trying to overcome this barrier by adding absorption enhancers that improve the passage of macromolecules across the barrier. Unfortunately, the transport structures available so far lack sufficient efficiency. Of course, a transport system that is sufficiently efficient and relatively affordable would be highly desirable.

SUMMARY OF THE INVENTION

The present invention provides a composition comprising a hydrogel carrier and a bioactive component, and the use of the composition to deliver the active agent in the active form to the intestine. A preferred hydrogel carrier is a network formed by crosslinking poly (methacrylate-g-ethylene glycol) with tetraethylene glycol dimethacrylate, "P (MAA-g-EG) hydrogels", which exhibit pH-dependent disintegration due to the presence of acid side groups and interpolymer complex formation between the ether groups of the attached chains and the protonated side groups. In acidic environments, these systems are substantially free due to the formation of interpolymer chains. In basic solution, the side groups are ionized and the complex dissociates. The pH-dependent disintegration of these hydrogels along with their bioadhesive properties make these hydrogels an ideal carrier for oral administration of proteins.

•

The present invention provides compositions for delivering biologically active proteins and drugs in mammals after oral administration.

The term bioactive component as used herein describes any substance that has an effect on living cells, for example, agents that induce biochemical effects in cells. In accordance with one variant of the present invention, an orally administered composition comprising a disintegrating gel skeleton and a labile protein contained within the hydrogel skeleton. The term labile protein as used herein describes any protein whose biological activity is destroyed or disrupted upon exposure to the protein at low pH. , or its exposure to enzymes present in the gastrointestinal tract of warm-blooded animal species.

Hydrogels disintegrate in water, which is well known to those skilled in the art for cross-linked polymer matrices. See, e.g., Dresback, U.S. Pat. Ptent No. No. 4,220,152, issued Sep. 2, 1980, which is incorporated herein by reference.

Hydrogels have been found to be an effective system for oral administration in vertebrates. The disintegration properties of the hydrogels can be used to protect the hydrogel content against adverse environmental effects in the stomach while the preparation passes through the gastrointestinal tract, and then the hydrogel content is released in the more favorable gastrointestinal tract environment, particularly in the more distal parts of the intestine. It has been found that the hydrogel composition of the present invention passes through the stomach without substantial disintegration and, when it arrives in the small intestine, disintegrates to allow release of the contents.

The hydrogels may be impregnated or filled with a variety of bioactive components, such as pharmaceuticals, growth factors, vaccines, vitamins, steroids and peptides, and may be used as vehicles for orally administered bioactive components. These compounds enclosed in the hydrogel can then be released in a controlled manner depending on the rate of hydration of the hydrogel within the digestive tract of the animals. In one variation of the present invention, the hydrogel backbone is in the form of pellets containing polymethacrylic acid, wherein long poly chain polymers such as polyethylene glycol (PEG) are attached to the polymethacrylic acid polymers.

The hydrogel pellets are synthesized primarily by the polymerization of methacrylic acid in the presence of a cross-linking component. The component may be a variety of biocompatible agents known to one of ordinary skill in the art, for example, tetraethylene glycol dimethacrylate, ethylene dimethacrylate, diethylene dimethacrylate, triethylene dimethacrylate, tetraethylene dimethacrylate, pentaethylene dimethacrylate, star methacrylate-containing polyacrylates, or corresponding diacrylates or methylene bis-acrylamido groups. Polymerization is • ·

initiated by a free radical initiator, such as an organic peroxide type thermal initiator or a UV radical initiator known to those skilled in the art.

In one variation, the hydrogel backbone comprises a copolymer of methacrylic acid and a polyalkylene glycol monomethacrylate (or monoacrylate) crosslinked with a biocompatible agent. The term "polyalkylene glycol monomethacrylate" as used herein describes poly (ethylene glycol) monomethacrylate, poly (propylene glycol) monomethacrylate, and poly (ethylene / propylene glycol) monomethacrylate, wherein poly (ethylene / propylene glycol) monomethacrylate is a polymer formed by hydroxyl-initiated polymerization. methacrylate functional groups in a mixture of ethylene oxide and propylene oxide. The resulting poly (alkylene glycol) groups have a molecular weight of from 200 to 4000, preferably from 200 to 2000, and in one variant, from about 200 to about 1200. The molar ratio of methacrylic acid to polyalkylene glycol monomethacrylate (or monoacrylate) monomers is from about 4: 1 to about 1: 4.

In one preferred variant, the hydrogel backbone comprises a polymer composed of methacrylic acid and poly (ethylene glycol) monomethacrylate crosslinked by tetraethylene glycol dimethacrylate, "P (MAA-g-EG) hydrogels". In preparing these polymers, the poly (ethylene glycol) monomethacrylate will have a molecular weight of from about 200 to about 2000, preferably from about 200 to about 1200, and will copolymerize with tetraethylene glycol dimethacrylate. The molar ratio of methacrylic acid to poly (ethylene glycol) monomethacrylate will range from about 4: 1 to about 1: 4. In one variation of the present invention, the molar ratio of methacrylic acid to poly (ethylene glycol) monomethacrylate is 1: 1. The crosslinking agent is added in an amount of from about 0.25 to about 10.00 mol%, preferably from about 0.25 to about 1.00 mol%, and in one variation is about 0.75 mol%.

The hydrogels may be loaded with the desired materials using standard techniques known to those skilled in the art. In one variation, the P (MAA-g-EG) hydrogels are produced in the form of microparticles having a diameter in the range of 50 μιη to about 500 μιη, preferably in the range of about 100 to 200 μιη. Polymer microparticles are produced according to one variant of the invention by forming a polymer backbone and comminuting it into hydrogel particles of the desired size. The hydrogel particles may be filled with the desired substances and enclosed in a standard tablet or capsule using standard techniques known in the art. In one variation, the hydrogel particles are enclosed in a gelatin capsule.

According to one variant of the present invention, the hydrogels are filled with a bioactive substance so that it is evenly distributed. More specifically, the hydrogels are hydrated in a solution of pH> 5.8 which contains the substance to be enclosed in the hydrogel. The hydrogels are then collected and washed with a solution of pH <5.8 and the filled hydrogels are dried and stored at 4 ° C. Next ·· ···· · · ·

I · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · The method of filling the hydrogels of the present invention is to gradually add an aqueous solution of the desired substance to the monomer solution and the crosslinking agent and then initiate the polymerization of the mixture. The ability of substances to diffuse through the polymer network depends on the degree of gel release and the size of the substance. As the hydrogel disintegrates, the polymer chains between the crosslinked points stretch and the mesh size in the mesh, or the corresponding length ξ, increases, allowing greater permeability of the dissolved material (see Fig. 1). In a balanced complex network, the rate of release is strongly dependent on the pH of the environment.

The P (MAA-g-EG) hydrogels of the present invention create temporary physical cross-linking when exposed to an acidic environment (typically pH < 5.8) due to hydrogen bridges formed between polymethacrylate groups and free poly (alkylene glycol) groups. These physical bonds are reversible by nature and depend on the pH and ionic strength of the environment. Thus, the degree of cross-linking and mesh size ξ is strongly dependent on the pH and ionic strength of the environment. In an acidic environment, these systems are essentially compact due to the formation of intermacromolecular complexes. In basic solution, the free groups ionize and dissociate the complexes. The disaggregation equilibrium of P (MAA-g-EG) hydrogels is shown in FIG. 2. Data are presented as the polymer volume fraction in hydrogels (PEG grafts 1000 MW and MAA: EG molar ratio 1: 1) as a function of pH. In the case of equimolar amounts of MAA and EG at low pHs, the degree of complexing was high and the fraction of polymer volume in the hydrogel in the dissolved state V20 was at least 0.70. Thus, as the pH of the release solution rose above pH-4.6, the complexes began to dissociate and the backbone began to expand, resulting in a significant reduction in the polymer volume fraction in the gel. Highly loose, uncomplexed hydrogels contain less than 5% of the polymer once more water is incorporated into the structure.

Due to the phenomenon of complex formation and decomposition of P (MAA-g-EG) hydrogels, the mesh size in the structure varies considerably as a function of pH. Moreover, samples of hydrogels with small deformations (less than 10%) were obtained in solutions of different pH. Using these data, it is possible to calculate the mesh size as a function of pH by determining the ends of the polymer chains between the crosslinked points, both covalent and physical. The average mesh size or the corresponding length is dramatically affected by the pH of the disintegrating solution (Fig. 3). At low pH when complexation occurs, the mesh size for P (MAA-g-EG) hydrogels is about 70 Å. With increasing pH, the physical bonds dissociate and the polymer chain stretches, resulting in an increase in mesh size up to three times to almost 210 Å. More importantly, assuming the mesh is ideal, the space available for diffusion corresponds to a square of mesh size. Thus, there is 9 times greater diffusion space in uncompleted hydrogels (pH greater than 5.2) than in complex hydrogels (pH less than 5.2). Due to the reversible nature of the complexing process, P (MAA-g-EG) hydrogels are ideal for oscillating drug release. In addition, due to large changes in the structure of the network at a small pH change, these materials can serve as very suitable carriers for peptides and proteins. In particular, the P (MAA-g-EG) hydrogels of the present invention can serve as carriers for substances having a molecular weight in the range of 1,000 to 100,000, more preferably in the range of 1,000 to 20,000.

An important parameter for evaluating the potential of a gel as a carrier for a particular drug is the ratio of effective molecular size (hydrodynamic diameter, dh) to mesh size. In order to study the permeability characteristics of these networks, the release of two substances of different molecular sizes, proxyphylin (molar mass 238 and dh = 4.3 Å) and vitamin Bi ₂ (molar mass 1355 and dh 17 17 Å) from complexed and uncomplexed hydrogels ( Fig. 4). In solutions of pH = 3.2, hydrogel polymers are highly complexed and drug release is greatly inhibited. From the polymer were released after 2 hours less than 10% of vitamin B] _{the second} Due to the smaller size, more than 30% of the proxyphylin has been released from the polymer in the same time. Once the gels have come into contact with a solution of pH = 7.4, the interchain hydrogel complexes dissociate due to ionization of adjacent acid groups. The result is a high degree of loosening of the hydrogel leading to a massive diffusion of vitamin B] ₂ and proxyphylinu polymer.

Data obtained from release studies were analyzed by short-term approximation from the source of classical Fickian expression for planar systems and the diffusion coefficient was calculated for the diffusion of proxyphylin and vitamin B ₂ through complexed and uncomplexed hydrogels (Table 1). Transport of larger molecules of vitamin B ₂ was much more influenced by complexation than proxyphylin due to the larger diameter of the dissolved molecules due to the mesh size of the hydrogel network. The diffusion coefficient of vitamin Bi ₂ from uncomplexed hydrogels was two orders of magnitude higher than that of complex hydrogels, whereas the diffusion coefficient of proxyphylin was only one order higher when diffused from hydrogels complexed to uncomplexed hydrogels.

Table 1. Diffusion coefficient of proxyphylin and vitamin Bi ₂ from complexed and uncomplexed P (MAA-g-EG) hydrogels.

molecule pH or A) dh / č D3.12xl0 ⁸ cm ² / s) proxyphylin 3.2 70.8 0,060 0.403 proxyphylin 7.4 194.4 0,022 9.38 Vitamin Bj ₂ 3.2 70.8 0.240 0.0168 Vitamin Bi ₂ 7.4 194.4 0,084 6.75

• 9 · 9 · · · · · · · · · · · · · · · · · · · · · · · · · · ·

6 · · · · · · ·

9 9 9

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To further investigate the ability of P (MAA-g-EG) hydrogels to function as carriers for oral delivery of vitamins, drugs, and other bioactive agents, sustained release of various agents was tested under conditions simulating the gastrointestinal tract environment. In vitro release experiments were performed with theophylin (MH = 180.2), vancomycin (MH = 1485.7) and insulin (MH = 5733.2) see Example 1. Each of these was uniformly filled into P (MAA-g). -EG) hydrogels, which were then immersed in 200 ml of liquid at pH = 1.2, simulating gastric fluid for 2 hours. The polymer microparticles were then transferred to a phosphate-saline solution at pH = 6.8. Concentrations of insulin released to the environment were monitored by HPLC and the results of theophylin, vancomycin and insulin release are shown in Figures 5a to 5c.

The release of these substances from hydrogels is considerably reduced in acidic environments (see Figure 2, first two hours). In contrast, a rapid release of these substances is evident in a solution of pH = 6.8. This trend is more noticeable with increasing molecular weight. For example, P (MAA-g-EG) hydrogels are an efficient insulin delivery system (MH = 5733.2). In the liquid simulating gastric fluid (pH = 1.2), less than 10% of the insulin was released from the polymer during the first phase of the experiment. Once polymer microparticles were transferred to a buffered solution at pH =

7.4, the hydrogel immediately relaxed, allowing massive insulin washout. These conclusions demonstrate that P (MAA-g-EG) hydrogels are useful for developing delivery systems for oral insulin delivery. As used herein, the term insulin includes purified human and natural animal insulin, as well as its derivatives, such as insulin lispro and recombinant forms of insulin and mono or divalent salts of insulin or insulin derivatives.

In addition, P (MAA-g-EG) hydrogels exhibit strong mucoadhesive properties due to the presence of fused PEG chains which serve as initiators of adhesion. These mucoadhesive properties of P (MAA-g-EG) hydrogels are strongly pH dependent (see Fig. 6). The adhesive characteristics between the gel and mucosa are considerably stronger under conditions simulating the intestinal environment (at pH = 7.4), relative to the conditions simulating the stomach environment. For a realistic comparison of the mucoadhesive properties of gels, the rate of adhesion was recalculated to the polymeric gel fraction. The recalculated rate of adhesion was two orders of magnitude higher for hydrogels in the uncomplexed state. This suggests that the mucoadhesive properties of the P (MAA-g-EG) hydrogels will be relatively weak as the hydrogels pass through the stomach and remain complex. Once they enter the intestine, the interchain complexes disintegrate, thereby increasing the adhesion of hydrogels to the intestinal mucosa relative to the gastric mucosa several times. Thus, the residence time of hydrogel carriers is much higher in an environment where insulin can be absorbed (i.e., in the intestine) after oral administration of hydrogels in mammals.

* · · Μ

9 9 ·· · · 9 9 9 9 • 9 9 · · 9 9 9 9 - 99 99 999 99999999/999999 99

99,999,999 «9,99

The differences in the hydrogel adhesion characteristics at different pH values are due to the mobility of the PEG chains in each material. In a highly relaxed, uncomplexed state, the attached PEG chains are free and capable of anchoring in the mucosa where they fold as anchors for adhesion. In the complexed state of the PEGu chain in P (MAA-g-EG) hydrogels, it forms complexes with carrier chains and is therefore unavailable for interaction with the mucosal surface.

In accordance with the present invention, hydrogel-containing formulations may be used to administer therapeutically effective amounts of a protein to a mammal. The method of administration comprises oral administration of a composition comprising a protein enclosed in a hydrogel carrier to a mammal. The therapeutic composition enclosed in the hydrogel carrier may further comprise protease inhibitors, pharmaceutically acceptable carriers, stabilizing agents, and biocompatible fillers, well known to those skilled in the art.

A preferred hydrogel carrier is P (MAA-g-EG), and in one particular variant, the P (MAA-g-EG) hydrogel comprises a pharmaceutically acceptable insulin-containing composition. In addition, according to one particular variant, the insulin preparation may comprise protease inhibitors or absorption enhancers. The formulation containing insulin encapsulated in P (MAA-g-EG) hydrogels showed surprisingly good potential for transporting insulin to the bloodstream of animals (see Examples 3 and 4). The hydrogel carrier is typically formulated in a particular form and rendered suitable for oral administration (eg, tablet, capsule, etc.) using techniques known to one of ordinary skill in the art.

In one particular variation, the microparticle delivery systems comprise interconnected copolymers of polymethacrylic acid and polyethylene glycol and are filled with insulin. This system is essentially effective because the structure of the copolymers exhibits pH-dependent disintegration and thereby protects the insulin as it passes through the adverse environment of the stomach. The PEG side groups also serve as adhesion promoters thereby increasing the resident time of the hydrogel carrier at the desired site. As shown in Example 2, the mucoadhesive properties of hydrogels are strongly influenced by the pH thereby improving adhesion to the mucosa in the intestine rather than in the stomach. The side groups of the PEG polymer also serve as peptide stabilizers and help maintain the biological activity of bioactive substances such as insulin.

The formation of interchain complexes in hydrogel copolymers is sensitive to the nature and pH of the environment as well as the composition of the copolymers and the chain length. In the acidic environment of the stomach, hydrogels are in a complexed state due to the formation of interchain complexes stabilized by hydrogen bonds between the carboxylic acid protons and the ether group of the chains. Under these conditions, substances with a molecular weight of at least 1000 (eg insulin) cannot essentially pass from the hydrogels due to the small mesh size, ξ, and thus they are protected from

9999 • · · ·····

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99 999 999 99 99 adverse stomach environment. As the particles pass through the stomach and enter the intestine, the pH of the environment rises above the transient pH of the gel. The complexes disintegrate immediately and the mesh size of the hydrogel network increases rapidly resulting in the release of substances with a molar mass of less than 100,000. It follows that P (MAA-g-EG) hydrogels can be used as efficient delivery systems for oral delivery of a molecular weight in the range of about 1000 to about 100,000.

BRIEF DESCRIPTION OF THE DRAWINGS

Giant. 1 Reversible complexing in P (MAA-g-EG) hydrogels. C represents the captured bioactive component.

Giant. 2 Corresponding volume of polymer fraction as a function of pH for samples containing PEG 1000 MV grafts and a 1: 1 MAA: EG ratio at 37 ° C.

Giant. 3 Corresponding mesh size as a function of pH for samples containing PEG 1000 MV grafts and a 1: 1 MAA: EG ratio at 37 ° C.

Giant. 4 Controlled cleavage proxyphylinu in solution at pH 3.2 (e) and 7.4 (), and vitamin B _I2 at pH 3.2 (o) 7.4 (o) in buffered saline at 37 ° C.

Giant. 5a Gradual release of theophyllin in-vitro from P (MAA-g-EG) hydrogel at 37 ° C.

Giant. 5b Gradual release of vancomycin in-vitro from P (MAA-g-EG) hydrogel at 37 ° C.

Giant. 5c Gradual release of insulin in-vitro from P (MAA-g-EG) hydrogel at 37 ° C.

Fig. 6 Adhesion behavior of the P (MAA-g-EG) hydrogel containing 1: 1 MAA / EG and PEG chains of 1000 molecular weight chains at pH 3.2 and 7.4 in contact with bovine mucin from the sub-jaw glands.

Giant. 7 Blood glucose concentration in rats after oral administration of P (MAA-g-EG) hydrogel containing insulin of 25 U / kg (o) and 50 U / kg (e) and insulin solution of 50 U / kg () (N = 5 ).

Giant. 8 Blood glucose concentration in rats after oral administration of 25 U / kg insulin-containing P (MAA-g-EG) hydrogel without aprotinin (o) and with aprotinin (♦) and insulin solution 50 U / kg () (N = 5) .

Giant. Blood glucose reactions in healthy (·) and diabetic (o) male Wistar rats following oral administration of P (MAA-g-EG) microspheres (25 IU / kg body weight insulin) using gelatin capsules.

Giant. Blood glucose reactions of diabetic male rats following oral administration of P (MAA-g-EG) microspheres (25 IU / kg body weight) using Eudragit capsules.

· · • »....«.

• ·· ·

• 9

Giant. Blood glucose response in healthy dogs (25 kg) following oral administration of P (MAA-g-EG) microspheres (25 IU / kg insulin dose relative to body weight) using gelatin capsules.

Giant. 12 Blood glucose response in diabetic dogs (25 kg) following oral administration of P (MAA-g-EG) microspheres (25 IU / kg body weight insulin) using gelatin capsules.

EXAMPLES OF EMBODIMENTS OF THE INVENTION

EXAMPLE 1. Release of P (MAA-g-EG) hydrogel content as a function of pH

The ability of the P (MAA-g-EG) hydrogel to function as a carrier has been tested for three substances with different molecular weights: theophylin (MH = 180.2), vancomycin (MH = 1485.7) and insulin (MH 5733.2).

P (MAA-g-EG) hydrogels were prepared at 37 ° C by polymerization of methacrylic acid and polyethylene glycol methacrylate in a non-radical solution, and the oligomer chains were cross-linked with tetraethylene glycol dimethacrylate. The resulting hydrogels were washed for one week in deionized water to remove unreacted monomer and uncrosslinked oligomeric chain, dried under vacuum to a powder with a particle diameter ranging from 100 to 150 µm. Drug incorporation experiments were tested for: theophylin (MH = 180.2), vancomycin (MH = 1485.7) and insulin (MH = 5733.2). Each drug was dissolved in phosphate buffer pH = 7.4 and P (MAA-g-EG) hydrogels were added to this solution to be evenly filled with the desired drug. The hydrogel was then transferred to an acidic medium to form interpolymer complexes and thereby reduce the mesh size of the hydrogel. The hydrogel microparticles were isolated by filtration and dried under vacuum. Incorporation efficiency was then calculated from the remainder of the drug in the solution remaining after microparticle filtration, relative to the original concentration, and was determined by HPLC.

Drug release experiments were performed according to the Japanese Pharmacopoeia (JP) method. The formulation was stirred with a stirrer at 100 rpm at 37 ° C in the first (pH = 1.2) and second (pH = 6.8) baths according to JP. After two hours of incubation in the first bath, polymer samples were collected by filtration and transferred to a second bath at pH = 6.8. Drug concentration was measured by HPLC. Drug release experiments were performed according to the Japanese Pharmacopoeia (JP) method. The formulation was stirred with a stirrer at 100 rpm at 37 ° C in the first (pH-1.2) and second (pH = 6.8) bath according to JP. After two hours of incubation in the first bath, polymer samples were collected by filtration and transferred to a second bath at pH = 6.8. Drug concentration was measured by HPLC.

The average incorporation efficiency of insulin into the hydrogel reaches 94% 30 minutes after the start of the experiment, indicating that this polymer is a suitable carrier for insulin. The results of theophylline, vancomycin and insulin release experiments from the P (MAA-g-EG) hydrogel are shown in Fig. 4. 5a, 5b and 5c. The release of these compounds from the hydrogel is reduced in an acidic environment (see first two hours of release). The fastest release rate is in a buffered solution of pH 6.8. This dependence is more evident for higher molecular weight substances, less than 10% of the insulin was released from the hydrogel in the simulated gastric fluid environment (pH = 1.3) during the first phase of the experiment. Upon transfer of the particles to a buffered solution of pH = 7.4, the hydrogels rapidly disintegrate to allow the release of insulin. These results indicate that P (MAA-g-EG) copolymers are useful for the development of carriers for oral administration.

EXAMPLE 2. In vitro mucoadhesion study

P (MAA-g-EG) hydrogels were prepared in the form of a thin film from solution by the polymerization technique. They were then evenly distributed in buffered DMGA at pH 3.2 and 7.4. The disintegrated hydrogels were cut into 20 cm diameter discs and placed in a tensile testing device at 25 ° C and 90% humidity. Polymer samples were fixed to the top tester holder using cyanoacrylate medical glue, while bovine submaxillary mucosa samples were placed on the bottom holder using the same glue. The tester jaws were clamped and the samples were in close contact for 15 minutes before the jaws were opened at 1 mm / min. The force required to detach was measured as a function of separation. The peel strength corresponding to the bioadhesion strength was calculated as the area under the curve.

P (MAA-g-EG) hydrogels function as insulin delivery devices for oral administration because they are able to suspend the effect of protease inhibitors and also because they adhere to the intestinal mucosa allowing close contact and improving drug absorption.

Once the insulin-containing hydrogels are placed in the intestinal fluid, the hydrogels are released almost immediately to allow insulin release. P (MAA-g-EG) microparticles were disintegrated for 1 hour in phosphate-saline and then transferred to intestinal fluid. Proteolysis of insulin in intestinal fluid was measured using an insulin EIA kit. More than 50% of the biological activity of insulin was maintained for more than 1 hour in the presence of proteolytic enzymes. In contrast, when only insulin is dissolved in the intestinal fluid, its biological activity is rapidly lost. P (MAA-g-EG) hydrogels protect insulin by binding calcium to ionized side groups, reducing the activity of proteolytic enzymes.

In addition, P (MAA-g-EG) hydrogels exhibit mucoadhesive characteristics due to the presence of PEG chains that serve as adhesion promoters. The mucoadhesive characteristics of the P (MAA-gEG) hydrogels are strongly dependent on the pH of the surrounding fluid (Fig. 6). The area under the curve in FIG. 6 corresponds to the adhesive force between the gel and the mucosa. Under the simulated intestinal pH (7.4) conditions, the adhesive force between the gel and the mucosa was significantly higher. For a real comparison of mucoadhesive • · · ·

Hydrogen characteristics, adhesion strength normalized to polymer gel fraction (see Table 2) Normalized adhesion rate was two orders of magnitude higher for uncomplexed hydrogels compared to complexed hydrogels. It follows that hydrogels adhere to the mucosa of the intestine to a much greater extent than to the gastric mucosa. Therefore, the resident time of insulin carriers is much longer in the area where insulin can be absorbed.

Table 2. Adhesion rate of P (MAA-g-EG) hydrogels containing 1: 1 MAA / EG and attached PEG chains of 1000 molecular weight.

pH adhesion rate fraction volume normalized adhesion rate

W 10 ⁶ (J) of polymer, Ο2 / 5_WZ (Q2 / 5) ^2/3 10 ⁶ (J)

3.2 5.38 0.693 62.1

7.4 9.34 0.049 6720

The differences in adhesion characteristics of hydrogels at different pH values are due to differences in the mobility of the PEG groups in each material. In a highly relaxed uncomplexed state, the PEG chains are free and able to penetrate the mucosa and can then serve as anchors for adhesion. In the complexed state of the PEG chain in P (MAA-g-EG) hydrogels, they form complexes with the backbone and cannot then adhere to the mucosa and form temporary anchors.

EXAMPLE 3. In vivo administration of insulin to rats

The copolymers were prepared by polymerizing methacrylic acid and polyethylene glycol methacrylate in a free radical solution. The resulting hydrogels were washed for one week in deionized water to remove unreacted monomer and un-amplified oligomer chains. The hydrogels were then dried under vacuum to a powder with a particle diameter ranging from 100 to 150 µm. Crystalline pig insulin (26.9 U / mg) was filled evenly into the gels. The drug loaded hydrogel particles were filtered and washed to remove drug residues from the surface, and then dried in vacuo.

Male Wistar rats (200 g) were kept without food for 24 hours. Then, the rats were fixed in a lying position and given insulin in polymeric microparticles using a gelatin capsule that dissolves in the stomach. Serum glucose was monitored by collecting 0.2 ml aliquots of blood samples from the jugular vessel before the experiment and 0.25, 0.5, 1, 2, 4, 6, and 8 hours after administration. Serum was collected by centrifugation at 3000 rpm for 3 minutes and frozen until analysis. Serum insulin levels were determined by enzymatic immunoassay using

• The insulin EIA kit. • • • • • • • • • • • • • • • • Serum glucose levels were determined by the glucose oxidase method using a glucose B-test kit.

Giant. 7 summarizes the rat glucose response to insulin administered in P (MAA-g-EG) microparticles. A strong hypoglycaemic effect (lowering blood glucose) was observed within 2 hours after administration of the polymeric drug form. The extent of this reduction in blood glucose depends strongly on the insulin dose. No reaction was observed in rats treated with insulin solution.

The effect of applying a formulation comprising P (MAA-g-EG) hydrogel gels containing insulin and the protease inhibitor aprotinin is shown in FIG. 8. A control group of rats received P (MAA-g-EG) hydrogel containing protease-free insulin (for comparison) and another group received an insulin solution of 50 U / kg (to serve as a control).

EXAMPLE 4. In vivo studies with diabetic rats and dogs

Diabetes was induced in healthy male Wistar rats by administration of straptozotocin. In healthy dogs, diabetes was induced by alloxan. P (MAA-g-EG) microspheres were prepared by polymerizing methacrylic acid and polyethylene glycol dimethacrylate (PEG MH = 1000) in a free radical suspension. Tetraethylene glycol dimethacrylate was added as a cross-linking agent. 2,2'-Azobis-isobutyronitrile (AIBN) was added in an amount of 0.5% of the total monomer as thermal initiator of the reaction.

P (MAA-g-EG) hydrogel microparticles were evenly filled with insulin. The bovine pancreatic insulin solution was dissolved in 200μ1 IN NaOH. The insulin solution was diluted with 20 ml of phosphate buffer (pH = 7.4) and normalized with 0.1 N NaOH. Dry P (MAA-g-EG) hydrogels were added to this solution for 24 hours. The particles were then filtered off and washed with 100 ml of a 0.1 N HCl solution to complex the hydrogels and remove the residual buffer. The insulin-filled microspheres were then dried under vacuum and stored at 4 ° C. The fill rate was measured by HPLC analysis of the insulin concentration in the original solution and in the filtrate after washing.

Prior to administration of insulin-loaded P (MAA-g-EG) hydrogels to Wistar rats (250 g), rats were kept free of food for 24 hours. Then, the rats were fixed in the supine position and were injected through the orifice with insulin in polymeric microparticles and control solution using a gelatin capsule and a capsule prepared with Eudragit L100. The gelatin capsule dissolves very quickly in the stomach, while the Eudragit capsule dissolves considerably more slowly.

During the experiment, rats were separated (four each) and fed with water. Samples of 0.2 ml aliquots of blood samples from the jugular vessel were taken before the experiment and 0.25, 0.5, 1, 2, 4, 6, and 8.

1J ······ · · · · · · · «·« · · hours after application. Blood serum was isolated by centrifugation at 3000 rpm for 3 minutes. Blood glucose level was determined by the glucose oxidase method using a glucose Btest kit.

Diabetic dogs (25 kg) were fasted for 24 hours prior to drug administration. The polymeric drug was administered using gelatin capsules. After application, the dogs were fed.

During the experiment, the dogs were housed and watered. Blood samples were collected by catheterization. The blood glucose level was determined by a portable glucose analyzer.

While many systems were effective in lowering blood glucose levels in healthy animals following administration of the insulin contained in the polymeric carrier, a similar result was not seen in diabetic animals. The blood glucose response in diabetic and healthy rats after administration of insulin-containing P microparticles (MAA-g-EG) using gelatin capsules (dose 25 IU / kg) is shown in Fig. 9. Blood glucose levels in diabetic rats were reduced to 40 % of the original level. The decreased blood glucose level remained for more than 8 hours, and the blood glucose lowering rate was higher in diabetic animals than in healthy animals. In addition, a strong hypoglycaemic effect was observed longer in diabetic animals. The blood glucose response of diabetic rats to oral administration of insulin-loaded Eudragit capsules loaded with P (MAA-g-EG) microparticles (dose 25 IU / kg) is shown in Figure 10. Blood glucose levels in these animals were reduced by more than 50% and remained reduced for at least 8 hours after administration. The microparticles encapsulated in the Eudragit capsule were far more effective than the gelatin capsules since the microparticles in the Eudragit capsules were exposed to the adverse environment of the upper gastrointestinal tract for a shorter time due to the slower dissolution of the Eudragit capsules. Blood glucose of healthy dogs was significantly reduced after oral administration of a single dose of polymeric drug (10 IU / kg dose). At time 0, the dogs were fed and the body's normal response is to maintain basal levels. However, after feeding, blood glucose levels decreased and for 2 hours after administration, blood glucose levels were reduced by more than 20% due to the penetration of insulin through the upper small intestine wall. In addition, the second reduction occurred about eight hours after administration, as previously observed in rats, probably due to the absorption of insulin in the colon. Blood glucose levels continued to decrease gradually after 8 hours, probably due to the absorption of insulin in the colon.

The blood glucose response in diabetic dogs also confirmed insulin uptake after oral administration. Blood glucose in diabetic dogs was also affected by oral administration of insulin-containing P (MAA-g-EG) hydrogels using gelatin capsules (dose 10 IU / kg). Subsequent feeding and administration of the polymer dosage form first rapidly increases blood glucose levels. Within one hour, glucose levels begin to stabilize as insulin is gradually absorbed. Levels) Blood glucose in diabetic dogs given a polymer dosage form is eventually reduced by 40% compared to dogs that have not received any insulin.

Insulin delivery systems must be able to protect the drug from adverse environmental effects in the stomach and deliver insulin in biologically active form over extended periods of time to areas with a more favorable environment for insulin absorption in the gastrointestinal tract, such as the upper intestine. Due to their properties, P (MAA-g-EG) hydrogels are ideal for this purpose. P (MAA-g-EG) hydrogels are capable of efficiently delivering biologically active insulin after oral administration. This drug has been shown to be effective in reducing blood glucose levels in diabetic dogs and rats and to maintain this level for more than 8 hours. These substances are effective because insulin enclosed in the hydrogel is not released until the hydrogel reaches the upper small intestine. Once it arrives there, it adheres immediately to the mucosa allowing for close contact between the carrier and the site of absorption. In addition, polymers serve to suppress the proteolytic activity of enzymes in the intestine, which is another factor extending the life of insulin in the intestine. The inhibitory effect of polymers on the function of enzymes is probably due to the ability of the polymer to form complexes between cations, such as calcium, which are necessary for the function of the enzymes.

Claims (17)

A pharmaceutical composition for oral administration comprising a disintegrating hydrogel carrier and a labile protein enclosed within a hydrogel carrier composed of a crosslinked copolymer of methacrylic acid and polyalkylene glycol monometactlate. A composition according to claim 1 wherein the polyalkylene glycol monomethacrylate is polyethylene glycol monomethacrylate. The composition of claim 1, wherein the hydrogel backbone is crosslinked with tetraethylene glycol dimethacrylate. The composition of claim 2, wherein the molar ratio of methacrylic acid to polyethylene glycol monomethacrylate is about 1: 1. The composition of claim 4, wherein the protein has a molecular weight of from about 1,000 to about 20,000. 6. A composition according to claim 4, wherein the protein is insulin. The composition of claim 4, wherein the polyethylene glycol monomethacrylate has a molecular weight of from about 200 to about 4,000. 8. A formulation according to claim 4, wherein the hydrogel is microparticles and enclosed in a capsule. The composition of claim 4, wherein the capsule is a gelatin capsule. 10. The composition of claim 4, further comprising a protease inhibitor. Composition for oral administration of insulin to mammals, characterized in that it comprises insulin encapsulated in a P (MAA-g-EG) hydrogel. 12. The composition of claim 11, further comprising a protease inhibitor. 13. The composition of claim 11, wherein the hydrogel is microparticles and enclosed in a capsule. The composition of claim 11, wherein the capsule is a gelatin capsule. · · · 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 15. A method of administering a therapeutically effective amount of a protein to a mammal comprising oral administration of said composition according to claim 1 to the mammal. 16. The method of claim 1, comprising the steps of polymerizing methacrylic acid and polyalkylene glycol dimethacrylate with a crosslinking agent to form a hydrogel carrier, transferring the hydrogel carrier to an aqueous protein solution, wherein the pH of the solution is greater than about 5.4. and finally lowering the pH of the solution to less than 5.4 and isolating the hydrogel carriers containing the protein. 17. The process of claim 16 wherein the polyalkylene glycol monomethacrylate is polyethylene glycol monomethacrylate.