KR20010005952A - Method for oral delivery of proteins - Google Patents

Abstract

A method and composition for administering a bioactive compound to an oral cavity to a vertebrate are described. The method consists in orally administering to the vertebrate a composition consisting of an expandable hydrogel matrix and a bioactive compound included in the hydrogel matrix.

Description

METHOD FOR ORAL DELIVERY OF PROTEINS

There are two main problems in transporting substances with proteins such as insulin to the oral system. First, most proteins are inactivated by the enzymes in the stomach, mostly in the gastrointestinal system (GI). This problem can be solved by designing a carrier that can protect the protein from the harmful environment of the stomach before the drug is released into the more beneficial environment of the GI tract, especially the lower region of the intestine. In addition, protease inhibitors capable of delaying the action of enzymes present in the GI system capable of decomposing orally administered proteins can be used. Another problem is the slow movement of large peptides across the bloodstream in the intestine. The researchers tried to get around these obstacles by adding absorption enhancers to help them cross these boundaries. However, currently used transport vehicles are less effective. Therefore, there is a need for an oral delivery system that can be made effective and at relatively low cost.

Summary of the Invention

The present invention relates to a composition consisting of a hydrogel matrix carrier and a bioactive compound, and to the use of such a composition to deliver an active form of the compound into the gut. One of the most preferred hydrogel matrices is a poly (methacrylic acid-g-ethyleneglycol) hybrid polymer structure crosslinked with tetraethylene glycol dimethacrylate, "P (MAA-g-EG) hydrogel", wherein the acidic pendant groups Since the interpolymer complex is formed between the graft chain and the pendant group to which the proton is added, it has a property of expanding according to pH. In acidic media such a system is relatively less inflated due to the interpolymer complex. In basic solutions, pendant groups are ionized and complexes decompose. These hydrogels expand in accordance with the pH of the hydrogel and their bioadsorption properties to create an ideal hydrogel that is used to transport proteins through the oral cavity.

The present invention relates to the use of such compositions for the delivery of an active bioactive compound to the oral cavity into a composition consisting of an expandable hydrogel matrix and the viscera of vertebrates.

1 shows reversible complex formation in P (MAA-g-EG) hydrogels. C represents the captured bioactive compound.

FIG. 2 shows a polymer volume aliquot at equilibrium as a function of pH for a sample comprising PEG graft (MW 1000) and MAA: EG (molar ratio 1: 1 at 37 ° C.).

3 shows the mesh size of the equilibrium as a function of pH for a sample comprising PEG graft (MW 1000) and MAA: EG (molar ratio 1: 1 at 37 ° C.).

4 shows pH 3.2 in a buffered salt solution at 37 ° C. ) And 7.4 ( Controlled release of proxiphylline in solution and pH 3.2 ( ) And 7.4 ( ) Is a controlled release of vitamin B ₁₂ .

5 shows in-vitro pulsatile release of theophylline from P (MAA-g-EG) hydrogel at 37 ° C.

6 shows in-vitro pulsatile release of vancomycin from P (MAA-g-EG) hydrogel at 37 ° C.

7 shows in-vitro pulsatile release of insulin from P (MAA-g-EG) hydrogel at 37 ° C.

FIG. 6 shows the adsorptive properties of P (MAA-g-EG) hydrogels comprising MAA / EG 1: 1 and graft PEG chains having a molecular weight of 1000 when contacted with bovine mandibular mucins at pH 3.2 and 7.4. .

7 is 25 U / kg ( ), 50 U / kg ( ), 50 U / kg ( Insulin dripping P (MAA-g-EG) hydrogel in the (N-5) insulin solution is administered to the oral cavity of mice, and then the blood glucose level is shown.

8 shows aprotinin ( ) And without aprotinin ) In the presence of 50U / kg ( ), 50 U / kg ( Insulin dripping P (MAA-g-EG) hydrogel in the (N-5) insulin solution is administered to the oral cavity of mice, and then the blood glucose level is shown.

9 shows healthy male Wistar mice after administration of P (MAA-g-EG) microspheres (25 U / kg insulin dose) into the oral cavity of mice using gelatin capsules. ) And diabetic Wistar mice ( In the blood glucose response.

Figure 10 shows the glycemic response in male mice with diabetes after administration of P (MAA-g-EG) microspheres (25 IU / kg) orally using Eudragit capsules.

FIG. 11 shows the blood glucose response in healthy dogs (25 kg) after administration of P (MAA-g-EG) microspheres (10 IU / kg insulin dose) to the oral cavity of mice using gelatin capsules.

FIG. 12 shows the blood glucose response in diabetic dogs (25 kg) after administration of P (MAA-g-EG) microspheres (10 IU / kg insulin dose) to the oral cavity of mice using gelatin capsules.

The present invention relates to compositions used to transport biologically active proteins and pharmaceutical substances after administration to the oral cavity of vertebrates. As used herein, a biologically active compound refers to any compound having an effect on living cells, for example, a compound capable of inducing a biochemical effect in a cell. In one embodiment, the compound administered orally consists of the expandable hydrogel complex and the labile protein included in the expandable hydrogel. Unstable proteins used herein are exposed to enzymes present in the digestive tract of warm-blooded animals or exposed to low pH to destroy or reduce the biological activity of the proteins.

Hydrogels are expanded in water and refer to crosslinked polymer matrices known in the art (see, Dresback, U.S. Patent No. 4,220,152; September 2, 1980). Hydrogels have been found to be effective delivery vehicles for delivering proteins to oral vertebrates. Using the expandable nature of the hydrogel, the composition first protects the hydrogel contents from the strong environment of the stomach as it passes into the digestive tract, and provides more favorable conditions in the GI organs, e.g. To be released. The hydrogel compositions of the present invention pass through the stomach without actual swelling, localize to the small intestine, swell in the small intestine, and release their contents.

Hydrogels can trap or drop various biologically active compounds, including, but not limited to, pharmaceutical compositions, growth hormones, vaccine compositions, vitamins, steroids, peptides, and the like. It is used as a delivery vehicle for administering the sexual compound to the oral cavity. When hydrated in the digestive system of an animal, the compound loaded on the hydrogel is controlled for release. In one embodiment, the hydrogel matrix of the present invention is in the form of pellets composed of polymethacrylic acid, wherein the polymethacrylic acid polymer is grafted with an ionic long chain polymer such as polyethylene glycol (PEG).

Hydrogel pellets are suitably synthesized by polymerizing with methacrylic acid in the presence of a crosslinking material. The crosslinker may be selected from a variety of biocompatible crosslinkers known in the art, for example tetraethylene glycol dimethacrylate, ethylene dimethacrylate, diethylene dimethacrylate, triethylene dimetha Acrylate, tetraethylene dimethacrylate, pentaethylene dimethacrylate, and corresponding diacrylate or methacrylate, star polymers composed of acrylate or methylene bis-acrylamido groups and the like. The polymerization starts with free radical initiators such as organic peroxides or UV radical initiators known in the art.

In one embodiment, the hydrogel matrix consists of methacrylic acid and poly (alkylene glycol) monomethacrylate (or monoacrylate) interpolymers crosslinked with biocompatible crosslinked materials. The "poly (alkylene glycol) monomethacrylate" as used herein includes poly (ethylene glycol) monomethacrylate, poly (propylene glycol) monomethacrylate, and poly (ethylene / propylene glycol) monomethacrylate. In this case, the poly (ethylene / propylene glycol) monomethacrylate is a polymer produced by a polymerization reaction in which a mixture of ethylene oxide and propylene oxide is initiated with hydroxy functional group methacrylate. The resulting pendant poly (alkylene glycol) groups have a molecular weight of about 200 to about 4000, more suitably about 200 to about 2000, and in one embodiment have about 200 to about 1200. The molar ratio of methacrylic acid and poly (alkylene glycol) monomethacrylate (or monoacrylate) is from about 4: 1 to about 1: 4.

In one suitable embodiment, the hydrogel matrix consists of methacrylic acid and poly (ethylene glycol) monomethacrylate, “P (MAA-g-EG) hydrogel” polymer, crosslinked with tetraethylene glycol dimethacrylate. do. In preparing the polymer, poly (ethylene glycol) monomethacrylates having a molecular weight of about 200 to about 2000, suitably about 200 to about 1200, are methacrylic acid and tetraethylene glycol dimethacrylate. The molar ratio of methacrylic acid and poly (ethylene glycol) monomethacrylate monomer is from about 4: 1 to about 1: 4. In one embodiment, the molar ratio of methacrylic acid and poly (ethylene glycol) monomethacrylate monomer is 1: 1. The crosslinker is added in an amount of about 0.25 to 10.00 mol%, more suitably about 0.25 to 1.00 mol%, and in one embodiment is about 0.75 mol%.

The desired compound can be loaded onto the hydrogel using standard techniques forgotten in the art. In one embodiment, the P (MAA-g-EG) hydrogel is formed from microspheres of about 50 μm to about 500 μm in diameter, suitably about 100-200 μm. Hydrogel microspheres are made according to one embodiment, making a polymerized matrix and grinding the matrix to produce hydrogel particles having a desired average particle size. The compound that oozes into the hydrogel microspheres is loaded and packaged into standard tablets or capsules using standard techniques known in the art. In one embodiment the hydrogel particles are packaged in gelatin capsules.

In one embodiment, the hydrogel is loaded with the bioactive compound by a balanced ratio. More specifically, the hydrogel is pH> 5.8 and hydrated with a solution containing the composition to be loaded. The hydrogel is then recovered, washed with a solution of pH <5.8, the loaded hydrogel is dried and stored at 4 ° C. Another method of dropping the hydrogel of the present invention consists in adding the desired aqueous solution of the compound to the monomer and crosslinker solution and initiating the polymerization of the mixture.

The ability of the compound to diffuse through the crosslinked polymer structure depends on the extent of gel expansion and the size of the compound. When the hydrogel expands, the polymer chain extends between the points of intersection, the network mesh size or the corresponding length,

When the P (MAA-g-EG) hydrogel of the present invention is exposed to acidic conditions (generally pH <5.8), a temporary physical crossover is due to hydrogen bonding between the polymethacrylate group and the pendant poly (alkylene glycol) group. A bond is formed. Such physical crosslinking is reversible in nature and depends on the ionic strength and pH of the environment. Thus, the degree of crosslinking and the network mesh size ε depends significantly on the pH and ionic strength of the surrounding environment. In acidic media, such systems have relatively less expansion because intramolecular complexes are formed. In basic solution, pendant groups are relaxed and complexes are dissolved. 2 shows the equilibrium expansion of P (MAA-g-EG). The data is presented as a polymer volume aliquot of hydrogel (PEG graft molecular weight 1000 MW, MAA: EG mole ratio 1: 1) according to pH. When the molar ratio of the same amount of MAA and EG at low pH is high, the degree of complex formation is high and the polymer volume aliquot on the gel in the expanded state ₂₀ is close to 0.70. However, when the pH of the expansion solution is increased to 4.6, the complex begins to decompose and the chain of the basic structure is extended, so that the equilibrium polymer volume aliquot in the gel is significantly reduced. Hydrogels that swell more and do not form complexes contain less than 5% of polymers because more water is bound to the structure.

Due to complex formation / complex separation phenomena in P (MAA: g-EG) gels, the mesh size of the network will change significantly if it exceeds the desired range of pH. In addition, modules for small variations (less than 10%) can be obtained in solutions of varying pH. Using this data, the mesh size is calculated as a function of pH by determining the end-to-end distance of the polymer chain between the nominal and physical crosslinking points. The average mesh size or length associated therewith is significantly affected by the pH of the expansion solution (FIG. 3). In the low pH solution where the complex is formed, the network mesh size of the P (MAA-g-EG) hydrogel is as low as 70 kPa. However, as the pH is increased, the physical crosslinks break down and the polymer chains extend, resulting in a threefold increase in the mesh size of the network to nearly 210 GPa. More importantly, given the ideal network, the area available for diffusion is equal to the mesh size. Therefore, the area is increased by 9 times or more in the case of diffusion in the hydrogel (pH is more than 5.2) than the complex formed hydrogel (pH is less than 5.2). Because of the natural phenomenon of forming a reversible complex, P (MAA-g-EG) hydrogels are ideal for vibrating the drug. In addition, even with slight changes in pH, there is a large change in network structure, so these substances function as carriers for peptides and proteins. In particular, the P (MAA-g-EG) hydrogel of the present invention may function as a carrier carrier of a compound having a molecular weight of about 1,000 to about 100,000, more specifically about 1,000 to 20,000.

In evaluating the gel's ability to function as a carrier for a particular drug, an important variable is the ratio of effective molecular size (fluid dynamic diameter, d _h ) to network mesh size. To study the size-extrusion characteristics of this network, two complexes of different molecular sizes, proxiphylline (molecular weight 238; d _h = 4.3 kPa) and vitamin B ₁₂ (molecular weight 1355; d _h = 17 kPa), The release of the formed hydrogel and the complex from the unformed hydrogel was studied (FIG. 4). In solutions with pH = 3.2, the hydrogel polymer complexed to a significant extent and drug transport was significantly hampered. However, because of the small molecular size, about 30% of proxiphylline is released from the gel at the same time. When the hydrogel is contacted with a solution of pH = 7.4, the interchain complexes of the hydrogel decompose due to the ionization of the pendant acid groups. As a result, the hydrogel has a significant expansion of the hydrogel, which actually diffuses vitamin B ₁₂ and proxiphylline from the polymer.

In a two-dimensional system, this release data is fixed in a short time approximation in a traditional Fickian solution, and the diffusion coefficient is proxiphylline and vitamin B through a complexed and uncomplexed P (MAA-g-EG) hydrogel. Can be calculated for a spread of ₁₂ (Table 1). When transporting a large molecular weight solute, vitamin B ₁₂ , the ratio of network mesh size to the diameter of the solute is increased, which is more affected by complex formation than proxiphylline. In the non-complexed hydrogel, the vitamin B ₁₂ diffusion constant is about 2 times higher than the diffusion constant of the complex-formed hydrogel, and the diffusion constant of proxiphylline is higher in the non-complexed hydrogel than in the complex-formed hydrogel. It is about 1 times the size.

Diffusion Constants for Proxiphylline and Vitamin B ₁₂ in Complexed and Non-complexed Hydrogels solute pH (Å) d _h / D _3.12 x 10 ⁸ (㎠ / s) Proxiphylline 3.2 70.8 0.060 0.403 Proxiphylline 7.4 194.4 0.022 9.38 Vitamin B ₁₂ 3.2 70.8 0.240 0.0168 Vitamin B ₁₂ 7.4 194.4 0.087 6.75

Conditions similar to gastrointestinal conditions to further investigate the ability of P (MAA-g-EG) hydrogels to function as oral transporters for vitamins, pharmaceutical compositions and other biologically active compounds. The pulsatile release of the various compounds under investigation was investigated. In vitro release experiments were performed using theophylline (MW = 180.2), vancomycin (MW = 1485.7) and insulin (MW = 5733.2) as in Example 1. Each compound was loaded onto P (MAA-g-EG) hydrogel by equilibrium splitting, and the loaded hydrogel was immersed in 200 ml (pH = 1.2) of gastric acid-like environment for 2 hours. The polymer microparticles are then transferred to a pH = 6.8 phosphate buffer solution. Insulin concentrations released into the peripheral solution were monitored using HPLC, and the results for thiophylline, vancomycin, and insulin are shown in FIGS. 5A-5C, respectively.

Release of the compound from the hydrogel matrix is reduced in acidic solutions (see FIG. 2, exposure for the first 2 hours). However, in the buffer solution of pH6.8 it can be seen that the compound is released quickly. As the molecular weight of the drug increases, this tendency is more pronounced. For example, P (MAA-g-EG) hydrogels are an effective vehicle for insulin (MW = 5733.2); Less than 10% of insulin is released from the polymer in the hypothetical gastric acid (pH = 1.3) during the first phase of the experiment. However, when the particles are placed in a pH = 7.4 buffer solution, the hydrogel is released quickly, releasing insulin rapidly. These results indicate that graft P (MAA-g-EG) is useful for systems that transport insulin to the oral cavity. As used herein, insulin includes purified human and animal natural insulins and derivatives thereof such as insulin lispro and recombinant insulins, monovalent or divalent insulin salts or insulin derivatives.

In addition, P (MAA-g-EG) hydrogels exhibit strong mucoadsorbents due to the presence of grafted PEG chains, which act as adsorption promoters. In addition, the mucoadsorbed crystals of P (MAA-g-EG) are significantly affected by the pH of the environmental fluid (see FIG. 6). The adsorption between the gel and the mucosa is significantly greater at the most visceral pH (pH = 7.4) compared to the uppermost environment. However, in order to actually compare the mucous membrane adsorption properties of the gel, the adsorption operation was standardized to describe the polymer gel aliquots. Standardization of the adsorption work is about twice the size of the hydrogel in the absence of complexes. Thus, the mucoadsorption properties of P (MAA-g-EG) hydrogels are relatively low as they pass through the gastrointestinal tract, and remain intact when the complex is formed. After arriving in the intestines, the interchain complexes decompose and enhance the adsorption of the hydrogel to the intestinal mucosa compared to the gastrointestinal mucosa. Thus, after administration to the oral cavity of vertebrates, the insulin carrier remains in the portion where insulin is absorbed (eg, intestines) for more time.

The difference in adsorption properties of these hydrogels at different pH values is due to the mobility of PEG chains in each material. Under conditions that do not form highly expanded complexes, the pendant PEG chains are free and directly penetrate the mucosa and act as adsorptive immobilizers. In the state where the complex is formed, P (pendant PEG of MAA-g-EG forms a complex with the basic structural chain so that it cannot interact with the mucosal surface.

According to the present invention, a therapeutically effective amount of protein is administered to a vertebrate using a hydrogel composition. The method consists in administering a composition consisting of a protein contained in a hydrogel carrier to the oral cavity of a vertebrate. Compositions included in the hydrogel matrix further include protease inhibitors, pharmaceutically acceptable carriers, stabilizers, biocompatible fillers known in the art.

One suitable hydrogel carrier is P (MAA-g-EG), and in one embodiment the P (MAA-g-EG) hydrogel matrix comprises a pharmaceutically acceptable composition consisting of insulin. In addition, according to one embodiment, the insulin composition further comprises a protease inhibitor or absorption enhancer. It can be seen that the composition consisting of insulin contained in the P (MAA-g-EG) hydrogel has a surprising effect in transporting insulin into the bloodstream of the animals (Examples 3 and 4). Hydrogels are generally prepared in a specific form and packaged in suitable oral delivery vehicles (eg, tablets, capsules, etc.) using techniques known in the art.

In one embodiment, the delivery system consists of microparticles of poly (methacrylic acid) and poly (ethylene glycol) crosslinked interpolymers and includes insulin. While the composition passes through the extreme conditions of the stomach, such a system is particularly effective because the copolymer exhibits pH-sensitive swelling properties to protect insulin. The pendant PEG chain also acts as an adsorbent promoter to increase the residence time of the hydrogel carrier at the desired delivery site. As described in Example 2, the adsorptive properties of the hydrogel are strongly influenced by pH, so they are more adsorbed on the viscera surface than on the gastrointestinal surface. In addition, pendant PEG polymers are present, which act as peptide stabilizers and help maintain the biological activity of bioactive compounds such as insulin.

Complex formation between chains in hydrogel interpolymers is sensitive to the characteristics and pH of the surrounding fluid and to the interpolymer and graft chain length. In the above acidic environment, the hydrogel is in a complex state because a complex is formed between the carboxylic acid protons and the polymer stabilized by hydrogen bonding between ether groups in the grafted chain. Under these conditions, at least a compound having a molecular weight of 1000 (eg insulin) does not diffuse through the membrane because of the small pore size, ε. Thus, such compounds are protected from the harsh environment of gastric acid. As the particles pass through the stomach into the intestines, the surrounding pH is increased above the gel's transition pH. The complex decomposes immediately, and the network pore size rapidly increases to release a compound having a molecular weight of 100,000. Thus, P (MAA-g-EG) hydrogels can be used as an effective vehicle for the delivery of compounds having molecular weights of 1,000 to about 100,000 to the oral cavity.

Example 1; Release of P (MAA-g-EG) hydrogel contents according to pH

Three different size compounds (theophylline (MW 180.2), vancomycin (MW 1485.7), insulin (MW 5733.2)) were investigated to see whether P (MAA-g-EG) hydrogels could act as transport vehicles.

Methacrylic acid and poly (ethylene glycol) monomethacrylate were free radical solution polymerized and the oligomer chain was crosslinked with tetraethylene glycol dimethacrylate to prepare P (MAA-g-EG) hydrogel at 37 ° C. . The hydrogel is then washed with deionized water for one week to remove unreacted monomers and uncrosslinked oligomer chains, dried under vacuum and ground to a powder having an average particle size of 100-150 μm.

Drug collection experiments were performed using theophylline (MW 180.2), vancomycin (MW 1485.7), and insulin (MW 5733.2). Each drug is dissolved in pH 7.4 phosphate buffer solution and P (MAA-g-EG) hydrogel is added to the drug solution to drop the drug solution onto the hydrogel by equilibrium partitioning. The hydrogel matrix is then contacted with an acid solution such that the polymer induces complex formation and reduces the hydrogel matrix pore size. Hydrogel microspheres are obtained by filtration and dried under vacuum. Collection efficiency is calculated from the filtrate obtained by washing the hydrogel separated from the drug concentration remaining in the initial solution, which is performed by HPLC analysis.

Drug release experiments are conducted according to the Japanese Pharmacopoeia (JP) paddle method. The composition is stirred with a paddle at 100 rpm, 37 ° C. first (pH 1.2) and second (pH 6.8) JP solutions. Two hours after treatment with the first solution, the polymer sample is collected using filtration and transferred to a second solution at pH6.8. Drug concentration is monitored by HPLC.

Thirty minutes after the start of the experiment, the average insulin concentration trapped in the hydrogel matrix reaches almost 94%, thus the polymer is considered to be a suitable carrier for carrying insulin. Experiments in which theophylline, vancomycin, and insulin are released from P (MAA-g-EG) hydrogels are shown in FIGS. 5A, 5B, and 5C, respectively. Release of the compound from the hydrogel matrix is reduced in acidic solutions (see 2 hours after initial exposure). However, it can be seen that the compound is released rapidly in pH 6.8 buffer solution. This tendency is more pronounced as the molecular weight increases; In the first experiment, less than 10% of insulin is released from the polymer in pseudogastric juice (pH = 1.3). However, when the particles are in pH = 7.4 buffer solution, the hydrogel expands rapidly, releasing insulin rapidly. These results indicate that graft interpolymer P (MAA-g-EG) is useful as a system for transporting insulin to the oral cavity.

Example 2; In vitro membrane adsorption study

Using a solution polymerization technique, P (MAA-g-EG) hydrogel is prepared in a thin film. The hydrogel is expanded to equilibrate in DMGA buffer solution (pH = 3.2 and 7.4). The expanded hydrogel is cut into 20 cm diameter flasks and placed in a tensile tester at 25 ° C., 90% RH. The polymer sample is adsorbed to the upper holder of the tester using cyanacrylate medical adhesive, while the gelled bovine mandibular mucin sample is fixed to the lower part when using the adhesive. Bring the two together for 15 minutes, then separate at 1 mm / minute. Measure the separation force as a function of travel. Rupture work equivalent to biosorption work is calculated under the curve area.

P (MAA-g-EG) hydrogels function well as oral insulin devices because they can delay the action of protease inhibitors and adsorb to the visceral walls, which makes them a significant contact. It helps to adsorb the drug.

If a hydrogel containing insulin is in the visceral fluid, the hydrogel expands rapidly and releases insulin. P (MAA-g-EG) microparticles containing insulin are expanded in phosphate buffer solution for 1 hour and then transferred to visceral fluid. Protein breakdown of insulin in visceral fluid is monitored using the insulin EIA kit. At least 50% of the biological activity of insulin was maintained for at least 1 hour in the presence of protease. For comparison purposes, biological activity is rapidly lost when insulin is dissolved in the visceral fluid. P (MAA-g-EG) hydrogel protects insulin by binding calcium to ionized pendant groups, delaying the action of protease enzymes.

Thus, P (MAA-g-EG) hydrogels exhibit mucoadhesive properties because of the presence of graft PEG chains acting as adhesion promoters. Mucosal adsorption characteristics of P (MAA-g-EG) hydrogels are strongly influenced by the pH of the surrounding environment fluid (FIG. 6). The area under the curve of FIG. 6 is equivalent to the adhesion between the gel and the mucosa. Under conditions similar to visceral pH (pH = 7.4), the adsorption force between the gel and the mucosa increases significantly. However, for the actual comparison with the mucoadhesion of hydrogels, the adsorption work was standardized to describe the polymer gel aliquots (see Table 2). The standardized adsorption operation is about twice as large for the hydrogel in the absence of the complex as compared to the hydrogel with the complex. Thus, hydrogels are better adsorbed to mucous membranes of the intestine than in the stomach. Thus, the advancing time of the insulin carrier at the site where insulin is absorbed is greater.

Adsorption Capacity of P (MAA-g-EG) Hydrogels with PEG of Molecular Weight 1000 and 1: 1 MAA / EG pH Adsorption work W ^* 10 ⁶ (J) Polymer Volumetric O _2'S Standardized adsorption work W / (O _2'S ) ^{2/3 *} 10 ⁶ (J) 3.2 5.38 0.693 62.1 7.4 9.34 0.049 6720

The adsorption properties of hydrogels differ at different pH values due to PEG mobility in each material. In the highly expanded state, without forming a complex, the graft PEG chains are free and directly penetrate the mucosa, acting as anchors for adsorption. In the complexed state, the graft PEG chain in P (MAA-g-EG) forms a complex with the basic structural chain and cannot penetrate between the gel / mucosa and only temporary fixation.

Example 3; In vivo Insulin Administration in Mice

Graft interpolymers are prepared by free radical solution polymerization of methacrylic acid and poly (ethylene glycol) monomethacrylate. The hydrogel is then washed with deionized water for 7 days to remove unreacted monomers and uncrosslinked oligomeric chains. The hydrogels are dried under vacuum and ground to a powder. The powder is filtered to obtain particles having a diameter of 100 to 150 mu m. Crystalline porcine insulin (26.9 U / mg) is added dropwise by equilibrium division. The drug loaded particle is filtered, washed to remove surface drug and dried under vacuum.

Tie Male Wlstar rats (200 g) for 24 hours. The rats are allowed to lie down, and gelatin capsules are used to administer insulin-containing polymer microparticles, which dissolve directly in the stomach. Blood glucose is monitored by taking 0.2 ml blood samples before and after the experiment at 0.25, 0.5, 1, 2, 4, 6 and 8 hours. Serum is separated at 3000 rpm for 3 minutes and stored until analysis. Serum insulin levels are determined by enzyme immunoassay using the insulin EIA kit. Serum glucose concentration was determined by the oxidized glucose method using the glucose B-Test kit.

Figure 7 summarizes the glycemic response of mice receiving insulin collected on P (MAA-g-EG) microparticles. Within 2 hours after receiving the polymer weak form, a significant hypoglycemic effect (lower blood glucose level) was observed. Reduced blood sugar levels are significantly affected by insulin dosage. No response was observed in mice receiving insulin solution.

The effect of administering a composition composed of P (MAA-g-EG) hydrogel comprising insulin and protease inhibitor aprotinin is shown in FIG. 8. Reference mice received a hydrogel containing insulin only, without proteases (for comparison), and one group received 50 U / kg insulin (used as reference). The two groups receiving the insulin polymer dosage form significantly reduced blood glucose within 2 hours after administration. Mice receiving the aprotinin complex, an insulin and protease inhibitor, had the lowest blood sugar levels. Aprotinin delays the action of degrading enzymes in the intestines and allows loaded insulin to remain locally active longer. Thus, insulin that is transported into the bloodstream is maximal in mice receiving hydrogel insulin and protease inhibitor compositions (captured in P (MAA-g-EG) hydrogels), which causes maximal reduction in blood glucose levels. .

Example 4; In Vivo Study in Diabetic Mice and Dogs

Streptozotocin is administered to healthy male Wistar rats to cause diabetes. Healthy dogs are administered diabetes to induce diabetes. P (MAA-g-EG) microspheres are prepared using methacrylic acid and poly (ethylene glycol) dimethacrylate (PEG MW = 1000) free radical macrosuspension polymerization. Tetraethylene glycol silver dimethacrylate is added as a crosslinking agent. As the thermal reaction initiator, 2,2'-azobisbutyronitrile (AIBN) is added in an amount of about 0.5% of the total monomers.

Insulin is loaded onto the P (MAA-g-EG) microspheres using the equilibrium splitting of the drug. Bovine pancreatic insulin is dissolved in 200 μl 1N NaOH. The insulin solution is diluted with 20 ml phosphate buffer solution (pH = 7.4) and then equilibrated with 200 μl 0.1 N NaOH. The drug is added dropwise by expanding the initially dried P (MAA-g-EG) in insulin solution for 24 hours. The particles are then filtered and washed with 100 ml 0.1N HCl to crush the microspheres and squeeze out the remaining buffer solution. The microspheres loaded with the drug are dried under vacuum and stored at 4 ° C. The dropping ratio is determined by HPLC analysis of the initial insulin concentration and the filtrate obtained from the wash liquor.

Before administering the P (MAA-g-EG) hydrogel loaded with insulin, male Wistar rats (250 g) were tied for 24 hours. Mice are left lying down and insulin loaded P (MAAA-g-EG) microspheres and reference solutions are made using gelatin capsules and Eudraglt L 100 capsules. Gelatin capsules dissolve directly in the stomach, while Eudraglt capsules dissolve fairly slowly.

During the experiment, the mice are separated (4 per cage) and the water is allowed to eat. At 0.25, 0.5, 1, 2, 4, 6 and 8 hours after dosing, 0.2 ml of blood is drawn from the cervical vein. Blood flow is separated by centrifugation at 3000 rpm for 3 minutes. Blood glucose levels are measured by the oxidized glucose method using the glucose B-test kit.

Dogs with diabetes (25 kg) are kept tied for 24 hours before administering the composition. Polymeric dosage forms are administered using gelatin capsules. Feed the dog at the time of administration.

During the experiment, dogs are kept in cages and water is available. Blood is obtained using an dilated catheter. Blood glucose levels are determined using a mobile glucose analyzer.

Many systems have been shown to be effective in lowering blood glucose levels in healthy animals after oral administration of a polymer carrier containing insulin, but similar results have not been observed in animals with diabetes. The glycemic response observed in diabetic and healthy mice using P (MAA-g-EG) microspheres containing insulin using gelatin capsules (25 IU / kg doses) is shown in FIG. 9. In diabetic rats, blood glucose levels were reduced by up to 40% at initial levels. Blood glucose levels last longer than 8 hours, and suppressed blood glucose levels are much greater in animals with diabetes than in healthy animals. Thus, strong mortgage effects lasted longer in diabetic animals.

Figure 10 shows the blood glucose response in mice with diabetes after oral administration of Eudragit capsules containing insulin loaded P (MAA-g-EG) microspheres. In rats receiving this dosage form, glucose levels were reduced by 50% or more within at least 8 hours after a single dose. The microspheres trapped in Eudragit are more effective than the gelatin trapped because the microspheres contained in Eudragit capsules are exposed to the environment of the upper GI organs in a shorter time, causing the Eudragit capsules to dissolve slowly.

Blood glucose in healthy dogs was significantly lower after oral administration of one polymer dosage form (10 IU / kg). Dogs should be fed at time zero, and the body's normal response should remain at baseline. After feeding, blood sugar levels increase within 2 hours, but because insulin is absorbed from the upper intestine, blood sugar levels decrease by more than 20%. There is also a second decrease at the 8 hour time point, which is consistent with what is previously seen in rats, probably because insulin is absorbed in the colon. In addition, after 8 hours the blood sugar content is constantly reduced because insulin is absorbed from the colon.

In dogs with diabetes, the glucose response demonstrates that they absorb insulin after oral administration. Blood glucose levels in diabetic dogs can be controlled by administering P (MAA-g-EG) microspheres containing insulin using gelatin capsules (10 IU / kg doses). After feeding the food, after administering the polymer dosage form, the blood glucose level in the dog initially rises sharply. However, after one hour, blood glucose levels stabilize for the next three hours because insulin is absorbed. Blood glucose levels in dogs with diabetic polymer dosages are 40% less than in dogs without any insulin.

The system for transporting insulin to the oral cavity carries biologically active insulin for a significant amount of time and can be protected from the stomach so that it can be moved along the GI organs, such as the upper small intestine, to a more desirable absorption zone. These inherent properties make the composite P (MAA-g-EG) hydrogels ideal for use in such places.

P (MAA-g-EG) hydrogels can effectively transport biologically active insulin through the oral cavity. These substances have been shown to lower blood sugar levels and maintain blood sugar levels at nearly normal levels for more than 8 hours in diabetic rats and dogs. Such substances function well because insulin trapped in the hydrogel does not release insulin until it reaches the upper part of the small intestine. In the small intestine, the hydrogel is strongly adsorbed to the mucosa so that the carrier can be adsorbed well at the adsorption site. In addition, the polymer acts to delay protease activity in the intestines. The inhibitory effect of polymers on enzyme function is due to the polymer's ability to complex with cations such as calcium, which are essential for enzyme function.

Claims (17)

In orally administrable pharmaceutical compositions, the composition comprises an expandable hydrogel matrix and a heat labile protein contained therein, wherein the hydrogel matrix crosses methacrylic acid and poly (alkylene glycol) monomethacrylate A pharmaceutical composition, characterized in that it consists of a bonded interpolymer. The pharmaceutical composition of claim 1, wherein the poly (alkylene glycol) monomethacrylate is poly (ethylene glycol) monomethacrylate. The pharmaceutical composition of claim 1, wherein the hydrogel matrix is crosslinked with tetraethylene glycol dimethacrylate. The pharmaceutical composition of claim 2, wherein the molar ratio of methacrylic acid and poly (ethylene glycol) monomethacrylate is about 1: 1. The pharmaceutical composition of claim 4, wherein the protein has a molecular weight of about 1,000 to about 20,000. The pharmaceutical composition of claim 4, wherein the protein is insulin. 5. The pharmaceutical composition of claim 4, wherein the poly (ethylene glycol) monomethacrylate has a molecular weight of about 200 to about 4000. 5. A pharmaceutical composition according to claim 4 wherein the hydrogel is in the form of particles and contained in a capsule. The pharmaceutical composition of claim 8, wherein the capsule is a gelatin capsule. 5. A pharmaceutical composition according to claim 4 further comprising a protease inhibitor. A composition for orally administering insulin to vertebrates, wherein the composition is comprised of insulin contained in a P (MAA-g-EG) hydrogel. The pharmaceutical composition of claim 11, further comprising a protease inhibitor. 12. The pharmaceutical composition of claim 11, wherein the hydrogel is particulate and contained in a capsule. The pharmaceutical composition of claim 13, wherein the capsule is a gelatin capsule. A method of administering a therapeutically effective amount of protein to a vertebrate, the method comprising administering the composition of claim 1 to the oral cavity of a vertebrate. A method for preparing a composition according to claim 1, Methacrylic acid and poly (alkylene glycol) dimethacrylate and crosslinker are polymerized to form a hydrogel matrix; The hydrogel matrix is brought into contact with the aqueous environment of the protein, wherein the solution has a pH of at least 5.4; The pH of the solution is adjusted to 5.4 or less; Separating the hydrogel matrix comprising the protein. 18. The method of claim 16, wherein the poly (alkylene glycol) monomethacrylate is poly (ethylene glycol) monomethacrylate.