Alternative approach to the treatment of diabetes mellitus.

The influence of insulin preparations (Actrapid and Ransulin) on the glucose and insulin blood level has been studied in patients with diabetes mellitus. It has been shown that comparable changes in the measured parameters are achieved in most patients with oral doses of Ransulin that are two to three times higher than the doses of Actrapid.

[Effect of the Hydrogel Carrier Structure on the Activity of Immobilized Trypsin].

The dependence of the activity of trypsin immobilized in polyacrylamide hydrogel on the hydrogel swelling ratio, the size distribution of its pores, and the means of enzyme binding has been studied. It has been shown that the most favorable conditions for immobilized trypsin are provided upon its binding to hydrogel via trypsin macromonomer copolymerization with acrylamide and a linking agent in the presence of a modifier that limits polymer chain growth.

[Novel antiproteinase hemosorbent].

A method has been developed for producing a biospecific hydrogel hemosorbent by the radical copolymerization of an unsaturated derivative of ovomucoid from duck egg white with acrylamide and N,N'-methylene bisacrylamide in an aqueous solution in the presence of mercaptoacetic acid serving as a chain transfer agent. The use of a chain transfer agent has been shown to result in changes in the structure of the hydrogel formed, namely, an increase in the degree of swelling in aqueous solutions and a decrease in the number of large pores. This creates favorable conditions for the functioning of immobilized ovomucoid and allows for an increase in the serine proteinase absorption capacity of the hemosorbent.

[Thromboresistance of glucose-containing hydrogels].

The thromboresistance of glucose-sensitive polymer hydrogels, modeling one of the functions of the pancreas, namely, the ability to secrete insulin in response to the introduction of glucose into the environment, has been studied. Hydrogels were synthesized by the copolymerization of hydroxyethyl methacrylate with N-acryloyl glucosamine in the presence of a cross-linking agent and subsequently treated with concanavalin A. Introduction of glucose residues into the hydrogel did not result in significant changes in either the number of trombocytes adhered to the hydrogel or the degree of denaturation of blood plasma proteins interacting with the hydrogel. Consequently, the biological activity of insulin did not change after release from the hydrogel. The use of glucose-sensitive hydrogels is supposed to contribute to the development of a novel strategy for the treatment of diabetes.

[Chemical modification of proteins by "smart" polymers].

The modification of proteinase inhibitor ovomucoid from duck eggs white by poly-N,N-diethylacrylamide having a low critical solution temperature (LCST) have been studied. Modification of free amino groups of lysine and N-terminal residue of ovomucoid is resulted in a significant decrease in the activity of the inhibitor toward trypsin and small decrease in the activity toward α-chymotrypsin. At heating of the solution of modified ovomucoid above the LCST transformation of the antitryptic centers of ovomucoid in antichymotryptic centers was observed. It was shown that this phenomenon is due to the hydrophobization the lysine residues localized in the reactive centers of the inhibitor while maintaining the structure of the "linkage loops". Therefore the α-chymotrypsine molecules began to interact with these residues, mistaking them for the residues of hydrophobic amino acids of antichymotryptic centers.

[Thermoregulation of catalytic activity of enzymes polymeric derivatives].

Trypsin was immobilized on polymeric carriers with low critical solution temperature (LCST). Homopolymer of N,N-diethylacrylamide (DEAA), random copolymers of DEAA and acrylamide (AA), and block copolymers polyDEAA-polyAA were used as the carriers. It was shown that at a temperature above LCST all carriers have a conformation change and trypsin's polymeric derivatives precipitate. The maximal activity after phase transition keeps trypsin, immobilized on polyDEAA block in polyDEAA-polyAA block-copolymer.

[Oral insulin preparations for the regulation of the glucose level in the blood].

Oral administration of diluted solutions of insulin results in the absorption of the hormone into bloodstream. After oral administration of diluted insulin solutions to healthy volunteers and animals with experimental diabetes insulin does not undergo hydrolytic degradation under the action of proteolytic enzymes and its absorption is sufficient to produce the hypoglycemic effect. This approach gives a chance of creating of novel strategy for diabetes treatment.

[Biodegradable polymeric derivatives of polypeptide drugs for targeting].

Relatively short polymer chains with lower critical solution temperatures were immobilized on protein macromolecules to obtain biodegradable polymeric derivatives of proteins (including those for heat-inactivated targeting of polypeptide drugs). Addition of a derivative to a multicomponent biological system and heating of the target to a temperature in excess of the lower critical solution temperature was followed by the carrier release into a separate phase and the transportation of the bound protein to the target. The protein molecule served as a biodegradable region and was progressively hydrolyzed, with the formation of low-molecular-weight fragments. These fragments were readily eliminated from the organism. The physiological activity of immobilized serum albumin was independent of the number of attached chains in the polymer carrier (the constant of bilirubin binding equaled 10 M(-1)). The biodegradation of synthetic systems, caused by alpha-chymotrypsin, was also studied. The more polymer chains were attached to serum albumin, the greater was the resistance of the protein to enzymatic hydrolysis.

[Immobilization of ovomucoid on chitosan].

The inhibitory activity of ovomucoid from duck egg white, immobilized on chitosan with the use of glutaraldehyde or carbodiimide as cross-linking agents, was studied. Glutaraldehyde proved to be a more preferable cross-linking agent than carbodiimide. When chitosan is used as a protein carrier, the possibility of shifting the pH optimum of these compounds should be taken into account.

[Oral administration of protein solutions].

Experiments on animals showed that native proteins may diffuse into the blood flow after oral administration of diluted protein solutions. An in vitro study led us to hypothesize that treatment with diluted solutions is accompanied by a decrease in the rate of protein proteolysis and accelerated protein diffusion through the intestinal mucosa.

[Structure and functional properties of temperature-sensitive derivatives of immobilized proteins].

Polydiethylacrylamides (degree of polymerization, 13-470) containing a terminal carboxyl group were obtained by the method of radical polymerization of N,N-diethylacrylamide in the presence of mercaptoacetic acid. In the presence of 1-ethyl-(3,3-dimethylaminopropyl)-carbodiimide, these polymers reacted with ovomucoid to produce its polymeric derivatives. The values of the lower critical mixing temperature of these derivatives and the inhibitory activities of immobilized ovomucoid were determined by the length and amount of polydiethylacrylamide macromolecules bound to the molecule of ovomucoid.

[New prospects for a method of biospecific chromatography]. / Novye vozmozhnosti metoda biospetsivicheskoi khromatografii.

The polyacrylamide hydrogels with covalently immobilized ovomucoid from the duck's egg white were synthesized by radical copolymerization. These hydrogels can defend the immobilized insulin against the action of proteolytic enzymes. Biospecific interaction of the polysaccharide component of ovomucoid with lectins leads to the targeting transport of the hydrogel particles onto the small intestine wall.

[Polymeric systems with antithrombin activity for thermally activated targeting]. / Polimernye sistemy s antitrombinovoi aktivnost'iu dlia termoaktiviruemogo napravlennogo transporta.

Copolymers of N,N-diethylacrylamide and N-acryloylphthalimide with lower critical solution temperature (LCST) were synthesized by radical copolymerization. Polymeric systems with antithrombin activity and LCST were prepared via a reaction of amino groups of hirudin with phthalimide groups of the copolymers. On increasing hirudin content, LCST of the polymeric systems increased. The antithrombin activity of polymeric systems obtained by hirudin immobilization on copolymer carriers was inversely related to the content of the copolymer, amounting to 6% of the activity of native hirudin.

Mucoadhesive polymers with immobilized proteinase inhibitors for oral administration of protein drugs.

A new approach to overcome the degradation of protein drugs by proteolytic enzymes and their targeting to the blood through the digestive apparatus was developed. The approach is based on the immobilization of drugs into the polymeric hydrogel containing glycoprotein--ovomucoid from duck egg whites. This glycoprotein inhibits the activity of proteolytic enzymes and acts as a biospecific ligand to lectins on the walls of the gastrointestinal tract.

[Stability of immobilized insulin preparation at various pH]. / Stabil'nost' preparata immobilizovannogo insulina pri razlichnykh pH.

The stability of polyacrylamide hydrogel, modified by ovomucoid with immobilized insulin was studied at different pH of external medium. At 3.8 < pH < 5.5 insulin binds to the gel, due to electrostatic interaction between ovomucoid and insulin molecules.

[Inhibitors of proteolytic enzymes in the treatment of diabetes mellitus]. / Ingibitory proteoliticheskikh fermentov v terapii sakharnogo diabeta.

A new approach to overcome the degradation of insulin by proteolytic enzymes and its targeting to the blood through the digestive apparatus was developed. The approach is based on the immobilization of insulin into the polymeric hydrogel which is modified by ovomucoid--glycoprotein, inhibitor of proteolytic enzymes. Oral administration of this system to rabbits and rats, (in contrast to the hydrogels modified by proteolytic enzymes inhibitors without polysaccharide part), statistically significantly lowered blood glucose level.

Chemical modification of polymers with physiologically active species using water-soluble carbodiimides.

The covalent immobilization of some proteins (ovomucoid from duck egg white, human serum albumin, aldolase and thiroglobulin) on the surface of polyethylene grafted with polyacrylic acid has been studied. Water-soluble carbodiimide was (1-ethyl-3-(3-dimethylaminopropyl carbodiimide) used as a condensing agent. It was shown that three different reactions can occur in the reaction mixture during immobilization: the reaction between carboxy groups of graft copolymer and amino groups of protein (the immobilization reaction itself) and reactions between carboxy and amino groups of protein molecules (reactions of intra- and intermolecular cross-linking). The reaction of intramolecular cross-linking leads to a decrease in the physiological activity of the immobilized substance, while other reactions do not affect it. The preliminary activation of this surface by carbodiimide before the modification allows to maintain the activity after immobilization on the insoluble surfaces.

[Interaction between globular proteins and water-soluble carbodiimides]. / O vzaimodeistvii globuliarnykh belkov s vodorastvorimymi karbodiimidami.

Interactions of a number of globular proteins with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide were studied. Under conditions of carbodiimide excess at a protein concentration lower than 88.5 x 10(-5) mol/l, the crosslinking reaction was found to proceed exclusively by an intramolecular mechanism. This resulted in conformational changes in the protein secondary structure and decreased its biological activity. At higher protein concentrations, the reaction of intramolecular crosslinking was always the first to proceed, and only then intramolecularly crosslinked proteins interacted. The reaction of intermolecular crosslinking was not followed by a further change in the protein conformation and activity.

[Synthesis and study of immobilized insulin]. / Sintez i issledovanie immobilizovannogo insulina.

Conjugates of insulin with duck egg-white ovomucoid and soybean trypsin inhibitor were synthesized. The conjugates are highly stable to alpha-chymotrypsin treatment. Oral administration of insulin-ovomucoid conjugates in rabbits significantly lowered blood glucose level unlike administration of insulin-soybean trypsin conjugate.

[Problems in creating insulin preparations with increased stability against proteolysis]. / Problemy sozdaniia preparatov insulina s povyshennoi ustoichivost'iu k proteolizu.

Chemical methods that accelerate the transport of insulin and other polypeptides across biological membranes and increase their resistance to enzymatic hydrolysis are reviewed. These methods include chemical modification of insulin macromolecules, the use of compounds that increase the permeability of biological membranes or inhibit enzymatic proteolysis, the hormone immobilization in a polymer coat protecting it against the aggressive environment, the incorporation of insulin into liposomes, etc. The advantages and drawbacks of these methods are analyzed, and promising lines of research in this field of applied biochemistry are described.