Wound bandage including protein polymer and polyfunctional spacer

FIELD: medicine.

SUBSTANCE: method of obtaining wound bandage material is claimed. Method involves obtaining protein polymer by protein reaction with polyfunctional spacer or its activated derivative. Preferred polyfunctional spacer is polycarboxylic acid, especially dicarboxylic acid. Protein polymers obtained with such spacers can be applied in a wide range of therapeutic purposes, including wound bandage materials, therapeutic agent delivery to organism, and bioadhesive and sealing substances.

EFFECT: obtaining material taking exact shape of wound filling wound hollow completely without irritation of exposable tissues.

27 cl, 2 dwg, 13 tbl, 12 ex

 

This invention relates to the field of wound care, in particular to the formation of a protein polymer gels for local application as dressings for wounds. The invention also relates to the field of drug delivery, in particular to methods and compositions for delivery of therapeutic agents or intravenously, or topically. The invention also describes methods for obtaining systems protein carriers for attaching or incorporating therapeutic agents for the treatment of disease States, eliminating bleeding and healing of tissue.

The invention relates to the formation of a number of drug delivery, from small soluble protein polymers to gel, using easy chemical methods. The process is simple and scalable for commercial use.

Soluble polymers can be used to target specific areas of the body and deliver one or more therapeutically active agents, from small drugs to large proteins. Attaching the active agent to the polymer is preferably by chemical bonding or by adsorption, or by incorporating the active agent into the polymer in the process of education. More than one agent can be delivered the same polymer.

The invention also associated with about what adowanie gels, suitable for local application, for example for surface wounds, burns and ulcers, among other applications. The application can be either as inclusion in a bandage or dressings, or as a spray or solution applied directly to the skin, which give the possibility to turn into a gel. Gels can also be used internally, as a vehicle for slow or controlled release of drugs, and can also be used for preventing or inhibiting adhesi tissues after surgical operations, by formation of a barrier between adjacent tissue membranes.

The invention also describes the formation of compounds suitable for coatings of surgical instruments, such as catheters or stents, and glass or plastic dice for diagnosis (e.g., ELISA, ELISPOT) or for the purposes of processing, for example for use for the growth of cells, including stem cells.

The invention also describes the formation of "natural" tissue sealants with or without added hemostatic and/or the blood-clotting agents.

Selection of the dressing material is difficult. The choice of a suitable dressing material for the patient requires a careful and thorough assessment of the wound, knowledge of the healing process and special knowledge of the properties of many dressings on the market. The patient and economic factors must also be taken into account.

Without careful consideration of all factors choice of dressing material will likely be arbitrary and potentially ineffective.

It is widely recognized that the warm and moist environment of the wound promotes healing and prevents dehydration of tissue and cell necrosis. Most modern means to care for wounds created to ensure these conditions.

There are several types available dressings for wound care. Among those most often used are hydrogels, hydrocolloids, alginates, polymer films and polymer foam. Each product type has common features, but the design and, thus, the performance of each brand can vary considerably within a single product type. Unified products unsuitable for use with wounds of all types and at all stages of healing.

The main characteristics of the dressing material, which determine its suitability for use for specific types of wounds include a snug fit to the body (desirable to maintain complete sealing of the wound), adsorption characteristics of the fluid and smell properties in use, the adhesive properties and the presence of antibacterial and cu is vostanavlivayet activity where you want. Other factors that can influence the choice of product include the ability of the dressing material to cause a reaction sensitivity, ease of application and removal (important to minimize pain and trauma of the wound) and the interval between shifts dressing material. Dressings should not lose particles or fibers, which can slow the healing process or cause the disposition of the wound to infection. They also must not contain extractable substances which can have an adverse effect on cell growth.

Full covering for deep wounds is important for most agreed shivlani RAS, ensuring bacterial barrier and a reduced rate of infection, reducing water loss and minimizing pain. To ensure that the cavity is completely covered, dressings often pressure on the wound, further damaging the fabric.

Hydrogel dressings for wounds practically useful for burns, wounds and deep wounds such as bedsores, because, among other effects, they relieve pain, give a cold feeling and provide control over the hydration of the surface of the wound. Unlike many alginate dressings, they do not stick to the wound and can be removed easily without pre-soaking. However, although they are easy to use, it is often difficult to completely fill the cavity of the hydrogel wound dressing material (for example, when the cover leg ulcers), so hydrogel dressings often provide insufficient barrier against bacteria and may not be suitable for use on infected wounds.

Undoubtedly, there is a need for improved dressing material for wounds that are more desired characteristics, being more universal, for this reason they are suitable for a wide range of types of wounds and stages of healing.

In particular, the dressing material with the advantages of hydrogel dressings, but with the best antibacterial properties and the ability to completely fill a cavity of any shape and size would provide an important improvement in relation to the hydrogels used in the present time.

Moreover, dressings for wounds, which also delivers active ingredients such as drugs, in place of the wound in a controlled manner will be an added advantage. The desired active ingredients can help fight or protect against infections, reduce pain, reduce inflammation and/or accelerate healing, for example, by maintaining the bleeding.

Protein serum albumin human (HSA), as b is lo detected, shows the number of properties that make it useful for healing of wounds. For example, by reversible binding of a wide range of drug molecules HSA can propose a mechanism of controlled release for drug delivery. HSA binds metal ions (e.g. zinc, copper and silver), which can be important in anti-infective treatment of wounds and can detoxify the wound site and to bind free radicals. Pathological platelet aggregation is inhibited HAS, and the levels of inflammatory chemicals (and, therefore, itching) is also reduced. HAS is non-allergenic and can naturally provide antibacterial/antiviral effect at the wound site.

Albumin is used for other medical purposes, for example to increase blood volume. WO 99/66964 belongs to based on albumin compositions for use as bioadhesive substances, surgical sealants and implantable devices for drug delivery and for prostheses. Adhesive properties of these compositions makes them unsuitable for use as dressings for external wounds, and although the songs are designed to disintegrate in the body, suitable for internal use is also limited to unwanted adhesion. After surgical p is ocedur adhesive substance, designed for the reunification of damaged tissues, may also be attached to the wound site to nearby tissues/organs and cause further damage.

WO 99/66964 discloses the use of auxiliary molecules to change the speed and/or extent of cross-links between molecules of albumin. It is argued that dicarboxylic acids are able to accelerate the gelation of bovine serum albumin. However, we found that the products produced in accordance with WO 99/66964 that are dry and brittle in comparison with the polymers of the present invention. Such fragile products unsuitable for use as dressings for wounds.

Currently has been developed a method of obtaining a dressing material for wounds, which overcomes or substantially reduces the above-mentioned and/or other disadvantages associated with the prior art.

In accordance with the first aspect of the invention is disclosed a method of obtaining a dressing material for wounds, where the method includes the formation of a protein polymer by reaction of the protein with a polyfunctional spacer or its activated derivative.

Dressings for wounds can be formed on site (in situ). The term "in place" in the context of this invention means that the reaction of the protein with half the functional spacer for the formation of the dressing material occurs at the wound site. The components of the composition can be applied to the wound site at the same time, or in rapid succession, or components may be mixed immediately before use and the mixture is then applied to the wound site.

Education bandages in place is practically advantageous because dressings takes the exact shape of the wound, completely filling the cavity of the wound without causing irritation of the tissue affected. Precise fit ensures that the wound is completely sealed.

Excipients can be entered into the dressing material formed in place by adding to the composition before gelation occurs, or in the process of gelation. In particular, it may be preferable to cover a song paropronitsaemoy membrane, which will protect the polymer gel from drying out and, more importantly, to keep the wound moist. Paraponera membrane could preferably be added at the end of the gelation process, so that it is tightly and evenly joined, but not absorbed too much in composition.

Dressings of the present invention may also be pre-formed (i.e. cross stitched before application to the wound site). Such dressings can taking the th form of bandages, saturated protein polymer, or gel layers, whether or not containing auxiliary substrate. Gels special shapes and sizes can be custom molded for specific types of wounds or body parts. Alternatively, dressings suitable size can be cut from larger sheets of gel directly prior to application.

Under "protein polymer" in the context of the present invention refers to a polymer samples, composed of a set of complete protein particles, bonded together by a linking group derived from a polyfunctional spacer. You need to understand that individual protein molecules are polymer in the sense that they are composed of chains of amino acid residues linked to each other. Such a single protein molecule is not "protein polymer" in the sense of this term, as used here. Instead, the protein polymer is a reaction product obtained by the connection together of individual protein molecules to the formation of the chain or matrix of such molecules covalently bonded together through linking groups.

Proteins that can be used in the present invention include globular and fibrillar proteins or structural proteins, and mixtures thereof.

Examples of globular proteins include various the s or the natural whey proteins, their natural or synthetic derivatives, salts, enzymatically, chemically or otherwise modified, split, short or cross-linked, oxidized or hydrolyzed derivatives or their subunits. Examples whey proteins are albumin, α-globulins, β-globulins, γ-globulins, fibrinogen, hemoglobulin, thrombin and other coagulation factors. Examples of fibrous or structural proteins include synthetic or natural collagen, elastin, keratin, fibrin and fibronectin, natural or synthetic derivatives, and mixtures thereof.

Particularly preferred proteins are albumins.

Where protein polymers obtained in accordance with the invention, are intended for introduction into the human body, used protein preferably is a protein of human origin, that is really derived from human or identical (or substantially so) on the structure of protein of human origin. Particularly preferred protein is serum albumin human.

Serum albumin may be serum origin, e.g. derived from donor blood. Serum albumin human is readily available as a product fractionated blood and safely used for many of the years for intravenous supply as a magnifier of blood volume. However, to eliminate or reduce the risk of transmission of potential contaminants, such as viruses and other harmful agents that may be present in products derived from blood, as well as potential constraints associated with material released from donor blood, protein, such as serum albumin person may be recombinant product released from microorganisms (including cell lines), from transgenic plants or animals that have been transformed or transliterowany for expression of the protein.

For veterinary use may be used corresponding nonhuman animal protein. Examples of such proteins include serum albumin horse serum albumin dogs, etc.

A mixture of proteins, i.e., more than one different protein, can be used.

Functional groups of protein molecules that can react the spacer include an amino group. Preferred proteins, thus, include proteins with a high degree of amino acid residues that contain free amino groups, especially NH2group. One example of such amino acid residue is lysine, and, thus, particularly preferred proteins for use in the invention include proteins containing residues Lisi is a, especially proteins with a high proportion of lysine residues, for example, more than 20 lysine residues on the protein molecule, more preferably more than 30 or more than 40 lysine residues.

Polyfunctional spacers that can be used in the present invention include polycarboxylic acids, polyamine, poly(carboxy/amino) compounds (i.e. compounds having multiple carboxyl and amino groups), a polyalcohol, polyketone, polyallelic and polyesters.

The spacers of the polycarboxylic acids or polyamines are preferred, more preferred dicarboxylic acids and diamines.

Polycarboxylic acids include citric acid and polyacrylic acid.

Preferred spacers are bifunctional spacers, especially homobifunctional the spacers.

Polyamine include poly(lysine) and chitosan.

Especially preferred spacers are dicarboxylic acid.

Of the dicarboxylic acid is more preferable alkylenediamine acid, especially unbranched molecules alkylenediamines acid formula

HOOC(CH2)nCOOH,

in which n is from 1 to 20. Preferably n is from 2 to 12, more preferably from 3 to 8.

Preferred spacers unbranched alkylenediamines acids are:

nCommon nameThe systematic nameFormula
2Succinic acidBatandjieva acidHOOC(CH2)2COOH
3Glutaric acidIntentionaly acidHOOC(CH2)3COOH
4Adipic acidHexandione acidHOOC(CH2)4COOH
5Emelyanova acidHeptanedionato acidHOOC(CH2)5COOH
6Cork acidAttentionby acidHOOC(CH2)6COOH
7Azelaic acidNonandiolov acidHOOC(CH2)7COOH
8Sabotinova acidCandinavia acidHOOC(CH2)8COOH

Unbranched alkylenediamine acids are especially useful spacers, since the properties of the resulting protein polymers can be changed simply by varying the length alkalinous chain. In General, at a fixed concentration of protein gelation time decreases, and polim the market become more solid, less rubber and more opaque with increasing chain length of the dicarboxylic acid. The chemistry is simple, and a wide range of protein polymer systems can be obtained by adjusting only a small number of variables. As well as activation of a high degree of control, properties of polymers can be predicted quite well from the composition and reaction conditions.

In order to accelerate the reaction of the spacers with the protein molecules, in General, it is desirable that the spacer has been activated, i.e. the functional group of the spacer were transformed into a group with greater reactivity towards protein groups. The chemistry matching activations well known to specialists in this field and includes the formation of the active ester groups.

One special class of activators suitable for use with the spacers of the dicarboxylic acids are carbodiimide compounds, and particularly preferred activator for use in the invention is ethyl[dimethylaminopropyl]-carbodiimide (EDC). In one embodiment of the invention dicarboxylic acid (preferably a length of C6-C10) are added to the protein solution. EDC is added to the mixture and allow the reaction to go. The concentration of the protein solution, the proportion of dicarboxylic acid and protein, the amount of EDC and time are all important for W is being result. EDC activates COOH groups and makes it possible for linking to free amino groups of the protein.

The control reaction indicates that the polymerization can be controlled, giving soluble polymers, insoluble particles or gels from the same reaction mixture. Can be obtained in more than 95% conversion of the initial concentration of protein in the polymer and up to 100% conversion in the gel.

The exception of the spacer of the dicarboxylic acid and the use of one EDC (described here) leads only to a partial polymerization for a period of from several hours to days with lower polymer yield in comparison with obtained when using dicarboxylic acid.

In General, a method in accordance with the invention will be carried out in solution. Preferably, the activating agent such as EDC are added to a solution of protein and a dicarboxylic acid. EDC can be in solution, for example, distilled water or it can be added to the protein solution of dicarboxylic acid in the form of solids such as powder. Although, in principle, it is also possible to first activate the EDC dicarboxylic acid, and then add the activated spacer to the protein solution, it was found in practice, does not give such good results as those obtained by addition of EDC to the mixture of protein and spacer.

To facilitate the application was is desirable to make the formulation of reagents as mixed dry powder, to which immediately before use, add water, saline or buffer solution. Protein and dicarboxylic acid cannot react without the addition of EDC, for this reason, in order to store the reagents as powders without the risk of premature reaction, it would be desirable to keep the protein powder/dicarboxylic acid separately from the powder EDC, for example placed in separate bags. In the preferred method of application is used with a syringe containing a solution of protein/dicarboxylic acid and powder EDC, and the solution and the powder is separated by a weak membrane. Pushing the plunger of the syringe, the user presses on the membrane to rupture, and the reagents are mixed immediately prior to use.

Application solutions on the site of the wound may occur through spillage, greasing or spraying solutions.

It would be desirable for the dressing material for wounds to deliver in the wound area a therapeutically active ingredients. Medications such as antibiotics, antiviral and anti-inflammatory agents, hemostatic agents, pain relievers and phages, can be added directly to the composition or through the media, which promote adsorption from wounds, such as liposomes. Active substances which promote or improve tissue healing, that can the same be entered for example, growth factors, agents that prevent the formation of scars, and agents that promote the development of blood vessels. By eliminating infection and adsorption of exudates can be reduced odor fetid wounds. However, the smell of the RAS may also be reduced/eliminated by introducing into dressings agents (e.g., charcoal), which adsorb volatile molecules responsible for the smell.

Entered active compound can be delivered to the wound site by leaching from the gel and by allocations from the gel during its decomposition. A key factor in determining the rate of release of the active substance is the softness/hardness of the protein polymer. Active compounds will be more easily flushed out of a softer polymer, because they don't stick with it as effectively as cross-linked protein molecules. Softer polymers will decompose at a faster rate due to the fact that the loosely coupled structure will allow moisture and enzymes to penetrate more easily.

In accordance with another aspect of the present invention provides nonwoven material obtained by the methods described above, i.e. dressings containing protein polymer formed by the reaction of protein with a polyfunctional spacer or its activated derivative.

Especially before Occitania chemistry of the present invention, as it was also found that gives protein polymers that are suitable for a number of other therapeutic applications.

Thus, in accordance with another aspect of the invention provides a method of formation of the protein polymer, which method comprises reacting the protein with a dicarboxylic acid or its activated derivative, provided that the protein is bovine serum albumin.

A further aspect of the invention is a method of obtaining a protein polymer, where the method comprises reacting the protein with alkylenediamines acid or its activated derivative.

The preferred protein is albumin, particularly serum albumin human.

With appropriate choice of reagents and reaction conditions can be derived products with a wide variety of properties. Thus, the protein polymer can be obtained in soluble form, in the form of insoluble particles or in the form of a gel. The shape of the gel can vary from very sticky until soft, but non-adhesive, and the rigidity can be increased with a certain step to very hard gels with low deformation. The parameters that can be modified to meet these differing results include the choice of protein source material, the choice of the spacer, the concentrations of various reactants, temperature, reactions and duration of the various reaction stages.

The rate of gelation can also vary in a wide range from seconds to minutes and hours by controlling the ratio of the reagents used for gel formation, and temperature.

The gelation reaction is best performed at moderately acidic pH (e.g. pH=5-6). However, it is often preferable to increase the final pH of the gel closer to physiological pH. There are several ways of controlling the pH of the final gel. One approach is to change the molar ratio of protein to dicarboxylic acid; low levels of dicarboxylic acids give gels with a pH close to physiological. The second approach consists in changing the molar ratio of protein to EDC; high level EDC results in gels with higher pH. For specialists in this area it is obvious that it is possible to find a balance conditions under which reaches the desired consistency of the gel for individual applications when the desired pH.

The gelation reaction can be bi-phase reaction in which the initial gelation followed by a second "cured" stage. The reaction will not proceed to the stage of vulcanization at a certain ratio HAS, dicarboxylic acid and EDC, for example, if the level of EDC is too low. Instead, the drop in pH observed after gelation, the gel is re-dissolved. Polaha is t, what is the minimum percentage of carboxylic acid groups must be activated with EDC in order to direct the reaction completely to the stage of vulcanization. Polymers with low pH generally less stable, because the spacer and HAS present unreacted carboxyl groups.

Adding additional connections can be useful. For example, adding a drug or other active compounds for controlled release (as described in relation to the bandages for wounds above) and/or other modifying agents, which modify the properties of the polymer, for example to remove water, to influence the flexibility to improve adsorption, feeling skin and aesthetics, mechanical and/or adhesive forces or to modify the profile of the degradation of the protein polymer.

Ethanol, glucose and glycerol are examples of compounds that can be added to protein gels of the present invention.

Ethanol is well known bacteriostatic agent may be added to improve the antibacterial properties of the gel, glucose to provide energy source and, therefore, promotion of cell growth, and glycerin to help prevent moisture loss and maintain the integrity of the gel in the wound.

Glucose can be especially useful for bandaging material according to the present image is the shadow for use in chronic wounds, because chronic wounds generally have insufficient blood supply, therefore, insufficient supply of energy and, thus, insufficient cell growth.

We found that the addition of ethanol, glycerol or glucose improves the consistency of the polymer by further reducing fragility.

Although it is possible to add the modifying agents to the HSA and dicarboxylic acid in one stage, in practice (using ethanol, glucose or glycerol), we found that it is more efficient to modify the percentage of HSA c modifying agent before mixing with the remaining unmodified HSA and the spacer of the carboxylic acid. Thus, the modifying agent is added to the aqueous HSA, and EDC is added to facilitate the reaction. The solution of the modified HSA and ethanol are added to a solution of unmodified HSA and dicarboxylic acid, and then this "gel-forming solution" is reacted with EDC to gel formation.

Since the modification of the protein is used to improve the physical properties of the protein polymer-modified HSA can be used as a therapeutic agent. Devoid of ligands albumin, for example, is available binding sites, which can trap and remove toxins, cytokines, etc.

The polymers can be obtained in a soluble form, and the uses low concentrations of protein. Soluble polymers are more easily obtained at neutral pH. Low concentrations and neutral pH is easily achieved by adding a suitable buffer, for example phosphate buffer solution. Soluble polymers suitable for parenteral delivery and have a number of applications as a means of delivery, such as drug delivery, delivery of contrast agents applicable to methods for forming images, or as a substitute or amplifier platelets (delivery of hemostatic agents).

Use for the treatment of cancer patients stimulates the need for substitutes platelet and/or amplifiers. One of the side effects of anticancer therapy is a sharp decrease in platelet count, or thrombocytopenia. The condition currently treated with transfusion of platelets isolated from the blood, but because of the chemotherapy regimens become more aggressive and because of increased use of bone marrow transplantation, requirements platelets grow. Moreover, platelets isolated from blood, have the potential transmission of viral infections, suffer from instability during storage and cause an immune response.

The terms "substitutes platelet" and "amplifiers platelets" are often used interchangeably or incorrectly, or for whom. Under the "substitutes platelets in the context of the present invention mean a complete replacement of platelets, which do not necessarily require the presence of platelets, obtained in a natural way. "Amplifiers platelets", on the other hand, may require natural formation of a tube of platelets at the wound site (and, therefore, the natural concentration of platelets may be required to be above the threshold level). Amplifiers platelets then aggregate in place trombotsitnoy tube, forming a clot, improving, thus, the activity of platelets in terms of blood clots. Substitutes/amplifiers platelets can be obtained in accordance with the present invention by immobilization coagulating agents or other active peptide derivatives on the surface of the polymer thus, in order to maintain their biochemical activity. In particular, protein polymers of the present invention can be conjugated with agents that promotirovat or regulate adhesion and platelet aggregation through specific receptors expressed on the surface of platelets. An example is GPllb/llla receptor, which interacts with fibrinogen, active peptides of fibrinogen and factor von Willebrand's disease. Methods of conjugating with fibrinogen include milirovanie Bel is a new polymer, activation of fibrinogen by hydrazide N-[multimediaphoto acid] and then conjugation of the activated fibrinogen through the thiol group with the protein polymer. Substitute/power platelets can be delivered by intravenous infusion and activated in place internal wounds in blood vessels.

As a means of delivery protein polymers suitable for slow and controlled release of drugs. Moreover, in the delivery of active agents or with good quality of their adsorption properties of protein polymers of the present invention can be useful for applying for detoxification.

Protein polymers may be naturally increase the delivery of medicines in the region of the body that are hard to aim independently. More preferably the protein polymers can be conjugated with one or more target components that have an affinity to a specific place in the body. Suitable target components can be antibodies. The antibody may act as a therapeutic agent in its own right, or one or more secondary agents may be attached, for example, cytokines radionuclides for targeted anticancer therapy, or vaccine, or genes. The target component may have the affinity to a single authority or estu disease, this may increase the delivery of secondary agent in this place and/or may alter the biodistribution of these agents, for example, forcing the agent to accumulate in a specific organ, for example in the liver, thereby allowing this body to be the target.

Similarly protein polymers of the present invention can be contacted with the target component and a contrasting agent. Contrast agents can be the metals used for the generation of magnetic resonance imaging or nuclear imaging or therapeutic agents in radiotherapy.

Insoluble protein particles can be obtained at higher concentrations of the spacer of the dicarboxylic acid with respect to an activated agent and/or increased reaction time while maintaining a low concentration of protein. Alternative insoluble particles can be obtained by dispersing soluble protein polymer of the present invention in organic solvents, such as acetone.

Using the method of the invention can be obtained gel protein polymer with different consistency from soft to hard) and with different adhesive force.

The non-adhesive protein gels of the present invention is suitable for preventing or inhibiting adhesion of the tissue after surgical procedures by way the of the barrier between neighboring tissue membranes. By adjusting the reagents and reaction conditions, the rate of degradation may be chosen, for example, that the polymer can be designed to degrade by the time of wound healing. The formation of the gel in place will ensure the full coverage of a separate area of the desired thickness. The gel can be applied as a thin film, or the composition may be molded into the cavity to fill the cavity.

Alternative adhesive gels of the present invention can be used for bonding fabrics together, for example for sealing inclusions, tears, holes and/or liquid or gaseous leaks in tissues. Well it is clear that the suturing and stapling the delicate tissues causes, in fact, their damage/weakening and subsequent problems such as leakage of fluids or bacterial infection. Were described adhesive agents, which provide a means of linking tissues. However, none of these compositions, as it was discovered, is not completely satisfactory. There is still a need for effective bioadhesive compositions that are truly safe and effective, and whose properties can easily be done in accordance with the nature of the tissue and the extent of the damage.

Protein gels are also suitable for coating prostheses and surgical instrumento is, for example, catheters or stents. Such a coating may have bioadhesive properties that contribute to the retention of the device in the desired position. The use of natural protein polymers and, in particular, HSA will reduce the risk of implant rejection of the natural protection of the body against the introduction of a foreign body.

Protein polymers of the present invention is suitable for coating glass and plastic dies for diagnosis (e.g., ELISA, ELISPOT) or for the purposes of processing, for example, when used for growth of cells, including stem cells.

Hard gels can be obtained using high levels of the spacer of the dicarboxylic acid and/or EDC. It is envisaged that hard gels of the present invention can be used to strengthen bone or cartilage, as implants, artificial bones or other prostheses. The gel can be obtained on site or preferowan in the form.

Hereinafter the invention will be described in greater detail for purposes of illustration, with reference to the following non-limiting Examples, which demonstrate that:

- Variation of the reaction conditions in terms of concentration and composition of components, pH and time can give different forms of polymers.

- Soluble polymers are more easily obtained at neutral pH with low con is antracene protein.

- Increase levels of the spacer and the activator in the reaction will be to give insoluble particles, which are also obtained when soluble polymers are mixed with organic solvents.

- An increase in the concentration of the protein and lowering the pH of the reaction network gels. Moreover, it is possible to modify the physical characteristics of the gel (soft-hard and adhesive properties) to change the ratio between the components of the gel, the protein concentration and pH or a combination of these factors. This is an important factor in obtaining gels for therapeutic applications, including dressings for wounds, gel implants and bioadhesive substances.

Reduction

DMSO - dimethyl sulfoxide

EDC - ethyl[dimethylaminopropyl]carbodiimide

EMCH - hydrazide, N-[multimediaphoto acid]

HSA serum albumin human

PBS - phosphate buffer solution

Description of the drawings

Figure 1 shows the allocation of the soluble polymer of the present invention, the gel-filtration on a column of Sepharose 6B, using standard conditions, in which the absorption was monitored at 280 nm.

Figure 2 shows the release of tetracycline from the gel of the present invention is a 45-hour period.

Example 1: Formation of water-soluble polymers

1.1The formation of the soluble polymer HSA using sabatinovka acid

Sabotinova acid (146 mg) in 2.5 ml of DMSO was added to 10 ml of 20% solution of HSA (BPL, Zenalb) and 20 ml of 0.01 M PBS buffer, pH=7,4 with stirring until then, until the solution became transparent. EDC (276 mg) in 7.5 ml of PBS buffer was added to the solution and stirred 16 hours (overnight). The resulting solution was centrifuged to remove a small amount of insoluble polymer. The soluble fraction was subjected to gel filtration on a column of Sepharose 6B, using standard conditions. The elution of protein was monitored by ANm. The results are shown in figure 1. Monomeric HSA eluted with ˜340 ml.

1.2To obtain soluble polymer HSA using adipic acid

Adipic acid, 26,3 mg in 1 ml of 50% ethanol was added to a mixed solution of 5 ml of 20% solution of HSA (BPL, Zenalb) and 25 ml of 0.01 M PBS buffer, pH=7,4 with stirring until then, until the solution became transparent. EDC, 69 mg in 4 ml of PBS buffer, was added dropwise to the solution with stirring. The resulting solution was stirred further for 2 hours. The resulting solution was centrifuged to remove a small amount of insoluble polymer. The soluble fraction was subjected to gel filtration on a column of Sepharose 6B (as in example 1.1 above)using standard conditions.

1.3The binding of fibrinogen to soluble HSA protein polymer for which Holocene substitute platelets

In this example, the replacement of platelets (amplifier) was obtained by immobilization coagulating factor, fibrinogen on the surface of HSA protein polymer thus, to save the biochemical activity of fibrinogen. Substitute platelets can be delivered by intravenous infusion and is activated in place internal wounds in blood vessels.

1.3.1To obtain soluble protein polymer

Sabotinova acid (30 mg) in 1.25 ml DMSO was added to 5 ml of 20% solution of HSA (BPL, Zenalb) and 15 ml of PBS buffer (0.01 M; pH=7,4) and stirred until then, until the solution became transparent. EDC (57 mg) in 4 ml of PBS buffer was added to a solution of HSA/spacer and stirred at room temperature for 3 hours.

Other dicarboxylic acids with different carbon chain lengths can be used instead sabatinovka acid in the above reaction.

1.3.2Milirovanie protein polymer

2-aminothiols (210 mg) was added as a solid to the polymer solution, followed by incubation in the dark at room temperature for 1.5 hours. The polymer was then absoluely gel-filtration in 0.01 M; pH=7.4 in PBS solution on a column of Sephadex G25 using standard conditions

1.3.3Activation of fibrinogen to connect with polymer

Fibrinogen (750 mg) in 10 ml of 0.05 M phosphate buffer was mixed with 2.5 ml of 100 mm periodate sodium is 0.1 M sodium acetate buffer, and incubated in the dark at room temperature for 30 minutes. The activated fibrinogen was then absoluely gel-filtration in 0.01 M; pH=7.4 in PBS solution on a column of Sephadex G25. Activated sugars reacted with hydrazide, in this example, the hydrazide N-[multimediaphoto acid] (EMCH), (11 mg) for 2 hours in the dark at room temperature.

1.3.4Conjugation of activated fibrinogen with protein polymer

The solution of activated EMCH-fibrinogen was added to the solution aminotoluene polymer and stirred over night. The resulting solution was centrifuged to remove a certain amount of insoluble material and then subjected to gel filtration in 0.01 M; pH=7.4 in PBS solution on a column of Sepharose 6B.

Example 2: Formation of insoluble particles

2.1The formation of insoluble particles in aqueous solutions

Insoluble polymer particles may be obtained by methods similar to the methods of example 1, but at higher concentrations of the spacer from a dicarboxylic acid and EDC and/or increased reaction time, while maintaining a low concentration of protein.

HSA (1 ml; 20%; BPL, Zenalb) and glutaric acid were mixed in 3 ml of distilled water in a molar ratio of 1/40. EDC in 1 ml of distilled water was added to the mixed solution in 1/20 molar ratio of HSA/EDC. The solution was stirred 3 hours at room temperature and then centrifuged. The precipitate was washed with distilled water and then dried.

2.2The formation of insoluble particles in organic solvents

Insoluble particles can also be obtained by dispersing soluble polymers obtained in example 1 above, in organic solvents, such as acetone.

One volume of a solution soluble polymer (example 1) was mixed with 10 volumes of acetone for 15 minutes at room temperature. The resulting particles can be collected by centrifugation or decantation.

Example 3: the Formation of gels of protein polymers

3.1Obtaining HSA polymer gel, using sabotinova acid and a high concentration solution of HSA

A solution of 48.5 mg sabatinovka acid in 1 ml of DMSO was added to 4 ml of 20% solution of HSA (BPL, Zenalb). The solution was stirred until then, until it became transparent. Solution was added 92 mg EDC in 2 ml of distilled water. The final concentration of HSA in the reaction was 114 mg/ml Final molar ratio of HSA/sabotinova acid/EDC was 1/20/40.

The resulting mixture formed a gel within 30 seconds after addition of EDC.

It was noted that in the experiment is equivalent to this example, but in the absence of a dicarboxylic acid, a gel was formed after 2 hours. Properties of the gels in this case were unsuitable for perawatan the x materials for wounds, which were hard, brittle properties that would make them difficult for application and removal. The gelation time on site would be too long for practical application.

3.2Obtaining HSA polymer gel, using sabotinova acid and a low concentration solution of HSA

Used the same experimental method, which is described in example 3.1, except that the final concentration of HSA was 72 mg/ml Final molar ratio of HSA/sabotinova acid/EDC was 1/20/40.

The resulting mixture formed a gel in less than 5 minutes.

3.3Obtaining HSA polymer gel, using adipic acid and a high concentration solution of HSA

Adipic acid, 35 mg, was dissolved in 4 ml of 20% solution of HSA (BPL, Zenalb). Solution was added 92 mg EDC in 2 ml of distilled water as before. The final molar ratio of HSA/adipic acid/EDC was 1/20/40.

The resulting mixture formed a soft gel after 2 minutes.

3.4Preparation of a gel containing hemoglobin

HSA (300 mg) and hemoglobin (100 mg), sabotinova acid (24,25 mg in 0.5 ml DMSO), EDC (46 mg in 1 ml distilled water) and 2 ml of PBSa (as above) were mixed together to obtain a final protein concentration of 80 mg/ml

The gel was formed after 10 minutes.

Example 4: Effect of the length of the spacer on the characteristics of a gel

In order to determine the effects of varying the chain length of the spacer from dicarboxylic acids, received the protein polymer gels using HSA at various concentrations and four different spacer from dicarboxylic acids, with EDC as activator.

The solution of dicarboxylic acid in DMSO (120 mcmole 250 ál or 90 mcmole 250 μl) was added to 1 ml of 20% aqueous solution of HSA in a molar ratio of dicarboxylic acid/HSA and 30/1 40/1. The solution was stirred at room temperature until, until it became transparent. An aqueous solution of EDC was then added in a molar ratio of EDC/dicarboxylic acid 2/1. The gelation time and the properties of the gels is described in detail in tables 1-3 below.

The gelation reaction is biphasic response: an initial gelation, there is an additional stage "vulcanization". The gelation time is associated with the original observed gelation, and the rigidity of the gel refers to the final state of the gel after the "vulcanization".

Table 1< / br>
The influence of the chain length of the dicarboxylic acid gelation time using 1/40/80 molar ratio of HSA/adipic acid/EDC
The HSA concentration (mg/ml)The gelation time (the EC)
Glutaric acid (C5)Adipic acid (C6)Cork acid (C8)Sabotinova acid (C10)
15140232119
14042252322
12749272724
10890453025
93130584230

Table 2< / br>
The influence of the chain length of the dicarboxylic acid gelation time using 1/30/60 molar ratio of HSA/adipic acid/EDC
The HSA concentration (mg/ml)The gelation time (sec)
Glutaric acid (C5)Adipic acid (C6)Cork acid (C8)Sabotinova acid (C10)
15146312923
14060353326
127754338 32
108140604537
93240956250

108
Table 3< / br>
The impact of the HSA concentration and chain length of the dicarboxylic acid on the properties of the gel when using 1/40/80 molar ratio of HSA/adipic acid/EDC
The HSA concentration (mg/ml)Properties gel
Glutaric acid (C5)Adipic acid (C6)Cork acid (C8)Sabotinova acid (C10)
151Hard rubber gel, slightly cloudyMuddy, hard rubber gelMuddy, very hard, brittle gelVery hard, dull, brittle gel
140Transparent, hard, rubber gelMuddy, hard rubber gelMuddy, very hard, brittle gelVery hard, dull, brittle gel
127Transparent, Srednerussky, rubber gelMuddy, hard rubber gelMuddy, hard, slightly gel rubberHard, white, brittle gel
Very soft transparent gelInitially very soft gel, hard after 3 minutes. Slightly cloudyOriginally Srednerussky muddy rubber. After 4 minutes of very hard, white, brittle gelHard, white, brittle gel
93Very soft transparent gelOriginal soft gel, medium, brittle after 3 minutes. Slightly cloudyInitially turbid, srednelanski, rubber. After 4 minutes of very hard, white gelSrednerussky, white, brittle gel

Each HSA concentration, the gelation time decreases, and the gels become in General more rigid, less rubber and more turbid with increasing chain length of the dicarboxylic acid. Increasing the concentration of HSA reduces the gelation time and increases the stiffness of the gels.

Example 5: Control time of gel formation and properties of gel

Various applications of gels require different times of gelation and gel consistencies. Gels can be formed in seconds or much longer periods of time. Gels can be extremely soft and sticky or hard rubber. There are several approaches to controlling these parameters, and for any application can be used by any or all of the following under the odes. All gels, described below, was transparent, if not stated otherwise.

5.1Control time of gel formation and properties of the gel by varying the molar ratio of HSA to the spacer of the dicarboxylic acid

The gels were obtained by dissolution of glutaric acid (GA) in an aqueous solution of HSA (20% USP) at room temperature, followed by addition of EDC solution in distilled water to activate the gelation reaction. The mixture in the process of gelation carefully turn it over several times to ensure complete mixing.

The molar ratio of HSA to glutaric acid ranged from 1/0 to 1/40 at two concentrations of EDC. Experimental results are presented in tables 4 and 5 below.

td align="center"> Soft
Table 4< / br>
The impact of changes in HSA/GA molar ratio (molar ratio of HSA to EDC 1:35)
HSA/GAThe gelation timepH gelProperties gel
1/0More than 2 hours7,1Soft
1/3,59 min6,8Average
1/55 min 25 sec6,5Average
1/103 min 15 sec5,6
1/203 min 40 secSoft gel, re-dissolved in 3 min
1/40The gel was not formed

Table 5< / br>
The impact of changes in HSA/GA molar ratio (molar ratio of HSA to EDC 1:70)
HSA/GAThe gelation timepH gelProperties gel
1/0About 30 min7,6Average
1/3,54 min 30 sec7,2Srednerussky
1/52 min 45 sec7,1Srednerussky
1/101 min 30 sec6,2Hard
1/201 min 5 sec5,3Average
1/401 min 15 secSrednelanski gel, re-dissolved in 30 min
1/501 min 45 secSoft gel, re-dissolved in 5 min

Initially, the increased levels of glutaric acid reduces the gelation time idet more rigid gels. However, at higher levels of glutaric acid gels become unstable, which can be compensated by higher levels of the EDC. This is discussed in example 6.

5.2Control time of gel formation and characteristics of the gel by varying the concentration of HSA

Gels were prepared using the method described in example 5.1. Used the molar ratio of HSA to glutaric acid 1/5 and the molar ratio of HSA to EDC 1/70. The HSA concentration was varied from 182 mg/ml to 120 mg/ml the Results are presented in table 6 below.

Table 6< / br>
The effect of the concentration of HSA on the gelation time and the rigidity of the gel
[HSA], mg/mlThe gelation timeThe rigidity of the gelpH gel
1822 min 27 secHard7,1
1662 min 45 secSrednerussky7,1
1504 min 12 secAverage7,2
1355 min 55 secSrednelanski7,3
1207 min 15 secAverage7,2

The decrease in the concentration of HSA leads to a longer time heliopaths the I and softer gels.

5.3Control time of gel formation and characteristics of the gel at varying molar ratio of HSA to EDC

Gels were prepared as described in example 5.1. Used the molar ratio of HSA to glutaric acid 1/10, and the final concentration of HSA was 166 mg/ml Molar ratio of HSA to EDC varied from 1/35 to 1/80. The results are presented in table 7 below.

Table 7< / br>
The effect of varying the molar ratio of HSA/EDC
The molar ratio of HSA/EDCThe gelation timepH gelThe rigidity of the gel
1/353 min 5 sec5,6Soft
1/501 min 50 sec5,9Average
1/601 min 32 sec6,1Hard
1/701 min 23 sec6,2Hard
1/801 min 5 sec6,6Very hard

Table 7 comparison of tables 4 and 5 above shows that higher levels of the EDC result in shortening the time of gelation and more rigid gels.

5.4Control time of gel formation and characteristics of the gel by adding ethanol, glucose and glycerol

Another important approach was to obtain "a gelling solution" HSA at initial modification of HSA by reaction with a reagent, such as ethanol, glucose or glycerol (each of which has-Oh groups), in the presence of low concentrations of EDC. Obtaining a gelling solution of HSA by the reaction with ethanol is described below.

5.4.1Obtaining a gelling solution of HSA by the reaction with ethanol

Ethanol was added dropwise to a stirred aqueous 20% solution of HSA. The solution was stirred until then, until it became transparent. To the solution was added solid EDC (in a molar ratio of HSA/EDC 1/15) and stirred at room temperature for at least 2 hours. Glutaric acid was dissolved in 20% aqueous HSA and stirred at room temperature for 30 minutes.

To obtain the final "gelling solution" the solution of the modified HSA/ethanol was mixed with a solution of unmodified HSA/glutaric acid in a volume ratio of 1/1 and stirred at room temperature for 30 minutes. The final molar ratio of HSA to glutaric acid was 1/5.

This mixture of gel-forming solution was reacted with EDC to gel formation, as in the previous examples.

The molar ratio solution of ethanol/HSA varied from 1/7 till 1/14, the results are presented in table 8 below.

Table 8< / br>
The effect of volume ratio of the modified HSA/ethanol on the gelation time and the rigidity of the gel
The volumetric ratio of HSA solution/ethanolThe molar ratio of HSA/EDC*The gelation timeThe rigidity of the gel
7/11/351 min 45 secHard
8/11/352 min 40 secHard
10/11/351 min 50 secSrednerussky
14/11/352 min 30 secSoft
Without ethanol1/355 min 30 secSrednerussky
(*Molar ratio of HSA to EDC added to the reaction gelation)

First, the reaction of HSA with ethanol leads to an overall reduction of the time of gelation. At low levels of ethanol are more soft gels. However, more than 10% vol./about. ethanol leads to more rigid gels. In these gels have not found any fragility; despite their rigidity, they remained elastic.

The experiments described in example 5.1, were repeated using a gelling solution of ethanol/HSA obtained as described is ANO above, with 10% vol./about. ethanol, and a molar ratio of HSA/glutaric acid in the range from 1/0 to 1/40. The results are presented in tables 9 and 10 below.

Table 9< / br>
The influence of molar ratio of HSA/glutaric acid using ethanol-modified HSA (at a molar ratio of HSA/EDC 1/35)
The molar ratio of HSA/GAThe gelation timepH gelThe rigidity of the gel
1/0About 30 min7,5Soft
1/3,53 min6,7Average
1/51 min 50 sec6,4Average
1/101 min5,5Srednelanski
1/2055 sec4,8Very soft
1/40The gel was not formed

Table 10< / br>
The influence of molar ratio of HSA/glutaric acid using ethanol-modified HSA (at a molar ratio of HSA/EDC 1/70)
The molar ratio of HSA/GAIn EMA gelation pH gelThe rigidity of the gel
1/08 min7,7Soft
1/3,51 min 30 sec7,3Average
1/51 min 5 sec6,9Srednerussky
1/1032 s5,6Hard
1/2025 sec5,1Average
1/4023 sec4,3Soft

5.4.2Obtaining a gelling solution of HSA in the reaction with glucose

Gel-forming solution prepared as in example 5.4.1, replacing the ethanol glucose, in the final molar ratio of HSA/glucose 1/15 and the final molar ratio of HSA/glutaric acid 1/5.

The resulting gels were softer than those without glucose, and the gelation time decreased.

5.4.3Obtaining a gelling solution of HSA adding glycerin

Glycerol was added to 20% HSA solution (USP) in volume percent of from 0 to 16.7. The gels were then obtained using the method described in example 5.1.

The addition of glycerol decreased the gelation time and, as has been shown, slowing the drying of the gel, when he was left nanocrystal at room temperature for a period of two weeks.

5.4.4u> Obtaining a gelling solution of HSA adding ethanol or glucose directly into the gelling solution

If the ethanol or glucose was added directly to the HSA solution and then used for the formation of gels by the method described in example 5.1, a similar but less pronounced effect was observed in comparison with examples 5.4.1 and 5.4.2, respectively, which was included preliminary modifications HSA additives.

Example 6: Effect of increasing level of dicarboxylic acids on the stability of the resulting gel

Increased levels of glutaric acid in gel-forming solution, as has been shown, leads either to the formation of the original medium to hard gels, which will eventually become soft gels, or soft gels, which re-dissolve, forming viscous solutions. The control of these processes of dissolution can be a useful way of controlling drug delivery in various applications described here.

The gels were obtained following the method described in example 5.1. The molar ratio of HSA to glutaric acid varied from 1/5 to 1/35 when two molar relationship HSA to EDC. The results are presented in table 11 below.

As the ratio of glutaric acid was increased, the gelation time decreased up to the point at which the gel is not formed. Prohm is filling levels gave gels, which was re-dissolved, forming viscous solutions when standing. This, as has been shown, is the result of changes in pH during the reaction. At low levels of glutaric acid pH gel-forming solution was raised to 6-7 after adding EDC, until then, until it formed a gel. At high levels of glutaric acid pH initially rose, then fell to acidic pH 5-6, forcing soft gel re-dissolving or preventing the formation of a gel.

Table 11< / br>
The influence of the level of glutaric acid on the stability of the resulting gel
The molar ratio of HSA/GA/EDCThe gelation timeProperties gel
1/5/359 min 20 secTransparent medium to soft gel
1/10/355 min 25 secSoft gel, becoming viscous solution after 10 min
1/15/35-----------The gel was not formed
1/10/503 minVery soft gel, becoming very hard after 5-25 minutes, returning to the average gel over night
1/15/502 min 45 secVery soft gel, becoming very indispute 4-8 min, returning to the soft gel over night
1/20/502 min 30 secSoft gel, becoming medium to hard after 4 min, forming a viscous solution in 1 hour
1/25/502 min 30 secSoft gel, becoming medium after 4 min, forming a viscous solution after 30 min
1/30/503 minVery soft gel, becoming viscous solution after 7 min
1/35/50-----------The gel was not formed

Example 7: Controlling the pH of the gel

The gelation reaction is best performed at acidic pH. It is possible to increase the pH of the final gel closer to physiological pH. There are two ways of controlling the pH of the gel. One approach is to change the molar ratio of HSA to dicarboxylic acid; low levels of dicarboxylic acids give gels with a pH closer to physiological. The second approach consists in changing the molar ratio of HSA to EDC, great levels EDC result in gels with higher pH. For specialists in this area it is obvious that it is possible to find a balance conditions under which reaches the desired consistency of the gel for individual use at the desired pH.

7.1Controlling the pH of the gels using a variety of concentric and dicarboxylic acids

Gels were prepared by dissolving glutaric acid 20% solution of HSA (USP) and adding a solution of EDC in distilled water to obtain final concentration of HSA 166 mg/ml Used the molar ratio of HSA to EDC 1:35 and 1:70. The results are presented in tables 4 and 5 above.

When all of these levels EDC gels can be formed using a molar ratio of HSA to glutaric acid 1:20 or less. At higher levels dicarboxylic acid gels are unstable if they are formed, as discussed in example 6. the pH Values of the gels in the range from 5.3 to 7.6 were obtained.

7.2Controlling the pH of the gels using different levels of EDC

Gels were prepared by dissolving glutaric acid 20% solution of HSA (USP) in a molar ratio of HSA to glutaric acid 1:10. The EDC solutions in distilled water were added to obtain a final concentration of HSA 166 mg/ml and the molar relationship HSA to EDC from 1:35 to 1:80. The results are presented in table 6 above.

the pH Values of the gels from 5.6 to 6.6 were achieved with varying levels of EDC. This is also supported by comparing the data of tables 4 and 5 above. Increasing levels of EDC in gelling of the mixture also leads to shorter gelation times and more rigid gels.

Example 8: Getting bioadhesive substances

Bioadhesive gels were prepared either as liquid or as dry powder. the DRS elasticity was measured, placing a liquid or powder between the two pieces of meat (3 cm3steak). One piece of meat was attached to the comb and held in place by a clamp and tripod. The cargo was attached to the second bottom piece of meat for measuring the strength of elasticity. Meat incubated at 37°C for 5 minutes before adding weights.

8.1HSA (4 ml 20%; BPL, Zenalb) was mixed with glutaric acid and EDC in the ratio 1/50/100 respectively.

The measured force of elasticity was 63 mg/mm2.

8.2The formulation is a dry powder was prepared by mixing 200 mg of liofilizirovannogo HSA (Sigma) with glutaric acid and EDC in a molar ratio or 1/50/100 or 1/60/120 respectively.

The force of elasticity increased with increasing ratio of the spacer and the EDC.

1/50/100 mixture gave strength elasticity ˜180 mg/mm2.

1/60/120 mixture gave strength elasticity ˜280 mg/mm2.

Example 9: the Release of drug from the gel (tetracycline)

To 1 ml of 20% solution of HSA was added 150 μl of 10 mg/ml tetracycline in ethanol. Gels were formed as described in the previous examples above, using the molar ratio of HSA/glutaric acid/EDC 1/30/60 or 1/40/80 respectively. The gel was left for the night, before to be placed in a vial containing 5 ml of distilled water. Release of tetracycline with time was measured at 364 nm (figure 2).

Note the p 10: Stability HSA gelling solutions

10.1Stability of modified ethanol HSA gelling solution at 4°and at room temperature

Modified ethanol HSA gelling solution (obtained as described in example 5.4.1) was filtered under sterile conditions through a 0.22 μm filter. Half of the solution was kept at 4°and the other half at room temperature in sealed vials. At 0, 7, 21 and 28 days, aliquots of the solutions were reacted with an aqueous solution of EDC, and the gelation time, the characteristics of the gel, the pH and the stability of the gel were compared.

Table 12< / br>
Storage gelling solution at 4°
DayThe gelation timeFeatures gel
02 min 10 secTransparent Srednerussky gel
72 min 15 secTransparent Srednerussky gel
212 minTransparent Srednerussky gel
282 min 15 secTransparent Srednerussky gel

All the obtained gels were kept in sealed vials at 37°From 14 days to compare the stability of the gels; none of them showed any signs of damage or bacterial what about the growth during this period.

Table 13< / br>
Storage gelling solution at 4°
DayThe gelation timeFeatures gelpH gel
02 min 10 secTransparent Srednerussky gel6,9
72 min 10 secTransparent Srednerussky gel6,9
212 minTransparent Srednerussky gel7,0
282 min 10 secTransparent Srednerussky gel6,9

These data demonstrate that the modified ethanol HSA gelling solutions are stable for at least 4 weeks at 4°and at room temperature.

10.2The stability of the glucose-modified HSA gelling solution at 4°and at room temperature

The above experiment was prepared using a modified glucose HSA gelling solution (as described in example 5.4.2). The solution is stored at room temperature, as has been shown, remained stable for 2 weeks. The solution is stored at 4°Since, as we have seen, remained stable for at least 4 weeks.

10.3the stability of the solution of HSA/glutaric acid at 4° C and at room temperature

The HSA solution (200 mg/ml) and glutaric acid (molar ratio of HSA/glutaric acid 1/37), as has been shown, using the techniques described in example 10.1, remained stable for at least 4 weeks at 4°and at room temperature.

10.4The stability of the solution of HSA/adipic acid at 37°and at room temperature

The HSA solution (200 mg/ml) and adipic acid (molar ratio of HSA/adipic acid 1/30), as has been shown, using the techniques described in example 10.1, remained stable for at least 3 weeks at 37°and at room temperature.

Example 11: the Stability of the resulting gels

Gels with molar ratio of HSA/glutaric acid/EDC 1/40/80, 1/50/100, 1/60/120 and 1/70/140 kept at 4°C, room temperature and at 37°C in sealed vials for 6-week period. All gels stored at 4°and at room temperature, remained stable for 6 weeks, although the turbidity of the gels with a high molar ratio slightly increased after 4 weeks. All gels stored at 37°C remained stable for 2 weeks. Within 3 weeks of these gels was increased rigidity and become more turbid. None of the gels did not show any signs of bacterial growth.

Example 12: Drawing of g is La to place

Holes (2 cm3and 0.5 cm deep) cut in a piece of pig skin in vitro. Gels (obtained as described in example 5.1 above) was formed in place in the hole, covering vapor permeable membrane (e.g., Tagaderm, 3M), and incubated at 37°C. the Gels remained soft and not dried out. They are easily removed from "wounds", being attached to the membrane.

1. A method of obtaining a dressing material for wounds, including the production of protein polymer by reaction of the protein with a spacer of alkylenediamines acid formula

in which n is from 3 to 8, or its activated derivative.

2. The method according to claim 1, in which the protein polymer is formed on site (in situ).

3. The method according to claim 1, in which the protein polymer is formed before application.

4. The method according to claim 3, in which the supporting substrate is introduced into the bandages.

5. The method according to claim 1, further comprising the application of dressings for wounds permeable membrane.

6. The method according to claim 1, wherein the protein is albumin.

7. The method according to claim 6, in which albumin is serum albumin human.

8. The method according to claim 1, wherein the protein is a recombinant product.

9. The method according to any of the preceding paragraphs, in which the dicarboxylic acid activeroles facilitate reaction with protein molecules.

10. The method according to claim 9, in which the dicarboxylic acid is activated with carbodiimide activating agent.

11. The method according to claim 3 or 4, in which dressings for wounds is in the form of a gel layer.

12. Dressings for wounds containing protein polymer obtained by reaction of the protein with a spacer of alkylenediamines acid formula

in which n is from 3 to 8, or its activated derivative.

13. Dressings for wounds indicated in paragraph 12, which contains a bandage impregnated with protein polymer.

14. Dressings for wounds indicated in paragraph 12, where the dressing material for wounds is in the form of a gel layer.

15. Dressings for wounds on any of PP-14, which further comprises one or more therapeutically active agents.

16. Dressings for wounds indicated in paragraph 15, in which therapeutically active agents selected from the group consisting of antibiotics, antiviral drugs, anti-inflammatory agents, analgesics, hemostatic agents, phages, growth factors, agents that prevent the formation of scars, odor adsorbing agents, and agents that promote angiogenesis.

17. A method of obtaining a protein polymer comprising the reaction of albumin with a spacer from alkyl is dicarbonate acid formula

in which n is from 3 to 8, or its activated derivative.

18. The method according to 17, in which albumin is serum albumin human.

19. The method according to any of 17 or 18, in which the dicarboxylic acid is activated with carbodiimide activating agent.

20. The method according to claim 19, in which the dicarboxylic acid is activated ethyl[dimethylaminopropyl]-carbodiimide.

21. Protein polymer obtained by the reaction of albumin with a spacer of alkylenediamines acid formula

in which n is from 3 to 8, or its activated derivative.

22. Protein polymer according to item 21, which is in the form of a solution.

23. Protein polymer according to item 21, which is in the form of insoluble particles.

24. Protein polymer according to item 21, which is in gel form.

25. Protein polymer according to item 21, where the protein polymer anywhereman with one or more coagulating agents or active peptide derivatives.

26. Protein polymer according to item 21, which anywhereman with a therapeutically active agent or its precursor, or with a contrasting agent and the target component having affinity to a specific place in the body.

27. Set for the manufacture of bandages for wounds in the under 12, where the kit comprises a first composition and a second composition, the first composition and the second composition are contained in separate containers in a manner to prevent reaction between the first composition and the second composition.



 

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15 cl, 4 dwg

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely treatment of wounds of soft tissues. For this purpose apply gel Kollapan on a traumatic surface in amount of 1 ml on 8-12 cm2. The way provides expansion of an arsenal of agents for treatment of wounds, by optimisation of reparative processes at healing of superficial and deep damages of soft tissues.

EFFECT: expansion of arsenal of agents for treatment of wounds, by optimisation of reparative processes at healing of superficial and deep damages of soft tissues.

1 ex

FIELD: nanotechnologies.

SUBSTANCE: invention relates to laser equipment used for the purposes of nanotechnologies, particularly, to the methods of nanostructurisation of bulk bio-compatible nanomaterials by laser radiation. Nanostructurisation of the aforesaid materials is performed by laser irradiation of colloidal water-protein solution of carbon nanotubes till evaporation of the solution liquid component.

EFFECT: varying the properties of produced materials in a wide range and remote irradiation provides for biological purity of products.

3 dwg

FIELD: medicine.

SUBSTANCE: device has metal, non-metal or ceramic body coated with active ingredients set usable for producing bones or has the ingredient set as a component. The active ingredients set has at least one structural ingredient based on extracellular substance, at least one ingredient for promoting cell migration, at least one ingredient as adhesive agent and at least one ingredient for supporting growth or maturation. Hollow metal body is shaped as cylinder of lattice structure and preferentially manufactured from titanium or titanium alloy.

EFFECT: high mechanical stability when preserving high active ingredient effectiveness; no adverse immune side responses.

5 cl, 9 dwg, 2 tbl

FIELD: medical engineering.

SUBSTANCE: device has a set of active ingredients. At least one structural ingredient based on extracellular substance, minimum one ingredient providing cell migration, minimum one adhesive ingredient, minimum one ingredient providing growth or maturation are used for producing the implantable prosthesis. External endoprosthesis surface is minimum partially coated with a set of active ingredients. The endoprosthesis has also minimum one internal cavity filled with a set of active ingredients.

EFFECT: long service life; accelerated engraftment in organism; greater load applied at earlier stage.

4 cl, 4 dwg, 2 tbl

FIELD: medicine.

SUBSTANCE: surgical thread is provided with inclined protrusions-notches formed as conical burrs with sharpened plastoelastic ends and sequentially positioned lengthwise of thread. Burrs are positioned at one or both sides of thread section (for example in staggered arrangement) with alternating inclination of burrs. Thread is not attached to surgical needle and is introduced into soft tissues through injection needle clearance.

EFFECT: increased efficiency by using protrusions formed as burrs allowing rupture strength of thread to be kept and functional designation, that is, usage in conducting of aesthetic cosmetic operations, to be fulfilled.

6 cl, 8 dwg

Surgical ligature // 2267332

FIELD: medicine, surgery.

SUBSTANCE: the suggested surgical ligature includes polyamide ligature with film covering based upon copolymer at additions of medicinal preparations. Ligature is being a nucleus out of polyamide twisted or woven thread with membrane out of copolymer being that copolyamide of E-caprolactam and hexamethylene diammonium adipinate and containing medicinal preparations - biologically active substances based upon plant biomass of Aralia family and doxicillin, at a certain ratio of components. The ligature in question is of high rupture loading and low lengthening that makes it more convenient in application. Ligature is more elastic and less traumatic.

EFFECT: higher efficiency of application.

10 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: medical bandage from chemically modified cellulose, which contains the immobilized chitosan, and which has the enzymes attached to it via chemical bonds, among those proteolytic, elastolytic and collagenolytic that constitute the enzyme complex of crab hepatopancreas. Also, the method of bandage preparation is described.

EFFECT: medical bandage shows improved mechanical properties.

2 cl, 1 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: disclosed are peptides derived from proenzyme forms of matrix proteinases which represent inhibitors of matrix proteinases. Amino acid sequence is disclosed in description. Described are composition for stimulation of healthy skin formation, containing therapeutically effective amount of peptides. Also disclosed are dressing for wounds, method for stimulation of healthy skin formation and wound healing. Disclosed is using of composition in production of drug for wound healing.

EFFECT: new anti-aging and wound-healing agents.

15 cl, 28 dwg, 6 tbl, 7 ex

FIELD: medicine, in particular agents for bone defect compensation.

SUBSTANCE: in the first embodiment claimed material includes hydroxyapatite with tricalcium phosphate and additionally it contains osteogenesis-inducing composition of non-collagen bone tissue proteins in specific component ratio per 100 g of material. In the second embodiment claimed material includes hydroxyapatite with tricalcium phosphate and/or collagen and additionally it contains osteogenesis-inducing composition of non-collagen bone tissue proteins. In the third embodiment claimed material includes hydroxyapatite with tricalcium phosphate and/or collagen and additionally it contains osteogenesis-inducing composition of non-collagen bone tissue proteins and double distilled water in specific component ratio per 100 g of material.

EFFECT: material with high osteoplastic ability.

9 cl, 3 ex, 6 dwg

FIELD: medicine.

SUBSTANCE: device has perforated polymer film from polycarboxyl-polycarbonate polymer having spongious coating. The spongious coating is not thicker than 1 mm and has native lyophilized collagen of type I impregnated with healthy donor blood serum having AB(IV) Rh- Kell- phenotypes. The biological bandage is applied 1-2 times a day after burn injury, that results in full-scale epithelium recovery without hypertrophic cicatrix being formed.

EFFECT: accelerated epithelialization when compared to self-standing treatment process.

3 dwg, 1 tbl

FIELD: medicine, pharmaceutical industry, pharmacy.

SUBSTANCE: invention relates to wound-healing collagen sponge. Wound-healing collagen sponge with the definite content of Bergenia pacifica black leaves extract is prepared by sublimation of mixture of 2% collagen solution, purified water and Bergenia pacifica black leaves dry extract for a definite time followed by additional drying of prepared plates and conditioning under the definite conditions. Sponge possesses the expressed wound-healing activity in wound injuries.

EFFECT: valuable medicinal properties of sponge.

7 tbl, 3 ex

FIELD: medicine, oncology.

SUBSTANCE: the present innovation deals with local chemotherapy of malignant cerebral tumors. Thus, after removing the tumor one should apply a collagen hemostatic sponge with a cavity inside it with a powder-like chemopreparation into tumor's bed, then the cavity should be covered with another fragment of collagen sponge. The innovation enables to minimize toxic manifestations of chemotherapy at applying maximal concentration of anti-tumor preparation.

EFFECT: higher efficiency.

6 dwg, 2 ex

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