Biogel

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics. There are developed agent for forming a biogel, biogels for hemostasis, wound closure, tissue engineering and targeted drug delivery. The agent contains a soluble carrier whereon a number of fibrinogen-binding groups is immobilised. The biogel that contains fibrinogen molecules and a number of soluble carriers applicable for intravenous and/or local administration; each carrier comprises a number of fibrinogen-binding groups immobilised on the carrier, and each fibrinogen molecule is bound to at least two fibrinogen-binding groups so that the fibrinogen molecules occurs to be bound to each other through the carriers by non-covalent bonds between the fibrinogen-binding groups and the fibrinogen molecules. The biogel containing fibrin monomers and a number of soluble carriers applicable for intravenous and/or local administration, wherein each carrier comprises a number of fibrinogen-binding groups immobilised on the carrier, while the fibrin monomers are bound to each other through the carriers by non-covalent bonds between the fibrinogen-binding groups and the fibrinogen monomers. The biogel containing fibrin and a number of soluble carriers applicable for intravenous and/or local administration, wherein each carrier comprises a number of fibrinogen-binding groups immobilised on the carrier with the fibrin monomers in fibrin are covalently bound to each other by peptide bonds, and the fibrin monomers in fibrin are bound to each other through the carriers by non-covalent bonds between the fibrinogen-binding groups and the fibrinogen monomers. A method for forming the biogel involving a contact of the fibrinogen molecules with a number of soluble carriers. A method for hemostasis by topical administration of the biogel at a haemorrhage or a wound. Using a number of soluble carriers applicable for intravenous and/or local administration. A pharmaceutical formulation for topical administration containing the biogel, agent or a number of soluble carriers.

EFFECT: using the declared invention enables preparing the agents requiring no toxic reagents to be used, have a minimal risk of allergic reactions, and are easy to prepare and use.

2 tbl, 4 dwg, 5 ex

 

The present invention relates to a Biogel and sets, the agents and methods of forming the Biogel. In a preferred aspect, the Biogel is a tissue adhesive. Specified Biogel or tissue adhesive can be used on a range of assignments, including hemostasis, closure of wounds, tissue engineering or targeted drug delivery.

In the process of blood clotting fibrinogen is transformed into fibrin, with the participation of thrombin. Fibrinogen consists of two sets of three different chains (α, β and γ), linked together by disulfide bonds. Together, these chains form a Central globular domain (E-domain), coupled with the two extreme globular domain (D-domain). Thrombin cleaves four peptide bond arginine-glycine in the Central E-domain of fibrinogen with the release of a peptide from each of the two α-chains and b-peptide each of the two β-chains. Peptides a and b are called fibrinopeptide. Molecule of fibrinogen without these fibrinopeptides, called fibrin monomer. The fibrin monomers spontaneously assemble in an ordered fibrous structures called fibrin. Fibrin is stabilized due to the formation of covalent cross-links between side chains of different molecules in the fiber of fibrin. Peptide bonds are formed between certain of the side chains of glutamine and lysine in the reaction is pereaminirovania, catalyzed by Factor XIIIa.

Platelets after activation also form an essential part of the blood clot. Platelets are attached to the open surface wounds and become activated. The glycoprotein GPIIb/IIIa platelet membrane undergoes a conformation change that allows it to bind fibrinogen. Fibrinogen can bind with more than one platelet, resulting in platelet aggregation with each other. Aggregates of platelets form the basic structure of the thrombus formed within the mesh of fibrin.

Fibrin tissue glue (FTC) is the name of the products obtained by simulating the last step of the coagulation cascade, resulting in the formation of fibrin clot. Available for purchase sets FTC provide you with quick, strong, biorstwami gels, which are used for hemostasis, drug delivery and as surgical adhesives and coatings for fabrics. Fibrinogen, Factor XIII, thrombin and calcium ions, usually delivered via injection device, in which the fibrinogen and Factor XIII is separated from calcium ions and thrombin during storage. The components are mixed during the injection of the syringe causes thrombolyse fibrinogen to form fibrin, which spontaneously going into the gel, which then activerow the hydrated ions of calcium Factor XIII to form a cross-linkage. However, in many FTC used bovine thrombin, which can cause allergic and autoimmune reactions in patients.

Zhang and others (Bioconjugate Chem. 2002 (13): 640-646) describe obtaining hydrogels on the basis of fibrinogen using photoactivated release of calcium ions from liposomes and subsequent activation catalyzed by transglutaminases formation of cross-links in fibrinogen. However, obtaining such hydrogels is difficult and requires specialized composition of the liposomes and Factor XIII.

Hidas and other (Urology 67(4), 2006: 697-700) describe the application of tissue adhesive based on albumin and glutaraldehyde in a seamless nephron-energy-saving surgery. Mixed bovine serum albumin and glutaraldehyde. Interaction with glutaraldehyde causes linking to each other lysine molecules of bovine serum albumin, extracellular matrix proteins and surfaces of the cells, which leads to the formation of strong covalent bonds.

However, the disadvantage of this glue is the toxicity of glutaraldehyde and the risk that bovine serum albumin can cause an allergic reaction in patients.

Accordingly, there is a need to create a gel or fabric glue, which does not require the use of toxic agents, which minimizes the risk of allergic reaction and which can easily in order to obtain the component, which can easily be stored in a stable condition.

According to the present invention proposed a kit form (receipt) of Biogel, which includes: the set of binding fibrinogen groups (molecules)immobilized on each carrier; and fibrinogen, with each molecule of fibrinogen can bind at least two binding fibrinogen molecule.

The term "Biogel" in this application includes a gel containing one or more components that are natural or recombinant biological molecules (or obtained by chemical synthesis of biological molecules, or molecules that are derived from biological molecules (for example, derivatives which retain one or more functions of biological molecules).

The tests can be obtained by making contact fibrinogen and media. Because each carrier immobilized many fibrinogen binding groups, and since each molecule of fibrinogen can bind at least two binding fibrinogen group, fibrinogen molecules are linked together via the carriers. Between the molecules of fibrinogen and fibrinogen binding groups are formed non-covalent connection.

Accordingly, the present invention also provides a method of forming a Biogel that VK is uchet implementation of contact of the molecules of fibrinogen with many carriers, each carrier includes a variety of binding fibrinogen groups immobilized on the specified carrier, and each molecule of fibrinogen can bind at least two binding fibrinogen molecules and, thereby, the binding molecules of fibrinogen through the media due to the formation of non-covalent bonds between the fibrinogen binding groups and molecules of fibrinogen.

Additionally, the present invention provides a Biogel, which includes molecules of fibrinogen and many carriers, with each carrier includes a variety of binding fibrinogen groups immobilized on the carrier, and each molecule of fibrinogen is associated with at least two binding fibrinogen groups thereby, the binding molecules of fibrinogen through the media due to the formation of non-covalent bonds between the fibrinogen binding groups and molecules of fibrinogen.

Instead of binding fibrinogen groups, the media can include many predecessors fibrinogen binding groups immobilized on each carrier, each precursor fibrinogen binding group may be converted into the binding fibrinogen group. For the formation of Biogel with the use of such media, you must turn predecessors svyazyvayus the fibrinogen groups in binding fibrinogen group so to bind fibrinogen group could then contact the fibrinogen molecules.

Accordingly, the present invention also provides a kit for forming a Biogel, which includes: multiple media, multiple predecessors fibrinogen binding groups immobilized on each carrier, each precursor fibrinogen binding group may be converted into the binding fibrinogen group; and fibrinogen, with each molecule of fibrinogen can bind at least two binding fibrinogen group.

Additionally, the present invention provides a method of forming a Biogel, which includes: providing a variety of carriers, each carrier includes many predecessors fibrinogen binding groups immobilized on the carrier; the transformation of the precursor fibrinogen binding groups in fibrinogen binding group; and implementation of contact of the molecules of fibrin from fibrinogen binding groups, with each molecule of fibrinogen can bind at least two binding fibrinogen group and, thereby, the binding molecules of fibrinogen through the media due to the formation of non-covalent bonds between the fibrinogen binding groups and molecules of fibrinogen.

Biogel according to us is oedema invention need not necessarily be capable of adhesion to a fabric substrate. However, in preferred aspects, the Biogel according to the present invention is a tissue adhesive. The term "tissue glue (adhesive) in this application refers to a substance that can stick to the fabric substrate, for example, to the skin or mucosal surface. Biogels or tissue adhesives according to the present invention can be used for hemostasis, as insulating coatings (sealants), tissue engineering (e.g., the substrate) or for targeted drug delivery.

The media can be a soluble or insoluble carrier, but is not a platelet. The carrier should be suitable for local injection into the tissue of the subject, for example, in the area of the bleeding wound or area of mucous membrane. Soluble media may be suitable rather for intravenous than for local introduction. The media may include soluble or insoluble protein, a therapeutic drug, polymer (e.g., a biocompatible polymer, such as polyethylene glycol), or a combination of any of the listed media.

Examples of protein carriers are enzyme or protein that is not an enzyme, such as serum albumin human.

The insoluble carrier may be a microparticle (including solid, aluu or porous microparticle, preferably essentially spherical microparticle). This particle may be composed of any suitable substance, for example, cross stitched protein.

One of the suitable protein is albumin (obtained from serum or recombinant having the sequence of human albumin or albumin, which is not human). Microparticles suitable for use as insoluble carriers, in the present invention can be obtained by spray drying serum albumin person using well-known techniques of spray drying, for example, described in WO 92/18164.

Alternatives to the use of microparticles as carriers include liposomes, synthetic polymer particles (such as polylactic acid, polyglycolic acid and polymer breast and glycol acid, or fragments of cell membranes.

The term "fibrinogen" in this application includes natural fibrinogen, recombinant fibrinogen or derived fibrinogen, able to transform under the action of thrombin to form fibrin (e.g., natural or recombinant fibrin monomer, or derivative monomer, fibrin, capable or incapable of spontaneous Assembly). Fibrinogen should be able to bind at least two connecting fibrinogenami. Fibrinogen can be obtained from any source and of any type (including bovine fibrinogen), but it is preferable to human fibrinogen. The human fibrinogen can be obtained from autologous or donor blood. Autologous fibrinogen is preferred because its use reduces the risk of infection with the introduction of Biogel (or adhesive) according to the present invention to a subject.

Preferably, binding of the fibrinogen group binds to the fibrinogen with the dissociation constant (KD)lying in the interval between 10-9and 10-6M, for example, approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400 or more nm. KDof approximately 100 nm is preferred. The dissociation constant can be measured in the equilibrium state. For example, radiolabelled fibrinogen in a known concentration can be incubated with the microspheres, with whom was associated transverse relationship linking the fibrin group. Typically, 5 μm peptide associated transverse relationship with 1 g of the microspheres, or 15-40 µm peptide associated transverse relationship with 1 g of the microspheres. Related peptide microspheres diluted to 0.5 mg/ml and incubated in isotonic buffer at pH 7.4 (for example, in the buffer 0.01 M Hepes, containing 0.15 M NaCl) with radiolabelled fibring the nom at the concentrations lying in the range between 0.05 and 0.5 mg/ml, for up to 1 h at 20°C. Fibrinogen associated with fibrinogen binding group on the microspheres can be separated from free fibrinogen by centrifugation and measure the amount of free and bound fibrinogen. The dissociation constant can then be calculated using analysis of Scatchard by drawing on a graph of the concentration of bound fibrinogen depending on the ratio of bound fibrinogen to the free, and the tangent of the angle to the x-axis tangent to the curve represents the KD.

In some embodiments of the present invention is preferred that the binding fibrinogen group selectively binds to fibrinogen. In other embodiments, the implementation is preferred that the fibrinogen binding group can communicate with fibrinogen and separately with fibrin monomer and/or fibrin. Linking of fibrinogen and fibrin monomer and/or fibrin preferably is selective.

Preferably, the fibrinogen binding group is a fibrinogen binding peptide or analogue of such a peptide. You can apply any suitable fibrinogen binding peptide. For example, the peptide may have the ability to bind with a plot of fibrinogen is a, which in nature is associated with fibrin or glycoproteins membrane of the platelet GPIIb-IIIa. Linking of fibrin from fibrinogen discussed in Mosesson and others 2001, Ann. N.Y. Acad. Sci., 936, 11-30. The binding of GPIIb-IIIa with fibrinogen discussed in Bennett, 2001, Annals of NY Acad. Sci., 936, 340-354.

The peptide may have the ability to bind with the carboxy - and/or aminocentesis domain of the α-chain of fibrinogen. In particular, the peptide may have the ability to bind to RGD-containing motif in the same domain, or both domains (such as RGDF (SEQ ID NO:1) amino acid 95-98 or RGDS (SEQ ID NO:2) amino acids 572-575). The peptide may have the ability to bind with carboxykinase domain of the γ chain of fibrinogen, preferably from 12 terminal amino acids in this domain (sequence HHLGGAKQAGDV (SEQ ID NO:3)). The peptide may have the ability to bind with the D-domain of the γ-chain of fibrinogen, for example, a segment of the β-chain of D-domain.

Binding fibrinogen peptide may include the sequence derived from the binding site of fibrinogen GPIIb or GPIIIa. For example, the peptide may include the sequence AVTDVNGDGRHDLLVGAPLYM (SEQ ID NO:4), which corresponds to the sequence of amino acid residues 294-314 GPIIb, or a fragment or a derivative thereof that retains the ability to bind fibrinogen. Fragments for which it is known that they have not lost the ability to binding the human fibrinogen, are TDVNGDGRHDL (296-306) (SEQ ID NO:5), GDGRHDLLVGAPL (300-312) (SEQ ID NO:6) and GAPL (SEQ ID NO:7). Suitable derivatives TDVNGDGRHDL include: T(D,E)VNG(D,E)GRH(D,E)L (SEQ ID NO:8); TD(V,L)NGDGRHDL (SEQ ID NO:9); TDV(N,Q)GDGRHDL (SEQ ID NO:10); TDVNGDG(R,K)HDL (SEQ ID NO:11).

Binding fibrinogen peptide may include the sequence of residues 95-223 GPIIIa, or a fragment or a derivative thereof that retain the ability to bind fibrinogen. For example, the remains of 211-222, including the sequence of SVSRNRDAPEGG (SEQ ID NO:12), seen as important to the fibrinogen-binding domain in GPIIIa. Other suitable areas GPIIIa include the remains 109-171 and 164-202.

Binding fibrinogen peptide may include a sequence of residues that under the action of thrombin are accessible for the binding of fibrinogen, and which bind fibrinogen in the first stage, the polymerization reaction with the formation of fibrin. Thrombin peptides it (releases fibrinopeptides a and b) from N-ends chains α and β fibrinogen, as a result becoming available sequence NH2-GPR- (SEQ ID NO:13) and NH2-GHR- (SEQ ID NO:14), respectively. Accordingly, a preferred example of the fibrinogen binding peptide comprises the amino acid sequence of NH2-G(P,H)RX- (SEQ ID NO:15) in aminobenzene region, where X represents any amino acid, and (R,N) means, in this position is either Proline, or histidine. Preference is sustained fashion peptide includes the sequence NH 2-GPRP- (SEQ ID NO:16) in aminobenzene area.

Preferably, the fibrinogen binding peptide has a length of 4-30, more preferably 4-10 amino acid residues.

The precursor fibrinogen binding group should not contact with fibrinogen binding molecules of fibrinogen to each other through the media, including immobilized precursor fibrinogen binding groups, contact media with fibrinogen.

Preferably, the dissociation constant predecessor linking of fibrinogen and fibrinogen greater than 1×10-6M Precursor fibrinogen binding group can be a peptide or analogue of the peptide, but preferably the peptide. The precursor fibrinogen binding group may not be the fibrinogen or include fibrinogen.

In preferred embodiments of the present invention, the precursor fibrinogen binding group comprises fibrinogen binding peptide coupled aminocentesis plot with a blocking component (preferably, a peptide), which blocks or inhibits (i.e. reduces) the linking of fibrin from fibrinogen binding peptide. Cleavage of the precursor fibrinogen binding group converting agent (preferably, a coagulation factor, such as thrombin) opens for usaimage the effects of fibrinogen binding peptide, associated with the carrier, thereby rendering the precursor fibrinogen binding groups in fibrinogen binding group. In such implementations, the blocking component blocks or inhibits the ability of fibrinogen binding peptide to bind fibrinogen until then, until its removal. Preferably the blocking component is a peptide length of 1-30 amino acid residues.

Obviously, in such embodiments of the precursor fibrinogen binding group should include the cleavage site, which is selectively recognized by transforming agent and is located between the fibrinogen binding peptide and a blocking component. Thrombin is the preferred converting agent. However, for cleavage of the precursor fibrinogen binding groups can use other serine protease or coagulation factors. It is known that thrombin cleaves peptide bonds on the carboxyl side of the end from the arginine residues and usually between residues arginine and glycine.

In especially preferred embodiments of the present invention, the precursor fibrinogen binding group is a peptide that includes the amino acid sequence of NH2-ZYXR/GPRP- (SEQ ID NO:17) in aminobenzene region, while the "/" before the hat is the site of cleavage by thrombin, and X represents any amino acid, but preferably is a Proline, Y is any amino acid, but is preferably aspartic acid or alanine, and Z represents at least one amino acid, which preferably is a leucine or Proline. Examples are: NH2-LVPR/GPRP- (SEQ ID NO:18), NH2-ADPR/GPRP- (SEQ ID NO:19), NH2-LDPR/GPRP- (SEQ ID NO:20), or NH2-LVPR/GPRV- (SEQ ID NO:21).

Binding fibrinogen groups or their precursors may be associated with the carrier by any suitable means, but, as a rule, they are bound covalently. Examples of preferred covalent bonds are disulfide linkage, thioester linkage, or amide linkage. Suitable covalent bond can be formed when connecting the fibrinogen group or their predecessors are peptides that include cysteine, and the carrier includes reactive Tilney group. This enables the binding of the peptide to contact the media by binding to-SH groups of cysteine reactive with Tilney group on the media. Preferably, in the fibrinogen binding peptide or peptide precursor enter end group of cysteine to ensure binding peptide cross links with tylenol reactive group on the carrier. As the e alternatives covalent bond can be formed when connecting the fibrinogen group or its precursor is a peptide that includes maleimido group (preferably a carboxyl end, for example, attached to carboxykinase lysine peptide), and the carrier includes a sulfhydryl group. The peptide can then be associated with the media by providing contact maleimide group of the peptide with the sulfhydryl group of the media.

As a rule, you must have a spacer between the fibrinogen binding groups or their precursors and the media to ensure that the fibrinogen-binding activity of the binding fibrinogen groups (if necessary, converted from precursor fibrinogen binding groups) is not exposed to the negative influences of the media. Suitable spacers are peptides or ones of the molecule, such as polyethylene glycol.

If fibrinogen binding groups or their precursors are peptides that include fibrinogen binding peptide, and these molecules or their precursors are associated with an insoluble carrier on terminal amino acid residue, spacer elements sequence preferably is located between the terminal amino acid residue and fibrinogen binding peptide of the above molecules or p is of electonica. Spacer elements sequence may, for example, have a length of 1-20, preferably 5-20 amino acid residues. Spacer elements sequence GGGGGG (SEQ ID NO:22) or GGGGG (SEQ ID NO:23) is preferred.

It is obvious that the number of binding fibrinogen groups or their precursors to the media and the relative number of media (with lots of fibrinogen binding groups or their precursors to the media) and fibrinogen, which are required for optimal Biogel (or fabric glue)may be different in different preparations of media and fibrinogen. Therefore, it may be necessary or desirable to test each new batch of media or fibrinogen to determine the optimal relative amounts of carrier and fibrinogen, which should be used for the formation of Biogel.

Preferably, each carrier has, on average, at least five binding fibrinogen groups or their precursors to the media. Theoretically, there is no upper limit for the number of binding fibrinogen groups or their precursors to the media. The optimal number probably depends on many factors such as the nature of the medium and the number on each medium reactive groups for attaching binding fibrinogen groups or their precursors. The n is less is preferred to each media contained on average up to 100 fibrinogen binding groups or their precursors to the media. The preferred range is 10-20 fibrinogen binding groups or their precursors to the media.

Preferably, use that amount of fibrinogen that there is at least one quarter (preferably at least half) of the number of moles of fibrinogen compared to the number of moles of binding fibrinogen groups or their precursors. Preferably, the number of moles of fibrinogen in relation to the number of moles of binding fibrinogen groups or their precursors is in the range from 1:4 to 4:1.

To evaluate the viscoelastic properties of Biogel obtained according to the present invention can be applied dynamic oscillation mode. The dynamic elastic modulus (G') and loss modulus (G") in viscoelastic solids are a measure of the accumulated energy, representing the elastic portion, and the energy dissipated in the form of heat, representing the viscous portion. Tan Delta is the ratio of the loss modulus (G") to the dynamic elastic modulus (G'). Therefore, it is a quantitative expression of the contributions of the elastic and viscous component, while a value above 1 indicates a similar viscous fluid behavior, and the value is s below 1 indicates an elastic behavior. Biogels according to the present invention preferably have a value of tan Delta less than 1. This suggests that components of the specified gel cross-linked bonds. Tan Delta can be defined at a frequency of 1 Hz, and a constant strain of 1%. Suitable methods of measurement of tan Delta are described in more detail in the examples below.

The components of the kit according to the present invention can be stored separately from one another, or some or all of the components of the kit can be stored together, provided that the components are stored together, will not react with each other. It is obvious that variants of realization of the present invention, in which a specified set includes carriers with immobilized fibrinogen binding groups, fibrinogen should be stored separately from the media, so that it does not react with native speakers. An important advantage of the kits according to the present invention, which include fibrinogen and carriers with immobilized precursor fibrinogen binding groups that do not bind fibrinogen, is that the media and fibrinogen can be stored together.

The components of the kit according to the present invention can be stored separately from each other in the device (e.g., syringe) for delivery of components to the fabric or the other the place. The device may be so designed that the components in contact with each other after delivery to the fabric or to another location.

Set according to the present invention may include instructions for use of kit components for the formation of a Biogel or tissue adhesive.

Kits according to the present invention have advantages in that for the formation of Biogel does not require the use of toxic agents, Biogel (or fabric glue) is easily obtained by applying the components of the kits, and components can easily be stored in a stable condition. It is also clear that for the formation of a Biogel or tissue adhesive to set according to the present invention does not require the presence of thrombin (or other enzymes). Kits according to the present invention that do not include thrombin, are particularly preferred because of the risk of allergic reaction on a Biogel or tissue adhesive obtained by the use of such sets, reduced compared to a Biogel or tissue adhesive, obtained with the use of exogenous thrombin. An additional advantage of the kits according to the present invention that do not include thrombin (or other enzymes), is that you do not want to store the components of the specified set under conditions which ensure the preservation and the activity of enzymes.

According to some variants of implementation of the present invention, the precursor fibrinogen binding groups can be turned into a binding fibrinogen group using the transforming agent, which is present in the place where the injected carriers, including immobilized precursor fibrinogen binding groups. This provides the advantage consisting in the fact that you do not want to include in the set turns the agent. Therefore, you do not need to store kit components at conditions that allow to preserve the activity of the transforming agent.

Preferably, the converting agent is an agent that is present in the damaged site (wound). The term "agent present in the wound" in this application means an agent that is present in the area of education of the wound. In a preferred implementation, the specified agent is present in the wound, is a coagulation factor. Examples of suitable coagulation factors include thrombin, Factor VIIa, Factor XA, or Factor XIa. Preferably, the coagulation factor is a thrombin.

According to other variants of implementation, set according to the present invention may further include converting agent for converting the precursor fibrinogen binding groups in the binding fibrinogen g is uppy. Specified converting agent should be separate from the media. In such implementations, converting agent can be an agent whose presence is not expected in the area of introduction of the media. Alternatively, converting agent can be an agent that is present in the area where the injected carriers. Converting agent may be a coagulation factor, such as thrombin, Factor VIIa, Factor XA, or Factor XIa.

Set according to the present invention, including carriers with immobilized fibrinogen binding groups may additionally include a coagulation factor. Set according to the present invention, including predecessors fibrinogen binding groups, which can be turned into a binding fibrinogen group using coagulation factor may include a coagulation factor, which does not make predecessors fibrinogen binding groups in fibrinogen binding group. In such implementations, the specified coagulation factor (not converting the precursor fibrinogen binding groups) can be presented as a separate component of the set or with the carrier and/or fibrinogen. The clotting factor can be immobilized on a carrier, for example, is associated with each before what Estonica fibrinogen binding group.

If set according to the present invention includes a coagulation factor (e.g., thrombin), the latter preferably is a coagulation factor, which preferably contains the sequence of the coagulation factor man, and not factor out other species (such as bovine coagulation factor), which helps to reduce the risk of allergic reactions to blood coagulation factor.

Biogel or tissue adhesive according to the present invention can be obtained (to be formed) before introduction into the tissue or in situ in the tissue, for example, in the education sector wounds. Biogel or tissue adhesive tissue preferably includes a carrier for topical administration.

Obviously, the thrombin may be present in the tissue, in which are introduced, or in which form the Biogel or tissue adhesive according to the present invention. For example, if the tissue is a site of bleeding wounds, thrombin owner, most likely present in the formation place of the wound. Of course, the thrombin may be present as alternatives or additions, if it comes together with a set according to the present invention.

The use of fibrinogen binding groups capable of contacting fibrinogen and separately with fibrin monomer and/or fibrin, mo is et to be pre-emptive, if Biogel or adhesive according to the present invention is formed in the presence of thrombin, or bring into contact with thrombin after education. If thrombin is present simultaneously with the fibrinogen and the media, including immobilized binding fibrinogen group, thrombin will make at least some amount of fibrinogen into fibrin monomers. Obviously, in such conditions, it is preferable that binds fibrinogen group was also associated with fibrin monomers, which enables linking of fibrin monomers formed by the action of thrombin with each other through the media.

If Biogel or adhesive according to the present invention is brought into contact with thrombin after formation, may be an advantage if the fibrinogen associated with fibrinogen binding groups capable of conversion into a monomer, fibrin, and fibrinogen binding groups remain linked with fibrin monomer. In such circumstances, non-covalent connection formed between fibrinogen and fibrinogen binding groups will not be destroyed in the conversion of fibrinogen to fibrin monomer. Can also be an advantage if at least some amount of the monomers of fibrin associated with fibrinogen binding groups capable of associating with the formation of fibrin, OST is as thus associated with fibrinogen binding groups. In such conditions, the formation of fibrin may increase the strength of Biogel or tissue adhesive.

In the presence of thrombin (e.g., from a set according to the present invention or thrombin owner in the area of education of the wound) is the preferred preliminary contact media and fibrinogen with each other before bringing them into contact with thrombin, or the implementation of the contact carriers, fibrinogen and thrombin with each other essentially at the same time.

Despite the possibility of forming a Biogel or tissue adhesive when making contact thrombin and fibrinogen with each other prior to contact with the media, it is expected that they will be less useful than a Biogel or tissue adhesive formed by the contact carriers and fibrinogen with each other prior to contact with thrombin, or by making contact carriers, fibrinogen and thrombin with each other essentially at the same time. This happens because the thrombin need to transform the molecules of fibrinogen into fibrin monomers, which are then aggregated with the formation of insoluble fibrin before contact with the media. However, under certain circumstances, it may be worthwhile getting a Biogel or tissue adhesive by contact of thrombin and fibrinogen before contact with the media, in the example, if thrombin is inactive before or after contact with the media.

Also according to the present invention, a method of forming a Biogel, which includes providing contact multiple media with molecules of fibrinogen and thrombin, with each carrier immobilized many fibrinogen binding groups, and each molecule of fibrinogen can bind at least two binding fibrinogen group, with the formation of Biogel, in which the fibrin monomers are linked to each other through the media of non-covalent bonds between the fibrinogen binding groups and the fibrin monomers.

The tests may include or not include fibrin. Therefore, the fibrin monomers can be part of the fibrin in the Biogel or monomers of fibrin may not be part of the fibrin in the Biogel.

Also according to the present invention proposed a Biogel, which includes monomers of fibrin and many carriers, each carrier includes a variety of binding fibrinogen groups immobilized on the carrier, and the fibrin monomers are linked to each other through the media of non-covalent bonds between the fibrinogen binding groups and the fibrin monomers.

Additionally, according to the present invention proposed a Biogel, which includes fibrin and many carriers, each carrier in the cancel many fibrinogen binding groups, immobilized on the carrier, while fibrin monomers specified fibrin connected to each other through the media of non-covalent bonds between the fibrinogen binding groups and the fibrin monomers.

In a preferred implementation, the Biogel is a fabric glue.

Set according to the present invention may include Factor XIII and possibly calcium ions and thrombin activation of Factor XIII with the formation of Factor XIIIa (in this case calcium ions and thrombin should be separate from Factor XIII). Alternatively or in addition, Factor XIIIa may be present in the tissue, where introduced, or in which form the Biogel or tissue adhesive according to the present invention. For example, if the tissue is a site of bleeding wounds, Factor XIIIa of the owner most likely present in the formation place of the wound.

If the Factor XIIIa is brought into contact with Biogel or tissue adhesive according to the present invention, which includes fibrin, the strength of Biogel or tissue adhesive can be further improved by means of the reaction of Factor XIIIa with fibrin, which provides a covalent cross-linking of fibrin.

Also according to the present invention, a method of forming a Biogel, which includes: the implementation of the contact set novtel the nd with the molecules of fibrinogen and thrombin, each carrier immobilized many fibrinogen binding groups, and each molecule of fibrinogen can bind at least two binding fibrinogen group, with the formation of Biogel, including fibrin, in which fibrin monomers specified fibrin connected to each other through the media of non-covalent bonds between the fibrinogen binding groups and fibrin monomers; and implementation of contact Biogel with Factor XIIIa, which provides covalent linking of fibrin monomers specified to each other by peptide bonds.

Additionally, according to the present invention proposed a Biogel, which includes fibrin and many carriers, each carrier includes a variety of binding fibrinogen groups immobilized on the carrier, while fibrin monomers specified fibrin covalently linked to each other by peptide bonds and fibrin monomers specified fibrin connected to each other through the media of non-covalent bonds between the fibrinogen binding groups and the fibrin monomers.

In a preferred aspect, the Biogel is a fabric glue.

Set according to the present invention may optionally include an agent that promotes wound healing. Suitable examples include growth factors, such as factor grew the platelets. The specified agent can be a separate component set, or may be immobilized on a carrier.

Set according to the present invention may optionally include an antimicrobial agent, for example, an antibiotic. Specified antimicrobial agent can be a separate component set, or may be immobilized on a carrier.

Obviously, if the media, including many fibrinogen binding groups immobilized on each medium is injected into the tissue, which contains fibrinogen host (for example, in the plot bleeding wounds), the media will react with fibrinogen owner with the formation of Biogel or tissue adhesive in situ in the specified section of the cloth.

Similarly, if the media, including many predecessors fibrinogen binding groups immobilized on each medium is injected into the tissue, which contains a transforming agent for the conversion of the precursor fibrinogen binding groups in fibrinogen binding group and fibrinogen host (for example, in the area of bleeding wounds), is the transformation of the precursor fibrinogen binding groups in fibrinogen binding groups, and the media will then react with fibrinogen owner with the formation of Biogel or tissue adhesive in situ in the specified phase is fabric. For example, the precursor fibrinogen binding groups can become binding fibrinogen group under the action of thrombin owner or other blood coagulation factor.

Accordingly, further according to the present invention proposed a Biogel, which includes carriers for topical administration, and each carrier includes a variety of binding fibrinogen groups immobilized on the carrier, and endogenous fibrinogen, with each molecule of endogenous fibrinogen is associated with at least two binding fibrinogen groups resulting in the binding of fibrinogen molecules to each other through the media of non-covalent bonds between the fibrinogen binding groups and molecules of endogenous fibrinogen.

The term "endogenous fibrinogen" in this application means that the fibrinogen is a fibrinogen master, who is present in area where the injected carriers. As a rule, fibrinogen owner will be present, because the blood of the host is present in the formation place of the wound.

Also according to the present invention it is proposed to use the media to generate Biogel, the media includes a variety of binding fibrinogen groups or precursors bind fibrinogen groups immobilized on the carrier.

Before occhialino, Biogel is a fabric glue.

The media can be used for forming a Biogel or tissue adhesive for hemostasis, as an insulating coating for targeted drug delivery or tissue engineering.

Introduction media for the formation of a Biogel or tissue adhesive has the advantage that the risk of allergic response of the host is minimized, because it does not require exogenous fibrinogen or thrombin, does not require toxic agents and media can be easily stored in a stable condition.

The present invention also provides a method of stopping the bleeding at the site which contains fibrinogen owner, which includes the topical introduction of many carriers in the specified location, with each carrier includes a variety of binding fibrinogen groups immobilized on the carrier, and molecules of fibrinogen master in this place can bind at least two binding fibrinogen group.

Additionally, the present invention provides a method to stop bleeding at the site where the fibrinogen of the owner and the coagulation factor, which includes the topical introduction of a variety of media in a specified area, with each carrier includes many predecessors connecting FIB is sometimes groups immobilized on the carrier and precursor fibrinogen binding groups capable of conversion into the binding fibrinogen group under the action of coagulation factor host, and molecules of fibrinogen owner in this area can bind at least two binding fibrinogen group.

Additionally, according to the present invention it is proposed to use a variety of media in the manufacture of a drug (for example, Biogel or tissue adhesive) to stop bleeding, or to treat or isolate (cover) wounds, with each carrier includes a variety of binding fibrinogen groups or precursors bind fibrinogen groups immobilized on the carrier.

Also according to the present invention it is proposed to use a variety of carriers, each carrier includes a variety of binding fibrinogen groups or precursors bind fibrinogen groups immobilized on the carrier, to form Biogel.

Additionally provided, in accordance with the present invention, a variety of media to use (for example, as a Biogel or tissue adhesive) to stop bleeding, or to treat or isolate the wound, with each carrier includes a variety of binding fibrinogen groups or precursors bind fibrinogen is Rupp, immobilized on the carrier.

The media can be insoluble or soluble. Media you can enter tapicerki.

If the carrier is a soluble carrier, a preferred dosage form for topical introduction of specified media, including immobilized binding fibrinogen group or precursor fibrinogen binding groups, is a liquid dosage form, preferably in isotonic buffer at physiological pH.

If the media is an insoluble carrier, a preferred dosage form for topical application specified media, including immobilized binding fibrinogen group or precursor fibrinogen binding groups is the form of powder that can be sprayed, for example, on the section of the cloth.

Media in powder form may be obtained by any suitable method, including methods, including spray drying, or lyophilization of the suspension media, including many fibrinogen binding groups or precursors bind fibrinogen groups immobilized on each medium (e.g., suspended in isotonic buffer at physiological pH). It is preferable to use spray drying, as this may be more quickly and easily mA is stairway the method of drying, than lyophilization. Suitable methods of spray drying is described in WO 92/18164.

According to the present invention provides an agent for the formation of Biogel, and the specified agent comprises a water soluble carrier, the set of binding fibrinogen groups or precursors bind fibrinogen groups immobilized on each medium.

Also according to the present invention proposed an agent for the formation of Biogel, and the specified agent comprises an insoluble carrier, including many fibrinogen binding groups or precursors bind fibrinogen groups immobilized on each carrier, the agent is in powder form, obtained otherwise than by lyophilization.

Also according to the present invention, a method for obtaining an agent in powder form, where the specified agent comprises an insoluble carrier, including many fibrinogen binding groups or precursors bind fibrinogen groups immobilized on each medium, and the method comprises spray drying a suspension of the specified agent.

Preferably, the specified agent suitable for topical application. If the agent comprises a soluble carrier with multiple predecessors fibrinogen binding groups immobilized on each carrier, MC is connected, the agent can be suitable for intravenous injection.

The carrier or agent can be represented as a component set. The specified set may additionally include a coagulation factor, an agent that promotes wound healing or antimicrobial agent. The coagulation factor, an agent that promotes wound healing or antimicrobial agent may be a separate kit components, or can be immobilized on the carrier. In some preferred embodiments of the coagulation factor, an agent that promotes wound healing or antimicrobial agent may be part of the precursor fibrinogen binding groups, so that they are released when the precursor fibrinogen binding group becomes binding fibrinogen group.

Set according to the present invention can be set with the departments in which the components of the kit, which are preferably stored separately from each other, are contained in separate cells (compartments or containers. Set according to the present invention can include instructions for use of kit components to implement the method according to the present invention.

Biogel or tissue adhesive according to the present invention can tapicerki to enter into the tissue, for example, in the skin or mucous membrane. Biogel or tissue adhesive according to Nast is Adamu invention can be used for hemostasis, as an insulating coating for targeted drug delivery or tissue engineering.

Biogel or tissue adhesive according to the present invention can be administered by the formation of gel or glue before putting gel or glue in contact with the site of injection, or by forming a Biogel or tissue adhesive at the site of injection.

It is also clear that the agent comprising a water soluble carrier, in which the set of predecessors of binding fibrinogen groups, each of which can be turned into binding fibrinogen group immobilized on each carrier, can be administered intravenously, for example, to stop bleeding or to deliver drugs. The preferred dosage form for intravenous represents a 20% aqueous isotonic solution. In a preferred implementation, the precursor is capable of turning under the action of thrombin.

Additionally, according to the present invention, a method for stopping bleeding, which includes intravenous agent comprising a water soluble carrier, the set of predecessors of binding fibrinogen groups, each of which can be turned into binding fibrinogen group immobilized on each medium.

In addition, the according to the present invention, a method for delivery of a drug to a subject, which includes intravenous agent comprising a water soluble carrier, the subject, the set of predecessors of binding fibrinogen groups, each of which can be turned into binding fibrinogen group immobilized on each carrier, and the carrier includes a drug, or medicine is immobilized on a carrier.

Of course, dosage forms for topical or intravenous administration must be sterile.

Biogel or tissue adhesive according to the present invention may optionally include an agent that promotes wound healing or antimicrobial agent.

Also according to the present invention, a method for stopping bleeding, which includes the topical introduction of Biogel or tissue adhesive according to the present invention in the area of bleeding.

Also according to the present invention, a method for treatment or isolation of a wound, which includes the introduction of (preferably topical) Biogel or tissue adhesive according to the present invention in the area of education of the wound.

Also according to the present invention proposed a Biogel or tissue adhesive according to the present invention for use in medicine.

Additionally, according to the present invention proposed PR is the application of Biogel or tissue adhesive according to the present invention in the manufacture of a medication to stop the bleeding, or to treat or isolate the wound.

Also according to the present invention proposed a Biogel or tissue adhesive according to the present invention to stop bleeding, or to treat or isolate the wound.

The Biogel or tissue adhesive according to the present invention can be a topical application. If Biogel or tissue adhesive obtained by using soluble carrier and binding fibrinogen group derived from precursor fibrinogen binding groups may be intravenous.

Preferred implementations of the present invention are described only as examples.

According to the first preferred implementation variant of the present invention, a variety of peptides, each of which includes a sequence of binding of fibrinogen in aminoclonazepam area (for example, peptides having the sequence GPRPGGGGGGC (SEQ ID NO:24)), are connected by their carboxyl ends of the carrier, which is a soluble protein (such as albumin). Biogel is obtained by implementation of the contact associated with the peptide carrier with fibrinogen. Specified Biogel you can then apply tapicerki, in the form of a bandage on the wound.

According to the second preferred implementation variant of the present invention, there is connected the output with the peptide carrier according to the first preferred implementation variant, and fibrinogen is mixed in the education sector wounds with the formation of Biogel in situ.

According to the third preferred implementation variant of the present invention associated with the peptide carrier according to the first preferred implementation variant injected into the site of the formation of the wound, which contains fibrinogen from the blood of the host with getting Biogel in situ.

According to the fourth preferred implementation variant of the present invention, a variety of peptides, each of which includes a sequence that binds fibrinogen, connected along aminobenzoic area with blocking peptide sequence (for example, peptides having the sequence LVPRGPRPGGGGGGC (SEQ ID NO:25)), are connected by their carboxyl ends of the carrier, which is a soluble protein (such as albumin). Associated with the peptide carrier can be combined with fibrinogen and then applying to the wound as a single mixture. Thrombin owner, who was present in the plot with the wound, cleaves peptides with the release of the blocking peptides, this opens the access sequence that binds fibrinogen. Media available sequence that binds fibrinogen reacts with fibrinogen with the formation of Biogel in situ.

Alternatively, associated with the peptide carrier of the fourth preferred embodiments can be administered intravenously. Tr is mbin owner at the wound site cleaves peptides with the release of the blocking peptides this provides access to the sequence,binding fibrinogen. Media with available sequences that bind fibrinogen reacts with fibrinogen owner to stop bleeding at the site of the wound.

The tests of the four preferred options of the implementation described above, may be a fabric glue.

The insoluble carrier (e.g., albumen microsphere) can be used instead of the soluble protein carrier in any of the above-described preferred implementation options (except intravenous Vvedenie).

As a soluble protein carrier in any of the above-described preferred implementation options you can apply branched polyethylene glycol (PEG) or any other biocompatible polymer.

In any of the above-described preferred variants of realization of the coagulation factor may be immobilized on a carrier, provided that if the precursor fibrinogen binding groups immobilized on the carrier, the coagulation factor does not make the precursor fibrinogen binding groups in fibrinogen binding group.

The carrier may optionally include an agent that promotes wound healing (e.g., a growth factor such as platelet growth factor) at any of opican the x above the preferred options for implementation.

Additional preferred implementations of the present invention are described in the following examples with reference to the accompanying figures, in which:

On Figure 1 is shown schematically: (a) albumen media, including many fibrinogen binding peptides immobilized on a carrier; (b) a molecule of fibrinogen; and (C) the tests in which molecules of fibrinogen are associated with each other through the media of non-covalent bonds between the fibrinogen binding peptides and molecules of fibrinogen;

The Figure 2 shows a graph of the dynamic modulus of elasticity (squares) and loss modulus (triangles), depending on the rate of deformation in a mixture of 25 µl of modified peptides serum albumin human (CSA) and fibrinogen at 32 mg/ml at 20°C and 37°C ();

The Figure 3 shows the dynamic modulus of elasticity (squares) and complex viscosity (triangles) as a function of frequency at 20°C (1% amplitude relative deformation), 25 µl of modified peptides, CSA mixed with fibrinogen at 32 mg/ml; and

Figure 4 shows pictures of a solution of fibrinogen to (a) and after (b) adding the modified peptides of CSA.

Example 1

The formation of a Biogel or tissue adhesive with application of fibrinogen and fibrinogen binding peptides immobilized on a carrier, including the next serum albumin human

Serum albumin human (CSA) was brought into contact with a 40-fold molar excess of linker Succinimidyl-4-(N-maleimidomethyl)sikorksy-1-carboxylate (SMCC) at pH 7.4. The modified protein was CSA-SMCC) was purified and determined that an average of one mol of CSA was modified 18 moles of SMCC. To CSA-SMCC was added or peptide GPRPGGGGGGC (B10; SEQ ID NO:24), or peptide LVPRGPRPGGGGGGC (TC-15; SEQ ID NO:25) with 1.5-molar excess relative to the molecules of maleimide. Modified peptides protein CSA then purified from unreacted peptide was stored at -80°C at a concentration of 7 mg/ml Lyophilized human fibrinogen was dissolved in water up to 16 mg/ml, as recommended by the supplier (Scottish National Blood Transfusion service).

Modified peptides of CSA (5 ml) was added to 0.3 ml of fibrinogen, education instant gel was observed for a 10-CSA, but for TC-15 did not observe the formation of gel. It is known that the open sequence of the peptide GPRP associated with pocket "and" in the carbonyl stretch of the two distal domains (D) fibrinogen (Figure 1b). I believe that CSA modified set of open sequences GPRP (B10), acts as the point of branching during polymerization of fibrinogen (Figure 1C). Described gel obtained in the absence of thrombin.

Methods for Examples 2-5

Synthesis of modified peptides, CSA

Whey and albumin person (CSA) at a concentration of 10 mg/ml was brought into contact with 5, 10, 20 or 40-fold molar excess of linker sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) at pH 7.4 in a total volume equal to 0.7 ml of Modified protein (CSA-SMCC) was purified using a centrifuge columns Zebra™ Desalt Spin Column (Pierce, Rockford, Il), and determined that an average of one mol of CSA was modified 5, 8, 16, or 18 moles of sulfo-SMCC, respectively. To determine the amount of bound peroxidase maleimide groups per 1 molecule of CSA, the sample with 3 nanomolar CSA incubated with known amounts of cysteine (100 nanomolar) buffer at room temperature for 30 minutes Remaining cysteine is then brought into contact with 1 millimole 5,5-dithiobis(2-nitrobenzoate) (DTNB) for 20 min and was measured by A412. The level of maleimide was calculated by comparing the absorbance of the control and protein samples. To CSA-SMCC was added or peptide GPRPGGGGGGC (B10; SEQ ID NO:24), or peptide LVPRGPRPGGGGGGC (TC-15; SEQ ID NO:25) with 1.5-molar excess relative to the molecules of maleimide. Modified peptides protein CSA then purified from unreacted peptide, using Zebra™ Desalt Spin Column (Pierce, Rockford, Il) and stored at -80°C at a concentration of 7 mg/ml Lyophilized human fibrinogen (Scottish National Blood Transfusion service, Edinburgh, Scotland) was dissolved in water up to 8, 16, 32 and 64 mg/ml Concentration of fibrinogen and albumin were determined by the absolute is bcii at 280 nm (A 280), applying the conversion factor for 1% Fibrinogen E280=15 and 1% CSA E280=13.8.

Rheological study

To evaluate the viscoelastic properties of the gels used dynamic oscillation mode. The mixture was allowed to balanced at room temperature over night and the gel was transferred into the lower bowl of the rheometer Physica MCR-501 (Anton Paar, Germany) with a cone and bowl diameter bowl 25 mm and a cone angle of 1°. Analyses were performed either at 20 or at 37±°C in a humidified atmosphere. Frequency sweep was performed between 0.01 and 50 Hz with constant amplitude relative deformation, equal to 1%. Investigated the rheological parameters were processed using specially developed software Anton Paar supplied with the rheometer.

Example 2.

Effect of the number of modified peptides, CSA and fibrinogen concentration on the gel formation

For the polymerization of fibrinogen was first observed visually after mixing fibrinogen solutions of CSA. Modified peptides of CSA modified 5, 8, 16, or 18 moles of binding fibrinogen peptides conjugatively with one mol of CSA. 25 μl of the sample modified with peptides CSA or unmodified, CSA was mixed with 0.2 ml of fibrinogen at 8, 16, 32, or 64 mg/ml of the gel Formation was visually detected after 0.5 h at room temperature the E. For the unmodified CSA or CSA modified 5 or 8 moles of peptide,did not observe the formation of gel at all tested concentrations of fibrinogen. The polymerization was observed only for 32 or 64 mg/ml fibrinogen with modified peptides CSA with 16 or 18 moles binding peptide.

Figure 4 shows pictures of a solution of fibrinogen (0.2 ml 32 mg/ml fibrinogen) before (a) and after (b) adding the modified peptides of CSA (25 μl, 18 mol of peptide). Biogel obtained according to the present invention, presented in Figure 4 (b).

Example 3.

Effect of the number of modified peptides, CSA

To determine the effect of the number of modified peptides, CSA and fibrinogen on the rheological properties of the mixtures were determined measure the sliding resistance for 25 or 50 µl of modified peptides, CSA with fibrinogen in 32 and 64 mg/ml In this experiment, we used the modified peptides of CSA, which was modified to 16 moles binding peptide 1 mol of CSA. To ensure that the experiments on dynamic measurement of fluctuations within the linear viscoelastic deformation of the gel was carried out by the study of a range of stresses (strains). The Figure 2 shows the sweep voltage at 20 and 37°C for 25 ál of modified peptides, CSA mixed with fibrinogen is ri 32 mg/ml As a dynamic elastic modulus (G')and loss modulus (G') have been obviously stable in the whole range and a constant strain of 1%was chosen within this range of linear viscoelastic deformation. Frequency sweep showed the value of G'was greater than G", which indicates the presence of cross stitched network (table 1). The values shown in this Table were obtained at a frequency of 1 Hz. Tan Delta is the ratio of loss modulus to dynamic modulus of elasticity. Therefore, it is a quantitative expression of the contributions of the elastic and viscous component, while a value above 1 indicates viscous behavior close to the liquid, and below 1 indicates an elastic behavior. The complex viscosity η*is defined as the complex modulus G*, divided by the circular frequency (ω)was determined under the same conditions. Apparently, under these conditions, the number of CSA, modified peptides, and fibrinogen has no significant influence on the mechanical strength of the gel.

Table 1
Fibrinogen (mg/ml)3264
Modified peptides H Is A (ál) 25502550
The dynamic elastic modulus (G')432439421588
Complex viscosity (η*)88707794
Tan Delta0.220.110.240.2

To the other party of the modified peptides of CSA 18 peptides associated with 1 molecule of CSA (25 μl), 32 mg/ml fibrinogen showed G', equal to 5200, complex viscosity, equal to 820, and tan Delta equal to 0.105.

Example 4.

The effect of temperature on rheological properties

Study of the effect of temperature on the gel is important for applications of this product in surgical body temperature or for topical application. We evaluated the mechanical strength of the gel at 20 and 37°C, and the values shown in Table 2, were obtained for a frequency equal to 1 Hz. Increasing the temperature of the gel obtained from fibrinogen at a concentration of 32 mg/ml and 25 μl of chca modified 16 moles of PEP is IDA, reduced as G, and the complex viscosity (table 2). For both temperatures, 20 and 37°C tan Delta was below 1, hence, cross-linked (non-covalent) bonds net structure was maintained at 37°C.

Table 2
Temperature (°C)2037
The dynamic elastic modulus (G')432109
Complex viscosity (η*)8821
Tan Delta0.220.72

Example 5

The gel formation in the plasma of human blood

Research whether the modified peptides of CSA to polimerizuet fibrinogen in plasma, it is important to apply this product as a surgical glue/adhesive. Modified peptides of CSA (75 μl), modified 18 moles of peptide was mixed with 0.8 ml of a mixed human plasma. Rheological study of the gel obtained in the plasma was performed at 37°C and obtained a value of G', equal to 6100, and tan Delta equal to 0.104. From this the final and, that modified peptides CSA can polimerizuet fibrinogen in the plasma of a person with the formation of cross-linked (non-covalent) bonds net structure at 37°C.

1. Agent to generate tests for hemostasis, wound closure, tissue engineering or the directed delivery of medicinal products containing soluble carrier suitable for intravenous and/or by local injection, with each carrier immobilized many fibrinogen binding groups, so that when making contact with fibrinogen, each molecule of fibrinogen is associated with at least two specific binding fibrinogen groups, which leads to the formation of Biogel, in which molecules of fibrinogen are associated with each other through the media, through the formation of non-covalent bonds between the molecules of fibrinogen and specified fibrinogen binding groups.

2. The agent according to claim 1 for use in medicine.

3. The tests for hemostasis, wound closure, tissue engineering or the directed delivery of medicinal products containing molecules of fibrinogen and many soluble media suitable for intravenous and/or by local injection, each carrier contains many binding fibrinogen groups immobilized on the carrier, and each of the molecules is of fibrinogen is associated with at least two binding fibrinogen groups thus that fibrinogen molecules are bound to each other through the media, through non-covalent bonds between the fibrinogen binding groups and molecules of fibrinogen.

4. The tests for hemostasis, wound closure, tissue engineering or the directed delivery of medicinal products containing monomers of fibrin and many soluble media suitable for intravenous and/or by local injection, in which each carrier contains many binding fibrinogen groups immobilized on the carrier, and the fibrin monomers are linked together via the carriers by non-covalent bonds between the fibrinogen binding groups and monomers of fibrin.

5. The tests for hemostasis, wound closure, tissue engineering or the directed delivery of medicinal products containing fibrin and many soluble media suitable for intravenous and/or by local injection, in which each carrier contains many binding fibrinogen groups immobilized on the carrier, and the monomers of fibrin to fibrin covalently linked to each other by peptide bonds, and the monomers of fibrin to fibrin connected to each other through the media of non-covalent bonds between the fibrinogen binding groups and monomers of fibrin.

6. Biogel on any of PP-5 for use as IU the of icament.

7. The method of forming Biogel, including the implementation of contact of the molecules of fibrinogen with many soluble media suitable for intravenous and/or by local injection, each carrier contains many binding fibrinogen groups immobilized on the carrier, and each molecule of fibrinogen can bind at least two binding fibrinogen group so that is the binding of fibrinogen molecules to each other through the media, through the formation of non-covalent bonds between the fibrinogen binding groups and molecules of fibrinogen.

8. The method of forming Biogel, including the implementation of contact of the molecules of fibrinogen with the agent for the formation of Biogel according to claim 1.

9. The way to stop bleeding, or treatment or isolation of a wound, comprising the topical introduction to the site of bleeding or wound Biogel on any of PP-5 or many soluble media suitable for intravenous and/or by local injection, with each carrier contains many binding fibrinogen groups immobilized on the carrier, and transferring the fibrinogen molecule in the specified location can bind at least two binding fibrinogen group.

10. The use of multiple soluble media suitable for intravenous and/or by local injection where each carrier contains many binding fibrinogen groups, immobilized on the carrier to form a Biogel or to obtain a drug containing Biogel, to stop bleeding or to treat or isolate the wound.

11. The use of claim 10 to form Biogel used for hemostasis, as an insulating coating for targeted drug delivery or tissue engineering.

12. The Biogel on any of PP-5 for hemostasis, as an insulating coating for targeted drug delivery or tissue engineering, or to obtain drugs to stop bleeding, or treatment or isolation of a wound.

13. The use of an agent according to claim 1 for hemostasis, as an insulating coating for targeted drug delivery or tissue engineering, or to obtain drugs to stop bleeding, or treatment or isolation of a wound.

14. Pharmaceutical composition for topical administration, containing Biogel on any of PP 5 agent according to claim 1, or many soluble media suitable for intravenous and/or by local injection, where each carrier contains many binding fibrinogen groups immobilized on the specified media.



 

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Organic compounds // 2496479

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a method for preparing an inhaled fine or crystalline glycopyrronium powder salt. The declared method involves suspending the crystalline glycopyrronium salt in acetone to prepare a suspension, homogenising the suspension at pressure 500-2000 bar to prepare glycopyrronium salt particles at average particle size less than 10 mcm, and drying the glycopyrronium salt particles to remove residual acetone if any.

EFFECT: invention provides preparing the aggregation and agglomeration resistant crystalline glycopyrronium powder salt.

7 cl, 2 tbl, 4 ex

FIELD: food industry.

SUBSTANCE: this invention relates to a crystalline maltitol powder composition; its specificity consist in the fact that the average particle size across the bulk (according to laser diffraction results) is equal to 10 - 150 mcm; the content of maltitol in the composition is 80 - 99.9 wt %; at least 50 wt % of the particles pass through a sieve having retention threshold equal to 2000 mcm according to A1 test; at least 35 wt % of the particles pass through a sieve having retention threshold equal to 2000 mcm according to A2 test; the composition includes 0.1 - 20 wt % of at least one water-insoluble anti-clogging agent; the said anti-clogging agent has hydroscopic property (determined according to Test B) equal to 2.5 - 25%; the said anti-clogging agent is selected from the group including pyretogenous silicon dioxide, sodium aluminosilicate, anhydrous tricalcium phosphate and dehydrated potato starch (especially dehydrated potato starch containing less than 12% residual water, preferably containing less than 10% residual water, preferably containing less than 8% residual water, preferably containing less than 6% residual water) and their mixtures.

EFFECT: this composition is not prone to clogging and finds application in the food and pharmaceutical branches.

13 cl, 5 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to pharmaceutics and medicine and concerns a composition for oral administration containing protein having molecular weight up to 100000 Da, an absorption intensifier specified in a group of: SNAC, SNAD, or salts thereof, a protease inhibitor; there are also declared methods of treating diabetes mellitus involving the administration of the above composition, and a method for oral administration of protein with enzymatic activity.

EFFECT: group of inventions provides substantially increased protein bioavailability and ease of use.

16 cl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and pharmaceutical industry and concerns a dry powder preparation containing micronised: salmeterol xinafoate and fluticasone propionate. The preparation contains a carrier consisting of lactose of average particle size 100-120 mcm and sodium benzoate. What is also described is a method for producing the preparation.

EFFECT: formulation possesses higher percentage of a respirable fraction of the active substances.

4 cl, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to dry powder-like pharmaceutical composition for inhalation and to method of its obtaining. Into composition of pharmaceutical composition included are two active ingredients, which represent salmeterol and fluticasone or their pharmaceutically acceptable salts, solvates or esters, two pharmaceutically acceptable excipients, which have value d50 respectively approximately 125-145 mcm and approximately 50-100 mcm and which represent mono- or disaccharides. Method of composition obtaining consists in formation of preliminary mixtures of each of active ingredients and each of pharmaceutically acceptable excipients and mixing two preliminary mixtures with obtaining with fraction of medication, with weight ratio of exipients in said two preliminary mixtures constituting from 1 to 5.

EFFECT: pharmaceutical composition is applied for treating asthma, COLD (chronic obstructive lung disease), allergies and infectious diseases.

6 cl, 7 dwg, 3 tbl, 3 ex

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