CZ2005668A3 - Two-component biologically degradable tissue adhesive for medicinal application - Google Patents

Abstract

The present invention relates to a bi-component biodegradable, medical-based bi-degradable adhesive, which is microbial and independent of the presence of calcium or other metal cations. It is present in the adhesive at a concentration of 0.05 to 10% by weight, together with a proteinaceous carrier from the group of denatured animal collagen, gelatin, milk proteins and albumin, optionally chitosan.

Description

(57)

The present invention relates to a biodegradable tissue adhesive for medical use based on transglutaminase, which is of microbial origin and is independent of the presence of calcium or other metal cations. It is present in the adhesive at a concentration of 0.05 to 10% by weight, together with a protein carrier from the group of denatured animal collagens, gelatin, milk proteins and albumin, and possibly chitosan.

CZ 2005 - 668 A3

Two-component biodegradable tissue adhesive for medical use

Technical field

The present invention relates to a two-component biodegradable tissue adhesive for veterinary and human medicine.

BACKGROUND OF THE INVENTION

Surgical sealants, or adhesives and adhesives, are preparations of synthetic or biological origin that stop bleeding, create an impermeable barrier to liquids and gases, or close wounds. Apart from targeted applications for targeted delivery of biologically active substances to different tissues, bioadhesive preparations used in medicine can be freely divided into the following categories:

Surgical sealants - stop bleeding, creating an impermeable barrier to liquids and gases.

Surgical Adhesives - creates a stronger bond than sealants, usually of biological origin, but non-resorbable.

Surgical Adhesives - creates the strongest bond, usually synthetic and non-resorbable, tightly seals the wounds. Cyanoacrylates are a typical example.

Sealants are the largest in the market, estimated to be used in 35 to 40% of internal surgeries in Europe and Asia, and the global annual market is estimated at hundreds of millions of US dollars.

Examples of some commercial preparations in these categories are given in the following tables:

Adhesive category

commercial name / manufacturer active ingredient or system advantages disadvantages INDERMIL ^'m / Loctite Corporation n-butyl cyanoacrylate high bond strength and solidification rate suspicion of cytotoxicity DERMABOND / Johnson & Johnson 2 - octyl cyanoacrylate high bond strength and solidification rate suspicion of cytotoxicity

• · ♦ · · • 2 • · · · · Adhesive category • · · · · · · · · · · · • · · · • · · · · · • · · · • ····· · · commercial name / manufacturer active ingredient advantages disadvantages BioGlue / Cryolife Inc. BSA with glutaraldehyde high binding strength and setting speed suspected cytotoxicity GRF / MedChem gelatin - resorcinol - formaldehyde high binding strength and setting speed suspected cytotoxicity

Category sealants

commercial name / manufacturer active ingredient advantages disadvantages CoSeal TM / Cohesion Technologies PEG polymers biocompatible and biodegradable they are not known to us Tisseel, Tissucol / Baxter fibrinogen - thrombin biocompatible and biodegradable weak binding strength, danger of viral infection Beriplast / Centeon fibrinogen - thrombin biocompatible and biodegradable weak binding strength, danger of viral infection FocalSeal / Focal synthetic components biodegradable, light activated need accessory equipment

Currently, there are several approaches for crosslinking natural or synthetic polymers that are used in the formation of gels, useful further as, for example, tissue adhesives, tissue engineering materials, or for covering and treating surface wounds.

The first approach utilizes the initiation of the polymerization reaction in the presence of multifunctional monomers. Since these multifunctional monomers are gradually incorporated into two or more growing polymer chains, a three-dimensional covalently crosslinked gel structure is gradually formed. Cyanoacrylate adhesives are an example of this approach. Since this procedure uses low molecular weight and highly reactive cytotoxic monomers, there are reasonable concerns about the adverse health consequences of these products. The gels thus prepared are poorly absorbed by the organism and are very difficult to biodegradable and resorb.

The second approach is the use of so-called "smell of polymers" which cross-link "in situ" due to changes in conditions after application, usually in response to changes in temperature or pH. These prospective materials are currently the subject of intensive research.

In the third approach, crosslinking of soluble linear polymers or macromolecules is initiated using light or low molecular weight crosslinking agents, such as formaldehyde or glutaraldehyde. When using these low molecular weight cytotoxic crosslinking agents, the same concerns apply as in the first approach.

These disadvantages resulting from the use of low molecular weight crosslinking agents can be eliminated by replacing them with enzymes catalysing the formation of three-dimensional hydrogel networks. An example of such an enzyme is transglutaminase.

Transglutaminase (EC 2.3.2.13; protein-glutamine γ-glutamyltransferase) is an enzyme with many important physiological functions that catalyzes the transfer of the acyl group of the glutamine residue (donor) of a peptide chain to several primary amines of the same or another chain as acceptors. When lysine residues serve as the acceptor, the enzyme can form intra- and intermolecular N-e- (γ-Glu) -Lys isopeptide bonds. Transglutaminases have been found in mammalian tissues, fish, plants and various microorganisms.

Transglutaminases of animal origin are dependent on calcium or other metal cations, which is not always desirable. Patented transglutaminase isolated from guinea pigs (JP Publication No. 1-50,382). The use of fish transglutaminase has also been proposed.

Microbial transglutaminases and processes for their preparation have also been patented (WO2004078973, US2002187525, US2002151703,

US2002173021, US6538122, US6190879, CA2237041, JP11075876, US6100053, WO9641866, JP3043080).

Commercial production of bacterial transglutaminase, carried out by the Japanese company Ajinomoto, has enabled the industrial use of the enzyme in many applications, especially in the food industry.

The following overview relates to patents for the use of transglutaminase primarily in biomedical applications:

US5736132 (Biological adhesive composition and method of promoting adhesion between tissue surfaces) discloses a biological adhesive comprising tissue transglutaminase in a pharmaceutically acceptable aqueous medium. Tissue transglutaminase is used in an effective catalytic amount to allow adhesion of two tissue surfaces by their cross-linking. The aforementioned aqueous medium must contain calcium or other divalent metal cations necessary for the functionality of the enzyme, and may also include an auxiliary protein mediating binding between tissue surfaces.

Ajinomoto (Japan) patent EP0686401 (Living-tissue adhesive and blood coagulant) relates to a microbial transglutaminase that is independent of calcium and other metal cations and is claimed herein for use as a tissue adhesive and blood coagulant. The method of the invention does not envisage the use of any helper protein or other polymer, but the direct coupling of tissue surfaces by a cross-linking reaction. Transglutaminase enables blood coagulation by direct cross-linking of fibrin, or the attachment of a blood clot to tissue by the formation of covalent bonds between the clot protein and the tissue surface.

WO2004082728 (Medical Material) uses transglutaminase to prepare biodegradable tissue engineering materials (scaffolds) from a suspension containing denatured collagen, tissue regeneration cells and some other components.

WO2004042068 (Injectable Bioadhesive Polymeric Hydrogels as well as related methods of enzymatic preparation) relates to the use of transglutaminase for the preparation of injectable bioadhesive polymer hydrogels using synthetically prepared peptides of a specific composition.

All of the above-mentioned products are subject to varying degrees of unwanted negative manifestations, so researchers are constantly trying to develop new and better products worldwide.

SUMMARY OF THE INVENTION

The above-mentioned drawbacks are overcome by the two-component biodegradable tissue adhesive for medical use based on transglutaminase according to the invention, which is characterized in that the transglutaminase is of microbial origin and is independent of the presence of calcium or other metal cations. It is present in the adhesive at a concentration of 0.05 to 10% by weight, together with a protein carrier from the group of denatured animal collagens, gelatin, milk proteins and albumin, and possibly chitosan.

The two-component biodegradable tissue adhesive of the invention is further characterized in that the adhesive preferably comprises 0.5 to 10 wt. gelatin or denatured collagen as carrier.

The two-component biodegradable tissue adhesive of the invention is further preferably characterized in that the adhesive comprises 0.1 to 4 wt. chitosan • ·

The principle of the adhesive according to the invention is to utilize catalytic crosslinking of gelatin, denatured collagen or other suitable protein by the action of a commercially available transglutaminase enzyme from microbial sources, independent of the presence of calcium or other metal cations. The crosslinking reaction leads to the formation of solid irreversible gels. This process is utilized in a tissue adhesive composition. Said enzyme catalyses transamidation reactions which lead to the formation of covalent bonds of the "N-? - (γ-glutamyl) lys" type. In a preferred embodiment, the proposed adhesive of the invention also comprises chitosan isolated from natural sources, which accelerates the rate of gel formation and its strength. Chitosan contributes to increasing the rate of gel formation, improving its firmness and improving the integrating, hemostatic, healing and regenerative properties of a product intended for use in human or veterinary medicine.

The two-component biodegradable tissue adhesive of the present invention has been successfully tested by the inventors, as the following examples and graphs in the accompanying drawings illustrate.

The advantage of the adhesive according to the invention is primarily commercial microbial transglutaminase independent of calcium or other metal cations and the high binding strength of the fully biocompatible and biodegradable stable irreversible gel. Furthermore, the adhesive provides coverage and structural integrity of the damaged tissue, accelerates the regeneration of the original tissue, allows sterilization by γ-radiation, and the initiation of gel formation requires no other factors such as light or other crosslinking agents and is sufficiently viscous and flexible for ease of application. Furthermore, the adhesive of the invention achieves the combined healing effects of chitosan and gelatin as well as the combined hemostatic and coagulatory effect of chitosan and transglutaminase. In some applications, the disadvantage may be the slower setting time of the adhesive of the invention. In view of the above classification, the adhesive of the invention can be categorized as "sealants" and adhesives for use in both human and veterinary medicine.

Examples

Example 1

Determination of the effect of transglutaminase concentration on the gel formation rate in a 10% (w / v) gelatin solution at 37 ° C.

A 10% (w / v) gelatin solution was prepared by dissolving 10 g of pork skin gelatin (Type A, 300 bloom, Sigma) in 100 ml demi-water at 45 ° C, pH was adjusted to 6.5 by addition sodium hydroxide (NaOH). Gel formation was initiated by the addition of 0.5 and 1% (w / v) ACTIVA WM transglutaminase (Ajinomoto Co., Inc, Tokyo, Japan) to gelatin solutions. Transglutaminase activity. 81 to 135 U / g (hydroxamate method).

Viscosimetric gel rate measurements were performed at a constant rotational speed and temperature of 37 ° C using a RHEOTEST 2.1 viscometer (MLW Prufgerate-Werke Medingen, NDR). The attached graphs show the breaking point of the experimental curves at a time at which a relatively rapid loss of fluidity occurs and the viscosity usually begins to rise sharply. The time at which this break occurs is indicated by the gel formation time of the experimental mixture.

The results of the experiment are shown graphically in Figure 1. From the graph in this figure, it is apparent that the rate of gel formation increases with the concentration of transglutaminase TGA. At an enzyme concentration of greater than 1% (w / v), gel formation times of less than 15 minutes can be achieved in a concentrated gelatin solution. This solidification time of the composition is sufficient to use the test composition as a surgical sealant and, in many cases, as an adhesive for firm tissue joining.

Example 2

Determination of the effect of gelatin concentration on the gel formation rate in the presence of 0.5% w / v. transglutaminases at 37 ° C.

Gelatin solutions (5 or 10% w / v) were prepared by dissolving 5 or 10 g of pork skin gelatin (Type A, 300 bloom, Sigma) in 100 ml demi-water at 45 ° C, pH was adjusted to 6.5 by addition of NaOH.

• ··· ··· ··· ··

Gel formation was initiated by the addition of 0.5% (w / v) ACTIVA WM transglutaminase (Ajinomoto Co., Inc., Tokyo, Japan) to gelatin-containing solutions. Transglutaminase activity: 81-135 U / g (hydroxamate method).

Viscosimetric measurements of the gel formation rate were performed in the same manner as in Example 1. The results of the experiment are shown graphically in Fig. 2. It is apparent from the graph in this figure that the gel formation rate increases with increasing gelatin concentration. In order to achieve the shortest gel formation time, the gelatin concentration in the formulation should be as high as possible.

Example 3

Determination of the effect of the presence of chitosan on the gel formation rate in a 5% (w / v) gelatin solution with 0.25% (w / v) transglutaminase at 37 ° C.

A 5% (w / v) gelatin solution was prepared by dissolving 5 g of pork skin gelatin (Type A, 300 bloom, Sigma) in 100 ml demi-water at 45 ° C, pH was adjusted to 6.5 by addition NaOH.

A chitosan solution (3.2% w / v) was prepared by dissolving A. niger fungal chitosan (SCF, molecular weight distribution: 5 to 50,000, degree of acetylation: 4 to 5%) in demi-water after pH adjustment by addition of HCl Finally, the pH of the solution was raised back to 6.5 by addition of NaOH.

Gel formation was initiated by the addition of 0.25% (w / v) transglutaminase ACTIVA WM (Ajinomoto Co., Inc., Tokyo, Japan) to gelatin-containing solutions. Transglutaminase activity: 81-135 U / g (hydroxamate method). Viscosimetric measurements of the gel formation rate were performed in the same manner as in Examples 1 and 2. The results of the experiment are shown graphically in Figure 3. It is apparent from the graph in this figure that the addition of chitosan increased the gel formation rate. However, direct covalent binding of chitosan to gelatin has not yet been demonstrated.

Example 4

Measurement of the binding strength of the proposed tissue adhesive compared to the GRF adhesive.

• · · · · · · · · · · · · · · ·

The binding strength of the proposed tissue adhesive (gelatin - transglutaminase (TGA) - chitosan) was compared with that of the gelatin - TGA mixture, pure gelatin and the commercially used gelatin - resorcinol crosslinked formaldehyde (GRF adhesive) mixture.

The composition of the tested adhesive mixtures was as follows:

Sample 1

3.3% (w / v) pig skin gelatin (Type A, 300 bloom, Sigma), 2% (w / v) TGA (ACTIVA WM) and 1% (w / v) fungal of chitosan (P. oxalicum) in demi - water, pH 5.5

A sample of 2% (w / v) gelatin and 3% (w / v) TGA in demi-water, pH 5.5.

Sample 3

3.3% (w / v) gelatin, 6.7% (w / v) resorcinol (Sigma) and 1.2% (w / v) formaldehyde (Sigma) in demi-water, pH 5.5.

A sample of 4% (w / v) gelatin in demi-water, pH 5.5.

The adhesion characteristics of all prepared samples were determined by measuring their binding forces between two thin slices (8 x 2.5 cm) prepared from fresh pork heart muscle. Each sample of 0.4 ml was applied immediately after mixing the ingredients between 2.5 x 2.5 cm of the area of the two slices. After loading the joint at 800 g.cm ^{< 2 >} for 60 minutes at 37 [deg.] C., the binding strength of each sample was measured using a TA-XT 2 texture analyzer (Stable Micro System, UK) at a constant speed of 48 mm.miri ¹ until to tear the glued muscle slices together.

The results of the experiment are shown graphically in FIG. 4. From the graph in this figure, it is clear that the adhesive strength of Sample 2 (gelatin + TGA) was comparable to that of Sample 3 (GRF mixture). The composition of Sample 3 was designed according to the literature to approximate the composition of commercial GRF adhesives.

• · · · · · ·

The addition of chitosan in sample 1 did not significantly affect the binding strength of the mixture. The lower measured tensile force of this sample can be explained by the lower concentration of gelatin and TGA in the sample.

Thus, using a sufficiently high concentration of gelatin and enzyme, the binding strength of the adhesive of the invention is comparable to that of a commercial GRF adhesive.

Industrial applicability

Two-component biodegradable tissue adhesive for medical use based on microbial transglutaminase, which is independent of the presence of calcium or other metal cations and is present in the adhesive at a concentration of 0.05 to 10% by weight, together with a protein carrier from denatured animal collagen, milk protein and albumin, possibly chitosan. This two-component adhesive is widely used in veterinary and human medicine.

Claims (4)

PATENT CLAIMS A two-component biodegradable tissue adhesive for medical use based on transglutaminase, characterized in that the transglutaminase is of microbial origin and is independent of the presence of calcium or other metal cations, wherein the vadhesive is present in a concentration of 0.05 to 10% by weight with protein. a carrier from the group of denatured animal collagens, gelatin, milk proteins and albumin, and possibly chitosan. A two-component biodegradable tissue adhesive according to claim 1, characterized in that the adhesive preferably comprises 0.5 to 10% by weight. gelatin or denatured collagen as carrier. A two-component biodegradable tissue adhesive according to claim 1, characterized in that the adhesive preferably contains 0.1 to 4% by weight. chitosan ·· ····· · · · · · t · · ··· Giant. 1: Determination of the effect of transglutaminase concentration (TGA) on gel formation rate in a 10% (w / v) gelatin solution ()) at 37 ° C. 450 ω 400 m O. 350 E and ³⁰⁰ 8 250 to _ΛΑΛ > 200 and ¹⁵⁰ | ^ιοθ 50 50 0 0 50 100 150 200 polymerization time (min) ÉAA A AAAA ^ AA A 10% W; 0.5% TGA A 5% W; 0.5% TGA Giant. 2: Determination of the effect of gelatin concentration (F) on the gel formation rate in the presence of 0.5% w / v Tg at 37 ° C. • 4 ···· I »» · * * 300 250 <0 CL E 200 (O + J N O E 150 A 5% Z; 0.25% TGA 5% W; 1.6% Ch; 0.25% TGA < B O 100 Ě ffi c> » Ό j IaIaIaI i Iaaaaa a 50 100 150 200 polymerization time (min) 250 Giant. 3: Determination of the effect of the presence of chitosan (Ch) on the rate of gel formation in a 5% (w / v) solution of gelatin ()) with 0.25% (w / v) transglutaminase (TGA) at 37 ° C. Giant. 4: Measurement of the binding strength of the proposed tissue adhesive compared to the GRF adhesive. Sample 1: 3.3% (w / v) W, 2% (w / v) TGA and 1% (w / v) CH Sample 2: 5% (w / v) W and 3% (w / v) TGA Sample 3: 3.3% (w / v) R, 6.7% (w / v) resorcinol and 1.2% (w / v) formaldehyde GRF adhesive Sample 4; 10% (w / v)