DE10212553A1 - Acceleration of the biodegradation of porous wound coverings based on polyhydroxyalkanoate and polyanionic hydrocolloids is effected using a buffered formulation containing a poly-(hydroxybutyrate)-depolymerase - Google Patents

Abstract

Flat and porous composite materials composed of biodegradative polyhydroxyalkanoate and polyanionic hydrocolliodes are used as wound coverings. They should dissolve after a while. However, the natural in situ biodegradation of the wound coverings is very slow. A targeted acceleration of the in situ biodegradation of the wound covering is achieved by using a formulation containing a poly (hydroxyalkanoate) -degrading depolymerase. Another component of the formulation is a physiologically compatible buffer system with a high buffer capacity. The formulation can be used in liquid, viscous, gel-like or pasty form.

Description

The field of application of the invention is the treatment of wounds. The Invention is used when for medical reasons the degradation a flat composite material used to cover wounds, which polyalkanoates, in particular the poly (3-hydroxybutyrate and / or poly- (4-hydroxybutyrate) and polycationic or polyanionic hydrocolloids such as contains, for example, hyaluronic acid or its salts or derivatives, should be accelerated.

The polyalkanoates are already used in the manufacture of medical Proposed products that are absorbed by the body after some time should or are biodegradable (Brandl, H., R. A. Gross, R. W. Lenz, and R. C. Fuller, 1990. Plastics from bacteria and for bacteria: Poly (beta-hydroxyalkanoates) as natural, biocompatible, and biodegradable polyesters. Adv. Biochem. Closely. Biotechnol. 41: 77-93; Lindsay, K.F., 1992. Truly degradable 'resins are now truly commercial. Modern Plastics 69: 62-64; Lashinsky, A., 1992. Fast-growing Chicago firm sees green in disappearing plastic. Crain's Chicago Business, Vol. 15, No. 2).

Biodegradation takes place in situ through hydrolytic reactions in the tissue, possibly also supported by the action of tissue enzymes such as Esterases, proteases or lipases. The use of the polyalkanoates as Material for implants and as a carrier material for living animal cells also proposed (Gogolewski S. Jovanovic M, Perren SM, Dillon JG, Hughes MK., 1993. Tissue response and in vivo degradation of selected polyhydroxyacids: polylactides (PLA), poly (3-hydroxybutyrate) (PHB), and poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB / VA J. Biomed Mater. Res. 1993, 27, 1135-48; Yasin M, Holland S.J., Tighe B.J., 1990. Polymers for biodegradable medical devices. V9. Hydroxybutyrate-hydroxyvalerate copolymers: effects of polymer processing on hydrolytic degradation. Biomaterials, 11, 451-4, Dawes E.A., 1988. Polyhydroxybutyrate: an intriguing biopolymer. Biosci. Rep. 1988, 8, 37-47; Hankermeyer CR, Tjeerdema RS., 1999. Polyhydroxybutyrates: plastic made and degraded by microorganisms. Rev Environ Contam Toxicol. 159 1-24).

It is known that more or less specific acting esterases that Depolymerases from microorganisms the hydrolytic degradation of Accelerate polyalkanoates. JP 9.191.887 refers to the application of the Enzyme in the degradation of waste plastics from polyalkanoates. depolymerases are caused by a number of microorganisms such as Alcaligenes faecalis, Comamonas acidovorans, Pseudomonas picketii, Pseudomonas lemoignei, Pseudomonas testosteroni, Pseudomonas aeruginosa, Penicillium pinophilum, Alcaligenes faecalis, Corynebacterium aquaticum, Arthrobacter sp. D6, Penicillium funiculosum u. a. educated. (Kasuya K, Inoue Y, Tanaka T, Akehata T, Iwata T, Fukui T, Doi Y., 1997. Biochemical and molecular characterization of the polyhydroxybutyrate depolymerase of Comamonas acidovorans YM1609, isolated from freshwater, Appl Environ Microbiol, 12 4844-52; Kasuya K, Ohura T, Masuda K, Doi Y., 1999. Substrates and binding specificities of bacterial polyhydroxybutyrate depolymera-ses. Int J Biol Macromol, 24, 4 329-36; Tanio et al. Tanio T, Fukui T, Shirakura Y, Salto T, Tomita K, Kaiho T, Masamune S., 1982. An extracellular poly (3-hydroxybutyrate depolymerase from Alcaligenes faecalis: Eur J Biochem; 124 (1): 71-7; Delafield, F.P., M. Doudoroff, N.J. Palleroni, C.J. Lusty, and R. Contopoulos, 1965. Decomposition of poly (beta-hydroxybutyrate) by pseudomonads, J. Bacteriol. 90: 1455-1466; T. Tanio, T. Fukui, Y. Shirakuru, T. Salto, K. Tomita, T. Kaiho, and S. Masamune, 1982. An extracellular poly (3- hydroxybutyrate) depolymerase from Alcaligenes faecalis, Eur. J. Biochem. 124: 71-77; Timm, A .; Steinbuechel, A., 1995. Poly (3-hydroxyalkanoic acid) depolymerase PA5057 Pseudomonas aeruginosa (strain PAO1). J Bacteriol. 177 (3): 596-607).

Depolymerases were also found in the gram-positive streptomycetes. Streptomyces exfoliatus K 10 forms a depolymerase that Poly (3-hydroxybutyrate) and poly (3-hydroxyoctanoate) hydrolyzed. The enzyme shows great Homology to the PHB Depolymerase of Comamonas sp. and a low one Homology to other PHA depolymerases. (Klingbeil B, Kroppenstedt RM, Jendrossek D .: Taxonomic identification of Streptomyces exfoliatus K10 and characterization of is poly (3-hydroxybutyrate) depolymerase gene. FEMS Microbiol Lett. 1996. 1, 142 (2-3) 215).

A depolymerase was found in a marine streptomycete poly (3-hydroxybutyrate) and the copolymer poly (3-hydroxybutyrate-co-3- hydroxyvalerate P degrades. (Mabrouk mm, Sabry SA., 2001. Degradation of poly (3-hydroxybutyrate) and its copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by a marine Streptomyces sp. SNG9. Microbiol Res 156, 323-35). The Molar masses of the microbial depolymerases are between 30 and 50 kD.

Polyhydroxyalkanoates are used in contact with human or animal Tissue degrades very slowly due to natural biodegradation. The Depolymerases catalyze when enzyme is applied to the surface of a pure one Polyalkanoate a degradation process that slows down more and more, making a extensive dismantling is not possible. Therefore depolymerases become Removal of massive or porous implants or wound covers not used. It is known that thin membranes made of PHB as a suspension in a test volume can be used to determine their weight loss to determine the enzymatic activity of depolymer preparations.

The aim of the invention is to propose formulations by which or repeated application to porous wound cover Surfaces consisting of a composite material, which is polyanionic Contains hydrocolloids and polyhydroxyalkanoates, produced in situ pores or be enlarged and thus ultimately extensive to complete dismantling according to the needs of treatment within one to two Weeks to reach. This is to ensure that the side facing the wound surface ongoing natural biodegradation processes by degradation from the outward-facing surface of the wound covering and in the area of the pores get supported. The healing process taking place on the wound bed may not be disturbed by the treatment.

Surprisingly, it was found that the acceleration of natural Biodegradation of the porous fabrics is achieved in that a effective physiologically compatible buffer system containing one or several enzymes, the polyhydroxyalkanoate especially that Degrading poly- (3-hydroxybutyrate), applied from the outside to the wound coverings in situ, by which the pH of the surface of the wound coverings in the area is set between 5.0 and 8.0, preferably 5.8 to 7.2. It will be without thereby restricting the scope of protection, preferably phosphate buffer systems with a phosphate content between 0.05 M to 1.0 M or acetate or Citrate buffers with an acetate content between 0.05 M and 1.0 M are suggested on the wound covers are applied. The enzyme activity of the Formulations range between 0.05 and 20 relative units.

Any poly (hydroxybutyrate) hydrolyzing, especially Poly (3-hydroxybutyrate) hydrolyzing enzyme can be used in the present invention. One of Streptomyces spec. (HKI No. 0243) excreted poly (3- hydroxybutyrate) -Depolymerase with a molecular weight of about 36 kD used.

Auxiliaries may be present. The auxiliary substances include substances which influence the consistency of the formulations and those which affect the Improve treatment. Other additives are additives that increase the stability of the Improve formulations and their effectiveness.

It has so far not become known that such formulations for Accelerating the degradation of porous fabrics leading to wound coverage are used and which consist of a hydrocolloidal polyanion and Polyalkanoates, especially 3-polyalkanoates, exist under in situ conditions be used.

The formulations used for the treatment are liquid, viscous, gel-like or pasty. In one embodiment, an appropriately buffered solution of the enzyme used, which is a polyanionic polysaccharide in Contains concentrations between 0.5 g / l and 20 g / l as thickeners. By the presence a thickener does not receive the formulation from the wound covering draining consistency. At higher levels there is a brushable formulation receive. Because of the biocompatibility and the known cheap A solution of. Is preferred to influence wound healing Hyaluronic acid with molecular weights between 10 kD and 2000 kD or a hyaluronic acid Uronids with molecular weights between 10 kD and 500 kD are used. The Hyaluronic acid uronide is caused by the action of the enzyme hyaluronate lyase Hyaluronic acid produced in a manner known per se. According to the z. Legs Embodiment of the invention in which the liquid in one Calcium carbonate compound slurried powder and the semi-solid mass on the wound cover is applied. The paste is made by mixing one Solution of the enzyme protein in a buffer between pH 5.0 and 7.0, preferably between 5.5 and 6.5, with the finely powdered Calcium carbonate made. The semi-solid mass is applied to the wound covering with a Layer thickness of about 0.5 cm applied.

All formulation are in the treatment according to the invention on the Wound dressing, which is located on the wound or in situ and stay there for up to several hours. A multiple treatment, interrupted by washing processes is also possible. Here, the Formulation together with the degradation products with buffered physiological Rinsed saline with a pH between 5.5 and 8.0.

The depolymerases are prepared in a manner known per se Submerged cultures of microorganisms, preferably a Streptomyces spec., Removal of microorganisms, cleaning and concentration by Affinity chromatography and fractional precipitation with ammonium sulfate.

The activity of the polyhydroxybutyrate depolymerase is determined by the release of the hydroxybutyric acid from a 1 cm ² large polyhydroxybutyrate film under standard conditions. measured.

The following examples serve to illustrate the invention, are intended but in no way restrict it.

example 1 Production of PHB depolymerase

The Streptomyces strain Streptomyces spec. (HKI No. 0243) is in 100 ml Heart-brain broth pre-cultivated for two days at 28 ° C with stirring (160 rpm). With this preculture, 1.5 L of a main culture is inoculated, the medium Contains nutrient salts and 1 g glucose / L. After the inoculation become this main culture 4.5 g finely ground 3-PHB suspended in 200 ml A. dest. and subsequently added sterilized. It is at a pH between pH 6.5 and 7.0, one Stirrer speed of 300 rpm and a temperature of 28 ° C for 5 days fermented. The biomass is then removed by separation and the Culture solution concentrated by ultrafiltration through a 5 kDa filter.

100 ml of concentrate solution are mixed with 20 g of ammonium sulfate, filtered clear and then the depolymerase is absorbed on 10 g of octyl sepharose. With 10% ethanolic Tris buffer 0.05 M pH 7.5 becomes the PHB depolymerase desorbed. The desorbate is dialyzed against water and lyophilized. 50 mg Lyophilisate are dissolved in 0.02 M Tris buffer pH 7.5 and over Sephadex 75 separated. The active fractions are pooled, dialyzed and again lyophilized. 5 mg of lyophilizate are dissolved in 50 μl of 0.05 M Tris buffer pH 7.5 and rechromatographed again on Superdex 75. The active fractions will be collected, dialyzed and lyophilized. 1 mg of product is obtained.

The relative activity of the depolymerase is determined by the release of D-3-hydroxybutyric acid against a film of 3-polyhydroxbutyric acid. For this purpose, 1 cm ^{2 of} an approximately 15 μm thick film of non-porous 3-PHB, prepared by spreading and drying a solution of 3-PHB in chloroform on a glass plate, in 3 ml of 0.05 M phosphate buffer of pH 6.5 and approximately Put 1000 µg in 1 ml of water or phosphate buffer. The mixture is incubated at 37 ° C. for one hour while shaking at 120 rpm. The released 3-hydroxybutyric acid is oxidized to acetoacetate in the presence of the enzyme poly- (3-hydroxybutyrate) dehydrogenase and NAD. The resulting NADH reacts with iodonitrotetrazolium chloride in the presence of diaphorase to formazan, which is measured at a wavelength of 492 nm (ε = 19.9 l × mmol ^-1 × cm) (Methods of Enzymatic Analysis, 3rd edition, Vol 1, Verlag Chemie Weinheim, p. 204).

In another variant, the content of a certain volume is determined by measured a 0.45 µm filter filtered solution of combustible carbon. The value is converted into 3-hydroxybutyrate with the factor 2.166 and to 3 ml extrapolated.

A release of 10 mg / h 3-hydroxybutyrate at 37 ° C within 1 hour corresponds to an activity of 1.0 relative unit.

Example 2

About 4 ml of a phosphate-buffered solution (0.5 M) with a pH of 6.5 and a depolymerase activity of 1.0 relative unit, containing 10 g / l, a hyaluronic acid with a molecular weight of 1200 kD, is applied to an approximately 10 µm thick and 10 cm ² large porous wound covering, which is located on a wound surface. The gel-like formulation is carefully stroked back and forth several times with a spatula. After 12 hours, the formulation is rinsed with pH 6.8 phosphate-buffered saline. Then, after removing the liquid residues, apply fresh formulation by dabbing with gauze and repeat the procedure one day later.

Example 3

In situ wound coverage is treated as in Example 2. Instead of However, viscous formulation becomes a pasty formulation on the wound covering painted. This is achieved by mixing 4 ml of acetate buffer (1 M) at pH 6.4 made with 6 g finely powdered calcium carbonate. The mixture remains 1 Hour on the wound covering located on the wound surface and then removed by rinsing.

Claims (11)

1. Use of a formulation comprising a poly (hydroxybutyrate) Depolymerase and a buffer system for pH stabilization in the area between pH 5.0 and 8.0 together with the usual auxiliaries to accelerate the Biodegradation of flat wound coverings made of polyhydroxyalkanoate and polyanionic hydrocolloid. 2. Use according to claim 1, characterized in that in the Formulating a depolymerase enzyme activity between 20 and 0.05 relative Units is available. 3. Use according to claim 1, characterized in that it is the enzyme around the poly (3-hydroxybutyrate) depolymerase with a Molar mass between 32 kD and 36 kD and an isoelectric point of approximately pl = 3.85 from a microorganism of the genus Streptomyces. 4. Use according to claim 1, characterized in that the Formulation containing a phosphate ion, citrate ion or acetate ion Contains buffer system. 5. Use according to claim 1, characterized in that the Formulation is a liquid. 6. Use according to claim 1, characterized in that the Formulation has a viscous, pasty or semi-solid consistency. 7. Use according to claim 1, characterized in that the enzyme in an aqueous viscous solution containing 0.5 to 20 g / l one polyanionic hydrocolloid is present. 8. Use according to claim 1, characterized in that the Depolymerase in a suspension of solid particles with average Diameters between 0.001 µm to 0.2 mm. 9. Use according to claim 1, characterized in that the Formulation as a paste, made by mixing the liquid buffered Formulation with a pH between 5.5 and 6.5 with calcium carbonate particles, is used. 10. Use according to claim 1, characterized in that the enzyme in Form of a semi-solid or gel-like paste containing hyaluronic acid, Pectin, xanthan or alginic acid or their pharmaceutically usable Salts are present. 11. Use according to claim 1, characterized in that the Formulation additionally one or more antibiotics and / or preservatives contains.