Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
  • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
  • A61L2/0082—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using chemical substances
  • A61L2/0088—Liquid substances
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/50—Isolated enzymes; Isolated proteins

Definitions

• the invention is concerned with a formulation or kit for enzymatically disinfecting damaged but living animal tissue, and also relates to a novel method of disinfecting such tissue.
• damaged tissue refers chiefly to open and infected wounds of in the first place. mammals (including man) .
• Enzyme systems coupled to one another and bound covalently to a solid phase such as e.g. glucose oxidase - myeloperoxidase
• thiocyanate is known to be similarly convertible to 0SCN ⁇ by means of hydrogen peroxide, the OSCN ion in turn being capable of interfering with vital processes in practically all kinds of bacteria (see for example Bj ⁇ rk L. et al., Biotechn. Bioeng. 18 (1976), p. 1463-72, especially p. 1471 last paragraph) .
• the object of this invention is to provide compositions which are stable on storage and permit disinfection to be performed with hydrogen peroxide.
• the active agents are formed in situ, which permits optimum control/regulation of their presence, at the same time minimizing the risk that the surrounding healthy tissue is damaged. In many cases one administration is sufficient.
• a solid-phase-bound enzyme we obtain a composition which is easy to apply. This form of enzyme in addition minimizes the risk of sensitizing (human) individuals to the enzymes employed, and consequently it becomes possible to use the most efficient and cheap enzymes from non-human sources.
• the disinfectant formu ⁇ lation of the present invention containing: an effective amount of an enzyme capable of producing hydrogen peroxide; an effective amount of a substrate for said enzyme; and an effective amount of a compound that is oxidizable by hydrogen peroxide.
• effective amount in the specification and claims means that the amount has to be sufficient for the purpose contemplated - that is, when the formulation is applied to damaged but living animal tissue the amount of enzyme together with its substrate has to be sufficient to produce a disinfectant effect with the corresponding amount of oxidizable compound plus, optionally, additional components of the formulation.
• the enzyme producing hydrogen peroxide is in an insoluble form when it is being applied.
• infected damaged but living tissue is contacted with effec ⁇ tive amounts of the formulation components for a time sufficient for disinfection, whereupon the components applied plus reaction products formed are removed. If required the treatment may be repeated.
• Enzyme system capable of producing hydrogen peroxide
• Oxidases Many enzymes producing hydrogen peroxide are known. They all belong to the general class of oxidoreductases and require as their substrate both an electron acceptor and an electron donor. Some of the oxidoreductases, for instance the oxidases, may potentially be employed in accordance with the invention. All oxidases utilize oxygen (0_) as the electron acceptor and will either give hydrogen peroxide (H-O-) directly or will in a first step produce the superoxide radical ion
• the donor substrate of the oxidases is usually a compound containing an OH-group bound to a carbon atom, such as a phenolic hydroxyl group
• Oxidases may be named according to their donor substrates, the name of which is then followed by "oxidase". Some common oxidases which may potentially be of great importance for the purpose of this invention are: glucose oxidase, gala ⁇ tose oxidase, xanthine oxidase, NADPH oxidase etc. It should be noted here that oxidases must not be confused with an entirely different type of enzymes, viz., the peroxidases which utilize hydrogen peroxide as the electron acceptor.
• the formu ⁇ lation will comprise at least two different enzymes.
• the enzymes in such coupled systems are bound to a solid phase.
• Such coupled "tethered" enzyme systems are known to be far more efficient than corresponding systems in soluble forms (Mosbach K. et al., Acta Chem Scand 24(1970) p. 2093- and Mattiasson B et al Biochem. Biophys. Acta 235 (1971) p. 253-).
• glucose oxidase + lactase both bound to solid phase an example of such a coupled system is glucose oxidase + lactase both bound to solid phase.
• the lactose substrate is converted by the lactase to galactose and glucose, whereupon the glucose can be utilized by the glucose oxidase of the system for producing hydrogen peroxide,
• the enzymes in the formulation of this invention may ad ⁇ vantageously be selected from among those that require low molecular substrates, e.g. substrates having molecular weights of less than 1000 dalton.
• the oxidizable compound is selected from among physiologically acceptable halide salts, for example bromide, chloride and preferably iodide salt, or a corresponding thiocyanate salt.
• Solid phases that may be employed according to the invention are various hydrophilic macromolecular materials capable of adsorbing water. Examples of these are gauzes, compresses and various swellable water adsorbent layers which may be and have been employed in the treatment of tissue lesions (for instance GB-A-2,048,292) .
• tissue lesions for instance GB-A-2,048,292
• One type of highly suitable materials are water-insoluble macromolecular compounds in a particulate state which on being contacted with water are capable of limited swelling to thus form discrete gel particles.
• GB-1,454,055 has been employed as carrier phases in gel and affinity chromato ⁇ graphy, because of the large contact area these materials will offer to a surrounding aqueous liquid phase.
• the hydrophilic particles consist usually of a polymer containing amine and/or hydroxyl, such as e.g. a polysac ⁇ haride in an insoluble form, for instance an insoluble dextran derivative, cellulose, -starch, agarose etc.; a polymerized mono-, di- or oligosaccharide in an insoluble form; or a correspondingly polymerized sugar alcohol.
• the polymer may optionally be crosslinked or provided with covalently bound ion exchanging or hydrophobi ⁇ or hydrophilic groups. Such derivatization may bestow the suitable physical and chemical properties on a given polymer starting material.
• the swelling capacity expressed as "water regain” (that is, the water uptake capacity of the dry particles as such) may be.within a range of from 0.5 to 30 g/g, e.g. less than 15 g/g. While particle size may vary it will usually lie within the range of from 10 to 1000 ,u.
• the formulation comprises a physiologically acceptable buffer system the purpose of which is to bestow on an applied composition an optimum pH for the antimicrobial effect, this pH being within the range of 4.5-8, preferably 5-6.5.
• the buffer substance is one selected from among physiologically acceptable salts of phosphate, citrate or acetate, and other similar salts that are capable of giving a sufficient buffering capacity.
• a catalyst for the reaction occurring between hydrogen peroxide and the oxidizable compound for example, it may be advisable to also incorporate a catalyst for the reaction occurring between hydrogen peroxide and the oxidizable compound to thus- produce an antimicrobial substance; but in many cases the formulation can be used without such a catalyst.
• the catalyst (if employed) should be capable of converting I ⁇ to HIO/IO- or SCN " to OSCN ⁇ if the oxidizable compound is an iodide or thiocyanate salt respectively.
• various peroxidases for instance myelo- or lactoperoxidase
• the use of such extra enzyme systems is particularly suitable in cases with infected wounds.
• the formulation may also contain some of the above-mentioned solid phases, such as for example gauzes and compresses, without any enzyme bound to them.
• the components of the formulation may be packaged individually to form separate small packets in order to be blended only at the moment of use, in proportions suitable to the occasion.
• the solid-phase-bound enzyme of the invention may preferably be in a dry form, e.g. a dry free-flowing powder, dry sponge or dry compress.
• known per se suspension ointment bases of various types may be used (for example oil in water, water in oil, or fatty ointment).
• the enzyme suBstrate and the oxidizable compound may be either in a dissolved or in a dry state. If the oxidizable substance is reactive with atmospheric oxygen the package should be airtight.
• the solid phase is moistened and then applied on the damaged tissue whereupon the substrate and oxidizable compound are added, both of them preferably in a dissolved state.
• the individual components may be present as an all-in-one composition mix. In this case again it is preferred that atmospheric oxygen and humidity/ moisture are excluded.
• the proportions of the components of the formulation may vary within wide limits, inasmuch as many different types of wounds, with different degrees of infection, are contemplated for disinfection by the present means. As a rule of thumb it may be mentioned that in any given wound the components of the formulation should be capable of buffering the fluid of the wound to pH 4.5-8.
• the amount of solid-phase-bound enzyme to be employed will depend on factors such as the specific activity of the starting material and the pro ⁇ portional amount that has been tethered to the solid phase.
• the invention will now be exemplified further by way of experiments in an accepted rat model.
• the examples are non-limitative and show that the formulation of the present invention can be employed for efficiently killing S. aureus in wounds.
• the active substance formed is HIO/IO which is known to have a very strong effect.
• S. aureus which is employed in the experiments is the most frequently occurring bacterium in open wounds.
• MPO Myeloperoxidase
• the enzyme treatment was carried out by applying combinations, as according to the below Table, of 200 ,ul MPO preparation according to Example 1A, 100 ,ul GO preparation according to
• Example IB 100 ,ul H-O., and 1 ml 0.2 M phosphate buffer, pH 5.5, containing potassium iodide and glucose.
• Debrisan ® dextran crosslinked with epichlorohydrin, water regain 2.5 g/g, Pharmacia AB, Sweden
• the reaction mixtures were rendered viscous so that they remained in place more properly.
• the gel was cautiously removed by means of a spatula, and the wound was washed several times with physiological saline.
• the gel and wash fluid were pooled in each experiment, whereupon the total number of bacteria was determined in accordance with standard technology by counting colony-forming units after a 24-hr incubation period at 37 °C. In control experiments (representing 100 % survival of bacteria) only buffer and Debrisan were added to the wound chamber.
• Table I shows results from experiments in which GO bound to solid phase and glucose were used to produce H_0_. It can be seen that the H 2 0 2 -producing system was necessary for obtaining a bactericidal effect and that I will intensify that effect.
• Sepharose Sepharose Glucose I (mM) (limits, (n)
• H 2 0_ was added instead of GO Sepharose ® plus glucose. It should be noted that the mixture of I ⁇ and H 2 0- is very unstable, so each of these reagents has to be added separately to the chamber.
• the Example also demonstrates how a formulation according to the invention may be produced which contains GO-Sepharose ® , MPO-Sepharose ® and phosphate buffer (0.2 M, pH 5.5) con ⁇ taining KI (0.1 mM or alternatively 1 mM) , and glucose, 1 mM.

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Citations (6)

• 1985
  • 1985-01-24 SE SE8500341A patent/SE8500341L/sv not_active Application Discontinuation
• 1986
  • 1986-01-24 WO PCT/SE1986/000028 patent/WO1986004213A1/en not_active Application Discontinuation
  • 1986-01-24 EP EP86901156A patent/EP0210250A1/en not_active Withdrawn

Patent Citations (6)

Non-Patent Citations (1)

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