WO1986004213A1

WO1986004213A1 - Disinfectant formulation and method of disinfection in vivo

Info

Publication number: WO1986004213A1
Application number: PCT/SE1986/000028
Authority: WO
Inventors: Karl-Erik Arfors; Stefan Marklund
Original assignee: Pharmacia Ab
Priority date: 1985-01-24
Filing date: 1986-01-24
Publication date: 1986-07-31
Also published as: SE8500341D0; EP0210250A1; SE8500341L

Abstract

A formulation possessing enzymatic activity and containing (a) an enzyme which is capable of producing hydrogen peroxide and is present in an insoluble form when the components of the formulation are applied onto the damaged tissue; (b) a substrate for said enzyme, and (c) a compound which is oxidizable by hydrogen peroxide and which together with components of the formulation has an antimicrobial effect, for the disinfection of damaged but living animal tissue.

Description

Disinfectant Formulation and Method of Disinfection in vivo

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. The term "damaged tissue" refers chiefly to open and infected wounds of in the first place. mammals (including man) .

A great number of different types of preparations and methods of applying them have been used heretofore for disinfection purposes. Simple and cheap means of disinfection have been those comprising various low molecular oxidizing compounds as e.g. σhloroamine, iodine in the form of tri- iodide, hydrogen peroxide (H-,0_o) etc. Dosing of these types of agents is usually difficult, both as regards obtaining optimum dosage quantities and as regards optimum duration of administration. These agents are highly potent, thus giving rise to a risk that also healthy tissue will be damaged. Often they have a short-time effect only, and thus often have to be administered several times in the course of one treatment. Improvements are desirable.

For enzymatically disinfecting burns it has been a practice to employ glucose oxidase (Chemical Abstracts 78 (1973)

67654 v) . Myeloperoxidase in combination with halide ions has been proposed as a further antimicrobial composition to be used in vivo (General Patent Index Basic Abstracts

Journal, Section B, Farmdoc, Derwent Publications Ltd,

London, England; Abstract No 57474K (1983), 49918K (1983),

23221K (1983) and 18313K (1983), US-A-4,379,141 and EP-A-J3L8.,073)

Enzyme systems coupled to one another and bound covalently to a solid phase such as e.g. glucose oxidase - myeloperoxidase

(or lactoperoxidase) in combination with a thiocyanate ion or suitable halide ion have been suggested as means for sterilizing milk and other liquids in vitro (Bjδrk L. et al., Biotechnol. Bioeng. 18 (1976), p. 1463-72; Tenovouo J. et al., Arch. Oral Biol. 26 (1983) p. 309-14, and Henry S. et al., Biotechnol. Bioeng. 16 (1974), p. 289-91). It has also been suggested to use insoluble crosslinked peroxidase - oxidase systems in internal medicine as an agent for controlling tumour growth and infectious diseases^' (EP-A-62434) . However, no halide or thiocyanate ions seems to have been administered simultaneously with the enzy system.

It is well known, thus, that some oxidizable compounds together with hydrogen peroxide will have a bactericidal effect when exposed to the action of a peroxidase, and that this effect is of a broad-spectrum type. Also it is .well known that an iodide ion together with hydrogen peroxide can be converted catalytically to hypoiodite which in turn has an iodinating/oxidizing effect on proteins in general and therefore is capable of exerting an antimicrobial effect of a relatively broad-spectrum type. Moreover thiocyanate (SCN~) 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 Bjδrk L. et al., Biotechn. Bioeng. 18 (1976), p. 1463-72, especially p. 1471 last paragraph) .

Our results have revealed that the enzyme preparations mentioned above have been employed heretofore in an un¬ practical manner so that their full potential could not be exploited optimally. Moreover where "foreign" enzymes were employed there was a risk of sensitization.

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. By using 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.

For the disinfection of damaged but living tissue it has not been the practice heretofore to apply a solid-phase-bound oxidase or peroxidase. It has now been found surprisingly that in many cases a very satisfactory effect is obtainable also without peroxidase, that is, with omission of an essential component of the composition used heretofore in vitro or in internal medicine. It has moreover been found that glucose oxidase in combination with an pxidizable compound (e.g. I~) may produce a better effect in vivo than glucose oxidase itself.

The above objects are attained with 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. The term "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. According to the invention, the enzyme producing hydrogen peroxide is in an insoluble form when it is being applied.

In the method proposed in accordance with this invention, 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

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

(0_?T) which then forms hydrogen peroxide. 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

(e.g. xanthine) or an alcoholic hydroxyl group (e.g. as in carbohydrates, for instance a mono- or disaccharide) . 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, galaσtose 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.

In some cases it may be suitable to employ an enzyme for forming donor substrate in situ. In such a case the formu¬ lation will comprise at least two different enzymes. In conformity with the present invention, 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-).

An example of such a coupled system is glucose oxidase + lactase both bound to solid phase. When this system is used, 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.

As regards the possibilities of covalently binding various enzyme systems to solid phases, these are described below under the Solid Phases heading.

Oxidizable Compound

This is usually a low molecular compound which is able together with desired components of the formulation to react with hydrogen peroxide and give a compound having a stronger antimicrobial effect than the corresponding amount of hydrogen peroxide. The oxidizable compound (type, amount) should thus be chosen such that it will not inhibit any enzyme present in the formulation. Advantageously, 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 for Binding Enzymes

All enzymes in principle are nowadays held to be capable of covalently binding to or being trapped in solid phases while retaining their enzyme activity. This applies to oxidases too. For enabling such a bound enzyme to unfold its activity it is necessary that the eazy e is accessible to a substrate from a surrounding liquid. Thus a large contact area per unit volume permits (i) a great amount of enzyme to be bound to a given amount of solid phase and (ii) obtaining a high degree of activity of the enzyme thus bound. Several review articles concerning this field are available (see for example Biomedical Applications of Immobilized Enzymes and Proteins; Ed. Chang TMS; Plenum Press, New York, Vol. 1, esp. p. 1-90, 1977) .

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) . 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. Earlier on this type of materials in addition to being used in the treatment of wounds (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 polysacσharide 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 hydrophobiσ 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.

Further Additives

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. Generally 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.

For some types of tissue lesions 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. Examples of such catalysts are, in a preferred embodiment, various peroxidases (for instance myelo- or lactoperoxidase) bound to a solid phase. 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.

Physical Forms for Kits

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. Thus 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. In cases where the solid phase is a partiσulate material, 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. For actual use, 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. In other types of kits, 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. It should be noted here that 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.

Example 1

Preparation of solid-phase-bound enzymes

A. Myeloperoxidase (MPO)

2 mg of MPO from human leucocytes (Olsson I. & Wenge P., Blood 44(1974) p. 235-46) and having an activity of 100-150 U/mg was added to 3 g of CNBr-activated Sepharose^® 4B (Pharmacia AB, Sweden) slurried in 0.1 M bicarbonate buffer, pH 8.7, containing 0.5 M NaCl. The reaction mixture was then left standing overnight at +4 C whereupon it was worked up. The product obtained was stored as a suspension in 50 ml of 0.1 M acetate buffer pH 4.5. This suspension had an MPO activity of 0.8 U/ml totally bound to the particles; no activity could be detected in the supernatant. MPO activity determined according to Lundberg C. & Arfors KE., Inflammation 7(1983) p. 247-55.

B. Glucose oxidase (GO)

100 mg of GO (from Aspergillus niger Type X, Sigma Chemical Co, 100 U/mg) was coupled to CNBr-activated Sepharose 4B and stored in the same way as in Example 1A. Adsorbance measurement (280 nm) in the supernatant revealed that 45 % of the GO charged had failed to couple. Thus the coupling degree and the amount of GO retrieved in the product was 55 % of the amount charged.

C. Effect of the enzyme systems on bacteria in open wounds In the thorax region on the back of a rat two open wounds were made in the skin. Then a wound chamber was affixed over each wound, the chamber walls being sewn and glued to the edges of the wound (Lundberg C. et al., Reconstr. Surg. 16 (1982), p. 123-31). The rats were treated one week after this operation. Some 18 to

24 hours before the treatment with enzyme, the wound

7 was inoculated with about 10 S. aureus bacteria in

0.1 M physiological saline. 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.

The concentrations set forth in the Table are final concentrations in the added volume and are not corrected for exudate that was present, in some cases, in the wound chambers. Finally in each test 0.25 g of dry

Debrisan^® was added (Debrisan^® = dextran crosslinked with epichlorohydrin, water regain 2.5 g/g, Pharmacia AB, Sweden) whereby the reaction mixtures were rendered viscous so that they remained in place more properly. After 60 minutes 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.

Results

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 H₂0₂-producing system was necessary for obtaining a bactericidal effect and that I will intensify that effect.

Table I

Addition (= X) of 200 ,ul MPO Sepharose^®, 100 ,ul GO Sepharose^®,

0.5 ml phosphate buffer (0.2 M, pH 5.5) containing glucose

(1 mM) and/or I~.

% bacteria MPO- GO- surviving

Sepharose Sepharose Glucose I (mM) (limits, (n)

X X X 1 0.7 - 1 ( X X X 0.1 85-300 ( X X X 0 17-30 (

X X 1 0.3 (

X X 0 150 (

X X 0 50 (

X - - 1 150 (

In the experiments presented in Table II, H₂0_ was added instead of GO Sepharose^® plus glucose. It should be noted that the mixture of I^~ and H₂0- is very unstable, so each of these reagents has to be added separately to the chamber.

In agreement with the results of Table I, only 1 mM I was required for obtaining a pronounced bactericidal effect; addition of MPO Sepharose^® did not intensify that effect.

Table II

Addition of 200 ,ul MPO Sepharose , H₂0₂ and I

^H2°2 I % bacteria

(mM) (mM) surviving

1 1 1 1 0.1_. 6 0.1 0.1 50

1 1 0.4*

* MPO Sepharose not added

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.

The invention is defined more specifically in the appended claims which form an integral part of this specification.

Claims

1. A formulation possessing enzymatic activity and con¬ taining

(a) an enzyme which is capable of producing hydrogen peroxide and is present in an insoluble form,

(b) a substrate for said enzyme, and

(σ) a compound which is oxidizable by hydrogen peroxide and which together with hydrogen peroxide has an antimicrobial effect,

for the disinfection of damaged but living animal tissue.

2. A formulation according to claim 1 wherein said enzyme is a solid-phase-bound oxidase for production of hydrogen peroxide.

3. A formulation according to claim 2 wherein said oxidase is a glucose oxidase bound covalently to a solid phase.

4. A formulation according to claim 3 wherein said solid phase is a hydrophilic polymer which is insoluble but capable of adsorbing water.

5. A formulation according to claim 4 wherein said polymer carries hydroxyl groups.

6. A formulation according to any of claims 1-5 wherein said oxidizable compound is selected from the group consisting of physiologically acceptable chloride, bromide, iodide and thiocyanate salts.

7. A formulation according to any of claims 1-6 wherein said compound which is oxidizable by hydrogen peroxide is a iodide salt that is physiologically acceptable in the disinfecting procedure.

8. A method of disinfecting damaged but living animal tissue wherein the tissue is contacted simultaneously with

(a) an enzyme which is capable of producing hydrogen peroxide and is present in an insoluble form during the disinfecting procedure,

(b) a substrate for- said enzyme, and

(c) a compound which is oxidizable by hydrogen peroxide and which together with hydrogen peroxide has an antimicrobial effect,

the amounts of said (a) , (b) and (c) being chosen such that a disinfectant effect results upon combination of such system and substance,

whereupon said reagents as according to (a) , (b) and (c) plus their reaction products, if any, are removed after a period of time suitable for disinfection.

9. A method according to claim 8 employing a formulation according to any of claims 2-7.