Field of the Invention The present invention relates to an improved method for disinfecting contact lenses using superoxide. The present invention also relates to kits and devices for carrying out the inventive method.

Background of the Invention The growth of the contact lens industry has led to a dramatic increase in the number of lenses and care regimens in the marketplace. Designing care regimens to meet the needs of all possible permutations has become a challenge to the industry. In particular, a goal of the lens care industry is to simplify the lens care regimen to obtain greater patient compliance. Contact lenses, especially those made from hydrophilic materials, must be continuously disinfected to kill any harmful microorganisms that may be present or grow on the lenses. Microorganisms that are incorporated in the panel of microorganisms required by the 1985 U.S. FDA guidelines for contact lens solutions for disinfection efficacy include Serratia marcescens ( " S.M. " ) (ATCC 14041) , Staphylococcus epidermidis ( " S. E. " ) (ATCC 17917) , Pseudomonas aeruginosa ( " P.A. " ) (ATCC 15442) and Candida albicans ( " C.A. " ) (ATCC 10231) . A number of methods for disinfecting contact lenses have been described, such as the use of high temperatures, the use of oxidative chemicals, and the use of antimicrobial agents. U.S. Patent Nos. 4,407,791 and 4,525,346 show the polyguaternary ammonium contact lens disinfecting agent 1-tris (2-hydroxyethyl) ammonium-2- butenyl-4-poly [1-dimethyl ammonium-2-butenyl] -ω-tris (2- hydroxyethyl) ammonium chloride salt. European patent application 89810477.3 discloses the disinfecting agent dodecyl-dimethyl- (2-phenoxyethyl) -ammonium bromide. U.S. Patent No. 4,029,817, assigned to Allergan, Inc., shows the contact lens disinfecting agent tallow triethanol

ammonium chloride. U.S. Patent No. 4,758,595 describes the hexamethylene biguanide contact lens disinfecting agent.

Generation of free radicals has been proposed for disinfecting contact lenses. For example, hydrogen peroxide and a metal can be combined via the Fenton reaction to generate hydroxyl radicals. The Fenton reaction, however, is not particularly efficient and requires high concentrations of hydrogen peroxide in order to be effective.

U.S. Patent No. 4,588,586, to Kessler et al . , discloses a method for disinfecting a contact lens involving the use of peroxidases to generate free radicals in a solution. A peroxidase, such as horseradish peroxidase, is used to oxidize substrates, or "donor molecules," such as phenols, aryl and alkyl amines, hydroquinones, NADH, NADPH, palmitate, halogens, glutathione, ferrocytochrome C and ascorbate to produce anti-bacterial free radicals. A continuing need exists for new agents useful in disinfecting contact lenses.

Summary of the Preferred Embodiments In accordance with one aspect of the present invention, there has been provided a method of disinfecting a contact lens comprising the steps of generating superoxide in a solution, and exposing a contact lens to the superoxide in the solution for a time effective to disinfect the lens.

In a more specific aspect of the present invention, superoxide is generated enzymatically. In a preferred embodiment, the superoxide generating step comprises oxidizing a peroxidase with a first hydrogen peroxide molecule in a solution to generate an oxidized peroxidase, oxidizing a substrate in the solution with the oxidized peroxidase to generate a cationic radical, and oxidizing a second hydrogen peroxide molecule in the solution with the cationic radical to generate superoxide.

In a second more specific aspect of the present invention, superoxide is generated electrochemically. In a preferred embodiment of this aspect of the invention, the superoxide generating step comprises applying an electrical potential to a solution comprising a compound capable of forming a cationic radical upon application thereof, thereby forming a cationic radical, and oxidizing a hydrogen peroxide molecule in the solution with the cationic radical to generate superoxide. Another more specific aspect of the present invention is directed to generation of superoxide chemically. Preferably, the superoxide generating step comprises the step of dissolving in the solution a superoxide salt.

An additional more specific aspect of the present invention relates to generation of superoxide in an aqueous solution under conditions permitting the disproportionation of superoxide to hydrogen peroxide. A contact lens is exposed to the aqueous solution for a time effective to disinfect the lens. Preferably, the superoxide is generated by dissolution of a superoxide salt, such as potassium superoxide, in the aqueous solution.

In accordance with another aspect of the present invention, there has been provided a kit for disinfecting a contact lens comprising a system for generating an effective contact lens disinfecting amount of superoxide in a solution, and preferably a container adapted to hold a contact lens and a solution.

Preferably, the kit further includes a solution, such as a saline solution, for the system for generating superoxide.

In a more specific embodiment, the kit includes an enzymatic system for generating superoxide. Preferably, the enzymatic system includes a peroxidase, a substrate for the peroxidase which is capable of being oxidized to generate a cationic radical, and a source of hydrogen peroxide.

In another more specific embodiment, the kit includes an electrochemical system for generating superoxide. Preferably, the electrochemical system includes an apparatus for applying an electrical potential to the solution, a compound capable of forming a cationic radical in the solution upon application of the electrical potential thereto, and a source of hydrogen peroxide.

In a third more specific embodiment, the kit includes a chemical system for generating superoxide. Preferably, the chemical system includes a superoxide salt.

In accordance with still another aspect of the present invention, there has been provided an article of manufacture for use in disinfecting a contact lens which comprises a surface upon which a peroxidase is immobilized in reactive form. The amount of the peroxidase is sufficient to initiate a series of reactions, in the presence of a solution comprising hydrogen peroxide and a substrate for an oxidized form of the peroxidase, which generates an amount of superoxide sufficient to disinfect the contact lens.

In more specific embodiments, the article is a container adapted to hold a contact lens and a solution, or a dipstick.

In accordance with another aspect of the present invention, there has been provided a dispenser for a system for generating superoxide which comprises an outer container, a plurality of inner bladders, in particular two inner bladders, disposed within the container, and a cap to which the outer container and plurality of inner bladders are removably affixed. The cap has defined therein a manifold which is in fluid communication with each of the plurality of inner bladders, and an external opening which is in fluid communication with the manifold. Each bladder of the dispenser can be at least partially filled with a different solution, such that the solutions combine within the manifold to form the system for generating superoxide.

Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.

Brief Description of the Drawings The invention may be more readily understood by referring to the accompanying drawings in which

FIG. 1 depicts an exemplary enzymatic reaction sequence for generating superoxide;

FIG. 2 is a graph showing the dependence of superoxide half-life on solution pH;

FIG. 3 depicts a container for use with an embodiment of the inventive method using an enzymatic system for generating superoxide;

FIG. 4 depicts a dipstick for use in an embodiment of the inventive method using an enzymatic system for generating superoxide;

FIG. 5 depicts an apparatus for use with an electrochemical disinfection method according to the invention; and

FIGS. 6-8 depict an article useful for dispensing a system for generating superoxide, wherein

FIG. 6 is a perspective view of the article showing the disposition of two inner bladders within an outer container,

FIG. 7 is a sectional view along line A-A' in FIG. 6 showing the disposition of the two inner bladders within the outer container, with the large arrows indicating the application of external pressure, such as by squeezing, and the small arrows indicating the action on the inner bladders of air within the article compressed as a result

of the external pressure, and

FIG. 8 is a sectional view along line B-B' in FIG. 6 showing the attachment of the outer container and inner bladders to a cap within which is defined a manifold for mixing solutions which are dispensed from the inner bladders.

In the drawings, identical features are numbered alike.

Detailed Description of the Preferred Embodiments The present invention can be used with all contact lenses such as conventional hard, soft, rigid, gas permeable, and silicone lenses but is preferably employed with soft lenses such as those commonly referred to as hydrogel lenses prepared from monomers such as hydroxyethyl methacrylate, hydroxyethylmethyl methacrylate, vinylpyrrolidone, glycerol methacrylate, methacrylic acid or acid esters and the like. Hydrogel lenses typically absorb significant amounts of water such as from 38 to 80 percent by weight. Care should be exercised when using the present invention with tinted contact lenses, as tinted lenses may be bleached to a slight extent by compositions of the invention.

Superoxide has been found to be cytotoxic. See, e.g., Gardner et al . , J . Biol . Chem. 266: 1478-1483 (1991) . It has now been discovered that superoxide is highly effective as a disinfectant for contact lenses.

The present invention in its broadest aspect affords disinfection of a contact lens through exposure of the contact lens to a solution comprising superoxide. Any system capable of generating superoxide in a solution which can subsequently be brought into contact with a contact lens to effect disinfection thereof is contemplated as being within the scope of the invention. The term "system" as used herein denotes any element or combination of elements, whether enzymatic, electrochemical, chemical or otherwise, which singly or

together are capable of producing superoxide in a solution (preferably a saline solution or other aqueous solution) . The term "enzymatic system" as used herein denotes a system including an enzyme or combination of enzymes which participates in a reaction or sequence of reactions leading to the generation of superoxide.

An "electrochemical system" as used herein is a system in which an electrical potential is applied to at least one chemical species in a solution to generate superoxide. An electrochemical system can also include means for applying the electrical potential to the chemical agent.

A "chemical system" as used herein is a system in which at least one non-enzymatic chemical species, but no enzyme, participates in a reaction or sequence of reactions leading to the generation of superoxide.

According to the invention, an effective disinfecting amount of superoxide is generated in the solution in which the contact lens to be treated is disposed. An effective disinfecting amount of superoxide is an amount which will at least partially reduce the microorganism population in the solutions employed. Preferably, an effective disinfecting amount is that amount which will reduce the microbial burden by two log orders in four hours and more preferably by one log order in one hour for all organisms with the exception of A . fumigatus . Most preferably, an effective disinfecting amount is an amount which will eliminate the microbial burden on a contact lens when used in a contact lens care regimen which includes a recommended soaking time (FDA Chemical Disinfection Efficacy Test-July, 1985 Contact Lens Solution Draft Guidelines, incorporated herein by reference) . Typically, superoxide is present in the working solution in concentrations ranging from about 0.01% to about 2% (w/v) , preferably about 0.2% to 1% (w/v) . A first aspect of the invention comprises enzymatic generation of superoxide in a solution. An exemplary enzymatic generation process is depicted in Figure 1. A

molecule of hydrogen peroxide in a solution, for example saline solution, activates (i.e., oxidizes) a peroxidase. The activated enzyme subsequently oxidizes a substrate to produce a cationic radical. In turn, the cationic radical so formed oxidizes another molecule of hydrogen peroxide to form superoxide. The superoxide so formed is then available for disinfection of the contact lens.

The hydrogen peroxide molecules can be provided in stabilized form in the solution according to well-known methods. Alternatively, the hydrogen peroxide molecules can be generated chemically or enzymatically in the solution. Chemical sources of hydrogen peroxide can include, for example, sodium perborate and sodium peroxide. Enzymatic sources include the glucose/glucose oxidase system. Other chemical and enzymatic systems known to those skilled in the art can also be employed to generated hydrogen peroxide.

Preferably, an amount of hydrogen peroxide is provided which is sufficient for generation of an amount of superoxide capable of disinfecting the contact lens.

Typically, about 0.05% to 1% (w/v) of hydrogen peroxide are provided.

The enzyme or combination of enzymes useful according to the invention is capable of oxidizing a substrate to produce a cationic radical, which subsequently oxidizes a second hydrogen peroxide molecule to produce superoxide. Any enzyme or enzyme combination capable of producing a cationic radical as described herein is contemplated as being within the scope of the instant invention. Exemplary enzymes include ferric peroxidases, horseradish peroxidase and lactoperoxidase. A preferred ferric peroxidase is lignin peroxidase. Other peroxidases can also be employed.

In general, it is preferred that the enzymatic generation of superoxide be carried out at room temperature. The pH of the solution preferably ranges from about 7 to 12, more particularly from about 8 to 11.

The particular pH for a given system will be determined by balancing the desired disinfection efficacy against enzyme activity. Superoxide half-life is strongly dependent on pH, as shown in Figure 2 (data from J. Rabini et al., J. Phys . Chem. , vol. 7, p. 3736 (1969); S. Marklund, J. Biol . Chem. , vol. 251, p. 7505 (1976)). At high pH (10+) , superoxide half-life is significantly increased. The increased half-life in turn tends to increase microbial kill. However, high pH may also reduce peroxidase activity. Preferably, therefore, a peroxidase which is active at high pH should be employed.

The enzyme can be provided in a variety of forms, for example in liquid or solid form, and can be provided in combination with a reducing agent and optional additional components. For example, a superoxide salt such as K0 ₂ is preferably provided in solid form, such as tablets or powders, together with optional additional agents. The solid compositions are dissolved in a suitable solution for use in the inventive method. Enzymatic systems for generating superoxide preferably are provided in liquid form. In a preferred embodiment, the enzyme or combination of enzymes and their substrates are provided in a plurality of separate solutions, which are combined at the time of use. The amount of enzyme provided typically is about 10-200 μg, preferably about 50-100 μg.

Reducing agents useful according to the instant invention are generally any non-toxic reducing agent, either dry or liquid, depending in part upon whether the delivery system is tablet or solution. Exemplary reducing agents include thiols, such as N-acetylcysteine.

Additional components that may be added to or incorporated into the enzyme tablets or the disinfecting solution include effervescing agents, stabilizers, buffering agents, chelating and/or sequestering agents, coloring agents, tonicity adjusting agents, surfactants and the like. In addition, binders, lubricants, carriers,

and other excipients normally used in producing tablets may be incorporated into the enzyme tablet when enzyme tablets are employed. Neutralizing agents for neutralization of residual hydrogen peroxide can also be included, or can be supplied separately. Such additives and their use are well known to those skilled in the art . Other additives can also be employed to provide desired characteristics. Radical scavengers (e.g., mannitol) can reduce or eliminate adverse effects due to side reactions of radicals, such as the hydroxyl radical, with lens hydrogel materials, tints, etc. Additional enzymes, such as superoxide dismutase, can be employed for system neutralization. Useful additives include chelating agents such as EDTA, surfactants membrane permeabilizers, etc. Additional preferred enzymes include cleaning enzymes, in particular proteases such as subtilisin A. Proteases which are active at high pH, e.g., pH of 8 or above, are preferred.

Various substrates for producing the cationic radical are useful in the inventive method, depending on the enzyme employed. Exemplary substrates include phenolic lignins, non-phenolic lignins, methoxybenzene derivatives, 2, 2-azino-bis- (3-ethylbenzthiazoline) -6-sulfonic acid (ABTS) , chlorpazamine, benzyl alcohols and polyphenols. Such substrates may be chosen based on molecular weight. Higher molecular weights tend to reduce toxicity and lens uptake, and thus are preferred.

Particularly preferred substrates are ABTS and veratryl alcohol. Any other substrate which is capable of being oxidized by an enzyme to yield a cationic radical is contemplated as being within the scope of the instant invention. The substrates can also be provided in a variety of solid or liquid forms for use in the inventive method. Depending on the cationic radical generated in the particular reaction sequence according to the invention, a color indication of the reaction process may be

provided. For example, ABTS is oxidized to a stable green cation radical having absorption maxima at 414 and 660 nm. Other oxidative indicators or pH indicators Which are ophthalmologically acceptable may also be employed. The enzymatic embodiment of the disinfection process can be carried out in a number of ways. For example, separate solutions including the enzyme, hydrogen peroxide or peroxide generator, and substrate can be combined in a container into which the contact lens to be disinfected is placed. Such solutions can be produced, as noted above, by dissolving a solid composition including the particular ingredient in an appropriate solution. Alternatively, one of the enzyme or substrate can be immobilized in reactive form on the surface of the container, and solutions including the other of the enzyme and substrate, and hydrogen peroxide or peroxide generator, can then be combined within the container. The former option (immobilization of the enzyme) is preferred, while the latter option is appropriate, for example, when the substrate is not ophthalmologically acceptable, or when disposable containers are desired.

An exemplary embodiment of this alternative system is shown in Figure 3. In Figure 3, container 10 has inner surface 12 upon at least a portion of which is immobilized in reactive form an enzyme 14 as described herein. Saline solution 16, containing a substrate for enzyme 14, and solution 18, containing a source of hydrogen peroxide, are added to container 10. If desired, the same solution can include both the substrate and the source of hydrogen peroxide. Contact lens 20 is placed within container 10 and is disinfected with the superoxide generated by the reaction sequence involving the enzyme, substrate and hydrogen peroxide.

Container 10 can be, for example, a vial for holding a contact lens in a solution, a contact lens case, or any other type of container useful for holding a contact lens. In another alternative embodiment, an article of

manufacture, such as a dipstick, has immobilized in reactive form on a least a portion of its surface one of the enzyme and substrate described above. The article is subsequently contacted with a solution or combination of solutions containing the other of the enzyme and substrate and a hydrogen peroxide source. The contact lens to be disinfected is exposed to the resulting solution and disinfected by the superoxide so generated.

Figure 4 illustrates this embodiment. Dipstick 22 has a surface 24 upon at least a portion of which is immobilized in reactive form an enzyme 14 as described herein. Dipstick 22 is contacted with solution 26 including a substrate for enzyme 14 and a hydrogen peroxide source within container 28, resulting in generation of superoxide. Contact lens 20 is disinfected by the superoxide so generated.

The contact lens to be disinfected is exposed to the solution containing superoxide for a time effective to disinfect the lens. Exposure time will depend on a number of factors, such as speed of kill, impact on the lens to be treated, pH neutralization considerations, the presence of other active agents such as proteases, etc., all of which can readily be evaluated by those skilled in the art. Typically, the contact lens is exposed to the solution for a time ranging from about 5 minutes to 12 hr, or overnight, depending on the particular embodiment selected. For example, a tablet including a superoxide salt such as K0 ₂ will achieve relatively rapid kill, requiring treatment times from about 15 minutes to 1 hour. If a protease is employed together with the salt, however, a longer time typically will be required in order to clean the lens after disinfection has been completed. Enzymatic systems for generating superoxide usually require longer times, typically at least 30 minutes to 4 hours. A second aspect of the invention comprises electrochemical generation of superoxide in a solution. In this aspect, superoxide is generated by applying an

electrical potential to a solution which includes a compound capable of forming a cationic radical upon application of the potential. The cationic radical so formed subsequently oxidizes a hydrogen peroxide molecule present in the solution. As with the preceding enzymatic method, this oxidation step generates superoxide in the solution, which then is available to disinfect a contact lens immersed within the solution.

An embodiment of the electrochemical aspect of the instant invention is illustrated in Figure 5. Container 30 contains a solution 32 including a compound capable of forming a cationic radical upon application of a potential thereto. Electrodes 34 and 36 extend into the solution. The electrodes can, for example, be affixed to a cap 38 which fits over container 30. An electrical potential is applied via electrodes 34 and 36 to the compound in the solution, thereby producing the cationic radical. The cationic radical then oxidizes a hydrogen peroxide molecule to produce superoxide for disinfection of contact lens 20 immersed within the solution.

Various compounds can be used in accordance with the electrochemical aspect of the inventive method. Exemplary compounds include ABTS.

Hydrogen peroxide can be supplied in the same manner as with the enzymatic aspect of the inventive method, either as stabilized hydrogen peroxide or as the product of a chemical or enzymatic system for generating hydrogen peroxide.

A third aspect of the invention comprises chemical generation of superoxide in a solution. In this aspect, superoxide is generated by dissolution of a superoxide salt in a solution. For example, salts such as potassium superoxide or sodium superoxide can be dissolved in a solution, such as a buffered saline solution, boric acid

_* buffer or other buffered solution, to provide a disinfecting solution for a contact lens. The superoxide salts can be provided in tableted form, powdered form or

another form which is readily dissolved in the selected solution.

As discussed above with respect to enzymatic generation of superoxide, tablets containing superoxide salts can also contain other conventional additives such as effervescing agents, stabilizers, reducing agents and buffering agents, along with radical scavengers, additional enzymes such as superoxide dismutase, etc. Additional neutralizing agents for neutralization of residual hydrogen peroxide can also be included, or can be supplied separately.

The superoxide salt, such as potassium superoxide, is employed in the solution in an effective amount, typically about 5 to 200 mg/10 ml, preferably about 50 to 100 mg/10 ml.

Since superoxide salts such as K0 ₂ are strong oxidizers, appropriate precautions must be taken when preparing tablets and other solid formulations which include such salts. These precautions include care in selection of additional ingredients to be used in combination with the superoxide salts, in order to avoid possible chemical reactions and consequent fire or explosion hazards. Manufacturing process parameters, such as temperature, dust levels and potential sources of ignition, must likewise be carefully controlled. Compositions including superoxide salts which are stable at room temperature, for example, may react at elevated temperatures. In many cases it may be advantageous to prepare two separate solid formulations for use together. One of the formulations includes the superoxide salt and optionally an inert excipient such as sodium chloride, while the other formulation includes any other desired additives as discussed above.

A variation of the foregoing methods includes the steps of generating superoxide, by any desired means, in an aqueous solution under conditions permitting the disproportionation of superoxide to hydrogen peroxide.

The contact lens to be disinfected is exposed to the aqueous solution for a time effective to disinfect the lens.

The disproportionation of superoxide is believed to take place according to the following mechanisms:

₂ H.O

0 ₂ ^-" + 0 ₂ ' ^~ > H ₂ 0 ₂ + 0 ₂ + 20H ^" H-0

H0 ₂ - + 0 ₂ - ^~ > H ₂ 0 ₂ + 0 ₂ + OH ^"

The resulting solution contains superoxide, hydrogen peroxide, hydrogen and oxygen. Further reaction involving the target microorganisms may generate additional radicals, for example hydroxyl. Disinfection occurs through the action of one or more of these species.

Chemical sources of superoxide, typically a salt such as K0 ₂ , are most suitable for practice the above-described variant. The superoxide is preferably generated in a solution at room temperature and at approximately physiological pH (e.g., tap water, distilled water, buffered saline solution) ; however, it is advantageous to allow the pH of the solution to rise during the disinfection process. Hydroxide generated in the disproportionation reactions results in a solution pH in the range of about 8-13, depending on the initial quantity of superoxide salt, the buffering capacity of the solution, etc. Preferably, the pH is allowed to rise to about 12, then is reduced the pH to approximately neutral, over a cycle time of about 1 hour.

The foregoing disinfection method can be employed alone or in combination with other methods which require generation of hydrogen peroxide as part of the disinfection regimen. One advantage of such a combination is the elimination of the need for stabilized solutions of hydrogen peroxide.

When the system for generating superoxide is produced in solid form, such as a tablet, a coating preferably is

employed to control the release and dissolution of individual components (e.g., superoxide salts, buffers, peroxide neutralizers, etc.), and thereby control disinfection solution conditions such as pH, peroxide concentration, etc. In a preferred embodiment, particles are formed by separately coating a superoxide salt, such as K0 ₂ , and an additive, such as citric acid, with a polyvinylpyrrolidone such as VA-64 (commercially available from BASF) . The coated particles are then compressed together to form a tablet.

Related systems for disinfecting contact lenses which employ superoxide in combination with nitrogen oxide (NO) to generate peroxynitrite radicals are described in co- pending, commonly assigned U.S. application Serial No. (attorney docket 6850 PD-2994) , to Peng et al . , filed simultaneously herewith and incorporated herein in its entirety by reference.

The present invention also provides kits for disinfecting contact lens according to the foregoing methods. The kits in general include a system for generating an effective contact lens disinfecting amount of superoxide in a solution, in particular a system as described herein, and a container adapted to hold a contact lens and a solution. Thus, in a first preferred embodiment, the inventive kit includes a system for the enzymatic generation of superoxide as described herein. The selected peroxidase, substrate and hydrogen peroxide source are included in tablet, powder, liquid or other desired forms and packaged in pre-measured amounts effective to disinfect a contact lens. Multiple packaged units of the tablet, powder, liquid, etc., can be included in the kit for user convenience.

When the peroxidase, substrate and/or hydrogen peroxide source are provided in solid form (tablets, powders, etc.), the inventive kit optionally can further include a solution for the superoxide generating system.

The solution can be, for example, a prepackaged spray canister of a saline or other buffered solution which the user squirts into the container.

Other additives, such as radical scavengers or superoxide dismutase, can also be provided with the inventive kit, either in separately packaged form or in admixture with one or more of the other elements of the superoxide generating system.

A kit according to the invention can include, as the container, a container having immobilized in reactive form therein one of the peroxidase and substrate, as described herein. Alternatively, the kit can include a dipstick or other article of manufacture having immobilized in reactive form thereon one of the peroxidase and substrate. The dipstick or other article would be used in conjunction with the container and other elements of the kit as described herein.

Another kit according to the instant invention includes an electrochemical system for generating superoxide. Such a kit can include, for example, an apparatus for applying an electrical potential to a solution such as shown in Figure 5, together with appropriate pre-packaged amounts of a compound capable of forming a cationic radical in the solution upon application of the electrical potential thereto, and of a source of hydrogen peroxide. These materials can be in solid or liquid form as described herein.

Another kit within the scope of the present invention includes a chemical system for generating superoxide. For example, appropriate pre-packaged amounts of a superoxide salt such as potassium superoxide can be provided in tablet or powder form, optionally together with a supply of a solution such as saline solution.

Another kit within the scope of the present invention includes a dispensing device pre-filled with a plurality of solutions which can be combined to produce a system for generating superoxide. An exemplary dispensing device,

which can be employed to dispense solutions used in systems for generating superoxide or other solutions, is illustrated in Figures 6-8. Dispenser 40 includes outer container 42. Juxtaposed within space 44 within outer container 42, without contacting outer container 42, are first inner bladder 46, having outer exterior surface 48 and inner exterior surface 50, and second inner bladder 52 having outer exterior surface 54 and inner exterior surface 56. Inner exterior surfaces 50 and 56 of first and second inner bladders 46 and 52, respectively, preferably are separated by a plurality of contact points 58, one or more of which are disposed on each of said inner exterior surfaces 50 and 56.

Outer container 42 and first and second inner bladders 46 and 52 are joined to cap 60, within which is defined manifold 62. First and second inner bladders 46 and 52 are in fluid communication with manifold 62 by attachment to necks 64 in the underside of cap 60. Manifold 62 in turn is in fluid communication with opening 66 defined in cap 60.

In use, inner bladders 46 and 52 are filled with separate solutions which form a system for generating superoxide when combined. For example, one of inner bladders 46 and 52 can filled with a first aqueous solution comprising a peroxidase, and the other inner bladder can be filled with a second solution comprising hydrogen peroxide and a substrate for the peroxidase which is capable of being oxidized to generate a cationic radical. External pressure is applied to outer container 42, for example by squeezing. Air within space 44 is compressed, resulting in application of pressure to inner bladders 46 and 52. The solutions within each of the two inner bladders are forced to flow out of the bladders and into manifold 62, where the two solutions mix and subsequently flow through opening 66 into an appropriate container (not shown) which preferably is provided in combination with dispenser 40, and within which superoxide

is generated. A contact lens is then disinfected within the container.

The flowrate of solution from each of the first and second inner bladders can be controlled by conventional means, such as by the use of appropriately sized orifices within necks 64. Optionally, one-way valves can be disposed within necks 64 to prevent cross-contamination of the solutions within the two inner bladders.

Opening 66 in cap 60 preferably is reclosable to prevent contamination of the solutions within the two inner bladders. Exemplary closing means include outer cap 68 attached to cap 60 via flexible tab 70.

The invention is further illustrated by reference to the following non-limiting examples. Example 1 Disinfecting tablet combination

A first tablet is produced by combining K0 ₂ (100 mg) with NaCl (100 mg) and compressing the resulting mixture. A second tablet is produced as follows: Citric acid (105 mg) is pan coated with polyvinylpyrrolidone VA-64 (20% in acetone) to form coated particles. The coated particles are combined with polyvinylpyrrolidone PVP-15 (105 mg) , PEG-3350 (10 mg) as a lubricant, and pan coated ascorbic acid (50 mg) as a neutralizer, and compressed into a tablet. The tablets are dissolved in 10 ml of a saline solution for use in disinfecting a contact lens.

Example 2 Cleaning and disinfecting tablet combination A first tablet is prepared by combining K0 ₂ (100 mg) and NaCl (100 mg) as described in Example 1.

A second tablet is prepared by combining the following ingredients:

Ingredient • Amount

KH ₂ P0 ₄ 300 mg

PEG-3350 5 mg Subtilisin A 0.036 AU

Catalase 1000 AU

The tablets are dissolved in 10 ml of a saline solution for use in disinfecting a contact lens.

Example 3 Cleaning and disinfecting tablet combination A first tablet is prepared by combining K0 ₂ (50 mg) and NaCl (50 mg) as described in Example 1.

A second tablet is prepared by combining the following ingredients:

Ingredient Amount

Boric acid 150 mg

PEG-3350 5 mg Subtilisin A 0.060 AU

Ascorbic acid 100 mg

The tablets are dissolved in 10 ml of a saline solution for use in disinfecting a contact lens.

In Examples 4-18, various test solutions according to the invention were prepared, as were comparison solutions.

Each solution was tested one or more times against a selected microorganism. Results from multiple test runs using a single solution are separately tabulated.

Testing of K0 ₂ tablets was carried out according to

the following protocol: 10 ml volumes of test solution were dispensed into glass centrifuge tubes. The solutions were inoculated to contain a selected number of viable colony forming units (CFU) per milliliter, after which inoculation each tube was immediately vortexed. A tablet was then added to each tube, and the tubes were stored at room temperature. At various time intervals, 0.5 ml from each tube was transferred to a test tube containing 4.5 ml of fluid thioglycolate broth containing 400 units of catalase per milliliter. The tubes were processed for aerobic plate count according to a standard protocol.

Testing of K0 ₂ powders was performed as follows: KQ ₂ powders were weighed directly into glass centrifuge tubes or lens cases. Test solutions were inoculated to contain a selected number of viable colony forming units (CFU) per milliliter, after which inoculation 10 ml volumes of the inoculated test solution were dispensed into glass centrifuge tubes or lens cases containing the weighed K0 ₂ powder. Each tube was immediately vortexed, and the tubes were stored at room temperature. At various time intervals, 0.5 ml from each tube was transferred to a test tube containing 4.5 ml of fluid thioglycolate broth containing 400 units of catalase per milliliter. The tubes were processed for aerobic plate count according to a standard protocol.

The effectiveness of the solutions against selected microorganisms was then evaluated. Example 4

The following test solutions were evaluated for antimicrobial efficiency against S. aureus (ATCC 6538) . Where tablets were used, they were dissolved in the indicated buffers:

1) 1 tablet (100 mg K0 ₂ ) + 10 ml 0.5 M K∑i PQ /\ HPQ buffer

2) 1 tablet (50 mg K0 ₂ ) + 10 ml 0.5 M K£_ Pp fγ_ HPp buffer

3) 10 ml 0.5 M KH ₂ P0 ₄ /K ₂ HP0 ₄ buffer (no tablet added)

4) 1 tablet (100 mg K0 ₂ ) + 10 ml phosphate buffered saline-tween

An initial inoculum of 7xl0 ⁵ CFU/ml was employed with each solution. Results are given in Table I. Log kills are indicated in parentheses.

Table I

Solution 0 min 60 min 120 min CFU/ml CFU/ml CFU/ml

1 5x10 ^s (0.15) 5x10 ^s (0.15) 8x10* (0.9.)

6x10 ^s (0.07) 5x10 ^s (0.15) 8x10' (0.94)

6x10 ^s (0.07) 5x10 ^s (0.15) 6x10' (1.07)

2 6x10 ^s (0.07) 6x10 ^s (0.07) 6x10 ^s (0.07)

6x10 ^s (0.07) 6x10 ^s (0.07) 4x10 ^s (0. ₂ 4)

7x10 ^s (NK") 6x10 ^s (0.07) 3x10 ^s (0.37)

₃ 7x10 ^s (NK) 7x10 ^s (NK) 7x10 ^s (NK)

4 3xl0 ² (3.37) <10 (5.85) <10 (5.85)

^* NK = no kill

Example 5

The test solutions of Example 1 were evaluated for antimicrobial efficiency against S. marcescens (ATCC

14041) . An initial inoculum of 2xl0 ⁶ CFU/ml was employed with each solution. Results are given in Table II. Log kills are indicated in parentheses.

Table II

Solution 30 min 60 min 120 min CFU/ml CFU/ml CFU/ml

1 8x10 ^s (0.40) 2.5x10 ^s (0.90) 2x10 ^s (1.00)

8x10 ^s (0.40) 4x10 ^s (0.70) 1x10 ^s (1.30)

1x10' (0.30) 5x10 ^s (0.60) 2x10 ^s (1.00)

2 2x10' (NK) 2x10* (NK) 2x10* (NK)

1x10' (0.30) 8x10 ^s (0.40) 3x10 ^s (0.82)

2x10* (NK) 2x10' (NK) 2x10* (NK)

3 2x10* (NK) 2x10* (NK) 2x10' (NK)

4 <10 (6.30) <10 (6.30) <10 (6.30)

Example _

The test solutions of Example 4 were evaluated for antimicrobial efficiency against C. albicans (ATCC 10231) . An initial inoculum of 6xl0 ⁵ CFU/ml was employed with each solution. Results are given in Table III. Log kills are indicated in parentheses.

Table III

Solution 60 min 120 min 180 min CFU/ml CFU/ml CFU/ml

1 5x10 ^s (0.08) 3x10 ^s (0.30) 1x10 ^s (0.78)

5x10 ^s (0.08) 3.5x10 ^s (0.23) 2x10 ^s (0.48)

5x10 ^s (0.08) 4x10* (0.18) 1.5x10 ^s (0.60)

2 5x10 ^s (0.08) SxlO ⁵ (0.08) 5x10 ^s (0.08)

5x10 ^s (0.08) 5x10 ^s (0.08) 3x10 ^s (0.30)

6x10 ^s (NK) 6x10 ^s (NK) 3x10 ^s (0.30)

3 6x10 ^s (NK) 6x10 ^s (NK) 6x10 ^s (NK)

4 3xl0 ³ (2.30) <10 (5.78) <10 (5.78)

Example 7

The test solutions of Example 4 were evaluated for antimicrobial efficiency against A. fumigatus (ATCC 10894) . An initial inoculum of 8x10 ^s CFU/ml was employed with each solution. Results are given in Table IV. Log kills are indicated in parentheses.

Table IV

Solution 60 min 120 min 180 min CFU/ml CFU/ml CFU/ml

1 8x10 ^s (NK) 8x10 ^s (NK) 8x10 ^s (NK)

8x10 ^s (NK) 8x10 ^s (NK) 8x10 ^s (NK)

8x10 ^s (NK) 8x10 ^s (NK) 8x10 ^s (NK)

2 8x10 ^s (NK) 8x10 ^s (NK) 8x10 ^s (NK)

8x10 ^s (NK) 8x10 ^s (NKI 8x10 ^s (NK)

8x10 ^s (NK) 8x10 ^s (NK) 8x10 ^s (NK)

3 8x10 ^s (NK) 8x10 ^s (NK) 8x10 ^s (NK)

4 2xl0 ³ (2.60) 1x10* (3.90) <10 (5.90)

^* NK = no kill

Example 8

The following test solutions were evaluated for antimicrobial efficiency against S. aureus (ATCC 6538) :

1) 1 tablet (100 mg K0 ₂ ) + 10 ml 0.5 M KP PQ f\ HPQ buffer containing 0.5% tween

2) 1 tablet (100 mg K0 ₂ ) + 10 ml 0.5 M KI$ PQ /T_ HPQ buffer

3) 1 tablet (100 mg K0 ₂ ) + 10 ml phosphate buffered saline-tween 4) 1 tablet (50 mg K0 ₂ ) + 10 ml 0.5 M Kg Pp / . HPp buffer containing 0.5% tween

5) 1 tablet (50 mg K0 ₂ ) + 10 ml 0.5 M Kg PQ /£ HPQ buffer

6) 1 tablet (50 mg K0 ₂ ) + 10 ml phosphate buffered saline-tween

7) 10 ml 0.68% H ₂ 0 ₂ prepared in phosphate buffered saline, pH adjusted to 12 with 10 N NaOH

An initial inoculum of 6xl0 ⁵ CFU/ml was employed with each solution. Results are given in Table V. Log kills are indicated in parentheses.

Table V

Solution 15 min 30 min 60 min CFU/ml CFU/ml CFU/ml

1 4.5x10 ^s (0.12) 4.5x10 ^s (0.12) 6x10' (1.00)

5x10 ^s (0.08) 4.5x10 ^s (0.12) 2x10' (1.48)

2 5x10 ^s (0.08) 5x10 ^s (0.08) 4x10 ^s (0.18)

3 2x10' (2.48) 8x10' (3.88) <10 (5.78)

4 4.5x10 ^s (0.12) 5x10 ^s (0.08) 5x10 ^s (0.08)

5x10 ^s (0.08) 5x10 ^s (0.08) 4.5x10 ^s (0.12)

5 5x10 ^s (0.08) 5x10 ^s (0.08) 5x10 ^s (0.08)

6 4.5x10* (1.12) 3x10" (2.30) <10 (5.78)

7 1.5x10 ^s (0.60) 7x10' (1.93) lxlO ¹ (4.78)

Example 9

The test solutions of Example 4 were evaluated for antimicrobial efficiency against S. marcescens (ATCC 14041) . An initial inoculum of 2xl0 ⁶ CFU/ml was employed with each solution. Results are given in Table VI. Log kills are indicated in parentheses.

Table VI

Solution 15 min 30 min 60 min CFU/ml CFU/ml CFU/ml

1 7x10 ^s (0.46) 5x10 ^s (0.30) 5x10 ^s (0.30)

8x10 ^s (0.40) 6x10 ^s (0.52) 6x10 ^s (0.52'

2 1.5x10* (0.12) 1x10' (0.30) 8x10 ^s (0.40>

3 <10 (6.30) <10 (6.30) <10 (6.30)

4 1.5x10' (0.12) 9x10 ^s (0.35) 8x10 ^s (0.40)

2x10' (NK) 2x10' (NK) 2x10' (NK)

5 2x10' (NK) 1x10' (0.30) 1x10* (0.30'

6 lxlO ² (4.60) <10 (6.30) <10 (6.30 ^<

7 <10 (6.30) <10 (6.30) <10 (6.30)

Example 1Q

The test solutions of Example 4 were evaluated for antimicrobial efficiency against C. albicans (ATCC 10231) . An initial inoculum of 6xl0 ⁵ CFU/ml was employed with each solution. Results are given in Table VII. Log kills are indicated in parentheses.

Table VII

Solution 15 min 30 min 60 min CFU/ml CFU/ml CFU/ml

1 4x10 ^s (0.18) 3x10 ^s (0.30) 1x10 ^s (0.78)

3x10 ^s (0.30) 2.5x10 ^s (0.38) 1.5x10 ^s (0.60)

2 4.5x10 ^s (0.12) 4x10 ^s (0.18) 3x10 ^s (0.30)

3 lxlO ³ (2.78) 3x10' (4.30) <10 (5.78)

4 4x10 ^s (0.18) 4x10 ^s (0.18) 3x10 ^s (0.30)

4x10 ^s (0.18) 3x10 ^s (0.30) 3x10 ^s (0.30)

5 4x10 ^s (0.18) 4x10 ^s (0.18) 3x10 ^s (0.30)

6 βxlO ¹ (1.88) lxlO ² (3.78) <10 (5.7B)

7 4xl0 ^! (0.18) 2x10 ^s (0.48) 8x10" (1.88)

Example 11

The test solutions of Example 4 were evaluated for antimicrobial efficiency against A. fumigatus (ATCC

10894) . An initial inoculum of 8xl0 ⁵ CFU/ml was employed with each solution. Results are given in Table VIII. Log kills are indicated in parentheses.

Table VIII

Solution 15 min 30 min 60 min CFU/ml CFU/ml CFU/ml

1 4x10 ^s (0.30) 4x10 ^s (0.30) 4x10 ^s (0.30)

4x10 ^s (0.30) 4x10 ^s (0.30) 4x10 ^s (0.30)

2 4x10 ^s (0.30) 4x10 ^s (0.30) 4x10 ^s (0.30)

3 3x10 ^s (0.43) 1x10 ^s (0.90) 1x10' ( ₂ .90)

4 4x10 ^s (0.30) 4x10 ^s (0.30) 4x10 ^s (0.30)

5x10 ^s (0.20) 5x10 ^s (0.20) 5x10 ^s (0. ₂ 0)

5 5x10 ^s (0.20) 5x10 ^s (0. ₂ 0) 4x10 ^s (0.30)

6 4x10 ^s (0.30) ₂ χlO ^s (0.60) 1x10 ^s (0.90)

7 4x10 ^s (0.30) 4x10 ^s (0.30) 3x10 ^s (0.43)

Example 12

The following solutions were evaluated for antimicrobial efficiency against C. albicans (ATCC 10231) :

1) 1 mg K0 ₂ /10 ml saline, pH 12

2) 10 mg K0 ₂ /10 ml saline, pH 12

3) 50 mg KO ₂ /l0 ml saline, pH 12

4) 100 mg K0 ₂ /lO ml saline, pH 12

5) 200 mg K0 ₂ /lO ml saline, pH 12

6) 10 ml saline containing 0.2% H ₂ 0 ₂ , pH 12

7) 10 ml saline containing 0.04% H ₂ 0 ₂ , pH 12

8) 10 ml saline, pH 12

An initial inoculum of 5x10 ⁵ CFU/ml was employed with each solution. Results are given in Table IX. Log kills are indicated in parentheses.

Table IX

Solution 0.5 hr 1 hr 2 hr 4 hr CFU/ml CFU/ml CFU/ml CFU/ml

1 2x10 ^s (0.40) 1.5x10 ^s (0.52) 5x10' (1.00) 2x10' (1.40)

2x10 ^s (0.40) 1x10 ^s (0.70) 7x10' (0.85) 4x10' (1.10)

2x10 ^s (0.40) 1x10 ^s (0.70) 5x10' (1.00) 3x10' (1.22)

2 3xl0 ³ (2.22) 2xl0 ² (3.40) <10 (5.70) <10 (5.70)

2x10' (1.40) 2.5xl0 ³ (2.30) 3xl0 ² (3.22) <10 (5.70)

7x10' (1.85) 3xl0 ² (3.22) <10 (5.70) <10 (5.70)

3 <10 (5.70) <10 (5.70) <10 (5.70) <10 (5.70)

<10 (5.70) <10 (5.70) <10 (5.70) <10 (5.70)

<10 (5.70) <10 (5.70) <10 (5.70) <10 (5.70)

4 <10 (5.70) <10 (5.70) <10 (5.70) <10 (5.70)

<10 (5.70) <10 (5.70) <10 (5.70) <10 (5.70)

<10 (5.70) <10 (5.70) <10 (5.70) <10 (5.70)

5 <10 (5.70) <10 (5.70) <10 (5.70) <10 (5.701

<10 (5.70) <10 (5.70) <10 (5.70) <10 (5.70)

<10 (5.70) <10 (5.70) <10 (5.70) <10 (5.70)

6 4x10' (1.10) 8xl0 ² (2.80) <10 (5.70) <10 (5.70)

6x10' (0.92) 3xl0 ² (3.22) <10 (5.70) <10 (5.70)

5x10' (1.00) 3xl0 ² (3.22) <10 (5.70) <10 (5.70)

7 9x10' (0.74) 2x10' (1.40) 2x102 (3.40) <10 (5.70)

1x10 ^s (0.70) 2x10' (1.40) 3xl0 ² (3.22) <10 (5.70)

9x10' (0.74) 2x10' (1.40) 1.5xl0 ² (3.52) <10 (5.70)

8 5x10' (1.00'

Example 13

The solutions of Example 12 were also evaluated for antimicrobial efficiency against S. aureus (ATCC 6538) . An initial inoculum of 8x10 ^s CFU/ml was employed with each solution. Results are given in Table X. Log kills are indicated in parentheses.

Table X

Example 14

The following solutions were evaluated for antimicrobial efficiency against C. albicans (ATCC 10231) :

1) 50 mg KO ₂ /10 ml saline, pH 7; testing in glass tube

2) 50 mg KO ₂ /l0 ml PBST, pH 7; testing in glass tube

3) 50 mg KO ₂ /10 ml saline, pH 7; testing in lens case

An initial inoculum of 3x10 ^s CFU/ml was employed with each solution. Results are given in Table XI. Log kills are indicated in parentheses.

Table XI

Solution 15 min 30 min 60 min CFU/ml CFU/ml CFU/ml

1 3xl0 ³ (2.00) 7x10' (3.63) <10 (5.48)

2xl0 ³ (2.18) lxlO ² (3.4B) <10 (5.48)

3xl0 ³ (2.00) 3xl0 ² (3.00) <10 (5.48)

2 2x10* (1.18) 1.5xl0 ² (3.30) <10 (5.48)

3x10' (1.00) 2xl0 ² (3.18) <10 (5.48)

3x10' (1.00) 3xl0 ² (3.00) <10 (5.48)

3 3xl0 ³ (2.00) 1.5X10 ² (3.30) <10 (5.48)

3xl0 ³ (2.00) 2xl0 ² (3.18) <10 (5.48)

3xl0 ³ (2.00) 2xl0 ² (3.18) <10 (5.48)

Example 15

The following solutions were evaluated for antimicrobial efficiency against A. fumigatus (ATCC 10894) :

1) 50 mg KO ₂ /l0 ml saline, pH 7; testing in glass tube

2) 100 mg KO ₂ /10 ml saline, pH 7; testing in glass tube

An initial inoculum of 3x10 ⁵ CFU/ml was employed with each solution. Results are given in Table XII. Log kills are indicated in parentheses .

Table XII

Solution 0.5 hr 1 hr 2 hr CFU/ml CFU/ml CFU/ml

1 1x10 ^s (0.48> 1x10 ^s (0.48) 1x10 ^s (0.48)

2x10 ^s (0.18) 2x10 ^s (0.18) 8x10' (0.57)

2xl0 ^! (0.18) 1.5x10 ^s (0.30) BxlO' (0.57)

2 1.5x10 ^s (0.30! 2x10' (1.18) 3x10' (2.00)

1x10 ^s (0.48) 4x10' (0.88) 2x10' (2.18)

1x10 ^s (0.48) 4x10' (0.88) 5x10' (1.7B)

Example 16

A solution of 5 mg K0 ₂ in 1 ml saline (pH 7) was

evaluated for antimicrobial efficiency against C. albicans (ATCC 10231) . Testing was carried out in glass tubes. An initial inoculum of 5xl0 ⁵ CFU/ml was employed. Results are given in Table XIII. Log kills are indicated in parentheses.

Example 17

The following solutions were evaluated for antimicrobial efficiency against A. fumigatus (ATCC 10894) :

1) 5 mg K0 ₂ /l ml saline, pH 7; testing in glass tube

2) 10 mg K0 ₂ /l ml saline, pH 7; testing in glass tube

An initial inoculum of 4xl0 ⁵ CFU/ml was employed with each solution. Results are given in Table XIV. Log kills are indicated in parentheses.

Table XIV

Example 18

A solution of 50 mg K0 ₂ plus 10 ml distilled water was

evaluated for antimicrobial efficiency against C. albicans

(ATCC 10231). Testing was carried out in glass tubes.

An initial inoculum of 3xl0 ⁵ CFU/ml was employed. Results are given in Table XV. Log kills are indicated in parentheses.

Table XV

Test run 15 min 30 min 60 min CFU/ml CFU/ml CFU/ml

1 1x10 ^s (0.48) 4x10' (0.88) 9xl0 ² (2.62)

2 1x10 ^s (0.48) 6x10' (0.70) 7xl0 ² (2.63)

3 1x10 ^s (0.48) 2x10' (1.18) 3xl0 ² (3.00)

The foregoing test results demonstrate the efficacy of superoxide disinfection. High activity was observed when K0 ₂ was tested in phosphate buffered saline tween. At 60 minutes, S. aureus, C. albicans, S. marcescens and A. fumigatus were reduced by 5.8, 4.0, 6.3 and 2.8 logs, respectively, when 100 mg K0 ₂ was tested in 10 ml of phosphate buffered saline tween. In contrast, reduced activity was observed when K0 ₂ was tested in 0.5 M KH ₂ P0 ₄ /K ₂ HP0 ₄ buffer. The latter buffer serves to maintain the pH of the solution at neutral pH, while the former buffer does not maintain the pH of the solutions at neutral pH but allowed the pH to rise to 12 and higher, levels at which the half-life of superoxide is significantly longer. No increase in activity was observed when tween was added to the 0.5 M KH ₂ P0 ₄ /K ₂ HP0 ₄ buffer, however. Use of superoxide according to the invention showed unexpectedly increased effectiveness against C. albicans compared to use of hydrogen peroxide when carried out at high pH. As shown in Example 12, 50 mg K0 ₂ in 10 ml of 0.9% saline (pH 12) achieved up to 5.9 logs at 30 minutes against this microorganism. In contrast, 0.2% hydrogen peroxide in 0.9% saline at the same pH (equivalent to the hydrogen peroxide concentration produced by 100 mg K0 ₂ in

10 ml saline) achieved only 1 log after 30 minutes against C. albicans .

No significant increase or decrease in antimicrobial efficacy was observed when K0 ₂ was tested in 0.9% saline in a commercially available lens case (see example 14) .

Superoxide thus does not appear to adversely interact with the plastic of the lens case.

Likewise, no significant increase or decrease in antimicrobial efficacy was observed when K0 ₂ was tested in small volumes of 0.9% saline (see examples 16 and 17) . Disinfection by superoxide thus does not appear to be limited by diffusion of the radical into contact with the microorganisms throughout the solution.

However, decreased antimicrobial efficiency was observed when K0 ₂ was tested in distilled water (compare examples 14 and 18) . The observed results suggest that tonicity may enhance antimicrobial activity.