WO1998022567A1

WO1998022567A1 - Stable liquid enzyme compositions for cleaning contact lenses

Info

Publication number: WO1998022567A1
Application number: PCT/US1997/020057
Authority: WO
Inventors: Barry F. Van Duzee; Ralph P. Stone; Bahram Asgharian
Original assignee: Alcon Laboratories, Inc.
Priority date: 1996-11-18
Filing date: 1997-11-03
Publication date: 1998-05-28
Also published as: AU5103698A

Abstract

Compositions containing an ophthalmically acceptable enzyme of high purity and integrity in a liquid medium and methods involving the use of these compositions for cleaning a contact lens, and in combination with an anti-microbial agent for the simultaneous cleaning and disinfecting of contact lens are disclosed.

Description

STABLE LIQUID ENZYME COMPOSITIONS FOR CLEANING CONTACT LENSES

Background of the Invention

The present invention relates to the field of contact lens cleaning and disinfecting. In particular, this invention relates to liquid enzyme compositions and methods for cleaning human-worn contact lenses with those compositions. The invention also relates to methods of simultaneously cleaning and disinfecting contact lenses by combining the liquid enzyme compositions of the present invention with a chemical disinfecting agent. Various compositions and methods for cleaning contact lenses have been described in the patent and scientific literature. Some of these methods have employed compositions containing surfactants or enzymes to facilitate the cleaning of lenses. The first discussion of the use of proteolytic enzymes to clean contact lenses was in an article by Lo, et al. in the Journal of The American Optometric Association, volume 40, pages 1106-1109 (1969). Methods of removing protein deposits from contact lenses by means of proteolytic enzymes have been described in many publications since the initial article by Lo, et al., including U.S. Patent No. 3,910,296 (Karageozian, et al).

Numerous compositions and methods for disinfecting contact lenses have also been described. Those methods may be generally characterized as involving the use of heat and/or chemical agents. Representative chemical agents for this purpose include organic antimicrobials such as benzalkonium chloride and chlorhexidine, and inorganic anti-microbials such as hydrogen peroxide and peroxide-generating compounds. U.S. Patents Nos. 4,407,791 and 4,525,346 (Stark) describe the use of polymeric quaternary ammonium compounds to disinfect contact lenses and to preserve contact lens care products. U.S. Patents Nos. 4,758,595 and 4,836,986 (Ogunbiyi) describe the use of polymeric biguanides for the same purpose.

Various methods for cleaning and disinfecting contact lenses at the same time have been proposed. Methods involving the combined use of proteolytic enzymes and peroxides to clean and disinfect contact lenses simultaneously, are described in U.S. Patent No. Re 32,672 (Huth, et al.). A representative method of simultaneously cleaning and disinfecting contact lenses involving the use of proteolytic enzymes and quaternary ammonium compounds is described in Japanese Patent Publication 57-24526 (Boghosian, et al.). The combined use of a biguanide (i.e., chlorhexidine) and liquid enzyme compositions to simultaneously clean and disinfect contact lenses is described in Canadian Patent No. 1,150,907 (Ludwig, et al.). Methods involving the combined use of dissolved proteolytic enzymes to clean and heat to disinfect are described in U.S. Patent No. 4,614,549 (Ogunbiyi). The combined use of proteolytic enzymes and polymeric biguanides or polymeric quaternary ammonium compounds is described in copending, and commonly assigned United States Patent Application Serial No. 08/156,043 and in corresponding European Patent Application Publication No. 0 456 467 A2 (Rosenthal, et al.), as well as in U.S. Patent No. 5,096,607 (Mowrey-McKee, et al.).

The commercial viability of most prior enzyme/disinfectant combinations has depended on the use of stable enzyme tablets. More specifically, the use of solid enzymatic cleaning compositions has been necessary to ensure stability of the enzymes prior to use. In order to use such compositions, a separate packet containing a tablet must be opened, the tablet must be placed in a separate vial containing a solution, and the tablet must be dissolved in order to release the enzyme into the solution. This practice is usually performed only once a week due to the cumbersome and tedious procedure and potential for irritation and toxicity. Moreover, the enzymatic cleaning tablets contain a large amount of excipients, such as effervescent agents (e.g., bicarbonate) and bulking agents (e.g., sodium chloride). As explained below, such excipients can adversely affect both cleaning and disinfection of the contact lenses.

There have been prior attempts to use liquid enzyme compositions to clean contact lenses. However, those attempts have been hampered by the fact that aqueous liquid enzyme compositions are inherently unstable. When a proteolytic enzyme is placed in an aqueous solution for an extended period (i.e., several months or more), the enzyme may lose all or a substantial portion of its proteolytic activity. Steps can be taken to stabilize the compositions, but the use of stabilizing agents may have an adverse effect on the activity of the enzyme. For example, stabilizing agents can protect enzymes from chemical instability problems during storage in an aqueous liquid, by placing the enzymes in a dormant physical conformation. However, such agents may also inhibit the ability of the enzymes to become active again at the time of use. Finally, in addition to the general problems referred to above, a commercially viable liquid enzyme preparation for treating contact lenses must be relatively nontoxic, and must be compatible with other chemical agents used in treating contact lenses, particularly anti-microbial agents utilized to disinfect the lenses.

The following patents may be referred to for further background concerning prior attempts to stabilize liquid enzyme formulations: U.S. Patents Nos. 4,462,922 (Boskamp); 4,537,706 (Severson); and 5,089,163 (Aronson). These patents describe detergent compositions containing enzymes. The detergent compositions may be used to treat laundry, as well as other industrial uses. Such detergents are not appropriate for treating contact lenses. U.S. Patent No. 5,281,277 (Nakagawa) and Japanese Kokai Patent Applications Nos. 92-370197; 92-143718; and 92-243215 describe liquid enzyme compositions for treating contact lenses. The compositions of the present invention are believed to provide significant improvements relative to the compositions described in those publications.

Summary of the Invention

The present invention provides stable liquid enzyme compositions for cleaning contact lenses and methods for using the compositions. The liquid enzyme compositions of the present invention contain enzymes of high purity and structural integrity. This high purity and integrity limits: 1) the number of different potential antigenic or other irritating substances on a contact lens; 2) the quantity of potential antigenic or other irritating substances on a contact lens and 3) the unstabilizing feature of cross-degradation by dissimilar enzymes in liquid compositions.

The liquid enzyme compositions of the present invention contain single enzymes of high integrity and purity in a vehicle. The vehicles may be concentrated aqueous or non- aqueous liquid compositions which are then diluted with an appropriate aqueous composition, e.g. a disinfecting solution. The enzymes of the present invention may also be included in ready-to-use multi-purpose compositions containing all the ingredients necessary to clean and disinfect a contact lens. In general, concentrated enzyme compositions will be preferred when longer term storage and hence longer term stability of the enzyme is necessary, and multi-purpose compositions will generally be preferred when issues of ease of use are more important than storage life. Concentrated enzyme compositions which are aqueous are generally preferred due to their ease of preparation and sterilization. In order to further enhance the stability of the aqueous compositions, the use of an aqueous vehicle which contains an amount of a water- miscible organic molecule is preferred. The water-miscible organic molecule is in a quantity sufficient such that it interferes with the protease 's interaction with water. This interference provides additional prevention of autolysis of the protease in the compositions of the present invention.

The present invention also provides methods for cleaning contact lenses with the above-described liquid enzyme compositions. In order to clean a soiled lens, the lens is placed in a few milliliters of an aqueous solution and a small amount, generally one to two drops, of a concentrated enzyme composition of the present invention is added to the solution. The lens is then soaked in the resultant cleaning solution for a time sufficient to clean the lens. Alternatively, if an enzyme containing multi-purpose composition of the present invention is employed, the soiled lens is directly soaked in the multi-purpose composition without addition of another composition.

The concentrated liquid enzyme compositions of the present invention are preferably combined with an aqueous disinfecting solution to simultaneously clean and disinfect contact lenses. As will be appreciated by those skilled in the art, the disinfecting solution must be formulated so as to be compatible with contact lenses. The anti-microbial activity of many chemical disinfecting agents is adversely affected by ionic solutes (e.g., sodium chloride). As explained below, the concentrated liquid enzyme compositions of the present invention are low in ionic content, and therefore do not adversely affect the anti-microbial activity of such disinfecting agents. This is considered to be a major advantage of the present invention.

The compositions and methods of the present invention provide greater ease of use. This ease of use enables contact lens users to clean their lenses 2 to 3 times a week, or more preferably, every day. It has been found that daily use of the liquid enzyme compositions of the present invention results in dramatically better cleaning, as compared to the once-a-week enzyme cleaning regimens currently being utilized. Detailed Description of the Invention

The enzymes which may be utilized in the compositions and methods of the present invention include all enzymes which: (1) are useful in removing deposits from contact lenses; (2) cause, at most, only minor ocular irritation in the event a small amount of 'enzyme contacts the eye as a result of inadequate rinsing of a contact lens; (3) are relatively chemically stable and effective in the presence of the anti-microbial agents described below; and (4) do not adversely affect the physical or chemical properties of the lens being treated.

The proteolytic enzymes used herein are further required to be of high purity and integrity. Various chromatographic procedures and other assay methods known in the art may be used to determine the purity of an enzyme sample. The purity of the enzyme may be determined chromatographically by quantitatively observing the mass ratio of enzyme to all other separated proteins. For example the purity can be determined by the use of capillary electrophoresis. SDS-polyacrylamide gel electrophoresis in combination with isoelectric focusing may also be used to determine the purity of an enzyme sample. Gel electrophoresis will separate the various proteins based on molecular weight. Since some impurities may have similar molecular weights, these impurities may be more difficult to discern on the resulting electrophoretic gel. Isoelectric focusing, which separates proteins based on their isoelectric point, instead of molecular weight, may then be employed to further delineate any sample suspected of containing similar weight impurities. Capillary electrophoresis, gel electrophoresis and isoelectric focusing are well known chromatographic techniques in the art. Other chromatographic assays known in the art, such as ion exchange chromatography, may also be used to determine the purity of an enzyme sample. The above listed chromatographic techniques are known in the art and the instrumentation and chemical supplies required for their employment can be obtained from various commercial sources, such as Millipore/Waters Corp. (Boston, Massachusetts), Pharmacia (Piscataway, New Jersey), Bio-Rad (Hercules, California), Sigma Chemical Co. (St. Louis, Missouri), Beckman Instruments, Inc. (Fullerton, California) and Scientific Resources, Inc. (Eatontown, New Jersey). Additional determinations of the enzyme purity may be obtained by using functional enzyme assay methods with specific substrates. Such assay reagents are commercially available from various sources such as Sigma Chemical Co. The percent activity of an enzyme quantity may be determined by various functional assays known in the art. The use of specific substrates may also be employed to differentiate enzymes of the present invention from active enzyme impurities contained in the enzyme sample. These methods are generally known in the art, and the substrates and reagents may be obtained from Sigma Chemical Co., or other research chemical suppliers.

The enzymes of the present invention are of such purity that they will exhibit a mass ratio of at least 95:5 with respect to all other proteinacious material. A mass ratio of 97:3 is preferred, and a mass ratio of 99: 1 is most preferred. As used herein, the term "high purity mass ratio" refers to an enzyme quantity wherein the mass of the enzyme protein of the present invention is 95% or more by weight of the total protein mass of the enzyme quantity. In other words, the enzymes of the present invention are 95% or more pure relative to proteinacious impurities. As stated above, the enzymes of the present invention are also substantially active. As used herein, the term "substantially active" refers to an enzyme quantity wherein 90 ± 10% or more of the total protein is active enzyme. High purity and substantially active enzymes may be obtained commercially. Various companies sell such enzymes including: NovoNordsk (Bagsvaerd, Denmark) and Sigma Chemical Co. Alternatively, a crude enzyme can be purified and selected for substantially undenatured portions by typical methods known by those skilled in the art. For example, the use of column chromatography and crystallization techniques can generally be used to purify enzymes of the present invention.

For purposes of the present specification, enzymes which satisfy the foregoing requirements are referred to as being "ophthalmically acceptable."

Examples of suitable proteolytic enzymes include but are not limited to trypsin, subtilisin, collagenase, keratinase, carboxylase, aminopeptidase, Aspergillo peptidase, pronase E (from £_. griseus and dispase (from Bacillus, polymyxa) and mixtures thereof.

Microbial derived enzymes, such as those derived from Bacillus. Streptomyces. and Aspergillus microorganisms, represent one type of enzyme which may be utilized in the present invention. Of this sub-group of enzymes, the most preferred are the Bacillus derived alkaline proteases generically called "subtilisin" enzymes. The identification, separation and purification of enzymes is known in the art. Many identification and isolation techniques exist in the general scientific literature for the isolation of enzymes. The enzymes contemplated by this invention can be readily obtained by known techniques from plant, animal or microbial sources.

With the advent of recombinant DNA techniques, it is anticipated that new sources and types of stable proteolytic enzymes will become available. Such enzymes should be considered to fall within the scope of this invention so long as they meet the criteria set forth herein.

Subtilisin and trypsin are preferred enzymes for use in the present invention. Subtilisin is derived from Bacillus bacteria and is commercially available from various commercial sources including Novo Industries (Bagsvaerd, Denmark), and Fluka Biochemika (Buchs, Germany). Trypsin is purified from various animal sources and is commercially available from Sigma Chemical Co. and Boehringer Mannheim. Trypsin, purified from porcine sources, is particularly preferred.

While Applicants do not wish to be bound by any theory, it is believed that the high purity of the enzymes may enhance the stability of the liquid enzyme compositions of the present invention. More specifically, the use of enzymes having high purity mass ratios substantially eliminates the risk of cross-degradation by dissimilar enzymes which may occur. The stability of the compositions can be further enhanced by placing the enzyme in a dormant state. The enzymes are placed in a dormant state by forming a complex with the stabilizing agents. The enzymes are complexed to a point where the enzymes are inactivated, but where renaturation is easily achieved by dilution of the enzyme/stabilizing agent complex in an aqueous medium.

The enzymes of the present invention may be stabilized in concentrated non-aqueous compositions or aqueous compositions. Either composition, however, will comprise water- miscible organic molecules as stabilizers. Concentrated non-aqueous enzyme compositions of the present invention generally comprise a crystalline enzyme uniformly dispersed in a water-soluble organic liquid. Typical organic liquids include polyoxyethylenes (e.g., PEG-400) and alkoxy polyoxyethylenes such as methoxy polyethylene glycols. In this composition, the enzyme is in a dormant state, suspended within the non-aqueous liquid. Following dissolution in an aqueous diluting composition of the present invention, the enzyme solubilizes and becomes active. Preferred non-aqueous enzyme compositions comprise an enzyme in PEG-400. In concentrated aqueous compositions of the present invention, it is believed that the stabilizing agents compete with water for hydrogen bonding sites on the proteins. Thus, a certain percentage of these agents will effectively displace a certain percentage of water molecules. As a result, the proteins will change conformation to an inactive (dormant state) and complexed (with the stabilizing agents) form. When the enzyme is in an inactive form, it is prevented from self-degradation and other spontaneous, chemically irreversible events. On the other hand, displacement of too many water molecules results in protein conformational changes that are irreversible. In order to obtain a stable liquid enzyme composition of significant shelf life and thus commercial viability, a delicate balance point of maximum stability and maximum reversible renaturation must be ascertained.

It has been found that the use of a concentrated aqueous vehicle which contains a water-miscible organic molecule further enhances the stability of the highly pure enzymes utilized in the present invention. The use of this type of vehicle is therefore preferred.

It has been found that the use of a water-miscible organic molecule achieves the stability and sustainable activity required in the concentrated aqueous liquid enzyme compositions of the present invention. As used herein, the term "water-miscible organic molecule" or "stabilizer," refers to an organic compound that forms one liquid phase with water when added to water. As stated above, it is believed that the stabilizers compete with water in the hydrogen bonding of the enzyme in solution, and thereby disrupt the active state of the enzymes. Therefore, the particular structure of the stabilizer is generally not an important factor to stabilization efficacy. It is rather the ability of the organic molecule to form one phase with water that determines its stabilizing utility in the present invention. Furthermore, the stabilizers must also be suitable for ophthalmic use and will thus exhibit minimal adverse effects on the cleaning or cleaning and disinfecting regimen. For example, the stabilizer will not contribute to ocular irritation/toxicity or interfere with the antimicrobial efficacy of an anti-microbial agent. Given the above criteria, various and numerous molecules may be used in the present invention to stabilize the enzyme.

The stabilizers of the present invention will be employed in an amount of from 10-90%) weight/volume ("w/v"), and preferably, in an amount of from 40-80 % (w/v). In general, the stabilizers will be polar, non-volatile, non-ionic or amphoteric molecules. Examples of stabilizers include polyols (polymers and monomers), and poorly metabolized sugars, including disacharride or monomeric sugars. The above stabilizers are well known in the art and are available from numerous commercial sources.

The preferred polyols utilized in the concentrated aqueous compositions of the present invention are 2-3 carbon polyols. As used herein, the term "2-3 carbon polyol" refers to a compound with 2 to 3 carbon atoms and at least two hydroxy groups. Examples of 2-3 carbon polyols are glycerol, 1,2-propane diol ("propylene glycol"), 1,3-propane diol and ethylene glycol. Propylene glycol is the preferred 2-3 carbon polyol.

Preferred concentrated aqueous compositions of the present invention may include an acidic compound. Examples of acidic compounds include alkyl (C_] to C₆) carboxylic acid compounds, such as formic, acetic and propionic acids, as well as other acids, such as aromatic carboxylic and aromatic sulfonic acids.

Other types of compounds related to acids may also be used in the concentrated or multi-purpose compositions of the present invention. These compounds include borate compounds, such as alkali metal salts of borate, boric acid, borax, as well as aromatic borates, such as phenylboronic acid. The borate compounds may also contribute to the anti-microbial preservation of the liquid enzyme compositions of the present invention to a level effective for multi-use dispensing. The acids described above will typically be employed in an amount of from 0.01-5.0% (w/v). The borates will typically be employed in an amount of from 0.01- 10.0%) (w/v). The solubility of the acids or borates may be limited in water. However, the solubility of these compounds may be increased by increasing the amount of stabilizer employed.

Other agents may also be employed in the compositions of the present invention. The use of calcium chloride or other suitable sources of calcium may be employed to further stabilize the enzyme. Other divalent cations such as magnesium may also be employed. The compositions may additionally contain preservatives when preparing compositions for multi- dosing. Examples of suitable preservatives include sorbic acid and salts of sorbic acid, chlorhexidine, parabens as well as those compounds detailed below in the description of antimicrobial agents useful as disinfectants in the cleaning and disinfecting methods of the present invention. Still other agents may be added to the present invention including pH adjusting agents such as triethanolamine, Tris, HCl, or NaOH, and surfactants such as cationic, non-ionic, or amphoteric compounds. The concentrated liquid enzyme compositions of the present invention will have an enzyme concentration sufficient to provide an effective amount of enzyme to clean a lens when a small amount of the composition is added to a diluent. Or alternatively, the multipurpose compositions of the present invention will have an enzyme concentration sufficient to provide an effective amount of enzyme to clean lens without further dilution. As used herein, such an amount is referred to as "an amount effective to clean the lens." The amount of enzyme used in the concentrated liquid enzyme compositions and multi-purpose compositions will generally range from 0.05 to 2% w/v and 0.0005 to 0.2% w/v, respectively. The selection of a specific concentration will depend on various factors, such as: the specificity and efficacy of the enzyme selected; the type of lenses to be cleaned; the intended frequency of cleaning (e.g., daily or weekly); potential interference of the enzyme with an anti-microbial agent; ocular irritation toxicity potential of the enzyme chosen; the intended duration of each cleaning; and with concentrated compositions, the amount of enzyme composition and amount of diluent used. During storage, some of the activity of the enzyme may be lost, depending on length of storage and temperature conditions. Thus, the concentrated liquid enzyme compositions of the present invention may be prepared with initial amounts of enzyme that exceed the concentration ranges described herein. The preferred concentrated compositions of the present invention will generally contain an enzyme in an amount of about 300-6000 PAU/mL. The concentrated compositions will most preferably contain about 900-2200 PAU/mL, which corresponds to subtilisin in a range of about 0.1 to 0.3%) w/v, and trypsin in the range of about 0.1 to 0.3%) w/v. When the enzymes of the present invention are employed in multi-purpose compositions, the concentration of enzyme will range from about 5-75 PAU/mL, and preferably from about 5-25 PAU/mL. For purposes of this specification, a "proteolytic activity unit" or "PAU" is defined as the amount of enzyme activity necessary to generate one microgram (meg) of tyrosine per minute ("meg Tyr/min"), as determined by the casein-digestion, colorimetric assay described below.

Casein-digestion assay A 5.0 mL portion of casein substrate (0.65% casein w/v) is equilibrated for 10 minutes (min) ± 5 seconds (sec) at 37°C. A 1.0 mL portion of enzyme solution (0.2 mg/ml) is then added to the casein substrate and the mixture vortexed, then incubated for 10 min ± 5 sec at 37°C. After incubation, 5.0 mL of 14% trichloroacetic acid is added and the resultant mixture immediately vortexed. The mixture is incubated for at least another 30 min, then vortexed and centrifuged for 15-20 min (approx. 2000 rpm). The supernatant of the centrifuged sample is filtered into a serum filter sampler and a 2.0 mL aliquot removed. To the 2.0 mL sample is added 5.0 mL of 5.3% Na₂CO₃. The sample is vortexed, 1.0 mL of 0.67 N Folin's Phenol reagent is added, and the sample is immediately vortexed again, then incubated for 60 min at 37°C. The sample is then read on a visible light spectrophotometer at 660 nanometers (nm) versus purified water as the reference. The sample concentration is then determined by comparison to a tyrosine standard curve.

The cleaning obtained with the liquid enzyme compositions of the present invention is a function of the time. The soaking times utilized will generally vary from about 1 hour to overnight. However, if longer soaking periods (e.g., 24 hours) are to be employed, lower enzyme concentrations than those described above may be utilized. The cleaning methods of the present invention involve the use of a small amount of a concentrated liquid enzyme composition or a soaking amount of an enzyme multi-purpose composition to facilitate the removal of proteins and other deposits from contact lenses. The amount of concentrated enzyme composition utilized in particular embodiments of the present invention may vary, as stated above, and may depend on various other factors, such as the specificity and efficacy of the enzyme utilized, the proposed duration of exposure of lenses to the compositions, the nature of the lens care regimen (e.g., the frequency of lens disinfection and cleaning), the type of lens being treated, and the use of adjunctive cleaning agents (e.g., surfactants). However, the cleaning methods of the present invention will generally employ an amount of the above-described concentrated liquid enzyme compositions sufficient to provide a final enzyme concentration of about 5-75 PAU/mL, following dispersion of the liquid enzyme compositions in a disinfecting solution or other aqueous solvent. A final concentration of about 5-25 PAU/mL is preferred.

As indicated above, the concentrated liquid enzyme compositions of the present invention contain relatively minor amounts of ionic solutes. More specifically, these compositions do not contain bulking agents, effervescent agents or other ionic solutes commonly contained in prior enzyme tablets. The present compositions may contain the ionic solutes of borate or boric acid compounds and hydrochloric acid and/or sodium hydroxide, but the concentration of these solutes in the present compositions is relatively low. The compositions are therefore substantially nonionic. Moreover, as a result of the fact that these compositions are formulated as concentrated, multi-dose liquids, only a small amount of the compositions, generally one or two drops, is required to clean a contact lens. The present compositions therefore have very little impact on the ionic strength of disinfecting solutions. As explained below, this feature of the present invention is particularly important when the liquid enzyme compositions are combined with disinfecting solutions which contain ionic anti-microbial agents, such as polyquaternium-1.

The anti-microbial activity of disinfecting agents, particularly polymeric quaternary ammonium compounds such as polyquaternium-1, is adversely affected by high concentrations of sodium chloride or other ionic solutes. More specifically, polymeric quaternary ammonium compounds, and particularly those of Formula (I), below, lose antimicrobial activity when the concentration of ionic solutes in the disinfecting solution is increased. The use of solutions having low ionic strengths (i.e., low concentrations of ionic solutes such as sodium chloride) is therefore preferred. Since both ionic solutes (e.g., sodium chloride) and nonionic solutes (e.g., glycerol) affect the osmolality and tonicity of a solution, osmolality and tonicity can be indirect measures of ionic strength. However, the low ionic strengths preferably utilized in the cleaning and disinfecting methods of the present invention generally correspond to tonicities/osmolalities in the range of hypotonic to isotonic. For comfort sake, the osmolality of the disinfecting composition or multi-purpose composition will be preferably in the range of 150 to 400 milliOsmoles per kilogram ("mOsm/kg"). A range of 200 to 300 mOsm kg is particularly preferred, and an osmolality of about 220 mOsm kg is most preferred.

The liquid enzyme compositions of the present invention demonstrate effective cleaning efficacy while exhibiting minimal adverse effects or, more preferably, enhanced effects on the anti-microbial activity of disinfecting solutions. It has unexpectedly been discovered that enzyme compositions of the present invention enhance the anti-microbial activity of polyquaternium-1, a polymeric quaternary ammonium disinfecting agent. It has also been discovered that compositions containing an enzyme and polyquaternium-1 become even more effective than the polyquaternium-1 disinfecting solutions alone when lenses are treated for extended periods of approximately one hour to overnight, with four to eight hours preferred. Since, for the sake of convenience, contact lenses are typically soaked overnight in order to be cleaned with enzymes or disinfected with chemical agents, this finding has practical significance. While Applicants do not wish to be bound by any theory, it is believed that the above-described enhancement of anti-microbial activity is due to the disruption or lysis of microbial membranes by the enzyme over time. The cleaning methods of the present invention utilize an aqueous solvent. The aqueous solvent may contain various salts such as sodium chloride and potassium chloride, buffering agents such as boric acid and sodium borate, and other agents such as chelating agents and preservatives. An example of a suitable aqueous solvent is a saline solution, such as Unisol® Plus Solution (registered trademark of Alcon Laboratories). The cleaning and disinfecting methods of the present invention utilize a disinfecting solution containing an anti-microbial agent. Anti-microbial agents can be oxidative, such as hydrogen peroxide, or non-oxidative polymeric anti-microbial agents which derive their antimicrobial activity through a chemical or physicochemical interaction with the organisms. As used in the present specification, the term "polymeric anti-microbial agent" refers to any nitrogen-containing polymer or co-polymer which has anti-microbial activity. Preferred polymeric anti-microbial agents include: polyquaternium-1, which is a polymeric quaternary ammonium compound; and polyhexamethylene biguanide ("PHMB") or polyaminopropyl biguanide ("PAPB"), which is a polymeric biguanide. These preferred anti-microbial agents are disclosed in U.S. Patent Nos. 4,407,791 and 4,525,346, issued to Stark, and 4,758,595 and 4,836,986, issued to Ogunbiyi, respectively. The entire contents of the foregoing publications are hereby incorporated in the present specification by reference. Other anti-microbial agents suitable in the methods of the present invention include: other quaternary ammonium compounds, such as benzalkonium halides, and other biguanides, such as chlorhexidine. The anti-microbial agents used herein are preferably employed in the absence of mercury- containing compounds such as thimerosal.

The most preferred anti-microbial agents are polymeric quaternary ammonium compounds of the structure:

wherein:

R_j and R₂ can be the same or different and are selected from: N⁺(CH₂CH₂OH)₃X\ N(CH₃)₂ or OH; X^" is a pharmaceutically acceptable anion, preferably chloride; and n = integer from 1 to 50.

The most preferred compound of this structure is polyquaternium-1, which is also known as Onamer M™ (registered trademark of Onyx Chemical Corporation) or as Polyquad^® (registered trademark of Alcon Laboratories, Inc.). Polyquaternium-1 is a mixture of the above referenced compounds, wherein X^' is chloride and R_1? R₂ and n are as defined above.

The above-described anti-microbial agents are utilized in the methods of the present invention in an amount effective to eliminate substantially or to reduce significantly the number of viable microorganisms found on contact lenses, in accordance with the requirements of governmental regulatory agencies, such as the United States Food and Drug Administration. For purposes of the present specification, that amount is referred to as being "an amount effective to disinfect" or "an anti-microbially effective amount." The amount of anti-microbial agent employed will vary, depending on factors such as the type of lens care regimen in which the method is being utilized. For example, the use of an efficacious daily cleaner in the lens care regimen may substantially reduce the amount of material deposited on the lenses, including microorganisms, and thereby lessen the amount of anti-microbial agent required to disinfect the lenses. The type of lens being treated (e.g., "hard" versus "soft" lenses) may also be a factor. In general, a concentration in the range of about 0.00001% to about 0.01%) by weight of one or more of the above-described anti-microbial agents will be employed. The most preferred concentration of the polymeric quaternary ammonium compounds of Formula (I) is about 0.001%» by weight.

Oxidative disinfecting agents may also be employed in the methods of the present invention. Such oxidative disinfecting agents include various peroxides which yield active oxygen in solution. Preferred methods will employ hydrogen peroxide in the range of 0.1 to 3.0 % to disinfect the lens. Methods utilizing oxidative disinfecting systems are described in U. S. Patent Nos. Re 32,672 (Huth, et al.) and 5,491,091 (Loshaek, et al.); the entire contents of the foregoing are hereby incorporated in the present specification by reference. As will be appreciated by those skilled in the art, the disinfecting solutions utilized in the present invention may contain various components in addition to the above-described anti-microbial agents, such as suitable buffering agents, chelating and/or sequestering agents and tonicity adjusting agents. The disinfecting solutions may also contain surfactants. The methods of the present invention may involve adding a small amount of a concentrated liquid enzyme composition of the present invention to about 2 to 10 mL of an aqueous solvent or disinfecting solution, placing the soiled lens into the enzyme/solvent or enzyme/disinfectant solution, and soaking the lens for a period of time effective to clean or clean and disinfect the lens. The amount of concentrated liquid enzyme composition utilized can vary based on factors such as the amount of disinfecting solution used, but generally will be about 1 to 2 drops. Preferred methods involve adding 1 drop (approximately 30 μL) to 5 mL of aqueous solvent or disinfecting solution. The soiled lens can be placed in the aqueous solvent or disinfecting solution either before or after the addition of the liquid enzyme composition. Optionally, the contact lenses may be first rubbed with a non-enzymatic daily cleaner prior to immersion in the enzyme/solvent or enzyme/disinfectant solution. The lens will typically be soaked overnight, but shorter or longer durations are contemplated by the methods of the present invention. A soaking time of 4 to 8 hours is preferred. The methods of the present invention allow the above-described regimen to be performed once per week, but more preferably, every day. The methods of the present invention may also involve placing the soiled lens in about 2-10 mL of a multi-purpose composition of the present invention and soaking the lens for a period of time sufficient to clean and disinfect the lens. The multi-purpose composition may optionally first be used as a cleaner in a mechanical rub and rinse step prior to soaking the lens. The following examples are presented to illustrate further, various aspects of the present invention, but are not intended to limit the scope of the invention in any respect.

EXAMPLE 1

A specific concentrated liquid subtilisin composition of the present invention, and a suitable disinfecting solution for use in combination with that composition, are described below: A. Liquid Subtilisin Composition

The following concentrated liquid enzyme formulation represents a preferred embodiment of the present invention:

Note: (w/v) means weight/volume and QS means quantity sufficient

The liquid subtilisin formulation of this example is generally prepared by first mixing glycerol, purified water, calcium chloride and benzoic acid together. The required amount of subtilisin is then dissolved in the above solution. During mixing, the contents of the receiving tank are then brought to volume with an appropriate amount of purified water. The pH can then be adjusted, or it can be adjusted before all of the additional purified water is added. The mixture is then sterile filtered (0.2 μm filter) into the sterile receiving tank. The preferred pH of the above formulation is in the range of 6-8; a pH of 7.5 is most preferred.

B. Disinfecting Solution

To prepare the above formulation, sodium citrate dihydrate, citric acid monohydrate, disodium edetate, sodium chloride and Polyquaternium-1, in the relative concentrations indicated above, were mixed with purified water and the components allowed to dissolve by stirring with a mixer. Purified water was added to bring the solution to almost 100%). The pH was recorded at 6.3 and adjusted to 7.0 with NaOH. Purified water was added to bring the solution to 100%. The solution was stirred and a pH reading of 7.0 was taken. The solution was then filtered into sterile bottles and capped.

EXAMPLE 2

A preferred liquid trypsin composition of the present invention for use in combination with a suitable disinfecting solution, e.g. EXAMPLE IB., is described below:

Liquid Trypsin Compositions

* corresponds to an amount to adjust the pH to 6.0 Note: "% (v/v)" refers to percent volume/volume

The above liquid trypsin composition is made in the same manner as the liquid subtilisin composition, described in EXAMPLE 1. EXAMPLE 3

The following is a multi-purpose composition of the present invention:

corresponds to an amount to adjust pH to about 6.5-8.0

EXAMPLE 4

A stability study comparing a concentrated liquid trypsin composition of the present invention with a liquid pancreatin composition was performed. The trypsin composition was the composition of Example 2, and the pancreatin composition was also the composition of Example 2, except pancreatin was substituted for trypsin in the amount of 1.7% (w/v). The data are shown in Table I below. Aliquots of the compositions were stored in a chamber held to 35° C. At the appointed time, aliquots were tested for enzyme proteolytic activity. Activity levels were compared with initial levels and expressed as percent remaining activity.

TABLE I: STABILITY OF CONCENTRATED LIQUID ENZYME COMPOSITIONS CONTAINING TRYPSIN OR CHYMOTRYPSIN STORED AT 35° C

EXAMPLE 5

The disinfecting efficacy of a composition of the present invention was evaluated by determining the rate and extent of kill achieved with an aqueous system formed by combining the liquid enzyme composition and disinfecting solution described in EXAMPLE 2 above, with the exception that subtilisin in the amount of 0.1% (w/v) was substituted for trypsin in the liquid enzyme composition. That system was tested against Serratia marcescens. The test procedures and results are described below.

A 0.1 ml volume of inoculum (10⁸ colony forming units/mL) was first added to a 10 ml volume of the disinfecting solution of EXAMPLE 1 , followed by the addition of 2 drops of the liquid enzyme composition described above. A similarly inoculated 10 ml volume of the disinfecting solution of EXAMPLE 1 was used as a control. The solutions were maintained at room temperature throughout the test. Each microorganism and test solution was tested individually. Sets of four replicate (n = 8) samples were tested for each organism.

At selected time intervals of 4 and 24 hours a 1 ml volume of the inoculated test solution was removed and appropriate serial dilutions were made in sterile 0.9% sodium chloride solution dilution blanks. Pour-plates were prepared with soybean-casein digest agar containing 0.07% Asolectin and 0.5%) Polysorbate 80. At Time 0, a 1.0 ml volume of the saline control was removed and serial dilution pour-plates were prepared using the same recovery medium and dilution blanks. The Time 0 saline control count was used as the initial count. The pour-plates were incubated at 30°-35°C for appropriate incubation periods. The number of surviving organisms at each time interval was then determined. The results are summarized in Tables II below.

TABLE II: EFFECTS OF A SUBTILISIN CONTAINING LIQUID ENZYME COMPOSITION ON THE ANTI-MICROBIAL ACTIVITY OF A POLYQUATERNIUM-1

DISINFECTING SOLUTION

EXAMPLE 6

The disinfecting efficacy of a further embodiment of the present invention was evaluated by determining the rate and extent of kill achieved with an aqueous system formed by combining the liquid trypsin composition of Example 2 and the disinfecting solution described in Example 1. The system was tested against Serratia marcescens. Staphylococcus aureus. Pseudomonas aeruginosa. Candida albicans and Fusarium solani. The test procedure in Example 4 was followed. Sample times were 4, 6 and 24 hours. The test results, expressed as log reductions are presented in Table III below.

TABLE III: EFFECTS OF A TRYPSIN CONTAINING LIQUID ENZYME COMPOSITION ON THE ANTI-MICROBIAL ACTIVITY OF A POLYQUATERNIUM-1

DISINFECTING SOLUTION

The invention in its broader aspects is not limited to the specific details shown and described above. Departures may be made from such details within the scope of the accompanying claims without departing from the principles of the invention and without sacrificing its advantages.

Claims

What is Claimed is:

1. A stable, concentrated, liquid enzyme composition for cleaning contact lenses comprising an enzyme having a mass ratio of at least 95:5, and a vehicle for said enzyme, wherein the enzyme is substantially active and in an amount effective to clean the lens upon dilution in an aqueous solvent.

2. A stable, liquid enzyme composition for cleaning contact lenses comprising an enzyme having a mass ratio of at least 95:5, and an aqueous vehicle for said enzyme, wherein the enzyme is substantially active and in an amount effective to clean.

3. A composition according to Claim 1, wherein the vehicle comprises 5-80% (w/v) of a water-miscible organic molecule, and water.

4. A composition according to Claim 3, wherein the water-miscible organic molecule is selected from the group consisting of monomeric polyols, polymeric polyols and sugars.

5. A composition according to Claim 1, wherein the enzyme is trypsin.

6. A composition according to Claim 4, wherein the enzyme is trypsin.

7. A method of cleaning a contact lens which comprises: placing the lens in an aqueous solvent; dispersing a small amount of a stable, concentrated, liquid enzyme composition in the aqueous solvent to form an aqueous enzymatic cleaning solution, said concentrated liquid enzyme composition comprising an enzyme having a mass ratio of at least 95:5, and a vehicle for said enzyme; wherein the enzyme is substantially active and in an amount effective to clean the lens upon dispersion in the aqueous solvent; and soaking the lens in the enzymatic cleaning solution for a period of time sufficient to clean the lens.

8. A method according to Claim 7, wherein the concentrated, liquid enzyme composition comprises 5-80% (w/v) of a water-miscible organic molecule, and water.

9. A method according to Claim 8, wherein the water-miscible organic molecule is selected from the group consisting of monomeric polyols, polymeric polyols and sugars.

10. A method according to Claim 7, wherein the enzyme is trypsin.

11. A method according to Claim 9, wherein the enzyme is trypsin.

12. A method according to Claim 7, wherein the method is performed daily.

13. A method for cleaning and disinfecting a contact lens which comprises: placing the lens in an aqueous disinfecting solution containing an amount of an antimicrobial agent effective to disinfect the lens; dispersing a small amount of a stable, concentrated, liquid enzyme cleaning composition in said disinfecting solution to form an aqueous disinfectant/enzyme solution, said concentrated liquid enzyme composition comprising an enzyme having a mass ratio of at least 95:5, and a vehicle for said enzyme; wherein the enzyme is substantially active and in an amount effective to clean the lens upon dispersion in the disinfecting solution; and soaking the lens in the aqueous disinfectant/enzyme solution for a period of time sufficient to clean and disinfect the lens.

14. A method according to Claim 13, wherein the concentrated, liquid enzyme composition further comprises 5-80% (w/v) of a water-miscible organic molecule and water.

15. A composition according to Claim 14, wherein the water-miscible organic molecule is selected from the group consisting of monomeric polyols, polymeric polyols and sugars.

16. A method according to Claim 13, wherein the enzyme is trypsin.

17. A method according to Claim 15, wherein the enzyme is trypsin.

18. A method according to Claim 13, wherein the method is performed daily.