ENZYMES WITH LOW ISOELECTRIC POINTS FOR USE IN CONTACT LENS CLEANING

The present invention relates to the field of contact lens cleaning and disinfecting In particular, this invention relates to compositions containing enzymes which have been

modified to exhibit a low isoelectric point and methods for cleaning human-worn contact lenses with those compositions The invention also relates to methods of sιmultaneousl cleaning and disinfecting contact lenses by combining the enzyme compositions of the

present invention with a chemical disinfecting agent

Background ofthe Invention

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 Optometπc Association, volume 40, pages 1 106- 109 (1969)

Methods of removing protein deposits from contact lenses by means of proteolytic enzymes

have been descπbed 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

antimicrobials 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 Such methods are described in U S Patents Nos 3,873,696 (Randeπ, et

al ) and 4,414, 127 (Fu), for example A representative method of simultaneously cleaning and disinfecting contact lenses involving the use of proteolytic enzymes to remove protein

deposits and a chemical disinfectant (monomeric quaternary ammonium compounds) is

described in Japanese Patent Publication 57-24526 (Boghosian, et al ) The combined use

of a biguanide (l e , chlorhexidine) and enzymes to simultaneously clean and disinfect contact lenses is described in Canadian Patent No 1, 150,907 (Ludwig) 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

Although the use of these enzymatic systems provides effective cleaning, a number

of problems associated with their use exist One problem is that residual amounts of the

enzyme can bind to the contact lens This binding can lead to less clarity of vision when

using the lens It can also lead to ocular irritation and immunogenicity, due to the eye's

sensitization to the foreign protein For example, Breen reported symptoms of ocular

irritation in patients with ocular sensitivity to contact lenses which have been cleaned with the enzyme subtilisin (Breen et al , Clinical Comparison of Pancreatm-Bascd and

Subtihsin-Based Enzymatic Cleaners, Contact Lens Forum, volume 15, pages 32-38

(1990)) Consequently, the use of enzyme cleaning is generally limited to a once-per-week

regimen As a result, daily supplemental cleaning, which involves the rubbing of the lens with a surfactant, is necessary to clean the lens satisfactorily during the interim period

between the weekly enzymatic cleanings Thus, the contact lens user is burdened by the purchase of two separate cleaners and the employment of them separately in order to

effectively clean his lenses Therefore, although enzyme cleaning systems provide effective

cleaning, they have not been fully exploited as a once-per-day regimen for the optimal

cleaning and convenience they would otherwise provide The modification of the enzyme to hinder its binding to the lens would reduce ocular irritation and immunogenicity, improve

visual clarity, and therefore enable a more regular use of the enzyme for cleaning contact

lenses

The use of modified enzymes for use in cleaning various articles has been proposed For example, enzymes have been modified by altered amino acid sequences, in an effort to

decrease adsorption to an insoluble surface and for greater hydrolysis of target proteins,

such enzymes have been disclosed in WIPO Publication No WO 95/07991 (assigned to Procter & Gamble).

Enzymes have also been modified by organic polymer linkage. The covalent linking

of proteins with polyethylene glycol (PEG), to yield a polyoxyethylene-protein product, is

disclosed by United States Patent No. 4, 179,337 (Davis et al.). A variety of publications

and patents have described numerous types of PEG-modified proteins and methods of

preparation Davis et al , above, discloses PEG-modified or polypropylene glycol-

modified, non-immunogenic polypeptides for use in the circulatory system of the human body European Patent Application No 0 584 876 A2 discloses low diol polyalkylene

oxide biologically active proteinaceous substances, including a Subtilisin Carlsberg

Another method of enzyme modification has involved organic monomer linkage to

the enzyme For example, Johansen discloses methods of succmylation and glutarylation of subtilisins in Chemical Derivatives of Subtihsms with Modified Proteolytic Activities II

Succinyl- and Glutarylsubtihsm Type Carlsberg, Compt Rend Trav Lab Carlsberg,

volume 37, pages 145-177 (1970)

Summary of the Invention

The present invention is based in part on the finding that particular modified

enzymes exhibit low binding to hydrophilic lenses The enzymes contained in compositions

of the present invention exhibit low isoelectric points relative to physiological pH

Enzymes exhibiting low pis of the present invention, are prevented from electrostatic

attraction to negatively charged hydrophilic lenses This lower binding of enzymes to the contact lens surface reduces possible ocular irritation of the bound enzyme when the

contact lens is reinserted in the eye The lower binding may also improve enzyme cleaning

efficacy Thus, the present invention has overcome issues of toxicity and efficacy to

provide a more effective cleaning system for contact lenses

The compositions of the present invention are formulated in either solid or liquid

form. Compositions formulated in liquid form may, for example, comprise a polyol and an

enzyme The methods of the present invention provide for cleaning of contact lenses with

the compositions of the present invention The methods of the present invention also

provide for the simultaneous cleaning and disinfecting of contact lenses, when compositions

of the present invention are combined with suitable disinfecting solutions, such as those containing polyquaternium- 1

Detailed Description ofthe Invention

The enzymes of the present invention exhibit low isoelectric points ("pi") relative to physiological pH This is significant as some hydrophilic contact lenses exhibit a net

negative charge at physiological pH As the enzymes are neutral or have a net negative charge at physiological pH, they will not electrostatically bind to the lenses Enzymes, especially those from microbial sources, can accumulate in or on lenses and cause ocular

irritation when they come into contact with the eye Therefore, the use of low

electrostatically binding enzymes, i e., those with low pis, provides a safe and more comfortable method for contact lens cleaning.

As used herein, the term "low pi" refers to electrochemical properties of an enzyme such that the enzyme has a net charge of zero within the pH range of 4-8 The pi of an

enzyme can be determined by methods known to those skilled in the art. In general, the use

of the technique of isoelectric focusing, as described in Example 3 below, may be used to determine the pl of an enzyme

The enzymes which may be used in the compositions and methods of the present

invention include those enzymes which have been modified to exhibit low pis, and 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 antimicrobial agents described below, and (4) do not adversely affect the

physical or chemical properties of the lens being treated For purposes of the present

specification, enzymes which satisfy the foregoing requirements are referred to as being "ophthalmically acceptable "

The proteolytic enzymes used herein must have at least a partial capability to

hydrolyze peptide-amide bonds in order to reduce the proteinaceous material found in lens deposits to smaller water-soluble subunits Examples of native enzymes which may be modified for use in the present invention,

include but are not limited to pancreatin, trypsin, chymotrypsin, subtilisin, collagenase,

elastase, keratinase, carboxypeptidase, papain, bromelain, aminopeptidase, Aspergillo peptidase, pronase E (from S_ griseus) and dispase (from Bacillus polvmyxa and mixtures thereof If papain, or any sulfhydryl protease is used, a reducing agent, such as N-

acetylcysteine, may be required

Microbially derived enzymes, such as those derived from Bacillus, Streptomyces,

and Aspergillus microorganisms, represent a preferred type of enzyme to be modified for use in the present invention Of this sub-group of enzymes, the most preferred are the

Bacillus derived alkaline proteases generically called "subtilisin" enzymes

Examples of subtilisin enzymes include subtilisin BPN' and subtilisin Carlsberg

Subtilisin is commercially available from various commercial sources including Novo

Industries (Bagsvaerd, Denmark), Fluka Biochemika (Buchs, Switzerland) and Boehringer

Mannheim (Indianapolis, Indiana)

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, including those enzymes having proteolytic and mixed

proteolytic/amylolytic/ polytic activity The native enzymes to be modified for use in 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 as long as they meet the criteria for

stability, activity and can be modified to exhibit a low pi as set forth herein

The amount of enzyme used in the compositions of the present invention will range

from about 0 01 to 5% w/v, due to various factors, such as purity, specificity and efficacy The preferred compositions of the present invention will contain a low pi subtilisin in a

range of about 0 01 to 1 0% w/v

The enzymes ofthe present invention may be selected from those that have had part

of their ammo acid sequence altered in favor of a lower pi In general, amino acid residues

exhibiting high (net positive charge at physiological pH) pKa's (pH at which half of the total quantity ofthe particular residue is charged) may be replaced with neutral or low pKa

amino acids For example, lysine or arginine (high pKa) residues may be replaced by alanine, leucine (non-ionic), aspartate, glutamate (low pKa) or other low pKa residues

This can be achieved by traditional genetic recombinant techniques like those described in WIPO Publication No WO 95/07991 (assigned to Proctor and Gamble), the contents of

which pertain to genetic recombinant techniques are incoφorated herein by reference As used herein, "genetic recombinant techniques" refer to any method of producing mutant

enzymes of the present invention through the manipulation of DNA Generally, a plasmid

of a host bacterium is transfected with DNA coding for the modified amino acid sequence desired. The plasmid is reinserted in the host, and the host is grown under set conditions

Broth from the fermenting process, containing the bacterial exudate, is then separated from

the bacterial colonies and is extracted for the target enzyme Separation techniques

including gel and affinity chromatography are generally employed to purify the mutant type

enzymes exhibiting low pis Enzymes with lysine residues replaced with lower pi amino

acids are preferred modified enzymes of this class of modified enzymes of the present

invention The enzymes of the present invention may be selected from those that have been

chemically modified, covalently, with organic monomer or polymer molecules As used

herein, "organic monomer covalent linkage" refers to the linking of small organic

monomers covalently to an enzyme; and "organic polymer covalent linkage" refers to linking large organic polymers covalently to an enzyme Examples of organic monomers

include succinate, and methyl, ethyl or proplyl acylates. Examples of organic polymers

include various polyethylene glycols (PEG), such as PEG 500, 1000 and 2000 Such

modifications have been discussed in United States Patent No 5, 122,614, the entire contents of which are incorporated herein by reference The use of this technique or similar

techniques known to those skilled in the art may be employed to modify various proteases

so that they exhibit low pis and are opthalmically acceptable as set forth above Commonly

assigned United States Patent Application No 08/491 ,754, filed June 19, 1995 discloses

novel PEG-subtilisins; the contents pertaining to these novel enzyme-polymer complexes is

incorporated herein by reference Examples of methods for monomeric modifications of

enzymes are discussed in Johansen, Chemical Derivatives of Subtihsms with Modified

Proteolytic Activities II. Succinyl- and Glutarylsubtύisin Type Carlsberg, Compt Rend

Trav Lab Carlsberg, volume 37, pages 145-177 (1970), the entire contents of which, are

incorporated herein by reference Preferred enzymes of this class are succinylated-

subtilisins and more generally, acylated-subtilisins As it is known to those skilled in the art, the degree of substitution can be

controlled by adjusting the ratio of modifying reagent to enzyme concentration It has been

found that enzymes that are modified extensively are less thermally stable in aqueous

vehicles (l e., they may not be easily stabilized in liquid form) Enzymes suitable for liquid stabilized vehicles may require minimal modification whereas all modified enzymes would be useful in a solid form (1 e , effervescent tablets)

The compositions of the present invention may be either in solid or liquid form Solid forms usually encompass a compressed tablet wherein various excipients are

employed For example, components such as effervescing agents, stabilizers, buffering

agents, chelating and/or sequestering agents, coloring agents, tonicity adjusting agents,

surfactants and the like can be employed 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

Examples of suitable buffering agents which may be incorporated into an enzyme tablet include, but are not limited to, alkali metal salts such as potassium or sodium carbonates, acetates, borates, phosphates and citrates, and weak acids such as

acetic and boric acids Preferred buffering agents are alkali metal borates such as sodium

or potassium borates Additionally, other pH adjusting agents may be employed such as

inorganic or organic acids and bases For example, hydrochloric acid, sodium hydroxide,

triethanolamine or Tris may be employed in concentrations suitable for ophthalmic uses

Generally, buffering agents are present in amounts from about 0 01 to about 2 5% (w/v) and preferably, from about 0 5 to about 1 5% (w/v), ofthe working solution

Effervescing agents are typically employed when the enzyme is provided in solid

form Examples of suitable effervescing agents include, but are not limited to, tartaric or

citric acid used in combination with a suitable alkali metal salt such as sodium carbonate

The tonicity adjusting agent which may be a component of a disinfecting solution and may optionally be incorporated into an enzyme tablet is employed to adjust the osmotic

value of the final cleaning and disinfecting solution to more closely resemble that of human

tears and to maintain a suitable level for optimum activity by the antimicrobial agent Typical tonicity adjusting agents are NaCl and KCl

Suitable surfactants can be either cationic, anionic, nonionic or amphoteric

Preferred surfactants are neutral or nonionic surfactants which may be present in amounts up to 5% (w/v) Examples of suitable surfactants include, but are not limited to, polyethylene glycol ether or esters of fatty acids, polyoxyethylene-polyoxypropylene block

copolymers of ethylene diamine (l e , poloxamine), polyoxypropylene-polyoxyethylene

glycol nonionic block polymers (l e , polaxamers such as Pluronic F-127) and p-

lsooctylpolyoxyethylene phenol formaldehyde polymers (I e , Tyloxapol)

Examples of preferred chelating agents include ethylenediaminetetraacetic acid

(EDTA) and its salts (disodium) which are normally employed in amounts from about

0 025 to about 2 0% (w/v) Other known chelating (or sequestering agents) can also be employed

The binders and lubricants for enzyme tableting purposes and other excipients

normally used for producing powders, tablets and the like, may be incorporated into

enzyme tablet formulations

A disinfecting agent may optionally be added to the enzyme tablet Such

disinfectants include those described below in the methods of the present invention

The above ingredients may be incorporated into tablet form by methods known to

those skilled in the art

Liquid compositions containing low pi modified enzymes are also contemplated by

the present invention Such compositions will be comprised of one or more low pi

enzymes of the present invention and a suitable liquid vehicle As used herein, the term

"suitable liquid vehicle" refers to any aqueous or non-aqueous carrier that provides

stabilization ofthe enzyme and preservation ofthe composition for multiple use dispensing

Stabilizing agents in the liquid compositions of the present invention will include

monomeric and/or polymeric polyols, and optionally, an enzyme inhibitor As used herein,

the term "monomeric polyol" refers to a compound with 2 to 10 carbon atoms and at least

two hydroxy groups Examples of monomeric polyols are glycerol, propylene glycol,

ethylene glycol, sorbitol and mannitol. As used herein, the term "polymeric polyol" refers

to a polyalkoxylated glycol with a molecular weight ranging from 200- 1000 Examples of

polymeric polyols are polyethylene glycol 200 (PEG 200) and PEG 400

The amounts of the components comprising the polyol will vary depending on

the particular combination of polyols used. In general, liquid enzyme compositions of the

present invention will require 10-70% v/v of at least one polyol to achieve the necessary

criteria for efficacious and commercially viable liquid enzyme compositions, as described

above While any of the polyols can be components of the compositions of the present

invention, particular polyols may be used depending on the particular intended use For

example, propylene glycol, which has preservative activity, is a preferred monomeric polyol

when the need for an additional preservative present in a liquid enzyme composition of the

present invention is desired

The liquid compositions of the present invention may optionally contain a reversible

enzyme inhibitor The inhibitor will be added in an amount necessary to inactivate the enzyme, but where reactivation is easily achieved by dilution of the inhibited

enzyme/stabilizing agent complex in an aqueous medium When the enzyme is in an

inactive form, it is prevented from self-degradation and other spontaneous, chemically

irreversible events Examples of reversible inhibitors include borates, phenylboronic acid and lower alkyl carboxylic acids such as propanoic and butyric acids As used herein, the

term "lower carboxylic acid" refers to a compound having a carboxylic acid group and from

2-4 carbon atoms in total Preferred inhibitors include phenylboronic acid and it derivatives The preferred range of a phenylboronic acid derivative used in the present

invention is 0 1 to 5 0% weight/volume ("%w/v")

A variety of preservatives may be employed to preserve a multi-dispensing liquid

enzyme composition of the present invention In general, any of the agents listed for use in the disinfecting solutions of the methods of the present invention, with the exception of

oxidative disinfecting agents, may be employed Particularly preferred, are the polymeric

quaternary ammonium compounds, the most preferred is polyquaternium- 1 The amount of

preservative used will depend on several factors including the anti-microbial efficacy of the particular agent and any synergistic interaction the agent may have with the liquid enzyme

composition In general, 0 0001 to 0 1% w/v ofthe preservative agent will be used

The liquid compositions may contain one or more surfactants selected from anionic, non-ionic or zwitterionic classes Examples of non-ionic surfactants include alkyl

polyoxyethylene alcohols, alkyl phenyl polyoxyethylene alcohols, polyoxyethylene fatty acid

esters, polyethylene oxide-polypropylene oxide copolymers such as polaxomers and

polaxamines Examples of anionic surfactants include alkyl sarcosinates and alkyl glutamates Examples of amphoteric surfactants include alkyliminopropionates and

alkylamphoacetates In general 0 to 5% w/v ofthe surfactant will be used

The liquid compositions may contain additional stabilizing agents. These include

the stabilizing multi-valent ions, such as calcium and magnesium and their halide salts. Calcium chloride is the most preferred multi-valent stabilizing agent

Other ingredients may optionally be added to the liquid enzyme compositions of the

present invention Such ingredients include buffering agents, such as, Tris, phosphate or

borate buffers, tonicity adjusting agents, such as NaCl or KCl; metal chelating agents, such as ethylenediaminetetraacetic acid (EDTA) and pH adjusting agents such as sodium

hydroxide, tris, triethanolamine and hydrochloric acid.

The cleaning methods of the present invention involve the use of an amount of

enzyme effective to remove substantially or to reduce significantly deposits of proteins, lipids, mucopolysaccharides and other materials typically found on human- worn contact

lenses For purposes of the present specification, such an amount is referred to as "an

amount effective to clean the lens " The amount of liquid enzyme cleaning composition utilized in particular embodiments of the present invention may vary, depending on various

factors, such as the purity 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)

The liquid enzyme compositions of the present invention must be formulated to

provide storage stability and antimicrobial preservation suitable for multiple use dispensing,

and must provide effective enzymatic activity to breakdown and hence remove

proteinaceous, sebaceous, and other foreign deposits on the contact lens The liquid

enzyme compositions must not contribute to the adverse effects of deposit formation on the

lens, ocular irritation, or immunogenicity from continuous use Additionally, when combined with a disinfecting solution containing an antimicrobial agent which is adversely

affected by high ionic strength such as polyquaternium- 1 , the compositions of the present

invention must have little or no impact on the ionic strength ofthe disinfecting solution

As used in the present specification, the term "low osmolality effect" is defined as an increase in osmolality of about 0-50 milliOsmoles/kg when 1 to 2 drops of the liquid

enzyme composition is added to the diluent solution Osmolality can be an indirect

measure of the ionic strength of a solution It is convenient to utilize osmolality measurements to define acceptable tonicity ranges for disinfecting solutions As indicated

above, the antimicrobial 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 excipients.

The ionic strength or tonicity of the cleaning and disinfecting solution of the present

invention has been found to be an important factor More specifically, polymeric

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 Such low ionic strengths generally

correspond to osmolalities in the range of hypotonic to isotonic, and more preferably in the range of 150 to 350 milliOsmoles per kilogram (mOs/kg) A range of 200 to 300 mOs/kg

being is particularly preferred and a tonicity of about 220 mOs/kg is most preferred.

The methods of the present invention utilize a disinfecting solution containing an

antimicrobial agent Antimicrobial agents can be oxidative, such as hydrogen peroxide, or non-oxidative polymeric antimicrobial agents which derive their antimicrobial activity

through a chemical or physicochemical interaction with the organisms As used in the

present specification, the term "polymeric antimicrobial agent" refers to any nitrogen-

containing polymer or co-polymer which has antimicrobial activity Preferred polymeric antimicrobial agents include polymeric quaternary ammonium compounds, such as disclosed in U.S Patents Nos 3,931,319 (Green, et al ), 4,026,945 (Green, et al.) and

4,615,882 (Stockel, et al ) and the biguanides, as described below The entire contents of the foregoing publications are hereby incorporated in the present specification by reference

Other antimicrobial agents suitable in the methods of the present invention include benzalkonium halides, and biguanides such as salts of alexidine, salts of chlorhexidine,

hexamethylene biguanides and their polymers The polymeric antimicrobial agents used herein are preferably employed in the absence of mercury-containing compounds such as

thimerosal The salts of alexidine and chlorhexidine can be either organic or inorganic and are typically gluconates, nitrates, acetates, phosphates, sulphates, halides and the like.

Particularly preferred are polymeric quaternary ammonium compounds of the structure

wherein

Ri and R can be the same or different and are selected from N ⁺ (CH ₂ CH ₂ OH),X ^" , N(CH ₃ ) ₂ or OH,

X is a pharmaceutically acceptable anion, preferably chloride, and n = integer from 1 to 50

The most preferred compounds of this structure is polyquaternium- 1, which is also known

Onamer M™ (registered trademark of Onyx Chemical Corporation) or as Polyquad®

(registered trademark of Alcon Laboratories, Ine ). Polyquaternium- 1 is a mixture of the above referenced compounds, wherein X is chloride and Ri, R ₂ and n are as defined above

The above-described antimicrobial 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 antimicrobially effective amount " The amount of antimicrobial 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 antimicrobial 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 000001% to about 0 01% by weight of one or more of the above-described

antimicrobial 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 3 to 3 0 % to disinfect the lens Methods utilizing an oxidative disinfecting system are

described in United States Patent No. Re 32,672 (Huth, et al ) the entire contents of which,

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

antimicrobial agents, such as suitable buffering agents, chelating and/or sequestering agents and tonicity adjusting agents The disinfecting solutions may also contain surfactants

The tonicity adjusting agents, which may be a component of the disinfecting

solution and may optionally be incorporated into the liquid enzyme composition, are

utilized to adjust the osmotic value of the final cleaning and disinfecting solution to more closely resemble that of human tears Suitable tonicity adjusting agents include, but are not

limited to, sodium and potassium chloride, dextrose, calcium and magnesium chloride, the

buffering agents listed above are individually used in amounts ranging from about 0.01 to

2.5% (w/v) and preferably, from about 0.5 to about 1.5% (w/v).

Suitable surfactants can be either cationic, anionic, nonionic or amphoteric

Preferred surfactants are neutral or nonionic surfactants which may be present in amounts up to 5% (w/v) Examples of suitable surfactants include, but are not limited to,

polyethylene glycol esters of fatty acids, poly oxy propylene ethers of Cι ₂ -d ₈ alkanes and polyoxyethylene-polyoxypropylene block copolymers of ethylene diamine (i.e. poloxamine)

Examples of preferred chelating agents include ethylenediaminetetraacetic acid

(EDTA) and its salts (e.g., disodium) which are normally employed in amounts from about

0.025 to about 2.0% (w/v)

The methods of the present invention will typically involve adding a small amount

of a liquid enzyme composition of the present invention to about 2 to 10 mL of disinfecting

solution, placing the soiled lens into the enzyme/disinfectant solution, and soaking the lens

for a period of time effective to clean and disinfect the lens The small amount of liquid enzyme composition can range due to various applications and the amount of disinfecting

solution used, but generally it is about 1 to 2 drops The soiled lens can be placed in the disinfecting solution either before or after the addition of the liquid enzyme composition

Optionally, the contact lenses are first rubbed with a non-enzymatic daily surfactant cleaner prior to immersion in the 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 following examples are presented to illustrate further, various aspects of the

present invention, but are not intended to limit the scope ofthe invention in any respect.

Example 1

A preferred enzyme composition ofthe present invention, and a suitable disinfecting

solution that may be used in combination with that composition, are described below

A Subtilisin Tablet Composition

The following enzyme composition represents a preferred embodiment of the

present invention

Ingredient mg/50 mg Tablet

Succinylated-Subtilisin 0 1 - 0.5

Citric Acid 5 95

Sodium Bicarbonate 13 135

Povidone (K 29-32) 0 415

Polyethylene Glycol (3350) 0 75

Compressible Sugar QS

Alcohol QS*

* evaporated during processing

The above ingredients are combined and formed into tablets of appropriate size and

hardness, according to methods known to those skilled in the art

This tablet may also be formulated with a seal coating and/or a delayed release

coating to provide for a delay in dissolution of up to about 2 hours

B Disinfecting Solution

The following formulation represents a preferred disinfecting solution

Ingredient w/v (%)

Polyquaternium- 1 0 001 + 10% excess

Sodium chloride 0 48

Disodium Edetate 0 05

Citric acid monohydrate 0 021

Sodium citrate dihydrate 0 56

Purified water QS

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

The method of isoelectric focusing was performed to determine the pl of enzymes

The method of Bio-Rad, as described in Bio-Rad's Model 1 1 1 Mini IEF Cell Instruction Manual, was followed

Briefly, a 1 to 2 μl aliquot (5 mg ml) of one or more proteases to be determined for

pl or control proteins of known pis were applied to a polyacrylamide gel slab containing

carrier ampholytes (pH ranging from 3.0 to 10.0) Electrofocusing separation of the

proteases in the gel was then carried out with a mini isoelectrofocusing cell (Model III, Bio-Rad Laboratories, Hercules, CA) according to the instruction manual provided by the

vender After staining the gel with a dye (Coomassie Brilliant Blue R-250), the protein

bands displayed on the gel slab were identified The pi of each protease was determined based on a calibration curve (pH versus migration distance) established with the control

proteins (IEF Standards, a mixture of nine natural proteins with known isoelectric points ranging from 4 45 to 9 60, Bio-Rad) The data of some representative proteases of the

present invention, some proteases outside the scope of the present invention and the control proteins is presented in Table 1 below.

Table 1

Migration distance on the gel (mm)

As illustrated in Table 1, succmylation and acylation successfully lowered the pl of

subtilisin and trypsin down within the pi range ofthe present invention

The invention m 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