NZ230910A - Composition and method for cleaning contact lenses using solution of peroxide and proteolytic enzyme - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £30910 c ,."" ; . .. pu?r -3-BCr cS.CaC.I.^/cr' '• Q, Aui L.a/JS, Lc.iic'l /..; !1'- ,lP ' jj ftPR Stt » L • *' • r'.- |-|fH%.--' Under the provisions of Regu lation 23 (1) the ..

NEW ZEALAND patents act. i95j Specification h^.s been ante-dated to (Xvg.kHX.... 19 $fc.. divided out of no. 22 22 nUcust 198b Date COMPLETE SPECIFICATION Ir.i'.id' 3 METHOD AND COMPOSITION FOR THE SIMULTANEOUS CLEANING AND DISINFECTING OK CONTACT LENSES X/We, allergan inc., a corporation organized under the laws of the State of Delaware, one of the United States of America, of 2525 Dupont Drive, Irvine, California 92715, United States of America, hereby declare the invention for which X / we pray tn.vt a patent may be granted to CCHK/us, and the method by which it is to be performed, to be particularly described in and by the following statement: - - 1 - (followed by Page 1A) - !A 23C?;o METHOD AND COMPOSITION FOR THE SIMULTANEOUS CLEANING AND DISINFECTING OF CONTACT LENSF.S Background This invention relates to a composition for cleaning and disinfecting contact lenses. More specifically, this invention covers a solution containing a mixture or peroxide and peroxidc-active enzymes, particularly proteolytic enzymes suitable for use in the sitnuitaneous cleaning and disir.f ectmy of contact lenses.

The invention also provides a metnod for simultaneously cleaning and disinfecting contact iensos navmc a hydrophilic surface.

Related Art The evolution of contact lenses from glass to the present extended wear lenses based on hydrophilic polymeric materials has provided a shifting and changing need for new and more effective means for cleaning and disinfecting such lens materials to ma intain opt clarity, wea rab i1i ty and p revent the transfer of v infectious agents into the eve. < , * 0 J4/v/o~ n f Glass and the early polymers such as \~? V* polymethylmethacrylate (PMMA) lenses could be re^i.^yvjj, cleaned by manual means using detergent bicause of their rigidity and hydrophobic character. While such materials are, to a certain degree, vetted by the naturally W; occurring aqueous layer on the eye and tears, they are lipophilic to a degree such that all soils, with the possible exception of lipids, are readily removed by manual cleaning with detergents. Hydrophilic materials, bo910G 165C2 230910 particularly polypeptides and enzymes such as lysozyme do not adhece significantly to these materials and are readily removed by cleaning wich surfactants and detergents.

Glass and PMMA based contact lenses are also readily disinfected by detergent cleaning means. Mechanical cleaning processes readily remove adhered infectious materials. Secondly, since these types of materials are non-porous, chemical disinfectants can be included in storage and cleaning solutions without absorption of the disinfectant into the lens and leaching of this disinfectant into the eye during wear. Thus, there is minimal concern about the physical removal of infectious agents and the maintaining of sterility by chemical means during storage and in maintaining the sterility of cleaning, wetting and storing solutions.

Advances in polymer technology have provided significant increases in wearer comfort and eye health, but have resulted in novel problems for cleaning and disinfecting such materials.

A lens is most comfortable on the eye when the surface is wettable by eye fluid and tear solution. In all contact lens polymers now in use, except for the PMMA lenses, the lens surface is naturally hydrophilic or treated to make it hydrophilic. This is achieved by means of multiple negative charges, usually carboxylate in form, and neutral groups which provide a hydrophilic environment readily wetted by the fluid layer covering the cornea.

Such negatively charged hydrophilic surfaces are present not only on the hydrogel lenses but also on more rigid lenses such as the organosi1oxane-methacrylate lenses (Polycon®) and silicone elastomer based lenses. In this latter category, the silicone elastomer lenses, the hydrophobic surface is coated or otherwise treated to render the surface hydrophilic.

Proteinaceous materials adsorbs to the hydrophilic b6910G L o 5 02 230910 3- lens surface during day-co-day wear. On all but purely PMMA lenses, Che adsorption is so strong that even with lenses such as the rigid po1ys i1oxa ne/me t hy1 me t hac ry La te copolymers, manual detergent cleaning methods do not adequately remove this accretion. So-called hydrogel Lenses, those materials prepared from hydroxyethylmethacrylate, hydroxyethylmethylmethacrylate, viny1pyrro1idone and glycerolmethacry1 ate monomers and methaccylic acid or acid esters, and which absorb a significant amount of water, i.e., 35-80 percent water, are so fragile that mechanical cleaning means is not a practical way of removing soilant, particularly the strongly absorbed proteinaceous materials.

The result is that over time, the buildup of such materials can result in wearer discomforts and, more importantly, interfere with the optical characteristics of the lenses, particularly reduced light transmission and increased light detraction. Also, protein buildup results in eye irritation, loss of visual acuity, lens damage and in certain instances there may result a condition called giant papillary conjunctivitis.

Research has determined that the primary source of this protein build-up is the lysozyme enzyme.

Additionally there may be lipoproteins and mucopolysaccharides adsorbed onto the lens surface, but proteins per se, particularly lysozyme materials are the major source of lens protein accretions. These enzymes, along with minor amounts of similar proteins, lipoproteins and mucopolysaccharides accumulate on the surface of hydrophilic lens materials.

The only safe and effective means found to date for removing this accretion is the use of enzymes, whose hydrolytic activity reduce the proteinaceous materials to small, water soluble subunits. Particularly useful are proteolytic enzymes, proteases, which hydrolyze amide bonds to break proteins down into amino acids and very b6 910G 16 5 0 2 230910 small polypeptides. These protein fragments are generally water soluble and thus are easily solubilized by the surrounding aqueous environment. U.S. Patent No. 3,910,296 discloses the use of proteases for cleaning contact lenses. See also U.S. patent No. 4,285,738. Enzymes with lipolytic and or mucolytic activity are also of use in discrete amounts with proteolytic enzymes for lens cleaning.

A second problem with gas permeable contact lenses, especially the hydrogel or high-water contact lenses made from HEMA, VP and GMA monomers, are concerns with disinfecting and maintaining the sterility of the lenses and lens storage solutions.

A number of methods have been devised for disinfecting lenses, including the use of high temperature, sterile saline solution washes and chemicals, e.g., antimicrobial drugs or oxidation processes.

Heat has been effective to a substantial degree but has the drawbacks of ma king additional cleaning mo re difficult, i.e., denaturization of protein and the solidification of protein and other deposits on the lenses.

Sterile saline can be used to clean and soak lenses. Such solutions are not always sterile though as certain microbes can live in a saline environment and spores are not totally inactivated by sterile saline solutions.

In the chemical means category, the use of so-called drugs, heavy metal-based antimicrobiaIs such as thimerisol and trialkylammonium halides and compounds such as benzylalkonium chloride or similar compounds, have the potential problem of wearer discomfort if used incorrectly. The characteristics of such drugs which make them good microbios ides, also carry the possible phenomena of eye irritability. This phenomena is particularly present with the hydrogel type lens materials since the drug accumulates in the lens and is then released onto the eye during wear. Such drugs may cause eye discomfort for b6910G 16502 2309 10 some people, sufficient to cause them to seek alternative means foe sterilizing lenses.

In response to the problems with maintaining sterility with drugs, heat and saline, the use of oxidants has become an area of substantial interest for disinfecting contact lenses. Several two and one step systems based on pecoxides have been developed for disinfecting contact lenses. One system is illustrated by U.S. patent No. 3,912,451 issued to C. Oaglia. Another is 4,473,550 issued to Kosenbaum, et al.

It has now been found that contact lenses may be simultaneously cleaned and disinfected by combining in one solution a peroxide for disinfecting and a peroxide-active enzyme for cleaning, particularly a peroxide-active proteolytic enzyme. Surprisingly, there is an increase in the effect of each individual component when presented in combination. That is, proteinaceous material removal is potentiated several fold by the presence of peroxide and the disinfecting rate is potentiated when the peroxide-active enzyme is present. The total result is that in one step, contact lenses can now be cleaned and sterilized more effectively than by independent use of the two components.

Peroxides and proteases have been combined in laundry detergents and for cleaning dentures. For example, U.S. Patent 3,732,170 relates to a biological cleaning composition containing an enzyme and a source of peroxide, particularly an alkali-metal monopersulfate triple salt. The essence of this invention is a process for cleaning "proteinic" blood stains from a material, a laundry aid. This combination is noted to be formulated preferentially with an anionic detergent.

As another example, U.S. patent 4,155,868 recites a water soluble, effervescent denture cleanser tablet containing an enzyme and an active oxygen compound. The essence of this invention is the formulation of a tablet b6910G 16502 - <5 - C L> "> 7 r o * ^ L . . u in such a manner as co prevent the premature inactivacion of the enzyme by c he oxidizing agent during storage.

Sodium perborate and enzymes are Known components of modern laundry detergents. A review of this art is given by OLdenroth. O. in the German publication F e_t_te' Se i f e n ' Anstrichmittel , 1970 (72(7)), 532-7.

This article indicates that the removal of denatured egg yolk from fabric is effected by bacterial proteases, but in the presence of perborates, the effectiveness of the proteases was decreased.

None of these disclosures teaches or contemplates tne use of such compositions for cleaning and disinfecting contact lenses or the enhancement effect one component has on the activity of the other.

SUMMARY OF THE INVENTION In one aspect, this invention relates to an aqueous composition for simultaneous cleaning and disinfecting a contact lens, particularly one having a hydrophilic surface, which composition comprises a disinfecting amount of peroxide and an amount of peroxide-active proteolytic enzyme effective to remove substantially all protein accretions and to disinfect saia lens.

In a second aspect, the invention provides a method for simultaneously cleaning and disinfecting contact lenses having a hydrophilic surface which method comprises contacting a lens having a hydrophilic surface with a solution comprising a disinfecting Jount of peroxide and an effective amount of peroxide-active roteolytic enzyme for a time sufficient to remove substantially ail protein accretions and to disinfect the lens. nil \\~23 JAN 1991 ^ /' SPECIFIC EMBODIMENTS The concept of combining an enzyme and peroxide, to effect disinfecting and cleaning in one step can be applied to proteolytic, lipolytic and mucolytic enzymes, individually or in combination.

A peroxide-active enzyme is any enzyme having measurable activity at 3% (w/v) hydrogen peroxide in aqueous solution at standard temperature and pressure as determined by such colorimetric assays as the Azocoll method, Tomarelli, R.M., et al., J. Lab. Clin. Med., 34, 428 (1949), or the dimethyl casein method for determining X c e O' * 2 3 0 9 10 pcoteolytic activity as described by Yaun Lin, et a 1 . . J• Biol. Chem., 2 4 4: (4) 7 89-793, ( 19 6 9 ) .

Enzymes may be derived from any plant or animal source, including microbial and mammalian sources. They may be neutral, acidic or alkaline enzymes.

A proteolytic enzyme will have in part or in total the capacity to hydrolyze peptide amije bonds. Such enzymes may also have some inherent lipolytic and/or amylolytic activity associated with the proteolytic activity.

Preferred proteolytic en-iymos are those which are substantially free of sulfhydryl groups or disulfide bonds, whose presence may react with the active oxygen to the detriment of bo;, h the activity of the active oxygen and which may result in the untimely inaetlvatlon of the enzyme. MetaIlo- proteases, those enzymes which contain a divalent metal ion such as calcium, magnesium or zinc bound to the protein, may also be used.

A more preferred 'jroup of proteolytic enzymes are the serine proteases, particularly those derived from Bac i 1 lus and Streptomyces bacteria and Asperqi1lus molds. Within this grouping, the more preferred enzymes are the Bacillus derived alkaline proteases genetically called subtilisin enzymes. Reference is made to Keay, L., Moser, P.W. and Wildi, B. S.. "Proteases of the Genus Bac i 1 lus. II alkaline Proteases." Biotechnology and Bioenqineerinq. Vol. XII. pp 213-249 (1970) and Keay, L. and Moser, P.W., "Differentiation of Alkaline Proteases form Bacillus Species" Biochemical and Biophysical Research Comm., Vol 34. NO. 5. pp 600-604, (1969).

The subtilisin enzymes are broken down into two sub-classes, subtilisin A and subtilisin B. In the subtilisin A grouping are enzymes derived from such species as B. subtilis, B. 1icheniformis and B. pumilis. Organisms in this sub-class produce little or no neutral protease or amylase. The subtilisin B sub-class is made up of enzymes from such organisms as B. subtilis, B. b69 10G 16502 m 230910 ^ subt i I is va r . amy losaccha r i t icus, B. amyloliquefaciens and B. subt i I is NRRL B 3 4 11■ These organisms produce neutral proteases and amylases on a level about comparable to their alkaline protease production.

In addition other preferred enzymes are, for example, pancreatin, trypsin, collaginase, keratinase, carboxylase, aminopeptidase, elastase, and aspergiIlo-peptidase A and B, pronase E (from S. q r i seus) and dispase (from Bac iIlus po1ymyxa).

The identification, separation and purification of i^n^ymes is an old arc. Many identification and isolation techniques exist in the general scientific Literature tor the isolation of enzymes, including those enzymes having proteolytic and mixed proteoIytic/a my 1o1ytic or proteolytic/lipolytic activity. The peroxide stable enzymes contemplated by this invention can be readily obtained by known techniques from plant, animal or microbial sources.

Wich the advent of recombinant DNA cechniques, ic is ancicipaced thac new sources and types of peroxide stable proteolytic enzymes will become available. Such enzymes should be considered to fall wichin the scope of this invention so long as chey meet Che criteria for stability and activicy sec forch herein.

An effeccive amount of enzyme is to be used in Che practice of chis invencion. Such amount will be thac amount which effects removal in a reasonable time (for example overnight) of substantially all proteinaceous deposits from a lens due to normal wear. This standard is stated with reference co concact lens wearers with a hiscory of normal paccern of procein accrecion, noc che very small group who may at one time or another have a significantly increased rate of procein deposic such that cleaning is recommended every two or three days. b6 910G 16 502 230 9 1 0 The -amount of enzyme esquired to make an effective cleaner wilL depend on several factors, including the inherent activity of the enzyme, the full extent of its synergistic interaction with the peroxide among several factors stand out as pertinent considerations.

As a basic yardstick, the working solution should contain sufficient enzyme to provide between about O.OOOl to 0.5 Anson units of activity per ml of solution, preferably between about 0.0003 and 0.05 Anson units, per single lens treatment. Higher or lower amounts may be u:;ed. However, enzyme concent ra l ions lower than these stated here may clean lenses buc wilL take so long as io be practically not useful. Solutions with higher amounts of enzyme should effect more rapid cleaning but may involve amounts of material which are too sizeable for practical handling purposes.

In weight/vo1ume terms, since enzyme preparations are seldom pure, it is expected that the enzyme source will be used in amounts between about 0.003 to 15% of the final working solution. The precise amount will vary with the purity of the enzyme and will need to be finally determined on a lot-by-lot basis.

Enzyme activity is pH dependent so for any given enzyme, there will be a particular pH range in which that enzyme will function best. The determination of such range can readily be done by known techniques. It is preferred to manipulate the working solution to an optimum pH range for a given enzyme but such is not an absolute r equ i rement.

The peroxide source may be any one or more compounds which gives active oxygen in solution. Examples of such compounds include hydrogen peroxide and its alkali metal salts, perborate salts, particularly monohydrates and tetrahydrates, persulfates salts, salts of carbonate peroxide, diperisophtha 1ic acid, peroxydiphosphate salts and aluminum aminohydroperoxide salts. Hydrogen peroxide b 6 910G 1 f) 5 0 2 • 230910 ^ and Che alkali metal sales of perborates and persu1 fates, particularly the sodium and potassium jalts, are most preferred.

A disinfecting amount of peroxide means such amount as will reduce the microbial burden by one log in three hours. More preferably, the peroxide concentration will be such that the microbial load is reduced by one log order in one hour. Most preferred are those peroxide concentrations which will reduce the microbial load by one log unit in 10 minutes or less.

A single peroxide concentration can not be made to appLy to all peroxides as the percentage of active oxygen varies substantia 1ly between peroxides.

For hydrogen peroxide, on the lower side, a 0.5% weight/volume concentrat lon will meet the first criteria of the preceding paragraph under most circumstances. It Is preferred to use 1.0% to 2.0% peroxide, which concentrations reduce the disinfecting and cleaning time over that of the 0.5% peroxide solution. It Is most preferred to use a 3% hydrogen peroxide solution though an amount of 10% may be used. No upper limit placed on the amount of hydrogen peroxide which can be used in this Invention except as limited by Che requiremenc that the enzyme retains proteolytic activity.

Where other peroxides are concerned, the only limieacion placed on cheir concentration is that they exhib i t synerg is t ic activity in combination with the peroxide-stable enzyme at a given concent rat ion with regard to cleaning and disinfeccing. For example, ic has been found thac sodium perborace at concentrations of 0.02% weighc/volume will pocenciace Che enzymacic removal of procein from concact lenses. The appropriace concencracions of any given peroxide is a matter which can be readily determined through routine testing.

Increasing the pH of peroxide/enzyme solutions has been found to have a material affect on the disinfecting b6 910G 1.6 5 02 '30910 -li- capacity of these solutions. At pH 5.22, the D value of a 3% hydrogen peroxide solution measured against A. n iqer was 8.0 4 versus 3.5 7 at pH 7.3 2 and 1.7 9 at pH 8.2 2 and 9.23. Accordingly, the most preferred pH range is 6 - 10 for these solutions, particularly 7.5 - 9.0. Correspondingly, it is preferred to use peroxide-stab1e enzymes which are active at a neutral or alkaline pH.

Additional materials may be added to tablets or liquid solutions of the enzyme and/or peroxide formuLations. For example, tonicity agents, effervescing agents. stabilizers, binders, buffering agents, enzyme co- factors, disulfide bond reducing agents such as water-soluble meLcaptans and dithionites and the like, agents to inactivate residual peroxide and the like.

Formulation of peroxide and enzyme may require stabilizing agents to prevent premature inactivation of both components. For solutions, it may be necessary or appropriate to add materials to stabilize the peroxide, particularly against meta1-induced catalytic degradation. It may also be appropriate to add buffering agents to these solutions to maintain pH within a particular given range. Salts or other materials such as polyalcohols or the like may be added to modify the tonic value of such solutions.

In tablets or powders, the same considerations may be in effect in the sense of adding in salts, buffers and stabilizers so that when the tablet is dissolved, the appropriate pH and tonic value will be present. With tablets and powders it may also be appropriate to add effervescing agents. In addition, binders, lubricants for tableting purposes and any other excipients normally used for producing powders, tablets and the like, may be incorporated into such formulations. Indicators, colorants which indicate the presence or absence of peroxides, may also be incorporated into these formula t ions. b6 910G 16502 ^09 10 To practice the invention, a solution ot" peroxide and enzyme is prepared and the lenses contacted with this solution, preferably by being immersed in the soLution. The lenses will be left in contact with such solution long enough so that substantially all protein is removed from the lenses surfaces and the lenses are disinfected.

The method or sequence of combining the essential components to make up the solution which contacts the lenses wiLl vary with the physical characteristics of the component employed; but order of addition is not critical to the practice of this invention. For example, if hydrogen peroxide is used it will not be reasonably possible to formulate a tablet or powder of all the components. Thus when hydcogen peroxide is the peroxide source, it will be necessary to mix enzyme and other dry ingredients with aqueous peroxide. It is most convenient to formulate the enzyme and other dry components as a powder or tablet and to dissolve such material in a peroxide solution, then introduce the lenses into this solution. The lenses could already be in the peroxide solution when the enzyme is introduced but practical considerations make the first method the preferred one.

There is no particularly preferred form for the manufacturing of these materials. The two essential components may be formulated as separate components in dry or aqueous form. They may be combined in a single tablet or powder or one may be in dry form while the other is manufactured as an aqueous solution.

The final form will depend in part upon the type of peroxide source used in the formulation. It is anticipated that the powder or tablet form of this invention could also be in an effervescent form to enhance tablet break-up and to enhance the solubility rate of the ingredients. If a granular peroxide is employed, it will be possible to prepare powders and/or tablets from the several components of this invention. Where the peroxide b6 910G 16502 3 0 9 1 0 L 3 - is in soLution form, it may be necessary to provide the enzyme from a second source in order to prevent Long-term degradation of the enzyme.

Other energy input may be employed to potentiate the solution's cleaning and disinfecting effect. For example, ultrasonic devices are known to potentiate the speed at which proteases work in such circumstances as the cleaning of contact lenses. Heat, depending on the amount nnd timing may also have a salutatory effect on cleaning and disinfecting rates.

The practice of this invention is not to be limited temperature-wise except by those temperature extremes which would substantially inactivate the proteolytic capability of the enzymes employed before useful hydrolysis of protein accretions is effected. Enzymatic activity is a function of temperature, some enzymes being considerably more labile than others to temperature extremes, par t icular i.y temperature increases. Other enzymes are heat stable and remain significantly active at temperatures of 70°C or higher. Other enzymes retain substantial amounts of activity at or just above the freezing temperature of water. While the preferred temperature range for practicing this invention is between 20 and 37°C, particularly about 22-25°C, it may be possible to practice this invention with certain peroxide-active enzymes in the temperature range between about 5°C to 100°C.

One embodiment of this invention is to prepare a room temperature solution of enzyme and peroxide and to place this solution, along with the contact lens, in a contact lens heat disinfecting unit and run the unit through its the normal heat cycle. This is but one example of the heat variable aspects of this invention.

It is also contemplated that certain components may be separately prepared in a manner to effect the timed release of that component or to prevent interaction of b6910G 16502 "3 0 9 1 0 component I wich component 2 during cablet and powder preparacion and subsequent storage. For example, in certain instances it may be appropriate to separately prepare the peroxide and the enzyme in a manner to prevent or reduce their interaction in a tableting process and upon subsequent storage thereafter.

In addition, solutions or powders may contain agents for detoxifying residual peroxide as part of the overall process of cleaning, disinfecting and ultimately the removal of rosidual peroxide. F.nzymes which catalyze the conversion of peroxides to oxygen and water can be included in these formulations to remove residual peroxide in a tit. ic i pa c i on of inserting the lens back into the eye. For example catalases, oryanic enzymes which catalyze the degradation of peroxides, can be incorporated into tablets and powders, particularly in time-release form. AdditionaIly, metals such as the heavy metal transition elements which catalyse the conversion of peroxide to oxygen and water, can be included in a powder or tablet formulation, again preferably in some delayed release form to provide a method for reducing to a non-toxic level any residual peroxide remaining in the solution after a given time interval. The use of transition metal catalysts for decomposing peroxides in a contact lens disinfecting solution is disclosed in United States Patent 3,912,451, which information and technology is incorporated herein by reference as if set forth in full herein.

The following examples are set out to illustrate, but not limit, the scope of this invention.

Example 1 Comparative Cleaning Effects Twenty Hydrocurve* II 55% water lenses (Barnes-Hind, Inc. Sunnyvale, California, U.S.A.) were coated with heat-denatured lysozyme by placing the lenses in a phosphate buffered saline solution to which was then added bfi> lJ LOG 16 5 0 2 • 7 0 9 1 0 ^ sufficient lysozyme to make a 0.1% solution by weight. The lysozyme was from egg white. Individual vials were set up to contain 5 ml of the lysozyme solution and one fully hydcated lens. Vials were then heated for about 30 minutes at about 95° C. The lens was then removed, and aftec being cooled, was rinsed with distilled water and viewed to determine the type of lysozyme accretion.

Deposit class ification: First the lens was wetted with normaL saline, rubbed between thumb and finger, then grasped by the edge with plastic tweezers and rinsed with saline again. The anterior surface (convex surface) of the lens was viewed under the microscpoe at 100X. A film or deposit detected under these conditions was classified according to the percentage of surface which was covered by t he film.

After the treatment described in the first paragraph, all lenses were found to have 100% of their anterior surface covered by tnLn-film protein deposits.

These lenses were then treated with solutions based on peroxide and the following enzyme formulations: Papain Tablet Ingredient Percentage (w/w) Sodium Borate, Dihydrate 13.03% Sodium Carbonate 21.25% Polyethylene glycol 3350 2.74% Papa in 6.28% Tartaric Acid 13.71% L-Cysteine HCL 6.86% EDTA 5.04% Sodium Chloride 30.64% Subtilisin A Tablet Ingred ient Percentage (w/w) Sorbito 1 29.99% N-acetyleysteine 22.49% Sodium Carbonate 38.98% Polyethylene glycol 3350 3.00% Subt ilisin A 0.30% Tartaric Acid 5.24% The subtilisin A was obtained from Novo Industries of b6910G 16 5 0 2 :0 9 10 De nma r k .

The lenses were divided into four groups of five. One group was created with 3% hydrogen peroxide. A second group was treated with the Subtilisin A containing formulation (133.4 mg. 0.4 mg subtilisin A) in 10 ml of a commercial saline product (Lensrins® made and sold by Allergan Pharmaceuticals. Inc.). A third group was treated with the Subtilisin A tablet dissolved in 10 ml of 3% hydcogen peroxide and the fourth gcoup was treated with a 3% hydrogen peroxide (10 ml) containing one papain enzyme tablet (146.8 mg).

The lenses wece aLLowed to soak for 3.5 hours. Then each group of lenses was treated appropriately to remove test solution and examined under a microscope to determine the extent of protein removal. The percent surface cleaned equaled the percent of the surface not covered by a protein film at 100X. The results are presented below.

Results were as tallows: LENS 3% Hydrogen Peroxide* ^SURFACE CLEANED A1 A2 A3 A4 A5 0 1 0 0 1 SUBTILISIN A/Saline ^SURFACE LENS CLEANED SUBTILISIN A/3% H7O7* %SURFACE LENS CLEANED B1 B2 B3 B4 B5 20 25 15 30 CI C 2 C 3 C 4 C 5 50 60 70 60 50 b6910G 16502 2309 10 l PAPAIN/3% HoO-, LENS • %SUHFACE CLEANED EL E2 E 3 E4 E5 0 0 0 0 0 *Oxysept® - 3% Hydcogen peroxide solution marketed by Allergan Pharmaceuti c a Is, Inc.

While the hydrogen peroxide and papa in/hydrogen peroxide cleaning activity was essentially nil. subtilisin in combination with 3% hydcogen peroxide cleaned between 50 and 70% of the contact lens surface area. Secondly, subtilisin A alone without peroxide cleaned between 15 and 15 30% of the lens surface while in comparison, subtilisin A with 3% peroxide cleaned between 50 and 70% of the lens surface. Subtilisin A and peroxide was approximately twice as effective ip its cleaning capacity in comparison with subtilisin without peroxide.

Fifteen Hydrocurve II® lenses (Barnes-Hind) were exposed to lysozyme and the presence of Type IV protein 25 accretion confirmed as described in Example 1.

Five lenses each were soaked for eight hours in the following solutions: 3% hydrogen peroxide (Oxysept 1 produced by Allergan Pharmaceuticals, Inc.); commercially available, pancreatin containing enzyme tablet (Opti-Zyrae® tab 30 Opti-Zyme® tablets dissolved in 10 ml of saline solution (Boi1-'n-Soak®, a normal saline solution produced by Alcon); and a solution of pancreatin enzyme (Opti-Zyme®) . two tablets, in 10 ml of 3% hydrogen peroxide (Oxysept® 1).

Following an 8 hour soak, lenses were treated to b6 910G 16502 EXAMPLE 2 Peroxide/Enzyme Activity 2309 10 remove cesidual soaking solution and the percentage of protein removal determined as described in Example I. The results were as follows: 31 Hydrogen Peroxide %Sur face Lens Cleaned Al 0 A 2 0 A3 0 A 4 0 A 5 0 Panccoat in/Per oxide Panereatin/Nocma1 SaLine Solut ion Lens B1 B2 B 3 B4 B 5 tSurface C1ea ned 90 85 85 90 80 Lens ci C2 C3 C 4 C5 %Su r face C leaned 0 0 o 0 0 The combination of the pancreatin-containing enzyme tablet and 3% peroxide effected substantial cleaning while the peroxide alone and the enzyme alone had no detectable protein removing effect in the 8 hours of soaking time used here.

EXAMPLE 3 Effect of Peroxide Concentration Hydrocurve* lenses were coated with lysozyme as per Example 1. The subtilisin tablet formulation used here was the same as in Example 1 except that the N-acetyleysteine was removed. Five different levels of hydrogen peroxide were used, beginning at a concentration Of 0.5% by weight/volume. The control was the tablet without peroxide with the tonicity value adjusted to approximately that of the 0.5% peroxide/enzyme solution b69 10G 16 502 23 0 9 1 0 with sodium chloride. The pH was adjusted to beLween about 9.0-9.03 in each solution with hydrochloric acid. Five lenses were treated foe three hours at room temperature with 10 ml of each solution. The amount of protein (percentage) removed from the lens surface is g i vi-?n in Tab 1 e I .

Table I Ef f e c ts of Peroxide Cone entration on Clo a n i n q Ff f i <; a cy h'n i yme Cone.

PH Ton ic i ty % peroxide Weight/vol l.ons Cleaning (s .E.) A 0 . 04 mg/ m I 9 . 025 3 18 mOsm/ kg 0 9 . 0 ( 5 • ) B 0 . 04 mg/m I 9 . 086 3 30 raOsm/ kg 0 . . 5 % 44 . 0 (8 . 9) C 0. 04 mg/ml 9. .016 390 mOsm / kg 1. . 0% 78 . 0 (2- 7) D 0 . 04 mg/m 1 9 . . 022 643 mOsm/kg 1. . 5 % 87 . 0 (2 . 7) E 0 . 04 mg/ml 9 . 023 796 mOsm/kg 2 . 0 % 94 . 0 (4 . 2) F 0 . 04 mg/ml 9 . 016 932 mOsm/ kg 2 . % 97 . 0 (2 . 7) (S.E.) means standard error Example 4 Evaluation of Antimicrobial Activity of Subtilisin in 3% Hydrogen Peroxide The effect of a tableted formulation containing subtilisin A (given in Example I) on the antimicrobial activity of hydrogen peroxide when dissolved in 3% hydrogen peroxide (Lensan A, Allergan Pharmaceuticals, Inc.) was tested against the panel of micro-organisms required by the U.S. FDA guidelines for testing contact lens solutions for disinfective efficacy. Standard culture methods, harvest and guantitative microbiological analysis techniques were used. The organisms used were S. marcescens. ATCC 14756 or 14041; S. aureus, ATCC 6538; P. aeruginosa, ATCC 9027 or 15442; E. coli, ATCC 8739 . C. albicans, ATCC 1023 1 and niger. ATCC 16404. A 133.4 b6910G 16502 ^ 10 230 9 1 0 - mg tablet of the subtiLisin A formulation (0.4 mg subtilisin/tablet) given in Example 1 was used.

The results of this study are given in Table I.

TABLE I COMPARISON OF EXTRAPOLATED D-VALUES*IN MINUTES Study I Study II 2% 1^2—2 3% H-,' ORGANISMS 3\_H2P2 + SUB. A 3 % H2O2 ^ SUB S. marcescens 2 . 5 1 . 7 3 . 1 . 3 S. aureaus 4 . O 3 . 0 4 . 0 2 . 0 p. aeruginosa 0 . 3 0. S 0. 3 0 . 1 E . <: o 1 i 2 . b 0 . 9 1 . 7 0 . 2 C. albicans 36.5 '.3.0 .0 9 . 0 A. niger 9 . 5 11.6 6 . 0 6 . 0 "D-value is the time required to reduce a microbial challenge of 5xl05ocganism per mi by 90% or 1 logarithm.

The control, an enzyme tablet in saline, showed no antimicrobial activity over a 24 hour period.

A second study similar in design and following the 20 same procedure as the first was performed. The results are also presented in Table I.

Table II lists the average kill rates for the data presented in Table I.

TABLE II AVERAGE KILL RATES (D-VALUES) IN MINUTES AT ROOM TEMPERATURE ORGANISMS 3% HO.. 3% H..O-./SUB. A S . marcescens 3 . 0 1.5 E . co 1 i 2 . L 0. 6 P . aeruginosa 0 . 3 0.3 S . aureus 4 . 0 2 . 5 C . aIbicans 26.0 11.0 A. niger 8 . 0 9 . 0 Since the lower the D value, the more effective the antimicrobia 1 activity, each of these studies demonstrates b6 910G 16502 230910 chat 3 % hydrogen peroxide and subtilisin A together are a substancia1ly more effective disinfecting composition than either of the two components acting separately.

Example 5 Tes ti ng of Preservative Efficacy Three panels of organisms, one based on the USP XXI panel, another soft contact lens paneL containing representative organisms required by the KDA for antimicrobial efficacy testing of contact lens disinfection products and a third "isolates" panel comprised of selected organisms which commonly are encountered as natural flora of either the human body or the environment and which may be deposited on contact lenses or become innoculated into contact lens solutions, were used in testing the differential between the extrapolated D-values of 3% hydrogen peroxide (Oxysept I, Allergan Pharmaceuticals. Inc.) with and without subtilisin A. The organisms tested are listed in the tables appended hereto.

The micro-organisms were prepared by standard microbiological techniques. Each sample was tested in duplicate. As a first step in the assay, 10ml of 3% hydrogen peroxide was pipetted into screw-cap test tubes. Into selected tubes was added one tablet of subtilisin A, whose composition is described in Example 1. The subti1isin-containing tubes were vortexed for approximately 2 minutes to dissolve the subtilisin tablet. Immediately the challenge organism was added to the tube. After a predetermined contact time interval, survivors were quantified in CFU/ml.

A D-value was calculated by extrapolation from kill curves using an aerobic plate count method. This method worked essentially as follows: An aliquot of test solution was removed immediately after the predetermined contact interval, divided in half and dispersed into two b6 910G 16 5 0 2 23 0 9 10 test cubes containing neutcalizec media. A serial ten-fold diLution of the neutcalizec media was prepared in a manner to compensate foe the expected level of cecovecy. For low level cecovecy, a small aliquot was tcansfecred directly onto a neutcalizec agac plate. Foe the othec thcee secial dilution tubes, an equal volume of sample was placed on neutcalizec agac plates. All plates wece incubated at 35- 37°C foe 2-7 days, oc longec if cequiced. Colony counts wece then cecocded and D-values calculated as follows: All plate counts foe each time interval wece avecaged. The avecaged data was plotted on a a emi- log gcaph papec with the numbecs of sucvivocs on the ocdinate and the contact time on the abscissa. The stacting point (inoculum level) was connected to the first point yielding less than 10 ocganisms pec ml by a stcaight line. The slope of this line extcapolated to zeco gives the D-value. This is othecwise cefecced to as "end-point analys is".

TABLE III Extcapolated Kill Rates (D-values) of 3% Hydcogen Pecoxide (Oxysept I) With and Without Subtilisin (1) Ocganism and ID# USP XXI Panel Wi thout Subtilisin With Subtilisin Sercatia maccescens. ATCC #14 75 6 Staphylococcus auceaus. ATCC #6538 Pseudomonas aeruginosa. ATCC #9027 Escherichia coli. ATCC #8739 Candida albicans, ATCC #10231 Aspergillus niger, ATCC #16404 1. 4 mi n. 1. . 0 min. 3 . 4 min. 2 . . 1 min. 3 . 2 min. 2 . . 6 min. 0 . 2 rain. 0 . . 2 min. 1. 0 min. 0 . . 3 min. . 0 rain. 13 . , 0 min. . 0 min. 8 . . 0 min. b6 910G 16502 230 9 1 0 ( 2 ) ^_Sof c Lens" Panel ( FDA) Se r ra t i a ma rcescens, ATCC #14 04 1 ScaphvLococcus epidecmidis. ATCC #17917 Pseudomonas aeruginosa, ATCC #154 4 2 Aspergillus fumigacus. ATCC #108 94 Cand ida a 1 b i cans. ATCC # 1023 1 (3) Va r ious Isolates 1. . 7 min. 1 . 5 min 0 , , 8 min. 1 . 5 min 0. , 4 in i n . 1 . . 0 min 0 . , 6 min. 0 . 3 min 13 . , 5 min. 2 . . 5 min . 0 min. 13 . . 0 m i n Klebsiella pneumoniae, ATCC #13883 1. . 1 hi i n. 0 . . 6 min Pseudomonas cepacia, ATCC #17765 0. , 4 min. 0 . . 2 min Proteus mirabilis, CSULB/VA 1. . 2 min. 1 . . 0 min 1. 3 min. 0 . . 9 m i n Pro teus vuIgar is, ATCC #17 313 0. 4 min. 0 . . 3 in i n Cand ida pa ra ps i lq_s i_s , PM 4 0 6 4 6 I . 0 min. 55 . . 0 min PeniciLlium sp. (AquaTar isolate II) 2 . min. 2 . . 1 min Examp1e 6 Comparative Enhancement of Peroxide Wich and Without_ Enzyme Comparative enhancement of the antimicrobia1 kill rates of various solutions of 3% hydrogen peroxide due to the addition of the subtilisin enzyme. The figures in Table IV represent the percentage of decrease in the D-value for a particular peroxide solution plus the subtilisin tablet of Example 1 over that of the particular peroxide solution alone. The AO-Sept system employed a heavy metal catalyst (platinum coated disc) in the vials to degrade peroxide as per U.S. patent 3,912,451.

Organism TABLE IV Lensan A (Data From Table II) Oxysept I (Data From Table III) AO-Sept Serratia marcescens 30 Escherichia co 1 i Pseudomonas aeruginosa Staphylococcus aureus Cand ida a lbicans Aspergillus niger 50% 71% 0 38% 58% 0% 29% 70% 0 28% 35% 20% 88% 90% 20% 60% 33% 32% These figures demonstrate that each of the 3% peroxide 35 solutions is a much more effective disinfectant when subtilisin A is present. The effect is particularly b6 910G 16 5 02 2309 10 m pronounced Ln the AO-Sept system.

Example 7 Effeet of Peroxide Concentration on Enzyme Activity 5 The enzymatic activity of the subtilisin A tablet described in Example 1 and trypsin was determined at different hydrogen peroxide concentrations using the Modified Azocoll method [Sigma Catalog]. Baker Chemical Company, 30% hydrogen peroxide was used. 10 Appropriate dilutions were made with a 0.02M borate buffer at about pH 8.4. AzocoLl substrate and trypsin were obtained from Sigma Corporation.

Peroxide was first diluted with buffer to the appropriate concentrations. One subtilisin enzyme tablet 15 was dissolved in 10ml of buffer to which had been added SOmg of Azocoll substrate. One ml of this solution was then added to each of the peroxide concentrations, the enzyme/substrate buffer solution being the control. After mixing, the reaction was run at room temperature for 2 20 minutes, then quenched with 2ml of 10% trichloroacetic acid, which precipitated the enzyme. Residual color measurements were measured at 520nm. Subtilisin results are given in Table IV, trypsin results in Table V.

TABLE IV Subtilisin Activity in Hydrogen Peroxide %_H202 OP 520 0 0 . 2 7 1 0 . 39 2 0 . 57 3 0 . 56 4 0 . 66 4 . 5 0 . 56 0 . 68 6 0 . 68 8 0 . 90 0 . 91 b6910G 16502 23 0 9 1 0 l TABLE V "Trypsin Activity in Hydcogen Peroxide % H7O7 OP 520 0 3 .5 . 6 •LOmg of trypsin powder were added to the H.,02 solution.

■ • Table IV indicates that subtilisin A is active in the A Azocoll assay throughout a broad range of peroxide concentrations. The activity at 30% peroxide is approximately the same as at the 8% concentration.

Enzyme activity for subtilisin A appears to be saturated at hydrogen peroxide concentrations between 2-6%. Table 15 V indicates that trypsin is active in hydrogen peroxide.

Example 7 Effects of Perborate on Enzyme Activity Hydcocurve II® lenses were coated with 20 heat-denatured lysozyme as per the procedure described in Example l. The following solutions based on subtilisin A (Novo Industries, Denmark) and sodium perborate were prepared to test the combined effects of perborate as a source of peroxide on the proteolytic activity of 25 subtilisin A. Solution A - 0.04 mg/ml subtilisin A, bicarbonate buffer to adjust the pH to 8.307; Solution B - 0.02% (w/v) sodium perborate, bicarbonate buffer, pH adjusted to 8.533; and Solution C - 0.04 mg/ml subtilisin A, 0.02% (w/v) sodium perborate, bicarbonate buffer, pH 30 adjusted to 8.532. Each treatment was done in a 10 ml volume.

Five protein coated lenses were soaked in each of these solutions (10 ml) for 3 hours at room temperature. All lenses were then rinsed and the amount of residual 35 protein determined. Table VI gives the average percentage of surface cleaned after these treatments. b6 910G 16502

Claims (31)

m 2309 10 -26- Table VI Comparative Cleaning of Enzyme With and Without Peroxide Average % 5 Solution Surface Cleaned A 9.0+5.6 B 0 C 30.0+12.2 10 15 20 25 30 35 b6 9 LOG 16502 m 2zc::Q WHAT WE CLAIM 15:

1. An aqueous composition for simultaneously c'ean mg and disinfecting a contact lens which composition comprises a disinfecting amount of peroxide and an amount of peroxice-active proteolytic enzyme effective to remove substantially all protein accretions and to disinfect said lens.

2. The composition of claim 1 wherein the proteolytic enzyme is present in an amount between 0.0001 and 0.5 Anson units per ml.

3. The composition of claim 1 or claim 2 wherein the peroxide is present in an amount of 0.027. by weight/volume or greater.

4. The composition of any one of claims 1 to 3 wherein the peroxide is hydrogen peroxide.

5. The composition of claim 4 wherein said hydrogen peroxide is present in an amount of between 0.02 and 10% by weight/volume.

6. The composition of claim 5 wherein said hydrogen peroxide is present in an amount of about 3% by weight/volume.

7. The composition of any one of claims 1 to 3 wherein the peroxide is a perborate, persulfate, percarbonate , diperisophthalic acid, peroxydiphosphate or aluminium aminohydroperoxide salt.

8. The composition of any one of claims 1 to 7 wherein the proteolytic enzyme is a subtilisin enzyme.

9. | The composition of claim 8 wherein the proteolytic enzyme is t HI : subti1i sin A. -3 JANlgyj fpjj o.,/ 10.

The composition of any one of claims 1 to 7 wherein the proteolytic enzyme is pancreatin or trypsin.

The composition of any one of claims 8 to 10 wherein the proteolytic enzyme is present in an amount between 0.0003 and 0.05 Anson units per ml . 23 .'J u U I) 1 U

12. The composition of any one of claims 1 to li buffered to a pH between 6 and 10.

13. The composition of claim 1 wherein the enzyme is present in an amount between 0.0001 and 0.5 Anson units per ml and the peroxide is hydrogen peroxide which is present in an amount between 0.05 and 10% by weight/volume.

14. The composition of claim 1 where the proteolytic enzyme is subtilisin, pancreatin or trypsin in an amount between 0.0003 to 0.05 Anson units per ml.

15. The composition of claim 14 which comprises about 2% hydrogen peroxide and 0.0012 Anson units per ml of subtilisin A.

16. The composition of claim 7 where the enzyme is present in an amount between 0.0001 and 0.5 Anson units per ml and the peroxide is present in an amount of 0.02% by weight/volume or greater.

17. The composition of claim 16 buffered to a pH between 6 and 10.

18. The composition of claim 17 where the proteolytic enzyme is subtilisin, pancreatin or trypsin present in an amount between 0.0003 and 0.05 Anson units per ml.

19. The composition of claim 18 where the peroxide is sodium perborate, potassium persulfate, sodium percarbonate, or sodium aluminium aminohydroperoxide and the enzyme is subtilisin A present in an amount of 0.0012 Anson units per ml. 20. r*yff An aqueous composition for the simultaneous cleaning and disinfecting of a contact lens having a hydrophilic surface which composition comprises a disinfecting amount of peroxide an amount of peroxide-active proteolytic enzyme effective to ove substantially all protein accretions and to disinfect s'aid lens.

The composition of claim 20 wherein said peroxide-active A ^•'proteolytic enzyme is a subtilisin enzyme.

L'uUvi i U

22. The composition of claim 2! 'wherein said subr i"! i j i n enzyme is subtilisin A.

23. The composition of claim 20 wherein jaio peroxide is hydrogen peroxide in an amount between 0.02 and 10°!, by weight/volume.

24. The composition of claim 23 wherein said hydrogen peroxide is present in an amount of about 3% by weight/volume.

25. The composition of claim 20 wherein said peroxide-active proteolytic enzyme is subtilisin A and said peroxide is hydrogen peroxide.

26. An aqueous composition as defined in claim 1 for simultaneously cleaning and disinfecting contact lenses substantially as herein described with reference to the Examples.

27. A method for simultaneously cleaning and disinfecting contact lenses having a hydrophilic surface which method comprises contacting a lens having a hydrophilic surface with a solution comprising a disinfecting amount of peroxide and an effective amount of peroxide-active proteolytic enzyme for a time sufficient to remove substantially all protein accretions and to disinfect the lens.

28. The method of claim 27 wherein said peroxide-active proteolytic enzyme is a subtilisin enzyme.

29. The method of claim 28 wherein said subtilisin enzyme is subtilisin A.

30. The method of claim 27 wherein said peroxide is hydrogen peroxide in an amount between 0.02 and 10% by weight/volume. I ^/99r;

31. The method of claim 30 wherein said hydrogen peroxide is present in an amount of about 3% by weight/volume. The method of claim 27 wherein said peroxide-active proteolytic enzyme is subtilisin A and said peroxide is hydrogen peroxide. r B2. By H'r^/TUcir authorised Agent A. J. Pfy,?»K & 5 /> ' / • y ON