Note: Descriptions are shown in the official language in which they were submitted.
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ENZYMES WITEI LOW ISOl~LECTRIC POINTS FOR USE IN
CONTACT LENS CLEANING
The present invention relates to the field of contact lens çle~ning and dishlfe-;Lillg.
In particular, this invention relates to compositions co~"~;";..~ enzymes which have been
modified to exhibit a low isoelectric point and methods for ~le~ning human-worn contact
lenses with those compositions. The invention also relates to methods of ~imlllt~neously
cleaning and disinfecting contact lenses by combining the enzyme compositions of the
present invention with a chemical di~hl~cLing agent.
Back~round of the Invention
~arious compositions and methods for ~.le~nin~ contact lenses have been described
in the patent and scientific literature. Some of these methods have employed compositions
co~ illg surf~t~nt~ or enzymes to f~ilit~te the cleaning of lenses. The first ~ cll~.~ion 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).
25 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., inc:ln~ing
U.S. Patent No. 3,910,296 (Karageozian, et al.).
Nu~ ,rous compositions and methods for disinfecting contact lenses have also been
described. Those methods may be generally characterized as involving the use of heat
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and/or chemical agents. Representative ~h~:mic:~l 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 qua~ la~y
ammonium compounds to disinfect contact lenses and to preserve contact lens careproducts. U.S. Patents Nos. 4,758,595 and 4,836,986 (Ogunbiyi) describe the use of
polymeric bi~nides 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 (Randeri, et
10 al.) and 4,414,127 (Fu), for example. ~ representative method of ~imlllt~neously cle~ning
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 (i.e., chlorhexidine) and enzymes to simlllt~neously clean and disinfect
15 contact lenses is described in ~n~ n 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. 4,614,54g (Ogunbiyi). The combined use of proteolytic
enzymes and polymeric bigl~nides or polymeric quaternary ammonium compounds is
described in copending, and commonly assigned United States Patent Application Serial
20 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
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using the lens. It can also lead to ocular irritation and imml-nngenicity, due to the eye's
s~n~ili7~tion 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 Pancreatin-Based andSubtilisin-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
o 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 immlm~genicity, improve
visual clarity, and therefore enable a more regular use of the enzyme for cleaning contact
lenses.
The use of modified el~yl-les 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;
20 such enzymes have been disclosed in WIPO Publication No. WO 95/07991 (~ igned to
Procter & Gamble).
Enzymes have also been modified by organic polymer linkage. The covalent linkingof 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
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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-immlln~-genic polypeptides for use in the circulatory system of the human
body European Patent Application No. ~ 584 876 A2 discloses low diol polyalkylene
5 oxide biologically active proteinaceous substances, incllltling a Subtilisin Carlsberg.
Another method of enzyme modification has involved organic monomer linkage to
the enzyme. For example, Joh~n.~n discloses methods of succinylation and glutarylation of
subtilisins in Chemical Derivatives of Su~filisins with Modif ed Proteolytic Activifies Il.
Succinyl- and Glufa~ylsubtilisin Type Carlsherg, Compt. Rend. Trav. Lab. Carlsber~.
o volume 37, pages 145-177 (1970).
Summarv 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 t;~libi~ g 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 leil1selLed in the eye. The lower binding may also improve enzyme rle~ning
efficacy. Thus, the present invention has overcome issues of toxicity and efficacy to
provide a more effective rlf~ning system for contact lenses.
The compositions of the present invention are form~ tecl in either solid or liquid
form. Compositions formulated in liquid form may, for example, comprise a polyol and an
--4 --
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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 ~imlllt~neous cle~nin~ and disinfecting of contact lenses, when compositions
of the present invention are combined with suitable disinfecting solutions, such as those
5 cont~ining polyquaternium-1.
Detailed Description of the Invention
The enzymes of the present invention exhibit low isoelectric points ~"pI") relative to
o 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 acc -m~ te in or on lenses and cause ocular
irritation when they come into contact with the eye. Therefore, the use of low
15 electrostatically binding enzymes, i.e., those with low pls, provides a safe and more
comfortable method for contact lens cleaning.
As used herein, the term "low pI" refers to electroch~mic~l 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
20 of the technique of isoelectric focusing, as described in Example 3 below, may be used to
determine the pI 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
-- 5 --
= = = ==== === ===
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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 ~ntimicrobial 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 m~tf~!ri~l found in lens
deposits to smaller water-soluble subunits.
o Examples of native enzymes which may be modified for use in the present invention,
include but are not limited to: pancreatin, trypsin, chymotrypsin, subtilisin, coll~gen~e7
~ t~, keratinase, carboxypeptidase, papain, bromelain, aminopeptidase, Aspergillo
peptidase, pronase E (from S. ~riseus! and dispase (from Bacillus polvm~a) and ~ x~ult;s
thereof. If papain, or any sulfhydryl protease is used, a re-1ur.ing agent, such as N-
acetylcysteine, may be required.
Microbially derived enzymes, such as those derived from Bacillus. StreptomYces~
and Asper~illus rnicroorg;3ni~ms, 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 in~ lin~ Novo
Tnt11l.~tries (Bagsvaerd, Denmark), Fluka Biochemika (Buchs, Switzerland~ and Boehringer
M~nnh~im (Tn~ n~rolis, ~ndiana).
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The identification, separation and purification of enzymes is known in the art.
~any identification and isolation techniques exist in the general scientiffc literature for the
isolation of enzymes, including those enzymes having proteolytic and mixed
proteolytic/amylolytic/lipolytic activity. The native enzymes to be modified for use in this
invention can be readily obtained by known teçhniques 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 ple~lled 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 of the present invention may be selected from those that have had part
of their amino acid sequence altered in favor of a lower pI. In general, amino acid residues
~hilitillg high (net positive charge at physiological pH) pKa's (pH at which half of the
total quantity of the particular residue is charged) may be replaced with neutral or low pKa
amino acids. For example, Iysine or arginine (high pKa) residues may be replaced by
alanine, leucine (non-ionic), aspartate, ~ t~m~te (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 incorporated herein by reference. As
used herein, "genetic recombinant techniques" refer to any method of producing mutant
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enzymes of the present invention through the manipulation of DNA. Generally, a plasmid
of a host b~ct~rillm 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 ferm~n~ing process, cont~ining the bacterial exudate, is then separated from
s the bacterial colonies and is extracted for the target enzyme. Separation techniques
including gel and affinity chromatography are generaliy 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
mventlon.
o 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. Suchmodifications have been ~li.cc~ ed 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
2û so that they exhibit low pIs and are opthalmically acceptable as set forth above. Commonly
ned 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 ~ cllssed in Johansen, Chemical Derivatives of Subtilisins with Modified
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Proteolyfic Acfivities II. Succinyl- and Glufarylsubfilisi~ Type Carlsberg, C~ompt. Rend.
Trav. Lab. Carlsber~. 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 (i.e., they may not be easily stabilized in liquid form). Enzymes suitable for liquid
stabilized vehicles may require minim~l modification whereas all modified enzymes would
be useful in a solid form (i.e., effervescent tablets).
The compositions of the present invention may be either in solid or liquid form.Solid forms usually encompass a colllpl~;ssed tablet wherein various excipients are
employed. For example, components such as e;Lrt;lvescil1g 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 incl~7t7ç, but are not limited to, alkali metal salts such as potassium or
20 sodium carbonates, ~cet~tes~ 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 ophth~lmir uses.
g
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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 m~int~in a suitable level for optimum activity by the antimicrobial agent.
o Typical tonicity adjusting agents are NaCI and K~l.
Suitable surfactants can be either cationic, anionic, nonionic or amphoteric.
Preferred surf~ct~nt.~ are neutral or nonionic surfactants which may be present in amounts
up to 5% (w/v). Examples of suitable surf~ct~nt~ incllldç, but are not limited to,
polyethylene glycol ether or esters of fatty acids, polyoxyethylene-polyu7~yl,lu,uylene block
copolymers of ethylene diamine (i.e., poloxamine), polyu~y~ropylene-polyoxyethylene
glycol nonionic block polymers (i.e., polaxamers such as Pluronic F-127) and p-
isooctylpolyoxyethylene phenol formaldehyde polymers (i..e., Tyloxapol).
Examples of ple~ll~d chel~ting agents include ethylene~ minetetraacetic acid
(~DTA) and its salts (disodium) which are normally employed in amounts from about
0.025 to about 2.0% (w/v). Other known ch~l~ting (or se~uestering 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.
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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 cont~inin~ low pI modified enzymes are also cnntempl?lted bythe present invention. Such compositions will be comprised of one or more low pIenzymes 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 of the enzyme and preservation of the composition for multiple use dispensing.
o 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 m~nnitol. 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
20 criteria for efficacious and co~ elcially 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 plere~lled monomeric polyol
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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
h~vel~ible 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~ carbon atoms in total. Preferred inhibitors include phenylboronic acid and it
derivatives. The pler~ d range of a phenylboronic acid derivative used in the present
invention is 0.1 to ~.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 p~ d, are the polymeric
quaternary ammonium compounds, the most preferred is polyquaternium-l. The amount of
preservative used will depend on several factors inc,l~-ling the anti-microbial efEcacy of the
20 particular agent and any synergistic interaction the agent may have with the liquid enzyme
composition. In general, 0.0001 to û. 1% w/v of the preservative agent will be used.
The liquid compositions may contain one or more s~ rt~nts selected from anionic,
non-ionic or zwitterionic classes. Examples of non-ionic s~ rt~n~ include alkyl
polyoxyethylene alcohols, alkyl phenyl polyo?~yelhylene alcohols, polyoxyethylene fatty acid
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esters, polyethylene oxide-polypropylene oxide copolymers such as polaxomers andpolaxamines. Examples of anionic surf~ct~nts include alkyl sarcosinates and alkyl
~ t~m~t~.~. Examples of amphoteric suff~ct~nts include alkyliminopropionates andalkylampho~ret~tes. In general 0 to 5% w/v of the surfactant will be used.
The liquid compositions may contain additional stabilizing agents. These includethe stabilizing multi-valent ions, such as calcium and m~gne~il7m and their halide salts.
Calcium chloride is the most pl~r~ d mu}ti-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
10 borate buffers; tonicity adjusting agents, such as NaCl or KCI; metal chelating agents, such
as ethylenediaminetetraacetic acid (EDTA) and pH adjusting agents such as sodiumhydroxide, 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 cle~ning composition
utilized in particular embo-liment.~ of the present invention may vary, depending on various
factors, such as the purity of the enzyme utili7e-l, the proposed duration of exposure of
20 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., surf~c.t~nt~).
The liquid enzyme compositions of the present invention must be form~ ted to
provide storage stability and antimicrobial preservation suitable for multiple use dispensing,
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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 immlln~)genicity from continuous use. Additionally, when
combined with a dish~rcclil~g solution cont~ining an antimicrobial agent which is adversely
affected by high ionic strength such as polyquaternium-l, the compositions of the present
invention must have little or no impact on the ionic strength of the 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
measulcl--cnLs to define acceptable tonicity ranges for disinfecting solutions. As intli~ted
above, the antimicrobial activity of di~hlrc~;ling agents, particularly polymeric qll~t.o.rn~ry
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 plcrcllcd. 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 plcrclled.
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The methods of the present invention utilize a dishlre~ g solution co.~ g an
antimicrobial agent. Antimicrobial agents can be oxidative, such as hydrogen peroxide, or
non-oxidative polymeric antimicrobial agents which derive their antimicrobial activity
r ; through a chemical or physicochemical interaction with the org~ni.qm~. As used in the
present speçific~tion~ the term "polymeric antimicrobial agent" refers to any nitrogen-
cont~ining 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 big--~nitle.~, 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 bigll~nicles such as salts of alexidine, salts of chlorhexidine,
hexamethylene bi~ni~es and their polymers. The polymeric antimicrobial agents used
herein are preferably employed in the absence of mercury-cont~ining compounds such as
thimerosal. The salts of alexidine and chlorhexidine can be either organic or inorganic and
are typically gluconates, nitrates, ~cet~tes, phosphates, sulphates, halides and the like.
Particularly pr~rel 1 ed are polymeric ~uaternary ammonium compounds of the
structu}e:
R, ~ CH CH ~ Rz
CH, CHJ
--n (n + l)X (I)
wherein:
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Rl and R2 can be the same or di~elt;n~ and are selected from:
N+(CH2CH2OH)3X, N(CH3)2 or OH;
X is a pharm~.el7tically acceptable anion, preferably chloride; and
n = integer from 1 to ~0.
The most ~ Çell~d compounds of this structure is polyquaternium-l, which is also known
Onamer l~a (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 Rl, R2 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 ~ imin~te substantially or to reduce ~ignifiç~ntly the
number of viable microorg~ni~m~ found on contact lenses, in accordance with the
requirements of government~l regulatory agencies, such as the United States Food and
Drug ~lmini~tration. For purposes of the present specification, that amount is referred to
as being "an amount effective to disinfect" or "an ~ntimiçrobially 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 utili7e-1 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, inc~lu-ling microor~ni~m.c7 and thereby lessen the amount
of antimicrobial agent required to disinfect the lenses. The type of lens being treated (e.g.,
20 "hard" versus "soft" lenses) may a.so 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 ~le~ell~d concentration of the polymeric
q~ .y ammonium compounds of Formula (I) is about 0.001% by weight.
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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 ~ltili~in~ 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
10 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 inclllde, 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 surf~ct~nt.~ are neutral or nonionic surfactants which may be present in amounts
Up to 5% (w/v). Exarnples of suitable surf~ct~nt~ include, but are not limited to,
polyethylene glycol esters of fatty acids, polyoxypropylene ethers of Cl2-CI8 alkanes and
polyo~yt;Lllylene-polyoxy~ ~Jpylene block copolymers of ethylene diamine (i.e. poloxamine)
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Examples of prert;ll~d ~h~l~ting agents include ethylenedi~minetetraacetic 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
a 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 pl~r~lled. 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 int~n~1ed to limit the scope of the invention in any respect.
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Example 1
A preferred enzyme composition of the 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 pl~rel.ed 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 appl.,pliate size and
hardness, according to methods known to those skilled in the art.
This tablet may also be form~ tecl with a seal coating and/or a delayed release
coating to provide for a delay in dissolution of up to about 2 hours.
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B. Disinfectin~ Solution
The following formulation represents a plerell~d disinfecting solution:
Ingredient w/v (%)
Polyquaternium-l0.001 + 10% excess
Sodium chloride 0.48
Disodium F.det~te 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-l, in the relative
concentrations indicated above, were rnixed with purified water and the components
10 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.
~,~S~mr)l~ 2
The method of isoelectric focusing was performed to determine the pI of enzymes.
The method of Bio-Rad, as described in Bio-Rad's Model 111 Mini IEF Cell Instruction
ManuaL was followed.
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Briefly, a 1 to 2 ,ul aliquot (5 mg/ml) of one or more proteases to be determined for
pI or control proteins of known pIs were applied to a polyacrylarnide gel slab co"~ g
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
o ranging from 4.45 to 9.~;0, 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:
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Tablel
Migration distance on the gel(m m)
# Protein Migration PI
(mm~
Phycocyanin 4 4.6
213-Lactoglobulin B 9 5.1
3Bovine carbonic an.ydrase :5 6.0
4Human carbonic an.ydrase ~ 8 6.5
5~quine myoglo ~in - 2 7.0
6~ uman myog .o ~. n A 23 7.1
7E.uman Iryog.o~ n C 25 7.5
8Lent l lectin 28 8.2
9Cy-ocnrome C 39 9.6
Subtilis n A (Carls ~erg~ 26 9.4
Succiny ated subtil sin A 5 4.60
Acetylated su ~tilisin A ~ 4.45
PEC~5000-subt lisin 3PN ' 4.45
Me- 'EG-5000-subtil.s n A ~ 4.45
Tryp~in ~3ovine) 38 9.37
Acety ateo Tryps n 2 4.16
Methyatec Tryps.n 39 951
As illustrated in Table 1 succinylation and acylation sllcc~fiilly lowered the pI of
subtilisin and trypsin down within the pI range of the present invention.
o 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.
,
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