U.S. patent number RE32,672 [Application Number 07/082,860] was granted by the patent office on 1988-05-24 for method for simultaneously cleaning and disinfecting contact lenses using a mixture of peroxide and proteolytic enzyme.
This patent grant is currently assigned to Allergan, Inc.. Invention is credited to Stanley W. Huth, Richard M. Kiral, Sam W. Lam.
United States Patent |
RE32,672 |
Huth , et al. |
May 24, 1988 |
Method for simultaneously cleaning and disinfecting contact lenses
using a mixture of peroxide and proteolytic enzyme
Abstract
A one step method for cleaning and disinfecting contact lenses
is accomplished by immersing the lenses in a solution containing
peroxide and a peroxide-active enzyme.
Inventors: |
Huth; Stanley W. (Mission
Viejo, CA), Lam; Sam W. (Irvine, CA), Kiral; Richard
M. (Irvine, CA) |
Assignee: |
Allergan, Inc. (Irvine,
CA)
|
Family
ID: |
26767940 |
Appl.
No.: |
07/082,860 |
Filed: |
August 4, 1987 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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Reissue of: |
774193 |
Sep 9, 1985 |
04670178 |
Jun 2, 1987 |
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Current U.S.
Class: |
435/264; 424/616;
510/114; 510/115; 510/372; 510/374; 514/840 |
Current CPC
Class: |
A61L
12/124 (20130101); C11D 3/3942 (20130101); C11D
3/38609 (20130101); C11D 3/0078 (20130101) |
Current International
Class: |
A61L
12/12 (20060101); A61L 12/00 (20060101); C11D
3/38 (20060101); C11D 3/386 (20060101); C11D
3/00 (20060101); C11D 3/39 (20060101); C11D
007/42 (); C11D 007/54 () |
Field of
Search: |
;252/95,106,174.12,DIG.12,DIG.14 ;424/130 ;514/840 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2527534 |
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Jan 1976 |
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DE |
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140669 |
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May 1985 |
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EP |
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64303 |
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May 1975 |
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JP |
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1500707 |
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Feb 1978 |
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GB |
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2129458A |
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May 1984 |
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GB |
|
2139260A |
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Nov 1984 |
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GB |
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Other References
Johansen et al., "The Degradiation of the B-Chain of Oxidized
Insulin by Two Subtilisins and Their Succinylated and
N-Carbamylated Derivatives," Compt. Rend., Trav. Lab. Carlsberg
1968; 36(21): 365-84. .
Okunuki et al., "Specificity of Crystalline Bacterial Proteinase,"
J. Biochem. (1956); 43(6): 857-65. .
Hagihara et al., "Crystalline Bacterial Proteinase III. Comparison
of Crystalline Proteinase of Bacillus Subtilis with Crystalline
Trypsin," J. Biochem. (1958) 45(5): 305-11. .
White et al, "Principles of Biochemistry," Fourth Edition New York,
McGraw-Hill 1968:144. .
Novo Industri Information Bulletin, 169b-GB 1978; March:8. .
Berg et al., "Enzymes as Detergent Components," Detergent
Chemistry: Current Topics from Research and Development,
(Dusseldorf, West Germany): Alfred Huthig Verlag Heidelberg, (1976)
pp. 155-178. .
Enkelund, "Interaction Between Proteolytic Enzymes and Detergent
Components," In: Chemistry, Biochemistry and Applications
Technology of Surfactants, vol. IIIC., Munich: Carl Hanser Verlag,
1973:251-65. .
Oldenroth, "Comparative Examination of Different Enzymes in a
Washing Agents" Fats, Soaps, Coatings, vol. 72, No. 7, 1970, pp.
582-587. .
Jaag, "On a New Practical Procedure for the Determination of
Activity of Enzymes Used in Washing Agents," Fats, Soaps, Coatings,
vol. 71, No. 11, 1969, pp. 961-966. .
Weig, "Enzymes in Washing Powders," Process Biochemistry, 1969;
Feb.: 30-34. .
Wedler, "Analysis of Biomaterials Deposited on Soft Contact
Lenses," J. Biomed. Mater. Res. 1977; 11:525-35. .
Brot et al., "Biochemistry and Physiological Role of Methionine
Sulfoxide Residues in Proteins," Arch. Biochem. & Biophysics
1983; 223(1): 271-81. .
Neher et al., "Interaction Between Blood and Oxygenating Bleaching
Agents and Problems Involving Enzyme-Containing Washing Agents,"
Fats, Soaps, Coatings, vol. 72, No. 3, 1970, pp. 192-199. .
Stauffer et al., "The Effect on Subtilisin Activity of Oxidizing a
Methionine Residue," J. Biol. Chem. 1969; 244(19): 5333-8. .
Lo, Jia-Ruey, Silverman, H. I., Korb, D. R., Am. Opt. Assoc., vol.
40, #11, 1106-1109 (1969)..
|
Primary Examiner: Willis; Prince E.
Attorney, Agent or Firm: Finnegan, Henderson, Farabow,
Garrett & Dunner
Claims
What is claimed is:
1. A method for the simultaneous cleaning and disinfecting of
contact lenses which method comprises contacting the lenses with a
solution comprised of 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 lenses.
2. The method of claim 1 wherein the solution is prepared by
combining the enzyme and peroxide at the time the lenses are
contacted with the solution.
3. The method of claim 2 wherein the enzyme is in a powder or
tablet form and is dissolved in the peroxide solution.
4. The method of claim 1 wherein the solution is prepared by
dissolving a dry peroxide and dry enzyme in an aqueous
solution.
5. The method of claim 4 wherein both components are combined in
powder or tablet form.
6. The method of claim 2 wherein the enzyme is present in an amount
between 0.001 and 5 Anson units and the peroxide is hydrogen
peroxide and is present in an amount between 0.02 and 10% by
weight/volume.
7. A method according to claim 6 where the proteolytic enzyme is
subtilisin.
8. The method of claim 7 wherein the peroxide is hydrogen peroxide,
sodium perborate, potassium persulfate, sodium percarbonate,
diperisophthalic acid, peroxydiphosphate salts or sodium aluminum
aminohydroperoxide.
9. The method of claim 8 where the .[.aqueous composition.].
.Iadd.solution .Iaddend.comprises 3% hydrogen peroxide and 0.30%
subtilisin A by weight/volume. .Iadd.
10. The method of claim 1 wherein said contact lenses have a
hydrophilic surface. .Iaddend. .Iadd.11. The method of claim 10
wherein said peroxide-active proteolytic enzyme is a subtilisin
enzyme. .Iaddend. .Iadd.12. The method of claim 11 wherein said
subtilisin enzyme is subtilisin A. .Iaddend. .Iadd.13. The method
of claim 10 wherein said peroxide is hydrogen peroxide in an amount
between 0.02 and 10% by weight/volume. .Iaddend. .Iadd.14. The
method of claim 13 wherein said hydrogen peroxide is present in an
amount of about 3% by weight/volume. .Iaddend. .Iadd.15. The method
of claim 10 wherein said peroxide-active proteolytic enzyme is
subtilisin A and said peroxide is hydrogen peroxide. .Iaddend.
Description
BACKGROUND
This invention relates to a method and composition for cleaning and
disinfecting contact lenses. More specifically, this invention
covers the simultaneous cleaning and disinfecting of contact lenses
by means of a solution containing a mixture of peroxide and
peroxide-active enzymes, particularly proteolytic enymes.
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 maintain optical
clarity, wearability and prevent the transfer of infectious agents
into the eye.
Glass and the early polymers such as polymethylmethacrylate (PMMA)
lenses could be readily cleaned by manual means using detergent
because of their rigidity and hydrophobic character. While such
materials are, to a certain degree, wetted by the naturally
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, particularly polypeptides and enzymes such
as lysozyme do not adhere significantly to these materials and are
readily removed by cleaning with 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
organosiloxane-methacrylate lenses (Polycon.RTM. 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 lens surface
during day-to-day wear. On all but purely PMMA lenses, the
adsorption is so strong that even with lenses such as the rigid
polysiloxane/methylmethacrylate copolymers, manual detergent
cleaning methods do not adequately remove this accretion. So-called
hydrogel lenses, those materials prepared from
hydroxyethylmethacrylate, hydroxyethylmethylmethacrylate,
vinylpyrrolidone and glycerolmethacrylate monomers and methacrylic
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 resultant 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 defraction. 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 small polypeptides. These protein fragments are generally
water soluble and thus are easily solubilized by the surrounding
aqueous environment. U.S. Pat. No. 3,910,296 discloses the use of
proteases for cleaning contact lenses. See also U.S. Pat. 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 making additional cleaning more 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 antimicrobials 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
microbiosides, 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 some people, sufficient to cause them to seek
alternative means for 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 peroxides have been developed for
disinfecting contact lenses. One system is illustrated by U.S. Pat.
No. 3,912,451 issued to C. Gaglia. Another is U.S. Pat. No.
4,473,550 issued to Rosenbaum, 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
removed 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. Pat. No. 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. Pat. No. 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 in such a manner as to prevent the
premature inactivation of the enzyme by the 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 Fette Seifen Anstrichmittel, 1970 (72(7)),
582-7. 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 the 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 a method for the
simultaneous cleaning and disinfecting of contact lenses,
particularly one having a hydrophilic surface, which method
comprises contacting the lenses with a solution comprises of 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
lenses.
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
proteolytic activity as described by Yaun Lin, et al., J. Biol.
Chem., 244: (4) 789-793. (1969).
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 amide bonds. Such enzymes may also have some
inherent lipolytic and/or amylolytic activity associated with the
proteolytic activity.
Preferred proteolytic enzymes are those which are substantially
free of sulfhydryl groups or disulfide bonds, whose presence may
react with the active oxygen to the detriment of both the activity
of the active oxygen and which may result in the untimely
inactivation of the enzyme. Metallo-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 group of proteolytic enzymes are the serine
proteases, particularly those derived from Bacillus and
Streptomyces bacteria and Asperigillus molds. Within this grouping,
the more preferred enzymes are the Bacillus derived alkaline
proteases generically called subtilisin enzymes. Reference is made
to Deayl, L., Moser, P. W. and Wildi, B. S., "Proteases of the
Genus Bacillus. II alkaline Proteases." Biotechnology and
Bioengineering, 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 are B. subtilis, B. licheniformis
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. subtilis var.
amylosacchariticus B. amyloliquefaciens and B. subtilis NRRL B3411.
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 aspergillo-peptidase. A and B, pronase E (from S.
griseus) and dispase (from Bacillus polymyxa).
The identification, separation and purification of enzymes is an
old 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 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.
With the advent or recombinant DNA techniques, it is anticipated
that new sources and types of peroxide stable proteolytic enzymes
will become available. Such enzymes should be considered to fall
within the scope of this invention so long as they meet the
criteria for stability and activity set forth herein. See Japanese
laid open application No. J6 0030-685 for one example of the
production of proteases by recombinant DNA from Bacillus
subtilis.
An effective amount of enzyme is to be used in the practice of this
invention. Such amount will be that 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 to contact lens wearers with a
history of normal pattern of protein accretion, not the very small
group who may at one time or another have a significantly increased
rate of protein deposit such that cleaning is recommended every two
or three days.
The amount of enzyme required 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 0.001 to 5 Anson units
of activity, preferably between about 0.01 and 1 Anson units, per
single lens treatment. Higher or lower amounts may be used. Enzyme
concentrations lower than these stated here probably will serve to
clean the lens if sufficient time is allowed but such time may be
so long as to be practically not useful in a usual lens cleaning
and disinfecting regimen. Solution with higher activity should
effect more rapid cleaning but may involve amounts of material
which are too sizeable for practical cleaning purposes.
In weight/volume 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
requirement.
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, alkali metal
perborate monohydrate and tetrahydrate, alkali metal persulfates,
alkali metal carbonate peroxide, diperisophthalic acid,
peroxydiphosphate salts and sodium aluminum aminohydroperoxide.
Hydrogen peroxide and the sodium salts of perborates and
persulfates 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. More 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 substantially
between peroxides.
For hydrogen peroxide, on the lower side, a 0.5% weight/volume
concentration 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 through 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
the requirement that the enzyme retains proteolytic activity.
So far as other peroxides are concerned, the only limitation placed
on their concentration is that they exhibit synergistic activity in
combination with the peroxide-stable enzyme at a given
concentration with regard to cleaning and disinfecting. For
example, it has been found that sodium perborate at concentrations
of 0.02% weight/volume will potentiate the enzymatic removal of
protein from contact lenses. The appropriate concentrations of any
given peroxide will be a matter finally to be determined through
routine laboratory testing.
Additional materials may be added to the formulations, for example,
tonicity agents, effervescing agents, stabilizers, binders,
buffering agents, enzyme co-factors, disulfide bond reducing agents
such as water-soluble mercaptans and salts of sulfites,
pyrosulfites 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 metal-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
formulations.
To practice the invention, a solution of 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 of 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 hydrogen peroxide is not 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 maufacturing 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 enchance tablet breakup 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 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 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. Enzymatic activity is a function of temperature, some
enzymes being considerably more labile than others to temperature
extremes, particularly temperature increases. Other enzymes are
heat stable and remain significantly active at temperatures of
70.degree. 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.degree. and 37.degree. C., particularly about
22.degree.-25.degree. C., it may be possible to practice this
invention with certain peroxide-active enzymes in the temperature
range between about 5.degree. C. to 100.degree. 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 component 1 with component 2 during
tablet and powder preparation 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 residual
peroxide. Enzymes which catalyze the conversion of peroxides to
oxygen and water can be included in these formulations to remove
residual peroxide in anticipation of inserting the lens back into
the eye. For example catalases, organic enzymes which catalyze the
degradation of peroxides, can be incorporated into tablets and
powders, particularly in time-release form. Additionally, metals
such as the heavy metal transition elements which catalyze 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
U.S. Pat. No. 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.RTM. II 55% water lenses (Barnes-Hind, Inc.
Sunnyvale, Calif., U.S.A.) were coated with heat-denatured lysozyme
by placing the lenses in a phosphate buffered saline solution to
which was then added 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 hydrated
lens. Vials were then heated for about 30 minutes at about
95.degree. C. The lens was then removed, and after being cooled,
was rinsed with distilled water and viewed to determine the type of
lysozyme accretion.
Deposit classification: 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
microscope at 100X. A film or deposit detected under these
conditions was classified according to the percentage of surface
which was covered by the film.
After the treatment described in the first paragraph, all lenses
were found to have 100% of their anterior surface covered by
thin-film protein deposits.
These lenses were then treated with solutions based on peroxide and
the following enzyme formulations:
______________________________________ Ingredient Percentage (w/w)
______________________________________ Papain Tablet Sodium Borate,
Dihydrate 13.03% Sodium Carbonate 21.25% Polyethylene glycol 3350
2.74% Papain 6.28% Tartaric Acid 13.71% L-Cysteine HCL 6.86% EDTA
5.04% Sodium Chloride 30.64% Subtilisin A Tablet Sorbitol 29.99%
N--acetylcysteine 22.49% Sodium Carbonate 38.98% Polyethylene
glycol 3350 3.00% Subtilisin A 0.30% Tartaric Acid 5.24%
______________________________________
The subtilisin A was obtained from Nova Industries of Denmark.
The lenses were divided into four groups of five. One group was
treated 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.RTM. made and
sold by Allergan Pharmaceuticals, Inc.). A third group was treated
with the Subtilisin A tablet dissolved in 10 ml of 3% hydrogen
peroxide and the fourth group was treated with a 3% hydrogen
peroxide (10 ml) containing one papain enzyme tablet (146.8
mg).
The lenses were 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 follows:
______________________________________ 3% Hydrogen Peroxide* %
SURFACE LENS CLEANED ______________________________________ A1 0 A2
1 A3 0 A4 0 A5 1 ______________________________________ SUBTILISIN
A/Saline SUBTILISIN A/3% H.sub.2 O.sub.2 * % SURFACE % SURFACE LENS
CLEANED LENS CLEANED ______________________________________ B1 30
C1 50 B2 20 C2 60 B3 25 C3 70 B4 15 C4 60 B5 30 C5 50
______________________________________ PAPAIN/3% H.sub.2 O.sub.2 *
% SURFACE LENS CLEANED ______________________________________ E1 0
E2 0 E3 0 E4 0 E5 0 ______________________________________ *Oxysept
.RTM. 3% Hydrogen peroxide solution marketed by Allergan
Pharmaceuticals Inc.
While the hydrogen peroxide and papain/hydrogen peroxide cleaning
activity was essentially nil, subtilisin and 3% hydrogen peroxide
cleaned between 50 and 70% of the contact lens surface area.
Secondly, subtilisin A alone without peroxide cleaned between 15
and 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 in
its cleaning capacity in comparison with subtilisin without
peroxide.
EXAMPLE 2
Peroxide/Enzyme Activity
Fifteen Hydrocurve II.RTM. lenses (Barnes-Hind) were exposed to
lysozyme and the presence of Type IV protein 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.); a commercially available, pancreatin
containing enzyme tablet (Opti-Zyme.RTM. tablet manufactured by
Alcon) dissolved in 10 ml of saline solution (Boil-'n-Soak.RTM., a
normal saline solution produced by Alcon); and a solution of
pancreatin enzyme (Opti-Zyme.RTM.) in 10 ml of 3% hydrogen peroxide
(Oxysept.RTM. 1).
Following an 8 hour soak, lenses were treated to remove residual
soaking solution and the percentage of protein determined as
described in Example 1. The results were as follows:
______________________________________ 3% Hydrogen Peroxide %
Surface Lens Cleaned ______________________________________ A1 0 A2
0 A3 0 A4 0 A5 0 ______________________________________
Pancreatin/Peroxide Solution Pancreatin/Normal Saline % Surface %
Surface Lens Cleaned Lens Cleaned
______________________________________ B1 90 C1 0 B2 85 C2 0 B3 85
C3 0 B4 90 C4 0 B5 80 C5 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.RTM. 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-acetylcysteine 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 with sodium chloride. The
pH was adjusted to between about 9.0-9.03 in each solution with
hydrochloric acid. Five lenses were treated for three hours at room
temperature with 10 ml of each solution. The amount of protein
(percentage) removed from the lens surface is given in Table I.
TABLE I ______________________________________ Effects of Peroxide
Concentration on Cleaning Efficacy Enzyme % peroxide % Lens Conc.
pH Tonicity Weight/vol. Cleaning
______________________________________ A 0.04 mg/ml 9.025 318
mOsm/kg 0 9.0 (5.5) B 0.04 mg/ml 9.086 330 mOsm/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/ml 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 2.5% 97.0 (2.7)
______________________________________
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 quantitative 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 10231 and A. niger.
ATCC 16404. A 133.4 mg tablet of the subtilisin A formulation (0.04
mg) given in Example 1 was used.
The results of this study are given in Table 1.
TABLE I ______________________________________ COMPARISON OF
EXTRAPOLATED D-VALUES* IN MINUTES Study I Study II 3% H.sub.2
O.sub.2 3% H.sub.2 O.sub.2 ORGANISMS 3% H.sub.2 O.sub.2 + SUB. A 3%
H.sub.2 O.sub.2 + SUB. A ______________________________________ S.
marcescens 2.5 1.7 3.5 1.3 S. aureaus 4.0 3.0 4.0 2.0 p. aeruginosa
0.3 0.5 0.3 0.1 E. coli 2.5 0.9 1.7 0.2 C. albicans 36.5 13.0 15.0
9.0 A. niger 9.5 11.6 6.0 6.0
______________________________________ *D-valve is the time
required to reduce a microbial challenge of 5 .multidot. 10.sup.6
organism per ml 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 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% H.sub.2
O.sub.2 3% H.sub.2 O.sub.2 /SUB. A
______________________________________ S. marcescens 3.0 1.5 E.
coli 2.1 0.6 P. aeruginosa 0.3 0.3 S. aureus 4.0 2.5 C. albicans
26.0 11.0 A. niger 8.0 9.0
______________________________________
Since the lower the D value, the more effective the antimicrobial
activity, each of these studies demonstrates that 3% hydrogen
peroxide and subtilisin A together are a substantially more
effective disinfecting composition than either of the two
components acting separately.
EXAMPLE 5
Testing 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 FDA 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, 10 ml 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
subtilisin-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 test tubes containing neutralizer media. A serial ten-fold
dilution of the neutralizer media was prepared in a manner to
compensate for the expected level of recovery. For low level
recovery, a small aliquot was transferred directly onto a
neutralizer agar plate. For the other three serial dilution tubes,
an equal volume of sample was placed on neutralizer agar plates.
All plates were incubated at 35.degree.-37.degree. C. for 2-7 days,
or longer if required. Colony counts were then recorded and
D-values calculated as follows: All plate counts for each time
interval were averaged. The averaged data was plotted on a semi-log
graph paper with the numbers of survivors on the ordinate and the
contact time on the abscissa. The starting point (inoculum level)
was connected to the first point yielding less than 10 organisms
per ml by a straight line. The slope of this line extrapolated to
zero gives the D-value. This is otherwise referred to as "end-point
analysis".
TABLE III ______________________________________ Extrapolated Kill
Rates (D-valves) of 3% Hydrogen Peroxide (Oxysept I) With and
Without Subtilisin Without With Organism and ID # Subtilisin
Subtilisin ______________________________________ (1) USP XXI Panel
Serratia marcescens. ATCC #14756 1.4 min. 1.0 min. Staphylococcus
aureaus. ATCC #6538 3.4 min. 2.1 min. 3.2 min. 2.6 min. Pseudomonas
aeruginosa. ATCC #9027 0.2 min. 0.2 min. Escherichia coli. ATCC
#8739 1.0 min. 0.3 min. Candida albicans. ATCC #10231 20.0 min.
13.0 min. Aspergillus niger. ATCC #16404 10.0 min. 8.0 min. (2)
"Soft Lens" Panel (FDA) Serratia marcescens. ATCC #14041 1.7 min.
1.5 min. Staphylococcus epidermidis. 0.8 min. 1.5 min. ATCC #17917
0.4 min. 1.0 min. Pseudomonas aeruginosa. ATCC #15442 0.6 min. 0.3
min. Aspergillus fumigatus. ATCC #10894 13.5 min. 2.5 min. Candida
albicans. ATCC #10231 20.0 min. 13.0 min. (3) Various Isolates
Klebsiella pneumoniae. ATCC #13883 1.1 min. 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 min. Proteus vulgaris. ATCC #17313
0.4 min. 0.3 min. Candida parapsilosis. PM 4064 63.0 min. 55.0 min.
Penicillium sp. (Aqua Tar isolate II) 2.5 min. 2.1 min.
______________________________________
EXAMPLE 6
Comparative Enhancement of Peroxide With and Without Enzyme
Comparative enhancement of the antimicrobial kill rates of various
solutions of 3% hydrogen peroxide due to the addition of the enzyme
subtilisin. 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.
Pat. No. 3,912,451.
TABLE IV ______________________________________ Lensan A Oxysept I
(Data From (Data From Organism Table II) Table III) AO Sept
______________________________________ Serratia marcescens 50% 29%
88% Escherichia coli 71% 70% 90% Pseudomonas aeruginosa 0 0 20%
Staphylococcus aureus 38% 28% 60% Candida albicans 58% 35% 33%
Aspergillus niger 0% 20% 32%
______________________________________
These figures demonstrate that each of the 3% peroxide solutions is
a much more effective disinfectant when subtilisin A is present.
The effect is particularly pronounced in the A-OSept system.
EXAMPLE 7
Effect of Peroxide Concentration on Enzyme Activity
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. Appropriate
dilutions were made with a 0.2M 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 enzyme tablet was dissolved in 10 ml of buffer
to which had been added 50 mg 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 minutes, then
quenched with 2 ml of 10% trichloroacetic acid, which precipitated
the enzyme. Residual color measurements were measured at 520 nm.
Subtilisin results are given in Table IV, trypsin results in Table
V.
TABLE IV ______________________________________ Subtilisin Activity
in Hydrogen Peroxide % H.sub.2 O.sub.2 OD 520
______________________________________ 0 0.27 1 0.39 2 0.57 3 0.56
4 0.66 4.5 0.56 5 0.68 6 0.68 8 0.90 30 0.91
______________________________________
TABLE V ______________________________________ *Trypsin Activity in
Hydrogen Peroxide % H.sub.2 O.sub.2 OD 520
______________________________________ 03 .5 30 .6
______________________________________ *10 mg of tryspin powder
were added to the H.sub.2 O.sub.2 solution.
Table IV indicates that subtilisin A is active in 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 apears to be
saturated at hydrogen peroxide concentrations between 2-6%. Table V
indicates that trypsin is active in hydrogen peroxide.
EXAMPLE 7
Effects of Perborate on Enzyme Activity
Hydrocurve II.RTM. lenses were coated with heat-denatured lysozyme
as per the procedure described in Example 1. 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
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 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 protein determined. Table VI
gives the average percentage of surface cleaned after these
treatments.
TABLE VI ______________________________________ Comparative
Cleaning of Enzyme With and Without Peroxide Average % Solution
Surface Cleaned ______________________________________ A 9.0 .+-.
5.6 B 0 C 30.0 .+-. 12.2 ______________________________________
* * * * *