Note: Descriptions are shown in the official language in which they were submitted.
. CA 02257648 1999-O1-15
PROCESS FOR CLEANING AND DISINFECTING CONTACT LENSES
This application is divided out of Application
Serial No. 2,146,860, filed August 24, 1994.
Background of the Invention:
The present invention relates to the field of
products for treating contact lenses. More particularly, the
invention relates to an improved process wherein contact
lenses can be cleaned and disinfected with a single product
containing one or more polycarboxylates, polysulfonates or
polyphosphates. It has been found that these agents
effectively remove deposits of proteins and other materials
from contact lenses.
Products for treating contact lenses have generally
been classified based on the intended use or function of the
products. Most products have been classified as either
cleaners or disinfectants. However, there are also a number
of associated products which can be generally classified as
rewetting drops or conditioning solutions. Although there
have been attempts to accomplish two or more functions with a
single product, such attempts have generally had limited
success, because combining the components require to perform
multiple functions in a single product tends to reduce the
effectiveness of those components. For example, combining
surfactants commonly used to clean contact lenses with
antimicrobial agents commonly used to disinfect contact lenses
may reduce the activity of one or both of these agents. Thus,
from a purely scientific standpoint, the combining of two or
more functions in a single product has been discouraged.
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CA 02257648 1999-O1-15
'O 95106099 PCT/US94/09
The perspective of the patient is much different from that of the scientist.
The
primary concerns of the patient are typically effectiveness and convenience.
The latter
concern is particularly important among wearers of disposable contact lenses,
who tend
to be highly motivated toward convenience. The emphasis that patients place on
s convenience has led to a rapid rise in the popularity of disposable contact
lenses capable
of supporting either daily or extended wear. Depending on the planned
replacement and
wearing schedules, these lenses are most commonly cleaned and disinfected
daily, and
occasionally weekly or biweekly. The result is that many convenience-driven
patients
who originally sought relief from the rigors of lens care are being fitted
with lenses that
require daily, albeit less rigorous, care. Such patients place a premium on
products that
are simple and straightforward to use. By their very nature, frequent
replacement lenses
worn for daily wear are presumed to require the use of fewer or milder
cleaning products.
Thus, both patient preference for convenience and the fitting of frequent
replacement
lenses for daily wear has created a desire for easy-to-use disinfectants that
can also be
is used to clean, soak and rinse lenses.
If the care of the patients' contact lenses becomes too complicated, the
patients
may fail to comply with the cleaning and disinfection instructions provided by
their
physicians. Such non-compliance is a major concern of ophthalmologists and
optometrists. Repeated failures to clean and/or disinfect contact lenses
properly can lead
Zo to serious vision problems, such as corneal abrasions, infections,
inflammation of the
conjunctiva, and so on.
The use of a disinfecting solution to also clean contact lenses has been
proposed
previously. However, such disinfecting solutions have typically included one
or more
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CA 02257648 1999-O1-15
surfactants as the active cleaning component. A product of
this type is currently marketed by Bausch & Lomb as ReNu R
Multi-Purpose Solution.
In view of the foregoing circumstances, there is a
need for improved products and processes for cleaning and
disinfecting contact lenses in an efficacious but convenient
manner.
Summary of the Invention:
The present invention is based on the surprising
finding that a single aqueous solution containing a
disinfecting agent and a cleaning agent selected from
polycarboxylates, polysulfonates and polyphosphates can be
effectively utilized to both clean and disinfect contact
lenses, provided that this solution is utilized in accordance
with the process described herein. That process includes the
critical steps of: rubbing a small amount of the solution on
both surfaces of the lens, rinsing the lens with the solution,
and then soaking the lens in the solution for a time
sufficient to achieve disinfection.
Accordingly, in one aspect the present invention
provides a process for removing protein deposits from a
contact lens which comprises: rubbing a small amount of a
cleaning solution over the surface of the lens; rinsing the
lens to remove debris loosened by said rubbing; and soaking
the lens in the cleaning solution; wherein the cleaning
solution comprises: a protein cleaning agent consisting
essentially of a compound selected from the group consisting
of polycarboxylates, polysulfonates and polyphosphates in an
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73498-17D
. CA 02257648 1999-O1-15
amount effective to facilitate the removal of protein deposits
from the lens; and an aqueous vehicle therefor.
In another aspect the present invention provides a
process for cleaning and disinfecting a contact lens with a
solution which comprises: rubbing a small amount of the
solution over the surface of the lens; rinsing the lens to
remove proteins and other debris loosened by said rubbing and
soaking the lens in the solution for a time sufficient to
disinfect the lens and further remove protein deposits from
the lens; wherein the solution comprises: an ophthalmically
acceptable antimicrobial agent in an amount effective to
disinfect the lens; a protein cleaning agent selected from the
group consisting of polycarboxylates, polysulfonates and
polyphosphates in an amount effective to facilitate the
removal of protein deposits from the lens; and an aqueous
vehicle therefor.
In another aspect the invention provides a cleaning
solution for removing protein deposits from a contact lens,
which cleaning solution comprises a protein cleaning agent
consisting essentially of a compound selected from the group
consisting of polycarboxylates, polysulfonates and
polyphosphates in an amount effective to facilitate the
removal of protein deposits from the lens; and an aqueous
vehicle therefor.
In another aspect the invention provides a solution
for cleaning and disinfecting a contact lens, which solution
comprises an ophthalmically acceptable antimicrobial agent in
an amount to disinfect a lens, a protein cleaning agent
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73498-17D
CA 02257648 1999-O1-15
selected from the group consisting of polycarboxylates,
polysulfonates and polyphosphates in an amount effective to
facilitate the removal of protein deposits from the lens; and
an aqueous vehicle therefor.
The invention also extends to a commercial package
containing a solution of the invention, together with
instructions for its use in removing protein deposits from
contact lenses in accordance with the invention.
The ability to clean contact lenses effectively by
l0 means of this process is surprising, since prior products for
cleaning contact lenses have typically contained one or more
surfactants. The solution utilized in the process of the
present invention need not contain a surfactant, nor an
enzyme. It has been discovered that a surfactant or an enzyme
is not necessary to achieve a significant degree of cleaning
on mildly deposited lenses. More specifically, it has been
discovered that a solution which contains polycarboxylates
(e.g., citrate), polysulfonates and/or polyphosphates, but no
surfactant and no enzyme achieves a significant degree of
20 cleaning when utilized in accordance with the process of the
present invention.
The use of citrate as a component of various types
of cleaning products is known. For example, it has been used
as a builder in laundry and dishwashing detergents, wherein it
has generally been combined with surfactants to achieve
cleaning. It has also been utilized in denture cleansers,
dentifrices and mouthwashes. The use of citrate in solutions
for disinfecting contact lenses is described in United States
- 4a -
73498-17D
CA 02257648 1999-O1-15
Patent No. 5,037,647. However, that patent describes the use
of citrate as a complexing agent, so as to prevent binding
between polymeric quaternary ammonium compounds and contact
lenses. It does not describe the use of citrate as a cleaning
agent in a process of the type described and claimed herein.
A rinsing, disinfecting and storage solution for contact
lenses known as Opti-Free R (marketed by Alcon Laboratories,
Inc.) contains a citric acid/sodium citrate buffer system, but
this product has not been utilized in a process for cleaning
and disinfecting contact lenses. Similarly, the following
patent publications mention citric acid and/or salts thereof
as possible components of products for treating contact
lenses, but do not disclose the use of these substances in
conjunction with a process for cleaning and disinfecting
contact lenses with a single, surfactant-free solution:
Japanese Patent Publication No. JP 59 45,399 (Kokai Tokkyo
Koho); French Patent No. 2,544,880; and United States Patent
Nos. 4,599,195; 4,609,493; and 4,614,549.
Citrate is believed to enhance the removal of
protein and other deposits through complexation/solubilization
actions. More specifically, citrates are known to complex
with some biological molecules and to render them more water
soluble because of such association. For example, the
interaction of citrate with cationic organic molecules is well
documented; this includes lysozyme, which is cationic at
physiological pH values. Citrate has also been shown to have
the ability to displace lysozyme bound by polymers. In
- 4b -
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WO 95/06099 PCTlUS94.~ :1
terms of cleaning soft contact lenses, citrate is believed to counteract the
binding of
lysozyme by the lens, rendering the protein more soluble in the aqueous media
of the
cleaning solution via complexes (i.e., ion pairs) or salt formation, thereby
facilitating its
removal from the lens when rubbed and rinsed. Additional removal of lysozyme
may also
s take place during the time when lenses are subsequently soaked in the
solution during the
disinfection stage of the process.
Calcium is another common component of soft lens deposits, occurring as
inorganic salts and/or as an element of mixed deposits. In the latter
instance, calcium ions
can act as a "cross bridge" through ionic bonding and link protein, lipid or
mucus-type
io soilants as well as microbial cells contaminating the surface. While
chelation of calcium
by citrate effects removal of discrete inorganic deposits, it is believed that
it may also
have an impact on mixed deposits by disrupting intermolecular bridging,
thereby
weakening the structural integrity of the deposits and making them more
susceptible to the
shearing/dispersing/solubilizing effects of rubbing the solution on the
lenses.
is The cleaning effect achieved with the above-described solutions is
accomplished
by combining: (1) the mechanical effect of rubbing the soiled lenses with a
small amount
of the above-described cleaning and disinfecting solution, (2) the solvent
action of the
water contained in the solution, and (3) the above-described chemical cleaning
mechanisms of citrate or the other polycarboxylates, polysulfonates and
polyphosphates
Zo described herein.
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CA 02257648 1999-O1-15
Description of Preferred Embodiments:
A preferred embodiment of the improved contact lens
cleaning and disinfecting process of the present invention
utilizes an aqueous solution which contains an antimicrobial
agent and a cleaning agent selected from polycarboxylates,
polysulfonates and polyphosphates. Although various
antimicrobial agents may be utilized in the process of the
present invention, the preferred antimicrobial agent is a
polymeric quaternary ammonium agent known as
"polyquaternium-1". This antimicrobial agent is also known as
"Onamer M R" (registered trade-mark of Millmaster Onyx Group)
and "Polyquad R" (registered trade-mark of Alcon Laboratories,
Inc.). The use of this antimicrobial agent to disinfect
contact lenses is described in United States Patents Nos.
4,407,791; 4,525,346; and 5,037,647.
The solutions utilized in preferred embodiments of
the present invention will contain one or more antimicrobial
agents in an amount effective to eliminate or substantially
reduce the number of viable microorganisms present on the
contact lenses being treated, in accordance with criteria
established by the United States Food and Drug Administration
and corresponding health authorities in other countries. Such
an amount is referred to herein as "an amount effective to
disinfect". The amount of antimicrobial agent required for
this purpose may vary depending on the relative activity of
the particular antimicrobial agent selected and other factors
familiar to those skilled in the art, such as the tonicity of
the solution. The preferred polymeric quaternary ammonium
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73498-17D
CA 02257648 1999-O1-15
compounds described above are utilized in concentrations of
from about 0.00001 to about 3.0 percent by weight, based on
the total volume of the solution ("w/v"), preferably from
about 0.0001 to 0.1 w/v%.
The solutions utilized in the process of the present
invention contain a cleaning agent selected from: (1)
polycarboxylates, polysulfonates and polyphosphates; (2) acids
corresponding to these salts; and (3) combinations thereof, in
an amount effective to facilitate the removal of proteins,
calcium and other materials from contact lenses. As utilized
herein, the prefix "poly-" means that the molecule contains
more than one acid/salt group. The polycarboxylates,
polysulfonates and polyphosphates which may be utilized in the
present invention include, for example, citrate, succinate,
tartrate, malonate, maleate, ethanol diglycinate, diethanol
glycinate, polystyrene sulfonate and hexametaphosphate, and
other such compounds having a molecular weight of
approximately 90 to 600. As utilized herein, the terms
"polycarboxylates", "polysulfonates" and "polyphosphates"
include both the acid and salt forms of these compounds, as
well as mixtures thereof. The terms "polysulfonate" and
"polyphosphate" are used in chemistry sometimes in a strict
literal sense and sometimes in a broader, general sense, to
identify compounds that are oxy-acids of sulfur and
phosphorus, respectively, and compounds of such oxy-acids.
For instance, the material that is available from Monsanto
under the trade-mark bequest 2016 is given several chemical
names by The American Chemical Society, including the names
_7_
73498-17D
CA 02257648 1999-OS-OS
tetrasodium (1-hydroxyethylidene)bis-[phosphonate],
tetrasodium 1-hydroxy-1,1-ethane-diphosphonate and tetrasodium
1-hydroxyethylidene-1,1-diphosphate. For the sake of clarity
applicant states that in this specification the terms
"polysulfonate" and "polyphosphate" are used in the broader,
general sense. Similarly, terms such as "citrate",
"succinate", "tartrate", "malonate" and so on, include both
the acid and salt form of the compound, as well as mixtures
thereof. The sodium, potassium and ammonium salts are
preferred. The most preferred polycarboxylate is citrate.
The above-described polycarboxylates, polysulfonates
and polyphosphates are utilized in an amount effective to
clean the lens. This amount will generally be a molar
concentration ranging from 0.013 to 0.13 moles/liter for the
salt forms of the compounds, and a molar concentration
equivalent to that range for the acid forms of the compounds.
The use of a concentration in this range is believed to be
necessary in order to achieve cleaning of contact lenses, as
described herein.
The cleaning and disinfecting process of the present
invention includes three basic steps: cleaning the lenses by
means of rubbing a small amount (e.g., one to four drops) of a
surfactant-free, aqueous solution containing a disinfecting
amount of an antimicrobial agent and one or more of the above-
described cleaning agents over the surfaces of the lenses for
at least 10 seconds, rinsing the lenses thoroughly to remove
all debris, and soaking the lenses in an amount of the same
solution sufficient to completely cover the lenses for a
_ g _
73498-17D(S)
CA 02257648 1999-O1-15
period of from at least four hours to overnight. Suitably the
rubbing is performed by placing the lens in the palm of one
hand, applying the cleaning solution to the lens, and then
rubbing the cleaning solution over both surfaces of the lens
with a finger of the other hand. The lenses are preferably
allowed to soak in a closed container, such as a contact lens
case, and are also preferably rinsed before being replaced in
the eye. The above-described process should be repeated on a
daily basis.
l0 The present invention and the invention of
Application Serial No. 2,146,860 are further illustrated by
means of the following examples, which are presented for
purposes of illustration only and should not be deemed to be
limiting in any way.
Example 1
The following formulation is the preferred
surfactant-free cleaning and disinfecting solution for use in
the process of the present invention.
w v%
20 Polyquad~ 0.001 + 10% excess
Sodium Chloride 0.52
Disodium edetate 0.05
Citric acid monohydrate 0.021
Sodium citrate dehydrate 0.56
Purified Water q.s.
This solution may be prepared as follows. The purified water,
sodium citrate dehydrate, citric acid monohydrate, disodium
edetate, sodium chloride and Polyquad~ are combined
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73498-17D
CA 02257648 1999-O1-15
WO 95106099 PCT/US94/~, .1
and then dissolved by stirring with a mixer. Additional purified water is then
added to
bring the solution to 100%. The pH is then adjusted (if necessary) to pH 7Ø
The following examples demonstrate the cleaning effect of the citrate-
containing
formulations utilized in the present invention.
s FX~~pl~~
An in vitro study was conducted to determine the cleaning effect of the
solution
described in Example 1 above (i.e., Opti-Free~ Rinsing, Disinfecting and
Storage
Solution) on lenses from the four FDA soft lens polymer groupings. The study
compared
the solution's cleaning ability to that of Bausch & Lomb's ReNu~ Multi-Purpose
Solution
to on heavily deposited soft contact lenses. ReNu~ Multi-Purpose Solution
(sometimes
referred to herein as "ReNu~-MPS") is a sterile, isotonic solution that
contains boric acid,
edetate disodium, poloxamine, sodium borate and sodium chloride; it is
preserved with
DYMED (polyaminopropyl biguanide) 0.00005%.
New (i.e., unworn) contact lenses of the following types were utilized in the
study:
is ~ L~ Maaufacttu~ P~lx>z ~atct
~am~ ~n>;ent
I Soflens Bausch & Lomb Polymacon 38%
II B&L 70 Bausch & Lomb Lidofilcon 70
A
Permatlez CooperVision Surfilcon 74
A
2o III Durasoft 2 Wesley-Jessen Phemfilcon 38
A
Hydrocurve II SBH Bufilcon A 45
N Durasoft 3 Wesley-Jessen Phemfilcon 55
A
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CA 02257648 1999-O1-15
'O 95/06099 PCT/US94/0~
*Group I - Low-water-content, nonioaic
polymers
Group II - High-water-content, nonionic
polymers
Group III - Low-water-content, ionic
polymers
Group IV - High-water-content, ionic
polymers
s The lenses were deposited with an artificial tear solution prepared to mimic
common lens deposits. This artificial tear solution was prepared by dissolving
sodium
phosphate and sodium biphosphate in a beaker containing 90% of volume of
purified
water, USP. Dissolution was achieved by stirring on a magnetic stir plate with
a magnetic
stir bar for 10-15 minutes. Lysozyme in an amount to equal 0.05% final
concentration
to by weight was added and allowed to dissolve, following which the solution
was brought
to 100% volume with purified water, USP The pH of the solution was
approximately 7.4
with no adjustment.
The test lenses were rinsed with sterile 0.9% sodium chloride solution and
blotted
dry with lint-free towels, after which the lenses were placed in clean glass
vials. Five
is milliliters (mL) of the artificial tear solution was added to each vial.
The vials were
stoppered and clamped and placed in a preheated water bath and heated at
90°C for 15
minutes. Following the water bath treatment, the vials were removed and
allowed to cool
to room temperature. The lenses were then removed from the vials, rubbed, and
rinsed
with 0.9% sodium chloride solution remove loosely bound protein. The lenses
were
ao placed in clean glass vials with 5 mL of OPTI-PURE~ sterile saline and
stoppered.
The test procedure consisted of an initial rating of the in vitro deposited
lenses by
two trained raters following the FDA recommended Rudko method. After initial
rating
the lenses were transferred to a third person who performed cleaning by
following a
procedure similar to that which is normally recommended for the daily cleaning
of human
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WO 95/06099 PCT/US94! '1
worn lenses: The lenses were placed in the palm of the hand and two drops of
cleaning
solution from the coded bottles were applied to each lens surface. The lenses
were then
gently rubbed for 40 seconds and subsequently rinsed thoroughly with the test
solution.
Deposited Group IV lenses were put through three cleaning cycles for both
solutions since
s neither produced significant cleaning with this group of lenses after just
one cycle of
cleaning. The lens deposits were then rated again using the Rudko method by
the two
raters not involved in the cleaning steps.
The in vitro deposited lenses were rated before and after rubbing and rinsing
by
two technicians who were masked with respect to solutions used. The ratings
were
io converted into numerical scores. The numerical scores for all lenses in a
set were then
added to obtain a cumulative score, and that score was divided by the number
of lenses
in that set to obtain an average score for a lens in that set. The overall
cleaning efficacy
of each solution was computed as a percentage utilizing the average rating of
cleaned
lenses and that of deposited lenses prior to cleaning. The results are
presented below:
is Comc~osite of Comparative Cleaning Efficacy
of O~ti-Free~ Rinsing. DisinfectinE and Storage Solution
and ReNu~ Multi-Purnose Solution
Using Converted Rudko Numerical Scores
Opti-Free~ 60
ReNu~ 36
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CA 02257648 1999-O1-15
O 95/06099 PCT/US94/09~
~u~I P~aflex
Opti-Free~ 10 86 48
ReNu~ 8 65 36
duct I2lua~ft~ ~c~s'~unc~~I ~mimsi~
Opti-Free~ 39 54 46
ReNu~ 77 45 61
to
P~u~.L ~YCI~..l. ~XG1~.2~ ~, c~le 3
Opti-Free~ 3 11 53
t5 ReNu~ 6 19 64
*NO'TE: In a prior study with Group I leases using the same procedures as
those described
herein, the composite cleaning scores for Opti-Free~ and ReNu~ were 58 and 90,
respectively. T6e average cleaning of the Group I lenses based on these two
studies
2o is therefore 59 (Opti-Free~) and 63 (ReNu~).
The foregoing results show that both the solution of Example 1 and ReNu~ Multi
Purpose Solution removed a substantial amount of protein from in vitro
deposited soft
contact lenses. The cleaning efficacy of these two solutions was found to be
dependent
on the lens brand for Groups II and III. Overall, the cleaning efficacy of the
two products
is in this study was comparable.
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CA 02257648 1999-O1-15
WO 95/06099 PCT/US9411 1
A study was conducted to evaluate the role of citrate in cleaning Group IV
contact
lenses (i.e., Durasoft 3). This study compared the cleaning effect of the
solution described
in Example 1 above (i.e., Opti-Free~ Rinsing, Disinfecting and Storage
Solution) with
s modified versions of that solution, and with ReNuO Multi-Purpose Solution.
The
composition of the solutions utilized in the study is presented in the
following table:
Table 1
Comparative Compositions of Test ions'
Solut
96 w/v tions
to 92-2545 in Solu 92-2601 Q Q~ i-Free~
Q~
Citric Acid 0.021 None None None 0.021
Monohydrate. USP
Sodium Citrate. 0.56 None None None 0.56
USP
Disodium EDTA 0.05 0.05 0.05 0.05 0.05
is (Edetate Disodium),USP
Sodium Chloride. 0.48 0.48 0.48 None 0.48
USP
Mannitol, USP None None 1.0 None None
Polyquad, NOC None None None None 0.001
(+10%
excess)
2o Sodium Hydroxide,q.s. to q.s. to q.s. to q.s. to q.s. to
NF
and/or adjust adjust adjust adjust adjust
Hydrochloric Acid, pH to pH to 7.0 pH to 7.0 pH to pH to
NF 7.0 7.0 7.0
Purified Water, q.s. 100 q.s. 100 q.s. 100 q.s. 100 q.s. 100
USP
is The test procedures used in the study are described below.
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CA 02257648 1999-O1-15
-'O 95/06099 PCT/US94/09'
I. Preparation of De~sition Solution
A. p~,~nhate Buffered Saline l"PBS"):
1.311 g of sodium phosphate (monobasic, monohydrate), 5.749 g
sodium phosphate (dibasic, anhydrous), and 0.9 g sodium chloride
s were dissolved in distilled water and the solution was brought to
volume ( 1000 mL) with distilled water. The final concentration of
each component in the solution was: sodium phosphate, 0.05 M;
sodiura chloride, 0.14 M. The final pH was 7.4 (drops of 5N
NaOH or phosphoric acid may be added if pH adjustment is
to needed).
B. ans,~,~~,yme Solution:
750 mg of dansyl-lysozyme was dissolved in 500 mL phosphate
buffered saline. The final concentration of dansyl-lysozyme was
1.5 mg/mL.
is II. ns Deposition Procedure
Each lens was immersed in a Wheaton glass vial (8 mL capacity)
containing 5 mL of dansyl-lysozyme solution (1.5 mg/mL in PBS). The
vials were closed with a plastic snap cap and incubated in a constant
temperature water bath at 37°C for 24 hours. After incubation, each
2o deposited lens was rinsed by dipping into three (3) consecutive beakers
containing 50 mL of distilled water to remove any excess of deposition
solution and loosely bound dansyl-lysoryme, and blotted gently with a
laboratory towel (KayPees). These lenses served as the soiled lenses for
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CA 02257648 1999-O1-15
~JVO 95106099 PCT/US94/ 1
total protein determination (control lenses) as well as for the evaluation of
cleaning efficacies.
III. Protein Determination on Control Lenses
Five (5) deposited lenses were used as control lenses to determine the total
s dansyl-lysozyme deposition on the lenses. For this purpose, each deposited
lens was extracted with 10 mL SDS extraction solution (0.1 M Tris-HCI,
pH 8.0, containing 2% SDS, 0.1 % Dithiothreitol, and 0.1 mM EDTA) in
a screw-capped glass scintillation vial (20 mL capacity). The extraction
was conducted by shaking the vial with a rotary shaker (Red Rotor) at
io room temperature for at least 48 hours. The amount of dansyl-lysozyme
extracted from each lens was assessed by fluorescence measurement with
a fluorospectrophotometer. The total protein was calculated based on the
standard curve established for dansyl-lysozyme solution.
N. Cleaning Procedure
is Lenses were placed in Wheaton glass vials ( 12 mL capacity) containing 5
mL of test solution, with five (5) lenses being used for each test solution.
The vials were capped with plastic snap caps and gently agitated on a
rotary shaker at room temperature for six (6) or twenty-four (24) hours.
The lenses were removed from their respective test solutions after soaking
2o and were rinsed with distilled water as described previously under "Lens
Deposition Procedure." The lenses were then subjected to an extraction
procedure as described under "Protein Determination on Control Lenses."
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CA 02257648 1999-O1-15
~O 95/06099 PCT/US94/09~
Both the test soaking solutions and the lens extraction solution were
subjected to fluorescence measurements for protein determination.
V. Protein Determination
Quantitative determination of protein for the soaking solutions and the lens
s extracts were carried out by use of a fluorospectrophotometer. Two (2) mL
of solution were required for each measurement. The fluorescence
intensity was measured by setting the excitation/emission wavelength at
252 nm/530 nm with excitation/emission slits of 2.0 nm/8.5 nm. Dansyl-
lysozyme concentrations for each solution and extract were calculated
~o based on the slope established from linear standard dansyl-lysozyme curves
developed under the identical instrumental conditions for SDS-extraction
buffer and phosphate buffered saline respectively.
The following table shows a summary of the cleaning results for the six (6)
and
twenty-four (24) hours soaking for each test solution.
is Table 2
Time 92-2600 92-2601 92-2603 ~ti:~.~ BeNa~-~s
6 Hours 65.08 24.26 23.57 24.30 71.83 36.79
t 1.66 t 0.90 t 1.25 t 1.60 t 2.15 t 2.53
20 24 Hours 87.67 28.01 28.64 37.92 100.92 44.53
* 1.60 t 0.85 t 1.23 t 3.08 t 1.85 t 3.61
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CA 02257648 1999-O1-15
WO 95106099 PCT/US941t .I
The results of this study indicate that Opti-Free~ and Solution 92-2545, both
containing
citrate, removed more dansyl-lysozyme from lenses than Solutions 92-2603, 92-
2b00, and
92-2601, all of which contained no citrate. The differences were significant
based upon
statistical analysis (p < 0.05). Opti-Free~ and Solution 92-2545 (Opti-Free~
minus
s Polyquad~) also removed more dansyl-lysozyme from the deposited lenses than
did
ReNu~ Multi-Purpose Solution, evaluated under the same experimental
conditions. These
results were also statistically significant (p < 0.05).
The cleaning results observed during a prior experiment involving the same
solutions and procedures were substantially similar to the results presented
above. The
io results observed during the prior experiment are presented in the following
table:
Time 92-2545 22~2t~ 9Z?.~l 92-2603 '-Free~ Bed
6 Hours 68.36 26.62 27.98 28.46 72.68 43.10
15 1 1.72 1 1.15 1 1.59 1 1.61 1 1.83 1 1.37
24 Hours 82.28 36.16 34.00 47.32 92.12 45.00
t 7.87 t 1.55 t 1.12 t 1.28 t 3.71 t 0.88
Another study similar to the study described in Example 3 above, but which
2o involved somewhat different procedures, was also conducted to further
evaluate the role
of citrate in cleaning Group IV contact lenses (i.e., Durasoft 3). The study
evaluated the
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cleaning effect of four of the same solutions tested in Example 3 (i.e., 92-
2545; 92-2600;
92-2601; and 92-2603). The procedures used in this study are described below.
I. Per .ration of Denotition Solution
A. Composition
s y~ W° ww
Lysozyme 0.15
Sodium Phosphate, Basic 0.689
Sodium Chloride 0.9
Sodium Hydroxide, SN q.s. pH
7.4
io Purified Water q.s. 100
B. Procedure
Sodium chloride was dissolved in a beaker containing 80% of the total required
volume of purified water. Sodium phosphate was added and dissolved while
stirring. Lysozyme was then added and allowed to dissolve. The pH of the
is solution was adjusted to 7.4 with sodium hydroxide, and the volume of the
solution was adjusted with water.
II. ~gRPrimental Procedure
A. $sd_x Design
Lenses were numbered from one to ninety. After deposition, ten lenses were
2o not cleaned and used as control lenses to determine average lysozyme uptake
per lens. The remaining eighty lenses were divided into four groups of twenty
lenses and each group was cleaned with the respective test solutions. Ten
cleaning solution samples from each group were pulled for analysis after six
hours and the remaining ten from each group after 24 hours.
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B. 1 ins DeRosition Procedure
Each lens was immersed in a glass vial containing 5 mL of the deposition
solution. Vials containing the deposition solution and lenses were incubated
for 24 hours at 37°C. Each deposited lens was rinsed by dipping into
three (3)
s consecutive beakers containing 50 mL of purified water to remove any excess
of deposition solution and loosely bound protein.
C. Control
Ten deposited lenses were used as control lenses to determine lysozyme
deposition on the lenses. After rinsing as described above these were gently
blotted to remove any excess water and placed into screw cap glass culture
tubes. The tubes were stored in the freezer until analysis. Additionally, five
(5) new lenses were included to provide non-deposited lens values for the
ninhydrin procedure described below.
D. CleaninE Procedure
is Six Hour Same
Ten lenses per test solution were gently blotted to remove excess water
following rinsing. The lenses were placed in plastic vials containing 5 mL of
test solution. The vials and their contents were gently agitated for six (6)
hours. The lenses were removed from their respective test solutions after
zo cleaning and the solutions refrigerated until analyzed.
Ten lenses per test solution were gently blotted to remove excess water
following rinsing. The lenses were placed in plastic vials containing 5 mL of
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test solution. The vials and their contents were gently agitated for twenty-
four
(24) hours. The lenses were removed from their respective test solutions after
cleaning and the 24 hour cleaning solutions along with the control lenses and
six hour cleaning solutions were assayed for protein using the ninhydrin
s procedure.
E. Protein A-n~3rcis of Solutions and Control Lenses
The samples were evaluated by means of a ninhydrin assay procedure. In this
procedure, proteins are hydrolyzed under alkaline conditions to their amino
acid components which react with ninhydrin to form a colored complex. The
io latter can be quantitated by measuring absorbance at 570 nm. For this study
a standard curve was prepared covering a range of 5.00 to 15.00 ~g lysozyme.
The curve had a slope of 0.050 AU/~g, a y intercept of 0.021 AU and a R-
squared value of 0.9811. The quantity of protein in a sample was determined
using this standard curve and multiplying by appropriate dilution factors.
is The following table shows a summary of the cleaning data for the six (6)
and
twenty-four (24) hours cleaning:
92-2545 92-2600 92-2601 ~2-2603
6 Hours 47.52 -6.40 15.02 2.20
20 24 Hours 52.92 -2.33 -11.08 19.62
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This study shows that significantly (p <0.05) more lysozyme is cleaned from
lenses
when citrate is present than when it is absent. Thus, the results demonstrate
that citrate
has cleaning properties that are effective in removing lysozyrae from contact
lenses.
In another cleaning study, which used a HPLC assay procedure instead of the
s above-described ninhydrin method, similar results were obtained, as shown in
the
following table:
6 Hours 39.0 6.6 6.2 3.7
,0 24 Hours 95.3 10.6 7.7 5.5
While there is not absolute agreement between these two studies (in part due
to
substraction of large blank value and the lesser precision associated with the
ninhydrin
assay) with all solutions, both studies clearly show that the 92-2545
solutions contain
significantly higher levels of lysozyme than the others. Thus, the importance
of citrate
is in removing protein deposits is demonstrated by these studies.
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