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Title:
A METHOD FOR REMOVING EXOGENOUS DEPOSITS FROM HYDROPHILIC CONTACT LENSES
Document Type and Number:
WIPO Patent Application WO/1991/001820
Kind Code:
A1
Abstract:
The present invention describes a method and composition for removing exogenous mucin and protein-mucin deposits from hydrophilic contact lenses. Alpha-amylase and a protease are used in the composition.

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Inventors:
WEDLER FREDERICK C (US)
Application Number:
PCT/US1990/004317
Publication Date:
February 21, 1991
Filing Date:
August 01, 1990
Export Citation:
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Assignee:
PENNSYLVANIA RES CORP (US)
International Classes:
C11D3/00; C11D3/386; (IPC1-7): B08B11/00
Foreign References:
US4614549A1986-09-30
Other References:
CHEMICAL ABSTRACTS, Volume 100, issued 1984, (Columbus, Ohio, USA), KREINER, "Enzymic Contact Lens Clearing Product with pH-Controlled Activity", see page 316, Col. 2, Abstract 39653y; & EP,A,93 784.
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Claims:
I CLAIM:
1. A method for removing mucin and mucincontaining deposits from a contact lens which comprises placing a contact lens having a mucin or mucin containing deposit in contact with a solution comprising an effective amount of a mucindegrading enzyme sufficient to remove the mucin or mucincontaining deposit and for a time sufficient to allow for said removal.
2. The method of Claim 1 wherein the lens is a hydrophilic contact lens.
3. A method according to Claim 1 wherein the mucin degrading enzyme is alphaamylase.
4. A method according to Claim 3 wherein the solution may further contain a protease suitable for removing a proteinaceous deposit from a contact lens.
5. A composition for removing a mucin or mucincontaining deposit from a contact lens which comprises an enzyme effective to remove mucin or a mucin containing deposit from said lens.
6. A composition according to Claim 5 which further comprises a protease suitable for removing a proteinaceous deposit from a contact lens.
7. A composition according to Claim 5 wherein the enzyme is alphaamylase.
Description:
"A METHOD FOR REMOVING EXOGENOUS DEPOSITS FROM HYDROPHILIC CONTACT LENSES "

With the advent of hydrophilic or soft contact lenses, following the successful experiments of Wichterle and Seiderman reported in United States patent numbers 2, 979, 576 and 3, 21, 657, respectively, the practitioner was given another means to correct visual impairments in his patient population. The main virtues of these lenses were their ease of manufacture, their complete transparency and their increased comfort to the user when compared with hard plastic lenses developed much earlier. The earliest soft lenses offered commercially in the 1970's were made from polymeric polar monomers, e.g., hydro∑yethylmethacrylate (HEMA), polymethyimethacrylate (PMMA), or polyvinyipyrrolidone (PVP) polymeric materials having the appearance of soft, transparent hydrogeis. Within the past several years, however, other materials such as various silicone based polymers have become available and used for the manufacture of soft lenses.

The increase comfort experienced by the user which has resulted in near universal acceptance of soft contact lenses is brought about by the ability of the lens to absorb water. These lenses, when viewed under high power microscopy, appear as a highly porous matrix. When the lens is swelled in aqueous solutions prior to its initial use by the patient, this polar matrix allows the lens to absorb large quantities of water, in excess of 100 per cent of the weight of the dry lens. Consequently when placed on the eye, the user does not experience the discomfort of a foreign object in the eye but rather experiences the somewhat cooling sensation of the additional fluid being added to the eye.

The water-compatible properties which provide user comfort also are the basis for binding of exogenous materials, leading to formation of deposits on the anterior (air-exposed) surface of the lens. Deposit buildup may be exacerbated by conventional aseptization methods, with hazing of vision, loss of optical acuity, and moderate to severe eye irritation. Deposit formation is primary cause of dissatisfaction by roughly 1/3 of the lens wearing population to adapt successfully to soft contact lenses. These statistics also make it clear that currently available cleaning methods are i adequate for dealing with problems experienced by patients classified as "heavy" depositors.

In the early 1970's it was first demonstrated that deposits on hydrophilic lenses contained proteins found in the normal human tear fluid. These data were summarized in a 1982 review article by F.C. Wedler & T. Riedhammer, "Soft Contact Lenses: Formation of Deposits," lin CRC Critical Re iews on Biocompatibility, Vol. II, chapter 3, pp. 31 -46, CRC Press, Boca Raton, FL] the disclosure of which is hereby incorporated by reference. These findings .also refuted the belief that these deposits were bacterial plaques which would, if the "contaminated" lens were not replaced, cause infection and damage to the eye. Until fairly recently, methods for separation, purification, detection, and quantification of sub-microgram levels of bio-materials obtained form soft contact lenses did not exist. In 1987, biochemical techniques were developed for quantitative analysis of biomaterials deposited on single patients lenses [F.C. Wedler, D. Horensky, B.L. Iilman, & M. Mowrey-Mckee. "Analysis of protein and mucin components deposited on

a single hydophilic contact lenses," Clin. Exptl. Opto ., 70: 59-68]. Substantial amounts of tear fluid proteins were detected on "normal" patient lenses with which hazing and eye irritation were not observed. The four major tear fluid proteins detected in these studies were albumin, lysozyme, lactoferrin, and pre-albumin. The most important discovery arising from this works was that "heavy" lens deposits did not correlate with deposition of tear fluid proteins, but did coordinate strongly with mucin, a heterogeneous mixture of derivatized polysaccharides.

Prior to this finding, it was believed that proteinaceous materials were the main cause of irritating lens deposits, and based on this, protein- degrading enzymes (proteases) were used in cleaning solutions to remove these irritating deposits. Indeed, United States patent 3.910, 296 discloses and claims the use of protease-containing solutions for soft contact lens cleaning. Included in this formulation were sulfhydryl-group containing compounds, needed to activate the protease (papain) and which could also reduce disulfide bonds in the protein substrate, but which have an offensive "rotten egg" odor.

A number of subsequent disclosures have sought to improve on the basic concept of protease-based cleaners. These additional disclosures suggest that other substituents be added to the cleaning solution, that the condition under which cleaning occurs be adjusted, or both. For example, United States patent 4,096,870 suggests the use of the digestive aid pancreatin, a crude mixture of hydrolytic enzymes extracted from hog pancreas, formulated in combination with boric acid and sodium chloride as a cleaning mixture. United States patent 4, 285.738 suggest the use of

a hypertonic solution of urea and/or a guanidine salt added to the protease formulation, along with a sulfhydryl compound or other suitable reducing reagent capable of cleaning disulfide bonds.

Commercially available products for enzymatic cleaning of soft(hydroρhilic) contact lenses include, for example, OPTI-ZYME"' (Alcon Laboratories) ' based on porcine pancreatin as the active ingredient, and ALLERGAN ENZYMATIC", EXTENZYME™, and PROFREE/GP™ (Aliergan Pharmaceuticals) based on papain.

Now that the major cause of extraneous heavy lens deposits is known to be mucin, not proteins, it becomes clear why the majority of currently available commercial enzyme-based cleaners fail to remove heavy deposits. Although the enzymes contained in these cleaners will specifically attack and degrade proteinaceous materials, they are ineffective against mucin, which is a heterogeneous mixture of complex carbohydrates (mucopolysaccharides) and carbohydrate surrounding a protein core (glycoproteins).

Since presently available enzyme-based cleaning solutions fail to degrade or remove mucin deposits from soft contact lenses, there is obviously a need to develop a new, second-generation cleaner, based on mucin-degrading enzymes. These mucin degrading enzymes could be used either alone or in combination with proteases to enhance the cleaning of heavily deposited hydrophϋic lenses.

Essential for proving the efficacy of any composition or method for degrading and removing mucin from hydrophilic lenses are sensitive, accurate assay methods. Two such methods were developed in the

present work, both based on use of radioactively labeled mucin or mucin/protein. The first involves deposition onto new hydrophilic lenses of a synthetic mixture of components designed to model human tear fluid, including radiolabeled mucin. The second is based on direct radiolabeling of mucin and other components deposited on patient-worn lenses.

Based on the known composition of human tear fluid, a synthetic solution to mimic human tear fluid was formulated with the following pure constituents - obtained commercially (Sigma Chemical Co.) or purified according to published procedures - in PBS (phosphate- buffered saline: 10 mM phosphate, pH 7.4, 3 mM C1, 120 mM NaCl):

0.225 mg/ml albumin (bovine serum) 20 mM C1

0.075 mg/ml lysozyme (hen egg white) 1.0 mM CaCl2

0.075 mg/ml lactoferrin (bovine colostrum) 0.5 M MgCl2 0.010 mg/ml pre-albumin (human serum) 0.1 mM MnCl2 40 mg/ml I 14 C] - labeled mucin 0.1 mM ZnCl2

The radiolabeled mucin was produced by succinylation of bovine submaxiiiary mucin (75 mg/ml) in pH 8.0 carbonate buffer with 0.25 M [ ! C]-succinic anhydride (250 Ci).

Protein-mucin model deposits were produced by applying this synthetic tear fluid mixture to new (unworn) hydrophilic contact lenses according to the following example:

EXAMPLE I

Preparation of Model Mucin/Protein-deoosits on Hydrophilic Lenses: Typically, a group of 12 lenses were presoaked for 1 hr in PBS.

Lenses were then blotted dry and placed individually in wells of a porcelain spot-test plate. Over the exposed concave surface of each lens, a total of 0.1 ml of the above synthetic tear fluid/mucin mixture was then applied in 5 x 0.02 ml aliquots, distributed evenly with the end of a fire pohshed glass rod and evaporated to dryness in a stream of warm air, without drying the interior portions of the lenses. Deposited lenses were soaked and stored individually in 2 ml of PBS solution.

Patient-worn hydrophilic lenses, classified as being "heavily" deposited, were subjected to direct in vitro labeling with [14 CJ- succinic anhydride. This approach, which [14 c]- labels both protein and mucin components, was carried out according to the following example:

EXAMPLE II

Radioactive Succinylation of Mucin/Protein on Patient -worn Lenses: Typically, 12 patient -worn, heavily deposited lenses were presoaked in pH 8.0 carbonate buffer for 1 hr, blotted dry, and placed individually, with the anterior (deposited) side down, in the wells of a porcelain spot-test plate, each of which contained 25 1 of 0.25M [14 fl- succinic anhydride (250 Ci/ml) dissolved in anhydrous dioxane, and were allowed to react for 30 min at room temperature. The lenses were then each soaked individually in 3 x 100 ml of PBS to dissolve and dialyze away any unreacted labeled small molecules, after which each was stored in a vial with 1.0 ml PBS.

Studies were undertaken to determine the efficiency with which certain enzymatic agents can remove mucin and other components from hydrophilic contact lenses, using radiolabled deposits produced according to the methods described in either Example I or Example II above. A number of the candidates tested for ability to remove mucin were enzymes, commercially available in quantity, that were specific for a variety of carbohydrate polymers. The most likely ones included two enzymes known to degrade the constituents of mucin (glycoproteins and mucopolysaccharides), neuraminidase and hyaluronidase, both isolated from pathogenic micro-organisms. In addition, a number of other hydrolytic enzymes known to degrade carbohydrate polymers were tested, including pectinase and two amylases.

The general assay procedure for these studies is outlined in the following example:

EXAMPLE III

Assay for Removal of Mucin from Hvdroohilic Lenses: In a typical test for mucin removal, model deposited lenses (5 per group), prepared as described in Example I, were presoaked in PBS for 1 hr, then cut into two equal pieces. As a control, the first ("before") half lens was soaked in PBS lacking enzyme for 1 hr at 30 * C. The second ("after") half was placed in 1.0 ml of PBS containing the desired mixture of enzymes (see below) and was then incubated for 1 hr at room temperature. The amount of [14 c]- mucin remaining on a half lens was determined by heating it at 95' C for 30 min in 2.0 ml PBS containing 1 % sodium dodecyl sulfate and 2 mM dithiothreitol, then counting this solution in a water-compatible liquid scintillation cocktail. The amount of I * 4 CJ- mucin remaining on the "before" portion of the lens was taken as 100% and was compared to that on the "after" sample.

A number of different hydrolytic enzymes were tested as ' possibie candidates for mucin removal. Surprisingly, the two enzymes reported to be specific for mucin components, neuraminidase (Sigma, type V) and hyaluronidase (Sigma, type II), used individually or in combination, removed less than 5% of the mucin in model deposits or on patient-worn lenses. These negative results, plus the difficulties involved in providing these enzymes as a safe commercial product, economically priced for the consumer, indicated the need to identify an alternative enzyme system, commercially available in quantity, that can effectively remove mucin deposits from hydrophilic contact lenses.

The data in Table I also indicate that protease alone was relatively ineffective in removal of these bio-materials, especially with patient- worn lenses. Of the commercially available hydrolytic enzymes tested for ability to remove mucin or protein/mucin deposits, the most dramatic (but unexpected) positive results were produced by alpha-amyiase. In addition, alpha-amylase combined with subtilisin (used in currently available enzyme-based lens cleaners) exhibits enhanced or synergistic removal of mucin/protein deposits.

TABLE I

Tests of Enzvmic Removal of Mucin/Protein from Hydrophilic Lenses

%Removed/hr odel deposit Patient lenses

Separate tests were performed with model deposited lenses (Example I) to determine the effect of time and increased units of enzyme on the extent of deposit removal for the enzymes alpha-amylase and subtilisin, as shown in Table II:

TABLE II

Time Dependence of Enzvmic Mucin Removal from Hydrophilic Lenses

% Removed

30 -

42

70

47 57 65 78 92

The data reported in Tables I & II indicate that the commercial protease-based cleaner removed less than 50% of the deposit in 2 hr, but that 10 mg of alpha-amyiase (specific activity lOOOU/mg) alone was capable of removing 65% of the deposited mucin in 2 hr and more than 90% in 5 hr. The time required for complete (100%) removal could, of course, be shortened by using an increased amount of alpha-amylase.

Alpha-amylase has been demonstrated to be specific for internal alpha- 1,4 glucan bonds of linear homologous polymers of underivatized D-glucose [T. Tagaki, T. Hiro, & T. Isemura ( 1971 ) in "The Enyzmes", 3rd edn. (P. D. Boyer, ed), vol. V, pp. 235-271, Academic Press, New York]. Mucin, however, is a highly heterogeneous, branched mixture of mucopolysaccharides and glycoproteins, composed of highly derivatized

saccharides linked with alpha- and beta- 1,3- and 1,4- type glucan bonds. Based on this, the finding that alpha-amylase is efficacious for removal of mucin and mucin/protein deposits from hydrophilic contact lenses is clearly not one that would be obvious to a worker of ordinary skill in the art.

As a preparation for the removal of mucin or mucin/protein deposits from hydrophilic contact lenses, the hydrolytic enzymes may be compounded alone or in combination with other existing cleaning systems for hydrophilic contact lenses. For example, the data in Table II, taken with those in Table I, indicate the enhanced effect of using alpha-amylase plus subtilisin for removal of heavy mucin/protein deposits. By "compounded" is meant that the mucin degrading enzymes may have additional materials such as conventional excipients, antimicrobial agents, buffers, stabilizers, or other materials conventionally used with hydrophilic contact lens cleaners in order to increase the shelf li e of a commercial product, prevent damage to the lens material, make the cleaning composition more acceptable to the user, or allow for the cleaning composition to be manufactured in a specific form such as tablets, liquids, or powders. Of course, since these lenses must be maintained in aqueous solution, the cleaning system according to the present invention will be used as a solution, so that should the enzyme be compounded and sold as a tablet or powder, it will be necessary for the tablet or powder to be dissolved into an aqueous solution prior to its use. The exact amount of mucin-degrading enzyme present in the cleaning system any vary over a wide range, the amount depending upon the

speed of cleaning desired. Neither the additional materials (which are well known to persons aware of the contact lens cleaning art) nor the amounts of mucin-degrading enzymes (which are a matter of choice to suit the specific purposes of the manufacturer) used in a hydrophilic contact lens . cleaning system according to the present invention are necessarily critical. The amount of mucin-degrading enzyme present in a given formulation will necessarily be determined by the amount needed to clean the lens within the period of time chosen, as preferred by the manufacturer to satisfy the customer's needs. Thus, while I have illustrated and described the preferred embodiments of my invention, it is to be understood that this invention is capable of variation and modification, and I therefore do not wish to be limited to precise terms set forth, but desire to avail myself of such changes and alterations which may be made for adapting the invention to various usages and conditions. Accordingly, such changes and alterations are properly intended to be within the purview of the following claims.

Having thus described my invention and the manner and process of making and using it, in such full clear, concise and exact terms so as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same: