(12) United States Patent
`Asgharian et al.
`
`I lllll llllllll Ill lllll lllll lllll lllll lllll 111111111111111111111111111111111
`US006228323Bl
`US 6,228,323 Bl
`May 8, 2001
`
`(10) Patent No.:
`(45) Date of Patent:
`
`(54) MULTI-PURPOSE COMPOSITIONS
`CONTAINING AN ALKYL-TRYPSIN AND
`METHODS OF USE IN CONTACT LENS
`CLEANING AND DISINFECTING
`
`(75)
`
`Inventors: Bahram Asgharian; Bor-Shyue Hong,
`both of Arlington, TX (US)
`
`(73) Assignee: Alcon Laboratories, Inc., Fort Worth,
`TX (US)
`
`( *) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl. No.: 09/144,673
`
`(22) Filed:
`
`Sep. 1, 1998
`
`Related U.S. Application Data
`
`(63) Continuation-in-part of application No. PCT/US97/21579,
`filed on Nov. 24, 1997.
`(60) Provisional application No. 60/032,839, filed on Dec. 13,
`1996, now abandoned.
`
`Int. Cl.7 ........................................................ A61L 2/00
`(51)
`(52) U.S. Cl. ........................... 422/28; 252/106; 424/94.1;
`424/94.3; 424/264; 435/264
`(58) Field of Search .............................. 422/28; 424/94.1,
`424/94.3, 264; 435/264; 252/106
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`5/1988 Huth et al. .
`Re. 32,672
`3,402,855 * 9/1968 Schroeder et al. ..................... 222/83
`3,613,955 * 10/1971 Wetherell, Jr .......................... 222/83
`3,731,844 * 5/1973 Baker ................................. 222/83 X
`3,910,296
`10/1975 Karageozian et al. .
`3,924,741 * 12/1975 Kachur et al. ....................... 206/221
`4,407,791
`10/1983 Stark.
`4,525,346
`6/1985 Stark .
`4,614,549
`9/1986 Ogunbiyi et al. .
`4,758,595
`7/1988 Ogunbiyi et al. .
`4,836,986
`6/1989 Ogunbiyi et al. .
`5,096,607
`3/1992 Mowrey-McKee et al. .
`
`5,281,277
`5,409,546
`5,419,445 *
`5,421,483 *
`
`1/1994 Nakagawa et al. .
`4/1995 Nakagawa et al. .
`5/1995 Kaesemeyer ..................... 206/220 X
`6/1995 Parise ..................................... 222/82
`
`(List continued on next page.)
`
`FOREIGN PATENT DOCUMENTS
`
`1 150 907
`0 384 666
`0 456 467
`0 646 641
`57-24526
`92-143718
`92-243215
`92-370197
`WO
`89/111878
`WO 94/06479
`WO 96/40854
`WO 97/18288
`WO 98/25650
`
`8/1983 (CA).
`8/1990 (EP).
`11/1991 (EP).
`4/1995 (EP).
`5/1982 (JP).
`5/1992 (JP).
`8/1992 (JP).
`12/1992 (JP).
`
`12/1989 (WO).
`3/1994 (WO).
`12/1996 (WO).
`5/1997 (WO).
`6/1998 (WO).
`
`OTHER PUBLICATIONS
`
`Lo, J.; Silverman, H.; and Korb, D.; Studies on cleaning
`solutions for contact lenses, Journal of the American Opto(cid:173)
`metric Association, vol. 40, pp. 1106-1109 (1969).
`Means, GE and Feeney, RE. Reductive alkylation of amino
`groups in proteins, Biochemistry, vol. 7, pp. 2192-2201
`(1968).
`Rice, RH, Means, GE and Brown, WD. Stabilization of
`bovine trypsin by reductive methylation, Biochimica et
`Biophysica Acta, vol. 492, pp. 316-321 (1977).
`
`Primary Examiner---Krisanne Thornton
`(74) Attorney, Agent, or Firm-Michael C. Mayo
`
`(57)
`
`ABSTRACT
`
`Two-compartment bottle assemblies useful in preparing
`multi-purpose compositions containing an Al-trypsin and
`disinfectant, methods of preparing these compositions and
`methods involving the use of these compositions are dis(cid:173)
`closed for cleaning and disinfecting of contact lenses.
`
`33 Claims, 7 Drawing Sheets
`
`25-d',
`:l-s
`
`24--
`
`- I - ~'-34
`39
`154_1_>---22
`
`"o"
`
`"--14
`I - s
`-'-1s
`
`z30--
`1s- -
`17'
`10/-,
`13--
`
`ANCESTRY EX. 1014
`
`1
`
`

`
`US 6,228,323 Bl
`Page 2
`
`U.S. PATENT DOCUMENTS
`
`5,576,278
`5,604,190
`5,605,661
`5,672,213
`
`11/1996 Van Duzee et al. .
`2/1997 Chowhan et al. .
`2/1997 Asgharian et al. .
`9 /1997 Asgharian et al. .
`
`2/1998
`5,718,895
`3/1998
`5,723,421
`7/1998
`5,785,767
`10/1998
`5,820,696
`* cited by examiner
`
`Asgharian et al. .
`Chowhan et al. .
`Kimura et al. ......................... 134/42
`Kimura et al. ......................... 134/42
`
`2
`
`

`
`U.S. Patent
`
`May 8, 2001
`
`Sheet 1 of 7
`
`US 6,228,323 Bl
`
`2
`
`FIG. 1
`
`3
`
`

`
`U.S. Patent
`
`May 8, 2001
`
`Sheet 2 of 7
`
`US 6,228,323 Bl
`
`~->~5
`
`4
`
`I 11
`
`, 1 I
`I
`- - -
`
`2
`
`FIG. 2
`
`'-----___;> .... 5
`
`4
`
`

`
`U.S. Patent
`
`May 8, 2001
`
`Sheet 3 of 7
`
`US 6,228,323 Bl
`
`----->-4
`
`-.,_'r=......,.,..__~34
`22
`~~36
`~::c::Ii~ 1 4
`5
`
`FIG. 3
`
`2
`
`L--->-4
`
`5
`
`

`
`U.S. Patent
`
`May 8, 2001
`
`Sheet 4 of 7
`
`US 6,228,323 Bl
`
`4
`
`2
`
`FIG. 4
`
`1 1
`
`9
`
`6
`
`

`
`U.S. Patent
`
`May 8, 2001
`
`Sheet 5 of 7
`
`US 6,228,323 Bl
`
`4
`
`1 1
`
`9
`
`2 ------ -----
`
`4
`
`2
`
`22 .· .
`. . . .
`
`---- 1a----
`
`---1--- --
`
`-
`· - -
`· - -
`- -
`- - - - - -
`- -- 49 - -- -
`-
`
`FIG. 5
`
`FIG. 6
`
`7
`
`

`
`U.S. Patent
`
`May 8, 2001
`
`Sheet 6 of 7
`
`US 6,228,323 Bl
`
`23
`
`29 27
`
`5
`
`7
`
`19
`
`19
`
`FIG. 8
`
`8
`
`

`
`U.S. Patent
`
`May 8, 2001
`
`Sheet 7 of 7
`
`US 6,228,323 Bl
`
`Comparison of the Proteolytic Stability of
`Me-trypsin, Trypsin and Pancreatln in a
`Multi-Purpose Solution at 30°C
`
`100
`
`80
`
`-+-Methyl Trypsin
`
`-Native Trypsin ,
`-+- Pancreatln
`
`0) c
`·-c ·;
`E 60
`G>
`0:::
`b ·:;:
`;:;
`CJ
`4(
`::I! D
`
`40
`
`20
`
`0
`
`0
`
`7
`
`14
`
`21
`
`28
`
`35
`
`42
`
`Days
`
`FIG. 9
`
`9
`
`

`
`US 6,228,323 Bl
`
`1
`MULTI-PURPOSE COMPOSITIONS
`CONTAINING AN ALKYL-TRYPSIN AND
`METHODS OF USE IN CONTACT LENS
`CLEANING AND DISINFECTING
`
`5
`
`This application is a continuation-in-part of patent appli(cid:173)
`cation Ser. No. PCT/US97/21579, filed Nov. 24, 1997;
`which claims priority to U.S. Provisional Patent Application
`Serial No. 60/032,839, filed Dec. 13, 1996, now abandoned.
`BACKGROUND OF THE INVENTION
`The present invention relates to the field of contact lens
`cleaning and disinfecting. In particular, this invention is
`directed to the provision of alkyl trypsin containing multi(cid:173)
`purpose compositions and methods for the preparation of
`these compositions. The invention is also directed to meth(cid:173)
`ods of simultaneously cleaning and disinfecting contact
`lenses by using alkyl trypsin and disinfectant containing
`multi-purpose compositions of the present invention.
`Various compositions and methods for cleaning contact
`lenses have been described in the patent and scientific
`literature. Some of these methods have employed composi(cid:173)
`tions containing surfactants or enzymes to facilitate the
`cleaning of lenses. The first discussion of the use of pro(cid:173)
`teolytic enzymes to clean contact lenses was in an article by
`Lo, et al. in the Journal of The American Optometric
`Association, volume 40, pages 1106-1109 (1969). Methods
`of removing protein deposits from contact lenses by means
`of proteolytic enzymes have been described in many pub(cid:173)
`lications since the initial article by Lo, et al., including U.S.
`Pat. No. 3,910,296 (Karageozian, et al.).
`Numerous compositions and methods for disinfecting
`contact lenses have also been described. Those methods may
`be generally characterized as involving the use of heat
`and/or chemical agents. Representative chemical agents for
`this purpose include organic anti-microbials such as benza(cid:173)
`lkonium chloride and chlorhexidine, and inorganic anti(cid:173)
`microbials such as hydrogen peroxide and peroxide(cid:173)
`generating compounds. U.S. Pat. Nos. 4,407,791 and 4,525,
`346 (Stark) describe the use of polymeric quaternary 40
`ammonium compounds to disinfect contact lenses and to
`preserve contact lens care products. U.S. Pat. Nos. 4,758,
`595 and 4,836,986 (Ogunbiyi) describe the use of polymeric
`biguanides for the same purpose.
`Various methods for enzymatic cleaning and disinfecting 45
`contact lenses at the same time have been proposed. Meth(cid:173)
`ods involving the combined use of proteolytic enzymes and
`peroxides to clean and disinfect contact lenses
`simultaneously, are described in U.S. Pat. No. Re 32,672
`(Huth, et al.). A representative method of simultaneously 50
`cleaning and disinfecting contact lenses involving the use of
`proteolytic enzymes and quaternary ammonium compounds
`is described in Japanese Patent Publication 57-24526
`(Boghosian, et al.). The combined use of a biguanide (i.e.,
`chlorhexidine) and liquid enzyme compositions to simulta- 55
`neously clean and disinfect contact lenses is described in
`Canadian Patent No. 1,150,907 (Ludwig, et al.). Methods
`involving the combined use of dissolved proteolytic
`enzymes to clean and heat to disinfect are described in U.S.
`Pat. No. 4,614,549 (Ogunbiyi). The combined use of pro- 60
`teolytic enzymes and polymeric biguanides or polymeric
`quaternary ammonium compounds is described in
`copending, commonly assigned U.S. patent application Ser.
`No. 08/156,043 and in corresponding European Patent
`Application Publication No. 0 456 467 A2 (Rosenthal, et 65
`al.), as well as in U.S. Pat. No. 5,096,607 (Mowrey-McKee,
`et al.).
`
`10
`
`2
`The commercial viability of most prior enzymatic clean(cid:173)
`ing products has depended on the use of stable enzyme
`tablets. More specifically, the use of solid enzymatic clean(cid:173)
`ing compositions has been necessary to ensure stability of
`the enzymes prior to use. In order to use such compositions,
`a separate packet containing a tablet must be opened, the
`tablet must be placed in a separate vial containing a solution,
`and the tablet must be dissolved in order to release the
`enzyme into the solution. This practice is usually performed
`only once a week due to the cumbersome and tedious
`procedure and potential for irritation and toxicity.
`The use of concentrated liquid enzyme compositions in
`combination with a diluent to clean contact lenses has been
`attempted in an effort to avoid the cumbersome use of
`enzyme tablets. Those attempts, however, have been ham-
`15 pered by the fact that concentrated aqueous liquid enzyme
`compositions are inherently unstable. When a proteolytic
`enzyme is placed in an aqueous solution for an extended
`period (i.e., several months or more), the enzyme may lose
`all or a substantial portion of its proteolytic activity. Steps
`20 can be taken to stabilize the compositions. For example,
`stabilizing agents can protect enzymes from chemical insta(cid:173)
`bility problems during storage in an aqueous liquid, by
`placing the enzymes in a dormant physical conformation.
`However, the use of liquid enzyme compositions, as with the
`25 use of enzyme tablet compositions described above, still
`requires a separate, additional mixing step each time the lens
`is to be simultaneously cleaned and disinfected.
`Furthermore, since the amount of liquid enzyme composi(cid:173)
`tion placed in a diluting composition is controlled by the
`30 user, user error may result in too much or too little of the
`concentrate being dispensed in the diluting solution.
`The following patents may be referred to for further
`background concerning prior attempts to stabilize concen(cid:173)
`trated liquid enzyme formulations: U.S. Pat. No. 5,281,277
`35 (Nakagawa) and Japanese Kokai Patent Applications Nos.
`92-370197; 92-143718; and 92-243215. Improved liquid
`enzyme compositions have been disclosed in U.S. Pat. Nos.
`5,576,278, 5,604,190, 5,605,661, 5,672,213 5,718,895 and
`5,723,421, all issued to Alcon Laboratories, Inc.
`A number of multi-purpose compositions for cleaning,
`disinfecting and storing contact lenses are commercially
`available. The main cleaning ingredients of these products
`generally comprise surfactants. Soft contact lenses become
`soiled by collecting various debris and also by accumulated
`protein deposition on the lens surface. Failure to remove the
`protein deposits results in opacification of the lens and lens
`spoilage. While surfactants are used to remove debris from
`the lens, they are not very efficacious in removing protein
`deposits. Proteolytic agents, in contrast, are very effective in
`removing protein deposits that form on the lens over time.
`Thus, cleaning regimens using multi-purpose compositions
`comprising surfactants still require the additional step of
`employing a proteolytic agent to remove protein deposits.
`The use of a single enzyme containing multi-purpose
`solution for the cleaning and disinfecting of contact lenses
`has been proposed in U.S. Pat. No. 5,409,546 (Nakagawa et
`al.) and European Patent Application No. 0 646, 641
`(Nakagawa et al.). Those patents disclose compositions
`wherein the enzyme is in a dilute concentration, and the
`compositions, therefore, require no dilution step prior to use.
`Those compositions, however, provide limited stability of
`the enzyme (1 or 2 months at room temperature). The limited
`shelf-life of these compositions generally does not permit
`their commercialization.
`What is needed, therefore, are multi-purpose composi(cid:173)
`tions which provide a commercially viable storage/use shelf(cid:173)
`life.
`
`10
`
`

`
`US 6,228,323 Bl
`
`3
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 is a perspective view of a preferred embodiment of
`the invention.
`FIG. 2 is an elevation view of a preferred embodiment of
`the invention.
`FIG. 3 is an exploded elevation view of a preferred
`embodiment of the invention.
`FIG. 4 is an exploded cross-section view of a preferred
`embodiment of the invention about line 4-4 of FIG. 3.
`FIG. 5 is a cross-section view about line 5-5 of FIG. 2
`of a preferred embodiment of the invention.
`FIG. 6 is a cross-section view about line 5-5 of FIG. 2
`of a preferred embodiment of the invention, illustrating the
`downward rotation of a cap/plunger assembly, the breaking 15
`of a membrane, and the egress of an enzyme composition.
`FIG. 7 is a top plan view of a housing of the invention.
`FIG. 8 is a bottom plan view of a cap and collar of the
`invention.
`FIG. 9 is a graph comparing the proteolytic stability of
`methyl trypsin, trypsin and pancreatin in a multi-purpose
`composition, at 30° C. through 42 days.
`
`4
`allowing for the realization of maximum cleaning benefits,
`and the avoidance of unnecessary ocular irritation/toxicity.
`Another feature of the present invention is that the
`Al-trypsin component is kept separate from the diluting
`5 solution prior to initial use. This feature minimizes enzyme
`activity loss, which naturally occurs over time in aqueous
`environments, by minimizing the time the enzyme is solu(cid:173)
`bilized in the diluent prior to initial use. This feature allows
`for ambient temperature shipping, and long shelf-life with-
`10 out significant loss of enzyme activity. When the user is
`ready to use the system, the two components are combined
`and mixed aseptically, forming the multi-purpose composi(cid:173)
`tion. The multi-purpose composition can then be used for a
`period of from about 1-3 months.
`Another feature of the present invention is the employ-
`ment of an Al-trypsin as the cleaning agent in the compo(cid:173)
`sitions. Al-trypsins have been discovered to possess superior
`stability in liquid concentrated and diluted multi-purpose
`solutions. Thus, the use of an Al-trypsin in the present
`20 invention compositions provides prolonged pre-mixed
`storage, as well as extended stability for the post-mixed,
`ready-to-use multi-purpose compositions of the present
`invention.
`The cleaning and disinfecting compositions of the present
`25 invention may utilize ingredients similar to known cleaning
`or disinfection formulations. Various modifications may be
`made, however, to enhance the anti-microbial efficacy of the
`multi-purpose composition. Other additional components
`may also be added to enhance the shelf-life of the mixed
`30 components, such as the use of enzyme stabilizers.
`The multi-purpose compositions and methods of the
`present invention provide greater ease of use. This ease of
`use enables contact lens users to clean their lenses daily,
`thereby achieving maximum proteolytic cleaning of their
`35 lenses. It has been found that daily use of a liquid enzyme
`cleaner results in dramatically better cleaning, as compared
`to the once-a-week enzyme cleaning regimens currently
`being used.
`
`SUMMARY OF THE INVENTION
`
`The present invention is directed to two-part systems
`which provide for the generation of multi-purpose compo(cid:173)
`sitions useful in the simultaneous cleaning and disinfecting
`of contact lenses. The present invention is also directed to
`stable multi-purpose compositions. The present invention is
`further directed to methods of simultaneously cleaning and
`disinfecting contact lenses using the two-part system. The
`two-part system comprises an alkyl trypsin ("Al-trypsin")
`cleaning composition, an aqueous composition and one or
`more anti-microbial agent(s). The Al-trypsin composition
`provides a concentrated amount of an Al-trypsin. The aque(cid:173)
`ous composition provides a diluting solution. The anti(cid:173)
`microbial agent is contained in either the Al-trypsin com(cid:173)
`position or the aqueous composition. The two-part system is
`initialized for use by admixing the Al-trypsin composition 40
`with the aqueous composition.
`The two-part system uses a two-compartment device
`capable of keeping separate an enzyme composition and a
`diluting composition prior to their initial use. One feature of
`this device is that it combines the separate components in a 45
`single bottle assembly. This feature has the advantage over
`prior art systems which have required the more difficult,
`tedious and cumbersome use of separate containers. Related
`to this feature is the fact that the enzyme is added only once
`to the disinfecting composition, and the resulting multi- 50
`purpose composition can then be used many times over a
`period of several months. With most prior art systems, the
`enzyme must be added to the disinfecting composition each
`time the user cleans his lenses. A further advantage of this
`feature is that the two compositions are admixed is asepti- 55
`cally. This is due to the fact that the bottle assembly
`containing the two compositions is assembled in an aseptic
`manner with an air-tight seal, thus sterile mixing is per(cid:173)
`formed within the closed sterile system of the bottle assem(cid:173)
`bly. Still another advantage of this feature is that it elimi- 60
`nates possible user error resulting from the addition of
`improper amounts of an enzyme composition to the diluting
`solution. This is important as improper amounts of enzymes
`or excipients (e.g., salts) in the resultant multi-purpose
`solution can lead to ineffective cleaning and disinfecting of 65
`the lens and/or ocular toxicity. Thus, user compliance with
`the cleaning regimen is perfected with the present invention,
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`The present invention is directed to the use of a sterile
`two-part system for the preparation of multi-purpose com(cid:173)
`positions useful for cleaning and disinfecting contact lenses.
`The present invention is also directed to stable, multi(cid:173)
`purpose compositions. The present invention is also directed
`to methods of cleaning and disinfecting contact lenses by
`using the two-part systems. The first part ("Part I") is a
`sterile Al-trypsin containing solid (powder or tablet) or
`liquid composition and the second part ("Part II") is a sterile
`diluting composition. An anti-microbial agent is further
`required, and may be included in either composition.
`The present invention two-part systems require the use of
`a two-compartment device to store and mix the sterile
`two-part system, and to dispense the resultant sterile multi(cid:173)
`purpose composition. Various devices may be employed, but
`the central features of the device are that it provides separate
`component storage, a means for aseptically adding one
`component to the other component, a mixing chamber and
`a dispensing means, all in a single bottle assembly.
`FIG. 1 illustrates a preferred two-part bottle assembly for
`use with the two-part/multi-purpose compositions of the
`present invention. The preferred two-part bottle assembly,
`bottle assembly 1, generally comprises bottle 2 and con(cid:173)
`tainer 4.
`As illustrated in FIGS. 2-4; bottle 2 comprises neck 13,
`opening 10, external rings 12 and internal rings 43. Neck 13
`
`11
`
`

`
`US 6,228,323 Bl
`
`6
`away from housing 5, a channel is formed which runs from
`bottle 2 through now open housing 5, plunger 6 and dis(cid:173)
`pensing end 25. With the removal of cap 8, the resultant
`multi-purpose composition may now be dispensed through
`this channel to an appropriate container for cleaning,
`disinfecting, rinsing and storing the contact lens.
`
`5
`
`Other embodiments of two-compartment bottle assem-
`10 blies may be employed in the present invention. For
`example, a blister pouch and piercing means may be utilized
`as the enzyme compartment and break-away membrane
`component, respectively, of a bottle assembly.
`
`20
`
`As stated above, the present invention two-part systems
`are comprised of two separate compositions, which are then
`combined before initial use. Part I comprises an Al-trypsin
`and Part II comprises an aqueous diluting solution. The
`resultant multi-purpose composition may contain various
`other agents, but must contain: 1) an anti-microbial agent, 2)
`an Al-trypsin, 3) a buffering agent, 4) a tonicity agent, and
`5) water. The multi-purpose compositions of the present
`invention are intended to function as storing, rinsing, clean(cid:173)
`ing and disinfecting solutions. Therefore, the multi-purpose
`compositions will be physiologically compatible with the
`eye.
`
`The Part I sterile enzyme composition of the present
`30 invention comprises an Al-trypsin and various carriers.
`
`5
`is formed such that rings 12 annularly protrude from neck
`13. Bottle 2 is made generally of molded polyethylene,
`although other materials such as polyethyleneterphlalate
`(PET) and polypropylene (PIP) may be used.
`As best seen in FIGS. 3 and 4, container 4 comprises
`housing 5, plunger 6, cap 8 and collar 28. Housing 5
`comprises hollow cylinder 14 and cap 16. Cylinder 14 has
`external threads 36 annularly protruding near top end 15 and
`membrane disk 18 covering bottom end 17. Stops 23 are
`disposed annularly about exterior 38 of cylinder 14 (FIG. 7).
`Membrane 18 has thin cross-sectional thickness 49 about its
`circumference. Cap 16 has protruding internal rings 20, and
`cylinder 14 is coaxially disposed within cap 16 such that top
`end 15 and bottom end 17 project out of cap 16. Plunger 6 15
`comprises hollow cylinder 24, open end 22, dispensing end
`25 and ribs 34. Open end 22 has tooth 39 and is thinner in
`cross-sectional thickness than the thickness of hollow cyl(cid:173)
`inder 24, thereby forming sharp point 41 of tooth 39. Cap 8
`comprises pin 33, hollow cone 32, internal threads 30 and
`stops 21. As best seen in FIG. 8, collar 28 has tab 29, spokes
`19 and perforation 27 which forms ends 31. Container 4
`components are generally made of molded high density
`polyethylene or polycarbonate, but other materials and 25
`methods of manufacture such as PIP, PET, polystyrene and
`acrylonitrile butadiene styrene (ABS) may be employed.
`As illustrated in FIG. 4, container 4 is put together by first
`adding Al-trypsin cleaning composition 7 to hollow cylinder
`14 of housing 5, ringing collar 28 over cylinder 14, inserting
`plunger 6 within cylinder 14 such that rings 34 of plunger 6
`compress against the interior of cylinder 14, and screwing on
`cap 8 over plunger 6, engaging pin 33 of cap 8 with notch
`26 of plunger 6, by threading internal threads 30 within 35
`threads 36 of housing 5. In this configuration, spokes 19 of
`collar 28 are interspersed between both stops 21 of cap 8 and
`stops 23 of housing 5. Cap 8 in this configuration (see FIGS.
`2 and 5), is only partially threaded within threads 36 of
`housing 5 due to the prevention of further downward rota-
`tion of cap 8 by collar 28. Aqueous composition 9 is added
`to bottle 2, container 4 is then placed over neck 13, and cap
`16 is forced down on neck 13 such that rings 20 compress
`radially against neck 13, and exterior rings 12 compress 45
`against interior 47 of cap 16, forming an air-tight seal.
`In operation, collar 28 is first removed from container 4
`by screwing cap 8 downward on housing 5. When cap 8 is
`rotated, stops 21 engage and pull spokes 19, while stops 23
`hold spokes 19 stationary. The resulting stress causes collar
`28 to split at perforation 27. Split collar 28 may then be
`removed by pulling on tab 29. Cap 8 is then further screwed
`down on housing 5. With cap 8 rotation, plunger 6, is
`simultaneously pushed downward causing plunger 6 to 55
`descend cylinder 14. When plunger 6 reaches membrane 18
`of housing 5, sharp point 41 punctures thin circumference 49
`of membrane 18. Further rotation of cap 8 causes open end
`22 of plunger 6 to further descend, slicing off membrane 18
`about its circumference from housing 5, similar to the 60
`operation of a punch press. At this point the Al-trypsin
`cleaning composition contained in housing 5 is exposed to
`interior 11 of bottle 2 and falls into aqueous diluting com(cid:173)
`position 9 of bottle 2. Bottle 2 may then be inverted and 65
`shook, thus affecting the mixing of the Al-trypsin and
`aqueous diluting compositions. When membrane 18 is cut
`
`40
`
`50
`
`As used herein, "Al-trypsin" refers to a covalently modi(cid:173)
`fied trypsin wherein one or more of its lysine epsilon-amino
`groups has been mono-alkylated or di-alkylated to form the
`corresponding monoalkylamino or dialkylamino group. The
`alkyl group attached to the amine may be a primary or
`branched C1 _12 group. Preferred Al-trypsins of the present
`invention are those wherein the alkyl group is a primary or
`branched cl-4 group. Alkylation of trypsin is generally
`performed by reductive alkylation. The degree of alkylation
`of the lysine epsilon-amino groups will depend on the
`reaction conditions of the reductive alkylation process. For
`example, if the reaction cycle is repeated a number of times
`and/or a higher reagent to enzyme ratio is used, then full
`alkylation, i.e., alkylation of all of the lysine epsilon-amino
`groups, will tend to be achieved. Al-trypsins of the present
`invention will preferably be fully dialkylyated at all of their
`lysine epsilon-amino groups. The most preferred Al-trypsin
`is methyl trypsin ("Me-trypsin"). The most preferred
`Me-trypsin of the present invention will be derived from
`porcine tissue sources and will be fully dimethylated, as
`described above.
`
`Trypsin is a 23,800 dalton protease with 6 disulfide
`bridges. Trypsin can be synthesized or obtained from vari(cid:173)
`ous sources, such as porcine, bovine or swine pancreatin.
`Trypsin is also available from commercial sources such as
`Sigma Chemical Co. (St. Louis, Mo.), Biofac Co. (United
`Kingdom) and Novo Nordisk (Denmark). Trypsin may vary
`from species to species, but in general will be highly
`homologous with porcine or human trypsin.
`
`Al-trypsins may be synthesized by the process of reduc(cid:173)
`tive alkylation of trypsin, as generally described in Scheme
`1, below.
`
`12
`
`

`
`US 6,228,323 Bl
`
`7
`
`8
`
`Trypsin-NH2
`
`H+
`
`+ R ) lH
`(1)
`
`Trypsin-N+-R
`
`Scheme 1
`
`R
`//
`Trypsin-N
`(2)
`
`R
`I
`Trypsin-N
`\
`R
`
`(5)
`
`Trypsin-NHR
`(3)
`
`Trypsin-N+-R
`H2
`
`(1)
`
`Trypsin-N'=R
`
`I
`
`R
`
`wherein, R is branched or unbranched C1 _12 alkyl.
`As illustrated in scheme 1, the epsilon amino group of the
`lysine residues of trypsin is reacted with aldehydic alkylat(cid:173)
`ing reagent (1) to afford the alkylimino product (2). The 25
`alkylimino product (2) reduces to the resonant alkylamino
`species (3,4). The product (3,4) may react with another mole
`of the alkylating reagent (1) to yield the dialkylamino
`trypsin (5). As illustrated above, the resultant alkylated
`trypsin may either be mono or dialkylated at the lysine 30
`epsilon-amino groups.
`
`compositions need to be stable for longer periods of time
`than liquids. Excipients which make up the enzyme powder
`compositions are known in the art. Generally, the Al-trypsin
`composition will include bulking agents to carry the rela(cid:173)
`tively small volume of an Al-trypsin into the diluting solu-
`tion. Such bulking agents typically include polyols (e.g.,
`mannitol or soribitol), polyethylene glycols (molecular
`weights greater than 1000) and sugars. Other excipients may
`include salts such as NaCl, chelating agents such as EDTA,
`and buffering agents such as Tris. Other additives may
`include surfactants to ease dispersion and dissolution of the
`powder in water. Preferred Al-trypsin compositions com(cid:173)
`prise mannitol and polyethylene glycol-5000 (PEG-5000).
`Enzyme tablet compositions and methods of manufactur-
`ing are known in the art. Enzyme tablets require the use of
`bulking agents and binding agents. Additionally, tablets may
`contain effervescing agents such as bicarbonate to expedite
`dissolution of the tablet into the diluting solution. Other
`excipients known in the art may be added to provide greater
`40 consistency and easier manufacture of the tablets. Preferred
`Al-trypsin tablet compositions comprise sodium
`bicarbonate, citric acid, PEG-8000, carboxymethyl cellulose
`and lactose.
`Liquid Al-trypsin compositions are preferred Part I com(cid:173)
`positions of the present invention due to their ease of
`preparation, sterilization and dispensing within the enzyme
`container of a bottle assembly. Liquid Al-trypsin composi(cid:173)
`tions are solubilized in a suitable liquid vehicle. As used
`herein, "suitable liquid vehicle" refers to an aqueous or
`non-aqueous composition which, when diluted with an
`aqueous solvent described below, is compatible with the
`requirements of contact lens care regimens.
`Non-aqueous Al-trypsin compositions employed as Part I
`compositions of the present invention generally comprise a
`crystalline enzyme uniformly dispersed in a water-soluble
`organic liquid. Typical organic liquids include polyoxyeth-
`ylenes (e.g., PEG-400) and alkoxy polyoxyethylenes such as
`methoxy polyethylene glycols. In this composition, the
`Al-trypsin is in a dormant state, and following dissolution in
`a Part II composition of the present invention, the Al-trypsin
`solubilizes and becomes active. Preferred non-aqueous
`enzyme compositions comprise an enzyme in PEG-400.
`Aqueous Al-trypsin compositions employed as Part I
`compositions of the present invention generally comprise a
`water-soluble organic liquid. It has been found that the use
`of a concentrated aqueous vehicle which contains a water(cid:173)
`miscible organic molecule further enhances the stability of
`the Al-trypsin. The use of this type of vehicle is therefore
`
`EXAMPLE 1
`
`35
`
`Me-trypsin may be prepared by the following synthesis:
`The following solutions are first prepared:
`1. Borate buffer: 0.2 M sodium borate buffer, pH 9.2
`containing 2 mg/ml benzamidine hydrochloride and a
`trace amount of n-octanol.
`2. Trypsin: 1 g in 150 ml borate buffer.
`To the 150 ml solution of trypsin, 10 ml of 1 M sodium
`borohydride is added followed quickly by 10 ml of 2.4 M
`formaldehyde. Three more volumes of sodium borohydride
`and formaldehyde are added at 10 minute intervals. The
`reaction solution is then acidified with glacial acetic acid to
`approximately pH 4.2 and then dialyzed extensively against 45
`2 mM HCl at 4° C. (8 changes of 2 Leach within 24 hours).
`The dialyzed solution is finally lyophylized for over 20
`hours.
`The above reactions are further described in Rice, R H,
`Means, GE and Brown, W D. Stabilization of bovine trypsin 50
`by reductive methylation, Biochimica et Biophysica Acta,
`volume 492, pages 316-321 (1977); and Means, G E and
`Feeney, R E. Reductive alkylation of amino groups in
`proteins, Biochemistry, volume 7, pages 2192-2210 (1968).
`Me-trypsin is also available from commercial sources such 55
`as Sigma Chemical Co. and Promega Corp: (Madison, Wis.).
`Other Al-trypsins may be prepared by methods analogous
`to Example 1, wherein formaldehyde is replaced by other
`alkylating reagents. For example, ethyl trypsin ("Et(cid:173)
`trypsin") may be synthesized by an analogous method
`described in Example 1 and Scheme 1 above, wherein 60
`acetaldehyde is used as the alkylating reagent in place of
`formaldehyde.
`As stated above, the Part I sterile enzyme composition of
`the present invention generally comprises an Al-trypsin and
`various carriers. The enzyme composition may be formu- 65
`lated as a powder, tablet or liquid. Dry powder or tablet
`compositions may be preferred when the Part I enzyme
`
`13
`
`

`
`US 6,228,323 Bl
`
`9
`preferred. As used herein, the term "water-miscible organic
`molecule" or "stabilizer," refers to an organic compound that
`forms one liquid phase with water when added to water.
`While not intending to be bound by any theory, it is believed
`that the stabilizers compete with water in the hydrogen 5
`bonding of the enzyme in solution, and thereby disrupt the
`active state of t