(12) United States Patent
`US 6,872,705 132
`(10) Patent N0.:
`(45) Date of Patent:
`Mar. 29, 2005
`Lyons
`
`US006872705B2
`
`(54) USE OF ANTIMICROBIAL PEPTIDES AS
`PRESERVATIVES IN OPHTHALMIC
`PREPARATIONS, INCLUDING SOLUTIONS,
`EMULSIONS, AND SUSPENSIONS
`
`(75)
`
`Inventor: Robert T. Lyons, Laguna Hills, CA
`(US)
`
`(73) Assignee: Allergan, Inc., Irvine, CA (US)
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 272 days.
`
`(21) Appl. N0.: 09/904,753
`
`(22)
`
`Filed:
`
`Jul. 13, 2001
`
`(65)
`
`Prior Publication Data
`US 2003/0092612 A1 May 15, 2003
`
`Int. Cl.7 ......................... A61K 38/04; A61K 38/16
`(51)
`(52) us. Cl.
`........................... 514/13; 514/14; 514/912;
`530/326; 600/563
`(58) Field of Search ............................ 514/13, 14, 912;
`530/326; 600/563
`
`(56)
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`.
`
`6/1985 Stark ........................... 424/80
`4,525,346 A
`12/1992 W'ong et al.
`422/28
`5,171,526 A
`4/1993 Janssen .....
`514/399
`5,200,453 A
`5,474,979 A * 12/1995 Ding et al.
`514/11
`5,549,894 A *
`8/1996 Hunt
`.........
`424/94.64
`5,736,165 A *
`4/1998 Ripley et al.
`424/661
`5,792,831 A *
`8/1998 Maloy
`530/326
`5,830,508 A
`11/1998 MacKeen
`424/602
`5,993,864 A * 11/1999 Kross ......................... 424/661
`6,372,234 B1 *
`4/2002 Deckers et al.
`............. 424/401
`Tuse et al.
`.................... 514/14
`6,482,799 B1 * 11/2002
`FOREIGN PATENT DOCUMENTS
`
`
`
`
`WO
`
`WO 96/25183
`
`8/1996
`
`OTHER PUBLICATIONS
`
`Darveau et al., “Beta—Lactam Antibiotics Potentiate Magai-
`nin 2 Antimicrobal Activity In Vitro and In Vivo,” Antimi-
`crobial Agents
`and Chemotherapy,
`Jun.
`1991,
`p.
`1153—1159.*
`Matsuzaki et al., “Mechanism of Synergism between Anti-
`microbial Peptides Magainin 2 and PGLa,” Biochemistry,
`1998, 37, 15144—15153.*
`Maria Bishop, “DG Dispatch—ACR: Topical Cyclosporin A
`Restores Clear Vision to Sjogren’s Syndrome Patients”,
`Doctor’s Guide Global Edition, www.pslgroup.com/dg/
`le96aa.htm, Nov. 3, 2000.
`J .H. Lee et al., “High—Level Expression of Antimicrobial
`Peptide Mediated by a Fusion Partner Reinforcing Forma-
`tion of Inclusion Bodies”, Biochemical and Biophysical
`Research Communications 277, 575—580, (2000).
`
`Stevenson, D., MD et al., “Cyclosporin A Ophthalmic Emul-
`sion in the Treatment of Moderate to Severe Dry Eye
`Disease”, Eye News Late 2000—Richmond Eye Associates
`from Ophthalmology May 2000; 107:967—974.
`Gao J, et al., Abstract of “The role of apoptosis in the
`pathogenesis of canine keratoconjunctivitis sicca: the effect
`of topical CyclosporinAtherapy”, Department of Biological
`Science, Allergan, Inc. Cornea Nov. 1998; 17(6): 654—63.
`Andrew Acheampong et al., “Distribution of cyclosporin A
`in ocular tissues after topical administration to albino rabbits
`and beagle dogs”, Current Eye Research 1999, vol. 18, No.
`2, pp. 91—103.
`Sall K, et al., Abstract of “Two multicenter, randomized
`studies of the efficacy and safety of cyclosporine ophthalmic
`emulsion in moderate to severe dry eye disease. ScA Phase
`3 Study Group”, Ophthalmology Apr. 2000;107(4): 631—9.
`Stevenson D., Abstract of
`“Efficacy
`and
`safety of
`cyclosporin A ophthalmic emulsion in the treatment of
`moderate—to—severe dry eye disease: a dose—ranging, ran-
`domized trial. The Cyclosporin A Phase 2 Study Group”,
`Ophthalmology May 2000; 107(5):967—74.
`Katsumi Matsuzaki, “Why and how are peptide—lipide inter-
`actions utilized for self—defence? Magainins and tachyples-
`ins as archetypes”, Biochimica et Biophysica Acta 1462
`(1999) 1—10.
`David Andreu, “Animal Antimicrobial Peptides: An Over-
`view”, Biopolymers (Peptide Science), vol. 47, 415—433
`(1998).
`Katsumi Matsuaki, “Magainins as paradigm for the mode of
`action of pore forming polypeptides”, Biochimica et Bio-
`physica Acta 1376 (1998) 391—400.
`W. Lee Malloy et al., “Structure—Activity Studies on Magai-
`nins and Other Host Defense Peptides”, Biopolymers (Pep—
`tide Science), vol. 37, 105—122 (1995).
`Michael Zasloff et al., “Antimicrobial activity of synthetic
`magainin peptides and several analogues”, Proc. Natl. Acad.
`Sci. USA, vol. 85, pp. 910—913, Feb. 1988, Microbiology.
`
`* cited by examiner
`
`Primary Examiner—Robert A. Wax
`(74) Attorney, Agent, or Firm—Stout, Uxa, Buyan &
`Mullins, LLP; Frank J. Uxa; Greg S. Hollrigel
`
`(57)
`
`ABSTRACT
`
`Methods for preserving ophthalmic compositions are dis-
`closed. In one embodiment, such compositions include a
`liquid medium and an antimicrobial component which is
`preferably substantially non-oxidative. Compositions which
`include a liquid medium and antimicrobial peptide
`magainins, present in an amount effective as a preservative,
`are also disclosed. Preserved compositions useful for admin-
`istering a therapeutic component to the eyes or caring for
`contact lenses are also included within the scope of the
`present invention.
`
`22 Claims, N0 Drawings
`
`Argentum Pharm. LLC V. Alcon Research, Ltd.
`Case IPR2017-01053
`
`ALCON 2039
`
`

`

`US 6,872,705 B2
`
`1
`USE OF ANTIMICROBIAL PEPTIDES AS
`PRESERVATIVES IN OPHTHALMIC
`PREPARATIONS, INCLUDING SOLUTIONS,
`EMULSIONS, AND SUSPENSIONS
`
`BACKGROUND OF THE INVENTION
`This invention relates to preserved ophthalmic composi
`tions. More particularly, the present invention relates to
`preserved ophthalmic compositions, for example, useful in
`administering a therapeutic component to the eyes, and for
`example, to care for contact lenses, Which include one or
`more peptides and/or peptide derivatives as antimicrobial
`agents.
`Various compositions, such as solutions, emulsions and
`suspensions are used in association With administering
`therapeutic components to the eyes. For example, an oil-in
`Water emulsion may be used as a carrier for a therapeutic
`component to be administered to the eyes.
`At present, no safe effective preservative exists for an
`oil-in-Water emulsion product. This is because the most
`acceptable preservative, benZalkonium chloride, loses its
`effectiveness due to partitioning into the oil phase. As a
`result only single dose containers of oil-in-Water emulsion
`ophthalmic compositions can be marketed up to this time.
`Use of single dose containers to store ophthalmic com
`positions prevents contamination and groWth of microor
`ganisms. HoWever, single dose containers are inconvenient
`to use and are expensive for the consumer. Appropriate use
`of an effective preservative Will alloW for production of
`multidose containers of preserved ophthalmic compositions
`such as oil-in-Water emulsions.
`Various compositions are used in association With contact
`lenses to ensure that the lenses may be safely, comfortably
`and conveniently Worn. Contact lens care compositions, for
`example, cleaning compositions, Wetting compositions, con
`ditioning compositions and the like, often utiliZe at least one
`preservative, depending on the type of composition, for
`preserving the lens care composition itself.
`A preserved contact lens care composition has sufficient
`antimicrobial activity so that When the composition is con
`tacted With a contact lens substantially no increase in the
`microorganism population on the lens or in the composition
`is obtained. A preserved contact lens care composition may
`be termed a microbiostatic composition. Contact lens care
`compositions are often preserved to prevent any substantial
`increase in, or to gradually decrease, the population of
`contaminating microorganisms in the compositions and,
`thereby, to extend their shelf life.
`Various compounds are knoWn for use as preserving
`agents in preserved ophthalmic compositions. Examples
`include thimerosal, benZalkonium chloride and chlorhexi
`dine. HoWever, these preserving agents are knoWn to exhibit
`ocular toxicity Which may result in irritation or sensitivity to
`the eye. Further, a soft contact lens, a rigid gas permeable
`contact lens (RGP) or a hard contact lens can absorb or
`adsorb these compounds. This causes the contact lens to
`retain the irritating compound and contributes to the eye
`irritation and eye sensitivity Which may result.
`Thus, it is readily apparent that a continuing need exists
`for safe and efficacious compositions that can be used to
`preserve ophthalmic compositions.
`
`15
`
`25
`
`35
`
`40
`
`45
`
`55
`
`65
`
`2
`SUMMARY OF THE INVENTION
`NeW preserved compositions and methods employing
`such compositions, particularly compositions and methods
`directed to eye care and contact lens care, have been
`discovered. The present compositions include effective pre
`servatives to protect against groWth of contaminating micro
`organisms. Importantly, such preserving activities are
`achieved using the present compositions With little or no risk
`of eye irritation or sensitivity.
`In one embodiment of the invention, compositions useful
`for preserving ophthalmic compositions are provided. Such
`compositions include a magainin antimicrobial peptide, an
`analog of a magainin antimicrobial peptide or a mixture
`thereof present in an amount effective as a preservative. This
`effective amount may be less than about 10 milligrams per
`milliliter or less than about 1 milligram per milliliter or less
`than about 0.1 milligram per milliliter. Also included in the
`compositions is a therapeutic component. In a particularly
`useful embodiment of the invention, the compositions com
`prise magainin antimicrobial peptides. In another particu
`larly useful embodiment of the invention, the compositions
`comprise an analog of a magainin antimicrobial peptide
`comprising the amino acid sequence GIGKFLKKAKKF
`GKAFVKILKK (SEQ ID NO: 4). The compositions may
`also include Water and an effective amount of a buffer to
`provide the compositions With a desired pH. Also, the
`compositions may include an effective amount of a tonicity
`component to provide the compositions With a desired
`osmolality.
`The compositions exist in various forms. For example, the
`compositions may be an oil-in-Water emulsion, a solution or
`a suspension. Also, provided is for a sole preservative to be
`used in accordance With the invention.
`The compositions may be applied onto or into the eyes.
`For example, the compositions may be used as a surgical
`irrigant.
`In another embodiment of the invention, compositions
`useful for preserving ophthalmic compositions are provided.
`Such compositions include a magainin antimicrobial
`peptide, an analog of a magainin antimicrobial peptide or a
`mixture thereof present in an amount effective as a preser
`vative. This effective amount may be less than about 10
`milligrams per milliliter or less than about 1 milligram per
`milliliter or less than about 0.1 milligram per milliliter. In
`this embodiment, a sole preservative is used in the compo
`sitions. In a particularly useful embodiment of the invention,
`the compositions comprise magainin antimicrobial peptides.
`In another particularly useful embodiment of the invention,
`the compositions comprise an analog of a magainin antimi
`crobial peptide comprising the amino acid sequence GIGK
`FLKKAKKFGKAFVKILKK (SEQ ID NO: 4). The com
`positions may also include Water and an effective amount of
`a buffer to provide the compositions With a desired pH. Also,
`the compositions may include an effective amount of a
`tonicity component to provide the compositions With a
`desired osmolality.
`The compositions exist in various forms. For example, the
`compositions may be an oil-in-Water emulsion, a solution or
`a suspension.
`The compositions may be applied onto or into the eyes.
`For example, the compositions may be used as a surgical
`irrigant.
`
`

`

`US 6,872,705 B2
`
`3
`In still another embodiment of the invention, composi
`tions useful for preserving ophthalmic compositions are
`provided. Such compositions include a magainin antimicro
`bial peptide, an analog of a magainin antimicrobial peptide
`or a mixture thereof present in an amount effective as a
`preservative. This effective amount may be less than about
`10 milligrams per milliliter or less than about 1 milligram
`per milliliter or less than about 0.1 milligram per milliliter.
`In this embodiment, the composition is an oil and Water
`emulsion. In a particularly useful embodiment of the
`invention, the compositions comprise magainin antimicro
`bial peptides. In another particularly useful embodiment of
`the invention, the compositions comprise an analog of a
`magainin antimicrobial peptide comprising the amino acid
`sequence GIGKFLKKAKKFGKAFVKILKK (SEQ ID NO:
`4). The compositions may also include Water and an effec
`tive amount of a buffer to provide the compositions With a
`desired pH. Also, the compositions may include an effective
`amount of a tonicity component to provide the compositions
`With a desired osmolality.
`The compositions may be applied onto or into the eyes.
`For example, the compositions may be used as a surgical
`irrigant.
`In still another embodiment of the invention, composi
`tions useful for preserving ophthalmic compositions are
`provided. Such compositions include an analog of a magai
`nin antimicrobial peptide comprising the amino acid
`sequence GIGKFLKKAKKFGKAFVKILKK (SEQ ID NO:
`4) present in an amount effective as a preservative. This
`effective amount may be less than about 10 milligrams per
`milliliter or less than about 1 milligram per milliliter or less
`than about 0.1 milligram per milliliter. The compositions
`may also include Water and an effective amount of a buffer
`to provide the compositions With a desired pH. Also, the
`compositions may include an effective amount of a tonicity
`component to provide the compositions With a desired
`osmolality.
`The compositions may exist as a solution or a suspension.
`The compositions may be applied onto or into the eyes.
`In still another embodiment of the invention, composi
`tions useful for preserving ophthalmic compositions are
`provided. Such compositions include a magainin antimicro
`bial peptide, an analog of a magainin antimicrobial peptide
`or a mixture thereof present in an amount effective as a
`preservative. This effective amount may be less than about
`10 milligrams per milliliter or less than about 1 milligram
`per milliliter or less than about 0.1 milligram per milliliter.
`These compositions are applied onto or into the eyes. In a
`particularly useful embodiment of the invention, the com
`positions comprise magainin antimicrobial peptides. The
`compositions also may include Water and an effective
`amount of a buffer to provide the compositions With a
`desired pH. Also, the compositions may include an effective
`amount of a tonicity component to provide the compositions
`With a desired osmolality.
`Also provided for are methods of preserving ophthalmic
`compositions. One such method comprises contacting an
`ophthalmic composition With a magainin antimicrobial
`peptide, analogs of magainin antimicrobial peptides or mix
`tures thereof present in an amount effective as a preservative
`
`15
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`25
`
`40
`
`45
`
`55
`
`65
`
`4
`in the composition. In one embodiment, the composition is
`an oil and Water emulsion.
`Also provided for are methods for treating an eye. One
`such method comprises contacting an eye With a liquid
`medium Which includes magainin antimicrobial peptides,
`analogs of magainin antimicrobial peptides or mixtures
`thereof in an amount effective as a preservative. In one
`embodiment, the composition is an oil and Water emulsion.
`The invention also provides for ophthalmic compositions
`Which comprise magainin antimicrobial peptides, analogs of
`magainin antimicrobial peptides or mixtures thereof in an
`amount effective as a preservative. In a particularly useful
`embodiment of the invention, the compositions comprise an
`analog of a magainin antimicrobial peptide comprising the
`amino acid sequence GIGKFLKKAKKFGKAFVKILKK
`(SEQ ID NO: 4). Also in a preferred embodiment, the
`composition is an oil-in-Water emulsion and the composition
`is provided in a multidose format.
`Any and all features described herein and combinations of
`such features are included Within the scope of the invention
`provided that such features of any such combination are not
`mutually exclusive.
`These and other aspects and advantages of the present
`invention are apparent in the folloWing detailed description
`and claims.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`The present invention is applicable to preserving oph
`thalmic compositions, such as eye care compositions and
`contact lens care compositions Which are bene?ted from
`being preserved.
`One important feature of the compositions of the present
`invention is the inclusion of one or more antimicrobial
`peptides in the compositions.
`In one embodiment, the present compositions include a
`suf?cient amount of an antimicrobial peptide to effectively
`preserve the compositions. In a preferred embodiment, the
`antimicrobial peptide is a magainin antimicrobial peptide.
`The antimicrobial peptides useful according to the present
`invention include naturally occurring antimicrobial
`peptides, preferably cytolytic peptides, synthetic antimicro
`bial peptides, antimicrobial peptide mimetics and nanotubes.
`Such peptides may be the L-form, the D-form or combina
`tions or mixtures of both forms. At least some of these
`antimicrobial peptides may be membrane active. One or
`more of these antimicrobial peptides may act by disrupting
`a cell membrane.
`Among the antimicrobial peptides preferably employed
`are those selected from defensins, peptides related to
`defensins, cecropins, peptides related to cecropins, and other
`amino acid polymers With antibacterial, antifungal and/or
`antiviral activities. Particularly preferred antimicrobial pep
`tides employed in the present invention are magainin anti
`microbial peptides and peptides related to magainin antimi
`crobial peptides and mixtures thereof.
`Magainin antimicrobial peptides Were ?rst reported in the
`literature in 1987 (Zasloff (1987) Proc. Natl. Acad. Sci. USA
`84, 5449—5453). Magainin antimicrobial peptides are a
`family of linear, amphipathic, cationic antimicrobial
`
`

`

`US 6,872,705 B2
`
`5
`peptides, and are approximately 21 to 27 residues in length.
`It is believed that magainin antimicrobial peptides may exert
`their antimicrobial effect by disruption of cell membrane
`permeability.
`
`Magainin antimicrobial peptides have numerous charac
`teristics that make them a superior preservative for use in
`ophthalmic compositions. For example, magainin antimi
`crobial peptides are broad-spectrum antimicrobial agents
`Which exhibit cidal activity against Gram-negative and
`Gram-positive bacteria, fungi and protoZoa. Also, magainin
`antimicrobial peptides display a reduced eye irritation com
`pared to existing preservatives for ophthalmic compositions.
`For example, benZalkonium chloride is knoWn to exhibit
`ocular toxicity Which may result in irritation or sensitivity to
`the eye. In addition, magainin antimicrobial peptides are
`highly Water-soluble alloWing effective antimicrobial action
`in an oil-in-Water emulsion. This high Water solubility also
`minimiZes loss of effectiveness due to adsorption to plastic
`containers. Further, numerous magainin antimicrobial pep
`tides and magainin antimicrobial peptide derivatives are
`available Which increases the opportunities for avoiding
`incompatibilities With speci?c drugs or excipients in a
`particular formulation of a composition of the invention.
`Still further, magainin antimicrobial peptides have a loW
`degree of bacterial resistance, are effective at very loW
`concentrations and are easily produced by chemical synthe
`sis or heterologous gene expression. Because of these and
`other factors magainin antimicrobial peptides are very Well
`suited for use in the present invention.
`
`Exemplary magainin antimicrobial peptides include the
`peptides having the folloWing amino acid sequences:
`
`6
`
`-continued
`
`Lys Lys Phe Gly Lys Ala Phe Val Lys
`
`Ile Leu Lys Lys
`
`Other useful magainin antimicrobial peptide analogs and
`derivatives include magainin antimicrobial peptides having
`N-terminal positively charged chain extensions (e.g., (Lys)
`lo-magainin Which enhances the antimicrobial activity of the
`peptides).
`Additional magainin antimicrobial peptides, magainin
`antimicrobial peptide analogs and derivatives Which are
`contemplated for use according to the present invention are
`described in US. Pat. Nos. 5,912,231, 5,847,047, 5,792,831,
`and 5,643,876 and in the publications Zasloff et al., Proc.
`Natl. Acad. Sci. USA 85, 910—913 (February 1988); Zasloff,
`Proc. Natl. Acad. Sci. USA 84, 5449—5453 (August 1987);
`and Bessale et al, Antimicrobial Agents, Chemotherapy 36
`(No. 2), 313—317 (February 1992), and Maloy and Kari,
`Biopolymers 37, 105—122 (1995) each of Which is incorpo
`rated in its entirety herein by reference.
`
`Cecropins useful according to the invention include the
`peptides having the folloWing amino acid sequences:
`cecropin A:
`
`15
`
`25
`
`Lys Trp Lys Leu Phe Lys Lys Ile Glu
`
`(SEQ ID NO:
`5)
`
`Lys Val Gly Gln Asn Ile Arg Asp Gly
`
`35
`
`Ile Ile Lys Ala Gly Pro Ala Val Ala
`
`Val Val Gly Gln Ala Thr Gln Ile Ala
`
`Magainin I
`Gly Ile Gly Lys Phe Leu His Ser Ala
`
`(SEQ ID NO:
`1)
`
`Lys;
`
`40
`
`and cecropin B:
`
`Gly Lys Phe Gly Lys Ala Phe Val Gly
`
`Lys Trp Lys Val Phe Lys Lys Ile Glu
`
`(SEQ ID NO:
`6)
`
`Glu Ile Met Lys Ser
`
`Magainin II
`Gly Ile Gly Lys Phe Leu His Ser Ala
`
`Lys Lys Phe Gly Lys Ala Phe Val Gly
`
`Glu Ile Met Asn Ser
`
`45
`
`Lys Met Gly Arg Asn Ile Arg Asn Gly
`
`(SEQ ID NO:
`2)
`
`Ile Val Lys Ala Gly Pro Ala Ile Ala
`
`Val Leu Gly Glu Ala Lys Ala Leu Gly
`
`Exemplary magainin antimicrobial peptide analogs
`include the peptides having the folloWing amino acid
`sequences:
`
`55
`
`MSI-78
`Gly Ile Gly Lys Phe Leu Lys Lys Ala
`
`(SEQ ID NO:
`3)
`
`Lys Lys Phe Gly Lys Ala Phe Val Lys
`
`Ile Leu Lys Lys-NH2
`
`MSI-344
`Gly Ile Gly Lys Phe Leu Lys Lys Ala
`
`(SEQ ID NO:
`4)
`
`65
`
`Cecropin D can also be employed.
`Cecropin derivatives having C-terminus modi?cations,
`substitutions, and/or truncations Which either enhance or do
`not inhibit antimicrobial activity are also contemplated for
`use according to the present invention. Useful derivatives
`include cecropin A amide (CA—NH2), and cecropin AWith
`a C-terminal ethylenediamine-modi?ed homoserine (CA
`Hse-NH-Et-NHZ). The general sequence homology of the
`N-terminus portion of the cecropins is necessary for activity
`and is therefore less suitable for truncation, modi?cation, or
`substitution. HoWever, analogs resulting from substitution of
`amino acids With similar chemical characteristics to the
`original can be designed. Maintaining an amphipathic heli
`cal structure similar to the original peptide Will result in
`conservation of antimicrobial activity. An example of a
`substitution analog of cecropin B is Shiva-1:
`
`

`

`US 6,872,705 B2
`
`Met Pro Arg Trp Arg Leu Phe Arg Arg
`
`SEQ ID NO: 7)
`
`Ile Asp Arg Val Gly Lys Gln Ile Lys
`
`Gln Gly Ile Leu Arg Ala Gly Pro Ala
`
`Ile Ala Leu Val Gly Asp Ala Arg Ala
`
`Val Gly.
`
`Shiva-1 and other cecropin substitution analogs having
`antimicrobial activity are contemplated as being useful
`according to the invention.
`Defensins useful according to the invention include:
`
`HNP-l (human neutrophil
`Ala Cys Tyr Cys Arg Ile
`
`peptide
`Pro Ala
`
`l) :
`Cys SEQ ID NO: 8)
`
`Ile Ala Gly Glu Arg Arg Tyr Gly Thr
`
`Cys Ile Tyr Gln Gly Arg Leu Trp Ala
`
`Phe Cys Cys;
`
`HNP-2:
`Cys Tyr Cys Arg Ile Pro Ala Cys Ile (SEQ ID NO: 9)
`
`Ala Gly Glu Arg Arg Tyr Gly Thr Cys
`
`Ile Tyr Gln Gly Arg Leu Trp Ala Phe
`
`Cys Cys;
`
`HNP-3:
`Asp Cys Tyr Cys Arg Ile Pro Ala Cys (SEQ ID NO: 10 )
`
`Ile Ala Gly Glu Arg Arg Tyr Gly Thr
`
`Cys Ile Tyr Gln Gly Arg Leu Trp Ala
`
`Phe Cys Cys;
`
`NP-l (rabbit neutrophil
`Val Val Cys Ala Cys Arg
`
`peptide
`Arg Ala
`
`l) :
`Leu (SEQ ID NO: 11)
`
`Cys Leu Pro Arg Glu Arg Arg Ala Gly
`
`Phe Cys Arg Ile Arg Gly Arg Ile His
`
`Pro Leu Cys Cys Arg Arg;
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`and the BNP-1 (bovine neutrophil peptide) sequence:
`
`45
`
`Arg Leu Cys Arg Val Val Ile Arg Val (SEQ ID NO: 12 )
`
`Cys Arg.
`
`Other defensins and defensin analogs, such as those
`described in Selsted et al, J. Clin. Invest. 76, 1436—1439
`(October 1985), and Kagan et al, Proc. Natl. Acad. Sci. USA
`87, 210—214 (January 1990), each of Which is incorporated
`in its entirety herein by reference, are also useful in the
`present invention.
`Tachyplesins, such as tachyplesin I and II, and
`polyphemusins, such as polyphemusin I and II, are defensin
`like peptides. See, e.g., Ohta et al, Antimicrobial Agents and
`Chemotherapy 36 (No. 7), 1460—1465 (July 1992), Which is
`incorporated in its entirety herein by reference. These pep
`tides and antimicrobially active derivatives thereof are also
`contemplated as being useful in the present invention.
`Other peptides, such as hybrids (peptides comprised of
`sequences from more than one antimicrobial class), e.g.,
`cecropin-melittin hybrids, and peptide analogs in Which one
`
`55
`
`65
`
`8
`or more of the L-amino acids are replaced With other
`L-amino acids, can also be used With advantage provided
`that they retain sufficient antimicrobial activity.
`Exemplary hybrid peptides include cecropin A-(1—8)
`melittin-(1—18)-NH2:
`
`Lys Trp Lys Leu Phe Lys Lys Ile Gly (SEQ ID NO:13)
`
`Ile Gly Ala Val Leu Lys Val Leu Thr
`
`Thr Gly Leu Pro Ala Leu Ile Ser—NH2;
`
`and cecropin A-(1—3)-melittin-(1—13)-NH2:
`
`Lys Trp Lys Gly Ile Gly Ala Val Leu (SEQ ID NO:14)
`
`Lys Val Leu Thr Thr Gly Leu—NH2.
`
`Melittin itself, hoWever, is unsuitable for use due to its high
`toxicity.
`Antimicrobial peptide mimetics are also contemplated for
`use With the present invention. Antimicrobial peptide
`mimetics may have a loWer molecular Weight than an
`average siZe antimicrobial peptide. These peptides may
`comprise components such as modi?ed thiaZole and/or
`oxaZole moieties. Antimicrobial peptide mimetics may be
`membrane active molecules that function by disrupting cell
`membranes. At least one type of antimicrobial peptide
`mimetic can be obtained from Genaera Corp., Plymouth
`Meeting, Pa.
`The antimicrobial agents must be compatible With the
`composition being preserved. The antimicrobial peptides
`should also be non-toxic to humans.
`Antimicrobial agents useful according to the present
`invention can be prepared using techniques Well knoWn to
`those skilled in the art. For example, antimicrobial peptides
`can be prepared by solid-phase synthesis or using heterolo
`gous gene expression. Exemplary processes for preparing
`antimicrobial peptides are given in Wade et al, Proc. Natl.
`Acad. Sci. USA 87, 4761—4765 (June 1990), Bessale et al,
`FEBS Letters 274, No. 1,2, 151—155 (November 1990), and
`Biochem. Biophys. Res. Commun. 277(3) 675—580
`(November 2000) each of Which is incorporated herein by
`reference in its entirety.
`A second antimicrobial component can be employed in
`the present invention that is other than the ?rst antimicrobial
`component. This second antimicrobial component can be
`selected from substantially non-oxidative antimicrobial
`components and mixtures thereof.
`As used herein, substantially non-oxidative antimicrobial
`components include effectively non-oxidative organic
`chemicals, for example, synthetic polymers, Which derive
`their antimicrobial activity through a chemical or physio
`chemical interaction With the microbes or microorganisms.
`Suitable non-oxidative antimicrobial components include,
`but are not limited to, quaternary ammonium salts used in
`ophthalmic applications such as poly[dimethylimino-2
`butene-1,4-diyl]chloride, alpha-[4-tris(2-hydroxyethyl)
`ammonium]-dichloride (chemical registry number 75345
`27-6, available under the trademark polyquarternium 1®
`from ONYX Corporation), benZalkonium halides, and bigu
`anides such as salts of alexidine, alexidine-free base, salts of
`chlorhexidine, hexamethylene biguanides and their
`
`

`

`US 6,872,705 B2
`
`9
`polymers, antimicrobial polypeptides, and the like and mix
`tures thereof. A particularly useful substantially non
`oxidative antimicrobial component is selected from poly
`hexamethylene biguanide (PHMB), N-alkyl-2-pyrrolidone,
`chlorhexidine, polyquaternium-1, hexetidine, bronopol,
`alexidine, ophthalmically acceptable salts thereof and mix
`tures thereof.
`The salts of alexidine and chlorhexidine can be either
`organic or inorganic and are typically gluconates, nitrates,
`acetates, phosphates, sulphates, halides and the like.
`Generally, the hexamethylene biguanide polymers, also
`referred to as polyaminopropyl biguanide (PAPB), have
`molecular Weights of up to about 100,000. Such compounds
`are knoWn and are disclosed in Ogunbiyi et al US. Pat. No.
`4,758,595, the disclosure of Which is incorporated in its
`entirety herein by reference.
`The substantially non-oxidative antimicrobial compo
`nents useful in the present invention are preferably present
`in the liquid aqueous medium in concentrations in the range
`of about 0.000005% or about 0.00001% to about 2% (W/v).
`More preferably the substantially non-oxidative antimi
`crobial component is present in the liquid aqueous medium
`at an ophthalmically acceptable or safe concentration.
`The concentration of preservative selected depends, for
`example, on the effectiveness of the speci?c preservative in
`preventing groWth, or the killing, of bacteria, fungi, and/or
`protoZoa in a preserved composition. Concentration of pre
`servative selected may also depend on the effectiveness of
`the speci?c preservative in reducing the microbial load on a
`contact lens.
`The present compositions may conveniently be presented
`as solutions or suspensions in aqueous liquids or non
`aqueous liquids, or as oil-in-Water or Water-in-oil liquid
`emulsions. The present compositions may include one or
`more additional ingredients Which are conventionally
`employed in compositions of the same general type.
`The present compositions in the form of aqueous suspen
`sions may include excipients suitable for the manufacture of
`aqueous suspensions. Such excipients are suspending
`agents, for example, sodium carboxymethylcellulose,
`methylcellulose, hydroxypropyl-methylcellulose, sodium
`alginate, polyvinylpyrrolidone, gun tragacanth and gun aca
`cia; dispersing or Wetting agents may be a naturally occur
`ring phosphatide, for example, lecithin, or condensation
`products of ethylene oxide With long chain aliphatic
`alcohols, for example, heptadeca-ethyleneoxycetanol, or
`condensation products of ethylene oxide With partial esters
`derived from fatty acids and a hexitol such as polyoxyeth
`ylene sorbitol mono-oleate, or condensation products of
`ethylene oxide With partial esters derived from fatty acids
`and hexitol anhydrides, for example, polyoxyethylene sor
`bitan mono-oleate, and the like and mixtures thereof.
`The present compositions in the form of oily suspensions
`may be formulated in a vegetable oil, for example, olive oil,
`castor oil, soy oil, sesame oil or coconut oil, or in a mineral
`oil such as liquid paraffin. Such suspensions may contain a
`thickening agent, for example beesWax, hard paraf?n or
`cetyl alcohol.
`The present compositions may also be in the form of
`oil-in-Water emulsions. The oily phase may be a vegetable
`oil, for example, castor oil, olive oil, soy oil, or arachis oil,
`
`10
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`15
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`20
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`25
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`30
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`35
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`40
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`45
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`55
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`60
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`65
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`10
`or a mineral oil, for example, liquid paraffin, and the like and
`mixtures thereof. Suitable emulsifying agents may be
`naturally-occurring gums, for example, gum acacia or gum
`tragacanth, naturally-occurring phosphatides, for example,
`soya bean lecithin, and esters or partial esters derived from
`fatty acids and hexitol anhydrides, for example, sorbitan
`mono-oleate, and condensation products of the said partial
`esters With ethylene oxide, for example, polyoxyethylene
`sorbitan mono-oleate.
`Also included Within the scope of this invention are
`preserved compounds Which increase in viscosity upon
`administration to the eye. For example, “gelling polysac
`charides” Which are disclosed in US. Pat. No. 5,212,162
`Which is incorporated in its entirety herein by reference.
`Also disclosed in this patent are ophthalmic formulations
`containing carrageenans and furcellarans Which are admin
`istered as partially gelled liquids Which gel upon instillation
`into the eye. Additionally, US. Pat. Nos. 4,136,173, 4,136,
`177, and 4,136,178, disclose the use of therapeutic compo
`sitions containing xanthan gum and locust bean gum Which
`are delivered in liquid form to the eye and Which gel upon
`instillation. US. Pat. No. 4,861,760 discloses ophthalmo
`logical compositions containing gellan gum Which are
`administered to the eye as non-gelled liquids and Which gel
`upon instillation. Each of these four patents is incorporated
`in its entirety herein by reference.
`Also Within the scope