0
`
`United States Patent [19]
`Lucero
`
`[54] ENHANCEMENT OF BENZALKONIUM
`CHLORIDE PRESERVATIVE ACTIVITY IN
`FORMULATIONS CONTAINING AN
`INCOMPATIBLE DRUG
`
`[75] Inventor: Jasmm C‘ Lucem’ Irvine’ Cahf'
`[73] Assigneez Allergan, Inc.’ Irvine, Calif.
`
`[21] APPl- N05 204,853
`[22] Filed:
`Mar. 2, 1994
`
`[51] Int. CI-G ....................... ..
`
`
`
`[52] US. Cl. [58] Field of Search
`
`AOIK 14
`514/554; 514/643; 514/912
`................................... .. 514/554, 643,
`514/912
`
`[56]
`
`References Cited
`
`PUBLICATIONS
`
`lllllllllllllllllllllllllllllwlslgllllll?llllllllllllllllllIIIIHIII
`
`[11] Patent Number:
`[45] Date of Patent:
`
`5,504,113
`Apr. 2, 1996
`
`Primary Examiner-—Zohreh Fay
`Attorney, Agent, or Firm—Walter A. Hackler
`
`ABSTRACT
`[57]
`Aformulation and method includes an acceptable drug, such
`as Prostaglandins, Flurbiprofen, Keterolac Tromethamine,
`Cetirizine HCl Indomethacin and Bufrolin, which are inter
`active with benzalkonium chloride to form a precipitate
`along with benzalkonium chloride acting as a preservative
`and an amino acid having enough positive charge at the pH
`of the formulation and/or Tromethamine present in an
`amount Su?cient to interfere with the interaction between
`the drug and benzalkonium chloride in order to maintain the
`preservative activity of the benzalkonium chloride. Further,
`the use of Lysine, L-arginine, or Histidine is also useful in
`reducing the cytotoxicity of the formulation.
`
`Chemical Abstracts, 110:219120 (1989). Fu et a1.
`
`23 Claims, 1 Drawing Sheet
`
`LUPIN EX 1064
`
`Page 1 of 6
`
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`Page 2 of6
`
`Page 2 of 6
`
`

`
`5,504,113
`
`1
`ENHANCEIVIENT OF BENZALKONIUM
`CHLORIDE PRESERVATIVE ACTIVITY IN
`FORMULATIONS CONTAINING AN
`INCOMPATIBLE DRUG
`
`The present invention generally relates to improved
`formulations and solutions and more particularly to
`improved preservative systems for acceptable drug formu
`lations which have an incompatibility with benzalkonium
`chloride (BAK) such as Prostaglandins, Flurbiprofen, Ket
`erolac Tromethamine, Cetirizine HCl and Indomethacin.
`More speci?cally, the present invention pertains to the
`preservative for an ophthalmologically acceptable drug such
`as Bufrolin having activity for treating seasonal allergies,
`allergic conjunctivitis, giant papillar conjunctivitis, and ver
`nal keratoconjunctivitis.
`Ophthalmologically acceptable drug formulations gener
`ally contain effective compounds and a number of ophthal
`mologically acceptable excipients. formulations generally
`include solutions, ointments, and suspensions, etc. The
`formulations may include excipients such as stabilizing
`agents, surfactants, buffering systems, chelating systems,
`viscosity agents, tonicity agents, and, importantly, a preser
`vative.
`Ophthalmic formulations, understandably, must be ster
`ile and if a multi-dose regimen is intended, the formulation
`must be preserved with an effective antimicrobial agent.
`As discussed in US. Pat. No. 5,110,493, organo-mercu
`rials have been used extensively as the preservatives in
`ophthalmic solutions. As reported in this reference, these
`compounds pose di?‘iculties due to potential mercury tox
`icity as well as poor chemical stability.
`Therefore, benzalkonium chloride, which is a quaternary
`ammonium compound, has been widely used in ophthalmic
`solutions. It is also well-known however, that benzalkonium
`chloride is considered incompatible with annionic drugs,
`forming insoluble complexes which cause the solution to
`turn cloudy.
`This is because of the fact that many annionic drug
`entities carry a negative charge at physiological pH. In fact,
`all acidic drug entities will carry a negative charge at all
`pH’s above their pKa’s.
`In the case of benzalkonium chloride, which is a posi
`tively charged preservative, insoluble complexes can be
`formed with acidic drug entities causing the drug to pre
`cipitate out of solution. Concomitant with the removal of the
`drug from solution is the removal of benzalkonium chloride,
`thereby rendering this quaternary gernricide incapable of
`performing its function as an antimicrobial agent.
`Benzalkonium chloride is a mixture of alkylbenzyldim
`ethylamrnonium chloride of the general formula as shown
`below in which R represents a mixture of the alkyls from
`C8H17 to C1sH37
`
`. e
`
`CH3
`
`CI
`
`As hereinbefore noted, it is well-known that benzalko
`nium chloride is generally incompatible with anionic deter
`gents or anionic drug compounds. See US. Pat. No. 5,110,
`493, and The Merck Index, 11th Edition, Merck & Co., Inc.,
`1989.
`The present invention speci?cally relates to the discovery
`that an additive having a positive charge at the pH of the
`formulation can be used to compete with benzalkonium
`chloride and reduce complexation of any anionic drug with
`the benzalkonium chloride and thereby enhance the preser~
`vative effectiveness of the benzalkonium chloride.
`
`2
`SUMMARY OF THE INVENTION
`
`A formulation in accordance with the present invention
`generally includes an acceptable drug which is interactive
`with benzalkonium chloride in combination with the very
`entity, benzalkonium chloride, with which the acceptable
`drug forms a complex, thereby removing the benzalkonium
`chloride from solution, and consequently reducing its effec
`tiveness as a preservative. As noted, the benzalkonium
`chloride is added as a preservative and is active in that
`regard. Examples of such drugs include, but not limited to,
`Prostaglandins, Flurbiprofen, Keterolac Trometharrrine,
`Cetirizine HCl Indomethacin and Bufrolin.
`In combination with the acceptable drug and the benza
`lkonium chloride is an additive, having a net positive charge
`at the pH of the formulation, and present in amounts
`su?icient to enhance preservative eifectiveness of the ben
`zalkonium chloride. An effective amount is suflicient for the
`additive to compete with the benzalkonium chloride for the
`interaction of the ophthalmologically acceptable drug,
`thereby freeing more benzalkonium chloride and providing
`overall enhancement of the preservative activity of the
`benzalkonium chloride.
`More particularly, an ophthalmologically acceptable drug
`may comprise bufrolin and the additive may comprise an
`amino acid having a net positive charge at the pH of the
`formulation present in su?icient amounts to interfere with
`the interaction between the drug and the benzalkonium
`chloride in order to maintain the preservative activity of the
`benzalkonium chloride.
`More particularly, the amino acid may be selected from a
`group consisting essentially of Lysine, L-arginine and His
`tidine.
`More speci?cally, the ophthalmic solution in accordance
`with the present invention includes bufrolin as the ophthal
`mologically acceptable drug and present in an amount of up
`to about 4% w/v. Preferably, the amino acid comprises
`L~arginine present in an amount between about 0.5% and
`about 5% w/v.
`Utilization of the L~arginine reduces the amount of ben~
`zalkonium chloride necessary as a preservative and accord
`ingly, in accordance with the present invention, the benza
`lkonium chloride may be present in an amount about 100
`ppm or less.
`As an alternative embodiment of the present invention,
`the additive comprises Tromethamine present in an amount
`of between about 0.3% and about 2% w/v.
`In another embodiment of the present invention, a plu
`rality of additives, each having a net positive charge at the
`pH of the formulation, are utilized in an amount su?icient to
`inhibit formation of an insoluble complex between the
`benzalkonium chloride and the ophthalmologically accept
`able drug. More particularly, in this last-mentioned embodi
`ment, the ophthalmologically acceptable drug formulation
`comprises both an amino acid having a net positive charge
`at the pH of the formulation and Tromethamine. The amino
`acid may comprise either Lysine, L-arginine, or Histidine, or
`combinations thereof, but preferably comprises L-arginine.
`In addition,
`the utilization of L-arginine and
`Tromethamine together reduces the amount of BAK neces
`sary as a preservative and accordingly, in accordance with
`the present invention, the BAK may be present in an amount
`about 100 ppm or less.
`
`15
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
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`55
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`60
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`Page 3 of 6
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`

`
`5,504,113
`
`3
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`The advantages and features of the present invention will
`be better understood by the following description when
`considered in conjunction with the accompanying drawing
`in which:
`FIG. 1 is a comparison of bacterial (R aeruginosa)
`recoveries of formulations with and without L-arginine and
`Tromethamine; and
`FIG. 2 is a comparison of inulin permeability (cytotox
`icity) of formulations with and without L-arginine.
`
`5
`
`DETAILED DESCRIPTION
`
`Bufrolin is a classic example of an anionic drug that forms
`an insoluble complex with benzalkonium chloride. Bufrolin
`is 6-n-Butyl, l,4,7,10-tetrahydro-4,10-dioxo~1,7-phenan
`throline-2,8-dicarboxylic acid. As hereinabove noted, this
`drug has activity for treating seasonal allergies, allergic
`conjunctivitis, giant papillar conjunctivitis and vernal kera
`toconjunctivitis. It is to be appreciated that while this
`particular drug is cited throughout here as an example, other
`anionic drugs that form an insoluble complex with benza
`lkonium chloride are to be considered to be within the scope
`of the present invention.
`It is also well-known that benzalkonium chloride (BAK),
`alone or in combination with disodium edetate (EDTA), has
`been widely used for many years as an ophthalmic preser
`vative. This preservative, through extensive testing and use,
`has been proven to be one of the most effective and
`rapid-acting preservatives which is stable over the pH range
`which most ophthalmic products are formulated.
`It is also known that the addition of between about 0.01%
`and about 0.1% EDTA increases the effectiveness of BAK
`against some resistant strains of the pseudomonas species.
`Unfortunately, since BAK is a cationic compound, incom
`patibility with anionic drugs limits its use as a preservative.
`The synergism expected from the combination of BAK and
`EDTA has not been successful in preserving formulations
`having high concentrations of anionic drugs such as Bufrolin
`which require up to 4 % w/v for the treatment of allergic
`conjunctivitis in phosphate-buffered solutions with a pH
`between about 6 and about 8, and preferably about 7.4.
`Amino acids suitable for use in the present invention
`having a positive charge at the pH of the formulation may
`include Lysine, Arginine and Histidine, having positively
`charged R groups, as shown below with the amino and
`carboxyl group ionized as they would occur at pH 7.0. These
`amino acids can be used individually, or in combination with
`one another and Trometharnine or Tris [2-Arnino-2—(hy
`droxymethyl)-1,3-propanediol], which is a well-known bio
`logical bu?cer.
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`4
`invention, as well as cytotoxicity tests, have been performed
`in accordance with the following methods:
`
`Preservative Efficacy Test
`
`Materials:
`a. Test Organisms
`S. aureus, R aeruginosa, E. coli, C. albicans, A. niger
`b. Recovery Media
`Trypticase soy broth with neutralizing phosphate buffer
`and Polysorbate 80
`Trypticase Soy Agar with 1.0% Glucose
`Method:
`1. 10 mL aliquots of the test samples are inoculated with
`50 uL of test organisms to yield a ?nal concentration of
`about 5 l06 CFU/mL.
`2. The samples are tested for survivors on D-O, 6 hour,
`D-l, D- 14, D21, D-28.
`
`Cytotoxicity—Inulin Permeability Assay
`2X105 MDCK (Madin-Darby Canine Kidney) cells on
`collagen-coated semi-permeable inserts are incubated for 2
`days prior to the assay. 0.4 pCi 14C-inulin is added to 400 uL
`of the test sample and 100 uL of this is placed in each insert.
`At each timepoint, 20 uL of sample is taken from outside of
`the insert and radioactivity measured using scintillation
`counting.
`The preservative criteria for ophthalmic preparations uti
`lized in the studies include British Pharmacopeia (BF-88),
`Deutsches Arzneibuch (DAB-9) and United States Pharma
`copeia (USP) as shown in Table I.
`
`TABLE I
`
`Preservative Criteria for Ophthalmic Preparations
`
`BP-88
`
`DAB-9
`
`USP
`
`S. aureus
`#65381’
`
`—3 log in 6 hrs
`0 in 24 hrs
`
`P. aeruginosa
`#9027
`
`~3 log in 6 hrs
`0 in 24 hrs
`
`E. coli
`#8739
`C. albicans
`#10231
`
`A. niger
`#16404
`
`N/A
`
`—2 log in 7
`days
`0 incr. 28 days
`—2 log in 7 days
`0 incr. 28 days
`
`—1 log in 14
`days
`
`—1 log in 14
`days
`
`—2 log in 24
`hrs
`—3 log in 7
`days
`—2 log in 24
`hrs
`—3 log in 7
`days
`N/A
`
`—3 log in 14
`days
`
`—3 log in 14
`days
`
`—3 log in 14
`days
`0 incr. in
`14-28 days
`
`0 incr. in
`14-28 days
`
`Table II shows the preservative ef?cacy test results of
`some formulations:
`
`55
`
`TABLE II
`
`Preservative E?icacy Test Results of Formulations
`
`Preservative System
`
`Buifer
`
`DAB-9 USP
`
`100 ppm BAK, 0.10% EDTA
`70 ppm BAK, 0.03% EDTA
`50 ppm BAK, 0.05% EDTA
`50 ppm BAK, 0.02% EDTA,
`0.50% L-arginine
`
`0.02 M Po4
`0.10 M Tris
`0.10 M Tris
`0.10 M Tris
`
`fail
`pass
`fail
`pass
`
`fail
`pass
`fail
`pass
`
`Microbiological studies assessing the preservative e?i
`cacy of the formulation made in accordance with the present
`
`Sample formulations utilizing L-arginine, Lysine or His
`tidine include:
`Anionic drug—e.g. Bufrolin-4% w/v
`
`Page 4 of 6
`
`

`
`5
`Cationic preservative—~e.g. BAK
`Chelators—e. g. NaQEDTA
`Buffers-cg. tris, phosphate
`Salts—e.g. NaCl for tonicity adjustment
`Dilute acid/base—e.g. HCl/NaOH for pH adjustment
`As shown in Table H, the preservative e?icacy test (PET)
`showed that, even with as much as 100 ppm BAK and 0.1%
`EDTA in 0.02M phosphate buffer (pH 7.4), the 4% w/v
`Bufrolin formulation failed to meet the USP criteria.
`The use of Tris (Tromethamine) as a buffer and counterion
`enhanced the e?icacy of the BAK/EDTA combination and a
`formulation of 4% w/v Bufrolin with 70 ppm BAK, 0.03%
`EDTA, and 0.1M Tris pass the DAB-9 test, which is a more
`stringent criterion than that of the USP.
`The addition of Lysine, L-arginine or Histidine further
`improves the activity of BAK. With as low as 0.5% w/v
`L-arginine in combination with 0.10M Tris buffer, the for
`mulation passed DAB-9 with only 50 ppm BAK and 0.02%
`EDTA with a much better R aeruginasa kill pro?le, as
`shown in FIG. 1. Thus, it is shown in accordance with the
`present invention that the combination of L-arginine and
`Tris lowers the amount of BAK necessary to preserve the
`resulting formulation.
`In the inulin permeation test, FIG. 2, the presence of
`L-arginine in the formulation signi?cantly decreased the
`permeability (cytotoxicity) to tight junctions between the
`epithelial cells by approximately 45-fold in comparison to
`other formulations.
`Thus, as shown in FIG. 2, formulations containing 100
`ppm BAK, 0.1% EDTA, and 5% L'arginine have less
`permeability (less cytotoxicity) than the 50 ppm BAK
`control.
`In accordance with the present invention, the addition of
`an amino acid, having a net positive charge at about neutral
`pH, such as Lysine, L-arginine, and Histidine, along with
`Tris, compete with and prevent BAK from complexing with
`an anionic drug such as Bufrolin. Thus, the amino acid
`and/or the Tris are effective in maintaining a stable and
`adequately preserved formulation. In addition, the presence
`of an amino acid having a net positive charge at 7.4 pH, such
`as L-arginine, greatly decreases the cytotoxicity of the
`formulation.
`It should be noted that the effectiveness of the formulation
`is well within the 5 to 7.6 pH, generally accepted for
`ophthalmic formulations, with an ideal pH of 7.4 for comfort
`in use of the formulation. Also, while it is preferable to
`maintain the concentration of BAK below 100 ppm, such
`as—for example—25 ppm, 50 ppm, or 75 ppm, formula
`tions may be effective with higher amounts of BAK corre
`sponding to greater amounts of Tris or L~arginine of more
`than about 4%.
`Although there has been hereinabove described a speci?c
`ophthalmic solution and method in accordance with the
`present invention, for the purpose of illustrating the manner
`in which the invention may be used to advantage, it should
`be appreciated that the invention is not limited thereto.
`Accordingly, any and all modi?cations, variations, or
`equivalent arrangements which may occur to those skilled in
`the art, should be considered to be within the scope of the
`present invention as de?ned in the appended claims.
`What is claimed is:
`1. A formulation comprising:
`a drug interactive with benzalkonium chloride; benzalko—
`nium chloride active as a preservative; and
`L-arginine present in an amount su?icient to interfere with
`the interaction between the drug and benzalkonium
`chloride in order to maintain the preservative activity of
`the benzalkonium chloride.
`
`35
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`
`5,504,113
`
`10
`
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`
`6
`2. The formulation according to claim 1 wherein the drug
`is selected from a group consisting essentially of Prostag
`landins, Flurbiprofen, Keterolac Tromethamine, Cetirizine
`HCl and Indomethacin.
`3. The ophthalmic formulation according to claim 1
`wherein said drug comprises Bufrolin.
`4. The ophthalmic formulation according to claim 3
`wherein the drug comprises Bufrolin present in an amount
`up to about 4% w/v.
`5. The formulation according to claim 1 wherein the
`L-arginine is present in an amount of between about 0.5%
`and about 5% w/v.
`6. The formulation according to claim 5 wherein the
`benzalkonium chloride is present in an amount less than
`about 100 ppm.
`7. A formulation comprising:
`a drug having a negative charge at the pH of the formu
`lation;
`benzalkonium chloride present in a preservative elTective
`amount; and
`L-arginine present in an amount su?icient to enhance the
`preservative effectiveness of the benzalkonium chlo
`ride.
`8. The ophthalmic formulation according to claim 7
`wherein the drug comprises Bufrolin present in an amount
`up to about 4% w/v.
`9. The ophthalmic formulation according to claim 8
`wherein the L-arginine is present in an amount of between
`about 0.5% and about 5% w/v.
`10. The formulation according to claim 8 wherein the
`benzalkonium chloride is present in an amount less than
`about 100 ppm.
`11. A formulation comprising:
`an anionic drug capable of forming an insoluble complex
`with benzalkonium chloride;
`benzalkonium chloride present in a preservative effective
`amount; and
`L—arginine present in an amount effective to reduce cyto
`toxicity of the formulation.
`12. The formulation according to claim 11 wherein said
`drug comprises Bufrolin.
`13. The formulation according to claim 11 wherein the
`drug comprises Bufrolin present in an amount up to about
`4% w/v.
`14. The formulation according to claim 13 wherein the
`L-arginine is present in an amount of between about 0.5%
`and about 5% w/v.
`15. The formulation according to claim 14 wherein the
`benzalkonium chloride is present in an amount less than
`about 100 ppm.
`16. A method for preserving a formulation comprising the
`step of combining a drug interactive with benzalkonium
`chloride with benzalkonium chloride active as a preservative
`and L-arginine and Tromethamine present in an amount
`su?icient to interfere with the interaction between the drug
`and benzalkonium chloride in order to maintain the preser
`vative activity of the benzalkonium chloride.
`17. The method according to claim 16 wherein the drug is
`selected from a group consisting essentially of Prostaglan
`dins, Flurbiprofen, Keterolac Tromethamine, Cetirizine HCl
`and lndomethacin.
`18. The method according to claim 17 wherein said drug
`comprises Bufrolin.
`19. The method according to claim 17 wherein the drug
`comprises Bufrolin present in an amount up to about 4%
`w/v.
`
`Page 5 of 6
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`

`
`5,504,113
`
`7
`20. The method according to claim 16 wherein the L-argi
`nine is combined in an amount of between about 0.5% and
`about 5% w/v.
`21. The method according to claim 20 wherein the ben
`zalkonium chloride is combined in an amount less than
`about 100 ppm.
`22. The method according to claim 16 further comprising
`the step of combining Tromethamine in an amount su?icient
`
`8
`to lower the amount of benzalkonium chloride necessary to
`preserve the combination.
`23. The method according to claim 22 wherein the amount
`of Tromethamine combined is between about 0.3% and
`about 2% w/v.
`
`Page 6 of 6