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`Journal of
`
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`
`Clinical Pharmacy
`1. nd Therapeutics
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`ALCON 2123
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`Argentum Pharm. LLC V. Alcon Research, Ltd.
`Case IPR2017-01053
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`Journal of Clinical Pharmacy and Therapeutics
`
`EDITORS
`A. Li Wan Po and M. J. Kendall
`
`ASSOCIATE EDITOR
`D. B.Jack
`
`EDITORIAL BOARD
`M. C. Allwood
`P. Amacker
`R. A. Anderson
`C. W. Barrett
`Joaquin Ronda Beltran
`H. Bundgaard
`Win L. Chiou
`T. D. Clarke
`A. M. S. Cullen
`
`TECHNICAL EDITOR
`S. E. Tibbetts
`
`P. F. D'Arcy
`S. S. Davis
`A. F. Fell
`C. Hetherington
`C. R. Hitchings
`J. H. Kilwein
`E. van der Klein
`E.J. Lien
`P. Lundgren
`
`A. Main
`B. Mulley
`T. Nagai
`Mogens Nedergaard
`C. J. Rolles
`D. Schaaf
`R. G. Shanks
`A. Wertheimer
`J. Wikstrand
`D. Worthen
`
`is designed to provide a forum for clinicians.
`This journal
`pharmacists and other health professionals to explore and report on
`areas of common interest in pharmacy, therapeutics and clinical
`pharmacology. lts scope embraces all aspects of drug development
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`and pharmaceutical practice are also encouraged.
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`Printed in Great Britain by Henry Ling Limited, The Dorset Press, Dorchester, Dorset
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`

`

`Journal of Clinical Pharmacy and Therapeutics (1992) 1 7, 51-54
`
`The antimicrobial action of zinc ion/ antioxidant combinations
`T. J. McCarthy, J. J. Zeelie* and D. J. Krause
`Pharmacy Department, University of Port Elizabeth, PO Box 1600 and• Pharmacy Department, PE Technikon, Private Bag
`X601 1, Port Elizabeth 6000, South Africa
`
`SUMMA RY
`
`The potentiation of action of antimicrobial
`preservatives/antiseptics by, respectively, anti(cid:173)
`oxidants and metal ions has been established. In
`this investigation the antimicrobial effect of two
`antioxidants
`(Buty lated hydroxyanisole
`and
`Propyl Gallate) and zinc ions, both separately
`and combined, was determine d against three or(cid:173)
`g anisms at 37°C. With the exception of Escheri(cid:173)
`chia coli at low zinc concentrations, definite
`potentiation occurred, as re flected by a decrease
`in killing times.
`
`INTRODUCT IO N
`
`The fields of disinfection, antisepsis, and preservation
`are closely related. Before the plethora of antimicrobial
`agents became available it was a valid assertion to state
`that phenol, at concentrations of 3, 1 and 0·5% m/ v,
`respectively, could classify as a disinfectant, antiseptic
`and a preservative. Today we have over 100 preserva(cid:173)
`tives alone, each with its own spectrum of antimicrobial
`activity and its own inherent problems.
`Over the last decade there has been an increasing
`tendency to employ combinations of such antimicrobial
`agents, both to broaden the spectrum of activity (Gram(cid:173)
`positive/ Gram-negative/ fungal) and to di scourage the
`emergence of adaptive resistance. There has also been a
`move away from potent synthetic chemical structures
`to consumer friendly or natural prod ucts.
`Our previous work has centred on t he antimicrobial
`action of combinations of selected preservatives and
`antioxidants (1), and on combinations of selected preser(cid:173)
`vatives. antiseptics and metal ions (2, 3). Here we report
`on combinations of zinc ions and two antioxidants
`(Butylated hydroxyanisole and Propyl gallate). These
`have been examined as preservatives (as all three may
`be orally ingested) and as potential topical deodorants/
`antiseptics. It is the latter aspect which is reported
`here.
`
`MATERIALS AND METHODS
`
`Materials
`
`The following materials were used in this study. Zinc
`sulphate (ZnS04.7H 20) as a source of zN2+ ions;
`butylated hydroxyanisole (BHA), (2- and 3-tert-butyl-
`4-methoxy phenol); propyl gallate (PG), (propyl-3,4,5-
`trihydroxygenzoate); sodium chloride; and ethanol. All
`materials were from reputable suppliers.
`
`Media
`
`Tryptone Soy Broth was used as the recovery medium,
`followed by 24-h incubation at 37°C.
`
`Micro-org anisms
`
`Each system was separately challenged with an over(cid:173)
`night culture of the following, suitably diluted after
`reading
`the absorbance at 600 nm to give a final
`I x 106 cells/ ml final test solution.
`concentration of -
`The following micro-organisms were used: Escherichia
`coli (NCTC 10418), Staphylococcus aureus (NCTC 6571),
`Candida albicans (patient isolate).
`
`M ethods
`
`Ethanolic solutions of BHA and PG were prepared and
`through sterile 0·22 µM filters. A ll other
`sterilized
`ingredients were sterilized by autoclaving at 116°C for
`30 min. Suitable dilutions of z n2+, BHA and PG were
`made into normal saline containing a final calculated con(cid:173)
`centration of ethanol. 4% v/ v, to keep the poorly soluble
`antioxidants in solution. All work was performed in dupli(cid:173)
`cate under a horizontal laminar airAow cabinet.
`
`Contact killing time determinations
`
`Prepared inoculated solutions were kept at 37°C and
`samples (O·l ml) were transferred via sterile pipette
`
`51
`
`

`

`52 T.]. McCartliy et al.
`
`Micro-organism
`
`zni+
`(µg/ml)
`
`1
`
`5
`
`10
`
`16
`
`18
`
`24
`
`48
`
`Time(h)
`
`Table 1. Contact killing times at
`37°C of zinc ions against three micro-
`organisms in normal saline containing
`4%ethanol
`
`$. QIIWIS
`
`E.coli
`
`C. albicans
`
`5
`10
`50
`
`5
`10
`50
`
`5
`10
`50
`
`+ + + + + + +
`+ + + + + + +
`+
`+ + + + + + +
`+ + + + + + +
`+ + + +
`+ + + + + + +
`+ + + + + + +
`+ +
`
`+ = Growth, - = no growth.
`
`Micro-organism
`
`Concentration (%)
`BHA
`
`180 230 250 270 340 360
`
`Time (min)
`
`Table 2. Contact killing times at
`37°C of butylated hydroxyanisole
`against three micro-organisms in
`normal saline containing 4% ethanol
`
`5. aum15
`
`E.coli
`
`C. albicans
`
`0·038
`0·040
`
`0·038
`0·040
`
`0·038
`0·040
`
`+ +
`
`+
`
`+ +
`+ +
`+ +
`+
`
`+
`
`+ +
`
`+
`
`+
`
`tips to 2 ml volumes of T ryptone Soy Broth in sterile
`containers, followed by incubation for 24 h at 37°C.
`(Tables 1-4).
`
`Dilution coefficient (TJ values) determinations
`
`The 17 values for the antioxidants were determined
`(Table 5) using the following formula:
`
`where C, and C2 are the first and second concentrations
`of the test substance; and t 1 and 12 are the killing times of
`cl and cl' respectively.
`
`+ = Growth. - = no growth.
`
`RES UL TS AND D ISCUSS ION
`
`It can be seen from Table I that zn2+ ions have
`little effect on microbials except at a concentration of
`50 µg/ ml. The same effect has been observed for
`Minimum Inhibitory Concentrations (MIC) when used
`alone (2). Furthermore, zn2+ ions have little effect
`against the troublesome Pseudomonas aeruginosa. Tables
`2 and 3, respectively, reflect the killing times of low
`concentrations of BHA and PG. Although these killing
`times would be considered as satisfactory and even
`desirable in topical cosmetic and pharmaceutical formu(cid:173)
`lations, they would not be considered adequate in a
`clinical situation as a topical antiseptic. Combinations of
`zn2+ ions with each of the antioxidants has, however,
`shown promising potentiation for S. aureus and C.
`
`

`

`Table 3. Contact killing times at
`3 7°C of propyl gall ate against three
`micro-organisms in normal saline
`containing 4% ethanol
`
`Micro-organism
`
`Concentration
`(pg)
`
`130 190 220 240 310 330
`
`Time(min)
`
`Antimicrobial action of zinc ion 53
`
`S. aureus
`
`E.coli
`
`C. albicans
`
`0·19
`0.20
`
`0·19
`0.20
`
`0·19
`0·20
`
`+ + + +
`+ +
`+
`
`+ + + + +
`+ + +
`
`+ = Growth, - = no growth.
`
`Table 4. Effect at 37°C of added zinc ions on the contact
`killing times of 0·04% butylated hydroxyanisole and 0·2%
`propyl gallate against three micro-organisms in normal saline
`containing 4% ethanol
`
`Micro-organism
`
`Added Znz+
`(µg/ ml)
`
`BHA
`
`PG
`
`S. aureus
`
`E. coli
`
`C. albicans
`
`0
`5
`10
`50
`
`0
`5
`10
`so
`0
`5
`10
`so
`
`180
`70-90
`60-70
`20- 30
`
`220
`130
`80-90
`50
`
`250
`250-270
`300
`160-170
`
`130
`150
`150-170
`60-80
`
`230
`15
`9
`6
`
`240
`160
`130
`60-80
`
`Table 5. A summary of the I'/ values at 37°C for butylated
`hydroxyanisole and propyl gallate against three micro(cid:173)
`organisms in normal saline containing 4% ethanol
`
`Micro-organism
`
`S. aureus
`E.coli
`C. albicans
`
`BHA
`
`7·7
`7·3
`7-7
`
`PG
`
`7·1
`8·1
`6·7
`
`albicans. With the latter the addition of only 10 µg / ml
`of z n2+ ions to 0·04% BHA reduced the killing time
`from 230 to 9 min (T able 4). Propyl gallate in general is
`less active than BHA but enhanced activity is observed
`when z n2+ ions are added. In the case of E. coli Table 4
`shows that with only 50 µ g/ ml an improved killing time
`is observed for both BHA and PG, with 5 and 10 µg/ ml
`actually delaying the killing time. Isobolograms plotted
`for the minimum lethal concentrations of Znz+ ions and
`BHA or PG, respectively, showed defin ite antagonism
`for BHA and slight antagonism between the median
`range of Znz+ ions and PG against E. coli.
`Table 5 shows that the antioxidants possess high r,
`values. As shown by the formula C'1 x f = k, an increase
`in concentration of an antimicrobial with a high r, value
`can dramatically reduce the killing time. For example, if
`phenol (r, = 6) at 1% concentration kills an inoculum in
`100 min, then doubling the concentration should reduce
`the killing time to 1·56 min (100/26). If the phenol should
`be decreased to 0·5% by complexation however, (sorp(cid:173)
`tion into plastic or rubber, volatization, etc), then the
`killing time lengthens to 4·5 days: (!6 x 106(h) = 100).
`This can give rise to contaminated products, nosocomial
`infections and adaptive resistance. Fortunately the
`QACs have r, values of 1, and chlorhexidine has an r,
`value of 2 so that dilution will not profoundly affect
`these classes of antimicrobial agents. We have pre(cid:173)
`viously determined brono pol and germall II as having
`values of 0·92 and 0·96, respectively, against E. coli (4),
`and benzoic acid as 1·75, however, the aromatic alcohols
`benzyl alcohol and phenethyl alcohol gave figures of 7
`and 9, respectively (4), so that the compounds, along
`w ith the phenolics, need to be carefully monitored fo r
`inactivation/ dilution effects.
`
`

`

`54
`
`T. ]. McCarthy et al.
`
`This preliminary work indicates potentially useful
`synergism between the consumer friendly antioxidants
`BHA and PG and zn2+ ions at low levels suitable
`for foodstuffs and oral pharmaceuticals. At
`the
`concentrations employed here, however, the killing rate
`does not indicate any beneficial clinical advantage over
`existing preparations combined with z n2+ ions at levels
`below 50 µg/ ml.
`
`REFERENC ES
`
`I. Zeelie JJ. McCarthy TJ. (1985) The antimicrobial activities
`of selected preservatives in the presence of phenolic
`
`antioxidants. South African Pharmacy journal, 52(4),
`161-163.
`2. McCarthy TJ. (1985) Metal ions as microbial inhibitors.
`Cosmetics and Toiletries, 100, 69-72.
`3. McCarthy TJ, Zeelie )J. (1989) The effect of zinc ions on
`antimicrobial activity of selected preservatives. Journal of
`Pharmacy and Pharmacology, 4 IS, 114P.
`4. Hurwitz SJ, McCarthy TJ. (1986) 2, 3, 5-Triphenyltetra(cid:173)
`zolium chloride as a novel tool in germicide dynamics.
`journal of Pharmaceutical Sciences, 75, 912- 916.
`5. Hurwitz SJ. McCarthy TJ. (1987) The effect of pH and
`concentration on the rated of kill of benzoic acid solutions
`against E. coli. journal of Clinical Pharmacy and Therapeutics,
`12, 107- 115.
`
`