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`
`International ]ournal of Dermatology
`
`Published on behalf of the International Society
`‘of Dermatology
`Editor-in—Chief
`Lawrence E. Gibson, Rochester, MN, USA
`Associate Editors
`Rokca A. el—Azhary, Rochester, MN, USA
`David A. Mehregan, Monroe, MI, USA
`‘Dariu_s'R.‘Meh regan, Monroe, MI, USA
`George T. Rcizner, Madison, WI, USA
`Associate Editor for Su plements
`Aditya K. Gupta, Lon on, Ont, Canada
`I
`Correspondence Editor
`J
`Roberto Cortés—Fran<;o, Mexico City, Mexico
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`Jeffrey D. Bernhard, Worcester, MA, USA
`Gunter Burg, Zurich, Switzerland
`Celalettin R. Celehi, Ankara, Turkey
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`Philip R. Cohen, Bellaire, TX, USA
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`llaria Ghersetich. Florence, Italy
`lvlauricio Goihman—Yahr, Miami, FL, USA
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`Aditya K. Gupta, London, Canada
`Jennifer L. Hand, Rochester, MN, USA
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`rance
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`Aims and scope
`Published monthly, the Internationaljournal of Dermatology is specifically designed to
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`

`
`International fournal of
`
`Volume 42, Supplement 1
`
`September 2003
`
`Ciclopirox: a broad spectrum antifungal with
`antibacterial and anti-inflammatory properties
`
`1 Ciclopirox gel: an update Aditya Gupta
`
`3 Ciclopirox for the treatment of superficial fungal infections: a
`review Aditya Gupta, Alayne Skinner
`
`1 1 Evaluation of in vitro activity of ciclopirox olamine, butenafine
`HCI and econazole nitrate against dermatophytes, yeasts and
`bacteria Katrina Kolejohn, Mary Bradley, Brian Griffiths,
`Mahinoud Ghannounz
`
`19 Ciclopirox gel for seborrheic dermatitis of the scalp Raza Aly,
`Irving Katz, Steven Kernpers, Donald Lookinghill, Nicholas Lowe,
`Alan Menter, Manuel Morrnan, Ronald Savin, Mitchell Wortzman
`
`23
`
`Interdigital tinea pedis (dermatophytosis simplex and complex) and
`treatment with ciclopirox 0.77% gel Aditya Gupta, Alayne Skinner,
`Elizabeth Cooper
`
`29 Ciclopirox gel in the treatment of patients with interdigital tinea
`pedis Razz: Aly, George Fisher, Irving Katz, Norman Levine,
`Donald Loo/einghill, Nicholas Lowe, Alan Menter, Manuel Morrnan,
`David Pariser, Harry Roth, Ronald Savin, ]oel Shavin, Daniel Stewart,
`Richard Taylor, Stephen Tucker, Mitchell Wortzrnan
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`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`Original Article
`
`Evaluation of in vitro activity of ciclopirox olamine,
`butenafine HCI and econazole nitrate against dermatophytes,
`yeasts and bacteria
`
`Katrina Kokjohn, D.C."‘, Mary Bradley, M.S.1', Brian Griffiths, B.S."‘,
`Mahmoud Ghannoum, PhD‘)-
`
`*Medicis Pharmaceutical Corporation,
`Scottsdale, AZ; -rcase Western, Center for
`Medical Mycology, Department of
`Dermatology, Cleveland, OH, US
`
`Correspondence
`Katrina Kokjohn,
`Medicis Pharmaceutical Corp.,
`8125 N. Hayden Road, Scottsdale,
`AZ 85258, Us
`E—mail: kkokjohn@medicis.com
`
`Abstract
`
`Background In many instances, a cutaneous fungal infection may exist concomitantly with
`bacterial involvement. In this study we compared the in vitro activity of three antlfungal agents
`against the dermatophytes, yeasts and bacteria recovered most commonly from cutaneous
`mycoses and bacterial infections.
`
`Methods Using a microdilution method adapted from the National Committee for Clinical
`
`Laboratory Standards (NCCLS), we determined the minimum inhibitory concentrations (MICS)
`of ciclopirox olamine, econazole nitrate and butenafine HCI against a panel of dermatophyte
`fungi and yeasts (n = 39) and bacterial isolates (n = 45).
`Results All three antifungals demonstrated comparable activity against the dermatophytes
`tested, with a MIC range of 0.03—0 .25 ug/ml for ciclopirox, < 0.001—O.25 ng/ml for econazole
`and 0.03-0.25 ug/ml for butenafine. For yeasts, ciclopirox showed activity against all isolates,
`with an MIC range of 0.001—O.25 pg/ml, whereas econazole had a broader range of
`O.125—> 0.5 ug/ml. Butenafine displayed limited activity against the yeast Candida albicans
`and no activity against Ma/assezia furfur. For the antibacterial activity studies, ciclopirox
`demonstrated activity against all isolates tested with a range of 0.06—2 ug/ml, while econazole
`showed activity against Gram—positive bacteria only, with a MIC range of 0.004—0.25 pg/ml.
`Butenafine HCI had a limited activity against bacterial isolates tested, showing activity
`against [3—hemo|ytic Streptococcus Group A and Corynebacterium only. Neither econazole
`nitrate nor butenafine HCI demonstrated activity against any of the Gram-negative strains
`evaluated in this study.
`
`Conclusions The data suggest that ciclopirox olamine has the broadest in vitro activity, in
`comparison to econazole and butenafine HCI, against bacteria, yeasts and bacteria. These
`findings may have implications in the use of these antimycotics in the treatment of mixed
`cutaneous infections where bacteria or yeasts are present in addition to dermatophytes.
`
`Introduction
`
`infections are among the world’s most
`Superficial fungal
`common diseases. Dermatophytes are a unique group of fungi
`that infect keratinous tissue, with the skin, hair and nails
`being the most common sites. Certain yeasts, such as Candida
`albicans, also have the capability of infecting the skin and
`causing superficial fungal infections. Gram—p0sitiVe and Grain-
`ncgative bacteria are commonly found as secondary infections
`at the site of fungal infections, with all organisms potentially
`contributing to the pathogenesis of many skin diseases.
`Secondary bacterial
`infection superimposed on a fungal
`infection, a noted sequela in interdigital tinea pedis, is known
`as dermatophytosis complex‘. Dermatophytosis complex
`
`is characterized by increases in the density of the resident
`bacterial population including aerobic diphtheroid bacteria,
`Gram—positive cocci and Gram-negative bacteria, particularly
`Brevibacterium epidermidis, Corynebrzcterium rrzinutissimum,
`Pseudomonas species, Staphylococcus aureus and Microwa-
`cus sedentarius. The primary event in these infections
`may be damage to the stratum corneum by dermatophytes.
`This is followed by an overgrowth of the resident bacterial
`population?
`
`The main classes of antifungals employed for the topical
`treatment of superficial fungal infections are polyenes, imida—
`zoles and allylaminc drugs? These agents differ in their mech-
`anism of action. The polyenes act by binding irreversibly to
`ergosterol, an essential component of fungal cell membranes.
`
`11
`
`© 2003 The International Society of Dermatology
`
`International Journal of Dermatology 2003, 42 (Suppi. 1),
`
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`
`Original Article
`
`In vlzro susceptibilities to clclopirox, bufenafine HCL and eoonazole nltrate
`
`Kokjohn et al_
`
`The interaction of the polyene with fungal membrane sterol
`results in production of aqueous pores; thus, altered permea-
`bility and leakage of vital cytoplasmic components“. Polyenes
`are not active against dermatophytes and their clinical use is
`limited to the treatment of infections caused by Candida
`
`species4. Because of their inactivity against dermatophytes,
`they were not included for testing in this study.
`The imidazoles, discovered in the late 1960s, are relatively
`broad—spectrum antifungals that are primarily fungistatic,
`and act by inhibiting ergosterol synthesis, causing defects in
`the fungal cell membrane. Specifically, azole antifungals
`interfere with the ability of the cytochrome F-450 enzyme
`lanosterol 14—demethylase to catalyze the conversion of
`lanosterol to ergosterol‘.
`Allylamines suppress the biosynthesis of ergosterol at an
`earlier stage of the metabolic pathway than the azoles, inde-
`pendent of the P—45o enzymes, by inhibiting the activity of
`squalene epoxidase. The resulting ergosterol deficiency is acc-
`ompanied by an accumulation of squalene in the fungal cell
`resulting in the disruption of fungal cell membranes. Ally-
`lamines are primarily fungicidal against dermatophytes and
`fungistatic against C. albiccms at therapeutic drug concentrations?
`In addition to these broad classes of antifungals, Ciclopirox,
`
`a hy droxypyridone, is also marketed worldwide for the treat-
`ment of superficial fungal infections. It differs from other
`antifungal agents in its chemical structure and its mechanism
`of action. Unlike most antifungal agents, ciclopirox olamine
`does not affect sterol biosynthesis. The mode of action of
`this drug is very complex, targeting a variety of metabolic
`processes in the fungal cell. Ciclopirox has a high affinity for
`trivalent metal cations such as Fe“. The trapping of this essen-
`tial enzymatic cofactor has an inhibitory effect on enzymes
`such as cytochromes, which are involved in mitochondrial
`electron transport processes? Additionally, the activity of
`catalase and peroxidase, which are responsible for the
`intracellular degradation of toxic peroxides, is strongly
`inhibited by the presence of this drug. Ciclopirox also affects
`the cytoplasmic membrane where it appears to impair active
`transport mechanisms resulting in reduced uptake of
`nutrients into the internal p0ol7. In growing cells, this intra-
`cellular depletion of essential amino acids and nucleotides
`secondarily contributes to the reduced synthesis of proteins
`or nucleic acids.
`
`The imidazoles, allylamines and Ciclopirox are reported to
`possess antibacterial activity as well as antifungal activity
`and, in light of the occurrence of secondary bacterial infec-
`tions, are often prescribed for skin infections that may have a
`bacterial component.
`The objective of this in vitro study was to compare three
`antifungals commonly used to treat superficial fungal infec-
`tions: ciclopirox (a substituted pyridone), econazole nitrate
`(an azole derivative) and butenafine HCl (an allylamine). The
`antifungal agents were tested against selected fungi and yeast
`
`isolates most comm only implicated in tinea infections as well
`as bacterial isolates typically found as secondary infections to
`primary fungal infections. Our data show that of the three
`antifungals tested, ciclopirox olamine had the broadest anti-
`microbial activity against both the fungi and bacteria tested.
`
`Materials and methods
`
`Antifungals
`The three antlfungal agents tested in this study were Ciclopirox
`olamine, econazole nitrate and butenafine hydrochloride (HCI).
`Ciclopirox and econazole were purchased from Sigma—Aldrich
`Chemical Company (St. Louis, MO). Butenafine HCl was isolated
`from Mentax 1.0% Cream (containing 10 mg butenafine HCl),
`(DPT Laboratories, San Antonio, TX).
`
`Organisms
`The antifungal susceptibility of the following organisms was
`determined: dermatophytes (five strains each): Trichophyton
`mentagrophytes, 77'lchophyton tonsurans, Trichophyton rubrum,
`Microsporum canls, Microsporum gypseum, and Epidermophyton
`floccosum; yeasts (five strains each): Candida albicans, and
`Malassezla furfur, Gram—positive bacteria (five strains each):
`Staphylococcus aureus, B—Hemolytic Streptococcus Group A,
`Micrococcus species, Brevibacterium species, and
`Corynebacterlum species; Gram-negative bacteria (five strains
`each): Pseudomonas aeruginosa, Proteus mirabilis, Escherichia
`coll, and Klebsiella pneumoniae. All organisms tested were from
`the culture collection available at the Center for Medical Mycology,
`
`University Hospital of Cleveland.
`
`Susceptibility testing of dermatophytes and yeasts
`Preparation of antlfungal agents: All antlfungal agents were
`standardized according to their weights and units of activity as
`determined by the assays of each batch lot. Ciclopirox olamine
`and econazole nitrate were dissolved in distilled water. RPMI-1 640
`
`media, without sodium bicarbonate and supplemented with
`
`L—glutamine_. was added to achieve a final stock solution
`containing 1280 uglml of antifungal agent. Butenafine was
`dissolved in ethanol and the final stock concentration was adjusted
`to the same concentration with RPMH640 media. Ten serial
`two—fold dilutions of each antifungal were prepared. For clclopirox
`and econazole, the concentration range was 0.001—O.5 pg/ml
`while the concentration range for butenaflne was 0.06—32 ug/ml.
`
`Preparation of dermatophytes and yeasts
`Dermatophyles and C. alblcans were grown on potato dextrose
`agar (Difco Laboratories, Detroit, MI) at 35 °C for 24-48 h. Five
`colonies 2 1 mm in diameter were selected from each culture and
`
`placed in 5 ml of 0.85% sterile saline. The suspensions were
`vortexed and then counted using a hemacytometer. M. furfurwas
`grown at 35 °C for 10 days on Sabouraud dextrose broth (Difco)
`containing 10 ul Tween 80. This cell suspension was counted
`
`lnternailonal Journal ofDerrnalology 2003, 42 (Suppl. 1),. 1147
`
`(3) 2003 The International Society of Dermatology
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`
`Kokjo/in et al.
`
`in vitro susceptibilities to Ciclopirox. butenaline HCL and econazole nitrate Original Article
`
`13
`
`using a hemacytometer and adjusted to a final working
`concentration of 2-5 x 103 cells per ml in RPMI 1640 medium.
`
`Microtiter p/ate preparation
`The method used to determine the antifungal susceptibilities of
`dermatophytes was developed at Center for Medical Mycology at
`University Hospital of Clevelandg. This method is an adaptation of
`the NCCLS document M27—A9 having a microdilution format that
`uses RPMI-1640 as the medium and 2-5 X103 conidia per ml as
`an inoculum. Using a multichannel pipette, 100 pl of 2X antifungal
`concentrations were dispensed into columns 2-11 of sterile
`disposable 96-well (U—shaped) microtiter plates. Column 2
`contained the highest concentration and column 11 the lowest
`concentration of drug. Columns 1 and 12 (controls) received
`100 pl of diluent (RPMl»1 640 media). Using a multichannel pipette,
`100 ul of working dermatophyte or yeast suspension prepared
`above were dispensed into each well of columns 2-12. Column 12
`served as the growth control while column 1 remained uninocu~
`lated and served as the sterility control. The plates were covered
`and incubated at 35 °C for 4-5 days for the dermatophytes,
`24-48 h for C. albicans and 5 days for M. furfur. Following the
`
`incubation period, the MlCs were read visually. MIC (ug/ml) end
`point was defined as the minimum concentration causing 80%
`inhibition compared to the growth control (see Tables 1 and 2).
`
`Susceptibility testing of bacteria
`
`Preparation of antifungal agents
`Same as described above.
`
`Preparation of bacteria
`At least three to five well—isolated colonies of the same
`
`morphological type were selected from an overnight culture.
`The top of each colony was transferred, using a wire loop, to a test
`tube containing 4-5 ml of a Mueller Hinton broth medium (Difco).
`The broth was incubated at 35 “C for 2 h. This actively growing
`broth culture was then adjusted with sterile saline to obtain
`turbidity optically comparable to the 0.5 McFarland standard.
`
`Microtiter plate preparation
`The microsdilution format used for measuring antibacterial
`suscepstibility of bacteria is the method developed by the NCCLS
`
`Table 1 Minimum inhibitory concentrations (MIC, ug/ml) of Ciclopirox olamine, econazole nitrate and butenafine HC1 against 30
`isolates representing different dermatophyte species
`
`Organism Name
`
`Isolate #
`
`Ciclopirox olamine
`
`Econazole nitrate
`
`Butenafine HCl
`
`M1
`M2
`MS
`M4
`M5
`32
`33
`34
`35
`
`18
`20
`
`Trichophyton mentagrophytes
`
`Trichophyton tonsurans
`
`Trichophyton rubrum
`
`Microsporum canis
`
`Microsporum gypseum
`
`Epidermophyton floccosum
`
`0.125
`0.125
`0.06
`0125
`0.125
`0.06
`0.03
`0.06
`0.03
`0.06
`0.125
`0.125
`0.125
`0.125
`0.125
`0.06
`0.03
`0.06
`0.004
`0.08
`0.125
`0.125
`0.125
`0.25
`0.06
`0.03
`0.03
`0.03
`0.03
`
`0.25
`0.125
`0.125
`0.25
`0.06
`0.25
`0.125
`0.25
`0.125
`0.125
`0.03
`0.06
`0.03
`0.03
`0,125
`0.25
`1
`_
`1
`1
`0.5
`
`< 0.001
`< 0.001
`< 0.001
`< 0.001
`< 0.001
`< 0.001
`< 0.001
`0.008
`< 0.001
`< 0.001
`0.25
`0.25
`0.125
`0.25
`< 0.001
`< 0.001
`<0.001
`< 0.001
`< 0.001
`< 0.001
`< 0.001
`< 0.001
`< 0.001
`< 0001
`< 0.001
`< 0.001
`< 0.001
`< 0.001
`< 0.001
`
`No growth obtained
`
`© 2003 The lnternationai Society of Dermatology
`
`International Journal of Dermatology 2003, 42 (Suppl. 1)) 1147
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`
`14
`
`Original Article
`
`In vitro susceptibilities to ciclopirox, butenatine HCL and econazole nitrate
`
`Kol</ohn et al.
`
`Organism Name
`
`Isolate #
`
`Ciclopirox olamine
`
`Econazole nitrate
`
`Butenafine HCI
`
`Candida albicans
`
`Malassezla furfur
`
`799
`798
`405
`648
`593
`5099
`5100
`2117
`3850
`2121
`
`0.06
`0.06
`0.25
`0.25
`0.06
`0.008
`0.001
`0.008
`0.008
`0.125
`
`> 0.5
`0.25
`> 0.5
`0.25
`> 0.5
`> 0.5
`0125
`> 0.5
`0.125
`0.25
`
`> 32
`16
`> 32
`16
`16
`> 32
`> 32
`> 32
`> 32
`> 32
`
`Table 2 Minimum inhibitory concentrations
`(MIC, pg/ml) of Ciclopirox olamine,
`econazole nitrate and butenafine HCI
`
`against IO isolates of two species of yeasts
`
`Table 3 Minimum, inhibitory concentrations (MIC, pg/ml) of ciclopirox olamine, econazole nitrate and butenafine HCI against 23
`isolates representing different Gran1—positive bacteria species
`
`
`Butenafine HCI
`>128
`>128
`>128
`
`> 128
`> 128
`
`18
`
`Organism Name
`
`Isolate #
`
`Ciclopirox olamine
`
`Econazole nitrate
`
`Staphylococcus aureus
`
`8-Hemolytic Strep. Group A
`
`Micrococcus /uteus
`
`Micrococcus sedentarius
`
`Brevibacterium linens
`Bret/lbacterium acetylicium
`Brevibacterlum linens
`Brevlbacterium helvolum
`Brevibacterium linens
`Corynebacterium species
`'
`
`730
`731
`732
`733
`734
`
`735
`736
`737
`738
`739
`783
`784
`495
`496
`497
`776
`777
`778
`779
`780
`781
`782
`
`0.5
`0.5
`0.5
`0.5
`0.5
`
`0.125
`0.06
`0.06
`0.125
`0.125
`2
`0.25
`0.25
`0.25
`0.25
`0.25
`0.125
`0.25
`0.060
`0 .25
`0.25
`0.25
`
`0.03
`0.03
`0.03
`0.03
`0.03
`
`0.03
`0.03
`0.25
`0.25
`0.25
`0.004
`0.004
`0.002
`0.015
`0.008
`0.008
`0.015
`0.008
`0.004
`0.008
`0.008
`0.004
`
`Corynebaclerium mlnulissimum
`
`0.015
`0.25
`492
`0.015
`0.25
`494
`0,008
`0.25
`495
`
`
`document M7—A5‘°. Briefly, drugs were added to sterile disposable
`96-well microtiter plates as described above. Within 15 min of
`preparation, the bacterial inoculum suspensions, containing
`approximately 5 x 105 colony forming units (CFU)/ml, were
`dispensed into the wells as described above. The plates were
`covered and incubated. S. aureus isolates, [3-Hemolytic
`
`Streptococcus Group A isolates and the Gram-negative bacteria
`were incubated at 35 “C for 24 h. All other bacterial isolates were
`
`incubated at 30 “C for 48 h. Following the incubation period, the
`MlCs were read visually. MIC (pg/ml) end point was defined as
`the minimum concentration causing 80% inhibition compared to
`the growth control (see Tables 3 and 4).
`
`Results
`The data show that all three antifungals tested demonstrated
`activity against the dermatophytes (Table 1). Ciclopirox olamine
`showed MIC ranging from o.o3 to 0.7.5 },Lg/ml; econazole
`nitr-ate’s range was < o.oo 1-0.2 5 pg/ml while butenafine I-ICl
`had a range of o.o3-—r.o ug/ml. A comparison of the MIC
`activity against the tested derrnatophytes is shown in Fig. I.
`For yeasts, Ciclopirox showed activity against C. albiczzns and
`M. furfur isolates (Table 2), with an MIC range of o.oor—
`o.25 pg/ml, whereas econazole had a broader range of 0.125
`to > o. 5 pg/ml and butenafine showed limited activity against
`
`International Journal of Dermatology 2003, 42 (Suppl 1), 11—17
`
`© 2003 The International Society of Dermatology
`
`CFAD V. Anacor, |PR201 5-01 776 ANACOR EX. 2087 - 7/“IO
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2087 - 7/10
`
`

`
`Kokjohn et ai.
`
`In vitro susceptibilities to Ciclopirox, butenafine HCL and econazole nitrate Original Article
`
`15
`
`Table 4 Minimum inhibitory concentrations (MIC, pg/ml) of ciclopirox olamine, econazole nitrate and butenafine HCI against 20
`isolates representing different Gram—negative bacteria species
`_M
`Organism Name
`Isolate #
`
`Ciclopirox olamine
`
`Econazole nitrate
`
`Bulenafine HCI
`
`Pseudomonas aeruginosa
`
`>128
`>128
`1
`720
`>128
`0.5
`721
`>128
`2
`722
`>128
`>128
`2
`723
`>128
`>128
`2
`724
`>128
`>128
`>128
`0.5
`723
`>128
`0.5
`724
`>128
`>128
`0.5
`725
`>128
`>128
`>128
`0.5
`726
`>128
`>128
`0.5
`727
`>128
`>128
`>128
`0.5
`744
`0.5
`745
`>128
`>128
`0.5
`746
`>128
`>128
`>128
`0.5
`747
`>128
`0.5
`748
`>128
`>128
`>128
`0.5
`740
`>128
`0.5
`741
`>128
`>128
`0.5
`742
`>128
`>128
`>128
`0.5
`743
`>128
`>128
`0.5
`749
`>128
`
`
`Proteus mirabilis
`
`Escherichia call’
`
`Klebsiella pneumoniae
`
`as03E3-._'4:c:
`
`<
`
` ] Range: onstow
`
`Range: < 0.001 to 0.25
`
`‘ Range: 0.03 to 0.25..,:
`
`0.2
`
`0:4
`
`0.8
`0.6
`MIC in ug/ml
`
`Figure 1 Minimum inhibitory concentrations for dermatophytes.
`
`C. albicans and no activity against M. furfur (Table 2). A
`comparison of these activities is depicted in Fig. 2.
`For the bacterial MIC studies, Ciclopirox olamine demon-
`strated activity against all isolates with a range of o.o6—2 }.Lg/
`ml. Econazole showed activity against Gram—positive bacte-
`ria, with a MIC range of o.oo4—o.z 5 pg/mL. Butenafine I-ICI
`showed activity against B-hemolytie Streptococcus Group A
`and Corynebacterium but failed to inhibit S. aureus (MIC >
`
`Range: 16 to > 32
`
`Antifungal
`
`Range: 0.125 to 0.5
`
`Range: 0.001 to 0.25
`:,
`
`5
`
`10
`
`15
`
`25
`20
`MIC in ug/ml
`
`30
`
`35
`
`40
`
`Figure 2 Minimum inhibitory concentrations for yeasts.
`
`128 ug/ml). Neither econ-azole nor butenafine had activity
`against any of the Gram-negative strains tested. Table 3
`shows MIC determinations for Gram-positive bacteria while
`Table 4 shows the results for the Gram—negative bacteria.
`
`Discussion
`
`This study reports that Ciclopirox olamine had MIC activity
`in a range of o.oo4—o. I2 5 pg/ml for the dermatophytes tested
`and a range of o.o6—o.25 pg/ml for the yeast isolates tested.
`The MIC data reported in this current study may include MIC
`values that are lower than previously reported in the litera-
`ture. In a review of the antimicrobial activity of ciclopirox
`olamine reported by Jue et al.“ in vitro studies report an MIC
`range of o. 5-3 .9 pg/ml for dermatophytes and a range of 0.9-
`3.9 pg/ml for various Candida species. The Jue study utilized
`a macrodilution method for testing antimicrobial suscepti-
`bility and the results are considerably higher than those
`observed in the current study“. Comparisons of MIC data
`obtained from the original agar macrodilution method vs. the
`more recent broth microdilution test reported in this study
`may not be valid since there are reported differences between
`the two methods.
`
`In a study reported by Niewerth et al.“ 50 dermatophyte
`strains obtained from clinical specimens were examined for
`their susceptibility to five systemic or topical antifungal
`agents using both an agar macrodilution and a broth micro-
`dilution method. A comparison of the MIC clearly showed
`differences between the two test methods applied. For all five
`
`© 2003 The lnternatlonai Society of Dermatology
`
`lnternat/‘ona/ Journai of Dermatology 2003‘ 42 (Suppl. 1), 11-17
`
`CFAD V. Anacor, |PR20’|5-01776 ANACOR EX. 2087 - 8/1 0
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2087 - 8/10
`
`

`
`Original Article
`
`In vitrosuseeptibilities to cielopirox, butenafine HCL and econazoie nitrate
`
`Kok/ohn et al.
`
`antifungal agents tested, MIC data were 3—7—fold lower in the
`microdilution test system. The lower MIC ranges reported
`in this study confirm Niewerth’s observation of lower MIC
`values with microdilution testing methods. These differences
`have to be taken into account when comparing MIC data in
`the literature. Physicians frequently rely on comparative MIC
`data to aid in the selection of an appropriate antifungal agent
`to treat tinea infections. This study, comparing MIC data
`performed with the same test method, in the same laboratory
`u11der the same test conditions may be a more appropriate
`comparison for comparing the antimicrobial activity of
`various agents.
`The current study, using a microdilution method, reports
`a MIC maximum of 2 ttg/ml for ciclopirox olamine for Gram-
`negative and Gram-positive bacteria while the ]ue study
`reports an MIC range of 0.25 to > 125 pg/ml for these same
`organisms. Comparisons between the two studies are difficult
`since the variables involved in determining the MIC results
`are not the same. However, there is accordance with Niew-
`
`erth’s report that microdilution testing methods will generate
`lower MIC Values.
`
`The current study also shows butenafine HCI having an
`MIC range against dermatophrtes of o.o3—o.25 ug/ml with
`limited activity against C. zzlbicarzs and no activity against
`M. furfur. An overview by Brennan and Leyden in 1997”
`reported MIC values from several different published studies
`and is complimented by the results obtained in the current
`study. This overview reported a MIC range for butenafine
`I-ICl of 0.12-0.24 ug/ml for dermatophytes and a considera-
`bly higher range of 64-128 rig/ml for C. albicans.
`Limbert and Ulbricht” investigated the activity of ciclopirox
`olamine and other antifungals against two Gram—negative
`bacteria: Proteus nzirabilis and Pseudomonas aeruginosa, con-
`
`firming the current study results by showing that ciclopirox is
`far more effective than econazole nitrate at killing Gram-
`
`negative bacteria. Although the method used was not the micro-
`dilution method reported in this current study, ciclopirox was
`clearly more active against the two Gram-negative organisms
`with MIC values of o.o2—o.5 g/l, compared to econazole
`nitrate’s MIC value reported as > i.oo g/l.
`In an article by Pierard et al.“ the reported antifungal activity
`of both ciclopirox olamine and econazole nitrate against der-
`matophytes and yeasts was comparable to that of the current
`work. However,
`the testing method was a combination
`method of culturing pathogenic dermatophytes and yeasts on
`human stratum corneum. Topical antifungals were applied in
`vivo and the stratum corneum was removed by cyanoacrylate
`skin surface strippings. After inoculation of the test organ-
`isms, the extent of fungal growth was measured, and the level
`of inhibition was determined by comparing the growth to a
`control. Even though MIC levels were not reported, the acti-
`vity of ciclopirox olamine and econazole against the tested
`organisms was similar to the activity reported in this study.
`
`If a