Dermatol Ther (Heidelb)
`DOI 10.1007/s13555-016-0093-x
`
`BRIEF REPORT
`
`Amorolfine 5% Nail Lacquer Exhibits Potent
`Antifungal Activity Compared to Three Acid-Based
`Devices Indicated for the Treatment
`of Onychomycosis: An In Vitro Nail Penetration Assay
`
`Mahmoud Ghannoum . Karine Sevin . Marlis Sarkany
`
`To view enhanced content go to www.dermtherapy-open.com
`Received: October 23, 2015
`Ó The Author(s) 2016. This article is published with open access at Springerlink.com
`
`ABSTRACT
`
`Introduction: Onychomycosis
`the most
`is
`common infectious disease involving nails.
`The aim of this study was to evaluate the
`antifungal activity of amorolfine 5% nail
`lacquer and three different acid-based medical
`devices
`indicated
`in the
`treatment
`of
`onychomycosis
`using
`an in vitro nail
`penetration assay.
`Methods: Four products were tested in vitro:
`(a) amorolfine 5% nail lacquer; (b) ethyl lactate
`and acetic acid; (c) citric acid and urea; (d) ethyl
`lactate, glycerin, lactic acid, and citric acid. Test
`products were applied to healthy human
`cadaver nails and allowed to dry. Disks were
`
`Electronic supplementary material The online
`version of this article (doi:10.1007/s13555-016-0093-x)
`contains supplementary material, which is available to
`authorized users.
`
`M. Ghannoum (&)
`Center for Medical Mycology, University Hospitals
`of Cleveland and Case Western Reserve University,
`Cleveland, OH, USA
`e-mail: mag3@case.edu
`
`K. Sevin
`Galderma R & D SNC, Sophia Antipolis, France
`
`M. Sarkany
`Galderma International, Paris, France
`
`cut from each piece of nail and placed on
`seeded agar plates of Trichophyton rubrum.
`Following incubation at 30 °C,
`zones of
`inhibition were measured.
`Results: Amorolfine-treated nails
`exhibited
`inhibitory activity against T. rubrum with a
`mean zone of
`inhibition of 59.2 mm in
`diameter.
`In contrast, all
`three acid-based
`medical devices and the untreated controls
`showed no zones of inhibition (mean effective
`zones of 0 mm).
`Conclusion: In this in vitro nail penetration
`model, head-on, comparative study, we showed
`that amorolfine 5% nail
`lacquer possesses
`potent
`antifungal
`activity, whereas
`no
`antifungal activity was detected for
`three
`commercially
`available
`acid-based medical
`devices under identical assay conditions.
`Funding: Galderma.
`
`Keywords: Amorolfine; Fungicidal; Low pH;
`Medical devices; Onychomycosis
`
`INTRODUCTION
`
`Onychomycosis is a common infectious disease,
`which if inappropriately treated can lead to
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`complications,
`life and may
`
`infection,
`the
`of
`spread
`diminished patient quality of
`result in stigmatization [1].
`A fungal nail psychosocial perception study
`carried out in Hong Kong revealed wide-ranging
`misconceptions
`about
`onychomycosis
`treatments since 26% of respondents thought
`disinfectant
`solutions,
`such as vinegar or
`alcohol, were
`effective
`treatments,
`42%
`incorrectly believed that antibiotics were an
`effective treatment, and 14% thought the only
`cure was complete removal of the nail
`[2].
`Another survey indicated that many people
`with mild, uncomplicated onychomycosis may
`never consult a physician [3], and hence are
`likely to rely on topical self-medication.
`for
`To be effective, a topical
`treatment
`onychomycosis must have potent fungicidal
`activity and be able to penetrate the nail plate,
`consisting of dense cross-linked keratin fibers
`held together by cysteine-rich proteins and
`disulphide bonds [4].
`(LocerylÒ,
`lacquer
`Amorolfine 5% nail
`Galderma SA, Lausanne, Switzerland)
`is a
`topical antifungal with proven clinical efficacy
`in the treatment of onychomycosis caused by
`dermatophytes, yeasts and moulds [5–7]. The
`active substance, amorolfine hydrochloride
`(5.574 g in 100 mL ethanol-based nail lacquer),
`has a fungicidal effect and broad antimycotic
`spectrum.
`A wide range of acid-based medical devices
`are commercialized to treat onychomycosis and
`common ingredients include acids to inhibit
`fungal
`growth,
`glycerin to hydrate
`and
`moisturize the nail, and urea to hydrate and
`gently dissolve the intercellular matrix of the
`nail plate, in addition to penetration enhancers
`and film-forming agents. However, there is a
`notable lack of published reports on their exact
`composition, mechanism of action and whether
`
`Dermatol Ther (Heidelb)
`
`they can penetrate through the nail down to
`the nail bed.
`The objective of this study was to compare
`the antifungal activity of amorolfine 5% nail
`lacquer with three different
`commercially
`available acid-based medical devices using an
`in vitro nail penetration assay.
`
`METHODS
`
`Four products were tested: (a) amorolfine 5%
`nail lacquer; (b) ethyl lactate and acetic acid
`(ExcilorÒ, Vemedia, Diemen, The Netherlands);
`(c) citric acid and urea (Scholl Fungal Nail
`Treatment, Bayer Healthcare AG, Leverkusen,
`Germany); and (d) ethyl lactate, glycerin, lactic
`acid, and citric acid (NailnerÒ, YouMedical,
`Amsterdam, The Netherlands).
`A total of 6 non-diseased big toe nails were
`taken from six fresh human cadavers and
`washed before use. For each test product, 3
`pieces of nail plate from different donors were
`treated with 25 lL test product/cm2. The
`compound was applied to the center of uncut
`nails and allowed to spread evenly. After
`air-drying for 24 h, the nail was inverted and
`two disks of 4 mm diameter (biopsy punch)
`were cut from the center of each piece of nail (a
`total of 6 assays per test product). Each disk was
`placed, with the treated side facing upwards, at
`the center of a seeded agar plate of Trichophyton
`rubrum (ATCCÒ MYA-4438TM, Manassas, VA,
`(2–5 9 105
`USA)
`conidia/mL). Disks
`from
`untreated nails were
`used
`as
`controls.
`Following 4-day incubation at 30 °C, the zone
`of inhibition was measured with an electronic
`caliper. Each test was performed in duplicate.
`The mean zone of inhibition ± the standard
`error of the mean (SEM) was calculated and
`compared between groups. A mean zone of
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`Dermatol Ther (Heidelb)
`
`inhibition [10 mm was chosen to indicate
`potent antifungal activity.
`No formal statistical testing was considered
`necessary to compare the amorolfine 5% nail
`lacquer group with the other treated groups
`(which gave values of zero without any variability).
`This article does not contain any new studies
`with human or animal subjects performed by
`any of the authors.
`
`RESULTS AND DISCUSSION
`
`The mean nail thickness ranging from 0.86 to
`1.09 mm was similar for assays for all test
`products
`(Table 1). Nail disks
`treated with
`amorolfine 5% nail
`lacquer showed a mean
`zone of inhibition ± SEM of 59.2 ± 3.4 mm in
`diameter (Table 1 and Fig. 1a). In contrast, the
`three acid-based medical devices (Fig. 1b–d) and
`the untreated control (Fig. 1e) all showed no
`zones of inhibition (mean effective zones of
`0 ± 0 mm)
`(Table 1). Amorolfine 5% nail
`lacquer
`demonstrated
`potent
`antifungal
`activity when compared to the three medical
`devices tested (Fig. 2).
`The disk-diffusion method has been widely
`used to demonstrate antifungal activity of various
`drugs [8, 9]. In this study, amorolfine 5% nail
`lacquer demonstrated effective antifungal activity
`(inhibition
`zone
`59.2 mm in
`diameter),
`corroborating previous findings showing that
`amorolfine 5% nail lacquer penetrates the nail to
`the
`site of
`infection in the
`subungual
`compartment
`[5, 10, 11]. Due to its high
`potency, even small amounts of amorolfine
`reaching the nail bed inhibit or kill
`the
`pathogens [12] and amorolfine concentrations
`deep in the nail bed still exceeded the minimum
`T.
`rubrum
`inhibitory
`concentrations
`of
`(\0.001–0.13 lg/mL) [6]. Conversely, the three
`acid-based medical products tested did not show
`
`any antifungal activity in the human nail
`penetration model used.
`This easy-to-use in vitro nail penetration
`model was developed taking care to ensure
`that the antifungal effects witnessed were from
`the test compound penetrating the nail plate
`and the test substance could not spread over the
`edge of the nail biopsy to influence the growing
`fungi beneath the nail plate. The compound
`was applied to the center of uncut nails, allowed
`to spread evenly and dry before biopsy punches
`were taken from the center of the nail specimen
`to ensure there was no spillage of the test
`compound. By inverting the nail before taking
`the biopsy, the disks were cut in the direction of
`unexposed nail surface to exposed nail surface
`to ensure that the test compound was not
`artificially dragged from the biopsy punch. It
`is noteworthy that it would also be possible to
`increase the biopsy size or seal the margins of
`the biopsied nail plate. However, presumably
`there was no spreading over the edges since no
`zones of inhibition were observed with the
`acid-based medical devices under identical assay
`conditions in this head-on comparative study.
`The role of acidity (low pH)
`in the
`pathogenesis of dermatophytes
`is complex
`[13]. Transmission electron microscopy has
`shown that many fungal cells were necrotic
`when T. rubrum or Candida albicans was treated
`for 60 min in direct contact with 50% K101 nail
`solution (Moberg Pharma, Bromma, Sweden), a
`topical formulation of pH 4 containing 50 g
`propylene glycol, 15 g lactic acid and 10 g urea
`[14]. The most prominent
`changes were
`observed with T. rubrum; the cell wall was
`clearly damaged, the membrane was disrupted
`and the content in the cytoplasm was degraded.
`The authors suggested that the presence of a
`diol
`(propylene glycol) disturbed cell wall
`integrity by an unspecific mode of action and
`the low pH may have contributed to the efficacy
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`
`Table 1 Measurement of zones of inhibition
`
`Table 1 continued
`
`Test compound Donor
`
`Nail
`disk
`
`Nail
`thickness
`(mm)
`
`Inhibition
`zone
`diameter
`(mm)
`
`Test compound Donor
`
`Nail
`disk
`
`Nail
`thickness
`(mm)
`
`Inhibition
`zone
`diameter
`(mm)
`
`(a) Amorolfine
`5% nail
`lacquer
`
`Mean (±SEM)
`
`Donor 1 1
`
`Donor 1 2
`
`Donor 2 3
`
`Donor 2 4
`
`Donor 3 5
`
`Donor 3 6
`
`Donor 4 1
`
`Donor 4 2
`
`0.83
`
`1.09
`
`1.08
`
`1.09
`
`1.25
`
`1.25
`
`1.09
`
`0.5
`
`0.54
`
`(e) Untreated
`control
`
`70
`
`51
`
`53
`
`68
`
`52
`
`61
`
`59.2 ± 3.4
`
`Mean (±SEM)
`
`0
`
`0
`
`Donor 1 1
`
`Donor 1 2
`
`Donor 2 3
`
`Donor 2 4
`
`Donor 3 5
`
`Donor 3 6
`
`1.26
`
`1.09
`
`1.33
`
`0.87
`
`0.75
`
`1.02
`
`1.05
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0 ± 0
`
`(b) Ethyl lactate
`and acetic
`acid
`
`Mean (±SEM)
`
`(c) Citric acid
`and urea
`
`Mean (±SEM)
`
`(d) Ethyl lactate,
`glycerin, lactic
`acid,
`and citric acid
`
`Mean (±SEM)
`
`Donor 2 3
`
`Donor 2 4
`
`Donor 5 5
`
`Donor 5 6
`
`Donor 1 1
`
`Donor 1 2
`
`Donor 6 3
`
`Donor 6 4
`
`Donor 3 5
`
`Donor 3 6
`
`Donor 4 1
`
`Donor 4 2
`
`Donor 6 3
`
`Donor 6 4
`
`Donor 5 5
`
`Donor 5 6
`
`1.02
`
`0.98
`
`0.98
`
`1.11
`
`0.86
`
`0.69
`
`0.83
`
`1.43
`
`1.46
`
`1.16
`
`0.96
`
`1.09
`
`0.5
`
`0.5
`
`1.01
`
`1.01
`
`1.15
`
`1.09
`
`0.88
`
`0
`
`0
`
`0
`
`0
`
`0 ± 0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0 ± 0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0 ± 0
`
`[14]. Although pH 4 is not low enough to
`inhibit growth, the production of T. rubrum
`arthroconidia has been shown to be dependent
`on pH with 85% less arthroconidia produced at
`pH 4.5 compared to pH 7.5 [15].
`A pH of B3 was found to be fungicidal when
`Sabouraud dextrose broths of different pH
`values were inoculated with T. rubrum and
`incubated for 14 days at 25 °C [16]. However, a
`penetration test in an in vitro porcine nail
`model showed that even after 120 applications
`of acetic acid (Excilor), the pH measured in the
`nails was only 3.37, i.e., not low enough to be
`fungicidal [16]. Furthermore, the lowest pH of
`3.37 was only measured in the superficial parts
`of the nail (0.5 mm depth) [16].
`Although
`acid-based
`solutions may
`penetrate and acidify the nails, as stated by
`the manufacturers, the difficulty in obtaining a
`low enough pH throughout the nail and in the
`nail bed may explain why the three acid-based
`medical devices demonstrated no fungicidal
`activity in the nail penetration model.
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`Dermatol Ther (Heidelb)
`
`Fig. 1 Representative plates showing zones of inhibition of
`Trichophyton rubrum growth in the nail penetration assay
`with a amorolfine 5% nail lacquer; b ethyl lactate and acetic
`
`acid; c citric acid and urea; d ethyl lactate, glycerin, lactic
`acid, and citric acid; e untreated control
`
`Fig. 2 Effect of amorolfine 5% nail lacquer and three medical devices against Trichophyton rubrum in a nail penetration
`assay
`
`Possible limitations of this in vitro study are
`that nails only received a single application of
`test product, and extrapolating from in vitro to
`in vivo may not be entirely accurate as in the
`nail environment in vivo, natural compounds
`may influence fungal growth. However, this
`in vitro nail penetration model has been shown
`to closely simulate in vivo testing and visually
`demonstrates
`both nail penetration and
`antifungal activity after a single application of
`amorolfine 5% nail lacquer [11].
`Clinical evidence of efficacy of acid-based
`solutions against onychomycosis remains scarce
`[17, 18]. The K101 nail solution (a mixture of
`
`propylene glycol, lactic acid and urea, pH 4) has
`been clinically demonstrated to be effective in
`the
`treatment
`of
`distal
`subungual
`onychomycosis
`[18–20].
`In a
`randomized
`(n = 346
`placebo-controlled
`study
`K101,
`n = 147 placebo), more patients with B50%
`nail
`involvement achieved mycological cure
`after 26 weeks in the K101 group (27.2%) than
`in the placebo group (10.4%; P = 0.0012) [20].
`
`CONCLUSION
`
`This study confirms penetration through the
`nail of amorolfine 5% nail lacquer with potent
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`antifungal activity in an in vitro human nail
`penetration model
`following
`a
`single
`application, whereas
`the
`three
`acid-based
`products
`tested had no antifungal activity
`under identical assay conditions.
`
`ACKNOWLEDGMENTS
`
`This study and article processing charges were
`funded
`by
`Galderma
`and Mahmoud
`Ghannoum received a contract. The authors
`wish to thank Helen Simpson of Galderma for
`editorial assistance. All named authors meet
`the
`International Committee of Medical
`Journal
`Editors
`(ICMJE)
`criteria
`for
`authorship
`for
`this manuscript,
`take
`responsibility for the integrity of the work as
`a whole, and have given final approval for the
`version to be published.
`
`Dermatol Ther (Heidelb)
`
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