Full text of DOI:10.1111/j.1348-0421.2002.tb02662.x

Microbiol. Immunol., 46(2), 83
―88, 2002
Development of a New Medium Useful for the
Recovery of Dermatophytes from Clinical
Specimens by Minimizing the Carryover
Effect of Antifungal Agents
,
1
1
1
1
2
Takuji Nakashima* , Akira Nozawa , Takao Ito , Toshiro Majima , and Hideyo Yamaguchi
1
2
Pharmaceutical R&D Laboratories, POLA Chemical Industries, Inc., Yokohama, Kanagawa 244
―
0812, Japan, and Teikyo
University Institute of Medical Mycology, Teikyo University, Hachioji, Tokyo 192―0352, Japan
Received September 19, 2001. Accepted November 28, 2001
Abstract: Two surface-active compounds, egg lecithin and polysorbate 80, usually used as the deactivators
of various preservatives were tested whether they also counteract either or all of the three major topical anti-
fungal drugs, bifonazole (BFZ), lanoconazole (LCZ) and terbinafine (TBF). Both egg lecithin and polysor-
bate 80, when added to culture media up to final concentrations of 1.0 and 0.7%, respectively, antagonized
the anti-dermatophytic activity of the three drugs in a concentration-dependent manner. A greater extent
of antagonistic action was exerted when the two deactivators combined at their maximal levels tested were
added; MIC’s of BFZ were increased more than 30-fold and those of LCZ and TBF more than 200-fold com-
pared with the values obtained in the absence of the deactivators. Using the agar medium supplemented with
the combined deactivators, culture studies were carried out with skin tissues specimens taken from
guinea pigs whose feet were infected with dermatophytes and subsequently treated with 1% topical
preparations of the three antifungal drugs. The experimental data from this animal study demonstrated that
the combined deactivators-supplemented medium yielded increased numbers of fungi compared with the
basal medium. It looks, therefore, likely that the fungal recovery on the former medium more correctly
reflects to actual fungal burden in the infected lesions than the latter. All these results suggest that the com-
bined deactivators-supplemented medium is more useful for mycological evaluation of therapeutic effica-
cy of imidazole and allylamine drugs against dermatophytoses in both preclinical and clinical studies.
Key words: Medium, Deactivator, Antifungal agents, Tinea pedis
Among a large number of antifungal drugs, currently
available in this country are two imidazole derivatives,
bifonazole and lanconazole, and an allylamine derivative
terbinafine which play a major role in the topical treat-
ment of dermatophytosis and several other superficial
dermatophytoses. These three drugs, particularly the
latter two, have potent in vitro anti-dermatophytic activ-
ity (4, 10, 17) and share a common pharmacokinetic
property that they remain or accumulate within the horny
layer of the epidermis at substantially high levels after
topical application in animals and/or humans (6, 8, 12).
The mycological evaluation of therapeutic efficacy
of a given antifungal drug has usually been made by
culture studies using scales or other skin tissues from the
infected sites as specimens. However, when the above-
mentioned drugs or others with a similar biological fea-
ture were used for the treatment, the fungal recovery
could be appreciably affected by the specimen-associated
drug residues. If this is the case, the fungal recovery may
be underestimated, misleading us about the efficacy of the
drug used.
A promising approach toward avoiding this problem
would be the use of a culture medium capable of can-
celing the putative carryover effect of the specimen-
associated antifungal drugs on the recovery culture. For
this purpose, we attempted to develop such a medium by
the supplementation of some appropriate deactivators
effective in counteracting imidazole and allylamine anti-
fungal drugs, including bifonazole, lanoconazole and
Abbreviations: BAM, basal agar medium; BFZ, bifonazole;
CDSAM, combined deactivators-supplemented agar medium;
LCZ, lanoconazole; MIC, minimal inhibitory concentration;
SDA, Sabouraud’s dextrose agar; SDB, Sabouraud’s dextrose
broth; TBF, terbinafine.
*
Address correspondence to Dr. Takuji Nakashima, Pharma-
ceutical R&D Laboratories, POLA Chemical Industries, Inc.,
60 Kasho-cho, Totsuka-ku, Yokohama, Kanagawa 244 0812,
Japan. Fax:＋81 45 826 7259. E-mail: takuji@pola.co.jp
5
―
―
―
―
8
3

8
4
T. NAKASHIMA ET AL
terbinafine. We chose egg lecithin and polysorbate 80 as
the deactivator candidates because these two surface-
active compounds have been used to deactivate various
preservatives contained in medical supplies and cos-
metics (5) and, moreover, because they were reported to
reverse the anti-Candida activity of imidazole drugs
clotrimazole and miconazolination, we examined the
antagonistic effect of egg lecithin and polysorbate 80,
used alone and in combination, on the anti-dermato-
phytic activity of bifonazole, lanoconazole and
terbinafine. The potential usefulness of culture medium
supplemented with a combination of the two deactivators
was compared its efficiency of the fungal recovery from
infected skin tissues previously treated each of the three
antifungal drugs with that of a conventional culture
medium in the guinea pig model of tinea pedis.
test drugs were prepared in SDB containing the indicat-
ed concentration of each of the following two deactiva-
tors or their combination, as well as in a deactivator-free
SDB. The deactivators used were egg lecithin (Wako
Pure Chemical Industries, Ltd.) and polysorbate 80
(Wako Pure Chemical Industries, Ltd.).
The drug dilutions at varying concentrations were
dispensed in a 100 µl-volume into wells of rows 1 to 20
of 96-well, flat-bottomed microdilution plates (Sumito-
mo Bakelite Co., Ltd., Tokyo). Row 1 wells received the
drug diluent at the highest concentration and row 20
wells did at the lowest concentration. Each well was then
inoculated with 100 µl of the conidial suspension. All
plates were incubated at 28 C for 7 days. The MIC
was defined as the lowest drug concentration at which
visual fungal growth was inhibited.
Final deactivator concentrations were 0.25 to 1% for
egg lecithin, 0.175 to 0.7% for polysorbate 80 and when
used in combination, 1.0 and 0.7%, respectively. The
final drug concentrations were in the range of 0.0005 to
25 µg/ml.
Materials and Methods
Antifungal drugs. Purified materials of bifonazole
(
BFZ) were purchased from Sigma-Aldrich Co., St.
Louis, Mo., U.S.A. and those of lanoconazole (LCZ) and
terbinafine (TBF) synthesized in our laboratory. Stock
solutions of these antifungal agents were prepared in
dimethyl sulfoxide at a concentration of 100 mg/ml.
For animal studies, commercial products of 1% BFZ
Animals. Female Hartley guinea pigs (Japan SLC,
Inc., Shizuoka, Japan) weighing approximately 300 g
were used. All animals were maintained in an air-con-
ditioned room at a temperature of 23±2 C and humidi-
ty of 55±5% and allowed access to feed and water ad
libitum and housed one per cage throughout the experi-
mental period.
Methods of infection and treatment. The procedure for
infecting and treating the animals was essentially iden-
tical to the method of Uchida et al. for producing the
guinea pig model of tinea pedis (13). In short, after
the sole of the hind feet of guinea pigs had been disin-
fected by alcohol-dipped cotton, the skin surface of the
local area was abraded with a sandpaper (#320, Sumi-
tomo 3M Co., Tokyo). A plaster (19×72 mm, 50A,
Nichiban Co., Ltd., Tokyo) was impregnated with 0.05
ml of the conidial suspension and fixed onto the guinea
pig foot with an elastic adhesive tape (SE-25F, Nichiban
Co., Ltd.). The plaster was removed on day-7 postin-
fection. The development of dermatophytic infection on
the inoculated feet was confirmed by the foot-press
method (7) on day-21 postinfection.
®
cream (Mycospor cream; Bayer Yakuhin, Ltd., Osaka,
®
Japan), 1% LCZ cream (Astat cream; Tsumura & Co.,
®
Tokyo), and 1% TBF cream (Lamisil cream; Novartis
Pharma K.K., Tokyo) were used.
Fungus and inoculum. Trichophyton mentagrophytes
TIMM 2789 was used as the test organism. Cultures
were grown on a Sabouraud’s dextrose agar (SDA) (Bec-
ton Dickinson and Co., Franklin Lakes, N.J., U.S.A.)
slant at 28 C for 2 weeks, and a conidial suspension of
the dermatophyte was prepared in sterile physiological
saline containing 0.05% (w/v) polysorbate 80 (poly-
oxyethylene sorbitan monooleate, Wako Pure Chemical
Industries, Ltd., Tokyo). Following filtration through a
sterilized gauze to remove hyphal fragments and agar
blocks, experimental conidial suspensions for suscepti-
bility testing and those for inoculum for animal experi-
4
ments were adjusted to concentrations of 2×10 and
8
1
0 conidia/ml, respectively.
Forty infected guinea pigs were randomised into the
following four groups: 1) untreated control group; 2)
BFZ-treated group; 3) LCZ-treated group; and 4) TBF-
treated group. In all animals of the three treated groups,
once-a-day topical treatment of the whole sole of infect-
ed feet with a 0.1 ml volume of active creams was start-
ed on day-28 postinfection and continued for 7 or 14 con-
secutive days.
Test for antagonism in broth media. The in vitro
activity of antifungal agents against T. mentagrophytes
TIMM 2789 was determined by a standardized microdi-
lution technique (3), expect that RPMI 1640 broth was
replaced by Sabouraud’s dextrose broth (SDB) (Becton
Dickinson and Co.) because of the too high electrolyte
concentrations of the former medium which may affect
the interaction of the deactivators with antifungal drugs.
A series of two-fold dilutions of the stock solutions of
Culture studies with infected skin tissues. All animals
were sacrificed and subjected to culture studies seven

NEW MEDIUM USEFUL FOR THE RECOVERY OF DERMATOPHYTES
85
Fig. 1. Experimental schedule for study in guinea pigs with experimental tinea pedis receiving 7 day- or 14 day-treatment with 1% cream
of bifonazole, lanoconazole and terbinafine.
days after the last treatment according to the experi-
mental schedule illustrated in Fig. 1. Two different agar
media were used for the recovery culture; one was SDA
containing (per ml) 500 µg cycloheximide (Wako Pure
Chemical Industries, Ltd.), 50 µg of sisomicin (Sigma-
Aldrich Co.), and 100 µg of chloramphenicol (Wako
Pure Chemical Industries, Ltd.). The other medium
was further supplemented with 1% egg lecithin and
deactivator was added to final concentrations of 0.25 to
1% (for egg lecithin) and 0.175 to 0.7% (for polysorbate
80) to SDB, in which the anti-dermatophytic activity
of the three drugs was tested using the microbroth dilu-
tion technique.
As shown in Table 1, both deactivators antagonized
each antifungal drug in a concentration-dependent man-
ner. With the addition of egg lecithin at a level of 1%, the
MICs of BFZ, LCZ and TBF was increased ＞32-, 17-
and 130-fold, respectively, as compared with the values
for each drug alone. Similarly, with the addition of
polysorbate 80 at 0.7%, MICs of the three drugs were
also increased ＞32-, 67- and 33-fold, respectively.
Moreover, a greater antagonistic effect was shown when
a combination of 1% egg lecithin with 0.7% polysorbate
80 was added; the combined deactivators caused ＞32-,
210- and 210-fold increases of MICs of BFZ, LCZ and
TBF, respectively.
0.7% polysorbate 80. These two media were referred as
the basal agar medium (BAM) and combined deactivators
supplemented agar medium (CDSAM), respectively.
At the end of the experimental period, the infected ani-
mals were sacrificed with a use of carbon dioxide (dry ice
evaporation), and the whole skin tissue extending from
the epidermis to the subcutaneous tissue was removed
from the soles of the feet. Excised whole sole skin tis-
sues were washed with 0.1% (w/v) benzalkonium chlo-
ride solution and rinsed in sterilized water. Then they cut
lengthwise to divide into a left-side part and a right-
side one. Each part was further cut into 10 blocks in an
almost equal size (approx. 1×2 mm). A set of ten tissue
blocks from either one-side part was implanted on BAM
plates and the other set of 10 tissue blocks from the
other-side part on CDSAM plates. All plates were incu-
bated at 28 C for 14 days. A tissue block yielding fungal
growth were assessed as culture-positive. Fungal burden
was given as a score ranging from 0 to 10 on the basis of
the number of culture-positive tissue blocks out of the 10
tissue blocks studied.
Based on these results, SDA itself and SDA contain-
ing this combination of the two deactivators, referred as
the basal agar medium (BAM) and the combined deac-
tivators-supplemented agar medium (CDSAM), respec-
tively, were used in the following culture studies.
Comparison between BAM and CDSAM of the Efficien-
cy of the Fungal Recovery from Infected Skin Tissues in
the Guinea Pig Model of Tinea Pedis after Antifungal
Treatment
Each group of infected guinea pigs were treated with
active creams for seven or 14 days or untreated (for
control) according to the experimental schedule (Fig.
1). Seven days after the termination of treatment, all ani-
mals were sacrificed and local skin tissues were removed
for culture studies, in which one of the two set of speci-
mens were implanted onto BAM and another onto
CDSAM.
The results of this animal study were summarized in
Table 2. It shows that the average fungal burden scores
for the control group in both 7 day- and 14 day-treatment
experiments were equally above 9.0 whichever medium
was used for the fungal recovery. It looks, therefore, like-
Statistical analysis. Data are presented as mean±S.D.
Statistical analysis was performed using the Mann-Whit-
ney U test for comparison of two groups.
Results
Effect of Egg Lecithin and Polysorbate 80 on the Anti-
Dermatophytic Activity of the Three Antifungal Drugs
Experiments were conducted to learn whether each of
the three antifungal drugs, BFZ, LCZ and TBF, is coun-
teracted by either or both of the two deactivators egg
lecithin and polysorbate 80. In these experiments, each

8
6
T. NAKASHIMA ET AL
Table 1. Effect of two deactivators egg lecithin and polysorbate 80, used alone and in
combination, on anti-dermatophytic activity of three antifungal agents
Deactivator added Concentration
%)
MIC (µg/ml)
Bifonazole Lanoconazole Terbinafine
(
a)
None (control)
Egg lecithin
―
0.25
0.78 (1.00)
12.5 (16)
25 (32)
0.003 (1.00) 0.0015 (1.00)
0.0125 (4.2) 0.0125 (8.3)
0
1
.5
.0
0.025 (8.3)
0.058 (17)
0.006 (2.0)
0.05 (17)
0.2 (67)
0.1 (67)
0.2 (130)
0.006 (4.0)
0.025 (17)
0.05 (33)
＞25 (＞32)
12.5 (16)
25 (32)
Polysorbate 80
0.175
0
0
.35
.7
＞25 (＞32)
Egg lecithin
＋
1.0
＞25 (＞32) 0.625 (210) 0.312 (210)
Polysorbate 80
0.7
a)
Numbers in the brackets show the increase rate of MIC relative to the value for the
control.
Table 2. Comparison between the basal agar medium (BAM) and combined deactivators
supplemented agar medium (CDSAM) of the efficiency of fungal recovery from skin tis-
sues of guinea pigs with receiving topical treatment with three antifungal drugs based
on the average fungal burden scores of infected feet
Average fungal burden
Topical
treatment with;
Treatment
period (days)
scores of 10 infected feet
determined on;
P value
BAM
CDSAM
Exp.A
untreated control
bifonazole
lanoconazole
terbinafine
7
7
7
7
9.4±1.26
4.0±2.45
0.0±0.00
0.3±0.48
9.7±0.67
6.9±2.58
1.4±1.51
2.1±1.45
0.619
0.037
0.005
0.003
Exp.B
untreated control
bifonazole
lanoconazole
terbinafine
14
14
14
14
9.4±0.48
4.3±2.58
0.0±0.00
0.0±0.00
9.6±0.84
6.5±3.17
0.0±0.00
0.3±0.48
0.842
0.054
1.000
0.029
ly that a dermatophytic infection almost fully devel-
oped and spread over nearly the whole sole part of the
feet in this animal model of tinea pedis during the exper-
imental period. The two culture media gave different
average fungal burden scores for the other three groups
of animals which had been treated with a 1% cream of
BFZ, LCZ or TBF. This was particularly the case when
animals received 7 day-treatment with such active
creams; average fungal burden scores for all the three
treated groups determined on CDSAM were signifi-
cantly higher than the corresponding values obtained
on BAM. On the other hand, such a significant differ-
ence in the average fungal burden scores was seen only
for the BFZ-treated group but not for the LCZ- or TBF-
treated group when animals received a 14 day-treat-
ment.
Discussion
Preservatives are usually supplemented to medical
supplies, cosmetics and foods as a gradient indispensable
for maintaining their quality. Therefore, when these
commodities are subjected to a microbial load testing to
examine the extent of bacterial and/or fungal contami-
nation, the supplemented preservatives must be inacti-
vated by some counteracting compounds, referred as
deactivators. Among a number of deactivators current-
ly available in these applications are a phospholipid
lecithin and a nonionic surfactant, polysorbate 80, which
have often been used to reverse the antimicrobial activ-
ity of substituted phenolic disinfectants and that of qua-
ternary ammonium chloride, respectively (1, 2, 11).
It looks likely that there is a similar need for such

NEW MEDIUM USEFUL FOR THE RECOVERY OF DERMATOPHYTES
87
deactivators when antifungal drug-containing clinical
specimen from the infected hosts are subjected to culture
studies to recover viable fungi for mycological evaluation
of efficacy of antifungal chemotherapy they received.
However, there is virtually no information available on
the deactivator of antifungal drugs for clinical use. The
only exception is previous papers reporting that several
phospholipids and nonionic surfactants lecithin with an
unsaturated fatty acyl group, including as egg lecithin and
polysorbate 80, are also effective as the antagonist of the
two first-generation imidazole-antifungal drugs, clotri-
mazole and miconazole (15, 16).
For these reasons, our initial studies were attempted to
see whether and how egg lecithin and polysorbate 80 are
antagonistic against the two second-generation imidazole
drugs BFZ and LCZ, as well as against an allylamine
drug TBF. The results of interaction studies demon-
strated that both deactivators dose-dependently reversed
the anti-dermatophytic activity of the three drugs, and,
moreover, that a combination of the two deactivators at
their respective highest concentrations tested (1% for
egg lecithin and 0.7% for polysorbate 80) showed a
greater level of antagonistic effect. These results led us
to the possibility that an agar medium supplemented
with this combination of deactivators (CDSAM) would
be more efficient in recovering dermatophytes from clin-
ical specimens than a conventional agar medium thus far
used, such as SDA (BAM). The antagonistic effect of
higher concentrations of either or both of the two deac-
tivators was not examined for a technical reason which
made it difficult to prepare culture media good enough
for the culture study. When added to the basal medium,
egg lecithin at levels above 1% formed insoluble aggre-
gates in the medium, and polysorbate 80 at levels above
the recovery of surviving fungi. Although there is no
direct evidence available for either possibility, the latter
looks more probable because our previous studies in
which experiments were conducted using the same ani-
mal model of tinea pedis as well as the same therapeutic
regimen demonstrated that the 7 day- to 14 day-treatment
with 1% LCZ or TBF resulted in complete inhibition of
relapse of the infection, suggesting achievement of
mycological cure or eradication of infecting fungi (9, 13,
14).
It remains to be answered as to the mechanism of
antagonistic action of egg lecithin and polysorbate 80
toward the imidazole or allylamine drugs. Since both
deactivators are surface-active, they form micelles in
an aqueous medium, into which these drugs of strong
hydrophobic and lipophilic nature may be entrapped,
eventually keeping the drug molecule from contact with
the surface of fungal cells. However, for the moment no
data are available for supporting this postulation, and fur-
ther physicochemical analyses of the deactivator-drug
interaction are warranted.
The results of the present study lead us to the possi-
bility that in vivo activities of various imidazole and
allylamine drugs or related antifungal compounds thus
for evaluated in animal model of dermatophytosis using
a conventional medium for recovery culture would lead
us to overestime their efficacy. And this culture problem
could be overcome partly, at least, by the use of CDSAM
or some other media equally capable of inactivating the
antifungal drug carried over for recovery culture in place
of a conventional medium. It is also suggested that
CDSAM may also be useful for mycological examina-
tions in the clinical settings. A clinical study of patients
with tinea pedis receiving treatments with BFZ and LCZ
to test the fungal recovery on this newly developed
medium is currently under way.
0.7% rendered the medium foamy.
The expectation of the usefulness of CDSAM was
met by subsequent animal studies using the guinea pig
model of tinea pedis. The results of recovery culture of
skin tissue specimens taken from infected animals
demonstrated that CDSAM gave a significantly increased
yield of viable fungi compared with BAM, when infect-
ed animals received a 7 day-treatment with BFZ, as
well as with the two more potent drugs LCZ and TBF.
This was also the case for animals with tinea pedis treat-
ed for 14 days with BFZ but not for those with LCZ or
TBF. All or almost all tissue specimens from infected
animals were culture-negative on both BMA and
CDSAM after the 14 day-treatment with LCZ or TBF,
leading us to the two possible explanations for the results.
One is that infecting fungi were actually eradicated.
The other is that prolonged treatment with such potent
drugs caused their accumulation to levels too high for the
deactivator combination to sufficiently inactivate to allow
We thank Shinichi Watanabe MD for his encouragement and
helpful discussions.
References
1) Brummer, B. 1976. Influence of possible disinfectant trans-
fer on Staphylococcus aureus plate counts after agar contact
sampling. Appl. Environ. Microbiol. 32: 80―84.
2) Erlandson, A.L., Jr., and Lawrence, C.A. 1953. Inactivating
medium for hexachlorophene (G-11) types of compounds and
some substituted phenolic disinfectants. Science 118: 274
―
2
76.
3
) Espinel-Ingroff, A., Dawson, K., Pfaller, M., Anaissie, E.,
Breslin, B., Dixon, D., Fothergill, A., Paetznick, V., Peter, J.,
Rinaldi, M., and Walsh, T. 1995. Comparative and collabo-
rative evaluation of standardization of antifungal suscepti-
bility testing for filamentous fungi. Antimicrob. Agents

8
8
T. NAKASHIMA ET AL
vitro. Antimicrob. Agents Chemother. 31: 1365―1368.
Chemother. 39: 314
―319.
4
) Hiratani, T., Uchida, K., Yamaguchi, H., Oka, H., Niwano, Y.,
Ohmi, T., and Uchida, M. 1992. In vitro antifungal activity of
NND-318, a new antimycotic. Jpn. J. Med. Mycol. 33:
11) Quisno, R., Gibby, I.W., and Foter, M.J. 1946. A neutralizing
medium for evaluating the germicidal potency of the qua-
ternary ammonium salts. Am. J. Pharm. 118: 320.
3
21
―
328.
12) Ritter, W., and Siefert, H. 1987. Biological disposition and
percutaneous absorption of bifonazole in animals and man,
5
) Ishizeki, C., Iwahara, S., Watanabe, T., Ono, F., Watanabe,
M., and Asaka, Y. 1973. Studies on the preservatives espe-
cially on the inhibitory activity of polysorbate 80 and lecithin.
p. 383―405. In Fromtling, R.A. (ed) Recent trends in the dis-
covery, development and evaluation of antifungal agents,
J. R. Prous Science Publishers, Barcelona.
Eisei Shikensho Hokoku 91: 82
) Jensen, J.C. 1989. Clinical pharmacokinetics of terbinafine
Lamisil). Clin. Exp. Dermatol. 14: 110 113.
―88.
6
7
13) Uchida, K., Kudoh, M., and Yamaguchi, H. 1994. A study on
effectiveness of treatment and prevention of relapse using top-
ical administration of terbinafine in a guinea pig model for
(
―
) Maruyama, R., Katoh, T., and Nishioka, K. 1998. Demon-
stration of dermatophyte dissemination from the infected
tinea pedis. Jpn. J. Antibiot. 47: 1407―1412.
soles using the foot-press method. Mycoses 41: 145
―
151.
14) Uchida, K., and Yamaguchi, H. 1996. Preclinical therapeu-
tic evaluation of agents for treating dermatophytosis. Jpn. J.
Med. Mycol. 37: 199―205.
15) Yamaguchi, H. 1978. Protection by unsaturated lecithin
against the imidazole antimycotics, clotrimazole and micona-
8
) Niwano, Y., Matsui, M., Tabuchi, T., Kanai, K., Hamaguchi,
H., Miyazaki, T., Uchida, K., and Yamaguchi, H. 1997.
Studies on the antifungal activity of the new imidazole
antimycotic lanoconazole in infected sites. Distribution in the
skin and in vitro activity in the presence of stratum corneum.
zole. Antimicrob. Agents Chemother. 13: 423―426.
Arzneimittelforschung/Drug Research 47: 1056
―
1060.
16) Yamaguchi, H., and Iwata, K. 1979. Effect of fatty acyl
group and sterol composition on sensitivity of lecithin lipo-
somes to imidazole. Antimicrob. Agents Chemother. 15:
9
) Niwano, Y., Tabuchi, T., Kanai, K., Hamaguchi, H., Uchida,
K., and Yamaguchi, H. 1995. Short-term topical therapy of
experimental tinea pedis in guinea pigs with lanoconazole, a
new imidazole antimycotic agent. Antimicrob. Agents
Chemother. 39: 2353―2355.
0) Petranyi, G., Meingassner, J.G., and Mieth, H. 1987. Anti-
706―711.
17) Yamaguchi, H., Hiratani, T., and Plempel, M. 1983. In vitro
studies of a new imidazole antimycotic, bifonazole, in com-
parison with clotrimazole and miconazole. Arzneimit-
1
fungal activity of the allylamine derivative terbinafine in
telforschung/Drug Research 33: 546―551.