Drug Development Research 8:309-316 (1986)

The In Vivo Antifungal Activity of BroadSpectrum Azoles

Jan Van Cutsem and Frans Van Gerven
Department of Bacteriology and Mycology, Janssen Pharmaceutica Research
Laboratories, Beerse, Belgium
ABSTRACT

Van Cutsem, J., and F. Van Gerven: The in vivo antifungal activity of broad-spectrum
azoles. Drug Dev. Res. 8:309-316, 1986.

The antifungal activities (topical and oral ) of miconazole, ketoconazole, and itraconazole
were compared in laboratory animals. Miconazole at concentrations of 1 and 2% was an
effective antifungal agent in the topical therapy of microsporosis and at 2% in vaginal
candidosis. Ketoconazolewas superior to miconazole in potency and efficacy, a concentration of 1% producing a 100% cure. With itraconazole the same efficacy was reached as
with ketoconazole but at concentrations 4 to 8 times lower. Miconazole, administered
orally, was marginally active in dermatophytosis and, after long-term treatment, in candidosis. In contrast, ketoconazole was found to be a broad-spectrum antifungal agent of
high efficacy at 10 mglkg. ltraconazole again was more potent (doses 1.25 to 2.50 rng/kg)
and more effective than ketoconazole in dermatophytosis, candidosis, and mycoses due
to dimorphic fungi. Moreover, itraconazole constitutes an important therapeutic progress
in the oral treatment of aspergillosis and cryptococcosis. The three compounds were free
of side effects in the present experimental conditions.
Key words: miconazole, ketoconazole, itraconazole, topical and oral activity
INTRODUCTION

The research of antifungal agents at Janssen Pharmaceutica started in 1962; etonam

(R 10100) was selected in 1965 [Vanbreuseghem et al., 19671. This compound was active in
vitro against dermatophytes and in vivo in experimental dermatophytosis after topical application. Clinical studies in man confirmed the laboratory data [Brugmans and Van Cutsem, 19691.
From 1967 on, several important broad-spectrum antifungals were synthesized [Godefroi
et al., 19691. Miconazole (R 14889) [Van Cutsem and Thienpont, 19721 became the archetype
of the first generation of broad-spectrum antifungals with potent in vitro activity and a broad-

Received final version March 3, 1986; accepted March 3, 1986.

Address reprint requests to J. Van Cutsem, Department of Bacteriology and Mycology, Janssen Pharmaceutica, Turnhoutseweg 30, B-2340 Beerse, Belgium.

0 1986 Alan R. Liss, Inc.

310

Van Cutsem and Van Gerven

spectrum in vivo activity after topical application and intravenous administration. Some of
these compounds were developed for veterinary use [Desplenter, 19791 while others found an
application in plant pathology [Van Gestel et al., 19811 and material preservation. Ketoconazole
(R 41400); Heeres et al., 1979; Van Cutsem, 19831 represented a new generation of antimycotics, since it was the first potent oral broad-spectrum antifungal. Itraconazole (R 51211)
[Heeres et al., 1984; Van Cutsem et al., 19831 was considered a third generation azole
antimycotic [Van Cutsem and Janssen, 19851, not only because it is the most potent and most
effective antifungal known, but also because the antifungal activity spectrum has again been
enlarged. The broad-spectrum in vitro activity of these three leading compounds was described
earlier [Van Cutsem, 19851. The purpose of the present paper is to describe the topical and
oral therapeutic antimycotic activity of miconazole, ketoconazole and itraconazole in several
animal models.

MATERIALS AND METHODS

Compounds

The chemical structures of miconazole, ketoconazole, and itraconazole are given in

Figure 1. For topical treatment of microsporosis, drugs were incorporated in carbowax
(polyethylene glycol 1,500-60% and polyethylene glycol 40040%). For topical treatment of
vaginal candidosis and for oral treatment the compounds were administered in polyethylene
glycol 200. Itraconazole was used in solution [see Van Cutsem et al., 1986bl.
Microsporosis

Albino guinea-pigs were infected with Microsporum canis on the scarified back. Rivaliers method as modified by Vanbreuseghem et al. [1967] was used. Topical treatment lasting
2 weeks (12 days) started 3 days after infection. One of the ointment at different concentrations
was applied daily. The once-a-day (0.d.) oral treatment (different doses) started on the day of
infection and lasted 14 days. Controls were treated with the solvent.
The assessment of the activity was based on clinical observations, microscopic examinations, and cultures. The guinea-pigs were considered to be cured if no lesions were present
and all were cultures negative. Animals with minor residual lesions or only positive upon
microscopic examination, or with only slightly positive cultures were considered to be markedly improved. The others were considered to be failures.

MICONAZOLE ( R 16889)

KETOCONAZOLE ( R 41600)

ITRACONAZOLE LR 51211)

Fig. 1. Chemical structure of miconazole, ketoconazole, and itraconazole.

Antifungal Broad-Spectrum Azoles

311

Vaginal Candidosis

Female ovariectomized and hysterectomized Wistar rats, in permanent pseudoestrus

after weekly subcutaneous administration of 0.1 mg oestradiol in 1 ml oily solution, were
infected intravaginally with Candida albicans and treated either topically twice daily (b. i d . )
or orally once daily for 3 consecutive days, starting 3 days after infection.
The animals were considered to be cured if C. ulbicans was absent from all cultures
performed 7 days after the 1st topical application, or 2 and 9 days after the last oral treatment.
If less than 26 colonies of C. albicans were isolated, the animals were considered to be
markedly improved. All others were considered to be failures.
Systemic Candidosis

Non-pretreated male Albino guinea-pigs were infected intravenously with 8,000 blastospores of C. albicans per gram body weight [Van Cutsem et al., 1985al. The animals were
treated orally, once daily, for 14 consecutive days, starting on the day of infection. Systemic
dissemination occurred and most of the organs were invaded. Three parameters for the
evaluation of the infection and of the activity of the administered drugs were used. Skin
folliculitis was scored during the whole experimental period and viable yeasts per gram tissue
were determined by culturing the whole left kidney and also large total skin samples, 3 days
after the last oral dose.
Sporotrichosis-Histoplasmosis

Male Albino guinea-pigs were infected in the left testicle with either Sporothrix schenckii
or Histoplasm duboisii. A number of 98,000 colony forming units (CFU) and 133,000 CFU
respectively were injected per animal. The animals were treated orally, once daily starting on
the day of infection for 28 days (Sp. schenckii) and for 14 days (H. duboisii) respectively. One
week after the last treatment all guinea-pigs were sacrificed and cultures performed from
complete organs: liver and left and right testicles.
Aspergillosis

Swiss mice were infected intravenously with spores of four strains of Aspergillus
jkmigutus (240,000 to 800,000 spores per mouse). They were treated orally 0.d. for 5 days.

Survival was noted and cultures were performed from dead animals and from animals sacrificed on day 42 after infection.
Cryptococcosis

Swiss mice were infected intracerebrally with 2,000 yeasts of Cryptococcus neofomans
and treated b.i.d. for 14 days, starting on the day of the infection or 2 days after infection.
Brain cultures from dead animals and from survivors, sacrificed 48 hours after the end of the
treatment period, were prepared. Mice were considered to be cured if all brain cultures were
negative and markedly improved if only a few residual yeasts were still present in the brain
tissue.
Non-pretreated male Albino guinea-pigs were infected intravenously with 200 yeasts of
Cr. neofomans per g body weight [Van Cutsem et al., 1986al. Invasion of organs was general
and progressive, resulting also in the formation of ulcerative skin cryptococcomas. Only Three
days after infection all brains were invaded and meningitis occurred. Oral treatment started
0.d. or b i d . on the day of infection and lasted for 28 days. A therapeutic treatment 0.d. or
b i d . started 3 days after infection for 35 consecutive days.
The skin granulomas were scored weekly. Dead animals were necropsied and cultures
were made. The number of CFU per gram organ was determined. The following 13 organs
were studied: skin, brain, spleen, lungs, liver, left kidney, heart, peritoneum, lymph node,
urinary bladder, thigh muscle, left eye, and left testicle. The same procedure was used for the
evaluation of the survivors, 3 days after the last oral dosage. The guinea-pigs were considered

312

Van Cutsem and Van Gerven

to be cured if all organs were negative, almost cured if only a strongly reduced cryptococcal
flora was present in the brain and improved if all organs, except the brain, were free of
cryptococcus.
RESULTS

In all experiments the solvent-treated animals acquired severe infection and were always
highly positive on cultures.
Microsporosis (Table 1)

Topical miconazole possessed high therapeutic efficacy at 1 and 2 % ; with topical

ketoconazole comparable results were obtained at 0.5 and 1% , while virtually the same efficacy
was reached with itraconazole at 0.125%. Oral miconazole was active at 80 and at 160 mg/kg.
The antidermatophyte activity of griseofulvin and of ketoconazole at 10 mg/kg were comparable. However, when administered at 20 mg/kg ketoconazole became more effective than
griseofulvin. With an oral dosage of 1.25 mg/kg of itraconazole, more than 90% of guineapigs were cured or markedly improved.
Vaginal Candidosis (Table 2)

In a short therapeutic topical course miconazole was active at 2 % , while ketoconazole

was more effective at a concentration 4 times lower and itraconazole at a concentration 16
times lower. The results of oral treatment with ketoconazole at 10 mg/kg and of itraconazole
at 2.5 mg/kg were comparable. Miconazole in this short-term oral treatment was inactive up
to very high doses. Oral treatment for longer periods provided some efficacy.
Systemic Candidosis (Table 3)

Skin folliculitis is a phenomenon associated with systemic candidosis in guinea-pigs

[Van Cutsem et al., 1985bl. The results obtained with miconazole at 160 mg/kg, ketoconazole
at 10 mg/kg, and itraconazole at 1.25 mg/kg were largely comparable, also with regard to the
efficacy in folliculitis, and the presence of C. albicans in skin and kidney cultures.
Other Systemic Mycoses (Table 4)

In guinea-pigs infected with Sp. schenckii or with H. duboisii, greater activity was
obtained with oral itraconazole at 20 and 10 mg/kg respectively than with ketoconazole in
previous experiments (unpublished data).
In aspergillosis the survival time of mice treated with 40 mg/kg of itraconazole was
doubled and even more than tripled with 80 mg/kg as compared with the controls. After 42
days, 15 out of 74 animals were still alive in the group treated at 80 mg/kg of itraconazole
versus none in the solvent-treated animals. With ketoconazole no survival was obtained in
aspergillosis in mice, even with a longer treatment schedule than that used for itraconazole
[Van Cutsem and Janssen, 19841.
Oral treatment with itraconazole of intracerebral cryptococcosis in mice resulted in
a high cure rate, both when treatment started simultaneously or when it started 48 hr after
infection. The anticryptococcal activity of itraconazole was also very pronounced in guineapigs, infected intravenously and treated 0.d. or b i d . for 28 or 35 days, starting on days 1 or
+ 3 respectively. Itraconazole at low dosages eliminated the cryptococci from almost all
organs. When administered therapeutically for 35 days at doses of 10 and 20 mg/kg, itraconazole was able to eliminate or to reduce the fungal meningo-cerebral flora and to make all other
organs free of yeasts.
Side-Effects

In these various experiments topical miconazole and oral and topical ketoconazole and
itraconazole were free of side-effects.

Solvent

Solvent
010

010

0.63
24/39

0.063
5/50

8716

1.25
-

0117
76/19

0.125

010
9812

2.5

0133
9017

0.25

11/28
10127
100/0

0.5
0125
5010
100/0

33/53
100/0

20
62/23
10OlO
-

10
47/33

2
54/46
83/17

1
25/67
100/0

% cured/ % markedly improved animals

33/67

80
50150

40
40150
010
100/0

160
67/33
71/29
-

Solvent
0.210.2
-

Oral (mglkg)
Ketoconazole
Itraconazole
317

0.31

1.25
010
51/10

2.5
1910
9114

613
8712

810
62/10

37/17
0.63
25/11

3113
9614

0.25
-

0.125

0.063

0.031
-

5
3814
9413

0.5
010
9213
9614

% cured/ % markedly improved animals

*Treatment started 3 days after infection topically b.i.d. and orally 0.d. for 3 consecutive days.

Solvent
012
-

Topical (%)
Miconazole
Ketoconazole
Itraconazole

Treatment

TABLE 2. Treatment of Vaginal Candidosis in Rats*

10
951 1
100/0

1M)lO
100/0

20
loo/O
-

2
56/13
10010
-

*Topical treatment (0.d.) for 2 weeks started 3 days after infection, and oral treatment (0.d.) started on the day of infection and lasted 14 days.

Oral (mglkg)
Griseofulvin
Miconazole
Ketoconazole
Itraconazole

Topical (%)
Miconazole
Ketoconazole
Itraconazole

Treatment

TABLE 1. Treatment of Microsporosis in Guinea-pigs.*

g.P.
g.P.
m.
m.
m.
g.p.
g.p.
g.p.
g.p.

Sporothrix'
Histoplasma'
Aspergillusd
Cryptococcus'
Cryptococcusc
cryptococcus'
Cryptococcus~
cryptococcuse
Cryptococcus~

i.t.
i.t.
,
l.v.
i.c.
i.c.
i.v.
i.v.
i.v.
i.v.

route'

Infection
28
14
5
14
14
28
28
35
35

0
0
0
0
0
0
+3
+3

+2

Days
0.d.
0.d.
0.d.
b.i.d.
b.i.d.
0.d.
b.i.d.
0.d.
b.i.d.

Schedule

Treatment days
Start
-

018

4.6174
0147
0124
018
0112
0112

018

o/ 10

0+4+4/8
0+6+1/7
0+4+2/6
0+7+1/8

Solvent

aAnimal species: g.p., guinea-pig; m., mouse.

'Infection route: i.t., intratesticular; i.v., intravenous; i.c., intracerebral.
'n cured + n markedly improved/total n of animals treated.
dMean survival in days for total n of animals treated.
en cured n almost cured n improvedltotal n of animals treated.

speciesa

Fungus

Animal

TABLE 4. Oral Treatment of Systemic Mycoses with Itraconazole

2+5+1/8

3 +0/10
5 318

10

40

1 +7+0/8

19.1/74
29 +26/55
10+28/38

80

10+0/10
8+0/8
9.1/10
0 14/20
-

8+2/10

20

Oral treatment with itraconazole (mg/kg)

Antifungal Broad-Spectrum h o l e s

315

TABLE 3. Svstemic Candidosis in Guinea-Pigs*

Treatment
w/kg

a) % cured/% markedly improved animals

b) % of negative skin cultures
c) % of negative kidney cultures
Solvent

0.63

1.25

2.5

10

20

40

100/0
100
61
-

50/50
50
33
-

Miconazole

a) 0

Ketoconazole

b) 0
c) 0.3
a)

Itraconazole

b)
c)
a)
b)
c)

42/15
0
5

0
0
0
93/5
95
27

20123
7
7

40/42
33
17

11/11
5
0
86/10
83
36

100/0

100/0

100/0

100
96

100
87

98
37

160

83/17
83
58
-

*Oral treatment 0.d. was given for 14 consecutive days, starting on the day of infection.

DISCUSSION

The results obtained in the present paper with miconazole, ketoconazole, and itraconazole reflect to a certain extent the historical evolution of antimycotic drugs over the past 2
decades. Chemical modification of the miconazole structure led to the development of the first
oral highly active broad-spectrum antifungal ketoconazole and to itraconazole, the youngest
member of the azole series. Itraconazole is characterized by high potency, high efficacy, and
broad-spectrum activity. The importance of miconazole consisted in its high broad-spectrum
topical antifungal activity; moreover, administered intravenously it became a valuable alternative for the more toxic amphotericin B. The topical antifungal activity was increased with the
introduction of ketoconazole, but more important oral treatment of various mycoses became
possible. Itraconazole in vitro was found more potent than any other reference compound. The
in vivo data, reported here, confirm the results obtained in experimental dermatophytosis,
candidosis, and various other fungal infections [Van Cutsem et al, 1986bl. The results obtained
with itraconazole in meningo-cerebral and in disseminated cryptococcosis, where oral antifungal therapy had only been marginally effective, are promising. The model of cryptococcosis in
guinea-pigs mimics a natural evolution of cryptococcosis in man, including its skin manifestations [Sarosi et al., 19721. This may have important consequences, especially in view of the
fulgurating proliferation of AIDS and the concomitant increase of cryptococcal infections
[Vandepitte et al., 19831. Also the oral therapeutic efficacy of itraconazole in sporotrichosis
makes the compound a valuable alternative for potassium iodide. The proven efficacy in animal
models makes itraconazole a valuable candidate for human treatment. Furthermore, the oral
activity of itraconazole in aspergillosis opens new possibilities for treatment of aspergillic
infections and of prophylaxis in patients at risk [Van Cutsem et al., 19841.

ACKNOWLEDGMENTS

This investigation was supported by the Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw (IWONL). The authors are grateful to R. Zaman,
M. Van der Flaes, and M. Oris for technical assistance, to L. Persegael, A. Van Breda, P. De
Backker, G . Clijmans, and K. Goossens for collaboration, to H. Vanhove for reviewing, and
to S. Sas for typing the manuscript.

316

Van Cutsem and Van Gerven

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