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Antimicrobial Agents and Chemotherapy, January 2000, p. 226-229, Vol. 44, No. 1
0066-4804/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
In Vitro Activities of the New Antifungal Triazole
SCH 56592 against Common and Emerging Yeast Pathogens
Francesco
Barchiesi,1,*
Daniela
Arzeni,1
Annette W.
Fothergill,2
Luigi Falconi
Di
Francesco,1
Francesca
Caselli,1
Michael G.
Rinaldi,2,3 and
Giorgio
Scalise1
Istituto di Malattie Infettive e Medicina
Pubblica, Università degli Studi di Ancona, Ancona,
Italy1; Fungus Testing Laboratory,
Department of Pathology, University of Texas Health Science Center, San
Antonio, Texas 72284-77502; Audie L. Murphy Memorial Veterans Hospital, San Antonio, Texas
78284-51003
Received 7 June 1999/Returned for modification 16 September
1999/Accepted 25 October 1999
 |
ABSTRACT |
A broth microdilution method performed in accordance with the
National Committee for Clinical Laboratory Standards guidelines was
used to compare the in vitro activity of the new antifungal triazole
SCH 56592 (SCH) to that of fluconazole (FLC), itraconazole (ITC), and
ketoconazole (KETO) against 257 clinical yeast isolates. They included
220 isolates belonging to 12 different species of Candida,
15 isolates each of Cryptococcus neoformans and
Saccharomyces cerevisiae, and seven isolates of
Rhodotorula rubra. The MICs of SCH at which 50%
(MIC50) and 90% (MIC90) of the isolates were inhibited were 0.06 and 2.0 µg/ml, respectively. In general, SCH was
considerably more active than FLC (MIC50 and
MIC90 of 1.0 and 64 µg/ml, respectively) and slightly
more active than either ITC (MIC50 and MIC90 of
0.25 and 2.0 µg/ml, respectively) and KETO (MIC50 and
MIC90 of 0.125 and 4.0 µg/ml, respectively). Our in vitro
data suggest that SCH has significant potential for clinical development.
| |
TEXT |
The risk of opportunistic infections
is greatly increased in patients who are severely immunocompromised due
to cancer chemotherapy, organ or bone marrow transplantation, or human
immunodeficiency virus infection (1, 8). Although
Candida albicans is the organism most often associated with
serious fungal infections, other Candida species have
emerged as clinically important pathogens associated with opportunistic
infections (1, 8). Recently, many yeasts belonging to genera
other than Candida have been reported as causative agents of
systemic fungal infections (8).
This rising incidence of fungal infections has made the pursuit of safe
and effective therapies an area of much activity over the past two
decades. Although amphotericin B remains the "gold standard"
therapy for life-threatening fungal infections, its use reveals
important clinical limitations, including toxic side effects and
inconvenience of intravenous dosing. Azole drugs represent a
therapeutic advance (2).
SCH 56592 (SCH) is a new broad-spectrum antifungal triazole currently
under development. SCH has been shown to have potent in vitro and in
vivo activities against Candida spp., Cryptococcus neoformans, Aspergillus spp., Blastomyces
dermatitidis, and Coccidioides immitis (3-6,
9-13).
The aim of the present study was to compare the in vitro activity of
SCH with those of three currently available azole drugs used in
systemic fungal infections: fluconazole (FLC), itraconazole (ITC), and
ketoconazole (KETO). The drugs were tested by a broth microdilution
method, performed as recommended by the National Committee for Clinical
Laboratory Standards (NCCLS), against common and emerging yeast pathogens.
Yeast isolates.
A total of 257 clinical yeast isolates were
used in this study. They included 220 isolates of Candida
spp. (84 strains of C. albicans; 20 strains of C. tropicalis; 15 strains each of C. glabrata and C. parapsilosis; 14 strains each of C. kefyr and C. krusei; 10 strains each of C. famata, C. guilliermondii, C. inconspicua, C. lusitaniae, and C. pelliculosa; and 8 strains of
C. lipolytica), 15 isolates of Cryptococcus
neoformans, 15 isolates of Saccharomyces cerevisiae,
and 7 isolates of Rhodotorula rubra. The isolates were
recovered from gastrointestinal, respiratory, urinary tract, blood,
cerebrospinal fluid, or other sterile body fluid specimens. With the
exception of nine strains of C. albicans isolated from four
patients with AIDS suffering from recurrent oropharyngeal candidiasis,
each of the other strains represented a unique isolate from a patient.
Yeast isolates were identified at the species level by conventional
morphological and biochemical methods and stored at 
70°C in 10%
glycerol (14). Before the initiation of the study, yeast
isolates were subcultured on antimicrobial agent-free medium to ensure
viability and purity. C. krusei ATCC 6258 was used as the
quality control and tested in each run of the experiments
(7).
Antifungal agents.
SCH was obtained as a standard powder from
Schering-Plough Research Institute (Kenilworth, N.J.). FLC, ITC, and
KETO were obtained from their respective manufacturers. Stock solutions were prepared in polyethylene glycol (SCH, ITC, and KETO) or water (FLC). Serial twofold dilutions were prepared as recommended in NCCLS
approved standard M27-A (7). Final dilutions were made in
RPMI 1640 medium (Sigma, Milan, Italy) buffered to pH 7.0 with 0.165 M
morpholinepropanesulfonic acid (MOPS) buffer (Sigma). The final
concentration of the solvent did not exceed 1% in any well.
Antifungal susceptibility testing.
Antifungal susceptibility
testing was performed by a broth microdilution method in accordance
with the NCCLS recommendations (7). The final concentrations
of the antifungal agents were 0.0078 to 4.0 µg/ml for SCH and ITC,
0.03 to 16 µg/ml for KETO, and 0.125 to 64 µg/ml for FLC. Plates
were incubated at 35°C for 48 h (72 h for C. neoformans and R. rubra). MICs of all drugs were
defined as the lowest concentrations resulting in 80% inhibition of
growth compared to that of the growth control. MIC ranges were obtained
for each species-drug combination tested. MICs for 50 and 90% of the
isolates of each species tested (MIC50 and
MIC90, respectively) were determined for yeast species with
10 isolates.
Determination of CFU per milliliter.
The antifungal activities
of SCH and FLC were compared by determination of CFU per milliliter of
two selected clinical isolates, C. albicans isolate 3474 and
C. neoformans isolate 486, incubated at multiples of the
respective MICs. Briefly, three to five colonies of the isolate from a
72-h growth plate were suspended in 10 ml of sterile distilled water
and the turbidity was adjusted according to spectrophotometric methods
to a McFarland turbidity standard (approximately 1 × 106 to 5 × 106 CFU/ml). One milliliter of
the adjusted fungal suspension was added to 9 ml of either RPMI 1640 medium buffered with MOPS or a solution of growth medium plus an
appropriate amount of antifungal stock solution. This resulted in a
1:10 dilution of the fungal suspension and yielded a starting inoculum
of approximately 1 × 105 to 5 × 105
CFU/ml. Drug concentrations in test solution were equal to 0.25, 0.5, 1, 2, 4, 8, and 16 times the MICs of both drugs. At predetermined time
points (0, 6, 12, 24, 48, and 72 h) following the introduction of
the isolate into the system, a 100-µl aliquot was removed from either
the control tube (drug-free) or each test solution. Tenfold serial
dilutions were performed on samples, and a 50-µl aliquot from each
dilution was streaked onto Sabouraud dextrose agar plates for colony
count determination. Following incubation at 35°C for 48 h, the
number of CFU on each plate was determined. Each experiment was
conducted in triplicate. The results were expressed as the logarithmic
variation of the ratio of CFU of treated cells per milliliter to CFU of
untreated cells per milliliter for each time point considered.
The median MICs of SCH, FLC, ITC, and KETO for C. krusei
ATCC 6258 were 0.25, 32, 0.25, and 0.5 µg/ml, respectively. Table 1 summarizes the in vitro
susceptibilities of 257 clinical yeast isolates to four azole drugs. In
general, a broad range of MICs was observed with each antifungal agent.
SCH was from 4- to 128-fold more active than FLC. The smallest
difference between drugs was observed for strains of C. lipolytica, for which SCH and FLC MIC50s were 1.0 and
4.0 µg/ml, respectively. The widest difference was observed for
strains of C. inconspicua, for which SCH and FLC MIC90s were 0.5 and 64 µg/ml, respectively. SCH was from
two- to fourfold more active than ITC against all the yeast isolates tested with the exception of strains of C. famata, C. pelliculosa, and C. lipolytica, for which no
differences in MIC90s (or MIC50 in the case of
C. lipolytica) were observed between drugs. SCH was from 2- to 16-fold more active than KETO against all the yeast isolates tested,
with the exception of strains of C. kefyr, C. guilliermondii, C. pelliculosa, S. cerevisiae, and R. rubra. For the first two
Candida spp. and S. cerevisiae, no differences in MIC90s were observed between drugs. On the other hand, KETO
was twofold more active than SCH against strains of C. pelliculosa and R. rubra (Table 1).
To compare the antifungal activity of the new triazole with that of
FLC, we determined the CFU per milliliter of two selected
isolates
incubated with the drugs at multiples of the MICs:
C. albicans 3474 (FLC MIC, 0.25 µg/ml; SCH MIC, 0.06 µg/ml) and
C. neoformans 486 (FLC MIC, 4.0 µg/ml; SCH MIC, 0.25 µg/ml). The
results are shown in Table
2. Overall, the antifungal activities
of
both drugs were higher for
C. neoformans than for
C. albicans.
For the latter isolate, a reduction in CFU per
milliliter of >1
log, compared with that of the growth control, was
reached at
12 h of incubation with FLC at concentrations
8 times
the MIC
and with SCH at a concentration equal to 16 times the MIC. The
anticryptococcal activity of SCH was superior to that of FLC.
At
concentrations equal to their respective MICs, SCH produced
a reduction
of >1 log of CFU/ml with respect to FLC from 24 to
72 h of
incubation. The same phenomenon was seen at concentrations
equal to 0.5 times the MICs at 24 h and of
2 times the MICs at
72 h of
incubation (Table
2).
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|
TABLE 2.
Antifungal activities of SCH and FLC against C. albicans 3474 and C. neoformans 486 at multiples
of MICsa
|
|
Overall, our data confirmed the potent in vitro activity of SCH against
the most common yeast pathogens and showed its potent
in vitro activity
against a wide spectrum of less commonly isolated
yeasts. Similar to
previous reports of other investigators (
3,
5,
9,
12), SCH
was found to be more active than both ITC
and FLC against isolates of
C. albicans. SCH was slightly more
active than both ITC and
KETO against strains of
C. glabrata and
C. krusei, two species of
Candida often reported, in vitro
as
well in vivo, to be resistant to FLC (
2). Our SCH MICs
for
the strains of
C. glabrata were similar to those
reported previously
by Pfaller et al., while SCH MICs for strains of
C. krusei were
slightly higher than those reported by the
same authors (
12).
The other three species of
Candida which have been reported as
relatively resistant to
FLC were included in this study:
C. famata,
C. inconspicua, and
C. pelliculosa (
1,
8). In
general, SCH
was as active as ITC against strains of
C. famata and
C. pelliculosa,
and it was slightly more
active than ITC against strains of
C. inconspicua. On the
other hand, KETO showed slightly more activity
than both SCH and ITC
against strains of
C. pelliculosa. Overall,
with the
exception of one isolate each of
C. famata and
C. inconspicua which were inhibited by SCH at a concentration of 4.0 µg/ml, all
the other strains belonging to these three species of
Candida showed an SCH MIC of
2.0 µg/ml. Interestingly,
preliminary data
on SCH pharmacokinetics showed that serum levels over
2.0 mg/liter
may be achieved in several animal models (
12).
Among the four azoles tested, SCH was the most active drug against
isolates of
C. neoformans. Our in vitro data on this yeast
paralleled those previously reported by Galgiani and Lewis
(
3),
having found SCH MICs ranging from 0.015 to 0.25 µg/ml. Interestingly,
the anticryptococcal activity of SCH, measured
by determination
of CFU per milliliter of cells incubated at multiples
of the MIC,
was higher than that observed with FLC. Recently, Perfect
et al.
showed that SCH was as effective as FLC in a rabbit model of
cryptococcal
meningitis (
11). Taken together, these data
suggest the potential
beneficial effect of this new triazole as an
alternative to FLC
for the treatment of cryptococcal
infections.
In conclusion, our study underlines the potent and broad-spectrum
activity of the new antifungal triazole SCH 56592. Clearly,
other in
vitro and in vivo studies to further elucidate its potential
for
clinical development are
warranted.
| |
ACKNOWLEDGMENTS |
This work was supported in part by grants from Istituto Superiore
di Sanità, Rome, Italy (I AIDS project, no. 50A.0.32), from
MURST, Rome, Italy (cofin. 98/99), and from Schering-Plough S.p.A.,
Milan, Italy.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Istituto di
Malattie Infettive e Medicina Pubblica, Università degli Studi di
Ancona, Ospedale Umberto I°, Largo Cappelli 1, 60121, Ancona,
Italy. Phone: 39.71.5963467. Fax: 39.71.5963468. E-mail:
cmalinf{at}popcsi.unian.it.
| |
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Antimicrobial Agents and Chemotherapy, January 2000, p. 226-229, Vol. 44, No. 1
0066-4804/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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