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Antimicrobial Agents and Chemotherapy, October 1998, p. 2726-2730, Vol. 42, No. 10
Departments of
Medicine1 and
Microbiology,
Received 24 November 1997/Returned for modification 27 January
1998/Accepted 15 July 1998
LY303,366 (LY) is a novel derivative of the echinocandin class of
antifungal agents. The in vitro activities of LY, itraconazole (ITZ),
and amphotericin B (AMB) were assessed against 60 Aspergillus isolates, including 35 isolates of A. fumigatus, eight isolates of A. terreus, eight
isolates of A. flavus, eight isolates of A. niger and one isolate of A. nidulans. Four A. fumigatus isolates were resistant to ITZ. Susceptibility testing
for all drugs was performed with a broth microdilution procedure. LY
was tested in two media: antibiotic medium 3 (AM3) and Casitone with
2% glucose (CAS) with an inoculum of 2 × 103
spores/ml. ITZ and AMB were tested in RPMI 1640 with 2% glucose with
an inoculum of 1 × 106 spores/ml. All tests were
incubated at 37°C for 48 h. A novel end point was used to
determine a minimal effective concentration (MEC) for LY, i.e., almost
complete inhibition of growth save a few tiny spherical colonies
attached to the microplate. MICs were measured for ITZ and AMB with a
no-growth end point. Ranges and geometric mean (GM) MECs were from
0.0018 to >0.5 and 0.0039 mg/liter and from 0.0018 to >0.5 and 0.008 mg/liter for LY in AM3 and LY in CAS, respectively. Differences between
species were apparent, with A. flavus being significantly
less susceptible to LY than any other species tested with both media
(P Invasive aspergillosis is now one of
the most common fungal infections found in immunocompromised patients
(1) and is also one of the most fatal (2).
Treatment of Aspergillus infections is still not ideal, and
the two currently used antifungal drugs have a variety of associated
problems. Amphotericin B (AMB) can cause serious side effects due to
its toxicity and itraconazole (ITZ) is not always absorbed in high
enough quantities to be therapeutic, especially in certain patient
groups, e.g., AIDS patients.
The rise in serious fungal infection over the past decade has prompted
the development of new antifungal agents with novel modes of action.
LY303,366 (LY) is a semisynthetic derivative of a natural product class
of antifungal agents belonging to the new class of drugs known as
echinocandins. Echinocandins are noncompetitive inhibitors of
(1,3)- In this study we evaluated the in vitro activity of LY against a
variety of Aspergillus species and compared it with the
activity of currently used antifungal agents, ITZ and AMB.
Isolates.
Sixty Aspergillus isolates were used in
the study, comprising 35 isolates of Aspergillus fumigatus,
eight isolates of Aspergillus terreus, eight isolates of
Aspergillus flavus, eight isolates of Aspergillus
niger, and one isolate of Aspergillus nidulans. The
isolates had been collected over a period of time in both the United
States and the United Kingdom. This included four ITZ-resistant A. fumigatus isolates (4, 5) and one isolate
resistant to AMB in vivo (11). The isolates were stored in
15% glycerol at Antifungal agents.
LY (Eli Lilly, Indianapolis, Ind.) was
supplied as pure drug powder and was dissolved in dimethyl sulfoxide
(Sigma, Dorset, United Kingdom) after adjusting for potency to a stock
concentration of 1,280 mg/liter. ITZ (Janssen, Beerse, Belgium) was
dissolved in 1:1 acetone-0.2 M HCl to a stock concentration of 1,280 mg/liter. The drug preparation was mixed vigorously and heated in a
60°C water bath until the drug had completely dissolved. AMB
(Fungizone; Squibb, Middlesex, United Kingdom) was dissolved in water
to a final concentration of 1,280 mg/liter after adjustment for sodium desoxycholate. All drugs were dispensed into small vials and stored in
the dark at Preliminary work.
To evaluate the growth and end point
determination with LY, four isolates were tested in several media with
both broth micro- and macrodilution procedures. The broth macrodilution
procedure was similar to the method used by Moore et al.
(7). The media tested were RPMI 1640 (Sigma) buffered with
morpholinopropanesulfonic acid (MOPS) (Sigma) and supplemented with 2%
glucose (herein referred to as RPMI), antibiotic medium 3 supplemented
with 2% glucose (Difco, Surrey, United Kingdom) (AM3), Casitone
(Difco) supplemented with 2% glucose (CAS), and yeast nitrogen base
(Difco) supplemented with 0.5% glucose (YNBG). The same batch of media
was used throughout the study, including reproducibility tests. Two
inocula were also evaluated: 1 × 106 conidia/ml and
2 × 103 conidia/ml.
Susceptibility testing.
Drug concentrations ranged from
0.0009 to 0.5 mg/liter for LY and from 0.03 to 16 mg/liter for ITZ and
AMB. The medium that was used for ITZ and AMB tests was RPMI 1640. Previous in vivo and in vitro correlation work had determined that RPMI
was the most suitable medium for ITZ (5).
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
In Vitro Activity of the Echinocandin Antifungal
Agent LY303,366 in Comparison with Itraconazole and Amphotericin B
against Aspergillus spp.
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
0.05). Ranges and GM MICs were from 0.125 to
>16 and 0.7 mg/liter for ITZ and from 0.25 to 16 and 1.78 mg/liter for
AMB. Minimal fungicidal concentrations (MFCs) were also determined for
all drugs. GM MFCs were 0.018, 0.09, 19.76, and 12.64 mg/liter for LY
in AM3, LY in CAS, ITZ, and AMB, respectively. LY in AM3 and LY in CAS
were fungicidal for 86.7 and 68% of isolates, respectively (98%
killing). In comparison, ITZ and AMB were fungicidal for 35 and 70% of
isolates, respectively (99.99% killing). A reproducibility study was
performed on 20% of the isolates. For 12 isolates retested, the MEC or
MIC was the same or was within 1 dilution of the original value for 11, 11, 10, and 9 isolates for LY in AM3, LY in CAS, ITZ, and AMB,
respectively. In conclusion, LY seems to be a promising antifungal agent with excellent in vitro activity against Aspergillus
spp.
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
-D-glucan synthase which produces glucan polymers,
a major component of the fungal cell wall (3). LY has been
reported to have excellent activity against a wide range of fungal
pathogens, including Aspergillus species (12) and Candida species (8, 10, 12).
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
70°C until required for susceptibility testing.
For susceptibility testing all isolates were grown on Sabouraud
dextrose agar (Lab M, Bury, United Kingdom) at 30°C for 3 to 4 days.
20°C.
MFCs.
For all drugs 100 µl was removed from all wells
showing no growth and was plated on horse blood agar. The liquid was
allowed to soak into the agar and the plate, once dry, was streaked
with a sterile loop. This was to separate any spores that were present and to remove any spores from the drug. The plates were then incubated at 37°C for 48 h. For LY the minimal fungicidal concentration (MFC) was defined as the lowest drug concentration allowing two or
fewer colonies to grow. This represented a killing of 
98% of the
original inoculum. For ITZ and AMB the MFC was defined as the lowest
drug concentration allowing five or fewer colonies to grow. This
represented a killing of 99.99% of the original inoculum.
End point determination. A typical MIC end point is not seen when LY is tested against Aspergillus species. There is instead a transition from a homogeneous mat of long, thin hyphae to subspherical colonies, most being attached to the bottom of the microtiter well. Therefore, the minimal effective concentration (MEC) was considered to be the concentration in the first well to show the small subspherical colonies and no hyphal growth. For ITZ and AMB a no-growth end point was used.
Data analysis. The differences between species were analyzed for each drug by a one-way analysis of variance with a Bonferroni correction for multiple comparisons (SPSS for Windows). A. nidulans was excluded from the statistical analysis because only one isolate of this species was being tested. For data analysis, MECs, MICs, and MFCs of >0.5 mg/liter were recorded as 1 mg/liter for LY and those of >16 mg/liter were recorded as 32 mg/liter for ITZ and AMB.
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RESULTS |
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Abstract
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Materials & Methods
Results
Discussion
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The preliminary study to assess the most appropriate susceptibility testing method for LY involved evaluating a variety of susceptibility methods, media, and inoculum sizes. The broth macrodilution method was found to be quite labor-intensive in comparison with the broth microdilution method, which was easier and quicker to perform. Also, growth of Aspergillus appeared only on the sides of the tubes and not in the media; hence, the tests were quite difficult to read. The broth microdilution method was therefore chosen in preference to the broth macrodilution method.
Two inoculum sizes were tested: 1 × 106 conidia/ml and 2 × 103 conidia/ml. No end point was found with the large inoculum, even after the range of drug dilutions was extended (0.0018 to 8 mg/liter). In comparison, the small inoculum showed a mass of hyphal growth gradually changing to small, subspherical colonies as the drug concentration increased. This end point correlated with in vivo in-house company data. Hence, the small inoculum (2 × 103/ml) was chosen for susceptibility testing.
Four media were initially tested with LY: AM3, RPMI, CAS, and YNBG. YNBG and RPMI showed poor growth when the small Aspergillus inoculum was used, and consequently these media were not chosen. The Aspergillus isolates were found to grow very well in both AM3 and CAS, producing a clear transition from hyphal growth to the formation of small colonies. Hence, AM3 and CAS were both chosen to test the in vitro activity of LY.
Figure 1 shows the type of growth that appears when Aspergillus is grown in the presence of LY. Two types of growth can be seen in the microtiter tray: hyphal growth in the lowest concentrations of LY, gradually changing to small, white colonies as the drug concentration increases. Differences in growth patterns between species are clearly seen in Fig. 2. A. terreus (Fig. 2, rows A and B) and A. niger (rows E and F) have only a small number of tiny colonies in the microplate wells, with a few wells having no visible growth at all. In comparison, the growth of A. fumigatus (Fig. 2, rows C and D) and A. flavus (rows G and H) is much greater and there are many more colonies present in the wells.
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The range of MECs and MECs at which 50% (MEC50) and 90% (MEC90) of the isolates are inhibited for different species of Aspergillus tested against LY are shown in Table 1, together with the range of MICs and MIC50 and MIC90 for ITZ and AMB. LY in both AM3 and CAS was more active in vitro than ITZ or AMB. The MECs for LY in both media ranged from 0.0018 to >0.5 mg/liter, compared to MICs ranging from 0.125 to >16 mg/liter for ITZ and from 0.25 to 16 mg/liter for AMB. For MECs there were differences between species, with A. flavus being significantly less susceptible to LY in AM3 and CAS (P < 0.01). A. niger seemed to be much more susceptible than other species, with MICs being obtained for five of eight isolates in AM3 and for two of eight isolates in CAS (i.e., the well was clear and contained no subspherical colonies). There were no significant differences between the values obtained with the two media, AM3 and CAS, for testing LY, although AM3 values did tend to be always slightly lower than CAS values.
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The range of MFCs, MFC50, and MFC90 for LY in both AM3 and CAS, ITZ, and AMB are shown in Table 2. MFCs ranged from 0.0018 to >0.5 mg/liter for LY. There was a significant difference in LY MFCs between species (P = 0.003 for AM3 and CAS). Among isolates tested in AM3, A. flavus was significantly less susceptible than A. niger and A. terreus. In comparison, among isolates tested in CAS A. niger, A. fumigatus, and A. flavus were significantly less susceptible than A. terreus. The ratio between MFC and MEC was much higher for A. fumigatus and A. flavus than for other species. LY in both AM3 and CAS was fungicidal for 86.7 and 68% of isolates, respectively (98% killing). In contrast, the MFCs of ITZ and AMB were much higher. For ITZ, MFCs ranged from 2 to >16 mg/liter and, for AMB, MFCs ranged from 1 to >16 mg/liter. ITZ showed no significant differences from species to species (P = 0.141) and was fungicidal for 35% of isolates tested. In comparison, there were several significant differences between species (P = 0.0001) for AMB. A. terreus was significantly less susceptible than either A. fumigatus or A. niger. Also, A. flavus was significantly less susceptible than A. niger. Overall, AMB was fungicidal for 70% of the isolates tested.
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DISCUSSION |
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Abstract
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Materials & Methods
Results
Discussion
References
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LY has been shown to have potent in vitro activity against a wide range of fungal pathogens, including Candida spp. (8, 10, 12) and Aspergillus spp. (12). Our in vitro studies with LY have shown similar results against Aspergillus spp., including ITZ-resistant isolates.
We found LY to have excellent activity against different species of Aspergillus and demonstrated that LY is more active at lower concentrations than either ITZ or AMB. One species of Aspergillus, A. flavus, was found to be slightly less susceptible than other species, and this was found to be statistically significant. It was also interesting that for some isolates of A. niger and A. terreus there were far fewer wells containing subspherical colonies and that in a small number of drug dilutions the wells were totally clear.
A typical MIC was not seen for Aspergillus spp. when they were tested against the echinocandin antifungal agent LY. Two distinct types of growth are apparent when Aspergillus is grown in the presence of LY. At the lowest concentrations of LY, a dense mat of hyphal growth completely covers the entire well. At higher concentrations of LY, wells containing purely white, subspherical colonies attached to the bottom of the microtiter plate were visible. Examination of these colonies (9) by electron microscopy has revealed that they appear to be cell wall-deficient microcolonies. However, subcultures in the absence of LY regain both their cell walls and their susceptibility to this drug (9). For the purposes of determining an inhibitory concentration that might be clinically useful, the transition from matted growth to microcolonies was taken as the first breakpoint and was recorded as the MEC. A second breakpoint of no growth at all was seen with some species of Aspergillus (i.e., A. niger and A. terreus). However, for other species of Aspergillus (i.e., A. fumigatus and A. flavus) initial MIC tests with the drug range extended up to 8 mg/liter still showed small colonies in the bottoms of the microtiter wells. One in vivo study with two A. fumigatus isolates showed that the MEC most closely correlates with the efficacy of LY in a murine model of invasive aspergillosis (11).
Phase I data from human volunteers following oral administration of LY showed concentrations in serum up to about 0.7 mg/liter and a half-life of 30 h (6). Such concentrations substantially exceed all the MECs and the majority of MFCs recorded here. Thus, LY has promising in vitro and in vivo activity against Aspergillus species and further investigation is warranted.
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ACKNOWLEDGMENTS |
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We thank Eli Lilly and the Fungal Research Trust for their financial support of this research project.
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FOOTNOTES |
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* Corresponding author. Present address: Department of Infectious Diseases and Tropical Medicine (Monsall Unit), North Manchester General Hospital, Delauneys Rd., Manchester, M8 6RB, United Kingdom. Phone: 44 161 720 2734. Fax: 44 161 720 2732.
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REFERENCES |
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| 1. | Bodey, G. P., and S. Vartivarian. 1989. Aspergillosis. Eur. J. Clin. Microbiol. Infect. Dis. 8:413-437[Medline]. |
| 2. | Denning, D. W. 1996. Therapeutic outcome in invasive aspergillosis. Clin. Infect. Dis. 23:608-615[Medline]. |
| 3. |
Denning, D. W.
1997.
Echinocandins and pneumocandins a new antifungal class with a novel mode of action.
J. Antimicrob. Chemother.
40:611-613 |
| 4. | Denning, D. W., K. Venkateswarlu, K. L. Oakley, M. J. Anderson, N. J. Manning, D. A. Stevens, D. W. Warnock, and S. L. Kelly. 1997. Itraconazole resistance in Aspergillus fumigatus. Antimicrob. Agents Chemother. 41:1364-1368[Abstract]. |
| 5. |
Denning, D. W.,
S. A. Radford,
K. L. Oakley,
L. Hall,
E. M. Johnson, and D. W. Warnock.
1997.
Correlation between in vitro susceptibility testing to itraconazole and in vivo outcome of Aspergillus fumigatus infection.
J. Antimicrob. Chemother.
42:401-414 |
| 6. | Lucas, R., K. Desante, B. Hatcher, J. Hemingway, R. Lachno, S. Brooks, and M. Turik. 1996. LY303366 single dose pharmacokinetics and safety in healthy male volunteers, abstr. F50, p. 108. In Abstracts of the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C. |
| 7. |
Moore, C. B.,
D. Law, and D. W. Denning.
1993.
In vitro activity of the new triazole D0870 compared with amphotericin B and itraconazole against Aspergillus spp.
J. Antimicrob. Chemother.
32:831-836 |
| 8. | Pfaller, M. A., S. A. Messer, and S. Coffman. 1997. In vitro susceptibilities of clinical yeast isolates to a new echinocandin derivative, LY303366, and other antifungal agents. Antimicrob. Agents Chemother. 41:763-766[Abstract]. |
| 9. | Rennie, R., C. Sand, and R. Sherburne. 1997. Electron microscopic evidence of the effect of LY303366 on Aspergillus fumigatus, abstr. P451. In Program and abstracts of the 13th Congress of the International Society for Human and Animal Mycology, Parma, Italy. |
| 10. | Uzun, Ö., S. Kocagöz, Y. Çetinkaya, S. Arikan, and S. Ünal. 1997. In vitro activity of a new echinocandin, LY303366, compared with those of amphotericin B and fluconazole against clinical yeast isolates. Antimicrob. Agents Chemother. 41:1156-1157[Abstract]. |
| 11. |
Verweij, P. E.,
K. L. Oakley,
J. Morrissey,
G. Morrissey, and D. W. Denning.
1998.
Efficacy of LY303366 against amphotericin B-susceptible and -resistant Aspergillus fumigatus in a murine model of invasive aspergillosis.
Antimicrob. Agents Chemother.
42:873-878 |
| 12. | Zhanel, G. G., J. A. Karlowsky, G. A. J. Harding, T. V. Balko, S. A. Zelenitsky, M. Friesen, A. Kabani, M. Turik, and D. J. Hoban. 1997. In vitro activity of a new semisynthetic echinocandin, LY-303366, against systemic isolates of Candida species, Cryptococcus neoformans, Blastomyces dermatitidis, and Aspergillus species. Antimicrob. Agents Chemother. 41:863-865[Abstract]. |
Antimicrobial Agents and Chemotherapy, October 1998, p. 2726-2730, Vol. 42, No. 10
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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