In vitro susceptibilities of clinical yeast isolates to a new echinocandin derivative, LY303366, and other antifungal agents.

LY303366 is a new semisynthetic echinocandin derivative with potent, broad-spectrum fungicidal activity. We investigated the in vitro activity of LY303366, amphotericin B, flucytosine (5FC), fluconazole, and itraconazole against 435 clinical yeast isolates (413 Candida and 22 Saccharomyces cerevisiae isolates) obtained from over 30 different medical centers. MICs for all five antifungal agents were determined by the National Committee for Clinical Laboratory Standards method with RPMI 1640 test medium. LY303366 was also tested in antibiotic medium 3 as specified by the manufacturer. Overall, LY303366 was quite active against all of the yeast isolates when tested in RPMI 1640 (MIC at which 90% of the isolates are inhibited [MIC90], 1.0 microg/ml) but appeared to be considerably more potent when tested in antibiotic medium 3 (MIC90, 0.03 microg/ml). When tested in antibiotic medium 3, LY303366 was 16- to >2,000-fold more active than itraconazole, fluconazole, amphotericin B, or 5FC against all species except Candida parapsilosis. When tested in RPMI 1640, LY303366 was comparable to amphotericin B and itraconazole and more active than fluconazole and 5FC. All of the isolates for which fluconazole and itraconazole had elevated MICs (> or = 128 and > or = 2.0 microg/ml, respectively) were inhibited by < or = 0.007 microg of LY303366/ml when tested in antibiotic medium 3 and < or = 0.5 microg/ml when tested in RPMI 1640. Based on these studies, LY303366 has promising antifungal activity and warrants further in vitro and in vivo investigation.     

Activity of MK-0991 (l-743,872), a new echinocandin,compared with those of LY303366 and four other antifungal agents tested against blood stream isolates of Candida spp.

MK-0991 (formerly L-743,872) is a water soluble semisynthetic echinocandin that possess potent, broad-spectrum antifungal activity. We evaluated the in vitro activity of MK-0991 and an echinocandin derivative LY303366, compared with that of itraconazole, fluconazole, amphotericin B and 5-flucytosine against 400 blood stream isolates of Candida spp. (nine species) obtained from more than 30 different medical centers. MICs for all antifungal drugs were determined by the NCCLS method using RPMI 1640 test medium. Both MK-0991 and LY303366 were very active against all Candida spp. isolates (MIC₉₀, 0.25 and 1 μg/mL, respectively). MK-0991 was twofold to 256-fold more active than amphotericin B, fluconazole, itraconazole (except against C. parapsilosis), and 5-flucytosine (except against C. glabrata and C. parapsilosis). LY303366 was comparable to MK-0991, but was fourfold less active against C. tropicalis (MIC₉₀, 0.5 versus 0.12 μg/mL) and C. parapsilosis (MIC₉₀, >2 versus 1 μg/mL). All of the isolates for which fluconazole and itraconazole had elevated MICs (≥64 μg/mL and ≥1 μg/mL, respectively) were inhibited by ≤0.5 μg/mL of MK-0991 and LY303366. These results suggest both MK-0991 and LY303366 possess promising antifungal activity and further in vitro and in vivo investigations are warranted.     

In Vitro Activities of Voriconazole (UK-109,496) and Four Other Antifungal Agents against 394 Clinical Isolates of Candida spp.

Voriconazole (formerly UK-109,496) is a new monotriazole antifungal agent which has potent activity against Candida, Cryptococcus, and Aspergillus species. We investigated the in vitro activity of voriconazole compared to those of fluconazole, itraconazole, amphotericin B, and flucytosine (5FC) against 394 bloodstream isolates of Candida (five species) obtained from more than 30 different medical centers. MICs of all antifungal drugs were determined by the method recommended by the National Committee for Clinical Laboratory Standards using RPMI 1640 test medium. Overall, voriconazole was quite active against all the yeast isolates (MIC at which 90% of the isolates are inhibited [MIC₉₀], ≤0.5 μg/ml). Candida albicans was the most susceptible species (MIC₉₀, 0.06 μg/ml) and Candida glabrata and Candida krusei were the least (MIC₉₀, 1 μg/ml). Voriconazole was more active than amphotericin B and 5FC against all species except C. glabrata and was also more active than itraconazole and fluconazole. For isolates of Candida spp. with decreased susceptibility to fluconazole and itraconazole MICs of voriconazole were also higher. Based on these results, voriconazole has promising antifungal activity and further in vitro and in vivo investigations are warranted.     

Influence of Human Serum on Antifungal Pharmacodynamics with Candida albicans

Antifungal susceptibilities (NCCLS, approved standard M27-A, 1997) were determined for the reference strain ATCC 90028 and 21 clinical isolates of Candida albicans with varying levels of fluconazole susceptibility using RPMI 1640 (RPMI) and 80% fresh human serum-20% RPMI (serum). Sixty-four percent (14 of 22) of the isolates tested demonstrated significant decreases (> or = 4-fold) in fluconazole MICs in the presence of serum, and the remaining eight isolates exhibited no change. Itraconazole and ketoconazole, two highly protein-bound antifungal agents, had MICs in serum that were increased or unchanged for 46% (10 of 22) and 41% (9 of 22) of the isolates, respectively. All 10 isolates tested against an investigational antifungal agent, LY303366, demonstrated significant increases in the MIC required in serum, while differences in amphotericin B MICs in the two media were not observed. Four of 10 isolates tested demonstrated fourfold higher flucytosine MICs in serum than in RPMI. Postantifungal effects (PAFEs) and 24-h kill curves were determined by standard methods for selected isolates. At the MIC, fluconazole, itraconazole, ketoconazole, flucytosine, and LY303366 kill curves and PAFEs in RPMI were similar to those in serum. Isolates of fluconazole-resistant C. albicans required lower MICs in serum than in RPMI, without relative increases in fungal killing or PAFEs. Isolates tested against amphotericin B demonstrated significantly reduced killing and shorter PAFEs in serum than in RPMI without observable changes in MIC. In conclusion, antifungal pharmacodynamics in RPMI did not consistently predict antifungal activity in serum for azoles and amphotericin B. Generally speaking, antifungal agents with high protein binding exhibited some form of reduced activity (MIC, killing, or PAFE) in the presence of serum compared to those with low protein binding.     

Structure-In Vitro Activity Relationships of Pentamidine Analogues and Dication-Substituted Bis-Benzimidazoles as New Antifungal Agents

Twenty analogues of pentamidine, 7 primary metabolites of pentamidine, and 30 dicationic substituted bis-benzimidazoles were screened for their inhibitory and fungicidal activities against Candida albicans and Cryptococcus neoformans. A majority of the compounds had MICs at which 80% of the strains were inhibited (MIC₈₀s) comparable to those of amphotericin B and fluconazole. Unlike fluconazole, many of these compounds were found to have potent fungicidal activity. The most potent compound against C. albicans had an MIC₈₀ of ≤0.09 μg/ml, and the most potent compound against C. neoformans had an MIC₈₀ of 0.19 μg/ml. Selected compounds were also found to be active against Aspergillus fumigatus, Fusarium solani, Candida species other than C. albicans, and fluconazole-resistant strains of C. albicans and C. neoformans. It is clear from the data presented here that further studies on the structure-activity relationships, mechanisms of action and toxicities, and in vivo efficacies of these compounds are warranted to determine their clinical potential.     

In Vitro Susceptibilities of Candida Bloodstream Isolates to the New Triazole Antifungal Agents BMS-207147, Sch 56592, and Voriconazole

BMS-207147, Sch 56592, and voriconazole are three new investigational triazoles with broad-spectrum antifungal activity. The in vitro activities of these three agents were compared with those of itraconazole and fluconazole against 1,300 bloodstream isolates of Candida species obtained from over 50 different medical centers in the United States. The MICs of all of the antifungal drugs were determined by broth microdilution tests performed according to the National Committee for Clinical Laboratory Standards method using RPMI 1640 as a test medium. BMS-207147, Sch 56592, and voriconazole were all quite active against all Candida sp. isolates (MICs for 90% of the isolates tested [MIC₉₀s], 0.5, 1.0, and 0.5 μg/ml, respectively). Candida albicans was the most susceptible species (MIC₉₀s, 0.03, 0.06, and 0.06 μg/ml, respectively), and C. glabrata was the least susceptible (MIC₉₀s, 4.0, 4.0, and 2.0 μg/ml, respectively). BMS-207147, Sch 56592, and voriconazole were all more active than itraconazole and fluconazole against C. albicans, C. parapsilosis, C. tropicalis, and C. krusei. There existed a clear rank order of in vitro activity of the five azoles examined in this study when they were tested versus C. glabrata: voriconazole > BMS-207147 = Sch 56592 = itraconazole > fluconazole (MIC₉₀s, 2.0, 4.0, 4.0, 4.0, and 64 μg/ml, respectively). For isolates of Candida spp. with decreased susceptibility to both itraconazole and fluconazole, the MICs of BMS-207147, Sch 56592, and voriconazole were also elevated. These results suggest that BMS-207147, Sch 56592, and voriconazole all possess promising antifungal activity and that further in vitro and in vivo investigations are warranted to establish the clinical value of this improved potency.     

Specific Activities of Dolastatin 10 and Peptide Derivatives against Cryptococcus neoformans

The biosynthetic peptide dolastatin 10 is currently in phase I and II cancer clinical trials. We evaluated the antifungal spectrum of dolastatin 10 and four structural modifications. In broth macrodilution assays, the peptides were fungicidal for American Type Culture Collection strains and clinical isolates (including fluconazole-resistant strains) of Cryptococcus neoformans but no other yeasts or filamentous fungi examined. Specificity for C. neoformans was also demonstrated in the solid-phase disk diffusion assay, and fungicidal activity was confirmed in time-kill experiments. For a methyl ester modification, the MICs at which 50 and 90% of 19 clinical isolates were inhibited (MIC₅₀ and MIC₉₀, respectively) were 0.195 and 0.39 μg/ml, respectively. The MFC₅₀ (50% minimum fungicidal concentration) for this peptide was 0.39 μg/ml, and the MFC₉₀ was 0.78 μg/ml. MICs and MFCs were identical or lower in the presence of human serum but increased with lowered pH. These peptides should be pursued as potential chemotherapeutics for C. neoformans, a leading cause of infection and mortality in immunocompromised patients.     

Antifungal activity of the echinocandin anidulafungin (VER002, LY-303366) against yeast pathogens: a comparative study with M27-A microdilution method

This study further evaluated the in vitro activity of anidulafungin (VER002, Versicor Inc.) (LY303366) against 460 clinical yeast isolates. MICs of anidulafungin, fluconazole and itraconazole were determined by following the NCCLS M27-A guidelines. Minimum fungicidal concentrations (MFCs) of anidulafungin were determined for 230 isolates of Candida spp. The activity of anidulafungin in vitro was significantly superior (P < 0.05) to those of itraconazole and fluconazole against Candida albicans, Candida tropicalis, Candida glabrata and Candida krusei, but anidulafungin was less active for Candida famata and Candida parapsilosis. The differences were not significant for the other species evaluated.     

Susceptibilities of Candida Species Isolated from the Lower Gastrointestinal Tracts of High-Risk Patients to the New Semisynthetic Echinocandin LY303366 and Other Antifungal Agents

Fifty-two percent of stool specimens collected from 1,200 high-risk patients were colonized with yeasts, primarily Candida albicans (53.6%) and Candida glabrata (35.7%). Susceptibilities to all antifungal agents tested, including LY303366, were similar to those reported previously for Candida species isolated from blood.     

Efficacy of LY303366 against Amphotericin B-Susceptible and -Resistant Aspergillus fumigatus in a Murine Model of Invasive Aspergillosis

LY303366 is a novel antifungal echinocandin with excellent in vitro activity against Aspergillus spp. We compared four doses (1, 2.5, 10, and 25 mg/kg of body weight) of LY303366 with amphotericin B (0.5 to 5 mg/kg) in a temporarily neutropenic murine model of invasive aspergillosis against an amphotericin B-susceptible (AF210) and an amphotericin B-resistant (AF65) Aspergillus fumigatus isolate based on in vivo response. Mice were immunosuppressed with cyclophosphamide (200 mg/kg) and infected 3 days later. Treatment started 18 h after infection and lasted for 10 days. LY303366 was given once daily intravenously for 10 days, and amphotericin B (at 0.5, 2, and 5 mg/kg) was given once daily intraperitoneally for 10 days, or only on days 1, 2, 4, and 7 (at 5 mg/kg). Kidneys and lungs from survivors were cultured on day 11. Control mice in both experiments had 90 to 100% mortality. Amphotericin B at 0.5 mg/kg and LY303366 at 1 mg/kg yielded 10 to 20% survival rates for mice infected with either AF210 or AF65. Amphotericin B at 2 and 5 (both regimens) mg/kg yielded a 70 to 100% survival rate for mice infected with AF210 but a 10 to 30% survival rate for mice infected with AF65 (P = 0.01 to 0.04 compared with AF210). Against AF210 and AF65, LY303366 at 2.5, 10, and 25 mg/kg produced a survival rate of 70 to 80%, which was as effective as amphotericin B for AF210, but superior to amphotericin B for AF65 (P < 0.03 to 0.0006). For AF65, LY303366 at 10 and 25 mg/kg/day was superior to amphotericin B at 2 and 5 mg/kg/day in reducing tissue colony counts (P = 0.01 to 0.003), and for AF210, amphotericin B at 5 mg/kg/day and at 5 mg/kg in four doses was more effective than all four regimens of LY303366 in reducing renal culture counts (P = 0.01 to 0.0001). The present study shows, for the first time, that in vivo resistance of A. fumigatus to amphotericin B exists, although this could not be detected by in vitro susceptibility assays. Furthermore, LY303366 appears to be effective against amphotericin B-susceptible and -resistant A. fumigatus infection in this model and should be further evaluated clinically.     

Susceptibility of fluconazole-resistant clinical isolates of Candida spp. to echinocandin LY303366, itraconazole and amphotericin B

The in vitro activity of LY303366 was compared with those of itraconazole and amphotericin B against 156 fluconazole-resistant (MIC > or = 16 mg/L) clinical isolates of CANDIDA: spp. An adaptation of the NCCLS reference method was employed for determination of MICs. LY303366 was more potent than either itraconazole or amphotericin B against Candida albicans, Candida glabrata, Candida krusei and Candida tropicalis, even against isolates with itraconazole MICs > or = 1 mg/L. LY303366 was less potent in vitro against Candida parapsilosis and Candida guilliermondii isolates. LY303366 has promising antifungal activity and warrants further investigation.     

In vitro activity of micafungin (FK-463) against Candida spp.: microdilution,time-kill,and postantifungal-effect studies

We evaluated the in vitro activity of the new echinocandin antifungal micafungin against Candida spp. using microdilution and time-kill methods. Additionally, we examined the postantifungal effect (PAFE) of micafungin. Finally, we evaluated the effect of the addition of serum and plasma on the MIC of micafungin. Four Candida albicans isolates and two isolates of each Candida glabrata, Candida krusei, and Candida tropicalis were selected for testing. The MICs of micafungin were determined in RPMI 1640 medium buffered with morpholinepropanesulfonic acid alone and with the addition of 10, 20, and 50% human serum and plasma. MICs were determined by using two endpoints: a prominent reduction in growth (the MIC at which 80% of isolates are inhibited [MIC(80)]) and complete visual inhibition of growth (MIC(100)). The minimum fungicidal concentration (MFC) of micafungin for each isolate was also determined. Time-kill curves were determined for each isolate in RPMI 1640 medium with micafungin at concentrations ranging from 0.125 to 16 times the MIC(80) to assess the correlation between MIC(80) and fungicidal activity. PAFE studies were conducted with each isolate by using concentrations ranging between 0.25 and 4 times the MIC(80). The MIC(80)s for the test isolates ranged from 0.0039 to 0.25 micro g/ml. Overall, the addition of serum or plasma increased the MIC 6 to 7 doubling dilutions for C. albicans and 3 to 4 doubling dilutions for C. krusei and C. tropicalis. Micafungin time-kill studies demonstrated fungicidal activity at concentrations ranging from 4 to 16 times the MIC(80). Micafungin is very potent agent against a variety of Candida spp., producing fungicidal activity against 7 of 10 isolates tested. A PAFE was observed against all isolates. The PAFE was influenced by the drug concentration, with the highest concentration resulting in the longest observed PAFE in each case. The highest concentration tested, four times the MIC, resulted in a PAFE of more than 9.8 h for 5 of 10 isolates tested (range, 0.9 to > or =20.1 h).     

Influence of Test Conditions on Antifungal Time-Kill Curve Results: Proposal for Standardized Methods

This study was designed to examine the effects of antifungal carryover, agitation, and starting inoculum on the results of time-kill tests conducted with various Candida species. Two isolates each of Candida albicans, Candida tropicalis, and Candida glabrata were utilized. Test antifungal agents included fluconazole, amphotericin B, and LY303366. Time-kill tests were conducted in RPMI 1640 medium buffered with morpholinepropanesulfonic acid (MOPS) to a pH of 7.0 and incubated at 35°C. Prior to testing, the existence of antifungal carryover was evaluated at antifungal concentrations ranging from 1× to 16× MIC by four plating methods: direct plating of 10, 30, and 100 μl of test suspension and filtration of 30 μl of test suspension through a 0.45-μm-pore-size filter. Time-kill curves were performed with each isolate at drug concentrations equal to 2× MIC, using a starting inoculum of approximately 10⁵ CFU/ml, and incubated with or without agitation. Last, inoculum experiments were conducted over three ranges of starting inocula: 5 × 10² to 1 × 10⁴, >1 × 10⁴ to 1 × 10⁶, and >1 × 10⁶ to 1 × 10⁸ CFU/ml. Significant antifungal carryover (>25% reduction in CFU/milliliter from the control value) was observed with amphotericin B and fluconazole; however, carryover was eliminated with filtration. Agitation did not appreciably affect results. The starting inoculum did not significantly affect the activity of fluconazole or amphotericin B; however, the activity of LY303366 may be influenced by the starting inoculum. Before antifungal time-kill curve methods are routinely employed by investigators, methodology should be scrutinized and standardized procedures should be developed.     

In Vitro Activities of Terbinafine against Cutaneous Isolates of Candida albicans and Other Pathogenic Yeasts

Terbinafine is active in vitro against a wide range of pathogenic fungi, including dermatophytes, molds, dimorphic fungi, and some yeasts, but earlier studies indicated that the drug had little activity against Candida albicans. In contrast, clinical studies have shown topical and oral terbinafine to be active in cutaneous candidiasis and Candida nail infections. In order to define the anti-Candida activity of terbinafine, we tested the drug against 350 fresh clinical isolates and additional strains by using a broth dilution assay standardized according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) M27-A assay. Terbinafine was found to have an MIC of 1 μg/ml for reference C. albicans strains. For 259 clinical isolates, the MIC at which 50% of the isolates are inhibited (MIC₅₀) of terbinafine was 1 μg/ml (fluconazole, 0.5 μg/ml), and the MIC₉₀ was 4 μg/ml (fluconazole, 1 μg/ml). Terbinafine was highly active against Candida parapsilosis (MIC₉₀, 0.125 μg/ml) and showed potentially interesting activity against isolates of Candida dubliniensis, Candida guilliermondii, Candida humicola, and Candida lusitaniae. It was not active against the Candida glabrata, Candida krusei, and Candida tropicalis isolates in this assay. Cryptococcus laurentii and Cryptococcus neoformans were highly susceptible to terbinafine, with MICs of 0.06 to 0.25 μg/ml. The NCCLS macrodilution assay provides reproducible in vitro data for terbinafine against Candida and other yeasts. The MICs for C. albicans and C. parapsilosis are compatible with the known clinical efficacy of terbinafine in cutaneous infections, while the clinical relevance of its activities against the other species has yet to be determined.     

Association of fluconazole pharmacodynamics with mortality in patients with candidemia

Recent studies of nonneutropenic patients with candidemia or candidiasis suggest that fluconazole pharmacodynamic parameters correlate with clinical outcomes; however, additional data of correlation to mortality in patients with candidemia would be valuable. We assessed the impact of MICs for Candida, fluconazole pharmacodynamics, and patient characteristics on all-cause mortality with use of a prospective cohort of 96 hospitalized patients with candidemia. Among 84 patients for whom Candida isolates were available for testing, the most frequent Candida species isolated were Candida albicans (44%), followed by Candida parapsilosis (20.2%), and Candida glabrata (20.2%). Fluconazole resistance (MIC of ≥64 μg/ml) was present in 7 (8.3%) to 10 (11.9%) of 84 isolates, depending on the MIC endpoint determination method (50% or 80% inhibition read at 24 or 48 h). Overall mortality occurred in 27 (28.1%) of 96 patients, and nonsurvivors were more likely to have fluconazole-resistant isolates (25% versus 6.7%; P = 0.02). Multivariable analysis demonstrated an association between fluconazole resistance and mortality, but it did not reach statistical significance (odds ratio, 5.3; 95% confidence interval, 0.8 to 33.4; P = 0.08). By pharmacodynamic analysis, a fluconazole area under the concentration-time curve/MIC of <11.5 or MIC of ≥64 was associated with increased patient mortality (P ≤ 0.09). These data support previous findings of an antifungal exposure-response relationship to mortality in patients with candidemia. In addition, similar MICs were obtained using a 24- or 48-h MIC endpoint determination, thus providing the opportunity to assess earlier the impact of isolate susceptibility on therapy.     

LY303366 exhibits rapid and potent fungicidal activity in flow cytometric assays of yeast viability

LY303366 is a semisynthetic analog of the antifungal lipopeptide echinocandin B that inhibits (1,3)-beta-D-glucan synthase and exhibits efficacy in animal models of human fungal infections. In this study, we utilized flow cytometric analysis of propidium iodide uptake, single-cell sorting, and standard microbiological plating methods to study the antifungal effect of LY303366 on Saccharomyces cerevisiae and Candida albicans. Our data indicate that an initial 5-min pulse treatment with LY303366 caused yeasts to take up propidium iodide and lose their ability to grow. Amphotericin B and cilofungin required longer exposure periods (30 and 180 min, respectively) and higher concentrations to elicit these fungicidal effects. These two measurements of fungicidal activity by LY303366 were highly correlated (r > 0.99) in concentration response and time course experiments. As further validation, LY303366-treated yeasts that stained with propidium iodide were unable to grow in single-cell-sorted cultures. Our data indicate that LY303366 is potent and rapidly fungicidal for actively growing yeasts. The potency and rapid action of this new fungicidal compound suggest that LY303366 may be useful for antifungal therapy.     

In vitro activity of doripenem, a carbapenem for the treatment of challenging infections caused by gram-negative bacteria, against recent clinical isolates from the United States

Doripenem, a 1β-methylcarbapenem, is a broad-spectrum antibiotic approved for the treatment of complicated urinary tract and complicated intra-abdominal infections. An indication for hospital-acquired pneumonia including ventilator-associated pneumonia is pending. The current study examined the activity of doripenem against recent clinical isolates for the purposes of its ongoing clinical development and future longitudinal analysis. Doripenem and comparators were tested against 12,581 U.S. clinical isolates collected between 2005 and 2006 including isolates of Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus pneumoniae, Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter spp. MICs (μg/ml) were established by broth microdilution. By MIC₉₀, doripenem was comparable to imipenem and meropenem in activity against S. aureus (methicillin susceptible, 0.06; resistant, 8) and S. pneumoniae (penicillin susceptible, ≤0.015; resistant, 1). Against ceftazidime-susceptible Enterobacteriaceae, the MIC₉₀ of doripenem (0.12) was comparable to that of meropenem (0.12) and superior to that of imipenem (2), though susceptibility of isolates exceeded 99% for all evaluated carbapenems. The activity of doripenem was not notably altered against ceftazidime-nonsusceptible or extended-spectrum β-lactamase screen-positive Enterobacteriaceae. Doripenem was the most potent carbapenem tested against P. aeruginosa (MIC₉₀/% susceptibility [%S]: ceftazidime susceptible = 2/92%S, nonsusceptible = 16/61%S; imipenem susceptible = 1/98.5%S, nonsusceptible = 8/56%S). Against imipenem-susceptible Acinetobacter spp., doripenem (MIC₉₀ = 2, 89.1%S) was twice as active by MIC₉₀ as were imipenem and meropenem. Overall, doripenem potency was comparable to those of meropenem and imipenem against gram-positive cocci and doripenem was equal or superior in activity to meropenem and imipenem against Enterobacteriaceae, including β-lactam-nonsusceptible isolates. Doripenem was the most active carbapenem tested against P. aeruginosa regardless of β-lactam resistance.     

Influence of Human Serum on Pharmacodynamic Properties of an Investigational Glycopeptide, LY333328, and Comparator Agents against Staphylococcus aureus

The MICs and MBCs of 15 antibiotics for two strains of Staphylococcus aureus were determined in Mueller-Hinton broth (MHB) and 90% serum–10% MHB. Subsequent experiments established that highly protein-bound antibiotics (≥80%), such as LY333328, demonstrated higher MICs and MBCs, less killing over an 8-h interval, and shorter postantibiotic effects in 90% serum–10% MHB than in MHB alone. Albumin was demonstrated to be almost solely responsible for changes in the aforementioned pharmacodynamic parameters of LY333328.     

In Vitro Activity of the Echinocandin Antifungal Agent LY303,366 in Comparison with Itraconazole and Amphotericin B against Aspergillus spp.

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 ≤ 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.

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)-β-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).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.     

Antifungal Susceptibilities of Paecilomyces Species

The MICs and minimum fungicidal concentrations (MFCs) of amphotericin B, miconazole, itraconazole, ketoconazole, fluconazole, and flucytosine for 52 isolates of Paecilomyces species were evaluated by the broth microdilution method, largely based on the recommendations of the National Committee for Clinical Laboratory Standards (document M27-A). The fungal isolates tested included 16 P. variotii, 11 P. lilacinus, 9 P. marquandii, 6 P. fumosoroseus, 4 P. javanicus, and 2 P. viridis isolates and 1 isolate of each of the following species: P. carneus, P. farinosus, P. fulvus, and P. niveus. The MFCs and the MICs at which 90% of isolates were inhibited (MIC₉₀s) for the six antifungal agents were remarkably high; the MIC₅₀s indicated that amphotericin B, miconazole, itraconazole, and ketoconazole had good activities, while fluconazole and flucytosine demonstrated poor efficacy. The ranges of the MICs were generally wider and lower than those of the MFCs. There were significant susceptibility differences among the species. All species with the exception of P. variotii were highly resistant to fluconazole and flucytosine; P. variotii was susceptible to flucytosine. Amphotericin B and the rest of the azoles showed good activity against P. variotii, while all the antifungal agents assayed showed low efficacy against P. lilacinus.