In Vitro Activity and In Vivo Efficacy of Icofungipen (PLD-118), a Novel Oral Antifungal Agent, against the Pathogenic Yeast Candida albicans

Media.

YNG, YPG, YNGW, YPD, LB, Sabouraud dextrose, and RPMI 1640 media were used and prepared as described elsewhere (4, 24, 30).

YNG medium was buffered to pH 6 or pH 7 using Soerensen buffer containing Na₂HPO₄ · 2H₂O (1.4 g/liter) and NaH₂PO₄ (8.0 g/liter) or Na₂HPO₄ · 2H₂O (7.2 g/liter) and NaH₂PO₄ (3.6 g/liter) (Merck, Germany). Under all conditions in which these buffers were used, no change in incubation pH was observed. To evaluate the influence of amino acids, YNG was supplemented with isoleucine, leucine, or valine at a final concentration of 0.1 mM, 1 mM, or 10 mM. RPMI 1640 powdered medium was prepared according to the CLSI (formerly NCCLS) standard M27-A (15). YNG media were adjusted to a given pH (4.0 to 8.0) by the addition of NaOH or HCl.

Strains.

A control strain was obtained from the American Type Culture Collection (C. albicans ATCC 90028). Clinical specimens were isolated from various tissues (blood, mucosal surfaces, and skin) at the University Hospital (KBC), Zagreb, Croatia, and from various clinical centers in Germany.

Clinical isolates of C. albicans were identified by chlamydospore formation on rice agar (Merck, Darmstadt, Germany). Strains unable to form chlamydospores were biochemically identified using the API ID 32C system (bioMérieux, Nuertlingen, Germany). Strains were preserved by being freeze-dried in milk powder (Struthmannn, Kleve, Germany) and stored at 4°C.

Isolates were stored at −70°C in a Cryobank (Mast Diagnostica, Germany) and cultured on Sabouraud agar at 35°C prior to being tested.

A clinical isolate of C. albicans, BSMY 212 (C. albicans strain, deposited at ATCC as ATCC 200498), from the Bayer strain collection was used to establish basic parameters of infection and efficacy. In addition, various clinical isolates were used as well as strains from the ATCC (Bethesda, Maryland), as indicated.

Inoculum preparation.

Candida species were grown overnight at 37°C in YPG medium without shaking. The overnight suspension was centrifuged at 1,000 × g for 10 min. The supernatant was decanted, and the pellet was resuspended in phosphate buffer, pH 8. Using a McFarland standard protocol, optical density was adjusted to approximately 10⁷ CFU/ml. Dilutions of fungal suspensions were made, as required, in YNG medium. A final inoculum of 1 × 10³ CFU/ml (resulting in 100 CFU/microtiter well) was used. Inoculum size was checked in every experiment by plating 10 μl of the suspension on Sabouraud 2% dextrose agar plates. Alternatively, inoculum was prepared by direct colony suspension from a 24-h Sabouraud 2% dextrose agar plate incubated at 37°C.

For inoculum consisting of logarithmic-phase cells, one to two colonies were resuspended in 5 ml of YPG and incubated for 2 h at 37°C with moderate shaking. From this culture, 1 ml was added to 50 ml of YNG and further incubated for 16 h.

Compound preparation.

Icofungipen was prepared at PLIVA in GLP quality. FLC was purchased from Chemoiberica, Spain, while clotrimazole, amphotericin B, miconazole, nystatin, and flucytosine were obtained from USP. Icofungipen was dissolved in phosphate buffer as a stock solution of 5 mg/ml. All other substances were dissolved in N,N-dimethylformamide, and final working solutions were prepared in YNG medium.

Broth microdilution assay.

The highest concentration in the broth microdilution assay (96-well plates; Greiner, Germany) was 64 μg/ml, and twofold dilutions were used subsequently. The total volume was 100 μl/well incubated at the indicated temperature. Optical density was determined at 590 nm using a spectrophotometer (SpectraFluor Plus; Tecan, Switzerland). MICs were determined as the lowest concentration at which a prominent decrease in turbidity (score 2 according to CLSI standard M27-A [15]) was observed (fluconazole and icofungipen), while the MIC of amphotericin B was determined as a clear endpoint.

Tests were repeated at least five times on different days, using different preparations of the individual strains.

Animals.

Six-week-old male, specific-pathogen-free, CFW1 mice and male, specific-pathogen-free, Sprague-Dawley rats (200 g body weight) were obtained from Harlan Winkelmann, Paderborn, Germany. Male Sprague-Dawley rats, 200 g body weight, were obtained from Zentral Institut fuer Versuchstiere, Hannover, Germany. Animals were adjusted to the housing conditions for 1 week prior to use. Water and food were given ad libitum. There were five animals per treatment group unless otherwise stated.

Infection models.

The infection models are described in more detail elsewhere (27). Briefly, the inoculum for infection was prepared from 24-h cultures of isolate on malt extract agar slopes (Difco, Sparks, MD) at 28°C. Yeast cells were scraped off, diluted with phosphate buffer, and vortexed. The inoculum was adjusted by turbidometric analysis and confirmed by quantitative plating.

Infection was induced by injection of Candida suspension (3 × 10⁵ or 1 × 10⁶ CFU per mouse and 5 × 10⁶ CFU per rat) via the lateral tail vein. The animals were observed for signs of disease and mortality after infection twice daily for 7 to 40 days, depending on the experiment. Candida infection in mice affected predominantly kidneys that showed extensive granulomatous nephritis. Intravenous infection of rats led to a more disseminated infection involving kidneys, lung, heart, brain, and liver, resulting in mortality within 5 to 7 days. Following an observation period, surviving animals were sacrificed by inhalative exposure to CO₂.

Various C. albicans strains were checked for pathogenicity in mice. Strains causing high mortality of 80 to 100% after intravenous injection at 1 × 10⁶ or 3 × 10⁵ were used. Survival curves (Kaplan-Meier plots) were generated using the GraphPad software package Prism, California.

Efficacy of icofungipen in systemic infection with FLC-resistant yeast.

Clinical isolates were obtained from various hospitals in Germany and were tested in vitro for susceptibility to FLC, as described above. Strains exhibiting an MIC toward FLC of ≤4 mg/liter were considered susceptible, whereas those with an MIC of ≥64 mg/liter were classified as resistant. Subsequently, strains were tested for pathogenicity in mice. About 75% of all strains caused a lethal infection in the model, and no differences (ratio of pathogenic versus nonpathogenic strains) were observed between FLC-sensitive and FLC-resistant strains. In total, 27 FLC-susceptible and 27 FLC-resistant strains causing 80 to 100% mortality in the systemic mouse infection model were identified.

Treatment.

Icofungipen, free base or hydrochloride salt, was dissolved in a solution containing 5% glucose and 0.2% agar (Sigma, St. Louis, MO). It was given orally twice daily (b.i.d.) by gavage at various doses and time intervals. Unless otherwise stated, treatment was initiated 30 min after infection. Control animals received vehicle.

Statistical analysis.

Statistical analysis was performed using GraphPad Prism Software version 4.0 (Graph Pad Software Inc., San Diego, CA) as indicated in the individual figures.

Parameters influencing in vitro susceptibility testing. (i) Influence of growth medium composition.

Results of repeated testing of three C. albicans strains in seven different growth media are shown in Table 1. The standard microdilution broth method was used, with the lowest possible inoculum still growing sufficiently in the drug-free control well (50 to 100 CFU/well). Testing in RPMI 1640 medium in these experiments was done according to the CLSI guideline M27-A (15). MICs for amphotericin B and FLC were within the recommended CLSI quality control range of 0.5 to 2 and 0.25 to 1 μg/ml, respectively.

Several complex (YPD, LB, YPG, Sabouraud, and RPMI 1640) and two chemically defined (YNGW and YNG) media were tested. Under these experimental conditions, only testing in YPG medium increased MICs for amphotericin B above those in RPMI 1640 medium. Icofungipen showed relatively high MICs (16 to >64 μg/ml) after 24 versus 48 h in all complex media, regardless of the read-out time. However, MICs for icofungipen were significantly lower if chemically defined media (YNGW and YNG) were used.

For further testing, therefore, YNG was chosen. We also tested whether MICs were altered by branched-chain amino acids (isoleucine, leucine, and valine) known to interfere with the uptake of icofungipen (31, 32). MICs increased in the presence of isoleucine, leucine, and valine (Table 2), particularly with isoleucine, which increased the mean MIC after 24 h from 2 to 256 μg/ml.

(ii) Influence of pH.

Susceptibility testing at different pH values was studied repeatedly on three different C. albicans strains in YNG medium. Strains were chosen on the basis of their susceptibility to icofungipen in YNG medium at pH 7 and differed in their susceptibilities at 24 h. C. albicans PSCF 0440 was the most sensitive strain (MIC, 0.5 to 4 μg/ml), ATCC 90028 showed moderate susceptibility (MIC, 4 to 8 μg/ml), and PSCF 0085 had the highest MICs (8 to 64 μg/ml). Incremental changes of 0.5 pH units of the YNG medium between 4.0 and 8.0 did not significantly influence the in vitro activity of standard antifungal compounds (data not shown). Apart from an increase in MIC at pH <5 or pH ≥7 after 48 h of incubation (about two- to fourfold), no significant differences were found for icofungipen. Testing was therefore performed at pH 6 to 7 after 24 h of incubation.

(iii) Influence of inoculum size.

Inoculum sizes of three different strains of C. albicans influenced the MICs of both icofungipen and several azole standards (Table 3) in the same way, irrespective of the method of inoculum preparation (stationary, logarithmic liquid, or plate culture) (data not shown). As expected, sensitivity to amphotericin B was not affected while FLC showed lower susceptibility with increasing inoculum size. Starting at 200 CFU/well and becoming pronounced at >1,000 CFU/well, sensitivity toward icofungipen was markedly reduced with increasing inoculum size. Slight differences were observed between the three strains tested. For example, C. albicans PSCF 0085 was more sensitive to changes in inoculum size than the other strains. Based on these findings, the inoculum size used for further testing was limited to 50 to 100 CFU/well.

(iv) Influence of temperature of incubation.

No difference was observed in growth of yeast either in the absence or in the presence of any antifungal substance between 30 and 37°C (data not shown).

In vitro testing of icofungipen against clinical isolates of C. albicans.

Sixty-nine C. albicans strains collected from patients with cutaneous and/or mucocutaneous infections were included in the study to analyze the MIC distribution for clinical isolates. MIC testing (n = 10 per group) was done by broth microdilution assay using YNG medium at pH 7, an inoculum size of 50 to 100 CFU/ml, and 24 h of incubation. Results for icofungipen were compared with those for amphotericin B and clotrimazole, the most active azole. Ninety percent of the strains of C. albicans showed susceptibility to icofungipen with MICs ranging from 8 to 32 μg/ml, giving an MIC₉₀ of 32 μg/ml. The total range of MICs in this set of strains varied between 4 and >64 μg/ml. MIC₉₀ values for amphotericin B and clotrimazole were 1 μg/ml and 0.5 μg/ml, respectively (Table 4).

In vivo efficacy of icofungipen in C. albicans infection in mice.

A typical experiment demonstrating the efficacy of icofungipen in the mouse model is summarized in Fig. 1. Mice infected with C. albicans strain BSMY 212 (ATCC 200498) succumbed to their infection within 7 days. A dose-dependent protection was observed, with icofungipen giving 100% protection at 10 mg/kg of body weight b.i.d.

We analyzed the efficacy of the compound in vivo against a variety of clinical isolates obtained from different clinical centers, for which MICs ranged between 0.5 and 32 mg/liter. After identification to the species level, C. albicans strains were tested for pathogenicity and strains showing 80 to 100% mortality in the infection model were used in treatment studies. In a first series of infection experiments, all strains were exposed to 5 mg/kg icofungipen b.i.d. (10 animals per strain and dose); 38% of the strains (n = 34 isolates) were highly susceptible, i.e., survival was >80% (Table 5). In a second series, strains with lower survival rates of less than 80% (n = 55) at 5 mg/kg b.i.d. were exposed to a higher dose of 10 mg/kg b.i.d. All strains responded to therapy, with 75% of strains achieving more than 80% survival. There was no correlation with the in vitro susceptibility.

Subsequently, the in vivo efficacy of icofungipen against FLC-susceptible and FLC-resistant C. albicans strains was tested according to CLSI guidelines. Two groups were established, one consisting of strains with an MIC₈₀ of ≤4 mg/liter and the other with an MIC₈₀ of ≥64 mg/liter. There was no correlation between the MICs for FLC versus those for icofungipen. The mean MICs for icofungipen were broadly distributed between 1 and 32 mg/liter (data not shown). Subsequently, these strains were tested for pathogenicity in mice (each strain was tested in groups of five mice); only strains which gave 80 to 100% mortality were used in the subsequent efficacy trials. Pathogenic strains were tested in a group of 15 mice, 5 per treatment cohort (control, FLC, and icofungipen). Treatment was performed orally twice daily over 4 days. FLC was given at dose of 2 mg/kg/day and icofungipen at 20 mg/kg/day. At these dose levels in vitro, FLC-resistant and -susceptible strains could be clearly separated.

A total of 54 strains, 27 FLC-susceptible and 27 FLC-resistant strains, were used in the experiments. As can be seen from the Kaplan-Meier plot in Fig. 2, the cumulative mortality in the control group was almost 100%, regardless of the susceptibility of the causative pathogen to FLC. FLC was highly effective against susceptible strains but did not decrease mortality above that in vehicle controls when tested against strains with an MIC of ≥64 mg/liter. In contrast, icofungipen was effective against both FLC-susceptible and FLC-resistant C. albicans strains.

In vivo efficacy of icofungipen in C. albicans infection in rats.

To demonstrate efficacy in rats, male Sprague-Dawley rats (n = 5) were infected by intravenous administration of 5 × 10⁶ CFU per animal from a freshly prepared 24-h C. albicans culture. In this experiment, lethality was achieved within 10 days at 100%. Oral administration of icofungipen to rats (n = 10/group) at a dose of 5 mg/kg b.i.d. over 5 days resulted in 100% survival over a period of 40 days. At a dose of 2.5 mg/kg b.i.d., 80% survival was achieved (Fig. 3).

The experiments were repeated with four different C. albicans strains (ATCC 10261, 44373, 44505, and 62342). All strains led to a lethal infection under the experimental conditions within 3 to 10 days, whereas oral treatment with icofungipen for 10 mg/kg b.i.d. (five rats/group) resulted in 100% survival over 10 days (data not shown).

A single oral icofungipen dose, given simultaneously with infection, was also effective in preventing mortality. As can be seen from Fig. 4, dose-dependent protection was observed, achieving 100% survival at 10 mg/kg.

We subsequently analyzed the protective effect when initiating treatment 24 h after the infection. For this purpose, rats (n = 5/group) were challenged on day 0 and icofungipen was given at 1, 5, and 10 mg/kg b.i.d. over 5 days, starting on day 1. All animals in the control group died within 10 days, while at a dose of 10 mg/kg b.i.d., all icofungipen-treated animals survived during the 20-day observation period; even at the lowest dose of 1 mg/kg b.i.d., more than half of the animals survived (data not shown).

Influence of isoleucine dosing on efficacy of icofungipen.

As demonstrated previously, the inhibition by icofungipen of isoleucyl-tRNA synthetase can be antagonized in vitro by the addition of l-isoleucine (29, 31). To test whether concomitant administration of l-isoleucine and icofungipen would antagonize the in vivo efficacy of icofungipen, l-isoleucine in equimolar mixtures with icofungipen was tested in the lethal model of C. albicans infection in mice and rats (10 animals/group). As can be seen from Fig. 5 and 6, no significant antagonism could be detected at any dose.