ABSTRACT
E1210 is a first-in-class, broad-spectrum antifungal with a novel mechanism of action—inhibition of fungal glycosylphosphatidylinositol biosynthesis. In this study, the efficacies of E1210 and reference antifungals were evaluated in murine models of oropharyngeal and disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis. Oral E1210 demonstrated dose-dependent efficacy in infections caused by Candida species, Aspergillus spp., and Fusarium solani. In the treatment of oropharyngeal candidiasis, E1210 and fluconazole each caused a significantly greater reduction in the number of oral CFU than the control treatment (P < 0.05). In the disseminated candidiasis model, mice treated with E1210, fluconazole, caspofungin, or liposomal amphotericin B showed significantly higher survival rates than the control mice (P < 0.05). E1210 was also highly effective in treating disseminated candidiasis caused by azole-resistant Candida albicans or Candida tropicalis. A 24-h delay in treatment onset minimally affected the efficacy outcome of E1210 in the treatment of disseminated candidiasis. In the Aspergillus flavus pulmonary aspergillosis model, mice treated with E1210, voriconazole, or caspofungin showed significantly higher survival rates than the control mice (P < 0.05). E1210 was also effective in the treatment of Aspergillus fumigatus pulmonary aspergillosis. In contrast to many antifungals, E1210 was also effective against disseminated fusariosis caused by F. solani. In conclusion, E1210 demonstrated consistent efficacy in murine models of oropharyngeal and disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis. These data suggest that further studies to determine E1210's potential for the treatment of disseminated fungal infections are indicated.
INTRODUCTION
The expanding population of immunocompromised patients receiving immunosuppressive or anticancer therapy has resulted in an increased incidence of opportunistic mycoses. Invasive fungal infections have become increasingly common among such immunocompromised and immunosuppressed patients, including solid-organ and hematopoietic stem cell transplant recipients and individuals on immunosuppressive drug regimens (3, 17, 19, 30, 32, 45). In response, the previously established guidelines for the treatment of invasive fungal infections have been recently updated (31, 43). There is still, however, a high rate of morbidity and mortality associated with invasive fungal infections (3, 17, 20, 45), because the currently available antifungal drugs, such as polyenes, azoles, and echinocandins, are limited in terms of their antifungal spectrum, side effects, and mode of action (7). In addition, there has been an increase in resistance to commonly used antifungal compounds, especially azoles, and an epidemiological shift toward more drug-resistant strains (19, 22, 33, 34, 35, 42). Thus, there is a critical need for new antifungal compounds with a novel mechanism of action that have a broad spectrum of activity and fewer side effects, although the development of such new antifungals is just starting to be reported (18, 23, 26).
With this in mind, we have directed our research toward the development of new promising antifungals with a novel mechanism of action. We have discovered a key compound, 1-(4-butylbenzyl) isoquinoline (BIQ) that inhibits the surface expression of glycosylphosphatidylinositol (GPI)-anchored proteins in Saccharomyces cerevisiae, resulting in inhibition of fungal growth, and then identified the GWT1 (GPI-anchored wall protein transfer 1) gene, the target molecule of BIQ, which encodes a new acyltransferase involved in an early step in the GPI biosynthetic pathway of fungi (39, 40). We have performed exploratory syntheses of many different compounds designed to enhance the antifungal activity of BIQ and finally discovered a candidate compound likely to be effective as a new antifungal (27, 38).
E1210, 3-(3-{4-[(pyridin-2-yloxy)methyl]benzyl}isoxazol-5-yl)pyridin-2-amine (Fig. 1), is a first-in-class, new antifungal compound that was discovered by the Tsukuba Research Laboratories of Eisai Co., Ltd. (Ibaraki, Japan). It has potent broad-spectrum antifungal activity with a novel mechanism of action, i.e., inhibition of fungal GPI biosynthesis, and favorable properties as a drug candidate (13, 24, 25, 29, 44). In the present study, the efficacies of oral E1210 and reference antifungal drugs, such as fluconazole (36), voriconazole (2), caspofungin (1), and liposomal amphotericin B (8), were evaluated in murine models of candidiasis, aspergillosis, and fusariosis. In addition, the initial pharmacokinetic and in vivo toxicological profiles of E1210 were also demonstrated.
Chemical structure of E1210.
(This work was presented in part at the 50th Interscience Conference on Antimicrobial Agents and Chemotherapy, abstracts F1-842 [12a] and F1-844 [29], Boston, MA, 12 to 15 September 2010.)
MATERIALS AND METHODS
Antifungals.E1210 was synthesized at Eisai Co. Ltd., Tokyo, Japan. The negative logarithm of the dissociation constant (pKa) of E1210 was determined by capillary electrophoresis. The pKa values for the conjugate acid of E1210 were 3 and 5.1 (I [ionic strength] = 0.05). The hydrophobicity (LC18; apparent log P determined by high-performance liquid chromatography [HPLC]) of E1210 at neutral pH was 3.48. Fluconazole and voriconazole were extracted at Eisai Co. Ltd. from commercial products obtained from Pfizer, Inc. (Tokyo, Japan). Caspofungin, amphotericin B, and liposomal amphotericin B were commercially obtained from Merck & Co., Inc. (Whitehouse Station, NJ), Bristol-Myers KK (Tokyo, Japan), and Dainippon Sumitomo Pharma Co., Ltd. (Osaka, Japan), respectively. All drugs were dissolved individually in dimethyl sulfoxide (DMSO) and then diluted with culture medium at required concentrations for in vitro studies. For in vivo efficacy studies in mice, E1210 was dissolved in 250 mmol/liter HCl at a concentration of 25 mg/ml and then diluted with vehicle to the required concentrations. For in vivo toxicological studies in rats, E1210 was dissolved in 400 mmol/liter HCl at a concentration of 100 mg/ml and then diluted with 400 mmol/liter HCl to the required concentrations. Voriconazole was dissolved in 1 mol/liter HCl to a concentration of 20 mg/ml and then diluted with vehicle to the required concentrations. The dosing formulations of E1210 and voriconazole were prepared and stored in a −40°C freezer until use. Other drugs were prepared on the day of use according to the manufacturer's specifications.
Organisms.In total, the following six fungal strains were used for these studies: Candida albicans IFM49971, Candida albicans IFM49738, Candida tropicalis E83037, Aspergillus flavus IFM50915, Aspergillus fumigatus IFM51126, and Fusarium solani IFM50956. These strains were provided by Chiba University (Chiba, Japan) and Gifu University (Gifu, Japan) and were stored as glycerin stock at −80°C.
Animals.Specific-pathogen-free female ICR mice (age, 5 weeks; weight, approximately 25 g; Charles River Japan Inc., Kanagawa, Japan), specific-pathogen-free female DBA/2N mice (age, 8 weeks; weight, approximately 18 g; Charles River Japan Inc., Kanagawa, Japan), or specific-pathogen-free male and female Sprague-Dawley rats (age, 8 weeks; weight, approximately 190 to 270 g; Charles River Japan Inc., Kanagawa, Japan) were used for these experiments. They were housed in cages of 5 to 10 animals per group and had access to food and water ad libitum. All procedures were performed in an animal facility accredited by the Center for Accreditation of Laboratory Animal Care and Use by the Japan Health Sciences Foundation. All protocols were approved by the Institutional Animal Care and Use Committee and carried out according to Eisai animal experimentation regulations.
In vitro susceptibility testing.The MICs of E1210 and the reference compounds were determined using the broth microdilution method detailed by the Clinical and Laboratory Standards Institute (CLSI) in documents M27-A3 (6) and M38-A2 (5). RPMI 1640 medium buffered to pH 7.0 with 0.165 M 3-(N-morpholino)-propanesulfonic acid (MOPS) was used. The results were expressed as the median MIC of each compound derived from three independent experiments.
The Candida spp. were subcultured in Sabouraud dextrose broth (SDB) at 35°C for 1 to 2 days. The Aspergillus spp. were subcultured onto potato dextrose agar (PDA) and then incubated at 35°C for 1 to 2 weeks. F. solani was subcultured onto PDA and incubated at 35°C for 2 to 3 days and then at 25°C for 4 to 5 days. The conidia were scraped from the PDA surface and suspended in sterile normal saline containing 0.05% Tween 80. The cell counts from the yeast cultures or conidial suspensions were determined with a hemocytometer by a modification of the CLSI method, and the cell suspensions were diluted with RPMI 1640 medium buffered to pH 7.0 with 0.165 M MOPS to obtain an inoculum size of 1.5 × 103 cells/ml for Candida spp. or 1.2 × 104 cells/ml for the filamentous fungi. The test organisms were cultured in medium containing E1210 (0.001 to 32 μg/ml), fluconazole (0.001 to 32 μg/ml), voriconazole (0.001 to 32 μg/ml), caspofungin (0.0005 to 16 μg/ml), amphotericin B (0.016 to 8 μg/ml), or 0.5% DMSO, and the growth inhibition induced by the test compounds was evaluated. For all the test compounds except caspofungin, the plates were incubated under the following conditions: at 35°C for 22 to 26 h for C. albicans and C. tropicalis and at 35°C for 46 to 50 h for the filamentous fungi. For caspofungin, the plates were incubated at 35°C for 22 to 26 h for all test strains.
For Candida spp., the reduction in growth was determined based on the changes in optical density of the medium at 660 nm (using a MTP-450 microplate reader; Corona Electric Co., Ltd., Ibaraki, Japan). The MICs of E1210, fluconazole, voriconazole, and caspofungin were defined as the lowest concentrations resulting in a prominent decrease in turbidity (that is, a 50% reduction in growth determined spectrophotometrically) relative to that in a control well by a modification of the CLSI method. The MICs of amphotericin B were defined as the lowest concentration resulting in complete growth inhibition determined visually.
For the filamentous fungi, the growth reductions were graded visually and expressed as a numerical score, ranging from 0 to 4, in accordance with the CLSI document for filamentous fungi (5). The MICs of amphotericin B and voriconazole were defined as the lowest concentration at which a score of 0 was observed, while those of E1210, fluconazole, and caspofungin were defined as the lowest concentration at which a score of 2 was observed. The MICs of caspofungin against the filamentous fungi corresponded to the minimal effective concentrations (MECs) defined in the CLSI guidelines (5).
Oropharyngeal candidiasis model.C. albicans was used to infect mice that were immunosuppressed with cortisone, and the number of C. albicans cells in the oral cavity of each mouse was measured following drug treatment (16, 37). ICR mice were immunosuppressed using 4 mg of subcutaneously administered cortisone acetate given 1 day before and 3 days after infection. The mice were also given 1 mg/ml tetracycline hydrochloride via their drinking water, starting on the day of cortisone administration and continuing throughout the experiment, in order to prevent bacterial infection. C. albicans IFM49971 was grown on Sabouraud dextrose agar (SDA) at 35°C for 2 days. The cells were suspended in sterile normal saline. The cells were counted with a hemocytometer and adjusted to the required density with sterile normal saline. The mice were then anesthetized with chlorpromazine hydrochloride (0.5 mg/mouse given subcutaneously). By use of a micropipette, aliquots (10 μl) of C. albicans IFM49971 suspension were inoculated into the oral cavities of the anesthetized mice. Then the challenge dose of 4 × 105 CFU of C. albicans (CFU)/mouse was given. This was followed with either E1210 orally administered twice daily (BID) or fluconazole orally administered once daily (QD) for three consecutive days starting 3 days after infection. The control group was given the equivalent volume of 5% glucose BID. The mice were anesthetized with chlorpromazine hydrochloride (0.5 mg/mouse subcutaneously) the day after the final dose of the study drug. Efficacy was assessed by determination of the number of C. albicans cells in the oral cavity of each mouse after study drug treatment. The oral cavity (that is, the cheek, tongue, and soft palate) was thoroughly swabbed using a fine-tipped cotton swab. After swabbing, the cotton end was placed into a test tube containing 1 ml sterile normal saline. The cells recovered were suspended in sterile normal saline by mixing them on a vortex mixer before being cultured, after serial 10-fold dilutions, on SDA plates supplemented with ampicillin (0.1 mg/ml). The SDA plates were incubated at 35°C overnight, and then the viable cells were counted as the number of CFU. The cell number was expressed in units of log10 CFU/swab. The lowest detectable number of cells in the oral cavity was 10 CFU (1 log10 CFU). The viable cell counts were performed in duplicate.
Disseminated candidiasis model.ICR mice were immunosuppressed utilizing 5-fluorouracil (5-FU) at 200 mg/kg of body weight subcutaneously administered 6 days prior to infection. These mice were also administered 0.1 mg/ml ciprofloxacin orally via their drinking water, from 2 to 3 days prior to infection to 5 to 7 days after infection, in order to prevent endogenous bacterial infections. C. albicans IFM49971, C. albicans IFM49738, and C. tropicalis E83037 were each cultured on an SDA plate at 35°C for 2 days. The cells from the surface of the agar plate were suspended in sterile normal saline, and the cells were counted with a hemocytometer. The final inoculum was adjusted to the required density using sterile normal saline. Infection was induced in the neutropenic mice by the intravenous administration of 0.2 ml of a C. albicans cell suspension (0.8 to 1.4 × 104 CFU/mouse or 5.3 × 104 CFU/mouse for IFM49971) or of a C. tropicalis cell suspension (3.0 × 105 CFU/mouse) injected into the lateral tail vein. Antifungal therapy was initiated 1 h or 24 h after infection and was continued for three consecutive days (days 0 to 2 or 1 to 3). E1210 or voriconazole was each orally administered two or three times daily, fluconazole was orally administered once daily, and caspofungin or liposomal amphotericin B was intravenously administered once daily. The control group received an equivalent volume of vehicle (5% glucose, 10 ml/kg) orally two or three times daily. In our preliminary studies, the survival curve of control mice receiving vehicle orally was similar to that of mice receiving vehicle intravenously. Therefore, we did not set up the control group to receive vehicle intravenously. The survival rate and survival period were determined over 14 days.
Pulmonary aspergillosis model.DBA/2N mice were immunosuppressed with subcutaneously administered 5-FU at 200 mg/kg, 5 to 6 days prior to infection. The mice were also administered 0.1 mg/ml ciprofloxacin orally via their drinking water, from 3 to 4 days prior to infection until 7 days after infection, in order to prevent endogenous bacterial infections. A. flavus IFM50915 and A. fumigatus IFM51126 were cultured on a PDA plate at 35°C for 7 days. The conidia from the surface of the agar plate were suspended in sterile normal saline containing 0.05% Tween 80, and the cells were counted with a hemocytometer. The final inoculum was adjusted to the required density with sterile normal saline containing 0.05% Tween 80. The mice were anesthetized with 0.1 ml ketamine hydrochloride (4.17 mg/ml) intravenously. Infection was induced in these neutropenic mice by the intranasal inoculation of 0.05 ml of an A. flavus conidial suspension (3.0 × 104 conidia/mouse) or 0.05 ml of an A. fumigatus conidial suspension (6.0 × 104 conidia/mouse). Antifungal therapy was initiated 1 h after infection and was continued for four or seven consecutive days (days 0 to 3 or 0 to 7). E1210 or voriconazole was administered orally twice daily, and caspofungin or liposomal amphotericin B was administered intraperitoneally once daily. The control group received an equivalent volume of vehicle (5% glucose, 10 ml/kg) orally twice daily. In our preliminary studies, the survival curve of control mice receiving vehicle orally was similar to that of mice receiving vehicle intraperitoneally. Therefore, we did not set up the control group to receive vehicle intraperitoneally. The survival rate and survival period were determined over 14 days.
Disseminated fusariosis model.DBA/2N mice were immunosuppressed with 200 mg/kg of subcutaneously administered 5-FU, 6 days prior to infection. The mice were also administered 0.1 mg/ml ciprofloxacin orally in their drinking water, from 3 days prior to infection until 7 days after infection, to prevent bacterial infections. F. solani IFM50956 was cultured on a PDA plate at 30°C for 7 days. The cells from the surface of the agar plate were suspended in sterile normal saline containing 0.05% Tween 80, and the cells were counted using a hemocytometer. The final inoculum was adjusted to the required density using sterile normal saline containing 0.05% Tween 80. Infection was induced in the neutropenic mice by the intravenous inoculation of a 0.2-ml F. solani cell suspension (5.0 × 103 cells/mouse) into the lateral tail vein. Antifungal therapy was initiated 1 h after infection and was continued for five consecutive days (days 0 to 4). E1210 was orally administered three times a day (TID). The control group received an equivalent volume of 5% glucose orally TID. The survival rate and survival period were determined over 14 days.
Pharmacokinetic study.E1210 was intravenously or orally administered to male ICR mice. After administration of E1210, blood samples were drawn from the vena cava of each mouse at designated time points (0.08, 0.25, 0.5, 1, 2, 4, 6, 8 h). Plasma samples were obtained by centrifuging blood. After deproteinization with methanol, the extracted sample was analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). The concentrations of E1210 in plasma were determined by an internal standard method using MassLynx (Waters, Milford, MA). The pharmacokinetic parameters of E1210 were calculated by model independent analysis.
Toxicology study.E1210 was administered orally by gavage once a day for 7 days to male and female Sprague-Dawley rats (3 animals/group/gender) at doses of 100, 300, or 1,000 mg/kg. A control group received an equivalent volume (10 ml/kg) of vehicle (0.4 mol/liter hydrochloric acid). All rats found dead or moribund were necropsied as soon as they were discovered, and all surviving animals were necropsied after 7 days of administration. The following were evaluated: mortality, clinical signs, body weight, food consumption, hematology, blood chemistry, toxicokinetics, hepatic drug-metabolizing enzymes, and macroscopic and microscopic pathologies.
Statistical analysis.In the oropharyngeal candidiasis model, data are expressed as the mean ± standard error of the mean (SEM). The differences in the viable cell counts between the control group and the E1210-treated groups or the fluconazole-treated groups were evaluated using one-way analysis of variance (ANOVA), followed by the Dunnett multiple-comparison test. The dose responsiveness of E1210 or fluconazole was determined using regression analysis.
The differences between the survival curves of the vehicle-treated (control) and antifungal-treated groups over 14 days postinfection were analyzed by the log rank test with the Bonferroni adjustment. Additionally, based on the survival rate at day 14 after infection, the 50% effective dose (ED50) and 95% confidence interval (CI) of each antifungal were estimated with a probit method. For the antifungals for which the 95% CIs were not calculated by a probit method, the 95% CIs of the ED50s were calculated based on the exact 95% confidence limits of the survival rate at each dose. Statistical analyses were performed with SAS version 8.2 software package (SAS Institute Japan Ltd., Tokyo, Japan). A probability (P) value of <0.05 (two-sided) was considered statistically significant.
RESULTS
In vitro antifungal activity.Table 1 shows the MICs of E1210 and reference comparator antifungal compounds against Candida spp., Aspergillus spp., and F. solani used in the in vivo efficacy studies of E1210. E1210 showed potent antifungal activity against C. albicans IFM49971 (MIC = 0.004 μg/ml). E1210 was more active than fluconazole, caspofungin, and amphotericin B and showed activity similar to that of voriconazole against C. albicans IFM49971. E1210 also had potent activity against both azole-resistant Candida strains, C. albicans IFM49738 (MIC = 0.008 μg/ml) and C. tropicalis E83037 (MIC = 0.016 μg/ml); E1210 was more active than all of the reference antifungals tested. E1210 showed potent antifungal activity against A. flavus IFM50915 (MIC = 0.03 μg/ml), A. fumigatus IFM51126 (MIC = 0.03 μg/ml), and F. solani IFM50596 (MIC = 0.06 μg/ml); E1210 was the most active compound tested against these strains of filamentous fungi.
Comparative in vitro antifungal susceptibilities of experimental infection strains to E1210 and reference compoundsa
Efficacy in the oropharyngeal candidiasis model.The efficacy of orally administered E1210 compared to that of fluconazole for the treatment of C. albicans-induced oral candidiasis is shown in Fig. 2. In this model, the control group showed a viable oral cavity cell count of 5.62 ± 0.11 log10 CFU. The oral cavity cell counts in mice treated with E1210 (BID) doses of 2.5, 5 and 10 mg/kg were 4.97 ± 0.16, 4.24 ± 0.31, and 3.08 ± 0.27 log10 CFU, respectively. The oral cavity cell counts in mice treated with doses of fluconazole (once daily [QD]) of 2.5, 5, and 10 mg/kg were 5.28 ± 0.18, 3.88 ± 0.23, and 2.75 ± 0.24 log10 CFU, respectively. Mice treated with E1210 or fluconazole at doses of 5 and 10 mg/kg showed significantly better resolution of oral candidiasis than control mice. E1210 or fluconazole reduced the number of viable C. albicans cells in the oral cavity in a dose-dependent manner.
Comparative efficacies of E1210 and fluconazole in a murine oropharyngeal candidiasis model. Mice, immunosuppressed with cortisone acetate administered subcutaneously 1 day before and 3 days after infection, were orally infected with 4 × 105 CFU of Candida albicans. E1210 was orally administered twice daily and fluconazole was orally administered once daily for three consecutive days starting 3 days after infection. The oral cavity of each mouse was thoroughly swabbed, and the recovered cells were cultured on SDA plates. After incubation at 35°C overnight, the viable cells were counted and expressed as the number of CFU. The viable cell counts were performed in duplicate. *, P < 0.05 versus control (one-way ANOVA with the Dunnett multiple-comparison test).
Efficacy in the disseminated candidiasis model.The efficacies of E1210, fluconazole, caspofungin, and liposomal amphotericin B on C. albicans-induced mortality in mice are shown in Fig. 3. In this model, all control mice died within 5 days. The mice treated with E1210 (BID) at 5 and 12.5 mg/kg, with fluconazole (QD) at 2 and 5 mg/kg, with caspofungin (QD) at 0.064 and 0.16 mg/kg, and with liposomal amphotericin B (QD) at 0.4 and 1 mg/kg (as amphotericin B) showed significantly higher survival rates than control mice. E1210 at doses of 0.8, 2, 5,and 12.5 mg/kg protected 0%, 11%, 56%, and 89% of the mice at day 14, respectively, and its ED50 was 4.8 mg/kg (95% CI, 3.0 to 7.9 mg/kg). Fluconazole at doses of 0.32, 0.8, 2, and 5 mg/kg protected 0%, 0%, 67%, and 100% of the mice at day 14, respectively, and its ED50 was 1.9 mg/kg (95% CI, 0.80 to 5.0 mg/kg). Caspofungin at doses of 0.01, 0.026, 0.064, and 0.16 mg/kg protected 0%, 11%, 100%, and 100% of the mice at day 14, respectively, and its ED50 was 0.030 mg/kg (95% CI, 0.026 to 0.064 mg/kg). Liposomal amphotericin B at doses of 0.064, 0.16, 0.4, and 1 mg/kg protected 0%, 22%, 56%, and 100% of the mice at day 14, respectively, and its ED50 was 0.31 mg/kg (95% CI, 0.20 to 0.49 mg/kg). The ED50s of all compounds tested in the disseminated candidiasis model are summarized in Table 2.
Efficacy of E1210 compared to that of reference antifungals in a murine model of disseminated candidiasis caused by C. albicans IFM49971. Mice (n = 9) were immunosuppressed with 5-FU administered subcutaneously 6 days prior to infection and then intravenously infected with 0.8 × 104 CFU of Candida albicans. E1210 was administered orally twice daily, fluconazole was administered orally once daily, and liposomal amphotericin B or caspofungin was administered intravenously once daily for three consecutive days starting 1 h after infection. The survival rate and survival period were determined over 14 days. (A) E1210; (B) fluconazole; (C) caspofungin; (D) liposomal amphotericin B. *, P < 0.05 versus control group (log rank test with Bonferroni's adjustment).
ED50s of E1210 and reference compounds based on day 14 survival ratesa
The efficacies of E1210 and voriconazole on azole-resistant C. albicans-induced mortality in mice are shown in Fig. 4. In this model, all of the control mice died within 5 days. The mice treated with E1210 (TID) at 2.5 mg/kg showed a significantly higher survival rate than the control mice. E1210 at doses of 0.313, 0.625, 1.25, 2.5, and 5 mg/kg protected 0%, 13%, 38%, 88%, and 100% of the mice at day 14, respectively, and its ED50 was 1.3 mg/kg (95% CI, 0.93 to 2.0 mg/kg). Voriconazole (TID) at a dose of 5 mg/kg protected 25% of the mice at day 14.
Efficacy of E1210 compared to that of voriconazole in a murine model of disseminated candidiasis caused by azole-resistant C. albicans IFM49738. Mice (n = 8) were immunosuppressed with 5-FU administered subcutaneously 6 days prior to infection and then intravenously infected with 5.3 × 104 CFU of Candida albicans. E1210 or voriconazole was administered orally three times daily for three consecutive days starting 1 h after infection. The survival rate and survival period were determined over 14 days. *, P < 0.05 versus control group (log rank test with Bonferroni's adjustment).
In addition, E1210 was effective in the model of disseminated candidiasis caused by C. tropicalis (data not shown). In this model, all of the control mice died within 4 days. The mice treated with E1210 (TID) at 2.5 mg/kg showed a significantly higher survival rate than the control mice. E1210 at doses of 1.25 and 2.5 mg/kg protected 20% and 100% of the mice at day 14, respectively.
The impact of E1210 administered orally for 3 consecutive days starting 1 h or 24 h after infection on C. albicans-induced mortality in mice is shown in Fig. 5. In this model, all of the control mice died within 5 days. The mice treated with E1210 (TID) at 2.5 mg/kg showed a significantly higher survival rate than the control mice regardless of the time that treatment was initiated. When therapy was started 1 h after infection, E1210 at doses of 0.625, 1.25, and 2.5 mg/kg protected 0%, 50%, and 100% of the mice at day 14, respectively, and its ED50 was 1.3 mg/kg (95% CI, 0.63 to 2.5 mg/kg). When therapy was started 24 h after infection, E1210 at doses of 0.625, 1.25, and 2.5 mg/kg protected 0%, 38%, and 75% of the mice at day 14, respectively, and its ED50 was 1.7 mg/kg (95% CI, 1.1 to 2.8 mg/kg). The efficacy of E1210 for the treatment of disseminated candidiasis was minimally affected by a treatment delay of 24 h.
Effect of treatment delay on the efficacy of E1210 in a murine model of disseminated candidiasis. Mice (n = 8) were immunosuppressed with 5-FU administered subcutaneously 6 days prior to infection and then intravenously infected with 1.4 × 104 CFU of C. albicans IFM49971. E1210 was administered orally three times daily for three consecutive days starting 1 h or 24 h after infection. The survival rate and survival period were determined over 14 days.
Efficacy in the pulmonary aspergillosis model.The efficacies of E1210, voriconazole, caspofungin, and liposomal amphotericin B on A. flavus-induced mortality in mice are shown in Fig. 6. In this model, all of the control mice died within 6 days. The mice treated with E1210 (BID) at 10 and 25 mg/kg, voriconazole (BID) at 4 and 10 mg/kg, and caspofungin (QD) at 0.4 and 1 mg/kg showed significantly higher survival rates than the control mice. E1210 at doses of 1.6, 4, 10, and 25 mg/kg protected 0%, 11%, 33%, and 100% of the mice at day 14, respectively, and its ED50 was 10 mg/kg (95% CI, 6.8 to 16 mg/kg). Voriconazole at doses of 0.64, 1.6, 4, and 10 mg/kg protected 0%, 11%, 44%, and 100% of the mice at day 14, respectively, and its ED50 was 3.7 mg/kg (95% CI, 2.5 to 5.9 mg/kg). Caspofungin at doses of 0.064, 0.16, 0.4, and 1 mg/kg protected 11%, 22%, 44%, and 78% of the mice at day 14, respectively, and its ED50 was 0.41 mg/kg (95% CI, 0.21 to 1.3 mg/kg). Liposomal amphotericin B (QD) at a dose of 10 mg/kg did not protect the mice against mortality. The ED50s of all compounds tested in the pulmonary aspergillosis model are summarized in Table 2.
Efficacies of E1210 and reference antifungals in a murine model of pulmonary aspergillosis caused by A. flavus IFM50915. Mice (n = 9) were immunosuppressed with 5-FU administered subcutaneously 5 days prior to infection and then intranasally infected with 3.0 × 104 of Aspergillus flavus conidia. E1210 or voriconazole was administered orally twice daily and caspofungin or liposomal amphotericin B was administered intraperitoneally once daily for four consecutive days starting 1 h after infection. The survival rate and survival period were determined over 14 days. (A) E1210; (B) voriconazole; (C) caspofungin; (D) liposomal amphotericin B. *, P < 0.05 versus control group (log rank test with Bonferroni's adjustment).
E1210 was also effective in treating pulmonary aspergillosis caused by A. fumigatus (data not shown). In this model, all of the control mice died within 6 days. The mice treated with E1210 (TID) at 10 and 20 mg/kg showed significantly higher survival rates than the control mice. E1210 at doses of 2.5, 5, 10, and 20 mg/kg protected 0%, 14%, 43%, and 100% of the mice at day 14, respectively, and its ED50 was 9.3 mg/kg (95% CI, 6.4 to 15 mg/kg).
Efficacy in the disseminated fusariosis model.The efficacy of oral E1210 on F. solani-induced mortality in mice is shown in Fig. 7. In this model, 80% of the control mice died within 6 days and 20% of the control mice survived at day 14. E1210 at doses of 5, 10, and 20 mg/kg protected 25%, 63%, and 100% of the mice at day 14, respectively. The mice treated with E1210 (TID) at 20 mg/kg showed significantly higher survival rates than the control mice.
Efficacy of E1210 in a murine model of disseminated fusariosis caused by F. solani IFM50956. Mice (n = 8–10) were immunosuppressed with 5-FU administered subcutaneously 6 days prior to infection and then intravenously infected with 5.0 × 103 of F. solani conidia. E1210 was administered orally three times daily for five consecutive days starting 1 h after infection. The survival rate and survival period were determined over 14 days. *, P < 0.05 versus control group (log rank test with Bonferroni's adjustment).
Pharmacokinetic profile.Mean plasma concentrations of E1210 after oral and intravenous administrations to mice are shown in Fig. 8. In mice, after intravenous administration, E1210 exhibited moderate clearance and volume of distribution and the elimination half-life was 2.2 h. E1210 dosed as an oral solution was rapidly absorbed and achieved a maximum concentration at 0.5 h after dosing. Oral bioavailability was calculated at 57.5% in mice.
Time-plasma concentration profiles of E1210 after oral and intravenous administrations of a single dose of 1 mg/kg to mice. After administration of E1210, blood samples were drawn from the vena cava of mice at designated time points. After deproteinization with methanol, the extracted sample was analyzed by LC/MS/MS. Each value represents the mean of the results for two animals.
Toxicological profile.At an E1210 dose of 1,000 mg/kg, mortality (1 male) and morbidity (1 male) were observed, which were caused by anorexia and gastrointestinal lesion. Adaptive hepatocellular hypertrophy resulting from liver enzyme induction was observed at 300 mg/kg and higher. No E1210-related change was observed at 100 mg/kg. E1210 was generally well tolerated when given orally to rats for 1 week at doses up to 300 mg/kg.
DISCUSSION
E1210 is a first-in-class, broad-spectrum antifungal with a novel mechanism of action—inhibition of fungal GPI biosynthesis. E1210 has a broad spectrum of potent activity against major pathogenic fungi, such as Candida spp., Aspergillus spp., and other filamentous fungi which are resistant to existing antifungals (24, 25). Invasive fungal infections, including disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis, are serious, life-threatening infections recognized increasingly more frequently in immunocompromised patients (17, 19, 28, 30, 45). Oropharyngeal candidiasis, the most commonly encountered opportunistic infection in human immunodeficiency virus-infected patients (10), is becoming increasingly more resistant to fluconazole and, soon, other azoles. We therefore developed experimental models of these fungal infections in immunosuppressed mice to evaluate the therapeutic efficacy of E1210. In our previous studies to evaluate the efficacy of ravuconazole, we showed that 5-fluorouracil treatment elicited a considerable reduction in the number of circulating leukocytes, especially neutrophils, in mice (41), and disseminated and pulmonary infections caused by Candida spp. or Aspergillus spp. were then readily induced in these mice (11, 12). The neutropenic disseminated or pulmonary infection model appears to more closely mimic the immunocompromised patient situation in clinical settings. Furthermore, a disseminated fusariosis model was also able to be established in these neutropenic mice, as part of this study. Oropharyngeal infection caused by C. albicans was able to be induced in cortisone acetate-treated mice.
In order to better evaluate the comparative efficacies of E1210 and reference antifungals, the experimental infection models were studied using various numbers of doses per day for each antifungal compound. It has been reported that the plasma half-lives of voriconazole (2), fluconazole (14), caspofungin (9), and liposomal amphotericin B (8) were 0.7 to 2.9 h, 5.1 h, 7.6 h, and 10.1 to 12.5 h, respectively, in mice. Therefore, the plasma half-life of E1210 (2.2 h) was noted to be similar to that of voriconazole and about two to five times shorter than those of the reference antifungals other than voriconazole. From these data, we determined the optimal frequency of dosing for E1210 and voriconazole to be BID or TID and those for other drugs to be QD for these murine models of infection. Thus, E1210 had a relatively shorter plasma half-life than those of the reference drugs, except for voriconazole, in mice. Also, the addition of serum (90%) greatly affected the MICs of E1210 for the strains tested; the MICs of E1210 increased 64-fold (24, 25). This result indicates that E1210 may have a high plasma protein binding ratio. For all these reasons, we expect that the higher doses of E1210 were needed for effective treatment in mouse models of infection. We are currently conducting further pharmacokinetic and metabolic studies for the future clinical development of E1210 in rats, dogs, and monkeys.
Orally administered E1210 demonstrated clear dose-dependent therapeutic responses in the various experimental infection models in mice. First, E1210 showed efficacy in treating oropharyngeal candidiasis in mice. Treatment with E1210 significantly reduced the number of Candida CFU in the oral cavity in comparison to that of the control treatment (P < 0.05), with the extent of eradication comparable to that of fluconazole. Second, E1210 increased the survival time in a dose-dependent manner in mice infected with Candida spp. or Aspergillus spp. E1210 was consistently effective in treating disseminated candidiasis caused by azole-susceptible C. albicans. Furthermore, E1210 had the additional benefit of an efficacy against azole-resistant C. albicans infections in mice comparable to that against azole-susceptible C. albicans infections. The frequency of clinical reports of azole-resistant C. albicans and azole-resistant Candida spp. other than C. albicans is increasing (22, 33, 35). E1210 proved to be effective against disseminated candidiasis caused by azole-resistant strains of C. albicans and C. tropicalis in mice. E1210 is further characterized by its efficacy against invasive pulmonary aspergillosis caused by A. flavus and A. fumigatus in mice. Invasive aspergillosis currently constitutes the most common cause of infectious pneumonic mortality in patients undergoing hematopoietic stem cell transplantation (HSCT) and is an important cause of opportunistic infection in other immunocompromised patients (3, 4, 17, 19, 30, 32). For primary treatment of invasive pulmonary aspergillosis, intravenous or oral voriconazole is recommended for most patients based on the Infectious Diseases Society of America's guideline published in 2008 (43). Interestingly, E1210 at doses of >20 mg/kg showed a maximum therapeutic efficacy (100% survival) that was comparable to that of voriconazole at 10 mg/kg in murine invasive pulmonary aspergillosis models. In addition, we confirmed that E1210 was effective against disseminated aspergillosis caused by A. fumigatus in mice (data not shown). In the future, it will be very important to evaluate the antifungal activity of E1210 against azole-resistant Aspergillus strains, which are increasingly being recognized in the clinic (34, 42).
From the viewpoint of antifungal spectrum of activity, one of the important characteristics of E1210 is its activity against non-Aspergillus filamentous fungi, such as F. solani and Scedosporium prolificans, which are intrinsically resistant to all currently approved or available antifungals (15, 21, 28). We therefore conclude that it will be very important to evaluate the in vivo efficacy of E1210 in animal models of infections attributable to these pathogens. We showed the efficacy of E1210 in a disseminated fusariosis model in this study and have already started to investigate the efficacy of E1210 in the same model for S. prolificans pulmonary infections.
Furthermore, E1210 proved to be effective in treating disseminated candidiasis, even if treatment was started 24 h after infection (Fig. 5). We also confirmed that E1210 was effective against disseminated aspergillosis caused by A. fumigatus even if treatment was started 24 h after infection in mice (data not shown). These results strongly suggest that E1210 has the potential for being effective if given during acute or subacute infections.
As a preliminary toxicological study, we have conducted a 7-day oral dose range toxicity study in rats. E1210 was generally well tolerated when given orally to rats for 1 week at doses up to 300 mg/kg. Further toxicological studies are needed to confirm the safety of E1210 in rats and other animals, such as dogs and monkeys.
In conclusion, these results suggest that E1210 has the potential for efficacy as a single-agent therapy in the treatment of oropharyngeal candidiasis, disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis. E1210 is thus a very promising drug for the treatment of fungal infections, and therefore, further studies on its pharmacokinetics/pharmacodynamics and toxicological characteristics are warranted.
ACKNOWLEDGMENTS
We thank Katsuhiko Kamei, Medical Mycology Research Center, Chiba University, Chiba, Japan, and Hiroshige Mikamo, Department of Infection Control and Prevention, Aichi Medical University, Aichi, Japan, for providing the fungal strains. We thank Takashi Owa, Eisai Product Creation Systems, Eisai Inc., NJ, for making constructive suggestions to the antifungal project team. We thank Frederick P. Duncanson, Eisai Product Creation Systems, Eisai Inc., NJ, for editing and proofreading this article.
FOOTNOTES
- Received 18 March 2011.
- Returned for modification 18 May 2011.
- Accepted 17 July 2011.
- Accepted manuscript posted online 25 July 2011.
- Copyright © 2011, American Society for Microbiology. All Rights Reserved.
