The Fungal Cyp51 Inhibitor VT-1129 Is Efficacious in an Experimental Model of Cryptococcal Meningitis.

Cryptococcal meningitis is a significant cause of morbidity and mortality in immunocompromised patients. VT-1129 is a novel fungus-specific Cyp51 inhibitor with potent in vitro activity against Cryptococcus species. Our objective was to evaluate the in vivo efficacy of VT-1129 against cryptococcal meningitis. Mice were inoculated intracranially with Cryptococcus neoformans Oral treatment with VT-1129, fluconazole, or placebo began 1 day later and continued for either 7 or 14 days, and brains and plasma were collected on day 8 or 15, 1 day after therapy ended, and the fungal burden was assessed. In the survival study, treatment continued until day 10 or day 28, after which mice were monitored off therapy until day 30 or day 60, respectively, to assess survival. The fungal burden was also assessed in the survival arm. VT-1129 plasma and brain concentrations were also measured. VT-1129 reached a significant maximal survival benefit (100%) at a dose of 20 mg/kg of body weight once daily. VT-1129 at doses of ≥0.3 mg/kg/day and each dose of fluconazole significantly reduced the brain tissue fungal burden compared to that in the control after both 7 and 14 days of dosing. The fungal burden was also undetectable in most mice treated with a dose of ≥3 mg/kg/day, even ≥20 days after dosing had stopped, in the survival arm. In contrast, rebounds in fungal burden were observed with fluconazole. These results are consistent with the VT-1129 concentrations, which remained elevated long after dosing had stopped. These data demonstrate the potential utility of VT-1129 to have a marked impact in the treatment of cryptococcal meningitis.

In Vivo Efficacy of VT-1129 against Experimental Cryptococcal Meningitis with the Use of a Loading Dose-Maintenance Dose Administration Strategy.

VT-1129 is a novel fungal enzyme-specific Cyp51 inhibitor with potent cryptococcal activity. Because of its long half-life (>6 days in mice) and our desire to quickly reach potent efficacy, we evaluated a VT-1129 loading dose-maintenance dose strategy against cryptococcal meningitis. VT-1129 plasma and brain pharmacokinetics were first studied in healthy mice, and these data were used to model loading dose-maintenance dose regimens to generate different steady-state concentrations. Mice were inoculated intracranially with Cryptococcus neoformans, and oral treatment began 1 day later. Treatment consisted of placebo or one of three VT-1129 loading dose-maintenance dose regimens, i.e., loading dose of 1, 3, or 30 mg/kg on day 1, followed by once-daily maintenance doses of 0.15, 0.5, or 5 mg/kg, respectively. In the fungal burden arm, therapy continued for 14 days and brains were collected on day 15 for fungal burden assessments. In the survival arm, treatment continued for 10 days, after which mice were monitored without therapy until day 30. VT-1129 plasma and brain concentrations were also measured. All VT-1129 doses significantly improved survival and reduced fungal burdens, compared to placebo. VT-1129 plasma and brain levels correlated with fungal burden reductions (R2 = 0.72 and R2 = 0.67, respectively), with a plasma concentration of 1 µg/ml yielding a reduction of ∼5 log10 CFU/g. With the highest loading dose-maintenance dose regimen, fungal burdens were undetectable in one-half of the mice in the fungal burden arm and in one-fourth of the mice in the survival arm, 20 days after the final dose. These data support a loading dose-maintenance dose strategy for quickly reaching highly efficacious VT-1129 concentrations for treating cryptococcal meningitis.

Modified release itraconazole amorphous solid dispersion to treat Aspergillus fumigatus: importance of the animal model selection.

Previously, modified release itraconazole in the form of a melt-extruded amorphous solid dispersion based on a pH dependent enteric polymer combined with hydrophilic additives (HME-ITZ), exhibited improved in vitro dissolution properties. These properties agreed with pharmacokinetic results in rats showing high and sustained itraconazole (ITZ) systemic levels. The objective of the present study was to better understand the best choice of rodent model for evaluating the pharmacokinetic and efficacy of this orally administered modified release ITZ dosage form against invasive Aspergillus fumigatus. A mouse model and a guinea pig model were investigated and compared to results previously published. In the mouse model, despite similar levels as previously reported values, plasma and lung levels were variable and fungal burden was not statistically different for placebo controls, HME-ITZ and Sporanox® (ITZ oral solution). This study demonstrated that the mouse model is a poor choice for studying modified release ITZ dosage forms based on pH dependent enteric polymers due to low fluid volume available for dissolution and low intestinal pH. To the contrary, guinea pig was a suitable model to evaluate modified release ITZ dosage forms. Indeed, a significant decrease in lung fungal burden as a result of high and sustained ITZ tissue levels was measured. Sufficiently high intestinal pH and fluids available for dissolution likely facilitated the dissolution process. Despite high ITZ tissue level, the primary therapeutic agent voriconazole exhibited an even more pronounced decrease in fungal burden due to its reported higher clinical efficacy specifically against Aspergillus fumigatus.

Effect of Antifungal Treatment in a Diet-Based Murine Model of Disseminated Candidiasis Acquired via the Gastrointestinal Tract.

Candida albicans, normally found as a commensal in the gut, is a major human fungal pathogen responsible for both mucosal and systemic infections in a wide variety of immunocompromised individuals, including cancer patients and organ transplant recipients. The gastrointestinal tract represents a major portal of entry for the establishment of disseminated candidiasis in many of these individuals. Here we report the development of a diet-based mouse model for disseminated candidiasis acquired via the gastrointestinal tract. Using this model, as well as an appropriate immunosuppression regimen, we demonstrate that dissemination of C. albicans from the gastrointestinal tract can result in mortality within 30 days postinfection. We also show a significant increase in fungal burden in systemic organs, but not gastrointestinal tract organs, upon immunosuppression. Importantly, we demonstrate that the administration of two widely used antifungals, fluconazole and caspofungin, either pre- or postimmunosuppression, significantly reduces fungal burdens. This model should prove to be of significant value for testing the ability of both established and experimental therapeutics to inhibit C. albicans dissemination from the gastrointestinal tract in an immunocompromised host as well as the subsequent mortality that can result from disseminated candidiasis.

Isavuconazole Is Effective for the Treatment of Experimental Cryptococcal Meningitis.

We evaluated the efficacy of isavuconazole against cryptococcal meningitis. Treatment with either oral isavuconazole (120 mg/kg and 240 mg/kg twice a day [BID]) or fluconazole as the positive control significantly improved survival in mice infected intracranially with either Cryptococcus neoformans USC1597 or H99 and significantly reduced brain fungal burdens for both isolates. Concentrations of isavuconazole in plasma and brain tissue also demonstrated that the greatest improvements in survival and fungal burden were associated with elevated exposures.

Comparison of Nonculture Blood-Based Tests for Diagnosing Invasive Aspergillosis in an Animal Model.

The EuropeanAspergillusPCR Initiative (EAPCRI) has provided recommendations for the PCR testing of whole blood (WB) and serum/plasma. It is important to test these recommended protocols on nonsimulated "in vivo" specimens before full clinical evaluation. The testing of an animal model of invasive aspergillosis (IA) overcomes the low incidence of disease and provides experimental design and control that is not possible in the clinical setting. Inadequate performance of the recommended protocols at this stage would require reassessment of methods before clinical trials are performed and utility assessed. The manuscript describes the performance of EAPCRI protocols in an animal model of invasive aspergillosis. Blood samples taken from a guinea pig model of IA were used for WB and serum PCR. Galactomannan and ß-d-glucan detection were evaluated, with particular focus on the timing of positivity and on the interpretation of combination testing. The overall sensitivities for WB PCR, serum PCR, galactomannan, and ß-d-glucan were 73%, 65%, 68%, and 46%, respectively. The corresponding specificities were 92%, 79%, 80%, and 100%, respectively. PCR provided the earliest indicator of IA, and increasing galactomannan and ß-d-glucan values were indicators of disease progression. The combination of WB PCR with galactomannan and ß-d-glucan proved optimal (area under the curve [AUC], 0.95), and IA was confidently diagnosed or excluded. The EAPRCI-recommended PCR protocols provide performance comparable to commercial antigen tests, and clinical trials are warranted. By combining multiple tests, IA can be excluded or confirmed, highlighting the need for a combined diagnostic strategy. However, this approach must be balanced against the practicality and cost of using multiple tests.

The novel arylamidine T-2307 maintains in vitro and in vivo activity against echinocandin-resistant Candida albicans.

We evaluated the in vitro and in vivo activities of the investigational arylamidine T-2307 against echinocandin-resistant Candida albicans. T-2307 demonstrated potent in vitro activity, and daily subcutaneous doses between 0.75 and 6 mg/kg of body weight significantly improved survival and reduced fungal burden compared to placebo control and caspofungin (10 mg/kg/day) in mice with invasive candidiasis caused by an echinocandin-resistant strain. Thus, T-2307 may have potential use in the treatment of echinocandin-resistant C. albicans infections.

Efficacy of the investigational echinocandin ASP9726 in a guinea pig model of invasive pulmonary aspergillosis.

ASP9726 is an investigational echinocandin with in vitro activity against Aspergillus species. We evaluated the pharmacokinetics and efficacy of this agent in an established guinea pig model of invasive pulmonary aspergillosis. ASP9726 plasma concentrations were measured in guinea pigs administered either a single dose or multiple doses of this agent at 2.5, 5, and 10 mg/kg of body weight/day by subcutaneous injection. Immunosuppressed guinea pigs were inoculated with A. fumigatus AF293, and ASP9726 (2.5, 5, and 10 mg/kg/day), voriconazole (10 mg/kg by oral gavage twice daily), or caspofungin (3 mg/kg/day by intraperitoneal injection) was administered for 8 days. Changes in fungal burden were measured by enumerating CFU and by quantitative PCR of specimens from within the lungs, as well as by analysis of serum (1 → 3)-ß-D-glucan and galactomannan. Lung histopathology was also evaluated. ASP9726 plasma concentrations increased in a dose-proportional manner, and the drug was well tolerated at each dose. Each dose of ASP9726, voriconazole, and caspofungin significantly reduced pulmonary fungal burden as measured by quantitative PCR and by determining (1 → 3)-ß-D-glucan and galactomannan levels, but only voriconazole significantly reduced numbers of CFU. ASP9726 at 5 mg/kg also significantly improved survival. Histopathology demonstrated morphological changes in hyphae in animals exposed to ASP9726 and caspofungin, consistent with the activities of the echinocandins. These results suggest that ASP9726 may be efficacious for the treatment of invasive pulmonary aspergillosis.

The investigational agent E1210 is effective in treatment of experimental invasive candidiasis caused by resistant Candida albicans.

The in vitro and in vivo activity of the inositol acyltransferase inhibitor E1210 was evaluated against echinocandin-resistant Candida albicans. E1210 demonstrated potent in vitro activity, and in mice with invasive candidiasis caused by echinocandin-resistant C. albicans, oral doses of 10 and 40 mg E1210/kg of body weight twice daily significantly improved survival and reduced fungal burden compared to those of controls and mice treated with caspofungin (10 mg/kg/day). These results demonstrate the potential use of E1210 against resistant C. albicans infections.

Limited activity of miltefosine in murine models of cryptococcal meningoencephalitis and disseminated cryptococcosis.

Miltefosine is an alkyl phosphocholine with good oral bioavailability and in vitro activity against Cryptococcus species that has gained interest as an additional agent for cryptococcal infections. Our objective was to further evaluate the in vivo efficacy of miltefosine in experimental in vivo models of cryptococcal meningoencephalitis and disseminated cryptococcosis. Mice were infected intracranially or intravenously with either C. neoformans USC1597 or H99. Miltefosine treatment (1.8 to 45 mg/kg of body weight orally once daily) began at either 1 h or 1 day postinoculation. Fluconazole (10 mg/kg orally twice daily) or amphotericin B deoxycholate (3 mg/kg intraperitoneally once daily) served as positive controls. In our standard models, miltefosine did not result in significant improvements in survival or reductions in fungal burden against either C. neoformans isolate. There was a trend toward improved survival with miltefosine at 7.2 mg/kg against disseminated cryptococcosis with the H99 strain but only at a low infecting inoculum. In contrast, both fluconazole and amphotericin B significantly improved survival in mice with cryptococcal meningoencephalitis and disseminated cryptococcosis due to USC1597. Amphotericin B also improved survival against both cryptococcal infections caused by H99. Combination therapy with miltefosine demonstrated neither synergy nor antagonism in both models. These results demonstrate limited efficacy of miltefosine and suggest caution with the potential use of this agent for the treatment of C. neoformans infections.