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.

ABSTRACT

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.

INTRODUCTION

Cryptococcosis is a significant cause of morbidity and mortality in immunocompromised patients, with the majority of infections being caused by the species Cryptococcus neoformans (1). Patient groups at the highest risk include those with HIV/AIDS, lymphoma, or leukemia; transplant recipients; and those receiving high-dose corticosteroids (2). Cryptococcal meningitis is the most common manifestation of disseminated disease, as Cryptococcus species are highly neurotropic following inhalation into the lungs (2, 3). This is a significant, life-threatening invasive fungal infection in many patient groups, including HIV/AIDS patients, for which the global burden has been estimated to be 223,100 cases annually (4), and the 3-month mortality with appropriate treatment is still unacceptably high (20% to 30%) (5–7). In regions where treatment options are limited, such as sub-Saharan Africa, mortality may reach as high as 70% (8). In the United States and other developed countries, recommendations for the treatment of cryptococcal meningitis typically include an induction phase utilizing an intravenous amphotericin B formulation plus flucytosine, followed by consolidation and maintenance phases with fluconazole (1). In resource-limited countries, intravenous medications are often unavailable for the induction phase, and in this setting, high-dose oral fluconazole may be used (9). However, this strategy has not been demonstrated to be as effective as induction therapy with a regimen containing intravenous amphotericin B. In addition, standard doses of fluconazole (200 to 400 mg per day) have resulted in higher rates of clinical failure and relapses when used as monotherapy (10, 11), while adverse effects as well as the potential for drug interactions may limit high-dose fluconazole therapy.

The tetrazole VT-1129 is a novel fungal Cyp51 inhibitor with potent in vitro activity against susceptible and fluconazole-resistant Cryptococcus species (12–14). This agent acts by inhibiting the fungal cytochrome P450 enzyme Cyp51 (lanosterol 14-α-demethylase), thus inhibiting the biosynthesis of ergosterol, but in a manner that is highly selective compared to that of clinically available azole antifungals (13, 15). This selectivity may markedly limit drug interactions and toxicities, which are associated with the azoles. The objective of this study was to evaluate the in vivo efficacy of VT-1129, administered orally as a benzenesulfonic acid salt (BSA), in an established murine model of cryptococcal meningitis.

RESULTS

In vitro susceptibility.

VT-1129 demonstrated potent in vitro activity against Cryptococcus neoformans var. neoformans isolate USC1597. The MIC of VT-1129 was ≤0.015 μg/ml at the 50% inhibition endpoint and 0.12 μg/ml when 100% inhibition of growth was used. In comparison, the MIC of fluconazole was 1 μg/ml at 50% inhibition.

Survival arm. (i) Survival.

Survival was assessed on day 30 and day 60 following 10 or 28 days of dosing, respectively. In the 30-day survival experiment, VT-1129 at doses of 10 mg/kg of body weight and 20 mg/kg once daily (QD) significantly improved the median duration of survival (>30 days for both) compared to that for the vehicle control group (10.5 days; P ≤ 0.0276) (Fig. 1A and B). In addition, VT-1129 at 20 mg/kg once daily resulted in a significant improvement in the percentage of mice surviving to day 30 (100%) compared to that for the control group (40%; P = 0.01). Although there was a trend toward a higher survival percentage in the 10-mg/kg once daily and twice daily (BID) groups, this difference did not quite reach the threshold of statistical significance (P = 0.0573). No significant improvements in survival were observed with the VT-1129 at 10 mg/kg twice daily group (median survival time, >30 days; 70% survival), although the survival in the control group was greater than expected on the basis of previous experience with this model. There was also no survival advantage observed with the VT-1129 at 20 mg/kg twice daily (13 days and 20%) and 40 mg/kg once daily groups (16.5 days and 40%) or with any dose of fluconazole (>28 days and 40% to 60%).

FIG 1.

Survival curves in mice inoculated intracranially with C. neoformans and treated with VT-1129 or fluconazole. Treatment with oral therapy started at 1 day postinoculation and continued for 10 (A and B) or 28 (C) days. Mice were followed off therapy until day 30 (A and B) or day 60 (C), respectively. Black squares, vehicle control; white triangles, fluconazole at 10 mg/kg BID; gray triangles, fluconazole at 20 mg/kg BID; gray circles, VT-1129 at 10 mg/kg QD; black circles, VT-1129 at 10 mg/kg BID; gray squares, VT-1129 at 20 mg/kg QD; inverted white triangles, VT-1129 at 20 mg/kg BID; inverted gray triangles, VT-1129 at 40 mg/kg QD. Data are for 10 mice per group.

Similarly, in the 60-day survival experiment in which mice received treatment for 28 days, no improvement in survival with VT-1129 at 20 mg/kg twice daily (14 days and 20%) compared to that for the control group (10.5 days and 10%) was observed (Fig. 1C). Although there was a trend toward improved median survival in the VT-1129 at 40 mg/kg once daily group (37 days; P = 0.0618) along with a significant improvement in percent survival (50%; P = 0.0325), early deaths were observed with both VT-1129 doses, similar to the findings for mice that received the vehicle control (between days 4 and 10). Finally, fluconazole significantly improved the median duration of survival (32 days; P = 0.01) compared to that for the control group but not the percentage of mice surviving to the study endpoint (0%).

The etiology for the lack of a survival benefit with the higher doses of VT-1129 in the 30-day and 60-day survival arms was not directly determined. However, it is noted that a preliminary tolerability study conducted in uninfected mice treated with 40 mg/kg VT-1129 once daily for 10 days showed that although all animals survived and showed no outward clinical adverse effects, the VT-1129-treated animals lost weight (mean loss, 1.5 g compared to their weight at the start of treatment); in contrast, the vehicle control-treated animals gained weight (mean gain, 3.6 g compared to their weight at the start of treatment). Because of the finding of a lower survival benefit with higher VT-1129 doses, lower doses were explored in the fungal burden arm.

(ii) Fungal burden.

In the survival experiments, the fungal burden was assessed in mice as they succumbed to infection and at the study endpoints (days 30 and 60 postinoculation). In the 30-day survival experiment, each dose of VT-1129 significantly reduced the CFU counts within the brain tissue (means, 1.65 to 2.14 log₁₀ CFU/g) compared to those in the control group (6.15 log₁₀ CFU/g; P < 0.0001 for each comparison) (Fig. 2A). Overall, 40 of the 50 mice administered VT-1129 in the 30-day survival experiment had CFU counts below the lower limit of quantification. Fluconazole did not result in a significant reduction in the fungal burden compared to that in the control group at either dose used (6.68 and 5.75 log₁₀ CFU/g in the 10-mg/kg and 20-mg/kg dose groups, respectively). Similar results were observed in the 60-day survival experiment (Fig. 2B). Here, the fungal burden observed in both the 20-mg/kg and 40-mg/kg VT-1129 groups (1.73 and 1.89 log₁₀ CFU/g, respectively) was significantly lower than that observed in the control group (5.96 log₁₀ CFU/g; P < 0.0001 for both comparisons), and the CFU counts were below the lower limit of quantification in 11 of the 20 mice administered VT-1129. The fungal burden in mice administered fluconazole was similar to that in mice in the control group.

FIG 2.

Brain tissue fungal burden (number of CFU per gram of brain tissue) in mice with cryptococcal meningitis secondary to C. neoformans in the survival arm studies. Treatment with oral therapy started at 1 day postinoculation and continued for 10 (A) or 28 (B) days. Mice were followed off therapy until day 30 (A) or day 60 (B), respectively. Lines represent mean values. *, P < 0.0001 versus the control (n = 7 to 10 mice per group). Black circles, mice that died prior to the day 30 or the day 60 endpoint; gray circles, mice that survived to the study endpoints.

Fungal burden arm.

The fungal burden was also assessed on day 8 and day 15 after 7 and 14 days of dosing, respectively. In these experiments, a wider range of VT-1129 doses was assessed, including doses lower than those used in the survival experiments. Following 7 days of treatment, VT-1129 significantly reduced the fungal burden, as the CFU counts in each of the VT-1129 dose groups (means, 1.66 to 5.37 log₁₀ CFU/g) were significantly lower than those in the control group (6.06 log₁₀ CFU/g; P ≤ 0.04 for all comparison) (Fig. 3A). In addition, a clear dose-response was observed with VT-1129, as the CFU counts were significantly lower in mice that received VT-1129 doses of ≥0.3 mg/kg QD than in mice that received a VT-1129 dose of 0.1 mg/kg QD (P ≤ 0.001). Likewise, the fungal burden was significantly lower in mice that received VT-1129 doses of ≥1 mg/kg QD than in mice that received a VT-1129 dose of 0.3 mg/kg QD (P < 0.001). Maximum reductions in the fungal burden were observed with VT-1129 total daily doses of 6 mg/kg and higher. Treatment with both doses of fluconazole also resulted in significant reductions in the fungal burden compared to that in the control group (2.42 and 2.12 log₁₀ CFU/g; P < 0.0001).

FIG 3.

Brain tissue fungal burden (number of CFU per gram of brain tissue) in mice with cryptococcal meningitis secondary to C. neoformans in the fungal burden arm of the studies. Treatment with oral therapy started at 1 day postinoculation and continued for 7 (A) or 14 (B) days. The number of CFU in brain tissue was measured on day 8 (A) or day 15 (B) postinoculation. Lines represent mean values. **, P < 0.05 versus the control; *, P < 0.0001 versus the control (n = 10 to 20 mice per group). Black circles, mice that died prior to the day 30 or the day 60 endpoint; gray circles, mice that survived to the study endpoints.

Similar results were also observed in mice that received treatment for 14 days (Fig. 3B). VT-1129 at total daily doses of 0.3 mg/kg and higher (1.60 to 4.93 log₁₀ CFU/g) significantly reduced the fungal burden compared to that in the control group (6.67 log₁₀ CFU/g; P < 0.0001 for each comparison). Both doses of fluconazole also significantly reduced the fungal burden compared to that in the controls following 14 days of therapy (1.87 and 3.03 log₁₀ CFU/g; P < 0.0001). A clear dose-response was also observed with VT-1129 with the longer treatment duration, as the fungal burden was significantly lower in mice dosed with VT-1129 at ≥0.3 mg/kg QD than in mice dosed with VT-1129 at 0.1 mg/kg QD (P ≤ 0.0001). Similarly, CFU counts were significantly lower in mice that received VT-1129 doses of ≥1 mg/kg QD than in mice that received a VT-1129 dose of 0.3 mg/kg QD (P < 0.0001). Maximum reductions in the fungal burden were achieved at total daily doses of 3 mg/kg and higher. In this experiment, 39 of the 40 mice that received VT-1129 daily doses of 3 mg/kg or higher had CFU counts below the lower limit of quantification. In contrast, in mice that received therapy for 7 days, 18 of 50 that received VT-1129 total daily doses of 3 mg/kg had CFU counts below the lower limit of quantification.

VT-1129 plasma and brain tissue concentrations.

VT-1129 plasma and brain tissue concentrations on days 8 and 15, 1 day after dosing had stopped, and on day 30, 20 days after dosing had stopped, are shown in Fig. 4. The day 8 mean VT-1129 plasma concentrations ranged from 238 ng/ml to 21,385 ng/ml, and the day 15 mean plasma concentrations ranged from 337 ng/ml to 32,919 ng/ml. The VT-1129 brain tissue concentrations measured on both day 8 and day 15 were higher than those found within the plasma at each dose level (day 8 range, 492 ng/g to 44,622 ng/g; day 15 range, 713 ng/g to 70,919 ng/g). These concentrations achieved within the plasma and brain tissue were above the VT-1129 MIC against the isolate used to establish infection. A clear concentration-response was also found by nonlinear regression analysis of VT-1129 levels measured on days 8 and 15 within both the plasma (R² = 0.87 on days 8 and 15) and brain tissue (R² = 0.87 and 0.82 on days 8 and 15, respectively) (Fig. 5).

FIG 4.

VT-1129 plasma and brain tissue concentrations in mice with cryptococcal meningitis due to C. neoformans on day 8 after 7 days of dosing and on day 15 after 14 days of dosing in the fungal burden arm and on day 30 in the survival arm 20 days after dosing had stopped. Lines represent mean values.

FIG 5.

Plasma and brain VT-1129 concentration-response curves on days 8 and 15 after 7 and 14 days of therapy, respectively.

Similar results were also observed in the 30-day and 60-day survival studies. At 30 days postinoculation, 20 days after treatment had stopped, VT-1129 plasma and brain tissue concentrations (plasma concentration range, 703 ng/ml to 28,211 ng/ml; brain tissue concentration range, 2,861 ng/g to 80,538 ng/g) remained elevated and above the MIC (≤15 and 125 ng/ml for 50 and 100% inhibition, respectively) against the infecting organism 20 days after therapy had stopped. In the 60-day survival experiment, 32 days after therapy had stopped, VT-1129 concentrations remained well above the MIC of the infecting organism in both the plasma and brain tissue (data not shown).

DISCUSSION

Cryptococcus species are highly neurotropic, dissemination to the central nervous system (CNS) is common in immunocompromised individuals infected with these fungi, and the global burden in HIV/AIDS patients remains elevated (4). Even with appropriate therapy, poor clinical outcomes may still result and mortality rates are unacceptably high (5–7). VT-1129 is an investigational tetrazole that is a highly selective inhibitor of the fungal Cyp51 enzyme but only weakly inhibits mammalian cytochrome P-450 enzymes (13). Previous studies have demonstrated the potent in vitro activity of VT-1129 against Cryptococcus species (12–14).

The results of the current study demonstrate that the in vitro anticryptococcal potency also translates to in vivo efficacy in this experimental model of cryptococcal meningitis. VT-1129 significantly improved survival in mice with infection caused by C. neoformans. The survival benefit reached 100% at the dose of 20 mg/kg VT-1129 once daily, but no benefit was observed at the highest doses tested (20 mg/kg twice daily and 40 mg/kg once daily). Although the explanation for this drop-off was not directly pursued, it is hypothesized that it may have been due to VT-1129 side effects (either alone or in conjunction with infection) at the very high drug levels achieved in these studies (both plasma and brain concentrations were >10 μg/ml [>10,000 ng/ml] after 20 days of drug washout; therefore, given the mouse half-life of ∼6 days, the maximum concentration in plasma [C_max] would be >40 μg/ml). The drop-off in survival was not due to a loss in antifungal activity, as the fungal burden decrease observed at the two highest doses was the same as that observed at the lower doses. In addition, in a preliminary tolerability study with VT-1129 at 40 mg/kg once daily for 10 days, although all animals survived, the mice did show significant weight loss compared to that seen in the vehicle control group. If this hypothesis is correct, this would set the in-study no-observable-adverse-effect level (NOAEL) at the dose of 20 mg/kg once daily with a C_max of ∼24 μg/ml (based on the mean plasma level of 3 μg/ml measured 20 days after the last dose). These data are consistent with the results of a 28-day toxicology study conducted with VT-1129 in the rat, where the NOAEL was the mid-high dose that gave a mean concentration at steady state (C_ss) of 10 μg/ml, with adverse effects (though no deaths) occurring at the high dose, which had a C_ss of 37 μg/ml (E. P. Garvey and R. J. Schotzinger, unpublished data).

In addition, significant reductions in the fungal burden were also observed with VT-1129 treatment. In the fungal burden arms, where dosing was continued for either 7 or 14 days, the reductions in fungal burden were dose dependent, and clear concentration-response relationships between the levels achieved within the plasma and brain tissue and the number of CFU within the brains were found. Furthermore, the fungal burden was below the lower limit of detection in many of the mice treated with VT-1129 at doses as low as 3 mg/kg/day, and the number of mice with undetectable CFU increased when the duration of treatment was extended from 7 to 14 days. In terms of efficacious plasma concentrations, a maximal or near-maximal fungal burden reduction occurred in the range of 1 to 2 μg/ml VT-1129 in the 14-day dose administration study. Therefore, using the in-study NOAEL of ∼24 μg/ml, this would translate into an ∼10-fold therapeutic index for achieving near-maximal antifungal suppression. The fungal burden was also undetectable in many of the mice in the survival arm. This is a significant finding, as treatment had been discontinued for between 20 and 32 days before the brains were collected for fungal burden analysis in these studies. These results can be explained by the long half-life of VT-1129, >6 days in mouse, and excellent CNS penetration, as the concentrations within the plasma and brain tissue remained above the MICs, both the 50% inhibition and 100% inhibition endpoints, measured against the isolate used to establish infection.

This study is not without limitations. The model used is an immunocompetent murine model in which infection is established by intracranial inoculation. Thus, the degree of in vivo efficacy in the setting of immunosuppression following natural dissemination from the lungs may be different. In addition, due to the long half-life of VT-1129 (estimated to be >150 h after administration of a single intravenous dose of 2 mg/kg), steady-state concentrations may not have been achieved during the relatively short dosing periods in the fungal burden arms (7 and 14 days) and the 30-day survival experiment (10 days). The fungal burden in these experiments may have been further reduced had a loading dose/maintenance dose strategy been used. The isolate used to establish infection in this model is susceptible to fluconazole. Thus, it is unknown if VT-1129 would have been as effective against infection caused by a strain with reduced azole susceptibility. However, in vitro potency was maintained in one study against isolates with high fluconazole MICs (14).

The results of this study are supportive of VT-1129 having an eventual role in the treatment of cryptococcal meningitis. Both improvements in survival and reductions in the fungal burden were observed in this experimental model. The fungal burden remained low or undetectable long after therapy had stopped, which is consistent with the long half-life of this tetrazole. These in vivo results are consistent with the in vitro potency of this agent against the isolates used in this study and previously reported by our group and others against Cryptococcus species. Additional work, including clinical studies, is needed to further understand the potential place in therapy of VT-1129 for cryptococcal meningitis.

MATERIALS AND METHODS

Isolate.

Cryptococcus neoformans var. neoformans isolate USC1597 was used in this study (16). The isolate was subcultured at least twice on Sabouraud dextrose agar prior to in vitro and in vivo experiments. The MICs of VT-1129 and fluconazole against this isolate were determined after 72 h of incubation at 35°C using the CLSI M27-A3 broth microdilution method (17). The concentration ranges tested were 0.015 to 8 μg/ml for VT-1129 and 0.125 to 64 μg/ml for fluconazole. Prior to in vivo inoculation, the colonies of each isolate were placed into brain heart infusion broth in a shaking incubator overnight at 37°C. The cells were collected the next morning by centrifugation and washed three times in sterile physiologic saline. The inoculum concentrations for each experiment were measured with a hemocytometer, and viable cell numbers were confirmed by plating of serial dilutions and CFU enumeration.

Antifungal drugs and dosing formulations.

VT-1129 benzenesulfonic acid salt (BSA), manufactured by the National Center for Advancing Translational Sciences (National Institutes of Health, Bethesda, MD), was dissolved in ethanol with heating at 40°C and stirring as necessary to facilitate dissolution. An equal volume of Cremophor EL was then added, and the mixture was mixed well. Sterile distilled water was then added with frequent vortexing. The composition of the stock solution was ethanol, Cremophor EL, and sterile distilled water at 1:1:8 parts, respectively. The maximum concentration of ethanol and Cremophor EL in the doses of VT-1129 BSA and placebo administered to mice was 4% (vol/vol) each. The desired concentration of VT-1129 BSA to obtain the equivalent oral doses of VT-1129 was achieved with further dilutions in sterile distilled water. The dosing solutions were prepared 1 day prior to the first dose and kept refrigerated (2 to 5°C). The pharmaceutical-grade intravenous formulation of fluconazole was used for oral administration.

Animal model.

An established murine model of cryptococcal meningitis was used to evaluate the in vivo efficacy of VT-1129 (18–20). Immunocompetent outbred male ICR mice (Envigo, Indianapolis, IN) weighing approximately 25 g were used and had access to food and water ad libitum. Following anesthesia with isoflurane, the mice were inoculated intracranially with 2,600 to 3,500 CFU/animal, which was delivered to each mouse as a 0.06-ml volume through a 27-gauge needle fastened to a tuberculin syringe with a cuff to prevent penetration of more than 1 mm (20, 21). The inoculum was injected approximately 6 mm posterior to the orbit through a midline puncture into the cranial vault. Mice were then randomized into experimental groups after inoculation. This animal protocol was approved by the Institutional Animal Care and Use Committee at the University of Texas Health Science Center at San Antonio, and all animals were maintained in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care.

Survival.

Both 30-day and 60-day survival was assessed in mice following 10 or 28 days of therapy, respectively. In the 30-day survival study, groups consisted of a control group treated with the vehicle used to prepare VT-1129 for dosing, as described above; animals treated with VT-1129 by oral gavage at doses of 10 mg/kg once and twice daily, 20 mg/kg once and twice daily, and 40 mg/kg once daily; and animals treated with fluconazole by oral gavage at 10 mg/kg and 20 mg/kg twice daily. In the 60-day survival study, the groups consisted of animals treated with the vehicle control, VT-1129 at doses of 20 mg/kg twice daily and 40 mg/kg once daily, and fluconazole at 20 mg/kg twice daily. In both studies, treatment began at 1 day postinoculation and continued through day 10 in the 30-day survival study and through day 28 in the 60-day survival study. Mice were monitored multiple times per day, any animal that appeared moribund prior to the designated endpoint was humanely euthanized, and death was recorded as occurring the next day.

Fungal burden.

In fungal burden studies, the groups consisted of animals treated with the vehicle control, VT-1129 by oral gavage at once daily doses of 0.1, 0.3, 1, 3, 10, and 20 mg/kg and twice daily doses of 3 and 20 mg/kg, and fluconazole at 10 and 20 mg/kg twice daily. Therapy began at 1 day postinoculation and continued for either 7 or 14 days. On days 8 and 15, the day after therapy had stopped, mice were humanely euthanized and the brains were collected for fungal burden analysis by quantification of the CFU. The brains were divided in half, with one half being weighed and homogenized in sterile saline and the other half being frozen for determination of drug concentrations. Serial dilutions of the homogenate for fungal burden analysis were prepared in sterile saline and plated onto Sabouraud dextrose agar. For samples in which no fungal growth was observed in the dilutions, the remaining undiluted brain tissue homogenates were plated. Following at least 72 h of incubation, colonies were counted and the number of CFU per gram of brain tissue was calculated for each animal. Fungal burden analysis was also performed in the same manner in the survival arm on day 30, on day 60, or on the day that the mice succumbed to infection or were humanely euthanized due to morbidity.

Pharmacokinetics.

Plasma and brain tissues were collected on day 8 or day 15, which was the day after therapy had stopped (i.e., ∼12 to 24 h after the last dose) in the fungal burden arms, and at the survival study endpoints on day 30 or 60, on the day that the mice succumbed to infection (brain tissue only), or on the day that the mice were humanely euthanized due to morbidity in the survival arm (brain tissue and plasma, if available). All samples were stored at −70°C prior to the analysis.

The plasma and brain concentrations of VT-1129 were measured by an established liquid chromatography-tandem mass spectrometry (LC/MS-MS) assay (Cyprotex US, LLC, Watertown, MA). For sample preparation, frozen plasma and brain tissues were thawed on ice and kept at 4°C during sample processing. Brain tissues were homogenized in 50 mM potassium phosphate, pH 7.4. An aliquot of a plasma or brain homogenate sample or a calibration sample was mixed with 3 volumes of methanol containing the internal standard (VT-406), and the mixture was incubated on ice for 5 min and centrifuged. The protein-free supernatant was used for analysis. The results for all plasma and brain samples were compared to a corresponding calibration curve prepared in blank mouse plasma and brain tissue.

The VT-1129 concentrations were quantified by LC/MS-MS using an Agilent 6410 mass spectrometer coupled with an Agilent 1200 high-performance liquid chromatograph (HPLC) and a CTC PAL chilled autosampler, all of which were controlled by MassHunter software (Agilent). After separation on a C₁₈ reverse-phase HPLC column (e.g., a Zorbax Eclipse 2.1-mm by 50-mm or Zorbax Poroshell 2.1- by 50-mm column; Agilent, Waters, or equivalent) using an acetonitrile-water gradient system, VT-1129 and internal standard peaks were analyzed by mass spectrometry (MS) using electrospray ionization in multiple-reaction-monitoring mode. Individual and mean drug concentrations for each treatment group were determined.

Data analysis.

Differences in the fungal burden between groups were assessed for significance by analysis of variance (ANOVA) with Tukey's posttest for multiple comparisons. Survival was plotted by Kaplan-Meier analysis, and differences in the median survival and percent survival were analyzed by the log-rank test and Fisher's exact test, respectively. Differences in plasma and brain tissue concentrations between groups were assessed for significance by ANOVA with Tukey's posttest for multiple comparisons. For mice in which no fungal growth was detected, the lower limit of quantification of each tissue (10 CFU/weight of the individual organ) was used for data analysis. Nonlinear regression was used to assess the relationship between the VT-1129 plasma concentrations and the brain tissue fungal burden. These data were fit to a four-parameter inhibitory sigmoid model (modified Hill equation) using curve-fitting software (Prism, version 6.0b; GraphPad Software, Inc., La Jolla, CA), and the goodness of fit was assessed by use of the coefficient of determination (R²).