The Novel Fungal Cyp51 Inhibitor VT-1598 is Efficacious in Experimental Models. of Central Nervous System Coccidioidomycosis Caused by Coccidioides

AAC Accepted Manuscript Posted Online 5 February 2018 Antimicrob. Agents Chemother. doi:10.1128/aac.02258-17 Copyright 2018 American Society for Microbiology. All Rights Reserved. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 The Novel Fungal Cyp51 Inhibitor VT-1598 is Efficacious in Experimental Models of Central Nervous System Coccidioidomycosis Caused by Coccidioides posadasii and Coccidioides immitis Nathan P. Wiederhold, 1 Lisa F. Shubitz, 2 Laura K. Najvar, 1,3 Rosie Jaramillo, 1,3 Marcos Olivo, 1,3 Gabriel Catano, 1,3 Hien T. Trinh, 2 Christopher M. Yates, 4 Robert J. Schotzinger, 4 Edward P. Garvey, 4 Thomas F. Patterson 1,3 1 University of Texas Health Science Center at San Antonio, San Antonio, TX 2 Valley Fever Center for Excellence, University of Arizona, Tucson, AZ 3 South Texas Veterans Health Care System, San Antonio, TX 4 Viamet Pharmaceuticals, Inc., Durham, NC Running Title: VT-1598 for CNS coccidioidomycosis Keywords: VT-1598, Coccidioides posadasii, Coccidioides immitis, murine model, coccidioidal meningitis, fluconazole Corresponding Author: Nathan P. Wiederhold, PharmD University of Texas Health Science Center at San Antonio Department of Pathology and Laboratory Medicine 7703 Floyd Curl Drive San Antonio, TX 78229 wiederholdn@uthscsa.edu 1

24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 ABSTRACT Coccidioidal meningitis can cause significant morbidity, and lifelong antifungal therapy is often required. VT-1598 is a fungal-specific Cyp51 inhibitor that has potent in vitro activity against Coccidioides species. We evaluated the in vivo efficacy of VT-1598 in murine models of CNS coccidioidomycosis caused C. posadasii and C. immitis. Infection was introduced via intracranial inoculation, and therapy began 48 hours postinoculation. Oral treatments consisted of vehicle control, VT-1598, and positive controls of fluconazole in the C. immitis study and VT-1161 in the C. posadasii study. Treatment continued for 7 and 14 days in the fungal burden and survival studies, respectively. Fungal burden was assessed in brain tissue collected 24 to 48 hours post-treatment in the fungal burden studies, and on the days the mice succumbed to infection or at the pre-specified endpoints in the survival studies. VT-1598 plasma concentrations were also measured in the C. posadasii study. VT-1598 resulted in significant improvements in survival in mice infected with either species. In addition, fungal burden was significantly reduced in the fungal burden studies. Plasma concentrations 48 hours after dosing stopped remained above the VT-1598 MIC against the C. posadasii isolate, although levels were undetectable in the survival study after a 4-week wash-out. Whereas fungal burden remained suppressed after a 2-week wash-out in the C. immitis model, higher fungal burden was observed in the survival arm of the C. posadasii model. This in vivo efficacy supports human studies to establish the utility of VT-1598 for the treatment of coccidioidomycosis. 2

47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 INTRODUCTION Coccidioidomycosis is a fungal infection that can occur in humans and nonhuman mammals, and is caused by dimorphic, saprobic fungal species, Coccidioides posadasii and C. immitis (1). Disseminated or progressive disease, including coccidioidal meningitis, develops in 1 to 3% of infected individuals, and risk factors for severe or disseminated infection include Filipino or African-American ethnicity, HIV/AIDS, pregnancy, cardiopulmonary disease, poorly controlled diabetes, and immunosuppression (2-4). Overall mortality in such patients with coccidioidomycosis has been reported to be high (~60%) (5, 6). Fluconazole remains the preferred triazole for meningitis and central nervous system (CNS) disease due to its excellent absorption following oral administration, a relatively low adverse effect profile, penetration into the central nervous system, and relative affordability as a generic medication (7). The disadvantage of this azole is its inability to clear the fungus from the host. In addition, there is concern for reduced in vitro susceptibility of Coccidioides isolates to fluconazole (8, 9). With the goal of reducing the potential for drug-drug interactions and other human toxicities, tetrazole-based fungal CYP51 inhibitors have been developed that are significantly more specific for inhibiting the target CYP51 compared with mammalian CYP enzymes (10, 11). VT-1598 is a structurally distinct CYP51 inhibitor with a broad spectrum of activity against a variety of yeast and moulds, as well as endemic fungi, including Coccidioides species (10, 12). The objective of this study was to evaluate the in vivo efficacy of VT-1598 against central nervous system (CNS) coccidioidomycosis in separate experimental models of disease caused by both C. posadasii and C. immitis. 3

70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 MATERIALS AND METHODS Fungal Isolates. For murine studies, C. posadasii, strain Silveira (ATCC 28868, CBS 113859), for the murine studies was grown to maturity on glucose-yeast extract agar, and arthroconidia were harvested as previously described at the University of Arizona (13, 14). Arthroconidia were enumerated by hemacytometer count and viability was quantified by fungal culture to adjust concentration of infecting suspensions. C. posadasii, strain Silveira, was also provided to the University of Texas Health Science Center at San Antonio for in vitro susceptibility testing. A contemporary clinical isolate collected from a CNS shunt that was confirmed to be C. immitis at the Fungus Testing Laboratory at the University of Texas Health Science Center San Antonio (UTHSCSA DI17-143) was also used for both animal studies and in vitro testing (15). At this institution, arthroconidia were harvested by the addition of sterile phosphate buffered saline (PBS) to the potato dextrose agar (PDA) and gentle scraping of the agar surface with a sterile disposable loop to dislodge the arthroconidia. The arthroconidia suspension was centrifuged, the supernatant discarded, and the arthroconidia were resuspended in PBS and filtered to remove hyphal fragments. The number of arthroconidia was adjusted to the desired inoculum concentration via dilution, which was verified by the use of a hemocytometer. The viability of the arthroconidia was evaluated by plating aliquots onto PDA and enumerating the number of colony-forming units (CFU) after incubation for 4 days at 35 C. All manipulations of Coccidioides species were performed at Biosafety Level 3 (BSL3). Antifungal Agents. VT-1598 and VT-1161 powders were provided by Viamet Pharmaceuticals, Inc. (Durham, NC) and were prepared for oral dosing using 4

93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 Cremophor EL (20% v/v). For the C. posadasii studies, the free base of VT-1598 was used, and for the C. immitis studies, a tosylate salt of VT-1598 was used, with the correction factor of 1.3 employed for dose amount. Pharmacokinetics studies have demonstrated that exposures of the doses used in this study were not different between the two forms of VT-1598 (Viamet, unpublished data). Pharmaceutical grade fluconazole was used for oral dosing in the C. immitis murine model. Pure powders of VT-1598 and fluconazole (Sigma, St. Louis, MO) were used for in vitro susceptibility testing. In vitro susceptibility tests. Antifungal susceptibility testing was performed by broth macrodilution according to Clinical and Laboratory Standards Institute M38-A2 standard (16). Isolates were adjusted by a spectrophotometer to a starting inoculum of 1 to 5 x 10 4 arthroconidia/ml and then added to tubes containing serial two-fold dilutions of VT-1598 (0.03 to 16 μg/ml) or fluconazole (0.125 to 64 μg/ml) in RPMI-1640 medium (0.165M MOPS ph 7.0, with L-glutamine and without bicarbonate). The tubes were incubated at 35 C for 48 hours and the minimum inhibitory concentration (MIC) recorded as the lowest concentration that resulted in >80% inhibition of growth compared to the drug-free control. Murine Models of CNS Coccidioidomycosis. The C. posadasii murine model was conducted at the University of Arizona, and the C. immitis murine model was conducted at the University of Texas Health Science Center at San Antonio. All animals were housed and utilized according to PHS guidelines under approved institutional animal care and use protocols. The experiments were independently designed and performed at the two institutions. For the C. posadasii studies, 8-week-old female 5

116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 Swiss-Webster mice were used, and 4-week-old male ICR mice were used in the C. immitis studies. Both Swiss-Webster and ICR mice were purchased from Envigo (Indianapolis, IN). Throughout the course of the experiments, animals were monitored at least twice daily to prevent and minimize unnecessary pain and distress. Any animal that appeared moribund prior to the scheduled endpoint was euthanized. Mice (N= 10/group) were infected according to a previously published protocol (13, 14, 17). Briefly, mice were anesthetized and inoculated intracranially with a lethal dose of Coccidioides arthroconidia (~90 arthroconidia of C. posadasii and ~137 of C. immitis). Both fungal burden and survival arms were used to evaluate the in vivo efficacy of VT-1598 against CNS coccidioidomycosis. Treatment with antifungals began 48 hours post-inoculation and continued for 7 days in the fungal burden arm and for 14 days in the survival arm. In the C. posadasii studies, VT-1598 doses of 4 mg/kg and 20 mg/kg administered by oral gavage once daily were used in the short-term fungal burden study, and doses of 3.2 mg/kg, 8 mg/kg, and 20 mg/kg were used in the survival study. Based on previously published data (13), VT-1161 20 mg/kg once daily by oral gavage served as the positive control in the C. posadasii studies. In the C. immitis model, VT-1598 doses of 5 mg/kg, 15 mg/kg, and 45 mg/kg once daily by oral gavage were used in both the fungal burden and survival studies, and fluconazole 25 mg/kg twice daily by oral gavage served as the positive control. In both models, Cremophor EL (20% v/v) served as the vehicle control and was administered by oral gavage once daily. Fungal Burden. Brain fungal burden was determined at the time of sacrifice, either 24 or 48 hours after final treatment in the fungal burden studies, or when 6

139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 euthanized due to moribundity or study termination in the survival studies. In the C. posadasii survival study, termination was 28 days after the last treatment dose (43 days post-inoculation), and in the C. immitis study, animals were terminated 15 days after final dosing (day 30 post-inoculation). Brains were collected, weighed, and homogenized in sterile saline. Dilutions of the homogenates were prepared and plated onto PDA or GYE agar, and the number of colony-forming units per gram of brain tissue (CFU/g) was determined after at least 48 hours of growth at 37 C. Drug Plasma Concentrations. Blood was collected terminally under anesthesia in the C. posadasii infected mice and plasma was frozen until analysis. The plasma concentration of VT-1598 and the positive control VT-1161 were determined by liquid chromatography / tandem mass spectrometry as previously described (13). Data Analysis. Survival was plotted by Kaplan-Meier analysis and differences in the median survival time and percent survival were analyzed by the log-rank test and chi-square test, respectively. As Coccidioides CFU data may be non-normally distributed among groups, the Kruskal-Wallis Test with Dunn s post-test for multiple comparisons was used to determine if differences between non-normally distributed fungal burden data were significant. For normally distributed fungal burden data, ANOVA with Tukey s post-test was used for multiple comparisons. For mice in which no fungal growth was detected, the lower limit of quantification of each tissue (10 colonies/weight of the individual organ) was used for data analysis. A p-value < 0.05 was considered statistically significant for all comparisons. Descriptive statistics were used for VT-1598 plasma concentration results. RESULTS 7

162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 In vitro Susceptibility. VT-1598 demonstrated in vitro activity against both the Coccidioides isolates used to establish infection in the murine models. The VT-1598 MICs were 1 g/ml and 0.5 g/ml against the C. posadasii and C. immitis isolates, respectively. In contrast, the MIC of fluconazole was 16 g/ml against both isolates. Coccidioides posadasii Fungal Burden. In mice infected with C. posadasii and treated with VT-1598, significant reductions in fungal burden were observed in the brain 48 hours post-treatment following 7 days of therapy (Figure 1A). In both the 4 mg/kg and 20 mg/kg VT-1598 groups, brain fungal burden (mean 2.64 and 1.34 log 10 CFU/g, respectively, at lower limit of quantification) was significantly lower than that observed in the vehicle control group (5.02 log 10 CFU/g; p < 0.0001 for both comparisons). In the VT-1598 20 mg/kg group, fungal burden was undetectable in 4 of the 10 mice prior to correction for the lower limit of quantification. Although brain fungal burden was also significantly reduced in mice treated with the positive control VT-1161 (3.83 log 10 CFU/g; p < 0.0001 vs. vehicle control), the fungal burden observed in this group was significantly higher than that observed with either dose of VT-1598 (p < 0.0001 for both comparisons). Coccidioides immitis Fungal Burden. In mice infected with C. immitis, brain tissue fungal burden was also significantly reduced. After 7 days of therapy, mean colony-forming unit counts ranged between 1.23 to 1.39 log 10 CFU/g in the VT-1598 groups and 1.37 log 10 CFU/g in mice treated with fluconazole, and fungal burden in each of these groups was significantly lower than that observed with the vehicle control (5.56 log 10 CFU/g; p < 0.001 for all comparisons) (Figure 1B). 8

184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 Coccidioides posadasii Survival. VT-1598 resulted in significant and doserelated improvements in survival in mice infected with C. posadasii. As shown in Figure 2A, median survival in each of the VT-1598 treatment groups (24 days, 34 days, and 39 days in the 3.2 mg/kg, 8 mg/kg, and 20 mg/kg groups, respectively) was significantly longer than in the vehicle control group (7.5 days; p < 0.001 for all comparisons). Mice treated with VT-1598 began to succumb to infection between days 21 and 31 postinoculation, which corresponded to 6 to 16 days after therapy was stopped. Percent survival in mice treated with VT-1598 at 4 weeks post-treatment was 0% in the 3.2 mg/kg group, 11.1% in the 8 mg/kg group, and 33.3% in the 20 mg/kg group. All mice in the vehicle control group succumbed to infection by day 8. Nine of ten mice treated with the positive control VT-1161 survived to day 43, 28 days post-treatment, which was significantly longer than mice given VT-1598 (p < 0.01 for both comparisons). To compare with the C. immitis study, survival was also evaluated at day 30 postinoculation, which was 15 days after therapy had stopped. When censored at day 30 in the C. posadasii study, median survival was >30 days in mice treated with VT-1598 at 8 and 20 mg/kg (Figure 2B). Survival percentage at this time point was also significantly higher in the VT-1598 8 mg/kg and 20 mg/kg groups (55.5% and 100%) compared to vehicle control (0%; p < 0.003 for both comparisons). In the C. posadasii survival study, fungal burden was also measured as a secondary endpoint at sacrifice, which for many mice was prior to scheduled study termination due to moribundity. In this study, mean fungal burdens in mice treated with VT-1598 (range 4.91-5.36 log 10 CFU/g) and VT-1161 (4.36 log 10 CFU/g) were similar to control mice (5.29 log 10 CFU/g) (Figure 3A). 9

207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 Coccidioides immitis Survival. Treatment with VT-1598 also resulted in significant improvements in survival in mice infected with C. immitis, and the survival advantage observed was also dose proportional (Figure 2C). Median survival was >30 days in each of the VT-1598 treatment groups, which was significantly greater than that observed with the vehicle control (9 days; p < 0.001 for all comparisons). Median survival was also significantly greater in mice treated with the positive control fluconazole (p < 0.001) compared to vehicle control. The percentage of mice surviving to day 30, the endpoint used in the C. immitis survival experiment, was higher in the VT- 1598 treatment groups (70% in the 5 mg/kg group and 100% in the 15 and 45 mg/kg groups) compared to vehicle control (0%; p < 0.003 for all comparisons). There was also a trend towards a higher survival percentage in the fluconazole group, although this did not reach statistical significance (40%; p = 0.087). In the C. immitis survival arm, fungal burdens were higher in mice treated with the lowest dose of VT-1598 (4.83 log 10 CFU/g) or fluconazole (5.73 log 10 CFU/g) (Figure 3B). However, mice treated with 15 mg/kg and 45 mg/kg VT-1598 had mean fungal burdens of 2.19 and 2.58 log 10 CFU/g, respectively, which were significantly lower than the vehicle control (6.11 log 10 CFU/g; p < 0.002 for both comparisons). VT-1598 Plasma Concentrations. Plasma drug concentrations of VT-1598 and VT-1161 were determined for mice infected with C. posadasii. Two days after the last treatment, VT-1598 plasma concentrations in the fungal burden study were 1.95 + 0.63 g/ml for the 4 mg/kg group and 17.6 + 5.50 g/ml for the 20 mg/kg group. Both were above the 1 g/ml MIC for C. posadasii. VT-1161 concentrations (20 mg/kg dose) were 13.9 + 3.35 g/ml. The half-life of VT-1598 in mice is ~24 hours, which is reflected in 10

230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 the lack of any measurable VT-1598 at the time of sacrifice in the survival study. This likely explains the dose-dependent survival time of mice after stopping treatment with VT-1598, with animals failing after the plasma concentrations fell below the MIC. In contrast, the mean VT-1161 concentration in the survival study 28 days post-treatment was 1.34 + 0.74 g/ml, consistent with its extended half-life of ~14 days in mice. Tolerability of VT-1598. In both the C. posadasii and C. immitis models, VT- 1598 was well tolerated. The weights of infected mice treated with VT-1598 in the fungal burden arms of both the C. posadasii and C. immitis models increased or remained the same over the 10-day period between inoculation and tissue collection (mean range of 0.05 to 2.9 gram weight gain), while mice in the vehicle control groups lost weight over the same period (4.0 to 6.39 gram weight loss). In the C. posadasii survival study, four mice in the VT-1598 groups died due to gavage injury (2 in the 3.2 mg/kg group and 1 each in the 8 and 20 mg/kg groups). This may have been due to the prolonged period of oral dosing (14 days) in this model, although no mice were lost to gavage injury in the C. immitis model. These tolerability results are consistent with those of a 28-day study in rats, where the no-observed-adverse-effect level (NOAEL) for VT-1598 was 100 mg/kg when dosed once daily by oral gavage (E. Garvey and R. Schotzinger, unpublished data). DISCUSSION Central nervous system involvement of disseminated coccidioidomycosis is a serious complication, and if left untreated coccidioidal meningitis is nearly always fatal (18-20). The current recommendation for the treatment of patients with coccidioidal meningitis is fluconazole, and therapy is life-long due to the suppressive rather than the 11

253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 curative effects of currently available antifungals in this disease (7, 21, 22). Although fluconazole is well tolerated relative to other antifungals, including other triazoles, prolonged therapy can be problematic due to drug-drug interactions that are mediated by interactions with CYP 450 isoenzymes, including CYP3A4, which can become problematic at doses greater than 200 mg (23). In addition, there is concern about a rise in the number of Coccidioides isolates with reduced susceptibility to fluconazole (8). Although the other triazoles have more potent in vitro activity (8), and there is clinical experience with itraconazole in the treatment of this Coccidioides infections (7), these agents have clinically significant CYP450 mediated drug-drug interactions, and issues with variable oral bioavailability, pharmacokinetics, and adverse effects may also limit their effectiveness (24-29). Thus, there is a need for newer agents in the treatment of coccidioidal meningitis. VT-1598 is a novel therapeutic antifungal candidate that has been designed to be a more specific inhibitor of fungal Cyp51 than mammalian CYP450 isoenzymes. This tetrazole-based agent has previously shown potent and near identical in vitro activities against both C. posadasii and C. immitis (12), which were confirmed herein for the specific isolates used in the murine studies. In the current studies, we demonstrate that VT-1598 is also similarly effective in murine models of CNS coccidioidomycosis caused by both species, with significant improvements in survival and reductions in brain tissue fungal burden. The suppression of fungal burden within the brain tissue 48 hours post-treatment, including undetectable fungal growth in some mice, was observed with VT-1598 plasma concentrations above the MIC for C. posadasii, demonstrating the in vivo potency of VT- 12

276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 1598 against Coccidioides in the brain. VT-1598 given at 8 mg/kg/day also significantly prolonged survival in 90% of mice infected with either species for 15 days beyond the last dose of drug. In the C. posadasii study, mice were observed out to 28 days past treatment, where length of survival became dose dependent, and mice began to succumb to infection between 21 to 31 days post-inoculation, or 6 to 16 days posttreatment. Presumably, deaths of mice that occurrred after stopping treatment were related to the progression of fungal burden following reduction of circulating drug, and not due to any drug-related intolerability, as VT-1598 was well-tolerated and overall survival was dose-dependent. With ongoing treatment, which is the current standard of care in patients with coccidioidal meningitis, VT-1598 would be expected to continue to potently suppress fungal burden. Some limitations in this study are noted as follows. The studies were conducted independently at two different institutions, using different sexes and strains of mice, and the same doses of VT-1598 were not used throughout. Thus, the exact threshold dose associated with in vivo efficacy is unknown, although the positive response observed over the range of doses that were evaluated is encouraging. This limitation may also be a strength insofar as the efficacy was reproducible with differing conditions and operators. As previously noted, residual fungal burden was observed in the survival arm of the C. posadasii model where mice were followed off therapy for 28 days. Since this survival endpoint was an additional 13 days after therapy had stopped relative to the C. immitis study, it is unknown if such a rebound would have been observed with VT-1598 in C. immitis model. In addition, VT-1598 plasma concentrations were not measured in the C. immitis model, so it is unknown how these may have compared to 13

299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 those measured in mice infected with C. posadasii. Furthermore, because mice were treated for only a relatively short period of time (14 days) in the survival arm where the subsequent rebound in fungal burden was observed in the C. posadasii study, it is unknown if this translates into VT-1598 being only suppressive rather than curative for the treatment of CNS coccidioidomycosis. Thus, additional in vivo studies could give valuable insight into these and other questions. The in vivo antifungal activity of VT- 1598 demonstrated in these studies lays the foundation for clinical investigation of this novel antifungal for the treatment of coccidioidomycosis. ACKNOWLEDGEMENTS We thank OpAns, LLC, for analysis of plasma concentrations of VT-1598 and VT-1161. FUNDING This project utilized preclinical services funded by the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Department of Health and Human Services, under Contract Nos. HHS272201100018I and HHSN272201000038I - Task Orders A28 and A98, respectively, to the University of Texas Health Science Center at San Antonio. The University of Arizona studies were funded by Viamet Pharmaceuticals, Inc., through a NIAID R21/R33 grant awarded to Viamet (AI 101497). VT-1598 and VT-1161 powder was provided by Viamet Pharmaceuticals, Inc. TRANSPARENCY DECLARATION NPW has received research support to the UT Health San Antonio from Astellas, biomerieux, Cidara, F2G, Merck, and Viamet, and has served on advisory boards for Merck, Astellas, Toyama, and Viamet. TFP has received research grants to UT Health 14

322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 San Antonio from Astellas, Merck, and Revolution Medicines, and has served as a consultant for Astellas, Gilead, Merck, Pfizer, Revolution Medicines, Toyama, Viamet, and Scynexis. LKN has received travel support from Viamet Pharmaceuticals, Inc. CMY, RJS, and EPG are employees of Viamet Pharmaceuticals, Inc. REFERENCES 1. Fisher MC, Koenig GL, White TJ, Taylor JW. 2002. Molecular and phenotypic description of Coccidioides posadasii sp. nov., previously recognized as the non- California population of Coccidioides immitis. Mycologia 94:73-84. 2. Brown J, Benedict K, Park BJ, Thompson GR, 3rd. 2013. Coccidioidomycosis: epidemiology. Clin Epidemiol 5:185-97. 3. Santelli AC, Blair JE, Roust LR. 2006. Coccidioidomycosis in patients with diabetes mellitus. Am J Med 119:964-9. 4. Stockamp NW, Thompson GR, 3rd. 2016. Coccidioidomycosis. Infect Dis Clin North Am 30:229-46. 5. Cohen IM, Galgiani JN, Potter D, Ogden DA. 1982. Coccidioidomycosis in renal replacement therapy. Arch Intern Med 142:489-94. 6. Nelson JK, Giraldeau G, Montoya JG, Deresinski S, Ho DY, Pham M. 2016. Donor-Derived Coccidioides immitis Endocarditis and Disseminated Infection in the Setting of Solid Organ Transplantation. Open Forum Infect Dis 3:ofw086. 7. Galgiani JN, Ampel NM, Blair JE, Catanzaro A, Geertsma F, Hoover SE, Johnson RH, Kusne S, Lisse J, MacDonald JD, Meyerson SL, Raksin PB, Siever J, Stevens DA, Sunenshine R, Theodore N. 2016. 2016 Infectious Diseases 15

345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 Society of America (IDSA) Clinical Practice Guideline for the Treatment of Coccidioidomycosis. Clin Infect Dis 63:e112-46. 8. Thompson GR, 3rd, Barker BM, Wiederhold NP. 2017. Large-Scale Evaluation of In Vitro Amphotericin B, Triazole, and Echinocandin Activity against Coccidioides Species from U.S. Institutions. Antimicrob Agents Chemother 61. 9. Ramani R, Chaturvedi V. 2007. Antifungal susceptibility profiles of Coccidioides immitis and Coccidioides posadasii from endemic and non-endemic areas. Mycopathologia 163:315-9. 10. Yates CM, Garvey EP, Shaver SR, Schotzinger RJ, Hoekstra WJ. 2017. Design and optimization of highly-selective, broad spectrum fungal CYP51 inhibitors. Bioorg Med Chem Lett 27:3243-3248. 11. Hoekstra WJ, Garvey EP, Moore WR, Rafferty SW, Yates CM, Schotzinger RJ. 2014. Design and optimization of highly-selective fungal CYP51 inhibitors. Bioorg Med Chem Lett 24:3455-8. 12. Wiederhold NP, Tran BH, Patterson H, Yates CM, Schotzinger RJ, Garvey EP. 2017. The novel fungal Cyp51 inhibitor VT-1598 demonstrates potent in vitro activity against endemic fungi, Aspergillus, and Rhizopus, abstr ASM Microbe, New Orleans, LA, June 1-5. 13. Shubitz LF, Trinh HT, Galgiani JN, Lewis ML, Fothergill AW, Wiederhold NP, Barker BM, Lewis ER, Doyle AL, Hoekstra WJ, Schotzinger RJ, Garvey EP. 2015. Evaluation of VT-1161 for Treatment of Coccidioidomycosis in Murine Infection Models. Antimicrob Agents Chemother 59:7249-54. 16

367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 14. Pappagianis D, Zimmer BL, Theodoropoulos G, Plempel M, Hector RF. 1990. Therapeutic effect of the triazole Bay R 3783 in mouse models of coccidioidomycosis, blastomycosis, and histoplasmosis. Antimicrob Agents Chemother 34:1132-8. 15. Tintelnot K, De Hoog GS, Antweiler E, Losert H, Seibold M, Brandt MA, Van Den Ende AH, Fisher MC. 2007. Taxonomic and diagnostic markers for identification of Coccidioides immitis and Coccidioides posadasii. Med Mycol 45:385-93. 16. CLSI. 2008. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi: Approved standard - Second edition.. Clinical and Laboratory Standards Institute, Wayne, Pennsylvania. 17. Hector RF, Zimmer BL, Pappagianis D. 1990. Evaluation of nikkomycins X and Z in murine models of coccidioidomycosis, histoplasmosis, and blastomycosis. Antimicrob Agents Chemother 34:587-93. 18. Winn WA. 1964. The Treatment of Coccidioidal Meningitis. The Use of Amphotericin B in a Group of 25 Patients. Calif Med 101:78-89. 19. Vincent T, Galgiani JN, Huppert M, Salkin D. 1993. The natural history of coccidioidal meningitis: VA-Armed Forces cooperative studies, 1955-1958. Clin Infect Dis 16:247-54. 20. Einstein HE, Holeman CW, Jr., Sandidge LL, Holden DH. 1961. Coccidioidal meningitis. The use of amphotericin B in treatment. Calif Med 94:339-43. 21. Mathisen G, Shelub A, Truong J, Wigen C. 2010. Coccidioidal meningitis: clinical presentation and management in the fluconazole era. Medicine (Baltimore) 89:251-84. 17

390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 22. Dewsnup DH, Galgiani JN, Graybill JR, Diaz M, Rendon A, Cloud GA, Stevens DA. 1996. Is it ever safe to stop azole therapy for Coccidioides immitis meningitis? Ann Intern Med 124:305-10. 23. Canafax DM, Graves NM, Hilligoss DM, Carleton BC, Gardner MJ, Matas AJ. 1991. Interaction between cyclosporine and fluconazole in renal allograft recipients. Transplantation 51:1014-8. 24. Pascual A, Calandra T, Bolay S, Buclin T, Bille J, Marchetti O. 2008. Voriconazole therapeutic drug monitoring in patients with invasive mycoses improves efficacy and safety outcomes. Clin Infect Dis 46:201-11. 25. Pascual A, Csajka C, Buclin T, Bolay S, Bille J, Calandra T, Marchetti O. 2012. Challenging recommended oral and intravenous voriconazole doses for improved efficacy and safety: population pharmacokinetics-based analysis of adult patients with invasive fungal infections. Clin Infect Dis 55:381-90. 26. Dolton MJ, Ray JE, Chen SC, Ng K, Pont LG, McLachlan AJ. 2012. Multicenter study of voriconazole pharmacokinetics and therapeutic drug monitoring. Antimicrob Agents Chemother 56:4793-9. 27. Levine MT, Chandrasekar PH. 2016. Adverse effects of voriconazole: Over a decade of use. Clin Transplant 30:1377-1386. 28. Wiederhold NP, Pennick GJ, Dorsey SA, Furmaga W, Lewis JS, 2nd, Patterson TF, Sutton DA, Fothergill AW. 2014. A reference laboratory experience of clinically achievable voriconazole, posaconazole, and itraconazole concentrations within the bloodstream and cerebral spinal fluid. Antimicrob Agents Chemother 58:424-31. 18

413 414 29. Sporanox. 2017. Product Information: SPORANOX oral capsules Janssen Pharmaceuticals, Inc., Titusville, NJ. 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 19

437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 FIGURE LEGENDS Figure 1. Brain fungal burden (CFU/g) in mice with coccidioidal meningitis secondary to C. posadasii (A) and C. immitis (B) in the fungal burden studies. Colony-forming units were measured on day 10 post-inoculation for C. posadasii (A) and on day 9 for C. immitis (B). FLU - fluconazole. Figure 2. Survival curves in mice inoculated intracranially with C. posadasii (A and B) and C. immitis (C) and treated with VT-1598 or controls. Treatment started two days post-inoculation and continued for 14 days. Mice were then followed until day 43 post-inoculation (28 days after therapy stopped) in the C. posadasii model (A) or until day 30 post-inoculation (15 days after therapy stopped) in the C. immitis model (C). (B) Survival for C. posadasii was also plotted out to day 30 for direct comparison to the C. immitis study. (A & B) black square - vehicle control; gray circle - VT-1598 3.2 mg/kg; gray square - VT-1598 8 mg/kg; gray triangle - VT-1598 20 mg/kg; white circle - VT-1161 20 mg/kg. (C) black square - vehicle control; gray circle - VT-1598 5 mg/kg; white square - VT-1598 15 mg/kg; gray triangle - VT-1598 45 mg/kg; gray rectangle - fluconazole 25 mg/kg. Figure 3. Brain tissue fungal burden (CFU/g) in mice with coccidioidal meningitis secondary to C. posadasii (A) and C. immitis (B) in the survival studies. Treatment continued for 14 days, and colony-forming units were measured at the time of moribundity or at the pre-specified study endpoints (day 43 in the C. posadasii survival study and day 30 in the C. immitis survival study). FLU - fluconazole. 20