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Isavuconazole Is Effective for the Treatment of Experimental Cryptococcal Meningitis Nathan P. Wiederhold,a Laura Kovanda,b Laura K. Najvar,a,c Rosie Bocanegra,a,c Marcos Olivo,a,c William R. Kirkpatrick,a,c Thomas F. Pattersona,c University of Texas Health Science Center at San Antonio, San Antonio, Texas, USAa; Astellas Pharma Global Development, Inc., Northbrook, Illinois, USAb; South Texas Veterans Health Care System, San Antonio, Texas, USAc
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.
I
savuconazole is a drug with broad-spectrum antifungal activity that is approved for the treatment of invasive aspergillosis and mucormycosis by the U.S. Food and Drug Administration. Although this agent has potent in vitro activity against Cryptococcus species (MIC50 and MIC90 values against Cryptococcus neoformans of ⱕ0.015 and 0.06 g/ml and against Cryptococcus gattii of 0.03 and 0.06 g/ml, respectively, per our experience) (1–5), data regarding the in vivo efficacy of isavuconazole against cryptococcosis are limited. The open-label, multicenter VITAL study had only 9 patients with infections caused by Cryptococcus species, including 3 patients with isolated pulmonary disease and 2 with central nervous system [CNS]-only infection (6). Overall, successful responses to therapy were observed in 6 of the patients who received isavuconazole, while therapy failed in 3 patients. Our objective was to assess the in vivo efficacy of isavuconazole using a murine model of cryptococcal meningitis. Cryptococcus neoformans clinical isolate USC1597 and isolate H99 were used in this study (7, 8). Isavuconazole demonstrated potent in vitro activity, as measured by broth microdilution susceptibility testing according to the CLSI M27-A3 reference standard (9), with MIC values of ⱕ0.03 g/ml against both isolates. The MICs for fluconazole against these isolates were 1 and 8 g/ ml, respectively. An established murine model of cryptococcal meningoencephalitis was used to determine the in vivo effectiveness of isavuconazole (7, 8, 10, 11). 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 guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care. Immunocompetent outbred ICR mice (Harlan) were inoculated intracranially with 2,600 to 3,500 CFU/animal as previously described (8), and treatment by oral gavage began 24 h after inoculation. Similar isavuconazole doses have shown efficacy in other animal models of invasive fungal infections (12–15). Isavuconazole concentrations were measured in infected mice. Plasma and brain tissue were collected after the 11th dose from three mice per group at various time points. Groups included isavuconazole equivalent doses (1.88 conversion factor between isavuconazole and isavucona-
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TABLE 1 Mean model pharmacokinetic parameters for isavuconazole Parametera
Mean
SD
Ka (h⫺1) Vmax (mol/h) Km (mol) V (liters) Vb (liters) Kcp (h⫺1) Kpc (h⫺1) Kcb (h⫺1) Kbc (h⫺1)
12.95 1.63 15.24 1.21 1.82 5.98 7.31 9.15 4.49
7.699 1.442 6.116 0.701 1.379 4.525 3.894 3.664 2.139
a Ka, absorption rate constant; Vmax, maximum potential difference; Km, MichaelisMenten constant; V, volume of the central compartment; Vb, volume of the brain compartment; Kcp and Kpc, rate constants for drug moving to and from the central and peripheral compartments; Kcb and Kbc, rate constants for drug flow to and from the central and brain compartments.
zonium sulfate) of 20, 40, 80, 120, and 240 mg/kg by oral gavage twice daily. Isavuconazole concentrations were measured using an established liquid chromatography-mass spectrometry (LC/ MS) assay (16, 17). Concentrations in plasma and brain tissue were modeled using the nonparametric estimation in Pmetrics software (18). Simulations were performed using ADAPT 5 from mean model parameters to generate a drug concentration-time profile and area under the curve (AUC) for each dose for both plasma and brain tissue. As nonlinear pharmacokinetics were observed, a 3-compartment Michaelis-Menten model fit the concentration data well. Mean model parameters (Table
Received 27 January 2016 Returned for modification 19 March 2016 Accepted 15 June 2016 Accepted manuscript posted online 20 June 2016 Citation Wiederhold NP, Kovanda L, Najvar LK, Bocanegra R, Olivo M, Kirkpatrick WR, Patterson TF. 2016. Isavuconazole is effective for the treatment of experimental cryptococcal meningitis. Antimicrob Agents Chemother 60:5600 –5603. doi:10.1128/AAC.00229-16. Address correspondence to Nathan P. Wiederhold, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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FIG 1 Simulated isavuconazole concentration-time profiles for brain tissue and plasma. Mean model parameters were used to generate drug concentration-time profiles for brain tissue and plasma: (A) 20 mg/kg; (B) 40 mg/kg; (C) 80 mg/kg; (D) 120 mg/kg; (E) 240 mg/kg. Each dose was administered by oral gavage twice daily.
1) were used to generate drug concentration-time profiles for plasma and brain tissue (Fig. 1). The data demonstrated a linear increase in AUC up to the 120 mg/kg dose but became nonlinear thereafter (Table 2). This may be of significance as the AUC/MIC is the parameter associated with isavuconazole efficacy in other animal models of invasive fungal infections (12, 13, 15, 19). The ratio of isavuconazole brain tissue to plasma exposure was approximately 1.35 for each dose level. As previously reported, the half-life of isavuconazole in this murine model was short (1.1 h) (19), and the trough levels were low to undetectable (data not shown). However, elevated conTABLE 2 Estimated AUCs for each dose from the pharmacokinetic experiment Dose (mg/kg BIDa)
AUCplasma (g ⫻ h/ml)
AUCbrain (g ⫻ h/ml)
20 40 80 120 240
7.9868 16.3214 34.065 53.286 120.13
10.8042 22.0776 46.074 72.064 162.414
a
BID, twice per day.
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centrations in the plasma and brain tissue were achieved shortly after the last doses (2.4 and 4.8 g/ml in the plasma and 8.4 and 17.3 g/g in the brain tissue for the 120 mg/kg and 240 mg/kg dosages, respectively). In both the survival and fungal burden arms, the in vivo efficacy of isavuconazole was evaluated at doses of 120 and 240 mg/kg twice daily and the in vivo efficacy of fluconazole at doses of 20 and 40 mg/kg twice daily. These doses were chosen based on the pharmacokinetic results in order to achieve exposures similar to that observed in the phase 3 clinical study (20). For the positive control, fluconazole doses were chosen based on our previous experience with this model (8, 11). In the fungal burden arm, mice were humanely euthanized on day 8 postinoculation, and the brains were removed, weighed, and homogenized. Dilutions of each homogenate were prepared and plated on Sabouraud dextrose agar (SDA) for incubation. After 72 h, colonies were counted and fungal burdens (CFU per gram of tissue) were determined. In survival studies, mice were treated for 10 days and then monitored off therapy until day 30 postinoculation. Survival was significantly improved in mice infected with
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FIG 2 Survival to day 30 in mice with cryptococcal meningitis due to either USC1597 (A) or H99 (B) and treated with isavuconazole (ISA) or fluconazole (FLU), both administered twice daily by oral gavage. Survival was plotted by Kaplan-Meier analysis, and differences in median survival and the percent survival between groups were analyzed by the log-rank test and Fischer’s exact test, respectively. The median survival P value was ⬍0.05 for all groups compared to the control. Against USC1597, percent survival increase significantly (P ⬍ 0.05) for 240 mg/kg ISA and both fluconazole doses.
USC1597 and treated with isavuconazole (median survival of 28 and ⬎30 days and percent survival of 40% and 70% for the 120 mg/kg and 240 mg/kg dosages, respectively) compared to the results for placebo (15.5 days and 0% survival) (Fig. 2A). Similar results were also observed for the positive-control fluconazole (⬎30 days and 60% survival for both doses). Against the H99 strain (Fig. 2B), survival was also increased in mice treated with isavuconazole (22 and 23 days, respectively) compared to that for placebo (15 days; P ⬍ 0.05). Similar results were also observed with fluconazole (survival of 21 and 22 days, respectively). However, percent survivals for isavuconazole and fluconazole were not significantly different from that for placebo (overall percent survival of 0 to 10%). CFU counts were also significantly lower in mice infected with the USC1597 isolate and treated with isavuconazole (mean range, 1.6 to 2.87 log10 CFU/g) and fluconazole (2.10 to 2.11 log10 CFU/g) compared to that for placebo (6.58 log10 CFU/g) (Fig. 3A). Brain tissue fungal burdens were also reduced in mice infected with C. neoformans H99 and treated with isavuconazole, but to a lesser degree (3.17 to 5.13 log10 CFU/g versus 6.87 log10 CFU/g), which is consistent with the reduced percent survival achieved with isavuconazole against this isolate. Similar results were also observed for fluconazole in that the fungal burden was reduced when this azole was used to treat
mice infected with the H99 isolate, but to a lesser degree than for mice infected with USC1597. These results suggest that isavuconazole may have a role in the treatment of cryptococcal meningitis, as improvements in survival and reductions in fungal burden were observed in this experimental model. Improvements in survival and reductions in fungal burden were also greater with the higher dose, which achieved higher isavuconazole exposures in the brain and plasma. These in vivo results are consistent with the in vitro potency of this agent (2–5, 21). Limitations of this study must be considered. We did not evaluate isavuconazole in combination with another agent (amphotericin B or flucytosine), nor did we assess the use of this agent as consolidation or maintenance therapy in place of fluconazole following induction treatment as currently recommended in the treatment guidelines (22). However, in the VITAL study, 6 of the 9 patients with cryptococcosis also received isavuconazole as the primary therapy (6). In addition, this is an immunocompetent model and thus does not fully mimic the clinical setting where cryptococcal meningitis is observed in immunocompromised individuals. However, this model has been shown to be beneficial in evaluating new treatment strategies against cryptococcosis (7, 8, 10, 11). Thus, while our results are encouraging, further studies are warranted.
FIG 3 Brain tissue fungal burden was assessed on day 8 postinoculation in mice with cryptococcal meningitis due to either USC1597 (A) or H99 (B) and treated with isavuconazole (ISA) or fluconazole (FLU), and all groups were dosed twice daily by oral gavage. Differences in brain fungal burden (CFU per gram) were assessed for significance by analysis of variance (ANOVA) with Tukey’s posttest for multiple comparisons. Mean log10 CFU/g P values were ⬍0.0001 for all groups compared to controls.
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ACKNOWLEDGMENTS We thank Arlene Farias for her help with the animal model and Dora McCarthy for assistance with the in vitro susceptibility testing. N.P.W. has received research support from Astellas, bioMérieux, Dow, F2G, Merck, Merz, Revolution Medicines, and Viamet and has served on advisory boards for Astellas, Merck, Toyama, and Viamet. T.F.P. has received research grants to the UT Health Science Center at San Antonio from Astellas, Merck, and Revolution Medicines and has served as a consultant for Amplyx, Astellas, Cidara, Gilead, Pfizer, Merck, Scynexis, Toyama, Viamet, and Vical. L.K.N. has received travel support from Viamet Pharmaceuticals, Inc. L.K. is an employee of Astellas. The other authors declare no conflicts of interest.
FUNDING INFORMATION This work, including the efforts of Nathan P. Wiederhold and Thomas F. Patterson, was funded by Astellas Pharma US (Astellas). Isavuconazonium sulfate and isavuconazole powders were provided by Basilea.
REFERENCES 1. Thompson GR, III, Wiederhold NP, Fothergill AW, Vallor AC, Wickes BL, Patterson TF. 2009. Antifungal susceptibilities among different serotypes of Cryptococcus gattii and Cryptococcus neoformans. Antimicrob Agents Chemother 53:309 –311. http://dx.doi.org/10.1128 /AAC.01216-08. 2. Pfaller MA, Messer SA, Rhomberg PR, Jones RN, Castanheira M. 2013. In vitro activities of isavuconazole and comparator antifungal agents tested against a global collection of opportunistic yeasts and molds. J Clin Microbiol 51:2608 –2616. http://dx.doi.org/10.1128/JCM.00863-13. 3. Pfaller MA, Rhomberg PR, Messer SA, Jones RN, Castanheira M. 2015. Isavuconazole, micafungin, and 8 comparator antifungal agents’ susceptibility profiles for common and uncommon opportunistic fungi collected in 2013: temporal analysis of antifungal drug resistance using CLSI species-specific clinical breakpoints and proposed epidemiological cutoff values. Diagn Microbiol Infect Dis 82:303–313. http://dx.doi.org/10.1016/j .diagmicrobio.2015.04.008. 4. Datta K, Rhee P, Byrnes E, III, Garcia-Effron G, Perlin DS, Staab JF, Marr KA. 2013. Isavuconazole activity against Aspergillus lentulus, Neosartorya udagawae, and Cryptococcus gattii, emerging fungal pathogens with reduced azole susceptibility. J Clin Microbiol 51:3090 –3093. http: //dx.doi.org/10.1128/JCM.01190-13. 5. Espinel-Ingroff A, Chowdhary A, Gonzalez GM, Guinea J, Hagen F, Meis JF, Thompson GR, III, Turnidge J. 2015. Multicenter study of isavuconazole MIC distributions and epidemiological cutoff values for the Cryptococcus neoformans-Cryptococcus gattii species complex using the CLSI M27-A3 broth microdilution method. Antimicrob Agents Chemother 59:666 – 668. http://dx.doi.org/10.1128/AAC.04055-14. 6. Thompson GR, III, Rendon A, Dos Santos RR, Queiroz-Telles F, Ostrosky-Zeichner L, Azie N, Maher R, Lee M, Kovanda L, Engelhardt M, Vazquez JA, Cornely OA, Perfect JR. Isavuconazole treatment of cryptococcosis and dimorphic mycoses. Clin Infect Dis, in press. http://dx .doi.org/10.1093/cid/ciw305. 7. Ding JC, Bauer M, Diamond DM, Leal MA, Johnson D, Williams BK, Thomas AM, Najvar L, Graybill JR, Larsen RA. 1997. Effect of severity of meningitis on fungicidal activity of flucytosine combined with fluconazole in a murine model of cryptococcal meningitis. Antimicrob Agents Chemother 41:1589 –1593. 8. Wiederhold NP, Najvar LK, Bocanegra R, Kirkpatrick WR, Sorrell TC, Patterson TF. 2013. Limited activity of miltefosine in murine models of cryptococcal meningoencephalitis and disseminated cryptococcosis. Antimicrob Agents Chemother 57:745–750. http://dx.doi.org/10.1128/AAC .01624-12. 9. Clinical and Laboratory Standards Institute. 2008. Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard—3rd ed. Clinical and Laboratory Standards Institute, Wayne, PA.
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10. Nguyen MH, Najvar LK, Yu CY, Graybill JR. 1997. Combination therapy with fluconazole and flucytosine in the murine model of cryptococcal meningitis. Antimicrob Agents Chemother 41:1120 –1123. 11. Thompson GR, III, Wiederhold NP, Najvar LK, Bocanegra R, Kirkpatrick WR, Graybill JR, Patterson TF. 2012. A murine model of Cryptococcus gattii meningoencephalitis. J Antimicrob Chemother 67:1432– 1438. http://dx.doi.org/10.1093/jac/dks060. 12. Lepak AJ, Marchillo K, VanHecker J, Diekema D, Andes DR. 2013. Isavuconazole pharmacodynamic target determination for Candida species in an in vivo murine disseminated candidiasis model. Antimicrob Agents Chemother 57:5642–5648. http://dx.doi.org/10.1128 /AAC.01354-13. 13. Lepak AJ, Marchillo K, Vanhecker J, Andes DR. 2013. Isavuconazole (BAL4815) pharmacodynamic target determination in an in vivo murine model of invasive pulmonary aspergillosis against wild-type and cyp51 mutant isolates of Aspergillus fumigatus. Antimicrob Agents Chemother 57:6284 – 6289. http://dx.doi.org/10.1128/AAC.01355-13. 14. Seyedmousavi S, Bruggemann RJ, Meis JF, Melchers WJ, Verweij PE, Mouton JW. 2015. Pharmacodynamics of isavuconazole in an Aspergillus fumigatus mouse infection model. Antimicrob Agents Chemother 59: 2855–2866. http://dx.doi.org/10.1128/AAC.04907-14. 15. Majithiya J, Sharp A, Parmar A, Denning DW, Warn PA. 2009. Efficacy of isavuconazole, voriconazole and fluconazole in temporarily neutropenic murine models of disseminated Candida tropicalis and Candida krusei. J Antimicrob Chemother 63:161–166. http://dx.doi.org/10.1093/jac /dkn431. 16. Schmitt-Hoffmann A, Roos B, Heep M, Schleimer M, Weidekamm E, Brown T, Roehrle M, Beglinger C. 2006. Single-ascending-dose pharmacokinetics and safety of the novel broad-spectrum antifungal triazole BAL4815 after intravenous infusions (50, 100, and 200 milligrams) and oral administrations (100, 200, and 400 milligrams) of its prodrug, BAL8557, in healthy volunteers. Antimicrob Agents Chemother 50:279 – 285. http://dx.doi.org/10.1128/AAC.50.1.279-285.2006. 17. Schmitt-Hoffmann A, Roos B, Maares J, Heep M, Spickerman J, Weidekamm E, Brown T, Roehrle M. 2006. Multiple-dose pharmacokinetics and safety of the new antifungal triazole BAL4815 after intravenous infusion and oral administration of its prodrug, BAL8557, in healthy volunteers. Antimicrob Agents Chemother 50:286 –293. http://dx.doi.org/10 .1128/AAC.50.1.286-293.2006. 18. Leary RH, Jelliffe R, Schumitzky A, Van Guilder M. 2001. An adaptive grid non-parametric approach to population pharmacokinetic/dynamic (PK/PD) population models, p 389 –394. In Proceedings of the 14th IEEE Symposium on Computer-Based Medical Systems. IEEE, New York, NY. 19. Warn PA, Sharp A, Parmar A, Majithiya J, Denning DW, Hope WW. 2009. Pharmacokinetics and pharmacodynamics of a novel triazole, isavuconazole: mathematical modeling, importance of tissue concentrations, and impact of immune status on antifungal effect. Antimicrob Agents Chemother 53:3453–3461. http://dx.doi.org/10.1128/AAC .01601-08. 20. Kovanda LL, Desai AV, Lu Q, Townsend RW, Akhtar S, Bonate P, Hope WW. Isavuconazole population pharmacokinetic analysis using non-parametric estimation in patients with invasive fungal disease: results from the VITAL study. Antimicrob Agents Chemother, in press. http://dx .doi.org/10.1128/AAC.00514-16. 21. Thompson GR, III, Fothergill AW, Wiederhold NP, Vallor AC, Wickes BL, Patterson TF. 2008. Evaluation of Etest method for determining isavuconazole MICs against Cryptococcus gattii and Cryptococcus neoformans. Antimicrob Agents Chemother 52:2959 –2961. http://dx.doi.org/10 .1128/AAC.00646-08. 22. Perfect JR, Dismukes WE, Dromer F, Goldman DL, Graybill JR, Hamill RJ, Harrison TS, Larsen RA, Lortholary O, Nguyen MH, Pappas PG, Powderly WG, Singh N, Sobel JD, Sorrell TC. 2010. Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the Infectious Diseases Society of America. Clin Infect Dis 50:291–322. http: //dx.doi.org/10.1086/649858.
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Isavuconazole Is Effective for the Treatment of Experimental Cryptococcal Meningitis Nathan P. Wiederhold,a Laura Kovanda,b Laura K. Najvar,a,c Rosie Bocanegra,a,c Marcos Olivo,a,c William R. Kirkpatrick,a,c Thomas F. Pattersona,c University of Texas Health Science Center at San Antonio, San Antonio, Texas, USAa; Astellas Pharma Global Development, Inc., Northbrook, Illinois, USAb; South Texas Veterans Health Care System, San Antonio, Texas, USAc
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.
I
savuconazole is a drug with broad-spectrum antifungal activity that is approved for the treatment of invasive aspergillosis and mucormycosis by the U.S. Food and Drug Administration. Although this agent has potent in vitro activity against Cryptococcus species (MIC50 and MIC90 values against Cryptococcus neoformans of ⱕ0.015 and 0.06 g/ml and against Cryptococcus gattii of 0.03 and 0.06 g/ml, respectively, per our experience) (1–5), data regarding the in vivo efficacy of isavuconazole against cryptococcosis are limited. The open-label, multicenter VITAL study had only 9 patients with infections caused by Cryptococcus species, including 3 patients with isolated pulmonary disease and 2 with central nervous system [CNS]-only infection (6). Overall, successful responses to therapy were observed in 6 of the patients who received isavuconazole, while therapy failed in 3 patients. Our objective was to assess the in vivo efficacy of isavuconazole using a murine model of cryptococcal meningitis. Cryptococcus neoformans clinical isolate USC1597 and isolate H99 were used in this study (7, 8). Isavuconazole demonstrated potent in vitro activity, as measured by broth microdilution susceptibility testing according to the CLSI M27-A3 reference standard (9), with MIC values of ⱕ0.03 g/ml against both isolates. The MICs for fluconazole against these isolates were 1 and 8 g/ ml, respectively. An established murine model of cryptococcal meningoencephalitis was used to determine the in vivo effectiveness of isavuconazole (7, 8, 10, 11). 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 guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care. Immunocompetent outbred ICR mice (Harlan) were inoculated intracranially with 2,600 to 3,500 CFU/animal as previously described (8), and treatment by oral gavage began 24 h after inoculation. Similar isavuconazole doses have shown efficacy in other animal models of invasive fungal infections (12–15). Isavuconazole concentrations were measured in infected mice. Plasma and brain tissue were collected after the 11th dose from three mice per group at various time points. Groups included isavuconazole equivalent doses (1.88 conversion factor between isavuconazole and isavucona-
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TABLE 1 Mean model pharmacokinetic parameters for isavuconazole Parametera
Mean
SD
Ka (h⫺1) Vmax (mol/h) Km (mol) V (liters) Vb (liters) Kcp (h⫺1) Kpc (h⫺1) Kcb (h⫺1) Kbc (h⫺1)
12.95 1.63 15.24 1.21 1.82 5.98 7.31 9.15 4.49
7.699 1.442 6.116 0.701 1.379 4.525 3.894 3.664 2.139
a Ka, absorption rate constant; Vmax, maximum potential difference; Km, MichaelisMenten constant; V, volume of the central compartment; Vb, volume of the brain compartment; Kcp and Kpc, rate constants for drug moving to and from the central and peripheral compartments; Kcb and Kbc, rate constants for drug flow to and from the central and brain compartments.
zonium sulfate) of 20, 40, 80, 120, and 240 mg/kg by oral gavage twice daily. Isavuconazole concentrations were measured using an established liquid chromatography-mass spectrometry (LC/ MS) assay (16, 17). Concentrations in plasma and brain tissue were modeled using the nonparametric estimation in Pmetrics software (18). Simulations were performed using ADAPT 5 from mean model parameters to generate a drug concentration-time profile and area under the curve (AUC) for each dose for both plasma and brain tissue. As nonlinear pharmacokinetics were observed, a 3-compartment Michaelis-Menten model fit the concentration data well. Mean model parameters (Table
Received 27 January 2016 Returned for modification 19 March 2016 Accepted 15 June 2016 Accepted manuscript posted online 20 June 2016 Citation Wiederhold NP, Kovanda L, Najvar LK, Bocanegra R, Olivo M, Kirkpatrick WR, Patterson TF. 2016. Isavuconazole is effective for the treatment of experimental cryptococcal meningitis. Antimicrob Agents Chemother 60:5600 –5603. doi:10.1128/AAC.00229-16. Address correspondence to Nathan P. Wiederhold, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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FIG 1 Simulated isavuconazole concentration-time profiles for brain tissue and plasma. Mean model parameters were used to generate drug concentration-time profiles for brain tissue and plasma: (A) 20 mg/kg; (B) 40 mg/kg; (C) 80 mg/kg; (D) 120 mg/kg; (E) 240 mg/kg. Each dose was administered by oral gavage twice daily.
1) were used to generate drug concentration-time profiles for plasma and brain tissue (Fig. 1). The data demonstrated a linear increase in AUC up to the 120 mg/kg dose but became nonlinear thereafter (Table 2). This may be of significance as the AUC/MIC is the parameter associated with isavuconazole efficacy in other animal models of invasive fungal infections (12, 13, 15, 19). The ratio of isavuconazole brain tissue to plasma exposure was approximately 1.35 for each dose level. As previously reported, the half-life of isavuconazole in this murine model was short (1.1 h) (19), and the trough levels were low to undetectable (data not shown). However, elevated conTABLE 2 Estimated AUCs for each dose from the pharmacokinetic experiment Dose (mg/kg BIDa)
AUCplasma (g ⫻ h/ml)
AUCbrain (g ⫻ h/ml)
20 40 80 120 240
7.9868 16.3214 34.065 53.286 120.13
10.8042 22.0776 46.074 72.064 162.414
a
BID, twice per day.
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centrations in the plasma and brain tissue were achieved shortly after the last doses (2.4 and 4.8 g/ml in the plasma and 8.4 and 17.3 g/g in the brain tissue for the 120 mg/kg and 240 mg/kg dosages, respectively). In both the survival and fungal burden arms, the in vivo efficacy of isavuconazole was evaluated at doses of 120 and 240 mg/kg twice daily and the in vivo efficacy of fluconazole at doses of 20 and 40 mg/kg twice daily. These doses were chosen based on the pharmacokinetic results in order to achieve exposures similar to that observed in the phase 3 clinical study (20). For the positive control, fluconazole doses were chosen based on our previous experience with this model (8, 11). In the fungal burden arm, mice were humanely euthanized on day 8 postinoculation, and the brains were removed, weighed, and homogenized. Dilutions of each homogenate were prepared and plated on Sabouraud dextrose agar (SDA) for incubation. After 72 h, colonies were counted and fungal burdens (CFU per gram of tissue) were determined. In survival studies, mice were treated for 10 days and then monitored off therapy until day 30 postinoculation. Survival was significantly improved in mice infected with
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FIG 2 Survival to day 30 in mice with cryptococcal meningitis due to either USC1597 (A) or H99 (B) and treated with isavuconazole (ISA) or fluconazole (FLU), both administered twice daily by oral gavage. Survival was plotted by Kaplan-Meier analysis, and differences in median survival and the percent survival between groups were analyzed by the log-rank test and Fischer’s exact test, respectively. The median survival P value was ⬍0.05 for all groups compared to the control. Against USC1597, percent survival increase significantly (P ⬍ 0.05) for 240 mg/kg ISA and both fluconazole doses.
USC1597 and treated with isavuconazole (median survival of 28 and ⬎30 days and percent survival of 40% and 70% for the 120 mg/kg and 240 mg/kg dosages, respectively) compared to the results for placebo (15.5 days and 0% survival) (Fig. 2A). Similar results were also observed for the positive-control fluconazole (⬎30 days and 60% survival for both doses). Against the H99 strain (Fig. 2B), survival was also increased in mice treated with isavuconazole (22 and 23 days, respectively) compared to that for placebo (15 days; P ⬍ 0.05). Similar results were also observed with fluconazole (survival of 21 and 22 days, respectively). However, percent survivals for isavuconazole and fluconazole were not significantly different from that for placebo (overall percent survival of 0 to 10%). CFU counts were also significantly lower in mice infected with the USC1597 isolate and treated with isavuconazole (mean range, 1.6 to 2.87 log10 CFU/g) and fluconazole (2.10 to 2.11 log10 CFU/g) compared to that for placebo (6.58 log10 CFU/g) (Fig. 3A). Brain tissue fungal burdens were also reduced in mice infected with C. neoformans H99 and treated with isavuconazole, but to a lesser degree (3.17 to 5.13 log10 CFU/g versus 6.87 log10 CFU/g), which is consistent with the reduced percent survival achieved with isavuconazole against this isolate. Similar results were also observed for fluconazole in that the fungal burden was reduced when this azole was used to treat
mice infected with the H99 isolate, but to a lesser degree than for mice infected with USC1597. These results suggest that isavuconazole may have a role in the treatment of cryptococcal meningitis, as improvements in survival and reductions in fungal burden were observed in this experimental model. Improvements in survival and reductions in fungal burden were also greater with the higher dose, which achieved higher isavuconazole exposures in the brain and plasma. These in vivo results are consistent with the in vitro potency of this agent (2–5, 21). Limitations of this study must be considered. We did not evaluate isavuconazole in combination with another agent (amphotericin B or flucytosine), nor did we assess the use of this agent as consolidation or maintenance therapy in place of fluconazole following induction treatment as currently recommended in the treatment guidelines (22). However, in the VITAL study, 6 of the 9 patients with cryptococcosis also received isavuconazole as the primary therapy (6). In addition, this is an immunocompetent model and thus does not fully mimic the clinical setting where cryptococcal meningitis is observed in immunocompromised individuals. However, this model has been shown to be beneficial in evaluating new treatment strategies against cryptococcosis (7, 8, 10, 11). Thus, while our results are encouraging, further studies are warranted.
FIG 3 Brain tissue fungal burden was assessed on day 8 postinoculation in mice with cryptococcal meningitis due to either USC1597 (A) or H99 (B) and treated with isavuconazole (ISA) or fluconazole (FLU), and all groups were dosed twice daily by oral gavage. Differences in brain fungal burden (CFU per gram) were assessed for significance by analysis of variance (ANOVA) with Tukey’s posttest for multiple comparisons. Mean log10 CFU/g P values were ⬍0.0001 for all groups compared to controls.
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Isavuconazole for Cryptococcal Meningitis
ACKNOWLEDGMENTS We thank Arlene Farias for her help with the animal model and Dora McCarthy for assistance with the in vitro susceptibility testing. N.P.W. has received research support from Astellas, bioMérieux, Dow, F2G, Merck, Merz, Revolution Medicines, and Viamet and has served on advisory boards for Astellas, Merck, Toyama, and Viamet. T.F.P. has received research grants to the UT Health Science Center at San Antonio from Astellas, Merck, and Revolution Medicines and has served as a consultant for Amplyx, Astellas, Cidara, Gilead, Pfizer, Merck, Scynexis, Toyama, Viamet, and Vical. L.K.N. has received travel support from Viamet Pharmaceuticals, Inc. L.K. is an employee of Astellas. The other authors declare no conflicts of interest.
FUNDING INFORMATION This work, including the efforts of Nathan P. Wiederhold and Thomas F. Patterson, was funded by Astellas Pharma US (Astellas). Isavuconazonium sulfate and isavuconazole powders were provided by Basilea.
REFERENCES 1. Thompson GR, III, Wiederhold NP, Fothergill AW, Vallor AC, Wickes BL, Patterson TF. 2009. Antifungal susceptibilities among different serotypes of Cryptococcus gattii and Cryptococcus neoformans. Antimicrob Agents Chemother 53:309 –311. http://dx.doi.org/10.1128 /AAC.01216-08. 2. Pfaller MA, Messer SA, Rhomberg PR, Jones RN, Castanheira M. 2013. In vitro activities of isavuconazole and comparator antifungal agents tested against a global collection of opportunistic yeasts and molds. J Clin Microbiol 51:2608 –2616. http://dx.doi.org/10.1128/JCM.00863-13. 3. Pfaller MA, Rhomberg PR, Messer SA, Jones RN, Castanheira M. 2015. Isavuconazole, micafungin, and 8 comparator antifungal agents’ susceptibility profiles for common and uncommon opportunistic fungi collected in 2013: temporal analysis of antifungal drug resistance using CLSI species-specific clinical breakpoints and proposed epidemiological cutoff values. Diagn Microbiol Infect Dis 82:303–313. http://dx.doi.org/10.1016/j .diagmicrobio.2015.04.008. 4. Datta K, Rhee P, Byrnes E, III, Garcia-Effron G, Perlin DS, Staab JF, Marr KA. 2013. Isavuconazole activity against Aspergillus lentulus, Neosartorya udagawae, and Cryptococcus gattii, emerging fungal pathogens with reduced azole susceptibility. J Clin Microbiol 51:3090 –3093. http: //dx.doi.org/10.1128/JCM.01190-13. 5. Espinel-Ingroff A, Chowdhary A, Gonzalez GM, Guinea J, Hagen F, Meis JF, Thompson GR, III, Turnidge J. 2015. Multicenter study of isavuconazole MIC distributions and epidemiological cutoff values for the Cryptococcus neoformans-Cryptococcus gattii species complex using the CLSI M27-A3 broth microdilution method. Antimicrob Agents Chemother 59:666 – 668. http://dx.doi.org/10.1128/AAC.04055-14. 6. Thompson GR, III, Rendon A, Dos Santos RR, Queiroz-Telles F, Ostrosky-Zeichner L, Azie N, Maher R, Lee M, Kovanda L, Engelhardt M, Vazquez JA, Cornely OA, Perfect JR. Isavuconazole treatment of cryptococcosis and dimorphic mycoses. Clin Infect Dis, in press. http://dx .doi.org/10.1093/cid/ciw305. 7. Ding JC, Bauer M, Diamond DM, Leal MA, Johnson D, Williams BK, Thomas AM, Najvar L, Graybill JR, Larsen RA. 1997. Effect of severity of meningitis on fungicidal activity of flucytosine combined with fluconazole in a murine model of cryptococcal meningitis. Antimicrob Agents Chemother 41:1589 –1593. 8. Wiederhold NP, Najvar LK, Bocanegra R, Kirkpatrick WR, Sorrell TC, Patterson TF. 2013. Limited activity of miltefosine in murine models of cryptococcal meningoencephalitis and disseminated cryptococcosis. Antimicrob Agents Chemother 57:745–750. http://dx.doi.org/10.1128/AAC .01624-12. 9. Clinical and Laboratory Standards Institute. 2008. Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard—3rd ed. Clinical and Laboratory Standards Institute, Wayne, PA.
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10. Nguyen MH, Najvar LK, Yu CY, Graybill JR. 1997. Combination therapy with fluconazole and flucytosine in the murine model of cryptococcal meningitis. Antimicrob Agents Chemother 41:1120 –1123. 11. Thompson GR, III, Wiederhold NP, Najvar LK, Bocanegra R, Kirkpatrick WR, Graybill JR, Patterson TF. 2012. A murine model of Cryptococcus gattii meningoencephalitis. J Antimicrob Chemother 67:1432– 1438. http://dx.doi.org/10.1093/jac/dks060. 12. Lepak AJ, Marchillo K, VanHecker J, Diekema D, Andes DR. 2013. Isavuconazole pharmacodynamic target determination for Candida species in an in vivo murine disseminated candidiasis model. Antimicrob Agents Chemother 57:5642–5648. http://dx.doi.org/10.1128 /AAC.01354-13. 13. Lepak AJ, Marchillo K, Vanhecker J, Andes DR. 2013. Isavuconazole (BAL4815) pharmacodynamic target determination in an in vivo murine model of invasive pulmonary aspergillosis against wild-type and cyp51 mutant isolates of Aspergillus fumigatus. Antimicrob Agents Chemother 57:6284 – 6289. http://dx.doi.org/10.1128/AAC.01355-13. 14. Seyedmousavi S, Bruggemann RJ, Meis JF, Melchers WJ, Verweij PE, Mouton JW. 2015. Pharmacodynamics of isavuconazole in an Aspergillus fumigatus mouse infection model. Antimicrob Agents Chemother 59: 2855–2866. http://dx.doi.org/10.1128/AAC.04907-14. 15. Majithiya J, Sharp A, Parmar A, Denning DW, Warn PA. 2009. Efficacy of isavuconazole, voriconazole and fluconazole in temporarily neutropenic murine models of disseminated Candida tropicalis and Candida krusei. J Antimicrob Chemother 63:161–166. http://dx.doi.org/10.1093/jac /dkn431. 16. Schmitt-Hoffmann A, Roos B, Heep M, Schleimer M, Weidekamm E, Brown T, Roehrle M, Beglinger C. 2006. Single-ascending-dose pharmacokinetics and safety of the novel broad-spectrum antifungal triazole BAL4815 after intravenous infusions (50, 100, and 200 milligrams) and oral administrations (100, 200, and 400 milligrams) of its prodrug, BAL8557, in healthy volunteers. Antimicrob Agents Chemother 50:279 – 285. http://dx.doi.org/10.1128/AAC.50.1.279-285.2006. 17. Schmitt-Hoffmann A, Roos B, Maares J, Heep M, Spickerman J, Weidekamm E, Brown T, Roehrle M. 2006. Multiple-dose pharmacokinetics and safety of the new antifungal triazole BAL4815 after intravenous infusion and oral administration of its prodrug, BAL8557, in healthy volunteers. Antimicrob Agents Chemother 50:286 –293. http://dx.doi.org/10 .1128/AAC.50.1.286-293.2006. 18. Leary RH, Jelliffe R, Schumitzky A, Van Guilder M. 2001. An adaptive grid non-parametric approach to population pharmacokinetic/dynamic (PK/PD) population models, p 389 –394. In Proceedings of the 14th IEEE Symposium on Computer-Based Medical Systems. IEEE, New York, NY. 19. Warn PA, Sharp A, Parmar A, Majithiya J, Denning DW, Hope WW. 2009. Pharmacokinetics and pharmacodynamics of a novel triazole, isavuconazole: mathematical modeling, importance of tissue concentrations, and impact of immune status on antifungal effect. Antimicrob Agents Chemother 53:3453–3461. http://dx.doi.org/10.1128/AAC .01601-08. 20. Kovanda LL, Desai AV, Lu Q, Townsend RW, Akhtar S, Bonate P, Hope WW. Isavuconazole population pharmacokinetic analysis using non-parametric estimation in patients with invasive fungal disease: results from the VITAL study. Antimicrob Agents Chemother, in press. http://dx .doi.org/10.1128/AAC.00514-16. 21. Thompson GR, III, Fothergill AW, Wiederhold NP, Vallor AC, Wickes BL, Patterson TF. 2008. Evaluation of Etest method for determining isavuconazole MICs against Cryptococcus gattii and Cryptococcus neoformans. Antimicrob Agents Chemother 52:2959 –2961. http://dx.doi.org/10 .1128/AAC.00646-08. 22. Perfect JR, Dismukes WE, Dromer F, Goldman DL, Graybill JR, Hamill RJ, Harrison TS, Larsen RA, Lortholary O, Nguyen MH, Pappas PG, Powderly WG, Singh N, Sobel JD, Sorrell TC. 2010. Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the Infectious Diseases Society of America. Clin Infect Dis 50:291–322. http: //dx.doi.org/10.1086/649858.
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