Isavuconazole (BAL4815) Pharmacodynamic Target Determination in an in vivo

にば Invasive pulmonary aspergillosis (IPA) continues to rise in concert with increasing にぱ numbers of immune suppression techniques to treat other medical conditions and にひ transplantation. Despite these advances, morbidity and mortality remain unacceptably ぬど high. One strategy utilized to optimize outcomes is antifungal pharmacodynamic (PD) ぬな examination. We explored the pharmacodynamics of a new triazole in development, ぬに isavuconazole, in a murine neutropenic IPA model. Ten A. fumigatus isolates were used ぬぬ including 4 Cyp51 wild type isolates and 6 Cyp51 mutants. MIC range was 0.125 8 ぬね mg/L. Following infection groups of mice were treated with 40 640 mg/kg/12 h oral ぬの prodrug (BAL8557) for 7 days. Efficacy was determined by quantitative PCR of lung ぬは homogenates. At the start of therapy mice had 4.97 log10 conidial equivalents (C.E.)/ml ぬば of lung homogenate and increased to 6.82 log10 C.E./ml of lung homogenate in untreated ぬぱ animals. The infection model was uniformly lethal in untreated control mice. PD target ぬひ endpoints examined included static dose AUC/MIC and 1 log10 kill AUC/MIC. A stasis ねど endpoint was achieved for all isolates with an MIC of ≤ 1 mg/L and 1 log10 kill in all ねな isolates with an MIC of ≤ 0.5 mg/L, regardless of presence or absence of Cyp51 ねに mutation. The static dose range was 65 617 mg/kg/12 h. The corresponding median ねぬ free drug AUC/MIC was near 5. The 1 log10 kill dose range was 147 455 mg/kg/12 h ねね and corresponding median free drug AUC/MIC of 11.1. These values are similar to ねの those previously reported with other triazoles. ねは ねば ねぱ ねひ のど のな のに INTRODUCTION のぬ Invasive pulmonary aspergillosis (IPA) is a common cause of morbidity and のね mortality in immunocompromised patients (1-7). The development of Aspergillus active のの triazoles was a major step forward in therapy; however, morbidity and mortality remain のは unacceptably high. Additionally, the emergence of Cyp51 mutant isolates that confer のば decreased susceptibility to triazoles is a threat to the efficacy of this drug class (8-13). のぱ Therefore, development of novel compounds and examination of the pharmacodynamic のひ relationships of drug exposure, MIC, and outcome are necessary for the optimal はど utilization of these drugs. はな Pharmacodynamic studies integrate the pharmacokinetic properties of a drug, in はに vitro potency (MIC), and treatment efficacy. Common goals of these PD studies are to はぬ maximize clinical outcome through dosing optimization and assist in susceptibility はね breakpoint determination. These investigations have been integral in the optimal use of はの antibiotics for bacterial infections and antifungal agents for mucosal and invasive はは candidiasis (14-18). However, only recently have pharmacodynamic investigations been はば utilized for filamentous fungal infections such as IPA (19-23). はぱ Isavuconazonium sulfate (BAL8557) is the water-soluble prodrug of はひ isavuconazole (BAL4815), a novel triazole compound, with potent activity against ばど numerous fungal pathogens including Aspergillus species (24-28). After intravenous or ばな oral administration the prodrug is rapidly cleaved by plasma esterases to form the active ばに drug isavuconazole (BAL4815) and an inactive cleavage product BAL8728 (25, 29, 30). ばぬ The drug is currently in clinical development, including two phase III trials examining it's ばね efficacy for patients with IPA (http://clinicaltrials.gov, NCT00634049 & NCT00412893). ばの The pharmacodynamic relationships of isavuconazole have been examined in ばは experimental models of invasive candidiasis, however, pharmacodynamic evaluation ばば and targets are unknown for IPA. The objectives of the current study were to [1] ばぱ examine the pharmacodynamic relationship of isavuconazole in a murine model of IPA ばひ and [2] define the optimal isavuconazole exposure for infection due to both wild type and ぱど Cyp51 mutant isolates. ぱな ぱに MATERIALS AND METHODS ぱぬ Organisms. Ten Aspergillus fumigatus isolates were chosen including 9 clinical isolates ぱね with and without Cyp51 mutations and one laboratory isolate with an Fks1 mutation. ぱの Organisms were grown and subcultured on Potato Dextrose Agar (PDA, Difco ぱは Laboratories, Detroit, MI). The organisms were chosen based on similar fitness as ぱば determined by growth in lungs and mortality in untreated animals. ぱぱ ぱひ Drug. Prodrug isavuconazonium sulfate (BAL8557) and isavuconazole (BAL4815) ひど powder were provided by the sponsor (Astellas) for in vivo and in vitro studies, ひな respectively. The prodrug was dissolved in sterile water and buffered to pH of 4.0 prior ひに to oral administration. Isavuconazole powder was dissolved in DMSO per sponsor ひぬ instructions prior to in vitro susceptibility testing. All dosages in the study were ひね administered by oral route and based upon oral prodrug dose. All concentration ひの measurements (i.e. PK data and calculated PK) are based upon active drug. A ひは conversion factor is necessary to compare equivalent prodrug and active drug on a ひば mg/kg basis. This conversion factor was determined based on a prodrug:drug ひぱ equivalency rating of 1.863 (provided by the sponsor) and 89% purity of the prodrug ひひ powder. Thus, the conversion factor for determining equivalent isavuconazole dose などど from prodrug dose was 0.48 (i.e. for every 1 mg/kg of prodrug administered orally, the などな equivalent in vivo isavuconazole dose would be 0.48 mg/kg). The purity of などに isavuconazole powder for in vitro susceptibility testing was >99%. などぬ などね In vitro susceptibility testing. All isolates were tested by broth microdilution in などの accordance with CLSI document M38-A2 (31). MICs were performed in duplicate three などは times. The median value is reported in Table 1. などば などぱ Animals. Six-week old swiss/ICR specific-pathogen-free female mice weighing 23 27 などひ g were used for all experiments (Harlan Sprague-Dawley, Indianapolis, IN). Animals ななど were housed in groups of five and allowed access to food and water ad libitum. Animals ななな were maintained in accordance with the American Association for Accreditation of ななに Laboratory Care criteria (32). Moribund animals showing distress were sacrificed prior ななぬ to study endpoint (7 days) in accordance with the humane treatment of laboratory ななね animals. The animal research committee of the William S. Middleton Memorial VA ななの Hospital and University of Wisconsin-Madison approved the animal studies. ななは ななば Infection Model. Mice were rendered neutropenic (polymorphonuclear cells <100/mm) ななぱ by injection of 150 mg/kg cyclophosphamide subcutaneously (SC) on days -4 and -1. ななひ Prior studies have shown this maintains neutropenia for 4 days; therefore, an additional なにど injection (150 mg/kg) was administered on day +3 to ensure neutropenia throughout the なにな entire 7-day experiment (20, 33, 34). Additionally, cortisone acetate 250 mg/kg SC was なにに administered on day -1 as previously described (20). Throughout the 7-day experiment なにぬ mice were also given ceftazidime 50 mg/kg/d SC to prevent opportunistic bacterial なにね infection. We have previously shown uninfected control animals given the above なにの immune suppression and antibiotic prophylaxis have 100% survival to study endpoint, なには whereas infected controls that were untreated have 100% mortality prior to study なにば endpoint (20). なにぱ Organisms were subcultured on PDA 5 days prior to infection and incubated at なにひ 37° C. On the day of infection, the inoculum was prepared by flooding the culture plate なぬど with 5 ml of normal saline and 0.05% Tween-20. Gentle agitation was applied to release なぬな the conidia. The conidial suspension was collected and quantified by hemocytometer なぬに (Bright-Line, Hausser Scientific, Horsham, PA). The suspension was diluted to a final なぬぬ concentration of 1.2 x 10 conidia/ml. Viability was confirmed by plating the suspension なぬね and performing CFU counts. なぬの An aspiration pneumonia model was utilized as previously described (20). なぬは Briefly, mice were anesthetized with a combination of ketamine and xylazine. Fifty なぬば microliters of the 1-2 x 10 conidia/ml suspension was pipetted into the anterior nares なぬぱ with the mice held upright to allow for aspiration into the lungs. As previously shown and なぬひ confirmed in the current study (data not shown) this results in invasive aspergillosis in なねど over 90% of animals and 100% mortality in untreated infected mice prior to the study なねな endpoint (20). Drug treatment commenced two hours after initiation of infection. なねに なねぬ Lung processing and organism quantitation. Processing and quantitation of なねね Aspergillus burden in the lungs of mice was performed as previously described (35, 36). なねの Briefly, at the time of sacrifice for moribund animals or at the end of therapy (7 days), なねは lungs were aseptically harvested and placed in a 2-ounce sterile polyethylene Whirl-Pak なねば bag (Nasco, Fort Atkinson, WI) containing 2 ml of sterile 0.85% NaCl. The lungs were なねぱ manually homogenized using direct pressure (37) to produce a primary homogenate. なねひ One ml of this primary homogenate was then placed in a sterile bead beating tube なのど (Sarstedt, Newton, NC) with 700 たl of 425-600 たm acid-washed glass beads (Sigmaなのな Aldrich, St. Louis, MO). The primary homogenate was bead-beaten in a Bio-spec bead なのに beater (Bartlesville, OK) for 90 s at 4200 rpm to yield a secondary homogenate. One なのぬ hundred たl of this secondary homogenate was mixed with 100 ul Buffer ATL (Qiagen, なのね Valencia, CA) and 20 たl proteinase K (Qiagen, Valencia, CA) and incubated overnight at なのの 56° C with gentle agitation. DNA was then isolated following the DNEasy Blood and なのは Tissue protocol (Qiagen, Valencia, CA). A final DNA elution step was performed with なのば 100 たl elution buffer (Qiagen, Valencia, CA). The DNA was stored at -20° C until the なのぱ day of PCR. なのひ Quantitative PCR (qPCR) plates were prepared on the day of assay. Standard なはど quantities of conidia were prepared by hemacytometer counts and were utilized to なはな generate standard curves. The results are reported as conidial equivalents (C.E.) per ml なはに of lung homogenate. Samples were assayed in triplicate using a Bio-Rad CFX96 Realなはぬ time system (Hercules, CA). A single copy gene, Fks1, was chosen for quantitation (38). なはね Primer sequences included: forward primer 5'-GCCTGGTAGTGAAGCTGAGCGT-3', なはの reverse primer 5'-CGGTGAATGTAGGCATGTTGTCC-3', and probe 5'-6-FAMなはは AGCCAGCGGCCCGCAAATG-MGB-3' (Integrated DNA Technologies, Coralville, IA). なはば The Fks1 mutation (EMFR S678P) was not located in the primer:probe area of the なはぱ genome. All isolates have been previously studied in a spiking experiment of known なはひ quantities of conidia into lung homogenate to ensure the primer:probe set performs なばど similarly for all isolates over the dynamic range (10 10) (20). なばな なばに Pharmacokinetics. Single dose pharmacokinetics of isavuconazole (BAL4815) were なばぬ determined in individual ICR/Swiss mice following oral administration of the prodrug なばね (BAL8557) at 10, 40, 160, and 640 mg/kg in 0.2 ml volumes by oral-gastric gavage なばの (OG). Plasma from groups of three isoflurane-anesthetized mice were collected at each なばは of 7 time points (0.5, 1, 2, 4, 8, 12, and 24 h). The plasma was stored at -80°C until day なばば of drug assay measurement. Drug concentration measurements were performed by the なばぱ sponsor using LC-MS as previously described. なばひ A non-compartmental model was used in the PK analysis. Pharmacokinetic なぱど parameters including elimination half-life and concentration at time zero (C0) were なぱな calculated via nonlinear least-squares techniques. The AUC was calculated by the なぱに trapezoidal rule. For treatment doses in which kinetics were not directly determined, なぱぬ pharmacokinetic parameters were estimated by linear interpolation for those doses なぱね between two measured doses and by linear extrapolation for doses above or below the なぱの highest and lowest measured doses. Protein binding (99%) was based on previous なぱは studies in mice by the sponsor (personal communication). なぱば なぱぱ Pharmacodynamic index and magnitude. The AUC/MIC was used as the PD index なぱひ for exploration of exposure response relationships based upon previous PK/PD なひど investigations with triazoles (19-22). Both total and free (non-protein bound) drug なひな concentrations were considered. Neutropenic mice were infected as described above. なひに Treatment consisted of 2-fold increases in prodrug (BAL8557) concentration (range 40 なひぬ 640 mg/kg) administered q12 h by OG gavage for 7 days. The doses were selected to なひね vary effect from maximal to no efficacy and included exposures expected with regimens なひの under study in clinical trials. Controls were utilized for each isolate and included a zero なひは hour and untreated controls. Four mice were included in each treatment and control なひば group. なひぱ なひひ Data analysis. The qPCR data was modeled according to a Hill-type dose response にどど equation: log10 D = log10 (E/Emax E)/N + log10 ED50, where D is the drug dose, E is the にどな growth over the study period as measured by qPCR and represented as C.E./ml lung にどに homogenate in untreated control mice, Emax is the maximal drug effect, N is the slope of にどぬ the dose-response curve, and ED50 is the dose needed to achieve 50% maximal effect. にどね The AUC/MIC for total and free drug was determined for each isolate. The coefficient of にどの determination (R) was used to estimate the percent variance in the change in log10 にどは C.E./ml of lung homogenate over the treatment period for the different dosing regimens にどば that could be attributed to the PD index (AUC/MIC). The dose necessary for net stasis にどぱ (static dose, SD) and 1-log kill were determined when these endpoints were achieved. にどひ Additionally, the PD target total and free drug AUC/MIC associated with these endpoints になど was calculated. The SD and PD target total and free drug AUC/MIC were compared になな between wild-type and Cyp51 mutant isolates by t-test for normally distributed data and になに by Mann-Whitney rank sum test for non-normally distributed data. になぬ になね RESULTS になの Organism susceptibility and in vivo fitness. Isavuconazole (BAL4815) susceptibility になは testing, genotype, and relative fitness in the in vivo murine model of each isolate are になば shown in Table 1. Cyp51 wild type MICs ranged from 0.25 1 mg/L and from 0.125 8 になぱ mg/L in Cyp51 mutants. The organisms exhibited similar in vivo fitness based on になひ increase in burden in untreated animals until the time of death or sacrifice. At the start of ににど therapy mice had 4.97 ± 0.33 log10 C.E./ml lung homogenate and the burden increased ににな to 6.82 ± 0.51 log10 C.E./ml lung homogenate in untreated animals. Each isolate ににに produced 100% mortality prior to study endpoint in untreated animals with death ににぬ occurring between days 3 and 6 in all untreated animals. ににね ににの Pharmacokinetics. The time courses for isavuconazole (BAL4815) in the plasma of にには mice following OG doses of 640, 160, 40, and 10 mg/kg of prodrug (BAL8557) are ににば shown in Figure 1. Peak levels were achieved within 2 h for each dosing regimen and ににぱ ranged from 0.51 to 25.4 mg/L. The elimination half-life in serum increased in a doseににひ dependent fashion from 1 to 5 h. The AUC from 0 h to infinity (AUC0-∞), as determined にぬど by the trapezoidal rule, ranged from 0.9 to 287 mg*h/L. The AUC was relatively linear にぬな over the dose range (R 0.98). にぬに にぬぬ Dose-response curves. A dose-response relationship was observed for each isolate にぬね with higher doses of isavuconazole achieving a larger microbiologic effect (Figure 2). にぬの However, higher doses were necessary to achieve similar microbiologic effect against にぬは isolates with elevated isavuconazole MICs. A net static outcome and cidal activity was にぬば achieved for all wild type isolates as well as 2 of 6 Cyp51 mutants. Maximal effect にぬぱ against wild type isolates was approximately a 2 log10 reduction in organism burden にぬひ compared to that in the lungs at the start of therapy and nearly a 4 log10 compared to にねど untreated controls at the end of therapy. にねな にねに PD index and target magnitude. The doses needed to produce net growth にねぬ suppression (i.e. static dose) and to produce 1 log10 kill are shown in Table 2. Net stasis にねね was achieved in all isolates that exhibited an MIC of ≤1 mg/L; whereas 1 log10 kill was にねの observed in all isolates with an MIC of ≤0.5 mg/L. Therefore, we were able to estimate にねは the static dose AUC/MIC target for all 4 Cyp51 wild type isolates and 2 of 6 Cyp51 にねば mutants, these latter two (F14403 and F14532) exhibiting lower MIC values than other にねぱ four mutant isolates. The static dose for Cyp51 wild type isolates ranged from 212 – 617 にねひ mg/kg/12 h. In comparison, the static dose for the two mutant isolates, F14403 and にのど F14532, were 65 and 515 mg/kg/12 h, respectively. The 1 log10 kill PD target was にのな achieved in 3 of 4 Cyp51 wild type isolates and only for a single mutant isolate. The 1 にのに log10 kill dose for Cyp51 wild type isolates ranged from 302 – 455 mg/kg/12 h; whereas it にのぬ was 147 mg/kg/12 h for the single mutant isolate (F14403) for which this endpoint was にのね achieved. にのの Total and free drug AUC/MIC PD targets are shown in Table 2. The free drug にのは AUC/MIC associated with net stasis for the Cyp51 wild type group ranged from 4.15 – にのば 11.1; whereas for the two mutant isolates for which this endpoint was slightly lower at にのぱ 3.61 – 3.67. The difference between the two groups was not statistically significant (p = にのひ 0.18). For all isolates where net stasis was achieved the median static dose free drug にはど AUC/MIC was 5.0. The 1 log10 kill free drug AUC/MIC was roughly 2-fold higher than the にはな static dose PD target, with a median value of 11.1. The AUC/MIC values and treatment にはに outcome for all organisms was fit to the Hill sigmoid dose-response model and the にはぬ relationship is shown in Figure 3. AUC/MIC was a strong predictor of observed にはね outcome with an R of 0.75. にはの にはは DISCUSSION にはば Aspergillus active triazoles have become the cornerstone for prevention and treatment of にはぱ invasive aspergillosis (39). Pre-clinical animal model にはひ pharmacokinetic/pharmacodynamics (PK/PD) investigation has proven useful for にばど optimizing therapy for numerous pathogens but has been under-utilized for filamentous にばな fungal pathogens such as Aspergillus. These pharmacodynamics studies are important にばに in the development of novel triazoles, such as isavuconazole, to provide a framework for にばぬ predicting drug exposures that are expected to achieve a successful therapeutic にばね outcome. Additionally, with the emergence of Aspergillus drug resistance to the triazole にばの class, mediated by mutations in the Cyp51 gene, these studies are integral in にばは examination of susceptibility breakpoints. にばば PK/PD studies for isavuconazole have been limited to two previous murine にばぱ disseminated candidiasis models (40, A. J. Lepak, K. Marchillo, J. vanHecker, and D. R. にばひ Andes, submitted for publication). Both showed a very strong relationship between the にぱど PD index AUC/MIC and treatment outcome. Results from the current study also にぱな demonstrated a strong relationship between total dose and effect in a murine invasive にぱに pulmonary aspergillosis model. While this is the first PK/PD examination for Aspergillus にぱぬ for the triazole isavuconazole, previous studies using these models with the triazoles, にぱね voriconazole and posaconazole, provide the opportunity to comparison across the class. にぱの Employing the same model and diverse group of isolates in the current study, we have にぱは previously shown a posaconazole free drug AUC/MIC of approximately 1 was にぱば associated with net stasis (20). Similar studies by other investigators using , pulmonary にぱぱ model and intravascular models found a posaconazole free drug AUC/MIC of 1.67 was にぱひ associated with 50% maximal efficacy (ED50) and a value of 3.2 was associated with にひど 50% increase in survival, respectively (19, 21). Previous study with voriconazole in a にひな murine pulmonary aspergillosis model observed a 50% maximal effect with a free drug にひに AUC/MIC of 11 (22). The static dose PD target free drug AUC/MIC identified in this にひぬ study for isavuconazole at a median value of 5.0 is congruent with these other in vivo にひね pharmacodynamic triazole studies. For comparison purposes, the ED50, which is shown にひの on Figure 3, was a free drug AUC/MIC of approximately 7. にひは In current model we utilized qPCR which has previously been shown to provide a にひば large dynamic range between effective and ineffective therapy, reproducibility among にひぱ biological replicates, and correlates very well with mortality (20). The static dose and 1 にひひ log10 dose were the primary PD target endpoints used in this study. It is unclear which ぬどど PD endpoint in the animal model correlates with optimal treatment effect in patients. ぬどな Further clinical PK/PD studies utilizing large sets of patient data, organism susceptibility, ぬどに and treatment outcome to delineate the optimal clinical PK/PD target are urgently ぬどぬ needed. ぬどね In summary, we have shown the isavuconazole PD index AUC/MIC correlates ぬどの well with treatment outcome in a murine model of IPA. MIC was a strong predictor of ぬどは success or failure regardless of presence or absence of a Cyp51 mutation. Mutations ぬどば that lead to elevated MICs to other triazoles did not universally correlate with elevated ぬどぱ isavuconazole MIC. The median total and free drug 24 h AUC/MIC PD target for net ぬどひ stasis was 503 and 5, respectively. This target correlates well with other triazole studies ぬなど utilizing this model. Further clinical study with isavuconazole for IPA is warranted and it ぬなな may be a useful addition to the triazole armamentarium for invasive aspergillosis. ぬなに ぬなぬ ACKNOWLEDGEMENTSぬなねAstellas provided funding for the studies. We kindly thank Dr. David Perlin for providingぬなのisolates DPL EC S 1 and EMFR S678P.ぬなはぬなばFigure 1. Plasma concentrations of isavuconazole (BAL4815) afterぬなぱadministration of oral prodrug (BAL8557) at 640, 160, 40, and 10 mg/kg. Eachぬなひsymbol represents the geometric mean ± standard deviation from three mice.ぬにどThe peak concentration (Cmax), 24 h AUC0-∞ (AUC), and elimination half-lifeぬにな(T1/2) are shown for each dose.ぬににぬにぬFigure 2. In vivo isavuconazole dose-response curves for multiple A. fumigatusぬにねisolates. Oral prodrug (BAL8557) was administered in 2-fold increasingぬにのconcentrations from 40 to 640 mg/kg by OG route every 12 h for a 7 d duration.ぬにはOpen symbols represent Cyp51 wild type isolates and closed symbols Cyp51ぬにばmutants. Each symbol represents the geometric mean ± standard deviation ofぬにぱorganism burden as measured by qPCR.ぬにひぬぬどFigure 3. Relationship between PD index total drug AUC/MIC and treatmentぬぬなefficacy for isavuconazole against ten A. fumigatus isolates. Open symbolsぬぬにrepresent Cyp51 wild type isolates and closed symbols Cyp51 mutants. Eachぬぬぬdata point represents the geometric mean of organism burden in four mice. Aぬぬね best-fit line based on the Hill equation is included. The PD parameters Emax,ぬぬのED50, slope (N), and coefficient of determination (R) are shown in the figureぬぬはlegend.ぬぬばぬぬぱぬぬひREFERENCESぬねど1. Baddley JW. 2011. Clinical risk factors for invasive aspergillosis. Medicalぬねなmycology : official publication of the International Society for Human and Animalぬねに Mycology 49 Suppl 1:S7-S12.ぬねぬ2. 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