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  • Isavuconazole (BAL4815) Pharmacodynamic Target Determination in an in vivo な Murine Model of Invasive Pulmonary Aspergillosis against Wild Type and Cyp51 に Mutant Isolates of Aspergillus fumigatus ぬ ね Running Title: の Isavuconazole Aspergillus Pharmacodynamics は ば Authors: ぱ Alexander J. Lepaka, Karen Marchilloa, Jamie vanHeckera, David R. Andesa# ひ など Author's Affiliation: なな aUniversity of Wisconsin, Madison, Wisconsin, USA なに なぬ #Corresponding Author: なね David R. Andes, MD なの Department of Medicine なは Department of Medical Microbiology and Immunology なば University of Wisconsin なぱ 1685 Highland Ave, MFCB, Room 5211 なひ Madison, WI 53705-2281 にど dra@medicine.wisc.edu にな にに にぬ にね にの には

    AAC Accepts, published online ahead of print on 7 October 2013 Antimicrob. Agents Chemother. doi:10.1128/AAC.01355-13 Copyright © 2013, American Society for Microbiology. All Rights Reserved.

  • ABSTRACT 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

  • 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 107 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 107 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