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Pharmacology

In Vitro and In Vivo Antifungal Profile of a Novel and Long-Acting Inhaled Azole, PC945, on Aspergillus fumigatus Infection

Thomas Colley, Alexandre Alanio, Steven L. Kelly, Gurpreet Sehra, Yasuo Kizawa, Andrew G. S. Warrilow, Josie E. Parker, Diane E. Kelly, Genki Kimura, Lauren Anderson-Dring, Takahiro Nakaoki, Mihiro Sunose, Stuart Onions, Damien Crepin, Franz Lagasse, Matthew Crittall, Jonathan Shannon, Michael Cooke, Stéphane Bretagne, John King-Underwood, John Murray, Kazuhiro Ito, Pete Strong, Garth Rapeport
Thomas Colley
aPulmocide Ltd., London, United Kingdom
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Alexandre Alanio
bInstitut Pasteur, CNRS, Molecular Mycology Unit, French National Reference Center for Invasive Mycoses & Antifungals, URA3012, Paris, France
cParis Diderot, Sorbonne Paris Cité University, Paris, France
dParasitology-Mycology Laboratory, Saint Louis Hospital, Assistance Publique-Hôpitaux de Paris (APHP), Paris, France
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Steven L. Kelly
eCentre for Cytochrome P450 Biodiversity, Institute of Life Science, Swansea University Medical School, Wales, United Kingdom
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Gurpreet Sehra
aPulmocide Ltd., London, United Kingdom
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Yasuo Kizawa
fNihon University, Chiba, Japan
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Andrew G. S. Warrilow
eCentre for Cytochrome P450 Biodiversity, Institute of Life Science, Swansea University Medical School, Wales, United Kingdom
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Josie E. Parker
eCentre for Cytochrome P450 Biodiversity, Institute of Life Science, Swansea University Medical School, Wales, United Kingdom
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Diane E. Kelly
eCentre for Cytochrome P450 Biodiversity, Institute of Life Science, Swansea University Medical School, Wales, United Kingdom
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Genki Kimura
fNihon University, Chiba, Japan
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Lauren Anderson-Dring
aPulmocide Ltd., London, United Kingdom
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Takahiro Nakaoki
fNihon University, Chiba, Japan
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Mihiro Sunose
gSygnature Discovery Ltd., Nottingham, United Kingdom
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Stuart Onions
gSygnature Discovery Ltd., Nottingham, United Kingdom
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Damien Crepin
gSygnature Discovery Ltd., Nottingham, United Kingdom
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Franz Lagasse
gSygnature Discovery Ltd., Nottingham, United Kingdom
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Matthew Crittall
gSygnature Discovery Ltd., Nottingham, United Kingdom
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Jonathan Shannon
gSygnature Discovery Ltd., Nottingham, United Kingdom
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Michael Cooke
gSygnature Discovery Ltd., Nottingham, United Kingdom
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Stéphane Bretagne
bInstitut Pasteur, CNRS, Molecular Mycology Unit, French National Reference Center for Invasive Mycoses & Antifungals, URA3012, Paris, France
cParis Diderot, Sorbonne Paris Cité University, Paris, France
dParasitology-Mycology Laboratory, Saint Louis Hospital, Assistance Publique-Hôpitaux de Paris (APHP), Paris, France
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John King-Underwood
hCompchem Resource, Pendock, United Kingdom
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John Murray
aPulmocide Ltd., London, United Kingdom
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Kazuhiro Ito
aPulmocide Ltd., London, United Kingdom
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Pete Strong
aPulmocide Ltd., London, United Kingdom
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Garth Rapeport
aPulmocide Ltd., London, United Kingdom
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DOI: 10.1128/AAC.02280-16
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  • FIG 1
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    FIG 1

    Efficacy of PC945 as an inhibitor of A. fumigatus sterol 14α-demethylases (CYP51 enzymes). (A) Structure of PC945. (B) Type II azole binding spectra for A. fumigatus CYP51A and CYP51B. Each experiment was performed 4 to 6 times, although data for only one replicate are shown. (C) Azole IC50 determinations for posaconazole (●) and PC945 (○). Mean relative velocity values are shown with standard deviations. (D) Sterol composition of A. fumigatus treated with PC945. The relative levels of lanosterol and eburicol are shown.

  • FIG 2
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    FIG 2

    Antifungal activity of PC945 against A. fumigatusin vitro and in vivo. (A) A549 cells treated with PC945 or posaconazole and infected with GFP-expressing A. fumigatus. Green, GFP-expressing A. fumigatus; blue, DAPI-stained nucleus; red, CellTracker-stained cytoplasm. (B) Persistence of action of PC945 and voriconazole on A. fumigatus hyphae. (C) Persistence of action of PC945 and voriconazole on human bronchial cells (BEAS2B cells) infected with A. fumigatus. (D and E) Effects of once-daily intranasal treatment with PC945 (0.56, 2.8, or 14 μg/mouse) and posaconazole (2.8, 14, or 70 μg/mouse) on survival of A. fumigatus-infected immunocompromised mice (n = 8 to 16). (F) Galactomannan levels in BALF. Each horizontal bar shows the mean ± SEM for 8 mice per group. #, dead before day 7; ***, significant difference from no infection (P < 0.001); †††, significant difference from infection control (P < 0.001).

Tables

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  • Additional Files
  • TABLE 1

    Azole Kd and IC50 determinations versus A. fumigatus CYP51 (AfCYP51) enzymes

    Test agentParameter value (μM)a
    AfCYP51AAfCYP51B
    KdIC50Kd1Kd2IC50
    PC9450.500.2319,2980.320.22
    Posaconazole0.960.160.012NA0.17
    • ↵a Kd determinations used 4 μM purified AfCYP51A and 4 μM AfCYP51B. IC50 determinations used 0.5 μM AfCYP51A and 0.5 μM AfCYP51B, recovered from the membrane fraction of E. coli expression clones. NA, not applicable.

  • TABLE 2

    Sterol composition of A. fumigatus treated with either posaconazole or PC945

    SterolSterol content in cells (%)
    DMSO-treated cellsPosaconazole-treated (μg/ml) cellsDMSO-treated cellsPC945-treated (μg/ml) cells
    0.00010.0010.010.110.00010.0010.010.11
    Ergosterol10094.587.274.767.867.410095.994.786.780.671.3
    Ergost-5,7-dienol03.33.900004.12.53.300
    Lanosterol003.07.08.88.80003.66.29.4
    Eburicol02.25.918.323.423.8002.76.513.219.3
  • TABLE 3

    Antifungal effects of PC945 and known antifungal agents in azole-susceptible and azole-resistant strains of A. fumigatusa

    StrainResistance mechanismIC50 (IC90) (μg/ml) of indicated agent
    PC945VoriconazolePosaconazoleItraconazoleAmphotericin BCaspofungin
    NCPF2010None0.0084 (0.010)0.16 (0.20)0.0086 (0.014)0.057 (0.085)0.23 (0.48)0.11 (>1)
    AF294None0.0020 (0.0043)0.082 (0.27)0.0056 (0.011)0.041 (0.052)0.21 (0.79)>1 (>1)
    AF293None0.0012 (0.0041)0.25 (0.74)0.010 (0.028)0.032 (0.23)0.24 (0.85)>1 (>1)
    AF72G54E mutation0.0061 (0.029)0.019 (0.062)0.032 (0.19)0.43 (>1)0.18 (0.64)0.10 (>1)
    AF91M220V mutation0.0081 (0.059)0.12 (0.38)0.024 (0.12)0.26 (>1)0.42 (>1)0.072 (>1)
    TR34/L98HTR34/L98H mutation0.034 (>1)>1 (>1)0.086 (0.13)0.22 (>1)0.14 (0.29)0.082 (>1)
    • ↵a IC50 and IC90 values were determined from optical density measurements.

  • TABLE 4

    In vitro activities of PC945, posaconazole, and voriconazole against 96 clinically isolated A. fumigatus strainsa

    Test agentMIC (μg/ml)b
    RangeGeometric meanModeMIC50MIC90
    PC9450.032–>80.17*0.1250.1251
    Voriconazole0.064–40.420.50.51
    Posaconazole0.016–20.10.0320.0630.5
    • ↵a All MICs were determined visually.

    • ↵b *, P < 0.05 for PC945 versus the results for posaconazole (one-way ANOVA with Tukey's test).

  • TABLE 5

    Antifungal effects of PC945 and known antifungal agents on four itraconazole-susceptible A. fumigatus strains, as determined using CLSI methodology

    Test agentMIC (μg/ml)a,cMIC50 (μg/ml)b,c
    MedianInterquartile rangeMedianInterquartile range
    PC9450.031*0.020–0.0310.011**0.0083–0.024
    Voriconazole0.50.5–0.50.140.082–0.15
    Posaconazole0.0470.031–6.00.0150.0095–0.016
    • ↵a Determined visually.

    • ↵b Determined using optical density measurements.

    • ↵c *, P < 0.05; **, P < 0.01 for PC945 versus the results for voriconazole (Kruskal-Wallis one-way ANOVA with Dunn's test).

  • TABLE 6

    Antifungal effects of PC945 and posaconazole on other fungal species

    Species (strain[s])No. of strains testedCulture methodMIC (μg/ml)a
    PC945VoriconazolePosaconazole
    Aspergillus carbonarius (ATCC 8740)1CLSI40.50.063
    Aspergillus flavus (ATCC 204304)1CLSI>820.13
    Aspergillus flavus (AFL8, NRRC3357)2EUCAST60.630.16
    Aspergillus niger (ATCC 1015)1EUCAST>810.20
    Aspergillus terreus (AT49, AT7130)2EUCAST0.07810.093
    Penicillium chrysogenum (ATCC 9480)1CLSI>820.13
    Penicillium citrinum (ATCC 9849)1CLSI>8>80.5
    Trichophyton rubrum (ATCC 10218)1CLSI0.0310.0630.031
    Aureobasidium pullulans (ATCC 9348)1CLSI>8>81
    Cladosporium argillaceum (ATCC 38013)1CLSI>80.50.25
    Candida albicans b (20240.047, ATCC 10231)2CLSI0.0810.140.081
    AR Candida albicansb,c (20183.073, 20186.025)2CLSI8.25108.13
    Candida glabrata b (ATCC 36583, R363)2CLSI0.58.130.5
    Candida krusei (ATCC 6258)1CLSI0.1250.250.125
    Chaetomium globosum (ATCC 44699)1CLSI>810.25
    Gibberella zeae (Fusarium graminearum) (ATCC 16106)1CLSI>8>8>8
    Cryptococcus gattii (clinical isolate)1EUCAST0.250.1250.5
    Cryptococcus neoformans (ATCC 24067)1CLSI0.0080.0160.016
    Lichtheimia corymbifera (ATCC 7909)1CLSI>8>8>8
    Mucor circinelloides (ATCC 8542)1CLSI>8>8>8
    Rhizomucor pusillus (ATCC 16458)1CLSI>8>8>8
    Rhizopus oryzae (ATCC 11145)1CLSI2>8>8
    • ↵a Due to the limited number of strains tested, the mean isolate MICs are presented.

    • ↵b The MIC given is the MIC50.

    • ↵c AR, azole resistant (fluconazole and voriconazole).

  • TABLE 7

    Potencies and persistence of action of PC945, posaconazole, and voriconazole

    Test agentHyphaeBEAS2B cells
    IC50 (μg/ml)aFold changeIC50 (μg/ml)Fold change
    No washoutWashoutNo washoutWashout
    PC9450.00010*0.0000860.870.00370.04311.5
    Voriconazole0.011>1>930.054>1>18.6
    Posaconazole0.000450.00224.900.00310.04614.7
    • ↵a *, P < 0.05 for PC945 versus the results for voriconazole (Kruskal-Wallis one-way ANOVA with Dunn's test).

  • TABLE 8

    In vivo activities of PC945 and posaconazole

    Test agentDose (μg/mouse)No. of survivors on day 7/total no. of mice (%)Median days of survivalLog rank (Mantel-Cox test) P valuea
    Vehicle 3/16 (19)5
    PC9450.564/8 (50)60.14
    2.85/8 (63)6.50.022*
    1410/16 (63)Undefined0.0095**
    Posaconazole2.81/8 (13)50.69
    147/16 (44)60.050*
    706/8 (75)Undefined0.0028**
    • ↵a *, P < 0.05; **, P < 0.01.

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In Vitro and In Vivo Antifungal Profile of a Novel and Long-Acting Inhaled Azole, PC945, on Aspergillus fumigatus Infection
Thomas Colley, Alexandre Alanio, Steven L. Kelly, Gurpreet Sehra, Yasuo Kizawa, Andrew G. S. Warrilow, Josie E. Parker, Diane E. Kelly, Genki Kimura, Lauren Anderson-Dring, Takahiro Nakaoki, Mihiro Sunose, Stuart Onions, Damien Crepin, Franz Lagasse, Matthew Crittall, Jonathan Shannon, Michael Cooke, Stéphane Bretagne, John King-Underwood, John Murray, Kazuhiro Ito, Pete Strong, Garth Rapeport
Antimicrobial Agents and Chemotherapy Apr 2017, 61 (5) e02280-16; DOI: 10.1128/AAC.02280-16

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In Vitro and In Vivo Antifungal Profile of a Novel and Long-Acting Inhaled Azole, PC945, on Aspergillus fumigatus Infection
Thomas Colley, Alexandre Alanio, Steven L. Kelly, Gurpreet Sehra, Yasuo Kizawa, Andrew G. S. Warrilow, Josie E. Parker, Diane E. Kelly, Genki Kimura, Lauren Anderson-Dring, Takahiro Nakaoki, Mihiro Sunose, Stuart Onions, Damien Crepin, Franz Lagasse, Matthew Crittall, Jonathan Shannon, Michael Cooke, Stéphane Bretagne, John King-Underwood, John Murray, Kazuhiro Ito, Pete Strong, Garth Rapeport
Antimicrobial Agents and Chemotherapy Apr 2017, 61 (5) e02280-16; DOI: 10.1128/AAC.02280-16
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KEYWORDS

antifungal agents
aspergillosis
Aspergillus fumigatus
Benzamides
Fungal Proteins
triazoles
Aspergillus fumigatus
azole
inhalation
CYP51
azole resistant
long acting

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