The Pharmacodynamics of Antifungal Agents Against Aspergillus

150 Pages·2013·37.65 MB·English

THE PHARMACODYNAMICS OF
ANTIFUNGAL AGENTS AGAINST
ASPERGILLUS

A thesis submitted to The University of Manchester for the degree of
Doctor of Medicine
in the Faculty of Medical and Human Sciences

2013

ADAM R JEANS
SCHOOL OF MEDICINE
CONTENTS

List of Figures 7
List of Tables 9
Abbreviations 10
Abstract 12
Declaration 13
Copyright 13
Acknowledgements 14
Preface 15
1. Introduction 16
1.1 Aspergillosis 16
1.1.1 Epidemiology 16
1.1.2 Acute invasive aspergillosis 17

1.1.3 Chronic aspergillosis 18
1.1.4 Treatment of aspergillosis 19
1.2 Susceptibility testing of Aspergillus species 20
1.2.1 Susceptibility testing methods 20
1.2.2 Clinical breakpoints 21
1.2.3 Epidemiological cut-off values 23
1.2.4 In vitro combination testing 23

1.3 Resistance to antifungal agents 26
1.3.1 Epidemiology of azole resistance 26
1.3.2 Mechanisms of azole resistance 27
1.3.3 Azole resistance in non-fumigatus Aspergillus species 29
1.3.4 Direct detection of azole resistance 30
1.3.5 Echinocandin and polyene resistance 30

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1.4 Antimicrobial pharmacokinetics and pharmacodynamics 31
1.4.1 Pharmacokinetics 31
1.4.2 Pharmacodynamics 32
1.5 Voriconazole 34
1.5.1 Pharmacology 34
1.5.2 Pharmacodynamics 36
1.5.3 Clinical studies 37
1.6 Anidulafungin 39
1.6.1 Pharmacology 39
1.6.2 Pharmacodynamics 39
1.6.3 Clinical studies 41
1.7 Voriconazole and echinocandin combination therapy 41
1.7.1 In vitro interactions 41

1.7.2 In vivo interactions 42
1.7.3 Clinical studies 43
1.8 Galactomannan 44
1.8.1 Galactomannan structure 44
1.8.2 Kinetics of galactomannan in vitro 45
1.8.3 Kinetics of galactomannan in vivo 46
1.8.4 Effect of antifungal treatment on galactomannan levels 47
1.8.5 Detection of galf antigen 48
1.8.6 Assay specificity 50
1.8.7 False positive results 51
1.8.8 False negative results 53
1.8.9 Clinical utility of the Platelia Aspergillus EIA 54
1.8.10 Galactomannan detection in urine 55
1.8.11 Galactomannan detection in BAL fluid 55

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1.9 In vitro model of invasive pulmonary aspergillosis 57
1.10 Project aims 60

2. Methods 61
2.1 Pharmacodynamics of voriconazole in a dynamic in vitro 61
model of invasive pulmonary aspergillosis
2.1.1 Aspergillus isolates and in vitro susceptibility testing 61
2.1.2 Dynamic in vitro model of the human alveolus 62
2.1.3 Bioreactors and construction of the dynamic model 63
2.1.4 Assessment of the integrity of the cellular bilayer 65
2.1.5 Inoculation of the dynamic model 65
2.1.6 Treatment with voriconazole 65
2.1.7 PK and PD of voriconazole 66
2.1.8 High performance liquid chromatography 66
2.1.9 Galactomannan 67
2.1.10 Mathematical modelling 68
2.1.11 Bridging to humans 69

2.2 Combination of voriconazole and anidulafungin in a 70
static in vitro model of invasive pulmonary aspergillosis
2.2.1 Aspergillus isolates and in vitro susceptibility testing 70
2.2.2 Static in vitro model of the human alveolus 70
2.2.3 Antifungal agents 71
2.2.4 Pharmacokinetics of voriconazole and anidulafungin 72
2.2.5 Pharmacodynamics of voriconazole and anidulafungin 72
combinations
2.2.6 Fluorescence microscopy 73
2.2.7 High performance liquid chromatography 73
2.2.8 Galactomannan 74
2.2.9 Data analysis and mathematical modelling 74
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3. Method development and preliminary data 76
3.1 Static in vitro model of the human alveolus 76
3.1.1 Exposure-response relationships 76
3.2 Dynamic in vitro model of the human alveolus 80
3.2.1 Initial experimental method 80
3.2.2 First preliminary experiment 81
3.2.3 Second preliminary experiment 82
3.2.4 Third preliminary experiment 85
3.2.5 Final experimental method 87

4. Pharmacodynamics of voriconazole in a dynamic in vitro model of 88
invasive pulmonary aspergillosis
4.1 Abstract 88

4.2 Introduction 89
4.3 Results 90
4.3.1 MICs 90
4.3.2 Dynamic in vitro model of the human alveolus 90
4.3.3 PK and PD of voriconazole in the dynamic model 91
4.3.4 Mathematical modelling 98
4.3.5 Bridging to humans, breakpoint determination and 100
targets for therapeutic drug monitoring
4.4 Discussion 102

5. Combination of voriconazole and anidulafungin for the treatment 106
of triazole resistant Aspergillus fumigatus in a static in vitro model
of invasive pulmonary aspergillosis
5.1 Abstract 106
5.2 Introduction 107

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5.3 Results 108
5.3.1 Susceptibility testing 108
5.3.2 Pharmacokinetics of voriconazole and anidulafungin 108
5.3.3 Pharmacodynamics of voriconazole and anidulafungin 110
combinations
5.3.4 Microscopy 110
5.4 Discussion 124

6. Discussion 128

7. References 133

Word count: 38,826

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LIST OF FIGURES

Figure 1 Macroscopic and microscopic appearance of A. fumigatus 16
Figure 2 Structure of galactomannan 44
Figure 3 The Platelia Aspergillus ELISA technique 48
Figure 4 In vitro model of invasive pulmonary aspergillosis 58
Figure 5 Bioreactor and circuit design 64
Figure 6 Exposure-response relationship of voriconazole for the 77
wild-type and TR/L98H carrying strains
Figure 7 Exposure-response relationship of voriconazole for the 78
G138C and G434C carrying strains

Figure 8 Exposure-response relationship of anidulafungin for the 79
wild-type and G138C carrying strains
Figure 9 First preliminary dynamic model experiment 82
Figure 10 Second preliminary dynamic model experiment 84
Figure 11 Third preliminary dynamic model experiment 86
Figure 12 Integrity of the cellular bilayer in the dynamic model 91
Figure 13 PK and PD of voriconazole against the wild-type strain 93
Figure 14 PK and PD of voriconazole against the strain carrying TR/L98H 94
Figure 15 PK and PD of voriconazole against the strain carrying G138C 95
Figure 16 PK and PD of voriconazole against the strain carrying G434C 96
Figure 17 Reproducibility of the PK and PD data 97
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Figure 18 Relationship between AUC:MIC and galactomannan 99
Figure 19 Fractional target attainments 101
Figure 20 PK of voriconazole and anidulafungin in the static model 109
Figure 21a Combination of voriconazole and anidulafungin for the 114
wild-type strain: galactomannan plotted against voriconazole
Figure 21b Combination of voriconazole and anidulafungin for the 115
wild-type strain: galactomannan plotted against anidulafungin
Figure 22a Combination of voriconazole and anidulafungin for the TR/L98H 116
carrying strain: galactomannan plotted against voriconazole
Figure 22b Combination of voriconazole and anidulafungin for the TR/L98H 117
carrying strain: galactomannan plotted against anidulafungin
Figure 23a Combination of voriconazole and anidulafungin for the G138C 118
carrying strain: galactomannan plotted against voriconazole

Figure 23b Combination of voriconazole and anidulafungin for the G138C 119
carrying strain: galactomannan plotted against anidulafungin
Figure 24 Fitted surfaces describing the effect of the combination of 120
voriconazole and anidulafungin for three strains of A. fumigatus
Figure 25 Effect induced by the combination of voriconazole and 121
anidulafungin expressed as the AUC:MIC ratio
Figure 26 Fluorescence microscopy of the wild-type GFP strain 123

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LIST OF TABLES

Table 1 EUCAST clinical breakpoints for A. fumigatus 23
Table 2 MIC distributions and ECVs for A. fumigatus 24
Table 3 Pharmacokinetic properties of voriconazole and anidulafungin 34
Table 4 Strains of A. fumigatus used in the dynamic in vitro model 90
Table 5 Strains of A. fumigatus used in the static in vitro model 108
Table 6 Galactomannan indices for voriconazole and anidulafungin 111
combinations against the wild-type strain
Table 7 Galactomannan indices for voriconazole and anidulafungin 112
combinations against the TR/L98H strain
Table 8 Galactomannan indices for voriconazole and anidulafungin 113
combinations against the G138C strain

Table 9 Parameter estimates describing the interaction of voriconazole 122
and anidulafungin
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ABBREVIATIONS

ABPA Allergic Bronchopulmonary Aspergillosis
AFG Anidulafungin
AUC Area Under the Curve
BAL Bronchoalveolar Lavage
CGD Chronic Granulomatous Disease
CI Confidence Interval
CLSI Clinical Laboratory Standards Institute
CPA Chronic Pulmonary Aspergillosis
CT Computed Tomography
CV Coefficient of Variation
DMEM Dulbecco’s Modified Eagle Medium
DNA Deoxyribonucleic Acid
EBM-2 Endothelial Basal Medium 2
ECM Extracellular Matrix
ECV Epidemiological Cut-off Value
EGM-2 Endothelial Growth Medium 2
EIA Enzyme Immuno Assay
ELISA Enzyme-linked Immunosorbent Assay
EUCAST European Committee on Antimicrobial
Susceptibility Testing
FBS Foetal Bovine Serum
FIC Fractional Inhibitory Concentration
FTA Fractional Target Attainment
GAG Galactosaminogalactan
GFP Green Fluorescent Protein
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