Table of contents Index Title Index Index of Tables Index of Figures Glossary Acknowledgements Page I VI X XIV XVII

Chapter 1 Introduction 1.1 Corticosteroids 1.2 Mechanism of action of corticosteroids 1.3 Formulation of corticosteroids 1.3.1 Inhaled corticosteroids 1.3.1.1 Inhaler devices 1.3.2 Intranasal corticosteroids 1.3.3 Oral corticosteroids 1.4 Pharmacokinetics 1.4.1 Absorption 1.4.1.1 Oral administration 1.4.1.2 Inhaled corticosteroids 1.4.1.3 Intranasal corticosteroids 1.4.2 Distribution 1.4.3 Metabolism 1.4.4 Elimination 1.5 Pharmacodynamics 1.5.1 Plasma protein binding 1.5.2 Bioavailability 1.5.3 Glucocorticoid receptor affinity 1.5.4 Lipophilicity 1.5.5 Lipid conjugation 1.6 Corticosteroids available in the United Kingdom 1.6.1 Prednisolone 1.6.2 Beclometasone dipropionate (BDP) 1.6.3 Budesonide (BUD) 1.6.4 Fluticasone propionate (FP) 1.6.5 Mometasone furoate (MF) 1.6.6 Betamethasone (BETA) 1.6.7 Triamcinolone acetonide 1.7 Problems with corticosteroids 1.7.1 Hypothalamic adrenal suppression 1.7.1.1 Basal adrenal function tests 1.7.2 Corticosteroid induced osteoporosis 1.7.2.1 Mechanism of corticosteroid induced osteoporosis 1.7.2.2 Bone density assessment 1.7.3 Corticosteroid induced diabetes 1.7.4 Corticosteroid induced high blood pressure 1.7.5 Other adverse effects

1 3 4 4 4 6 7 7 7 8 8 8 10 10 11 12 12 12 13 14 15 15 15 16 17 20 23 25 26 27 27 30 31 34 35 37 37 38

1.7.5.1 1.7.5.2 1.8 1.8.1 1.8.2 1.8.2.1 1.8.2.2 1.9 1.9.1 1.9.2 1.9.3 1.10 1.10.1 1.10.2 1.10.3 1.11 1.12

Cataracts Local side effects Asthma definition Pathology of asthma Pharmacological management of asthma Stable asthma Acute exacerbations of asthma Rhinitis Pathology of rhinitis Pharmacological management of rhinitis. Corticosteroids in rhinitis Nasal Polyposis Pathology of nasal polyposis Pharmacological management of nasal polyposis Corticosteroids in nasal polyposis Aim Objectives

38 39 39 39 41 42 45 47 48 49 50 51 52 53 54 56 56

Chapter 2 Method Development 2.1 Detection of corticosteroids in biological fluids 2.2 Sample preparation 2.2.1 Protein precipitation 2.2.2 Solid-phase extraction (SPE) 2.2.2.1 Pre-Conditioning 2.2.2.2 Sample Loading 2.2.2.3 Sample Washing 2.2.2.4 Elution of the Analytes 2.2.3 Liquid-liquid extraction (LLE) 2.3 Methods utilised in the quantification of corticosteroids 2.3.1 Stationary phase 2.3.2 Mobile phase 2.3.3 Internal and external standards 2.3.4 Detector 2.3.4.1 Wavelength of detection 2.4 Experimental 2.4.1 Materials 2.4.1.1 Chemicals and reagents 2.4.1.2 Stationary phases and solid phase extraction cartridges 2.4.1.3 Instrumentation 2.4.1.4 Mobile phases 2.4.2 Methods 2.4.2.1 Stock solution and standards 2.4.2.2 Preparation of samples for analysis of corticosteroids 2.4.2.3 High Performance Liquid Chromatography method development 2.4.2.4 Extraction method optimisation

57 57 59 59 61 62 62 62 63 63 65 66 67 68 69 70 70 70 70 71 75 75 75 82 83 84

II

2.5 2.5.1 2.5.1.1 2.5.1.2 2.5.1.3 2.5.1.3.1 2.5.1.3.2 2.5.1.3.3 2.5.2 2.5.2.1 2.5.2.2 2.5.2.2.1 2.5.2.2.2 2.6 2.6.1 2.6.2 2.6.3 2.6.4 2.6.5 2.6.6 2.6.7 2.6.7.1 2.6.7.2 2.6.7.3 2.6.7.4 2.6.8 2.7

Results and Discussion Optimisation of the chromatographic method for the analysis of corticosteroids. Selection of wavelength of detection Selection of mobile phase and stationary phase Selection of internal standard Dexamethasone acetate Dexamethasone Mometasone and Betamethasone Analysis of plasma samples Liquid liquid extraction (LLE) Solid-phase extraction (SPE) SPE Method 1 SPE Method 2 Analytical method validation Resolution Accuracy Precision Linearity Asymmetry Factor Specificity Selectivity Relative retention time (RRT) Relative response factor (RRF) Capacity factor (k) Selectivity () Limit of detection (LOD) and limit of quantification (LOQ) Summary of the method development study

86 86 86 91 112 1123 1167 1189 1201 121 123 123 124 127 128 128 130 131 134 134 136 137 138 138 140 140 145

Chapter 3 Clinical Data 3.1 Introduction 3.1.1 Markers of disease 3.1.1.1 Sino Nasal Outcome Test (SNOT-22) 3.1.1.2 Asthma Control Questionnaires (ACQ) 3.1.1.3 Fraction of Exhaled Nitric Oxide (FENO) 3.1.1.4 Nasal endoscopy score and nasal polyp grade 3.1.2 Markers of adrenal suppression 3.1.2.1 Salivary cortisol 3.1.3 Markers of osteoporosis 3.2 Aim 3.3 Objectives 3.4 Methods 3.4.1 Study design 3.4.2 Study population 3.4.2.1 Clinical sites 3.4.2.2 Selection of participants 3.4.2.3 Process of recruitment

148 148 148 149 150 151 151 152 153 154 154 155 155 155 155 155 157

III

3.4.3 3.4.4 3.4.4.1 3.4.4.1.1 3.4.4.1.2 3.4.4.1.3 3.4.4.2 3.4.4.2.1 3.4.4.2.2 3.4.4.2.3 3.4.4.2.4 3.4.4.2.5 3.4.4.2.6 3.4.4.2.7 3.4.5 3.5 3.5.1 3.5.2 3.5.2.1 3.5.2.2 3.5.2.3 3.5.2.4 3.5.2.5 3.5.2.6 3.5.2.6.1 3.5.2.6.2 3.5.2.7 3.5.2.8 3.6 3.6.1 3.6.1.1 3.6.1.2 3.6.1.3 3.6.1.4 3.6.1.5 3.6.1.6 3.6.1.7 3.7 Chapter 4 4.1 4.1.1 4.1.2 4.2 References

158 Ethical considerations 158 Data collection Questionnaires 158 Sino Nasal Outcome Test (SNOT-22) 158 Asthma Control Questionnaire (ACQ) 158 Clinical Study Questionnaire 158 Assessment of disease progression 159 Collection of salivary cortisol 159 Plasma analysis 159 Nasal polyp grade 161 Dual Energy X-ray Absorptiometry (DEXA) scan 162 Measurement of blood pressure 162 Measurement of blood glucose 162 Fraction exhaled nitric oxide (FENO) 163 1634 Data analysis 1645 Results 1645 Demographics 165 Clinical findings and effects of corticosteroids Salivary cortisol 165 Measurement of plasma concentration of 171 corticosteroids Correlation between corticosteroid dose and the risk 175 of osteoporosis Hypertension 180 Hyperglycemia 188 The symptom scores 190 Sino Nasal Outcome Test (SNOT-22) score 190 Asthma Control Questionnaire (ACQ) score 192 Steroid treatment card 193 First visit against all visits 194 207 Discussion 207 Principal findings Adrenal suppression 207 Osteoporosis 210 Hypertension 212 Diabetes mellitus 214 Steroid treatment card 216 Symptoms score 216 Fraction of exhaled nitric oxide (FENO) 217 218 Conclusion

Conclusions Strength and limitations of study Strengths Limitations General conclusion and future work

219 219 221 225 227

IV

Appendices Appendix 3.1 Appendix 3.2 Appendix 3.3 Appendix 3.4 Appendix 3.5 Appendix 3.6 Appendix 3.7

Sino Nasal Outcome Test (SNOT-22) Asthma Control Questionnaires (ACQ) Patient information leaflet Consent form Clinical study questionnaire Salivary sample collection instruction Steroid treatment card

Index of Tables

Index Table 1.1 Table 1.2 Table 1.3 Table 1.4 Table 1.5 Table 1.6 Table 2.1 Table 2.2 Table 2.3

Title The structure of corticosteroids Classification of bone mass density (BMD) and T-score (NOS, 2008; SIGN, 2003) Comparison of asthma and rhinitis (Bousquet et al., 2003; Meltzer et al., 2004) Comparison of seasonal and perennial rhinitis (adapted from: Meltzer, 2004; Bousquet et al., 2003) Pharmacological management of rhinitis The stages of nasal polyposis (Fokkens et al., 2005) Properties of columns used in the analysis High Performance Liquid Chromatography conditions. (a) isocratic programme and (b) gradient programme An example of the final concentration for preparation of calibration standard solutions (0.5 g ml-1 - 10 g ml-1) from Reference Standard A An example of the final concentration for preparation of calibration standard solutions (0.5 g ml-1 - 10 g ml-1) from Reference Standard F An example of the final concentration for preparation of calibration standard solutions (0.5 g ml-1 - 10 g ml-1) from Reference Standard G Actual concentration of UV stock solutions of each analyte for ultraviolet analysis Comparison between reported and measured maximal absorbance values and wavelength spectra of each analyte Chromatographic data of the analytes in the corticosteroid mixture (20 g ml-1) at two different wavelengths obtained using C18 Kromasil; gradient mobile phase (method GRAD D4); Flow rate: 1.0 ml min-1. UV: 239 nm The proportions of HPLC mobile phase for spiked solution containing corticosteroids (40 g ml-1) performed on column C18 ACE (150 x 4.6 mm (i.d), 5 m (par.)); Flow rate of 1.5 ml min-1 Resolution of the compounds performed on column B, C18 ACE column with mobile phase (a) methanol-water (ISO B1) and (b) acetonitrile-water (ISO B2). Flow rate: 1.5 ml min-1 The capacity factor of a spiked solution containing corticosteroids (40 g ml-1) performed on column B, C18 ACE (150 x 4.6 mm (i.d), 5 m (par.)) with isocratic mobile phase, ISO B1 [methanol-water (59:41 v/v)]

Page 2 36 47 48 50 52 71 72 79

Table 2.4

80

Table 2.5

81

Table 2.6 Table 2.7 Table 2.8

82 89 90

Table 2.9

94

Table 2.10

97

Table 2.11

98

VI

Table 2.12

Table 2.13

Table 2.14

Table 2.15

Table 2.16

Table 2.17

Table 2.18

Table 2.19

Table 2.20

Table 2.21

Table 2.22

Gradient programme used with C18 ACE column in the chromatographic analysis of a spiked solution containing corticosteroids (40 g ml-1) Gradient method used with C18 Hypersil BDS column in the chromatographic analysis of a spiked solution containing corticosteroids (40 g ml-1) Gradient programme used with the C18 Kromasil column in the chromatographic analysis of spiked solution containing corticosteroids (40 g ml-1) Gradient programme used with the C18 Hypersil BDS column in the chromatographic analysis of a spiked solution containing corticosteroids (40 g ml-1) The relative response factor (RRF) of Reference Solution A1 spiked with the internal standards at concentration of 0.5 g ml-1 Recovery of corticosteroids from plasma using liquidliquid extraction (n = 3).HPLC conditions: C18 Kromasil column (250 x 4.6 mm (i.d), 5 m (par.)); gradient mobile phase GRAD D4; detector wavelength: 239 nm; flow rate: 1 ml min-1 Mean recovery (%) of corticosteroids from human plasma after SPE method 1 at 40 g ml-1 with ethyl acetate: heptane (35:65) as the eluting solvent. HPLC condition: C18 Kromasil column (250 x 4.6 mm (i.d), 5 m (par.)); gradient mobile phase GRAD D4; detector wavelength: 239 nm; flow rate: 1 ml min-1 The extraction efficiency of solid phase extraction (SPE method 2) at different drying times after loading step. HPLC conditions: C18 Kromasil column (250 x 4.6 mm (i.d), 5 m (par.)); gradient mobile phase GRAD D4; detector wavelength:239 nm; flow rate: 1 ml min-1 The extraction efficiency of solid phase extraction with different elution solvent ratios. HPLC conditions: C18 Kromasil column (250 x 4.6 mm (i.d), 5 m (par.)); gradient mobile phase GRAD D4; detector wavelength: 239 nm; flow rate: 1 ml min-1 The mean recovery (%) of corticosteroids from plasma spiked with internal standard. HPLC conditions: C18 Kromasil column (250 x 4.6 mm (i.d), 5 m (par.)); gradient mobile phase GRAD D4; detector wavelength: 239 nm; flow rate: 1 ml min-1 Chromatographic data obtained for Reference solution A1 containing a corticosteroid mixture (10 g ml-1) spiked with internal standard (betamethasone or mometasone) (0.5 g ml-1)

100

103

110

111

120

122

123

125

126

127

128

VII

Table 2.23

Table 2.24

Table 2.25

Table 2.26

Table 2.27 Table 2.28

Table 2.29

Table 2.30

Table 2.31

Table 2.32

Table 2.33

Table 2.34 Table 2.35 Table 2.36 Table 2.37

The mean recovery (%) of a series of corticosteroid concentrations from plasma spiked with betamethasone (0.5 g ml-1) as the internal standard The mean recovery (%) of a series of corticosteroid concentrations from plasma spiked with mometasone (0.5 g ml-1) as the internal standard The precision (% RSD) of a series of corticosteroid concentrations from plasma spiked with betamethasone (0.5 g ml-1) as the internal standard The precision (% RSD) of a series of corticosteroid concentrations from plasma spiked with mometasone (0.5 g ml-1) as the internal standard Asymmetry factor of each analyte spiked with the internal standard (0.5 g ml-1) Chromatographic data obtained for a spiked solution of Reference solution A1 with the internal standards, betamethasone or mometasone (0.5 g ml-1) Chromatographic data obtained for a spiked solution of Reference solution A1 with the internal standards, betamethasone or mometasone (0.5 g ml-1) Chromatographic data obtained for a spiked solution of Reference solution A1 with the internal standard betamethasone (0.5 g ml-1) Chromatographic data obtained for a spiked solution of Reference solution A1 with the internal standard mometasone (0.5 g ml-1) Limit of detection and limit of quantification (LOD and LOQ) determined from Reference solution A1 spiked with the internal standard, betamethasone (0.5 g ml-1) Limit of detection and limit of quantification (LOD and LOQ) determined from Reference solution A1 spiked with the internal standard, mometasone (0.5 g ml-1) Validation parameters for the corticosteroid mixture with betamethasone (0.5 g ml-1) as the internal standard Validation parameters for the corticosteroid mixture with mometasone (0.5 g ml-1) as the internal standard Analytical method for the analysis of corticosteroids in plasma Sample extraction method for the analysis of corticosteroids in plasma

129

130

131

131

134 137

138

139

139

141

142

143 144 146 147

Table 3.1 Table 3.2 Table 3.3

Classification of bone mass density (BMD) and T-score 153 (NOS, 2008; SIGN, 2003) Inclusion and exclusion criteria for the clinical study 156 Sample extraction method for the analysis of 160 corticosteroids in plasma

VIII

Table 3.4 Table 3.5 Table 3.6 Table 3.7 Table 3.8 Table 3.9

Analytical method for the analysis of corticosteroids in plasma Nasal polyp grade Baseline demographics of the study participants Subset of patients prescribed intranasal corticosteroids alone (n = 47) Subset of patients treated with inhaled and intranasal (n = 85) Correlation between SBP and DBP and corticosteroid dose

161 162 164 169 170 187

IX

Index of Figures Index Figure 1.1 Figure 2.1 Figure 2.2 Figure 2.3 Title The basic corticosteroid structure Solid Phase Extraction Overlay UV Absorption spectra of the mixture of the eight corticosteroids under investigation Representative chromatogram of a spiked solution (Reference Solution A5) containing corticosteroids (0.5 g ml-1) using C8 Ultrasphere 150 x 4.6 mm (i.d), 5 m (par.); isocratic mobile phase [methanol-water (59:41 v/v)]; Flow rate: 1.5 ml min-1. Dexamethasone acetate was used as the internal standard (IS) Representative chromatograms of the spiked solution containing corticosteroids (40 g ml-1) performed on C18 ACE column with mobile phase (a) ISO B1 and (b) ISO B2. Flow rate: 1.5 ml min-1. Dexamethasone acetate was used as the internal standard Representative chromatogram of a spiked solution containing corticosteroids (40 g ml-1) performed on C18 ACE column with gradient mobile phase (methanol-water) (method GRAD B3). Dexamethasone acetate was used as the internal standard (IS) Representative chromatogram of spiked solution corticosteroids (40 g ml-1) performed on C18 Hypersil BDS column with isocratic mobile phase [methanol-water (59:41 v/v)] (ISO C1) Representative chromatogram of a spiked solution containing corticosteroids (40 g ml-1) performed on C18 Hypersil BDS column with gradient mobile phase (methanol-water) (method GRAD C3) Representative chromatogram of BDP (40 g ml-1) performed on the C18 Hypersil column with the gradient mobile phase (methanol-water) (method GRAD C4) Representative chromatographic data of BDP and its degradants using the C18 Hypersil column (100 x 4.6 mm (i.d), 5 m (par.)); [methanol 0.1 % formic acid in water (62:38 v/v)]; Flow rate of 1.0 ml min-1. (a) Chromatogram of beclometasone dipropionate (tR = 18.73 min) with its degradation product at tR = 6.95 min and 7.64 min. Mass spectra of the (b) BDP; (c) 17-BMP and (d) 21-BMP Representative chromatogram of a spiked solution containing corticosteroids (40 g ml-1) performed on the C18 Kromasil column with an isocratic mobile phase [(methanol-water (62:38)] (method ISO D2). Flow rate: 1 ml min-1. UV detector: 239 nm. Dexamethasone acetate was the IS Page 2 61 88 93

Figure 2.4

95

Figure 2.5

100

Figure 2.6

102

Figure 2.7

104

Figure 2.8

105

Figure 2.9

107

Figure 2.10

109

Figure 2.11

Figure 2.12

Figure 2.13 Figure 2.14

Figure 2.15

Figure 2.16

Figure 2.17

Figure 2.18

Figure 2.19

Representative chromatogram of a spiked solution containing corticosteroids (40 g ml-1) performed on the C18 Kromasil column with the gradient mobile phase (methanol-water) (method GRAD D4) Representative chromatogram of a solution spiked with dexamethasone-21-acetate which eluted at tR = 29.8 min and dexamethasone which eluted at tR = 12.2 min using a Kromasil C18 column (250 x 4.6 mm (i.d), 5 m (par.)) at 25 C with an isocratic mobile phase [methanol-water (62:38 v/v)] (method ISO 4b); Flow rate: 1 ml min-1 Representative mass spectra of the dexamethasone-21acetate degradation product dexamethasone (m/z 391.33) The degradation of dexamethasone-21-acetate over time using a Kromasil C18 column (250 x 4.6 mm (i.d), 5 m (par.)) at 25 C with an isocratic mobile phase [methanolwater (62:38 v/v)] (method ISO D2) The peak area of dexamethasone, the degradant of dexamethasone-21-acetate using the Kromasil C18 column (250 x 4.6 mm (i.d), 5 m (par.)) at 25 C with an isocratic mobile phase [methanol-water (62:38 v/v)] (method ISO D2) Representative chromatogram of dexamethasone and betamethasone which coeluted at tR = 17.89 min. The analysis was run on a C18 Kromasil column (250 x 4.6 mm (i.d), 5 m (par.)) with an isocratic mobile phase [methanol-water (62:38 v/v)] Dexamethasone and betamethasone share a similar chemical structure: the only difference is at position C-16 (as shown). Calibration curves of spiked solutions (Reference solution A1 A5) containing the corticosteroid mixture (0.5 10 g ml-1) spiked with (a) betamethasone (0.5 g ml-1); (b) mometasone (0.5 g ml-1) as the internal standard Representative chromatograms for (a) blank plasma; (b) plasma spiked with 10 g ml-1 corticosteroid mixture with the internal standard (0.5 g ml-1) (bi) betamethasone; or (bii) mometasone Flow chart of patient recruitment, enrolment and continuation in the clinical study Correlation between the measured salivary cortisol and daily corticosteroid burden (n = 155) The salivary cortisol according to administration route of corticosteroids Comparison between salivary cortisol from patients on oral corticosteroid and patients not on oral corticosteroids

112

114

115 116

116

118

118

133

136

Figure 3.1 Figure 3.2 Figure 3.3 Figure 3.4

157 165 167 168

XI

Figure 3.5

Figure 3.6 Figure 3.7 Figure 3.8 Figure 3.9 Figure 3.10 Figure 3.11 Figure 3.12

Figure 3.13

Figure 3.14

Figure 3.15

Figure 3.16

Figure 3.17 Figure 3.18 Figure 3.19

Figure 3.20

Figure 3.21

Correlation between salivary cortisol and nasal polyp grade in patients attending the GRI clinic (n = 130). The p value is in comparison to polyp grade C Correlation between plasma concentration of prednisolone and daily prednisolone dose (n = 23) Correlation between plasma concentration of prednisolone and corticosteroid burden (n = 23) Correlation between salivary cortisol and plasma concentration of prednisolone (n = 10) Correlation between lumbar spine (a) Z-score and (b) Tscore with daily corticosteroid burden (n = 16) (p = 0.786) Correlation between femoral neck (a) Z-score and (b) Tscore with daily corticosteroid burden (n = 16) (p = 0.878) Correlation between total hip (a) Z-score and (b) T-score with daily corticosteroid burden (n = 16) (p = 0.433) Correlation between (a) systolic blood pressure (SBP) and (b) diastolic blood pressure (DBP) with daily corticosteroid burden in all recruited patients (n = 259) Correlation between (a) systolic blood pressure (SBP) and (b) diastolic blood pressure (DBP) with daily corticosteroid burden in patients not on antihypertensive medications (n = 229) Correlation between (a) systolic blood pressure; (b) diastolic blood pressure (DBP) and daily corticosteroid burden in patients on antihypertensive medications (n = 30) Correlation between random blood glucose (RBG) and daily corticosteroid burden in all recruited patients (n = 255) Correlation between random blood glucose (RBG) and daily corticosteroid burden in patients not prescribed antidiabetic medications (n = 251) Correlation between salivary cortisol against sino nasal outcome test (SNOT-22) score (n = 129) Correlation between salivary cortisol and the asthma control questionnaire (ACQ) score (n = 18) Salivary cortisol against daily corticosteroid burden at (a) first visit (n = 66) and (b) all visit (n = 155) (Fisher transformation r-to-z; p = 0.448) Systolic blood pressure against daily corticosteroid burden at (a) first visit (n = 104) and (b) all visits (n = 259) (Fisher transformation r-to-z; p = 0.352) Diastolic blood pressure against daily corticosteroid burden at (a) first visit (n = 104) and (b) all visits (n = 259) (Fisher transformation r-to-z; p = 0.337)

171

173 174 175 177 178 179 182

184

186

189

190

191 193 195

197

199

XII

Figure 3.22

Figure 3.23

Figure 3.24

Random blood glucose against daily corticosteroid burden 201 at (a) first visit (n = 98) and (b) all visits (n = 256) (Fisher transformation r-to-z; p = 0.264) Salivary cortisol against SNOT-22 at (a) first visit (n = 59) 203 and (b) all visits (n = 129) (Fisher transformation r-to-z; p = 0.125) Salivary cortisol against ACQ score at (a) first visit (n = 6) 205 and (b) all visits (n = 18) (Fisher transformation r-to-z; p = 0.452)

XIII

Glossary Abbreviation 17-BMP 21-BMP ACN ACQ ACTH ALP AP As ATS AUC0-24h BDP BETA BHS BMD BMI BOH BP BTS BUD CFC CRS CT DBP DEXA DPI EDQM ENT EP ERS ESI FACET FDA FENO FESS FEV1 FP GINA GLUT-4 GM-CSF GOAL GRAD GRI HFA HPA HPLC Text in full Beclometasone-17-monopropionate Beclometasone-21-monopropionate Acetonitrile Asthma control questionnaires Adrenocorticotropic hormone Alkaline phosphatase Activator protein Symmetry factor American Thoracic Society 24 - hour plasma area under curve Beclometasone dipropionate Betamethasone British Hypertension Society Bone mineral density Body mass index Beclometasone British Pharmacopoeia British Thoracic Society Budesonide Chlorofluorocarbon Chronic Rhinosinusitis Computed tomography Diastolic blood pressure Dual Energy X-ray Absorptiometry Dry powder inhalers European Directorate for the Quality of Medicines and Healthcare Ear, Nose and Throat European Pharmacopoeia European Respiratory Society Electrospray ionisation Formoterol and Corticosteroids Establishing Therapy Food and Drug Administration Fraction of exhaled nitric oxide Functional Endoscopic Sinus Surgery Forced expiratory volume in 1 second Fluticasone Global Initiative for Asthma Glucose transporter 4 Granulocyte-macrophage colony-stimulating factor Gaining Optimal Asthma Control Gradient Glasgow Royal Infirmary Hydrofluoroalkane Hypothalamic adrenal suppression High performance liquid chromatography

XIV

i.d ICAM IgE IL IQR IS ISO k kg m-2 LABA LC LC-MS LLE LOD LOQ LTC LTRA m/z MeOH MF mg mmHg mmol L-1 MHRA MS N n NF-kB NHS NIH nmol L-1 NMR NO NOS NOS NP-LC P1NP Par. PaO2 PEFR PgD pMDI PNIF ppb PRED RSD RANTES RBG

Internal diameter Intercellular adhesion molecule Immunoglobulin Interleukin Interquartile range Internal standard Isocratic Capacity factor Kilogram per square metre Long-acting beta-agonist Liquid chromatography Liquid chromatography mass spectrometry Liquid-liquid extraction Limit of detection Limit of quantification Leukotriene Leukotriene receptor antagonists Mass-to-charge ratio Methanol Mometasone furoate Milligram Millimeters of mercury Millimoles per litre Medicines and Healthcare products Regulatory Authority Mass spectrometry Theoretical plates Number of samples Inhibitor of nuclear factor National Health Service National Institutes of Health Nanomoles per litre Nuclear magnetic resonance Nitric oxide Nitric oxide synthase National Osteoporosis Society Normal phase liquid chromatography Procollagen I N-terminal extension peptide Particle Partial pressure of oxygen in the blood Peak expiratory flow rate Prostaglandin Pressurised metered dose inhaler Peak nasal inspiratory flow Parts per billion Prednisolone Relative standard deviation Regulated on Activation Normal T Cell Expressed and Secreted Random blood glucose

XV

RIA RP-HPLC RP-LC RRF RRFIS RRFUV RRT Rs s.d S/N SABA SBP SF-36 SH SIGN SMART SNOT-20 SNOT-22 SPE SpO2 SST TGF TH2 THF TNF- tR UV v/v VCAM VD WHO max

Radioimmunoassay Reverse phase high performance liquid chromatography Reverse phase liquid chromatography Relative response factor Response factor relative to the internal standard Response factor relative to the UV standard Relative retention time Resolution Standard deviation Signal-to- noise ratio Short-acting beta-agonist Systolic blood pressure Medical Outcomes Study 36-Item Short Form Health Survey Stobhill Hospital Scottish Intercollegiate Guideline Network Symbicort Maintenance And Reliever Therapy Sino Nasal Outcome Test -20 Questionnaire Sino Nasal Outcome Test -22 Questionnaire Solid-phase extraction Oxygen saturation Short synacthen test Transforming growth factor T helper cells type 2 tetrahydrofuran tumor necrosis factor- Retention time Ultraviolet Volume-per-volume Vascular cell adhesion molecule Volume of distribution World Health Organisation Selectivity factor Wavelength at maximal UV absorption

XVI

Acknowledgement
It is pleasure to thank the many people who made this thesis possible.

I would like to first and foremost acknowledge and thank my supervisors, Dr Anne Boyter and Dr Oliver Sutcliffe for their mentorship, assistance and patience with me for the past 3 years. They have taught me many lessons, both scientific and life, and for this I am deeply indebted to both of them.

I also express my thanks to Mr Gerald McGarry and Dr Christine Bucknall, who allowed me to conduct my research at their clinics, the Ear, Nose and Throat (ENT) clinic, Glasgow Royal Infirmary (GRI) and the Problem Asthma clinic, Stobhill Hospital (SH) and to all hospital staff for their contribution and kindness during the study. I record grateful thanks to the volunteered participants who willingly

sacrificed their time and energy to participate in this research, and frankly shared their views and experiences and who gave me so much important data for analysis.

I would like to thank Dr RuAngelie Edrada-Ebel and Norman McIver for their technical assistance and friendship throughout my PhD process and my many colleagues for supporting me through the difficult times and for all the emotional support, entertainment, and caring they provided.

Lastly, and most importantly, I wish to thank my family, my beloved parents, Ahmad Mazlan Osman and Zainun Mohd Yusoff, my brother, Ahmad Naddi and my sister,

XVII

Natrah Amira. They bore me, raised me, supported me, taught me, and loved me. I hope that I can continue to make them proud.

To them I dedicate this thesis.

XVIII