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Pharmacokinetic and pharmacodynamic evaluation of mometasone furoate

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Pharmacokinetic and pharmacodynamic evaluation of mometasone furoate
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Sahasranaman, Srikumar
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iv, 138 leaves : ill. ; 29 cm.

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Asthma ( jstor )
Corticosteroids ( jstor )
Glucocorticoid receptors ( jstor )
Inhalation ( jstor )
Lungs ( jstor )
Metabolism ( jstor )
Metabolites ( jstor )
Plasmas ( jstor )
Rats ( jstor )
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Dissertations, Academic -- Pharmaceutical Sciences-Pharmacy -- UF ( lcsh )
Pharmaceutical Sciences-Pharmacy thesis, Ph. D ( lcsh )
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Thesis (Ph. D.)--University of Florida, 2004.
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by Srikumar Sahasranaman.

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PHARMACOKINETIC AND PHARMACODYNAMIC EVALUATION OF
MOMETASONE FUROATE














By

SRIKUMAR SAHASRANAMAN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2004




PHARMACOKINETIC AND PHARMACODYNAMIC EVALUATION OF
MOMETASONE FUROATE
By
SRIKUMAR SAHASRANAMAN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2004


Copyright 2004
by
Srikumar Sahasranaman


For my Matha, Pitha, Guru, Deivam and Sangi.


ACKNOWLEDGMENTS
I wish to convey my sincere thanks and appreciation to my mentor, Dr. Guenther
Hochhaus, for his patient guidance, constant encouragement and constructive criticism
throughout the course of my work. I would like to thank the members of my supervisory
committee, Dr. Hartmut Derendorf, Dr. Jeffrey Hughes and Dr. Scott Powers, for their
advice and availability. I take this opportunity to thank Dr. Staffan Edsbacker and
AstraZeneca for funding this work. I also thank Prof. Anne Tattersfield and Dr. Kevin
Mortimer, University of Nottingham, for their collaboration.
I am thankful to Manish for his help and support during the course of this work. I
also appreciate the laboratory help provided by Mrs. Yufei Tang, Ms. Anke Czerwinski
and Ms. Diana Biniasz. I owe many thanks to Dr. Margaret James for allowing the use of
her laboratory facilities and to Dr. Gabor Toth and Dr. Gyula Horvath for the NMR
analysis in this work. 1 am grateful to the secretaries of the Department of
Pharmaceutics, Mr. James Ketcham. Mrs. Patricia Khan, Ms. Andrea Tucker and Ms.
Vada Taylor, for their technical and administrative assistance. I thank my group
members, Vikram. Yaning. Intira, Kai, Elanor and Zia. and other graduate students and
post-docs in the department for their assistance and friendship. Special thanks go to
Kula, Hari, Vijay, Manan, Subbi and all my other friends for their help and support that
have made my stay in Gainesville a memorable one.
IV


Finally, I want to express my deep gratitude and love for my parents, sister and
other family members for their unwavering support and encouragement of my academic
pursuits over the years.
v


TABLE OF CONTENTS
page
ACKNOWLEDGMENTS iv
LIST OF TABLES x
LIST OF FIGURES xi
ABSTRACT xiii
CHAPTER
1 INTRODUCTION 1
Asthma 2
Corticosteroids in Asthma Therapy 3
Mechanism of Action of Corticosteroids 4
Disposition of Inhaled Corticosteroids 8
Clinical Effects and Systemic Effects of Inhaled Corticosteroids 10
Pharmacokinetic and Pharmacodynamic Aspects of Mometasone Furoate 12
Clinical Efficacy of Mometasone Furoate 13
Pharmacokinetic Properties of Mometasone Furoate 15
Systemic Effects of Mometasone Furoate 18
Objectives of the Study 18
2 STABILITY OF MOMETASONE FUROATE IN AQUEOUS SYSTEMS 23
Introduction 23
Materials and Methods 23
Chemicals 23
Stability of MF in Simulated Lung Fluid (SLF) 24
Effect of pH on Stability of MF in SLF 25
Isolation of Degradation Products of MF in SLF 26
Mass Spectrometry of MF and its Degradation Products 26
Structure Elucidation of Degradation Products Using NMR 27
Glucocorticoid Receptor Binding Assay Experiments in Rat Lung Cytosol. 27
Results and Discussion 29
Stability of MF in SLF 29
pH Effects on Stability of MF in SLF 33
MS Analysis of MF and the Degradation Products 34
vi


NMR Analysis of MF and the Degradation Products 35
Chemical Structures of the Degradation Products 36
Glucocorticoid Receptor Binding of MF and its Degradation Products 36
3 IN VITRO EXTRA-HEPATIC METABOLISM OF MOMETASONE FUROATE40
Introduction 40
Materials and Methods 40
Stability of MF in Plasma 41
Metabolism of MF in Lung 42
Preparation of homogenizing buffer 42
Preparation of NADPH generating system 42
Tissue metabolism using unlabeled MF 43
Metabolism in lung tissue using labeled [1,2-3H]-MF 44
Results and Discussion 44
Stability in Plasma 44
Stability in Lung 47
4 IN VITRO DISSOLUTION PROFILE OF MOMETASONE FUROATE 53
Introduction 53
Materials and Methods 54
Chemicals 54
Particle Size Distribution 54
In Vitro Dissolution 54
Data Analysis 55
Results and Discussion 56
Particle Size Distribution 56
In Vitro Dissolution 56
5 HEPATIC METABOLISM AND CHARACTERIZATION OF METABOLITES
OF MOMETASONE FUROATE 61
Introduction 61
Materials and Methods 61
Chemicals 61
In vitro Hepatic Metabolism of MF 62
In vivo Metabolism and distribution of MF 62
Glucocorticoid Receptor Binding Assay Experiments in Rat Lung Cytosol. 63
Results and Discussion 65
Metabolism of MF in Rat Liver S9 Fractions 65
Metabolism of MF in Human Liver S9 Fractions 69
In vivo Metabolism and Disposition 73
Glucocorticoid Receptor Binding of MF and Metabolites 75
Vll


6 A SENSITIVE LC-MS/MS METHOD FOR THE QUANTIFICATION OF
MOMETASONE FURO ATE IN HUMAN PLASMA 79
Introduction 79
Materials and Methods 80
Chemicals and Reagents 80
Preparation of Calibration Standards and Quality Control Samples 80
Sample Processing 81
HPLC-MS-MS Conditions 81
Method Validation 82
Selectivity 82
Recovery 82
Accuracy and precision 83
Stability 83
Results and Discussion 83
Mass Spectrometry/Chromatography 83
Internal Standard Selection 84
Selectivity 86
Recovery 86
Precision and Accuracy 87
Stability 88
7 SINGLE DOSE PHARMACOKINETICS OF INHALED MOMETASONE
FUROATE 89
Introduction 89
Materials and Methods 90
Subjects 90
Protocol 90
Bioanalytical Methods 91
Pharmacokinetic Data Analysis 91
Results and Discussion 94
Noncompartmental Analysis 94
Compartmental Analysis 97
8 SELECTIVITY OF MOMETASONE FUROATE AND OTHER INHALED
CORTICOSTEROIDS TO THE GLUCOCORTICOID RECEPTOR 101
Introduction 101
Materials and Methods 102
Chemicals 102
Glucocorticoid Receptor (GR) Binding Assay Experiments 102
Progesterone Receptor (PR) Binding Assay Experiments 103
Data Analysis 105
Molecular Dynamic Modeling 106
Results and Discussion 106
vm


CONCLUSIONS
117
APPENDIX NMR ANALYSIS OF MF AND DEGRADATION PRODUCTS 120
LIST OF REFERENCES 124
BIOGRAPHICAL SKETCH 138
IX


LIST OF TABLES
Table page
1-1. Cellular and transcriptional effects of corticosteroids 6
1-2. Pharmacokinetic and pharmacodynamic parameters of inhaled corticosteroids 17
2-1. Electrospray ionization mass spectral data of MF and its degradation products 35
2-2. Relative binding affinities (RBA) to the glucocorticoid receptor of rat lung tissue.. 37
4-1. Particle size distribution (in %) of MF and BUD micronized dry powders 56
5-1. Percentage of dose distributed into different tissues two hours after intravenous
administration of [1,2-3H]MF in male Sprague-Dawley rats 74
5-2. Average relative binding affinities (RBA) to the glucocorticoid receptor of rat lung
tissue 76
6-1. Intra-day and inter-day accuracy/precision for MF in human plasma 88
6-2. Stability (%) of MF in human plasma 88
7-1. Median (% coefficient of variation) pharmacokinetic parameters of MF obtained by
noncompartmental analysis following oral inhalation of 800 pg of MF by Asmanex
Twisthaler dry powder device 95
7-2. Estimates of the pharmacokinetic parameters of MF after fitting using a one-
compartment body model with first order absorption 97
8-1. Relative receptor affinities to the progesterone receptor of sheep uterus tissue 107
8-2. Chemical structures of various corticosteroids and metabolites 111
A-l. 'H and l3C chemical shifts, characteristic couplings (Hz), 13C, !H long-range
correlations (HMBC) and 'h/H steric proximities NOE of compound MF 121
A-2. 'H and l3C chemical shifts, Characteristic couplings (Hz), I3C,'H and long-range
correlations (HMBC) of compound D2 122
x


LIST OF FIGURES
Figure page
1-1. Disposition of corticosteroids after inhalation 10
1-2. Molecular structure of mometasone furoate 12
2-1. A schematic degradation pathway for conversion of MF into its degradation
products 31
2-2. Representative chromatograms of drug free SLF and SLF incubated with MF 32
2-3. Graphical representation showing nonlinear curve fitting of the concentration-time
data of MF and its degradation products D1 and D2 33
2-4. The degradation profiles for MF in phosphate buffer 34
2-5. Competitive binding experiments to the glucocorticoid receptor 38
3-1. Representative chromatograms of drug free rat plasma and rat plasma incubated with
MF 45
3-2. Concentration-time profile of MF and its degradation products following incubation
of MF rat plasma and human plasma 46
3-3. Representative chromatograms for blank S9 fraction of rat lung and S9 fraction of rat
lung incubated with MF 48
3-4. Representative radiochemical elution profiles of [1,2-3H]-MF incubated with S9
fraction of rat lung 49
3-5. A schematic degradation pathway for conversion of MF into its degradation products
on incubation of MF in plasma 51
4-1. Dissolution profiles of micronized dry powders of MF and BUD 58
4-2. Dissolution profiles of micronized dry powders of MF and BUD 59
5-1. Representative radiochemical elution profiles of [1,2-3H]-MF incubated in S9
fraction rat liver 66
xi


67
5-2. Concentration-time profile of [1,2-3H]-MF and the metabolites formed after
incubation of 3H-MF in S9 fraction of rat liver
5-3. Representative chromatograms for blank S9 fraction of rat liver and S9 fraction of
rat liver incubated with MF 68
5-4. Representative radiochemical elution profiles of 3H-MF incubated in S9 fraction of
human liver at 37C 70
>
5-5. Concentration-time profile of [1,2- H]-MF and the metabolites formed on incubation
of MF in S9 fraction of human liver 71
5-6. Radiochemical elution profiles of intestinal contents two hours after intravenous
administration of 5 pCi [1,2-3H]-MF in male Sprague-Dawley rats 75
5-7. Competitive binding experiments to the glucocorticoid receptor 77
6-1. Full scan and daughter scan spectra of MF 85
6-2. Representative calibration curve of MF in human plasma 85
6-3. Chromatograms of blank human plasma for the I.S and MF channels 86
6-4. Chromatograms of human plasma spiked with 1000 pg/ml I.S and 15 pg/ml MF
channels 87
7-1. Mean (+ SD) plasma concentration-time profile of MF after oral inhalation of 800 pg
of MF by Asmanex Twisthaler dry powder device 95
7-2. Representative fit using a one compartment body model for one subject following
oral inhalation of a 800 pg dose of MF 97
7-3. Absorption profile of MF after inhalation of a single dose of 800pg determined using
the Loo-Riegelman method 99
8-1. Competitive binding experiments to the progesterone receptors 108
8-2. Competitive binding experiments to the glucocorticoid receptors 109
8-3. Relationship between Log P value of different steroids to the observed steroid
selectivity 110
8-4. The structure of mometasone furoate superimposed over that of progesterone bound
to the ligand-binding domain of the human progesterone receptor 114
8-5. The structure of R-budesonide superimposed over that of progesterone bound to the
ligand-binding domain of the human progesterone receptor 115


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
PHARMACOKINETIC AND PHARMACODYNAMIC EVALUATION OF
MOMETASONE FUROATE
By
Srikumar Sahasranaman
August 2004
Chair: Guenther Hochhaus
Cochair: Hartmut Derendorf
Major Department: Pharmaceutics
Mometasone furoate (MF) is a corticosteroid that is being evaluated in the
treatment of mild-to-moderate persistent asthma. Clinical studies with orally inhaled MF
have shown that the systemic exposure and bioavailability (<1%) of MF are unusually
low in comparison to other inhaled corticosteroids. This study evaluated possible reasons
for the low systemic exposure of MF.
MF was found to be stable in the lung and blood, and this suggests that extra-
hepatic metabolism might not be a reason for the low systemic exposure of MF. MF
showed a slow in vitro rate of dissolution that indicates that MF could be removed by
mucociliary clearance mechanisms leading to the low systemic availability.
In vitro and in vivo studies showed that MF was efficiently metabolized by the
liver, and the 6(3-hydroxy-mometasone furoate metabolite showed strong binding to the
glucocorticoid receptor. MF was also found to be unstable in aqueous systems and one of
the degradation products showed significant activity to the glucocorticoid receptor. The


in vivo formation of these active products in humans needs to be evaluated and this is
important to assess the true pharmacological profile of MF.
A specific and sensitive HPLC-MS/MS assay was developed and was applied in a
clinical study to estimate the pharmacokinetic parameters of MF in asthmatic patients.
The systemic bioavailability of MF was found to be 3.2% in this study. This value was
assessed using robust methodology and is a more accurate estimate than the previously
reported value of <1%. This systemic availability along with the active metabolite could
be responsible for the systemic effects of MF. MF was also found to have a long mean
absorption time of 4.1 h that strongly suggests that MF could be removed from the lung
by mucociliary mechanisms.
It was of interest to study the glucocorticoid receptor selectivity of MF and other
corticosteroids as part of the pharmacodynamic evaluation of MF. The binding affinities
of MF and other corticosteroids to the glucocorticoid receptor and progesterone receptor
were assessed. MF showed the highest binding affinity to the progesterone receptor and
was the least selective among the glucocorticoids studied.
xiv


CHAPTER 1
INTRODUCTION
Asthma is a disease that afflicts 100 to 150 million people in the world and about
5% to 6% of the population in the United States (US) [1-3]. The prevalence of asthma in
the US has been increasing over the past decades and this disease has been reported to
cause more than 470,000 hospitalizations and 5000 deaths [2-5], Asthma also is a huge
economic burden, with a total cost of more than $ 11 billion being incurred because of this
disease in the US in 1998 [3].
Although there is no cure for asthma, advances in the understanding of the
pathophysiology of asthma have led to significant improvements in the management and
control of this disease [2], One of the most important developments has been the use of
inhaled corticosteroids (ICS) as the first-line therapy in patients with chronic asthma [2,
6]. Beclomethasone dipropionate (BDP) was developed in the early 1970s and was the
first ICS marketed for the treatment of asthma. Since then, other potent topical
corticosteroids have been developed and currently flunisolide (FLU), triamcinolone
acetonide (TA), budesonide (BUD) and fluticasone propionate (FP) along with BDP are
available in the US [2], The clinical efficacy and side effects associated with these drugs
have been widely documented. Mometasone furoate (MF) and ciclesonide (CIC) are
among the new ICS that are currently under evaluation for the treatment of asthma. In
this chapter, an overview of the pathophysiology of asthma, the mode of action of ICS
and the pharmacokinetic and pharmacodynamic issues related to MF have been
presented.
1


2
Asthma
Bronchial asthma is defined as a chronic inflammatory disease of the airways that
causes airway hyperresponsiveness and the appearance of variable and reversible airflow
obstruction [7, 8]. The inflammation is associated with hypersecretion of mucus, injury
to the epithelial cells, and deposition of fibrous material in the subepithelial basement
membrane. Asthma is characterized by the interaction between a multitude of cell types
that are involved in the pathogenesis of the disease. These interactions between mast
cells, eosinophils, epithelial cells, macrophages, and lymphocytes result in the release of
different mediators leading to bronchoconstriction, mucus hypersecretion, epithelial
damage, microvascular leakage and stimulation of neural reflex [7, 9].
An asthma attack is generally triggered upon the inhalation of an antigen and the
subsequent binding of the inhaled antigen to IgE antibodies on the mast cells in lungs
[10], The binding promotes the exocytosis of the mast cells leading to the release of
histamine and leukotrienes that may cause an acute episode of airway obstruction.
Histamine causes contraction of the smooth muscle cells of the bronchi that leads to
constriction of the lumen of the bronchi. The release of histamine and leukotrienes leads
to production of mucus and also the attraction and accumulation of inflammatory cells
like eosinophils [9], Eosinophils enhance the degranulation and release of granular
proteins, promote the formation of cytokines and are the main effector cells in the
inflammatory process. The major basic protein and other granular proteins contribute to
airway epithelial and tissue injury and airway hyperresponsiveness [10, 11]. The mast
cells also release proteases that are inflammatory enzymes, which exacerbate airway
inflammation and hyperresponsiveness [10]. Mast cells express and release cytokines that
lead to the infiltration of inflammatory cells into the airways. Several classes of


3
cytokines participate in allergic cell recruitment. These include specific endothelial
activators (IL-4 and IL-3), non-specific endothelial activators (TNF-a and IL-1),
miscellaneous activators (IL-5, granulocyte-macrophage colony-stimulating factor (GM-
CSF), and interferon gamma), and direct cell migrators like RANTES [10, 11].
Macrophages and activated lymphocytes are other cell types that contribute to airway
inflammation. Macrophages produce IL-1 that may prime eosinophils to respond to other
secretory stimuli [9]. The T helper lymphocyte, Th2, helps B lymphocyte cells make IgE
antibodies by synthesizing IL-4. Th2 also releases IL-5 that acts as a chemo-attractant for
eosinophils and other inflammatory cells.
Corticosteroids in Asthma Therapy
Inhaled corticosteroids represent the best available option in the treatment and
management of asthma because of their excellent therapeutic ratios [2], ICS are preferred
in the treatment of chronic asthma over other classes of medication like short and long
acting (32-adrenergic agonists (albuterol, salbutemol), anticholinergics (ipratropium
bromide) and other anti-inflammatory drugs like cromolyn sodium and nedocromil
sodium because of their ability to affect many of the inflammatory pathways involved in
asthma [12, 13]. ICS have been known to significantly improve lung functions and
reduce symptoms in comparison with newer therapeutic agents like leukotriene
antagonists [8]. Various dosing strategies have been used to maximize the therapeutic
effects of ICS at the pulmonary target site while minimizing the systemic side effects.
The National Institutes of Health and the National Heart, Lung and Blood Institute
(NHLBI), in their guidelines for asthma management and prevention, recommended
dosing strategies for inhaled steroids based on the severity of the disease determined


4
using lung function tests [2], This is an empirical approach and alternate rational
methods using pharmacokinetic/pharmacodynamic (PK/PD) modeling techniques have
been described to optimize the therapeutic ratios of treatment using ICS [14-18], In this
context, it is important to first understand the mode of action and the various
pharmacokinetic and pharmacodynamic concerns with ICS.
Mechanism of Action of Corticosteroids
Among the various therapies available, only corticosteroids are able to suppress
inflammation in the airways. Corticosteroids achieve this by targeting epithelial cells and
by suppressing the production of chemotactic mediators and adhesion molecules and also
by inhibiting the survival of inflammatory cells like mast cells, eosinophils, T
lymphocytes and dendritic cells [19]. This results in an inhibition of recruitment of
inflammatory cells into the airway. These cellular effects of corticosteroids are
summarized in Table 1-1.
Corticosteroids affect many of the inter-cellular interactions during the
pathogenesis of asthma that cause airway inflammation. Corticosteroids are lipophilic
molecules and diffuse easily across the cell membranes where they bind with high
affinity to the glucocorticoid receptor (GR) in the cytoplasm. The glucocorticoid
receptors (GR) are distributed throughout the body in many tissues and belong to the
steroid superfamily of cytosolic receptors [20]. The human GR is known to have two
isoforms: hGR-a and hGR-[3 [21], Even though both isoforms are transcriptionally
active, only the hGRa isoform is activated by corticosteroids. The hGR(3 isoform
provides an antagonistic effect to the glucocorticoid-activated hGRa isoform [21], In the
inactive state, the GR is bound by two molecules of 90 kD heat shock protein (hsp90) at


5
the C-terminal binding domain and one molecule of immunophilin p59 [22, 23]. This
dimeric complexation of the GR with hsp90 is required to maintain the C-terminal
domain of the GR in a favorable conformation for ligand binding [24]. Binding of
corticosteroids to the GR activates the receptor by dissociating hsp90 and the other heat
shock proteins and exposes a pair of DNA-binding zinc fingers [25, 26], This results in
a conformational change that allows the GR-ligand complex to either translocate into the
nucleus or to interact with transcription factors in the cytoplasm [27], Upon
translocation, the GR-ligand complex binds as a dimer to specific glucocorticoid response
elements (GREs) on the DNA and this leads to the modulation DNA transcription [28], It
has been believed that the binding of the GR-ligand complex to DNA results in either an
increase (transactivation) or decrease (transrepression) in the transcription of responsive
genes through the GREs depending on whether the GREs are positive (GRE+) or
negative (GRE-) [29-31]. However, recent studies indicate that negative GREs are
generally not a feature of the promoter region of the proinflammatory genes that are
repressed by corticosteroids [19].
The transrepression of the different genes responsible for encoding pro-
inflammatory proteins is thought to be responsible for the clinical anti-inflammatory
effects of corticosteroids. Corticosteroids repress the production of cytokines (IL-1, IL-2
IL-3,11-4, IL-5, IL-6, IL-11,11-13, GM-CSF, tumor necrosis factor-a (TNF-a)),
chemokines (IL-8, MCP-1, MCP-3, MCP-4, MIP-la, RANTES), mediator-synthesizing
enzymes (COX-2, cytoplasmic PLA2) and adhesion molecules (ICAM-1, VCAM-1, E-
selectin) [32-36].


6
Table 1-1. Cellular and transcriptional effects of corticosteroids (adapted from Barnes
and Adcock [19]).
Cellular effects
Cell Type
Cell
Effect
Inflammatory
Mast cells
Reduce numbers
Inflammatory
Eosinophils
Reduce numbers
Inflammatory
T-lymphocyte
Decrease cytokine production
Inflammatory
Macrophage
Decrease cytokine production
Inflammatory
Dendritic cell
Reduce numbers
Structural
Epithelial
Decrease cytokine production
Structural
Airway smooth
Decrease cytokine production
muscle
Increase [^-adrenergic receptor numbers
Structural
Endothelial
Decrease leakage
Structural
Mucus gland
Decrease mucus secretion
Effects on gene transcription
Decreased transcription
Cytokines
Chemokines
Adhesion molecules
Inflammatory receptors and enzymes
Peptides
Increased transcription
Annexin-1 (lipocortin-1, phospholipase A2 inhibitor)
IL-1 receptor antagonist
IL-1R2
IicBa (inhibitor of NF-kB)
P2-adrenergic receptor
Secretory leukocyte inhibitory protein
Clara cell protein (CC1Q, phospholipase Aj inhibitor)
Recent advances in the understanding of the mechanisms of corticosteroids have
shown that the activated glucocorticoid receptors cause deacetylation of histones and this
results in transrepression. Histones along with nucleosomes make up chromatin, and
alterations in the structure of chromatins are responsible for the regulation of gene
expression [19]. The closed conformation of the chromatin structure is associated with
suppression of gene expression and activated proinflammatory transcription factors such
as NF- kB cause the opening of the chromatin structure by acetylation of the core
histones [19]. The ability of corticosteroids to deacetylate these histones produces the


7
decrease in proinflammatory gene transcription at the therapeutically relevant low
concentrations of corticosteroids. Methylation, phosphorylation and ubiquitination, apart
from acetylation, have also been known to modify histones and these modifications may
regulate gene transcription. Corticosteroids may also inhibit or reverse these processes to
produce gene suppression.
In the case of genes encoding for proinflammatory proteins that lack GRE, the GR-
ligand complex modulates gene transcription through indirect protein-protein interactions
with transcription factors such as activating protein-1 (AP-1) and nuclear factor kB (NF-
kB) [37-40], These transcriptional factors induce many genes that encode
proinflammatory proteins like metalloproteinases and cytokines such as IL-2.
Corticosteroids may also produce nontranscriptional effects like inhibiting proteins
responsible for the stabilization of messenger RNAs of genes coding for GM-CSF [41].
Corticosteroids increase the expression of genes (transactivation) coding for anti
inflammatory proteins like annexin-1, IL-10, the inhibitor of NF-kP and IicB-a [19].
Corticosteroids also increase the production of lipocortin-1, a protein that inactivates
phospholipase A2 leading to the inhibition of proinflammatory lipid mediators like
leukotrienes, prostaglandins and the platelet activating factor [42], However, the
widespread anti-inflammatory actions of corticosteroids cannot be explained by
transactivation of limited numbers of anti-inflammatory genes [19]. The transactivation
of genes by corticosteroids is, however, believed to be responsible for producing the side
effects like growth retardation, bone and skin disorders that are associated with
corticosteroids [19]. Corticosteroids also enhance the transcription of the gene encoding
the P2-adrenergic receptor, thereby reversing or/and preventing the down-regulation


8
induced by long-term therapy with [fy-adrenergic agonists [43], This increase in P2-
adrenergic response has led to the development of combination therapy of corticosteroids
with long-acting P2-adrenergic agonists like salmeterol-fluticasone propionate (Advair
Diskus) and formoterol-budesonide (Symbicort) combinations [44],
The transactivation and transrepression effects of corticosteroids are summarized in
Table 1-1. Current research in the development of corticosteroids has shown increasing
attempts to identify corticosteroids with specific transrepression pharmacology. Such
steroids may be devoid of the side effects associated with transactivation. However, no
successful trials with these transrepression specific steroids have been reported in humans
yet.
Disposition of Inhaled Corticosteroids
The schematic representation of the disposition of corticosteroids after inhaled
administration is given in Figure 1-1. Upon inhalation of a glucocorticoid, only 10% to
60% of the administered dose is deposited in the lungs [13]. The majority of the dose
(40% to 90%) impacts on the oropharyngeal region and is swallowed. The swallowed
fraction of the dose is absorbed from the gastrointestinal tract and undergoes first pass
inactivation by the liver and the orally bioavailable fraction then enters the systemic
circulation. The currently marketed inhaled corticosteroids, except the prodrug BDP, are
metabolized by the liver into their inactive products [45]. The drug particles deposited in
the lungs dissolve according to the rate of dissolution of the drug. Only the dissolved
drug interacts with the glucocorticoid receptors (GR), as described in the previous
section, present in the lung to produce the anti-inflammatory effects. Following the
interaction with the GR, the drug is absorbed into the systemic circulation. The


9
absorption processes are rapid and it is believed that dissolution is the rate-limiting step
involved in pulmonary absorption of ICS [46]. The total systemic bioavailability of an
inhaled glucocorticoid is, therefore, the sum of the oral and pulmonary bioavailable
fractions. The non-protein bound, free drug in the systemic circulation is able to interact
with the systemic GR to produce the systemic side effects associated with corticosteroids
[47], The drug is eliminated from the systemic circulation mainly by hepatic clearance
mechanisms. Mucociliary clearance is an important mechanism that removes the
undissolved drug particles from the lung into the gastrointestinal tract [48]. Mucociliary
clearance acts as a primary defense mechanism that removes foreign particles and
aerosolized pollutants from the nose and the airways. This clearance mechanism is more
pronounced in the central airways of the lung compared to the peripheral airways and
plays an important role in reducing the pulmonary availability of highly lipophilic
corticosteroids that have a slow rate of dissolution.
The goal of treatment using ICS in asthma is to maximize the clinical anti
inflammatory effects in the lung while minimizing the systemic side effects. This can be
achieved by increasing the concentrations of the drug in the lung and decreasing the
levels in the systemic circulation and this is known as pulmonary targeting. Based on the
systemic disposition of ICS, computer simulations have been used to determine the
factors that are important in achieving good pulmonary targeting with ICS [49], These
factors are high pulmonary deposition, location of pulmonary deposition, optimal
pulmonary residence time, low oral bioavailability, high protein binding and high
systemic clearance. It is also important that the ICS bind specifically to the


10
glucocorticoid receptor as binding to the other members of the steroid family like the
progesterone receptors might result in undesired side effects.
40 90 % swallowec
Â¥ Mouth and Pharynx
10 60 % Lungl | Mucociliary clearance
Deposition X
Gl tract
Lun
Absorption
from Gut
Pulmonary^ Absorption
Systemic
Circulatio
Liver
Orally absorbed
Fraction
Clearance
First-pass
Inactivation
Systemic
Side Effects
Figure 1-1. Disposition of corticosteroids after inhalation (adapted from Derendorf et al.
[50])
Clinical Effects and Systemic Effects of Inhaled Corticosteroids
The clinical anti-asthmatic effects of inhaled corticosteroids and the systemic side
effects associated with these drugs have been widely documented. Because of the
excellent anti-inflammatory properties of these drugs, treatments with ICS have been
shown to result in attenuation of symptoms and improved pulmonary function, reduction
in usage of supplemental P-adrenergic agonist and fewer exacerbations of asthma that
require the use of oral corticosteroids [51]. The assessment of lung function is normally


11
monitored using parameters such as peak expiratory flow (PEF) and forced expiratory
volume in one second (FEVi). Treatments with ICS also result in improvement in the
quality of life and reduction in hospitalizations in asthmatic patients. Studies have also
shown that ICS are useful in protecting against life-threatening and fatal asthma and there
is a possible reduction in the mortality rates in these cases [52, 53].
Even though most of the dose is swallowed upon inhalation and the ICS have low
oral bioavailability, these drugs are not without side effects as they are able to bind to
glucocorticoid receptors in the systemic circulation after pulmonary absorption. The
potential systemic side effects associated with ICS are suppression of growth in children,
bone demineralization and bone metabolic disorders, muscle atrophy, cataracts and
glaucoma [51]. Other rare adverse events that have been reported with ICS are dermal
thinning, psychosis, hypoglycemia, development of Cushingoid features and Addisonian
crisis. Corticosteroids may also produce topical side effects like dysphonia and oral
candidiasis upon inhalation as most of the dose is deposited in the oropharyngeal region.
However, these topical side effects can be minimized by using spacer devices and by
mouth rinsing after inhalation [54], The systemic side effects are commonly assessed by
monitoring the suppression of the hypothalamus-pituitary-adrenal (HPA) axis. The HPA
axis, which maintains homeostasis in both basal and stress-related states, is a sensitive
and convenient biomarker to detect the presence of exogenous corticosteroids [55, 56],
The administration of exogenous corticosteroids results in a negative feedback effect on
glucocorticoid receptors in the hypothalamus and anterior pituitary gland, resulting in the
suppression of corticotrophin and corticotrophin-releasing hormone, respectively. This
leads to a decrease in the secretion of the endogenous corticosteroid cortisol from the


12
adrenal cortex [57], The exogenous corticosteroids that are systemically available may
sometimes lead to prolonged adrenal suppression resulting in atrophy of the adrenal
cortex [56]. The presence of low endogenous cortisol levels may not be clinically
relevant as long as there is compensation from the additional glucocorticoid activity
because of the exogenous corticosteroids in the body. However, the presence of adrenal
cortical atrophy becomes clinically relevant if there is a sudden termination of exogenous
glucocorticoid therapy, or if there is unusual stress resulting in acute adrenal
insufficiency crisis [57]. Several PK/PD models have been reported to predict the
expected cortisol suppression after administration of inhaled corticosteroids [14, 58].
Pharmacokinetic and Pharmacodynamic Aspects of Mometasone Furoate
Mometasone furoate (MF, C27H30CI2O6, 9a,21-dichloro-l 1(3,17a-dihydroxy-16a-
methylpregna-l,4-diene-3,20-dione 17-(2-furoate), Figure 1-2) is a synthetic
glucocorticoid with anti-inflammatory activity. MF has been used in the treatment of
topical dermatological disorders [59]. MF has also been approved in the prophylaxis and
treatment of allergic rhinitis [60]. MF is currently available in the European Union and is
being evaluated in the United States as an oral inhalation powder in the treatment of
patients with mild-to-moderate persistent asthma [61-63],


13
MF is a potent glucocorticoid with a higher relative binding affinity (Table 1-2) to
the glucocorticoid receptor than the currently available inhaled corticosteroids [64],
However, higher receptor affinity does not necessarily translate into a higher activity with
MF and only indicates that lower doses of MF are required to produce the same degrees
of receptor occupancies. MF also shows a higher degree of plasma protein binding
(98%-99%) than the other ICS [65],
In vitro functional assays have shown that MF inhibits the various proinflammatory
processes in asthma such as the release of cytokines (IL-4, IL-5, interferon-y) by the T-
helper cells, adhesion molecule expression (VCAM-1) on human bronchial epithelial
cells, the proliferation of mononuclear cells, eosinophil survival and the release of
histamine and cysteinyl leukotriene by basophils [66-70]. The production of IL-1, IL-6
and TNF-a in murine myelomonocytic leukemia cells was also inhibited by MF in vitro
[71], MF has also been shown to transactivate GR-mediated transcription of the
chloramphenicol acetyltransferase reporter gene with greater potency than the other
inhaled corticosteroids in an in vitro study using kidney cells of African green monkeys
[72],
Clinical Efficacy of Mometasone Furoate
The use of MF in patients with mild-to-moderate persistent asthma results in a
significant improvement in lung function, reduction in symptoms and reduction in
bronchial hyper-responsiveness. Administration of 50 to 400 pg bid MF showed
significant improvement in the FEV i values after 6 days of treatment in a clinical study
based on allergen-induced decrease in FEVi [73].
In patients with mild-to-moderate asthma previously using short-acting inhaled P2-
adrenergic agonists, the use of MF administered as 200 pg bid, 400 pg once daily in the


14
morning or 200 pg once daily in the evening for 12 weeks showed significant
improvement in asthma symptoms and lung function [74, 75], Significant improvements
were seen in FEV \ and PEF values when compared to placebo in three randomized,
double blind, multi-center clinical trials. Significant improvement in the time to
worsening of asthma was also seen in patients using MF compared to those using
placebo. In moderate to severe asthmatic patients dependent on oral prednisolone,
treatment with 400 pg or 800 pg bid MF for twelve weeks improved asthma control,
quality of life, lung function and decreased dosage of the oral steroids [76]. Clinical
trials have also assessed MF to be equally or more efficacious than currently used inhaled
corticosteroids like fluticasone propionate (FP), budesonide (BUD) and beclomethasone
dipropionate (BDP) in patients with mild to severe persistent asthma [63, 77-80], Patients
with mild-to-moderate asthma showed similar improvement in lung functions determined
by FEV i and PEF, when treated with 100 or 200 pg bid MF or 200 pg bid of BDP over
12 weeks [77], MF 200 or 400 pg bid showed significant improvements in lung function
when compared to BUD 400 pg bid [78], There was also a significant improvement in
mean P2-adrenergic agonist usage and mean wheezing score during treatment with MF
compared to budesonide [78], Similar improvements in lung function and asthma
symptoms were seen with treatment using 200 pg bid MF and 250 pg bid fluticasone
propionate in patients with moderate persistent asthma [81].
The recommended starting dose of MF in adolescent and adult patients previously
on [^-adrenergic agonists or inhaled glucocorticoid therapy is 400 pg once daily
administered by a dry powder inhaler (Twisthaler) [82], Once-daily administration of
MF has been shown to be more effective when administered in the evening than in the


15
morning [79], Mometasone furoate administered by dry powder inhaler has been shown
to be well tolerated. The most common adverse events associated with MF were oral
candidiasis, headache, pharyngitis and dysphonia [74, 75, 77, 78, 80], Clinical trials have
shown that MF <1600 pg/day is well tolerated up to one year and the incidence of
adverse events was similar with 100 to 400 pg bid MF, 200 pg bid BDP, 400 pg BUD or
250 pg bid FP [77, 78, 81].
Pharmacokinetic Properties of Mometasone Furoate
The pulmonary deposition of MF in asthmatic patients has been evaluated by
administering a 200 pg dose of technetium 99m ("mTc)-radiolabeled MF using a metered
dose inhaler [83]. Using gamma scintigraphy techniques, it was found that the mean
whole lung deposition was 13.9% while the mean deposition in the central, intermediate
and peripheral areas of the lung was 5.3%, 4.7% and 4.0% respectively [83], This pattern
of deposition is typical of inhaled drugs in patients with asthma [84]. However, the
deposition pattern of MF after administration by the marketed dry powder inhaler
formulation (Twisthaler) has not yet been reported.
The pharmacokinetic (PK) data available, after oral inhalation of MF, is limited.
Affrime et al. reported the PK in healthy subjects after a single dose administration of
400 pg of MF given intravenously (IV), by metered dose inhaler (MDI) and by dry
powder inhaler (DPI) [62], Following the administration by MDI, MF was undetectable
in plasma with mean plasma concentrations being below the lower limit of quantification
(LLOQ, 50 pg/mL) at all time-points. After the DPI administration, plasma
concentrations of MF were also extremely low and close to the LLOQ. The mean
bioavailability of MF after DPI was estimated to be <1% (0.96%) [62]. The


16
pharmacokinetic parameters after the inhalation administration were unable to be
determined because of the low concentrations that were below the LLOQ. The systemic
clearance and volume of distribution of the terminal phase determined after the IV
administration were 54 L/h and 332 L respectively [62]. This systemic clearance of MF
is consistent with the high systemic clearance values seen with the other inhaled
corticosteroids. The pharmacokinetic parameters of MF and other commonly used
inhaled steroids are given in Table 1-2. Minimal systemic exposure to MF was also seen
after multiple-dose administration by DPI with mean Cmax values close to or below the
LOQ (50 pg/mL) after a 200 to 800 pg/day dose administered by DPI for 4 to 52 weeks
[63, 77], These initial studies on the PK of MF suggest that the systemic levels and
bioavailability of MF are extremely low after an oral inhalation when compared to other
inhaled corticosteroids (Table 1-2).
There is very limited information available on the metabolism of MF. An abstract
reported that MF undergoes extensive metabolism in vitro by liver hepatocytes and the
metabolites were tentatively identified [85]. Other studies have suggested that MF
undergoes hydroxylation at the 6-position to form 6|3-hydroxy-MF; hydrolysis of the
furoate ester and the substitution of the C-21 chlorine with a hydroxyl group [60, 63].
The above-mentioned routes of metabolism are tentative and there is no conclusive
evidence of a single major metabolite and none of the metabolites have been
unequivocally identified [61]. Two reports, by the same group, on the metabolism of MF
in liver and intestinal microsomes reported the formation of one metabolite (tentatively
identified as 6p-hydroxy-MF) while mentioning that other parallel and subsequent
metabolism pathways may be present [86, 87].


17
Table 1-2. Pharmacokinetic and pharmacodynamic parameters of inhaled corticosteroids.
Drug
BDP
17-BMP
FLU
TA
BUD
FP
MF
Fora! (%)
15-20
72a
7-20
23
6-1 lb
<1
< 1
Finh (%)
25(CFC)
27(CFC)
70 (HFA)
39
(MDI)
22-25
(MDI)
26 (MDI)
38 (TBH)
26 (MDI),
12 (DH)
17(DSK)
< 1
(MDI)
(DPI)
fu (%)
13
1.6
20
29
12
10
1-2
CL (L/h)
230
54c
58
37
84,
67 (22S),
117 (22R)
69
54
Vdss (L)
NA
84c
96
103
183,
245 (22S),
425 (22R)
356
NA
Vd^L)
NA
NA
134
107
339
776
332
IV T'A (h)
0.1-0.5
0.6-1.7
1.6
2.0
2.8,
2.7 (22S),
2.7 (22R)
5-13
4.5
Inh T'A (h)
0.1
1.5-6.5
1.6
3.6
3.0
8-14
NA
MRT-IV
(h)
NA
1.6
1.7d
2.7
2.2
4.9
NA
MAT (h)
NA
NA
<1
2.9
0.3-2.6
5
NA
RRA
(Dexa=100)
NA
1022
190
233
935
1800
2900
References
[49, 88-
91]
[49, 88,
92, 93]
[49, 88,
94, 95]
[18, 49,
88, 96]
[49, 88, 97-
99]
[49, 88,
100-102]
[62, 64]
Abbreviations: BDP- Beclomethasone dipropionate; 17-BMP- Beclomethasone-17-monopropionate (active
metabolite of BDP); FLU-Flunisolide; TA- Triamcinolone acetonide; BUD- Budesonide; FP- Fluticasone
propionate; MF- Mometasone fiiroate; Foral- Oral bioavailability; Finh- Overall systemic bioavailability after
inhalation; fu- Fraction unbound; CL- Clearance; Vdss- Volume of distribution at steady state; Vdarea-
Volume of distribution of the terminal phase; IV T'A- Elimination half-life; Inh T'A- Terminal half-life after
inhalation; MRT-IV Mean residence time after intravenous administration; MAT Mean absorption time
after inhalation; RRA Relative receptor affinity; Dexa Dexamethasone; MDI- Metered dose inhaler;
DH- Diskhaler; DSK- Diskus; TBH- Turbohaler; CFC- Chlorofluorocarbon propellant based MDI; HFA-
Hydrofluoroalkane-134a propellant based MDI; NA- Not available;a -In rats;b- Radiolabeled budesonide;
c- Assuming complete conversion after intravenous administration of BDP; d- Calculated from Vdss and CL
after intravenous administration; 22S and 22R Epimers of Budesonide.


18
Systemic Effects of Mometasone Furoate
Even though MF has minimal systemic exposure, clinical studies have shown that
MF produces considerable systemic effects as assessed by suppression of the
hypothalamus-pituitary-adrenal (HPA) axis after an oral inhalation. Mean serum cortisol
under the plasma concentration-time curve over 24 hours (AUCC24) was used as the
surrogate parameter to determine the HPA axis suppression in these studies. In a clinical
study involving 64 patients with mild-to-moderate asthma, 400 pg or 800 pg bid of MF
administered by a metered dose inhaler for 28 days produced a 10%-40% decrease the
AUCC24 compared to placebo (,P<0.05) [63], In comparison, 880 pg bid of fluticasone
propionate produced a 43%-56% suppression and an oral dose of 10 mg/day of
prednisolone caused a 64% to 72% suppression of AUCC24 compared with placebo
(P<0.01) [63, 103], Higher systemic levels of MF are observed when administered with
the dry powder inhaler than with the metered dose inhaler [62], Therefore, it is expected
that administration with the former formulation would result in greater cortisol
suppression. Unfortunately, there are no reports of direct comparisons between the two
formulations with respect to HPA axis suppression. Administration of 400 pg bid and
800 pg bid of MF by dry powder inhaler resulted in significant lowering of AUCC24 by
10% to 25% and 21% to 40% respectively compared to placebo treatment [63]. The
results from these studies have led to suggestions that the reported bioavailability of MF
is spurious or that MF has at least one active metabolite responsible for producing the
systemic effects [104-107],
Objectives of the Study
As mentioned earlier, the systemic exposure of mometasone furoate is extremely
low (systemic bioavailability <1%) when compared to other corticosteroids after oral


19
inhalation (Table 1-2). The deposition efficiency of MF has been reported to be
comparable to other ICS and therefore, the low systemic bioavailability cannot be a result
of poor deposition of MF into the lungs [62, 83]. Efficient hepatic metabolism might be
suggested as a reason for the low systemic levels of MF. However, hepatic metabolism
alone is insufficient to explain the unusually low systemic levels of MF compared to
other ICS as high clearances have also been reported for FP, BUD, triamcinolone
acetonide (TA) and flunisolide (FLU) (CL in Table 1-2). The systemic bioavailabilities
(Fjnh in Table 1-1) of these other steroids are much higher than the <1% estimate
proposed for MF. Since systemic clearances of MF and the other corticosteroids are
close to the hepatic blood flow rates, efficient hepatic metabolism alone does not explain
the difference between the bioavailability of MF and the other corticosteroids.
The other reasons that might explain the unusually low systemic exposure of MF
after an oral inhalation are:
1. MF is efficiently metabolized by the lung and therefore is not absorbed into the
systemic circulation leading to low concentrations in the blood.
2. MF is metabolized by the enzymes present in blood.
3. MF is a highly lipophilic drug [103] and could have a slow rate of dissolution, leading
to the drug being removed by mucociliary action out of the lung into the
gastrointestinal tract.
In the present study, these three reasons would be evaluated as possible factors
responsible for the low systemic exposure of MF.
In vitro metabolism of MF would be conducted in fresh rat plasma and in the S9
fraction of rat lung tissue and the metabolic profile of MF would be studied under
comparable conditions in human plasma and S9 fraction of human lung tissue. Stability
of MF in simulated lung fluid (SLF) would also be studied. The purpose for conducting


20
stability of MF in buffer systems is primarily to delineate degradation effect from
metabolism. This is to ensure that any change occurring in the parent structure (MF) is a
result of enzymatic action and not a result of degradation due the buffer system
employed.
Even though the systemic bioavailability of MF has been reported to be extremely
low, the drug shows considerable systemic effects as manifested by the suppression of the
hypothalamic-pituitary-adrenal axis [63], While the high potency of MF may be
suggested as a reason for the systemic effects in spite of the low availability, this is not
likely because MF also has high plasma protein binding (98%-99%) thereby reducing the
free levels available to produce the systemic effects. This contradiction could therefore
be possibly explained by the metabolism of MF into at least one active metabolite that
produces the systemic effects. To assess this hypothesis, this study also evaluates the
hepatic metabolism and the biological activities of the metabolites thus formed.
The validity of the reported <1% bioavailability estimate for MF has also been
questioned as the estimate was based on an assay of inadequate sensitivity (lower limit of
quantification, 50 pg/mL) and at a low dose (400 pg), which resulted in most of the
concentrations after oral inhalation being below the limit of quantification [106, 107],
The concentrations below the limit of quantification were then substituted as zero for area
under the curve calculations and this leads to an underestimation of the area under the
curve and the resulting systemic bioavailability. Therefore, another objective of this
study was to develop and validate a specific and sensitive analysis method using tandem
liquid chromatography-mass spectrometry (LC-MS/MS) for the quantification of MF in
human plasma. This assay would be used in a clinical study to determine the


21
pharmacokinetics of MF in patients with asthma after oral inhalation of a single dose of
800 pg of MF. It is expected that the use of a sensitive assay and administration of a
higher dose would enable the robust and accurate estimation of the systemic
bioavailability and other pharmacokinetic parameters of MF.
Austin et al. recently reported that MF and fluticasone propionate are agonists of
the progesterone receptor (PR) and weak antagonists of the androgen receptor [108],
Binding to the PR might be of clinical importance since inappropriate activation of the
progesterone receptor might result in dysregulation of the oestrus cycle and could lead to
a worsening of the symptoms of premenstrual tension. These effects may be exacerbated
in women with pre-existing difficulties. An excessive activation of PR will ultimately
have a contraceptive effect. Therefore, specificity to the glucocorticoid receptor (GR) is
an important requisite for the safety of inhaled corticosteroids. The specificity of MF and
other commonly inhaled corticosteroids to the GR will be assessed by comparing their
binding affinities to the GR and PR respectively.
Based on the above discussions, the overall objectives of the study are summarized
below:
1. Evaluate the stability of mometasone furoate in biological buffers and to characterize
the structure and the pharmacological properties of the degradation products.
2. Study the metabolism of mometasone furoate in rat and human lung, and plasma.
3. Assess the in vitro dissolution rate of mometasone furoate.
4. Study the hepatic metabolism of mometasone furoate and characterize the
pharmacological properties of the metabolites.
5. Develop and validate a specific and sensitive LC-MS/MS assay for the quantification
of mometasone furoate in human plasma.
6. Assess the pharmacokinetics of mometasone furoate after administration of a single
dose of 800 pg by oral inhalation in patients with asthma.


22
7. Investigate the specificity of mometasone furoate and other inhaled corticosteroids to
the glucocorticoid receptor.


CHAPTER 2
STABILITY OF MOMETASONE FUROATE IN AQUEOUS SYSTEMS
Introduction
There is very limited information available on the stability of MF in biological
tissues/fluids and aqueous systems. Teng and coworkers have characterized the
degradation of MF in simulated lung fluid (SLF) and have shown that MF is unstable in
SLF resulting in three degradation products [109]. They have also recently reported the
degradation of MF in aqueous systems and have hypothesized the structure of the
degradation products based on previous studies conducted with other corticosteroids
[110]. The present study aimed to clearly identify the structures of these degradation
products by MS and NMR analysis. It was of further interest to assess their potential
biological activity, by performing rat lung glucocorticoid receptor binding studies,
because of the possibility of these degradation products being formed in vivo.
Materials and Methods
Chemicals
Mometasone furoate (MF) was purchased from USP (Rockville, MD).
Dexamethasone was purchased from Sigma Chemicals Co (St Louis, MO). 3H-
Triamcinolone acetonide (specific activity 38 Ci/mmole) was purchased from Perkin
Elmer Life Sciences (Boston, MA). The degradation products of MF: D1 and D2 were
isolated and purified using HPLC and solid phase extraction in our laboratory. All other
chemicals and solvents were obtained from Sigma Chemicals Co. (St Louis, MO) and
Fisher Scientific Co. (Cincinnati, OH).
23


24
Stability of MF in Simulated Lung Fluid (SLF)
Composition of the simulated lung fluid buffer was adopted from the report of
Kalkwarf [111]. In brief, SLF comprised of MgCl2.6H20 (1 mM), NaCl (103 mM), KC1
(4 wM), Na2HP04.7H20 (1 mM), Na2SO4(0.5 mM), CaCl2.2H20 (2.5/wM),
NaH3C202.3H20 (7 mM), NaHC03 (31 mM) and Na3H3C60y.2H20 (0.33 mM) dissolved
in a liter of distilled water. The pH of the SLF buffer solution was adjusted to 7.4 using
dilute HC1 (20% v/v). 10 ml of the SLF was pre-incubated at 37C for 10 min, and
spiked with MF (1 mg/mL stock solution) to achieve a concentration of 2.5 pg/mL.
Blank buffer was used in a control experiment. 500pL aliquot samples were withdrawn
at 0, 5, 10, 15, 30, 45 min and then at 1, 1.5, 2, 4, 6, 7, 24, 48 and 72 h into pre-chilled
tubes and the samples were diluted with 500pL of mobile phase (63:37% v/v MeOH-
H20). The samples were analyzed by HPLC-UV.
A LDC/Milton Roy CM4000 multiple solvent delivery system using a Milton Roy
SM 4000 programmable wavelength detector set at A.max 254 nm, a CR-3A Chromatopac
integrator (Shimadzu Corporation, Japan) and an automatic injector (Perkin Elmer,
Boston, MA) was used as HPLC system. Chromatographic separation of the analytes
was achieved on Waters 5-pm symmetry RP-18 (150 x 4.6 mm i.d) column (Milford,
MA) preceded with a guard column (10 x 4.6 mm i.d, filled with reverse phase pre
column material) using a mobile phase of 63:37% v/v methanol-water at a flow rate of 1
ml.
Calibration curves based on MF peak areas over the concentration range of 0.125 to
5.0 pg/mL using unweighted least squares linear regression analysis resulted in r2 values
of at least 0.999. The limit of quantification of the assay was 0.125 pg/mL based on
good accuracy (> 90%) and precision (< 10% coefficient of variation).The good


25
reproducibility of the system allowed quantification without an internal standard as
potential internal standards either cross-eluted with degradation products or were unstable
in the buffer systems used.
Because of the lack suitable standards of D1 and D2, quantification of the
degradation products was based on the assumption that the molar absorptivities were
similar to that of MF. Results for MF and degradation products are presented in pM and
expressed in % of MF starting concentrations in graphical representations.
The concentration-time data obtained for MF and its degradation products were
fitted using SCIENTIST software (version 2.0, Micromath, Salt Lake City, UT)
using the model shown in Figure 2-1. Alternative degradation pathways (broken arrows,
Figure 2-1) allowing first for the cyclization of MF followed by epoxide formation was
not indicated by the number of degradation products identified. The half-lives for the
different compounds were calculated using the equation below:
Ti/2 = ----- -, where kapp is the apparent degradation constant for that compound.
kapp
Effect of pH on Stability of MF in SLF
To assess the pH dependence, degradation process experiments were conducted in
50 mM K2HPO4 buffer at three different pH values (pH 7.0, 7.5 and 8.0). A fifty times
higher buffer strength (compared to SLF) of K2HPO4 was selected to improve the
buffering capacity. The pH of the buffer at the higher buffer strength did not change over
an incubation period of 24 h at 37C. MF at a concentration of 2.5 pg/mL (4.8 pM) was
incubated at 37C in a shaker bath. 200pL aliquot samples were withdrawn at 0, 5, 30,
min and then at 1,2, 4, 6, 8, and 10.6 h into pre-chilled tubes and the samples were


26
diluted with 200 pL of mobile phase (63:37 MeOH-IrhO). The samples were analyzed
using HPLC-UV as described above.
Isolation of Degradation Products of MF in SLF
For the purpose of isolation of degradation products, 4 vials containing 10 mL of
SLF buffer were spiked with MF (1 mg/mL stock solution) to give a concentration of 10
pg/mL. After incubation, the contents of the vials were pooled and 1 mL aliquots of the
spiked SLF were transferred to individual solid-phase extraction (SPE) cartridges and
extracted as reported previously [112]. The extracted and reconstituted samples were
injected onto the HPLC and the peaks corresponding to the various degradation products
were collected using a fraction collector (Gilson Model 203, Middleton, WI). Several
HPLC runs had to be performed to purify sufficient amount of material. The fractions
corresponding to each degradation product were pooled and extracted using the SPE
procedure described above. Purity (greater than 95%) and content of the isolated
degradation products were determined by HPLC. The isolated fractions were analyzed
by MS and NMR for structure determinations (see below). In addition, the activity of
these isolated degradation products was assessed in glucocorticoid receptor binding
experiments (see below).
Mass Spectrometry of MF and its Degradation Products
LC-MS analysis was used to identify the molecular mass of the degradation
products of MF. The chromatographic separation of the analytes was achieved on a
Waters 5-pm symmetry RP-18 (50 x 4.6mm i.d) column (Milford, MA) with an aqueous
mobile phase containing 70:30% v/v MeOH-H20 and flow rate of 0.3 ml/min. The LC
system was connected to a Micromass Quattro-LC-Z (Beverly, MA) triple quadrupole
mass spectrometer equipped with ES+ (electro-spray) ion source. The source


27
temperature was set to 120C and the ES probe temperature was 400C. Capillary and
cone voltages were set to 3kV and 25 V respectively. The samples were detected using a
full scan mode. The mass spectrometer was linked to a Perkin Elmer ISS-200 auto
sampler via a contact closure and the operation was controlled by computer software
MASSLYNX 3.1.
Electron ionization (El) mass spectra were taken on a Finnigan MAT 8430 mass
spectrometer under the following operating conditions: resolution, 1250; ion accelerating
voltage, 3 kV; electron energy, 70 eV; electron current, 500 pA, ion source temperature,
250 C, evaporation temperature, 140 C.
Structure Elucidation of Degradation Products Using NMR
NMR analysis was conducted at the IVAX Drug Research Institute, Budapest,
Hungary. The NMR spectra were recorded in CDCI3 at room temperature using Shigemi
sample tubes (180 pi) on Bruker Avance-500 spectrometer at 500.13/125.7 MHz. The
pulse programs were taken from Bruker software library.
Glucocorticoid Receptor Binding Assay Experiments in Rat Lung Cytosol
The various dilutions for MF (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0, 100 pM)
were prepared in methanol. Dilutions for degradation product D1 and dexamethasone
(0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1.0 pM) and degradation product D2 (0.001, 0.003,
0.01, 0.03, 0.1, 0.3, 1.0, 0.3 pM) were also prepared in methanol.
The animal protocol was approved by the local Institutional Animal Care and Use
Committee (IACUC) at the University of Florida, Gainesville, Florida. Sprague Dawley
rats (250 25gms) were obtained from Harlan (Indianapolis, IN). The rats were
anesthetized using a cocktail mixture of ketamine, xylazine and acepromazine (3:3:1 v/v)


28
and were decapitated. The lungs were removed and homogenized in 8 volumes of ice-
cold incubation buffer (10 mM Tris/HCL, 10 mM sodium molybdate, 2 mM 1,4-
dithioerythritol). The homogenate was incubated with 5% w/v charcoal suspension (in
deionized water) for 10 min. The homogenate was then centrifuged for 20 min at
40,000X g in J2 rotor of Beckman centrifuge to obtain the cytosol. Fresh cytosol was
prepared and used for all the individual experiments.
10 nM of 3H-labeled triamcinolone acetonide (TA) solution was used as a tracer,
based on previous saturation binding experiments performed in our laboratory (data not
shown). 20 pL of the drug solution in methanol was added to pre-chilled tubes. Blank
methanol was used for the determination of total binding. Non-specific binding was
determined after addition of 20 pL of 100 pM unlabeled triamcinolone acetonide (10 pM
in the final incubation mixture). 20 pL of 100 nM3H-triamcinolone acetonide solution
(10 nM in final incubation mixture) was then added. 160 pL of the lung cytosol was
added and the tubes were vortexed and incubated at 4C for 24 h. After the incubation,
200 pL of 5% w/v charcoal suspension (in water) was added to the tubes to remove the
excess unbound radioactivity. The tubes were vortexed and 300 pL of the supernatant
was transferred to the scintillation vials. 5 mL of the scintillation cocktail was added and
the scintillation vials were read in a liquid scintillation counter (Beckman Instruments LS
5000 TD, Palo Alto, CA). The stability of MF and the degradation products under the
incubation conditions (4C), was demonstrated by HPLC after extraction of the
incubation mixture by solid phase extraction.
The data obtained were fitted by SCIENTIST (Micromath, Salt Lake City, UT)
using the following Emax model to obtain the estimates of Bmax and IC50.


29
CN
DPM = Bmax -Bmax h NS where DPM represents the total tracer
IC50 +C
binding obtained at any given competitor concentration, NS represents non-specific
binding and N the Hill coefficient. Bmax is the specific binding by the ligand in the
absence of competitor.
The IC50 obtained for dexamethasone (ICso.dex) was used to calculate the relative
binding affinity (RBAtest) of the test compound form its IC50 value (ICso.test) as:
RB A =-de-xl00
test rn
50,test
The stability of MF and its degradation products in the rat lung cytosol at 4C was
checked by spiking the compounds separately in cytosol and incubating the samples for
24 h at 4C. Blank cytosol was considered as control. One set of spiked cytosol was
analyzed at zero time and the other set was analyzed after 24 h (time period of
incubation).
Results and Discussion
Stability of MF in SLF
In agreement with findings of Teng and coworkers [109], incubation of MF (Co =
2.5 pg/ mL) in SLF at 37C and subsequent HPLC analysis of the incubation mixture
suggests the formation of two major degradation products, named D1 and D2 (Figure 2-
2). The concentration-time profiles of MF, D1 and D2 (Figure 2-3) indicate that more
than 90% of MF undergoes conversion to either D1 and or D2 at the end of 4 h of
incubation with D1 being formed faster than D2. After 12 h, the majority of the starting
material entered the D2 pool with D1 being undetectable; therefore one can conclude that
D1 seems to be converted into D2. Consequently, it appears that MF is first converted


30
into Dl, which is subsequently converted into D2 (see Figure 2-3). Indeed, fitting of the
data to this model resulted in satisfying fits (Figure 2-3); small under or overestimations
might be related to the changes of the pH during incubation, which affected the
degradation rate constants (see below). The apparent degradation rate constant (ki = 0.52
h'1) for the conversion of MF to Dl was found to be nearly 4 times higher than the
degradation rate constant (k2 = 0.143 h'1) for the conversion of Dl to D2. The
degradation half-life for MF was found to be 1.3 h and the half-life of Dl was 4.8 h.
Thus, degradation profile shown in the upper part of Figure 2-1 (denoted by solid lines) is
supported by the data. This suggests that there are qualitative and quantitative
differences between this study and that conducted by Teng et al. who reported the
formation of another minor degradation product, D3 [109], However, D3 is not seen in
this study even though a more sensitive assay was used. The half-life of MF in SLF in
this study was found to be 3.5 times shorter compared to the half-life reported by Teng et
al. The concentration of D2 after 72 h in this study was approximately 80% as that of
parent MF compared to 30% by Teng et al. It might be speculated that differences in the
buffer composition, ionic strength or other factors might be the reason for the somewhat
different degradation pathways.
The rate of degradation of MF was found to vary as a function of pH of the buffer
solution. During incubation at 37C, the starting pH of the SLF buffer solution was 7.4
that gradually became alkaline (pH 8.9) at the end of 72 h. The pH shift of the SLF
buffer solution was also monitored at ambient temperature (25C) and it was observed
that the pH gradually changed from 7.4 to 7.9 in 4 h and was distinctly alkaline (pH 9.1)
at the end of 72 h. It is proposed that the buffer system gradually turns alkaline due to the


31
loss of acid (loss of CO2 from the buffer system). This could be due to the presence of
sodium bicarbonate in the buffer (~30 mM) and a low buffering capacity of phosphate
buffer (~1 mM) present in the SLF. However, the cause of the pH raise during incubation
is uncertain.
Figure 2-1. A schematic degradation pathway for conversion of MF into its degradation
products D1 and D2 on incubation of MF (Co = 2.5 pg mL'1) in SLF at 37C
for 72 h. Broken arrows indicate the possible formation of structure E (D3),
which was not observed in this study. Structures A, B, D and E represent MF,
DI, D2 and D3 respectively. Structure C shows only D-ring of the steroid for
presenting the proposed reaction mechanism for the conversion of B to D. A
similar reaction mechanism would be possible for formation of structure E
from A.


32
Figure 2-2. Representative chromatograms of (a) drug free SLF at 24 h and (b) SLF
incubated with MF (C0 = 2.5 pg mL'1) for 24 h at 37 C.


33
Figure 2-3. Graphical representation showing nonlinear curve fitting of the concentration
time data of MF and its degradation products D1 and D2 generated after
incubation of MF in SLF at 37C for 72 h (N=2). The inset graph shows the
expanded time scale up to 12 h.
pH Effects on Stability of MF in SLF
In order to assess the pH dependence, degradation process experiments were
conducted in 50 mM K2HPO4 buffer at three different pH values (pH 7.0, 7.5 and 8.0,
Figure 2-4). The degradation rate of MF increased with increased pH. The degradation
rate constants were 0.03, 0.06, and 0.15 h'1 for pH 7.0, 7.5 and 8.0 respectively. The
results in this study are in agreement with the report by Teng et al. that was recently
published [110]. It may be noted that the degradation rate constant of MF (0.52 h'1) in
SLF was higher than the degradation rate constant observed in 50 mM K2HPO4 buffer at


34
pH 8.0. This suggests that ionic effects, because of the presence of salts in the
composition of SLF, might play a significant role in the degradation of MF.
Figure 2-4. The degradation profiles for MF (Co = 2.5 pg ml/1) in phosphate buffer at
pH 7.0, 7.5 and 8.0 at 37C.
MS Analysis of MF and the Degradation Products
Based on the ESI+ mass spectra (Table 2-1), the molecular formulae of D1 and D2
agree with the general structures C27H29CIO6 and C27H27CIO5 respectively. The
molecular masses of D1 and D2 are in agreement with those reported by Teng et al.
[110]. The mass spectra data indicated a loss of HC1 for D1 when compared with MF.
Similarly, D2 differed from D1 by a loss of H2O. The presence of only one chlorine
atom in D1 and D2 was confirmed by the relative abundance of 37C1 isotope (Table 2) in


35
D1 and D2 (35%) compared to the relative abundance in MF (70%) that has two chlorine
atoms.
As electron ionization (El) mass spectra supply in general more structural
information, the El mass spectra of MF and D2 have also been taken. The El mass
spectrum of MF is dominated by the furoyl ion at m/z 95, while in the El mass spectrum
of D2 this peak is much less abundant and a very specific, chlorine-containing ion at m/z
211/213 gives rise to the base peak. The formation of this ion, which consists of the Cl 6-
C17 atoms of the steroid skeleton (with their substituents + 1 H) strongly support the
structure proposed here for D2.
Table 2-1. Electrospray ionization mass spectral data# of MF and its degradation
Compound
MF
D1
D2
Molecular formula
C27H3oC1206
c27h29cio6
C27H27C105
[MH]+
521(100)&
485(15)&
467 (60)&
[MH]+ (37C1 isotope)
523(70)
487 (5)
469 (20)
[M+Na]+
543 (30)$
507 (100)$
489 (35)$
[MH-H20]+
503 (10)$
-
-
[MH-HC1]+
485 (15)$
-
-
Others
Ur-. ; ^ i
441 (5)
413 (15)
413(5)
413(10)
Listed are m/z. % relative abundance to nearest 5% is given in parenthesis.
1 Corresponding 37C1 isotope ion was also observed.
& This was determined to be the parent compound based on independent chemical
ionization mass spectrometry analysis (data not shown).
NMR Analysis of MF and the Degradation Products
The NMR analysis of MF and the degradation products were performed at the
IVAX Drug Research Institute, Hungary and the detailed report on the NMR analysis is
given in Appendix A.


36
Chemical Structures of the Degradation Products
As a result of the MS and NMR analysis, the structure of D2 is shown in Figure 2-
1. This structure of D2 is different from the structure proposed by Teng et al who
hypothesized a furoate-ester migration between Cl7 and C21 positions [110]. Even
though such ester migrations have been reported for other steroids like betamethasone 17-
valerate [113], beclomethasone monopropionate [114] and hydrocortisone butyrate [115],
this phenomenon cannot be expected to happen in steroids like MF that have a halogen
atom at C21 [59, 116], Based on these MS and NMR analyses, the proposed structure for
D2 in this study is accurate and unambiguous.
As mentioned earlier, D2 appears to be formed upon degradation of Dl. Based on
this hypothesis, the structure of D2 and the MS analysis of Dl, Dl is 9, 11-epoxide
mometasone furoate (Figure 2-1). The complete degradation pathway of MF in SLF is
summarized in Figure 2-1. The results of this study do not completely preclude the
formation of compound D3 (pathway shown by dashed lines in Figure 2-1) under
somewhat different experimental conditions even though the formation of this compound
is not seen in this study.
Glucocorticoid Receptor Binding of MF and its Degradation Products
The relative binding affinities of MF, mometasone and the isolated degradation
products D1 and D2 were determined in a competition binding experiment in rat lung
cytosol. Table 2-2 compares the average IC50 values and relative binding affinity (RBA)
values for dexamethasone, MF, Dl and D2. The data shows that MF is 29 times more
potent than dexamethasone. The binding affinity of Dl to the rat glucocorticoid receptor
was 4 times higher than dexamethasone. The IC50 value of the degradation product D2


37
could not be determined precisely as D2 did not show binding to the glucocorticoid
receptors in the concentration range studied.
Table 2-2. Relative binding affinities (RBA) to the glucocorticoid receptor of rat lung
tissue.
Compound
ICso(pM)
RBA
Dexamethasone
0.046 0.01
100
Mometasone Furoate
0.0016 0.0001
2938
Dl
0.011 0.0002
433
D2*
-0.290 0.034
-15.8
* Indicates rough estimates of IC50 obtained by fixing the hill coefficient to 1.
Bledsoe et al. recently determined the crystal structure of the glucocorticoid
receptor ligand-binding domain in a ternary complex with dexamethasone [117]. The
glucocorticoid receptor ligand-binding domain has a unique side pocket that may account
for the selectivity of corticosteroids with substituents at the C-17a position [117, 118].
This may explain the strong binding of MF and Dl, which have a furoate moiety at the
Cl7a position, to the glucocorticoid receptor. However, the structural changes in D2,
with its condensation between the furoate ester carbonyl and the methylene group at C21
leading to the formation of the five-member ring, seem to interfere with the interaction of
the steroid with the receptor in this region of the receptor pocket resulting in a
significantly reduced binding affinity of D2.
The competition curves for dexamethasone, MF, Dl and D2 are shown in Figure 2-
5. In all binding experiments, non-linear curve fitting revealed slope factors (Hill
coefficient) for dexamethasone, MF and Dl were close to 1. The RBA value for MF in


38
this study is slightly higher but in agreement with earlier reported values [119]. 9,11-
epoxide-mometasone furoate (Dl) has a reduced binding affinity of 433, but this RBA
value is comparable to and even greater than many of the currently used inhaled
corticosteroids like flunisolide (RBA 180), triamcinolone acetonide (RBA 233) and
budesonide (RBA 935) [88], These results also indicate that the presence of an 11P-OH-
group is favorable but not absolutely necessary for receptor binding. The binding affinity
of 9,11-epoxide-MF compared to MF in this study to the receptor in rat lung is in
agreement with the values reported by Isogai et al for the glucocorticoid receptor of rat
skin [120].
Log of Competitor concentration (nM)
Figure 2-5. Competitive binding experiments to the glucocorticoid receptors in rat lung
cytosol. Nonlinear regression of non-transformed data was used for the
determination of IC50 values of dexamethasone, MF, degradation products Dl
and D2. An approximate IC50 value for D2 was determined to be 0.29 0.034
pM (n=2). The estimated parameters for D2 were then used to predict bound
tracer at competitor concentrations higher than 0.3 pM. The hill coefficient
was fixed to 1.


39
The results from this show that MF is unstable in aqueous systems resulting in two
epoxidated degradation products. One of the degradation products shows a substantial
glucocorticoid receptor activity and is unstable, the other representing a new cyclized
stable structure whose pharmacological and toxicological properties have not been
described. These instabilities are not typically seen for corticosteroids that do not have
the specific features of MF (furoate group in Cl 7, Cl group at C21 and C9). Further
studies need to investigate whether these degradation products also occur in vivo or in
aqueous based MF formulations.


CHAPTER 3
IN VITRO EXTRA-HEPATIC METABOLISM OF MOMETASONE FUROATE
Introduction
The systemic levels and bioavailability (<1%) of mometasone furoate have been
reported to extremely low in comparison to other corticosteroids after an oral inhalation
[62]. Even though a major portion of the dose is swallowed upon oral inhalation [50],
efficient hepatic metabolism alone is insufficient to explain the low systemic exposure of
MF since the currently used corticosteroids also have high hepatic clearance values
approaching the liver blood flow. Efficient extra-hepatic metabolism may therefore, be
proposed as a hypothesis to explain the low systemic exposure of MF. This study
investigated the stability and metabolism of MF in the lung and in blood, the two major
organs apart from the liver that the drug encounters after an oral inhalation. In this study,
the metabolism was studied by using both non-labeled and tritium-labeled mometasone
furoate in rat and human lung S9 fractions. The use of radio-labeled drug enables the
detection of highly polar metabolites that might otherwise go undetected using HPLC-
UV because of co-eluting endogenous polar interferences present in the tissues.
Materials and Methods
Mometasone furoate (MF) was purchased from USP (Rockville, MD, USA) and
j
[1,2- H]- mometasone furoate (specific activity: 0.56 Mbeq/mmole) was provided by
AstraZeneca (Lund, Sweden). All other chemicals and solvents were obtained from
Sigma Chemicals Co. (St Louis, MO) and Fisher Scientific Co. (Cincinnati, OH).
40


41
The use of male Sprague Dawley rats (Harlan, Indianapolis, IN) was approved by
the local Institutional Animal Care and Use Committee (IACUC) at the University of
Florida, Gainesville, Florida. Approval was obtained from the Institutional Review
Board, University of Florida for the use of human lung tissues. Human lung tissue
samples were obtained from persons aged between 18-60 years undergoing lung resection
surgery. Cigarette smoke has been known to induce metabolic enzymes like CYP1A1
and therefore, smokers were excluded in the study [121], Pregnant women were also
excluded for ethical reasons.
Stability of MF in Plasma
Male Sprague Dawley rats weighing 25025g were euthanized to harvest fresh
blood. The blood was transferred to heparin sodium containing vacuutainers (Becton
Dickinson, 100 U.S.P. I.U.). The tubes were then centrifuged at 3500 rpm for 20 min to
obtain plasma. 4 ml of freshly collected rat plasma was pre-incubated at 37C for 10 min
and then was spiked with MF (from a stock of 100 pg/mL) at a concentration of 3 pg/mL
(5.75 pM). Experiments were conducted in triplicates. 200 pL aliquots were removed at
regular intervals up to 72 h into pre-chilled tubes and the samples were precipitated with
600 pL of methanol. Blank rat plasma was used in a control experiment. Identical
stability studies were performed using fresh human plasma also. The samples were
stored at -20C and were analyzed by HPLC within 48 h of storage.
The precipitated plasma samples were centrifuged at 10000 rpm for 5 min and 100
pL of the supernatant was injected directly for HPLC-UV analysis. A LDC/Milton Roy
CM4000 multiple solvent delivery system using a Milton Roy SM 4000 programmable
wavelength detector set at kmax 254 nm, a CR-3A Chromatopac integrator (Shimadzu
Corporation, Japan) and an automatic injector (Perkin Elmer, Boston, MA) was used as


42
the HPLC system. Chromatographic separation of the analytes was achieved on Waters
5-pm symmetry RP-18 (150 x 4.6 mm i.d) column (Milford, MA) preceded with a guard
column (10 x 4.6 mm i.d, filled with reverse phase pre-column material) using a mobile
phase of 65:35% v/v methanol-water at a flow rate of 1 ml.
Calibration curves based on MF peak areas over the concentration range of 0.125 to
5.0 pg/mL using unweighted least squares linear regression analysis resulted in r2 values
of at least 0.999. The limit of quantification of the assay was 0.125 pg/mL based on
good accuracy (> 90%) and precision (< 10% coefficient of variation). The good
reproducibility of the system allowed quantification without an internal standard as
potential internal standards cross-eluted with either the endogenous interferences or the
degradation products formed.
Because of the lack of suitable standards, quantification of the degradation products
was based on the assumption that their molar absorptivities were identical to that of MF.
Results for MF and degradation products expressed in % of MF starting concentrations in
the graphical representations.
Metabolism of MF in Lung
Preparation of homogenizing buffer (1.15% KC1 in 50 mM K2HPO4 pH 7.4)
870 mg of dipotassium hydrogen orthophosphate (K2HPO4) and 1.15 g of
potassium chloride (KC1) was dissolved in 100 ml of deionized water and the pH was
adjusted with 20% v/v orthophosphoric acid to 7.4. The buffer was chilled to 4C before
use.
Preparation of NADPH generating system
NADPH generating system, which comprised of cofactors salts: NADP (2 mM),
glucose-6-phosphate (8 mM), nicotinamide (200 mM) and magnesium chloride (200


43
mM) was prepared in the homogenizing buffer. This cofactor solution was incubated at
37C for 15 min to allow generation ofNADPH and then was used immediately.
Tissue metabolism using unlabeled MF.
S9 fractions of rat and human lung were used for studying the metabolism of MF.
Frozen human tissue samples were obtained from the Molecular Tissue Bank at the
University of Florida and the samples were pulverized before homogenizing. Rat tissues
were obtained by sacrificing the rats by decapitation and removing the lung tissue
immediately. The tissues were then rinsed in ice-cold homogenizing buffer, blotted dry
and weighed.
The lung tissues were homogenized in ice-cold homogenizing buffer to obtain 20
w/v of tissue. Lung tissue homogenate was then centrifuged at 4C at lOOOOx g for 45
min to obtain the S-9 fraction for lung. S9 fractions were pre-incubated with equal
volumes of cofactor solution and incubated in a shaker bath to equilibrate the mixture to
37C. Unlabeled MF was added to initiate the reaction and the final drug concentration
was 2.5 pg/mL (4.8pM) and final lung tissue concentration was 10% w/v. The lung S9
fractions were incubated at 37C for up to 24 h under atmospheric air. Drug free S9
fraction of lung was used in a control experiment. Serial samples of250pL were
withdrawn at 0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 90, 120, 240 min and 24 h into pre
chilled tubes. The samples were precipitated with 250pL of acetonitrile and analyzed by
HPLC. The precipitated samples of lung homogenates were centrifuged at 10000 rpm for
5 min. 100 pL of the supernatant was injected directly into the column for HPLC
analysis. A mobile phase composition of 50:50% v/v of acetonitrile-water was used for
the analysis and the other chromatographic conditions for analysis of samples from S9
studies was identical to those mentioned earlier for analysis of the plasma samples.


44
Results for MF and metabolites are expressed in % of MF starting concentrations in
graphical representations. Semi logarithmic plots of % MF remaining versus time were
used to determine the apparent disappearance rate constant of MF. The disappearance
half-life was determined using the equation ti/2 = 0.693/kdiSS, where kdiss is the apparent
disappearance rate constant for that compound.
Metabolism in lung tissue using labeled (1,2-3H]-MF
An identical parallel incubation was performed simultaneously with 5 mL lung S9
fractions in which [1,2-3H]-MF (concentration <125 ng/mL) was added in addition to the
unlabeled MF (4.8 pM). These samples containing labeled MF were incubated as
mentioned in the previous section. The homogenizing buffer was also spiked with
identical concentrations of labeled and unlabelled MF and was used in a control
experiment. 250 pL aliquots were withdrawn at every 20 min for the first two hours and
finally at 24 h. The samples were withdrawn into pre-chilled tubes and were precipitated
with 250 pL of acetonitrile to terminate the reaction. The samples were immediately
analyzed using HPLC using the conditions mentioned for the analysis of unlabeled MF. 1
min fractions were collected using a fraction collector and transferred to individual
scintillation vials and counted using a liquid scintillation counter (Beckman Instruments
LS 5000 TD, Palo Alto, CA) for the presence of radioactivity. The total counts present
for MF at time zero were considered as 100%. The percentages of metabolites formed
were estimated as the ratio of dpm for metabolite to dpm of parent drug at time zero.
Results and Discussion
Stability in Plasma
The stability of MF was studied in fresh plasma harvested from rat blood.
Incubation of MF in rat plasma at 37C for 72 h led to the formation of three degradation


45
products DI, D2 and D3 (Figure 3-1). However, MF was found to be stable in plasma
until 6 h of incubation. The formation of degradation products DI, D2 and D3 occurred
after 20 h of incubation (Figure 3-2). The decline of MF and the formation of DI, D2
and D3 are shown in Figure 3-2(a). The degradation half-life of MF as calculated form
the terminal phase of the log concentration-time curve was found to be 85 h.
A
B
MF
4
ix.
, ix.
] i co
. io
a
IV
D1
1
* CO
3
D2 D3
44
CO LO
co ^ -cr
eo
c*J
I
1 1
i
0
Min
25 0
Min
25
Figure 3-1. Representative chromatograms of (a) drug free rat plasma at 48 h and (b) rat
plasma incubated with MF (Co = 3 pg/mL) until 48 h at 37C.
MF was also found to be stable in human plasma until 6 h of incubation at 37C.
However, prolonged incubation of MF in human plasma for 72 h led to the formation of
identical degradation products (DI, D2 and D3, Figure 3-2(b)) as observed in rat plasma.
The degradation half-life calculated for MF in human plasma was found to be 24 h which


46
was 3.5 times shorter compared to that in rat plasma and this might be attributed to
possible differences in protein binding of the compound between the two species.
0 8 16 24 32 40 48 56 64 72
Time (h)
(B)
Figure 3-2. Concentration-time profile of MF and its degradation products following
incubation of MF (Co = 6.2 pM) at 37C in (a) rat plasma and (b) human plasma for 72 h
(n=3).


47
Using a mass spectrometry (positive electro-spray ionization) analysis method
reported in the previous chapter, the molecular masses ([MH+]) of MF, DI, D2 and D3
were determined to be 521, 485, 467 and 503 respectively. The structure elucidations of
these compounds are presented later in the chapter.
Stability in Lung
Incubation of MF in rat lung S9 fractions showed no metabolic conversion until 2
h. However, incubation of MF for 24 h resulted in a degradation product, D2 that was
found to be less polar than the parent drug itself (Figure 3-3). This degradation product
had identical retention times as the product D2 seen upon incubation of MF in plasma
and in the simulated lung fluid (chapter 2). Parallel control incubations of labeled MF in
the incubation buffer also showed formation of D2. This indicated that the formation of
D2 was a result of slow break down of MF in the homogenizing buffer and not a result of
metabolism in the lung. Incubations of labeled MF in the S9 fraction of lung were also
conducted, which also proved that MF was stable in lung fractions. The representative
radiochemical elution profiles for MF in rat lung incubations are shown in Figure 3-4.
Incubation of MF in human lung S9 fractions also indicated that MF was stable with no
metabolic conversion until 2 h of incubation. These results indicate that MF does not
undergo significant pulmonary metabolism.


48
Figure 3-3. Representative chromatograms for (a) blank S9 fraction of rat lung and (b) S9
fraction of rat lung incubated with MF (Co = 2.5pg mL'1) for 24 h at 37C.
As the results of this study indicate, MF is stable in the plasma and lung S9
fractions up to 2 h. The degradation products (DI, D2 and D3 in plasma; D2 in lung) seen
upon prolonged incubation in plasma and lung appear to be a result of chemical
degradation and not metabolism, as these products (Figure 3-4) were observed in the
incubation buffer also. The degradation of MF in simulated lung fluid (an aqueous
system) and the determination of the structure and the pharmacological activity of the
two resulting degradation products have been reported in detail in chapter 2. Based on
the identical molecular masses and the retention times of the degradation products, it is
expected that the degradation products D1 and D2 observed in this study are identical to
the products reported earlier.


49
T
Figure 3-4. Representative radiochemical elution profiles of [1,2- H]-MF incubated with
S9 fraction of rat lung at 37C (A) at time zero, (B) after 2 h at 37C and (C)
after 24 h.


50
The structures of DI, D2 and D3 in plasma along with the proposed pathways of
degradation are provided in Figure 3-5. The conversion of D1 to D2 has been reported in
chapter 2. However, there is no conclusive evidence for the conversion of D3 to D2 in
this study and this pathway has been denoted by broken arrows in the figure. D1 has
been identified to be 9,11 -epoxide mometasone furoate and has strong glucocorticoid
receptor activity with RBA values of 433 while D2 is a condensed product of MF that
does not show significant binding to the glucocorticoid receptor (chapter 2). The
structure of D3 has been proposed based on the structures of D1 and D2 and the
molecular mass of D3. Based on the similarities in structure (condensed five-member
ring at position Cl7) between D2 and D3, it is expected that D3 would not show
significant binding to the glucocorticoid receptor either.


51
Figure 3-5. A schematic degradation pathway for conversion of MF into its degradation
products DI, D2 and D3 on incubation of MF (Co = 6.2 pM) in plasma at
37C for 72 h. Structures A, B, D and E represent MF, DI, D2 and D3
respectively. Structure C shows only D-ring of the steroid for presenting the
proposed reaction mechanism for the conversion of B to D. A similar reaction
mechanism would be possible for formation of structure E from A. Broken
arrows indicate the possible formation of D2 from D3.
Even though degradation products of MF are observed in lung and plasma in vitro,
these products might not be seen in vivo since they are formed by relatively slow
processes and therefore might be superceded by the faster pulmonary absorption and
hepatic metabolic processes. The relative stability of MF in plasma and the lung rules out
the possibility of extra-hepatic metabolism in these organs being a cause for the low
systemic exposure of MF after an oral inhalation. Other reasons such as the removal of


52
slow-dissolving, highly lipophilic MF particles deposited in the lungs and airways by
mucociliary clearance mechanisms might be likely to explain the low levels.


CHAPTER 4
IN VITRO DISSOLUTION PROFILE OF MOMETASONE FURO ATE
Introduction
Extra-hepatic metabolism and clearance of MF by mucociliary mechanism are two
possible causes for the low systemic exposure of MF after an oral inhalation. Studies
described in the previous chapter indicate that extra-hepatic metabolism might not be the
reason for the low systemic exposure. Therefore, it is hypothesized that MF, being a
highly lipophilic drug [103], has a slow rate of dissolution leading to the drug being
removed by the mucociliary clearance mechanisms from of the lung resulting in the
unusually low systemic levels of MF seen after oral inhalation [62], To assess the effect
of the dissolution rate the absorption characteristics, the in vitro dissolution profile of dry
powder formulation of MF were compared to that of budesonide (BUD).
Pharmacokinetic studies in humans have shown that the major portion of the dose of
BUD is rapidly absorbed after oral inhalation [122]. If MF has a significantly slower rate
of dissolution than BUD, MF can be expected to undergo mucociliary clearance. The
dissolution studies were performed in a phosphate buffered medium with surfactant at
physiological temperature (37C) and pH 7.4. Micronized powders of MF and BUD
were used in these studies. A 50 mM phosphate buffered medium was preferred over
simulated lung fluid in this study because of the increased stability of MF in the former
medium (chapter 2).
53


54
Materials and Methods
Chemicals
Budesonide was provided by AstraZeneca. Sodium dodecyl sulfate was obtained
from Fischer Chemicals (Fair Lawn, NJ) and lactose monohydrate NF extra-fine (particle
size 10-30 microns) was purchased from EM Industries (Hawthorne, NY). All other
chemicals and equipment have been described in method section in chapter 2.
Particle Size Distribution
The particle size of MF and BUD powders were checked using an Olympus BX60
optical microscope (Tokyo, Japan) equipped with SPOT RT digital camera and image
analysis software.
In Vitro Dissolution
In vitro dissolution of the dry powder formulations (500 mg of 2% drug in lactose)
was studied using a USP dissolution bath (VanKel Technology Group, Cary, NC) in 900
mL of pH 7.4 phosphate-buffered saline (50 mM) at 37C. To ensure that the powders
dissolved completely (MF aqueous solubility 125 ng/mL vs. 140 pg/mL with surfactant),
2% sodium-dodecyl sulfate (SDS) was added to increase solubility and wetting and to
more closely mimic in vivo pulmonary surfactant levels [123], 1 mL samples were
removed and filtered through a 0.2 pM syringe filter at 0, 0.5, 1, 1.5, 2, 2.5, 5, 10, 15, 30,
45, 60 and 240 minutes and analyzed using HPLC. The concentrations of MF and BUD
were be determined by HPLC using the instruments described in the methods section in
chapter 2. For analysis of MF, BUD (2 pg/mL) was used as an internal standard and for
analysis of BUD; MF (1 pg/mL) was used as the internal standard. A mobile phase
composition of methanol-water (60:40, v/v) was used at a flow rate of 1 mL/min in the


55
analysis. The experiment was repeated using 0.5% SDS as surfactant while maintaining
all other conditions as described above.
Data Analysis
Half-life of dissolution in all cases, except the dissolution of MF in 0.5% SDS, was
calculated by non-linear curve fitting using SCIENTIST (Micromath, Salt Lake City,
UT) program and using the monoexponential formula:
Cumulative % Dissolved = 100 (A*exp'at)
where a is the dissolution rate constant. Half-lives of dissolution (T5o%) were
calculated as 0.693/a.
The dissolution of MF in 0.5% SDS was characterized by a biphasic profile given
by the equation:
Cumulative % Dissolved = 100 ((A*exp~at )+(B*exp~pt)),
where a and P are the rate constants for the initial rapid and latter slow phases of
dissolution. The corresponding half-lives for a and P were obtained by multiplying their
reciprocals with 0.693.
Differences in dissolution between drug formulations were tested for significance
using the FDA scale-up and postapproval changes (SUPAC) similarity test (f2):
f2 = 50 x LOG ([1+1/n S(Rt-Tt)2]'0 5 x 100)
where n=12 time points (0, 0.5, 1, 1.5, 2, 2.5, 5, 10, 15, 30, 45 and 60 minutes), Rt
is the reference % drug dissolved at time t and Tt is the test % drug dissolved at time t.
An f2 value between 50 and 100 suggests the two dissolution profiles are similar and an f2
value less than 50 indicates a lack of similarity [124],


56
Results and Discussion
Particle Size Distribution
The particle size distributions of MF and BUD micronized dry powders are given
in Table 4-1. The particle size distributions were comparable for the two compounds and
more than 88% of the particles had aerodynamic diameters in the range 1-5 pm. This is
in accordance with the particle size distribution of dry powder formulations of inhaled
corticosteroids, as particles with diameters 1 -5 pm have been known to cause maximum
pulmonary deposition [125], Particles with diameter greater than 5 pm impact on the
back of the throat after an oral inhalation while particles with diameter smaller than 1 pm
will be exhaled out.
<1 pm
1-5 pm
5-10 pm
>10 pm
MF
3.5
88.5
5.8
2.2
BUD
2.1
94.3
3.1
0.5
In Vitro Dissolution
Cumulative dissolution profiles for MF and BUD powders using 2% SDS as
surfactant are depicted in Figure 4-1. From the release profiles, it was observed that
steady state was reached (10 pg/ml) at 30 minutes. The dissolution half-life (T5o%) for
MF was calculated to be 4.20.3 min (n=6) and Tso% for BUD was 2.10.2 min (n=6).
Dissolution half-lives of release was significantly longer for MF when compared to BUD
and the dissolution profiles for the two drugs were not similar (f2=42). Previous studies
have shown that the dissolution half-lives was significantly longer for FP when compared
to BUD also under identical conditions (f2=34) [126],


57
The dissolution profiles of MF and BUD using 0.5% SDS as surfactant is shown in
Figure 4-2. MF exhibited a biphasic dissolution profile when lower concentration of the
surfactant was used. The Tso% for MF for the initial phase of dissolution was 3.50.2 min
while the Tso% for the latter phase of dissolution was 37.10.9 min. 66% of the total MF
powder dissolved at the faster rate while the remaining 34% showed the slower phase of
dissolution. The biphasic dissolution profile of MF could be because of different
polymorphs of MF in the powder studied. The overall dissolution of MF in the medium
with 0.5% SDS was slower than in the 2% SDS containing medium. BUD followed a
mono-phasic profile and there was no significant change (p>0.5) in the Tso% (1.90.3
min) of BUD compared to using 2% SDS. The dissolution profiles for MF and BUD
were not similar with an ii value of 28. The results clearly indicate that the concentration
of the surfactant used in the dissolution medium plays an important role in assessing the
dissolution profile of the compounds. While the use of surfactants has been
recommended in performing dissolution studies of water-insoluble compounds, there is
no recommendation on the exact concentration of the surfactant to be used [123]. The in
vitro model used in this study also utilizes a large volume of buffer and surfactant
compared to in vivo conditions. Considering the above-mentioned constraints, this
method is primarily used to qualitatively compare the dissolution rates of the
corticosteroids studied.


58
Time (min)
Figure 4-1. Dissolution profiles of micronized dry powders of MF and BUD in pH 7.4
PBS (50 mM, 2% SDS) at 37C (n=6).


59
Figure 4-2. Dissolution profiles of micronized dry powders of MF and BUD in pH 7.4
PBS (50 mM, 0.5% SDS) at 37C (n=6).
The lipophilic corticosteroids are more prone to mucociliary clearance because of
their slow dissolution rates. Removal of beclomethasone dipropionate from the lung by
mucociliary mechanisms has been suggested to explain the low systemic exposure of the
drug after administration by a chloro-fluoro-carbon pressurized MDI [127]. Mucociliary
clearance also appears to play a significant role in reducing the pulmonary uptake of
fluticasone propionate in patients with moderate to severe asthma [128].
The results of this study show that MF has slower in vitro rates of dissolution than
BUD. The in vitro dissolution rates of MF are comparable with that of FP [126] and


60
since mucociliary clearance has been proposed for FP, it is expected that MF would also
be cleared by mucociliary mechanisms resulting in lower systemic availability of MF.
This needs to be investigated by assessing the in vivo dissolution rate in terms of the
absorption profile and pulmonary residence times MF. A clinical study assessing the
absorption profile and the systemic availability of MF in asthmatic patients is presented
in chapter 7.


CHAPTER 5
HEPATIC METABOLISM AND CHARACTERIZATION OF METABOLITES OF
MOMETASONE FUROATE
Introduction
Although the systemic bioavailability of MF has been reported to be extremely low,
the drug shows considerable systemic effects as manifested by the suppression of the
hypothalamic-pituitary-adrenal axis [63]. This contradiction could possibly be explained
by the metabolism of MF into at least one active metabolite. To assess this hypothesis,
this section of the study evaluated the hepatic metabolism and the biological activities of
the metabolites thus formed. The pharmacological activity of the metabolites formed was
tested by assessing their binding to the rat lung glucocorticoid receptors. The evaluation
of the activity of the metabolites is important in estimating the correct pharmacological
profile of MF. The in vivo formation of these metabolites was also assessed by
performing disposition studies using radiolabeled MF in rats.
Materials and Methods
Chemicals
Mometasone furoate (MF) was purchased from USP (Rockville, MD, USA) and
[1,2-3H]- mometasone furoate (specific activity, 0.56 Mbeq/mmole) was provided by
AstraZeneca (Lund, Sweden). [l,2,4-3H]-dexamethasone (specific activity, 38
Ci/mmole) was purchased from Amersham Biosciences (Piscataway, NJ). All other
chemicals and solvents were obtained from Sigma Chemicals Co. (St Louis, MO) and
Fisher Scientific Co. (Cincinnati, OH). The use of Sprague Dawley rats (Harlan,
61


62
Indianapolis, IN) was approved by the local Institutional Animal Care and Use
Committee (IACUC) at the University of Florida, Gainesville, Florida. Approval was
obtained from the Institutional Review Board, University of Florida for the use of human
liver tissues. Human liver tissue samples were obtained from persons aged between 18-
60 years undergoing liver resection surgery. Cigarette smoke has been known to induce
metabolic enzymes like CYP1A1 and therefore, smokers were excluded in the study
[121]. Pregnant women were also excluded for ethical reasons.
In vitro Hepatic Metabolism of MF
The in vitro hepatic metabolism of MF was studied using the same protocol
described for lung metabolism in the methods section of chapter 3. Liver tissues of rat
and humans were processed as reported to obtain 40% w/v (20% w/v in final incubation
mixture) S9 fractions. The metabolism of MF was assessed using both unlabeled MF
(4.8 pM) and a mixture of labeled and unlabeled MF in rat and human lung S9 fractions.
The experiments were performed in triplicates. The sample collection and HPLC
analysis were performed as described in chapter 3.
In vivo Metabolism and distribution of MF
A total of 5 pCi of [1,2-3H]MF per 250 mg of bodyweight, in normal saline was
administered by injection into the tail vein of male Sprague-Dawley rats. The animals
were sacrificed by decapitation after two hours. The abdominal and thoracic cavities
were opened immediately and blood from the heart was withdrawn. Urine was collected
by puncturing the urinary bladder. The organs (Table 1) were dissected, rinsed with ice-
cold normal saline, blotted, weighed and then homogenized in methanol. 1 cm2 skin and
the vastus lateralis muscle from the right leg were resected and homogenized without
being rinsed. The contents of the stomach and the intestines (both small and large) were


63
flushed out of the organs before homogenization and collected. 30 mL of methanol was
used to homogenize the liver and 20 mL was used to homogenize the other tissues. 1 mL
of the homogenates was transferred to scintillation vials and 10 mL of the scintillation
cocktail was added. The scintillation vials were read in a liquid scintillation counter
(Beckman Instruments LS 5000 TD, Palo Alto, CA).
Glucocorticoid Receptor Binding Assay Experiments in Rat Lung Cytosol
As described earlier in the metabolism experiments, liver S9 fractions were spiked
only with unlabeled MF (4.8 pM) and incubated at 37C in a shaker bath for 2 h. The 2 h
samples were analyzed by HPLC-UV analysis and the metabolites (MET1-MET5) were
isolated by collecting the fractions corresponding to the retention times of the
metabolites. The fraction corresponding to the elution of MF (denoted by MFfrac) in the
samples was also collected. These fractions were then evaporated under vacuum and
reconstituted in methanol. The concentrations of the MFfrac and the metabolites in the
fractions were calculated from % conversion of MF into the metabolites data obtained
from the previous section. The dilutions for MF (from a stock of 1 mg/mL), MFfrac and
the metabolites were prepared in methanol. Dexamethasone (DEXA) was used as a
reference compound for the receptor binding experiments. The concentration ranges for
the compounds in the study were 0.01 to 100 nM for MF, METI and MET3; 0.01 to 30
nM for MFfrac and MET2; 0.01 to 50 nM for MET4 and MET5 and 0.01 to 1000 nM for
DEXA.
A previously described method [129], with slight modifications, was used for
performing the receptor binding assays. Sprague Dawley rats were anesthetized using a
cocktail mixture of ketamine, xylazine and acepromazine (3:3:1 v/v) and were
decapitated. The lungs were removed and homogenized in 8 volumes of ice-cold


64
incubation buffer (10 mM Tris/HCL, 10 mM sodium molybdate, 2 mM 1,4-
dithioerythritol). The homogenate was incubated with 5% w/v charcoal suspension (in
deionized water) for 10 min. The homogenate was then centrifuged for 20 min at
40,000X g in J2 rotor of Beckman centrifuge to obtain the cytosol. Fresh cytosol was
prepared and used for all the individual experiments.
10 nM of 3H-labeled dexamethasone solution was used as a tracer, based on
previous saturation binding experiments performed in our laboratory (data not shown).
10 pL of the dilutions of the test compound in methanol was added to pre-chilled tubes.
10 pL of methanol instead of the test compound was used for the determination of total
binding. Non-specific binding was determined after addition of 10 pL of 100 pM
unlabeled DEXA (10 pM in the final incubation mixture). 10 pL of 100 nM 3H-
dexamethsone solution (10 nM in final incubation mixture) was then added. 80 pL of the
lung cytosol was added and the tubes were vortexed and incubated at 4C for 24 h. After
the incubation, 100 pL of 5% w/v charcoal suspension (in water) was added to the tubes
to remove the excess unbound radioactivity. The tubes were vortexed and then
centrifuged and 150 pL of the supernatant was transferred to the scintillation vials. 5 mL
of the scintillation cocktail was added and the scintillation vials were read in the liquid
scintillation counter. Control experiments were also conducted in which receptor binding
assays were performed on fractions collected during HPLC-UV analysis of blank S9
fractions and on fractions collected during blank mobile phase runs.
The data obtained were fitted by SCIENTIST (Micromath, Salt Lake City, UT)
using the following Emax model to obtain the estimates of Bmax and IC50.
CN
DPM = B -B
max max
IC5oN+Cn
+ NS


65
where DPM represents the total tracer binding obtained at any given competitor
concentration, NS represents non-specific binding and N the Hill coefficient. Bmax is the
specific binding by the ligand in the absence of competitor.
The IC50 obtained for dexamethasone (ICso.dex) was used to calculate the relative
binding affinity (RBAtest) of the test compound form its IC50 value (ICso^est) as:
RBA =^5^x100
^50, test
Results and Discussion
Metabolism of MF in Rat Liver S9 Fractions
Incubation of a mixture of non-labeled and labeled MF with rat liver S9 fractions
over 1 h showed formation of five metabolites (Figure 5-1). The retention times for the
metabolites METI, MET2, MET3, MET4 and MET5 were between 1-2, 5-6, 7-8, 11-12
and 12-13 min respectively. A semi logarithmic plot of % dpm versus time displayed a
linear relationship with a correlation coefficient of 0.956 indicating first order elimination
kinetics for MF. The disappearance half-life of labeled MF was 18.5 min in rat liver S9
fractions. The concentration-time profiles of MF and its metabolites are shown in Figure
5-2. At the end of 1 h, METI was observed as the major conversion product with
MET/MF ratio of 32% and 7% of MF was converted to MET2. The sum of the MET/MF
ratios for metabolites 3, 4 and 5 were less than 10%. The control buffer sample did not
show any formation of polar degradation products indicating that the formation of
metabolites was a result of enzymatic activity present in the S9 fraction of liver sample
and not a result of degradation in the buffer.


66
A
Collection time interval (min)
B
Figure 5-1. Representative radiochemical elution profiles of (a) [1,2-3H]-MF incubated in
S9 fraction rat liver at time zero and (b) [1,2-3H]-MF incubated in S9 fraction
of rat liver for time 20 min at 37C (bars in black and gray represent MF in S9
fraction of rat liver and homogenizing buffer respectively).


67
A
Time (min)
Figure 5-2. Concentration-time profile of [1,2- H]-MF following incubation of [1,2- H]-
MF in S9 fraction of rat liver and (b) concentration-time profile of the
metabolites formed after incubation of 3H-MF in S9 fraction of rat liver at
37C for 1 h.
The HPLC-UV profiles (Figure 5-3) observed after incubation of unlabeled-MF
with S9 fraction of rat liver indicated the formation of only one polar metabolite (MET2)
that eluted at 5.1 min. The disappearance half-life of unlabelled MF (16 min) as


68
determined by HPLC-UV analysis was found to be similar to the half-life of labeled MF
(18.5 min) that was estimated from the radiochemical elution profiles. The metabolite
MET 1 (retention time between 2-3) could not be viewed using a UV-VIS detector due to
the presence of endogenous interferences at the same retention time. MET3, MET4 and
MET5 could not be quantified using UV due to the very low concentrations of these
metabolites.
figure 5-3. Representative chromatograms for (a) blank S9 fraction of rat liver, (b) S9
fraction of rat liver incubated with MF (Co = 2.5pg/mL) until 0.25 h, (c) S9
fraction of rat liver incubated with MF until 1 h.


69
Metabolism of MF in Human Liver S9 Fractions
Incubation of a mixture of labeled and unlabelled MF in human liver S9 fractions
also showed formation of five metabolites (Figure 5-4). The retention times for these
metabolites were identical to the ones formed with rat liver S9 fractions. A semi
logarithmic plot of % dpm versus time displayed a linear relationship with a correlation
coefficient of 0.9584 and the disappearance half-life of 3H-MF in the S9 fraction was
found to be 0.76 h'1 with a corresponding half-life of 55 min. The concentration-time
profile for the disappearance of MF and appearance the metabolites are shown in Figure
5-5.
The HPLC-UV profiles observed after incubation of MF with S9 fraction of human
liver showed an endogenous tissue impurity eluting at around 5 min. Because of this
interference, MET2 could not be observed in human liver tissues. Hence, only the
decline of MF could be monitored under UV detection over time.


70
A
E
Q.
"D
30000
25000
20000
15000
10000
5000
ir>
CO
r--
00
o>
o
-r-
OsJ
00
'i-
lO
to
r--
oo
cr>
o
C\l
OO
-
i
c
00
cr>

-
Ovl
O
U
to
r-1
OO
OJ

Human liver 0 hr
Buffer 0 hr
MF
Collection time interval (min)
B
12000.0
10000.0
8000.0
E
% 6000.0
4000.0
2000.0
0.0
MET1
Human Liver 20 min
Buffer 20 min
MET2 MET3
n
MET4 MET5
MF
y(M0T|-lDffiNm0-W0 + l)NCr)0
'4 coO-Csio/>'i-iotDr--cocri
Collection time interval (min)
Figure 5-4. Representative radiochemical elution profiles of 3H-MF incubated in S9
fraction of human liver at 37C (a) at time zero and (b) after 20 min (bars in
black and gray represent MF in S9 fraction of human liver and homogenizing
buffer respectively).


71
A
B
Time (min)
Figure 5-5. Concentration-time profile of [1,2-3H]-MF following incubation of [1,2-3H]-
MF in S9 fraction of human liver and (b) concentration-time profile of the
metabolites formed on incubation of MF in S9 fraction of human liver at 37C
for 1 hr.


72
This study shows that MF is efficiently metabolized into at least five metabolites in
S9 fractions of both rat and human liver tissues. There were, however, quantitative
differences in the metabolites between the two species. The apparent half-life of MF in
the S9 fraction of human liver was found to be 3 times greater compared to that in rat.
METI, the most polar metabolite, was the major metabolite in rat liver fractions where as
both METI and MET2 were formed to an equal extent in human liver fractions. Such
species differences have been noted for other corticosteroids like budesonide also [130],
The molecular mass ([MH+]) of MET2 was estimated to be 538 using a mass
spectrometry method reported previously in chapter 2. Based on the molecular mass and
comparing the chromatographic elution profile of MET2 to putative metabolites [62],
MET2 is expected to be 6P-OH-MF. This is in agreement with other reports that have
hinted the formation of this metabolite [63, 85-87], Hydroxylation at the 6p-position is a
common route of metabolism among corticosteroids with budesonide, triamcinolone
acetonide and flunisolide getting metabolized into their respective 6p-hydroxy
derivatives [95, 130-133]. Using HPLC-UV detection in the present study, only the
decline of MF and the formation of MET2 could be viewed. This agrees with the report
by Teng et al., who monitored the formation of only one metabolite in human liver using
UV and concluded that other metabolites also should be formed to explain the loss of MF
[87], The use of the tritium-labeled drug in this study provided valuable information
about the formation of the other metabolites as well. METI is a metabolite that has not
been identified before and is not among the putative metabolites of MF that were
proposed by Affrime et al [62]. The formation of METI, however, is consistent with the
bioavailability study by the same group, who reported that majority of the radioactivity


73
eluting in the urine after administration of 3H-MF in humans eluted in the very polar
region [62]. However, no conclusive decision could be made about indications of
sequential metabolism in the present study. METI was extremely polar to be extracted
for further analysis, and therefore could not be identified. MET3, 4 and 5 were formed in
low amounts and could not be identified.
In vivo Metabolism and Disposition
The radioactivity (expressed as % of dose administered) in the tissues 2 h after an
intravenous injection of [1,2-3H]-MF in rats is given in Table 5-1. The data shows that
most of the radioactivity (90%) is present in the stomach, intestines and the intestinal
contents. This strongly suggests that there is biliary excretion of MF and its metabolites.
Biliary excretion has also been reported for other corticosteroids like triamcinolone
acetonide and prednisolone [134-136]. The bioavailability study by Affrime et al in
humans after the inhalation of 3H-MF reported the presence of radioactivity associated
with metabolites in the feces also suggesting biliary excretion of the metabolites [62].
There was no significant concentration of MF into the other tissues. The amount of
radioactivity associated with the brain was negligible and the least among the organs
studied. This is consistent with results from other studies that show that the uptake of
corticosteroids into the brain is limited by the multidrug resistance (mdr) P-glycoprotein
(Pgp) efflux transporter at the blood-brain barrier [137, 138].
Radiochemical elution profiles of the intestinal contents show the formation of
three metabolites that have identical retention times as the metabolites METI, MET2 and
MET3 (Figure 5-6) that were seen upon incubation of MF with liver S9 fractions. There
was no radioactivity corresponding to MF and this is consistent with the efficient hepatic
metabolism of MF that was seen in vitro. Most of the radioactivity in the intestinal


74
contents was associated with METI (Figure 5-6) while MET2 and MET3 were minor
metabolites. MET4 and MET5 were either not formed or formed in extremely low
concentrations to be seen in the intestinal contents. The results of this study clearly
indicate that the MF is metabolized efficiently in vivo and the metabolites are
predominantly eliminated by biliary excretion into the intestinal tract.
Table 5-1. Percentage of dose distributed into different tissues two hours after
intravenous administration of [1,2-3H]MF in male Sprague-Dawley rats.
Tissue % of dose
Brain
0.050.01
Thymus
0.080.02
Heart
0.090.02
Spleen
0.240.16
Lung
0.320.01
Kidney
0.320.01
Stomach
1.3U0.39
Liver
2.01.36
Large intestine
2.47.98
Small intestine
4.550.11
Intestinal contents
81.574.66
Muscle
0.200.05a
Fat
0.390.17a
Skin
0.050.02b
Plasma
0.260.08c
Urine
0.080.02c
a % of dose/gram tissue
b % of dose/cm2
c % of dose/ml


75
Collection time interval (min)
Figure 5-6. Radiochemical elution profiles of intestinal contents two hours after
intravenous administration of 5 pCi [1,2-3H]-MF in male Sprague-Dawley
rats.
Glucocorticoid Receptor Binding of MF and Metabolites
The relative binding affinities of DEXA, MF and the isolated metabolites were
determined in a competition binding experiment in rat lung cytosol. Table 5-2 gives the
relative binding affinities (RBA) values for DEXA, MF, MFfrac and the metabolites. The
competition curves for the compounds are shown in Figure 5-7. In all binding
experiments, non-linear curve fitting revealed slope factors (Hill coefficient) for all the
compounds were close to unity. The fractions collected during HPLC-UV analysis of
blank S9 fractions and during blank mobile phase runs did not show any binding to the


76
glucocorticoid receptor. The data shows that MF is 29 times more potent than
dexamethasone. The RBA of MFfracwas found to be 2700. The good agreement between
the RBA values of MF using a standard solution and MF collected from fractions (MFfrac)
indicates that the concentrations of the compounds in the fractions collected are accurate.
Table 5-2. Average relative binding affinities (RBA) to the glucocorticoid receptor of rat
lung tissue.
Compound
RBA
Dexamethasone
100
Mometasone furoate
2900
MFfrac
2700
METI
<25
MET2
700
MET3
25
MET4
<50
MET5
<50
The binding affinity of MET2 to the rat glucocorticoid receptor was 7 times higher
than dexamethasone. This RBA value of MET2 is greater than the RBA values of some
commonly used inhaled corticosteroids like flunisolide (RBA 180), triamcinolone
acetonide (RBA 233) and is comparable to budesonide (RBA 935) [88]. The strong
affinity of 6P-OH-MF is surprising because the 6P-hydroxy derivatives of other
corticosteroids do not show significant activity towards the glucocorticoid receptor. The
RBA values of 6p-OH-budesonide and 6p-OH-flunisolide have been reported to be 6 and
less than 1 respectively [119, 129]. The IC50 value of the metabolites METI, 4 and 5


77
could not be determined, as they did not show any binding to the glucocorticoid receptors
in the concentration range studied.
120 ,
Figure 5-7. Competitive binding experiments to the glucocorticoid receptor in rat lung
cytosol (n=2). Nonlinear regression of non-transformed data was used for the
determination of IC50 values of dexamethasone, MF, MFfrac, metabolites
MET2 and MET3.
Even though MF has minimal systemic exposure, MF produces significant systemic
effects after an oral inhalation. 800 pg BID of MF administered by a metered dose
inhaler for 28 days produced a 20%-30% suppression of the hypothalamic-pituitary-
adrenal axis (HPA) compared to placebo (PO.05) [63], In comparison, a 880 pg BID of
fluticasone propionate produced a 43%-56% suppression of HPA compared with placebo
(P<0.01) [63, 103]. The formation of the active metabolite, 6(3-OH-MF, might explain
the systemic effects of MF in spite of its low systemic exposure. Therefore, the


78
measurement of this metabolite would be critical in obtaining a true measure of the
systemic bioavailability of mometasone furoate. It would be of further interest also to
assess the plasma protein binding of the active metabolite to estimate the free levels of
the metabolite that would be able to interact with the systemic glucocorticoid receptors.


CHAPTER 6
A SENSITIVE LC-MS/MS METHOD FOR THE QUANTIFICATION OF
MOMETASONE FUROATE IN HUMAN PLASMA.
Introduction
Inhaled corticosteroids are now the drugs of choice in the treatment of chronic
asthma because of their distinct anti-inflammatory effects in the lung and minimal
systemic side effects. One of the goals of treatment with inhaled corticosteroids is to
reduce the levels of the drug in the blood to minimize the side effects. Therefore, the
quantification of these drugs in plasma demand a highly specific and sensitive assay.
HPLC-UV methods are not suitable because of their poor sensitivity [109]. Alternative
methods like radioimmunoassays (RIA) and HPLC/RIA have been used for the
quantification of inhaled corticosteroids in biological matrices. These methods are either
time-consuming or are limited by low selectivity [139, 140]. Liquid chromatography-
tandem mass spectrometry (LC-MS) techniques have become popular in the
determination of corticosteroids in biological matrices because of their increased
specificity and sensitivity [112, 141-145].
Affrime and coworkers estimated the pharmacokinetics of mometasone furoate
(MF) after an oral inhalation and plasma levels of MF were quantified using an LC-MS-
MS method with a lower limit of quantification of 50 pg/ml [62]. However, the details of
the assay are not available. It was found that the plasma levels of MF were undetectable
in most samples and there have been suggestions that a more sensitive assay is needed to
obtain a better estimate of the pharmacokinetic parameters of MF [106, 107], This paper
79


80
presents a specific and sensitive atmospheric pressure chemical ionization (APCI) based
LC-MS-MS assay using a multiple reaction monitoring technique to quantify MF in
human plasma after oral inhalation of therapeutic doses.
Materials and Methods
Chemicals and Reagents
Mometasone furoate was purchased from USP (Rockville, MD). 13C3-Fluticasone
propionate (l3C3-FP) was used as an internal standard (I.S.) and was provided by
GlaxoWellcome R&D, Ware, Hertz, UK. HPLC grade methanol, ethanol, ethyl acetate
and heptane were purchased from Fischer chemicals (Springfield, NJ, USA). The solid
phase LCis (3 ml) cartridges for sample extraction were obtained from Supelco
(Bellefonte, PA, USA). Drug-free human plasma was obtained from the Civitan regional
blood system (Gainesville, FL, USA).
Preparation of Calibration Standards and Quality Control Samples
A primary stock solution of MF was prepared by dissolving 20 mg of MF in 50 ml
of methanol and was stored at -20C. The working solutions used in preparing quality
control samples (QC) and plasma calibration standards were 100 and 10 ng/ml of MF in a
mixture of methanol-water (85:15, v/v). The calibration standards (CC) ranged from 15 to
1000 pg/ml. Quality control (QC) samples in plasma (15, 30, 50, 80, 120 and 400 pg/ml)
were prepared from the stock solutions and were used against the independently prepared
plasma calibration standards. Two different series of stock solutions were prepared
separate weightings for the CCs and QCs. The I.S. stock solution was prepared by
dissolving 200 pg of C3-FP in 1 ml of methanol, and working solutions of 20 ng/ml
were prepared by diluting the stock solution with methanol.


81
Sample Processing
Plasma samples were thawed at room temperature and 50 pi of I.S. (corresponding
to approximately 1 ng/ml of 13C3-FP) working solution was added to 1 ml of plasma. The
compounds were then extracted using a previously reported method [112, 146], Briefly, 1
ml of 30% ethanol was added to 1 ml of the plasma sample and centrifuged. 2 ml of the
supernatant was then extracted using a 6 ml end-capped Ci8 cartridge and the analytes
were then eluted with 3 ml of a mixture of ethyl acetate-heptane (35:65, v/v). The residue
was then evaporated under vacuum and reconstituted in 100 pi of a mixture of methanol-
water (85:15, v/v) and a sample volume of 80 pi was injected into the HPLC-MS/MS
system.
HPLC-MS-MS Conditions
Isocratic high performance liquid chromatographic separations at ambient
temperature were achieved with a Waters 5-pm column (50mm x 4.6 mm i.d., Milford,
MA, USA) preceded by a Whatman 5-pm ODS Cig guard column cartridge (20mm x 2.0
mm i.d., Clifton, NJ, USA). The mobile phase consisted of methanol-water (85:15, v/v)
and was delivered at a flow rate of 1 ml/min using a LDC Analytical
constaMetric35000 solvent delivery system (LDC/Milton Roy, Riviera Beach, FL,
USA). The analysis of MF was performed using a Micromass Quattro-LC-Z (Beverly,
MA, USA) triple quadrupole mass spectrometer equipped with an APCI ion source.
Negative APCI mode was chosen to acquire the mass spectra after tuning with MF.
Multiple reaction monitoring was used for the quantification. The source temperature and
the APCI probe temperature were set to 120C and 500C respectively. Argon was used
as the collision gas and the mass resolution was set to unit mass. The corona voltage for


82
both MF and I.S. transitions was set to 3.5 kV. The cone voltages for the MF and I.S.
transitions were set to 35 V and 20 V respectively. A dwell time of 0.7 s was used for
monitoring MF transition and dwell time of 0.1 s was used for monitoring the I.S
transition. The MS-MS signals were optimized by injecting 1 pg/ml solution of MF in
methanol at a flow rate of 100 pl/min using a Kd-Scientific (Holliston, MA, USA)
infusion pump. The mass spectrometer was linked to a Perkin Elmer ISS 200 autosampler
by contact closure and was controlled by the software, Masslynx 3.1. Data analysis was
performed using the same software. Peak area ratios of MF to I.S. were plotted against
MF concentrations to obtain the calibration curves for MF. A weighted (1/x) linear
regression model with ten concentration points (including blank plasma) ranging from 15
to 1000 pg ml'1 in duplicates was used to plot the calibration curves.
Method Validation
Selectivity
Drug-free plasma from eight humans were extracted and screened for any potential
endogenous interference. Any apparent response from interference co-eluting at the
retention times of MF and the I.S. were compared to the response at the lower limits of
quantification for MF and to the response at the working concentration of the I.S.
respectively.
Recovery
The recovery of MF was determined at low (40 pg/ml), medium (80 pg/ml) and
high (400 pg/ml) concentrations by comparing the responses from plasma samples spike
prior to extraction with those from plasma samples extracted and spiked after extraction.
The recovery of the I.S., using an identical extraction method and analytical conditions,
has been reported earlier [112].


83
Accuracy and precision
Intra-day accuracy and precision were determined by analyzing the quality control
samples at concentrations of 15, 30, 50, 80, 120 and 400 pg/ml (n=6 at each
concentration) on the same day. Inter-day accuracy and precision were assessed by
repeating the experiment on three different days. Accuracy was expressed as the
percentage ratio of the measured concentration to the nominal concentration. Precision
was calculated as the coefficient of variation. The lower limit of quantification (LLOQ)
of the assay was defined as the lowest drug concentration that can be determined with an
accuracy of 80-120% and a precision <20% [147],
Stability
Freeze-thaw cycle stability was assessed by analyzing quality control samples at
concentrations of 50, 120 and 400 pg/ml in triplicate, following three cycles of freezing at
-80C and thawing. Short-term temperature stability was evaluated for the quality control
samples at the same concentrations by thawing the samples and keeping them at room
temperature for 4 hours. Bench-top stability for 7 hours at the same concentrations was
determined after extraction and reconstitution. Stability was evaluated in terms of
accuracy, as the percentage ratio of measured concentration to the nominal concentration.
Results and Discussion
Mass Spectrometry/Chromatography
Preliminary tuning experiments were performed for MF in APCI and electrospray
ionization (ESI) modes. Negative APCI (APCF) was chosen because of better sensitivity
seen by the increased signal-to-noise ratio for the product ion peak. The full scan mass
spectra of the parent and daughter ions of MF in APCF under multiple reaction
monitoring mode are shown in Figure 6-1. The best sensitivity was observed with the


84
transition m/z 484.0 to m/z 389.2 and was chosen as the transition channel for MF. The
parent molecular ion {m/z 484.0) corresponds to a difference of 36 mass units from the
actual mass of MF {m/z 520.0) and this can be attributed to the loss of an HC1 molecule
from the structure of MF in the APCI source. The transition for l3C3-FP was selected as
m/z 503.3 to m/z 380.0 based on a previous report and the tuning conditions for this
compound has been described in the same report [112], Both MF and the I.S eluted at
0.97 min and the total analysis time for each sample was 4 min. The resulting calibration
curves for the validation were linear in the range 15-1000 pg/ml with correlation
coefficients (r2) >0.99 (Figure 6-2).
Internal Standard Selection
Isotope-labeled MF was unavailable for use as an internal standard. Therefore,
inhaled steroids structurally related to MF were screened and fluticasone propionate was
chosen as the I.S. because of similar lipophilicity values for MF and FP and identical
retention times for both compounds. Moreover, FP has excellent sensitivity under
negative ion mode APCI and shows good recovery with the extraction method used in the
assay [112], However, interference in the MF transition was seen when unlabeled
fluticasone propionate was used as the internal standard. No interferences in the MF
transition were observed with 13C3-FP and hence this compound was used as the internal
standard.


85
Figure 6-1. Full scan (top panel) and daughter scan (bottom panel) spectra of MF.
Compound 2 name: MF Method File: MF4-12-04
Coefficient of Determination 0.997646
Calibration curve: 0.121256 x + 0.000341955
Response type: Internal Std ( Ref 1 ), Area ( IS Cone. / IS Area )
Curve type: Linear, Origin: Include. Weighting. 1/x. Axis trans: None
Figure 6-2. Representative calibration curve of MF in human plasma.


Full Text
UNIVERSITY OF FLORIDA
3 1262 07332 068 0



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PHARMACOKINETIC AND PHARMACODYNAMIC EVALUATION OF
MOMETASONE FUROATE
By
SRIKUMAR SAHASRANAMAN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2004

Copyright 2004
by
Srikumar Sahasranaman

For my Matha, Pitha, Guru, Deivam and Sangi.

ACKNOWLEDGMENTS
I wish to convey my sincere thanks and appreciation to my mentor, Dr. Guenther
Hochhaus, for his patient guidance, constant encouragement and constructive criticism
throughout the course of my work. I would like to thank the members of my supervisory
committee, Dr. Hartmut Derendorf, Dr. Jeffrey Hughes and Dr. Scott Powers, for their
advice and availability. I take this opportunity to thank Dr. Staffan Edsbacker and
AstraZeneca for funding this work. I also thank Prof. Anne Tattersfield and Dr. Kevin
Mortimer, University of Nottingham, for their collaboration.
I am thankful to Manish for his help and support during the course of this work. I
also appreciate the laboratory help provided by Mrs. Yufei Tang, Ms. Anke Czerwinski
and Ms. Diana Biniasz. I owe many thanks to Dr. Margaret James for allowing the use of
her laboratory facilities and to Dr. Gabor Toth and Dr. Gyula Horvath for the NMR
analysis in this work. 1 am grateful to the secretaries of the Department of
Pharmaceutics, Mr. James Ketcham. Mrs. Patricia Khan, Ms. Andrea Tucker and Ms.
Vada Taylor, for their technical and administrative assistance. I thank my group
members, Vikram. Yaning. Intira, Kai, Elanor and Zia. and other graduate students and
post-docs in the department for their assistance and friendship. Special thanks go to
Kula, Hari, Vijay, Manian, Subbi and all my other friends for their help and support that
have made my stay in Gainesville a memorable one.
IV

Finally, I want to express my deep gratitude and love for my parents, sister and
other family members for their unwavering support and encouragement of my academic
pursuits over the years.
v

TABLE OF CONTENTS
page
ACKNOWLEDGMENTS iv
LIST OF TABLES x
LIST OF FIGURES xi
ABSTRACT xiii
CHAPTER
1 INTRODUCTION 1
Asthma 2
Corticosteroids in Asthma Therapy 3
Mechanism of Action of Corticosteroids 4
Disposition of Inhaled Corticosteroids 8
Clinical Effects and Systemic Effects of Inhaled Corticosteroids 10
Pharmacokinetic and Pharmacodynamic Aspects of Mometasone Furoate 12
Clinical Efficacy of Mometasone Furoate 13
Pharmacokinetic Properties of Mometasone Furoate 15
Systemic Effects of Mometasone Furoate 18
Objectives of the Study 18
2 STABILITY OF MOMETASONE FUROATE IN AQUEOUS SYSTEMS 23
Introduction 23
Materials and Methods 23
Chemicals 23
Stability of MF in Simulated Lung Fluid (SLF) 24
Effect of pH on Stability of MF in SLF 25
Isolation of Degradation Products of MF in SLF 26
Mass Spectrometry of MF and its Degradation Products 26
Structure Elucidation of Degradation Products Using NMR 27
Glucocorticoid Receptor Binding Assay Experiments in Rat Lung Cytosol. 27
Results and Discussion 29
Stability of MF in SLF 29
pH Effects on Stability of MF in SLF 33
MS Analysis of MF and the Degradation Products 34
vi

NMR Analysis of MF and the Degradation Products 35
Chemical Structures of the Degradation Products 36
Glucocorticoid Receptor Binding of MF and its Degradation Products 36
3 IN VITRO EXTRA-HEPATIC METABOLISM OF MOMETASONE FUROATE40
Introduction 40
Materials and Methods 40
Stability of MF in Plasma 41
Metabolism of MF in Lung 42
Preparation of homogenizing buffer 42
Preparation of NADPH generating system 42
Tissue metabolism using unlabeled MF 43
Metabolism in lung tissue using labeled [1,2-3H]-MF 44
Results and Discussion 44
Stability in Plasma 44
Stability in Lung 47
4 IN VITRO DISSOLUTION PROFILE OF MOMETASONE FUROATE 53
Introduction 53
Materials and Methods 54
Chemicals 54
Particle Size Distribution 54
In Vitro Dissolution 54
Data Analysis 55
Results and Discussion 56
Particle Size Distribution 56
In Vitro Dissolution 56
5 HEPATIC METABOLISM AND CHARACTERIZATION OF METABOLITES
OF MOMETASONE FUROATE 61
Introduction 61
Materials and Methods 61
Chemicals 61
In vitro Hepatic Metabolism of MF 62
In vivo Metabolism and distribution of MF 62
Glucocorticoid Receptor Binding Assay Experiments in Rat Lung Cytosol. 63
Results and Discussion 65
Metabolism of MF in Rat Liver S9 Fractions 65
Metabolism of MF in Human Liver S9 Fractions 69
In vivo Metabolism and Disposition 73
Glucocorticoid Receptor Binding of MF and Metabolites 75
Vll

6 A SENSITIVE LC-MS/MS METHOD FOR THE QUANTIFICATION OF
MOMETASONE FURO ATE IN HUMAN PLASMA 79
Introduction 79
Materials and Methods 80
Chemicals and Reagents 80
Preparation of Calibration Standards and Quality Control Samples 80
Sample Processing 81
HPLC-MS-MS Conditions 81
Method Validation 82
Selectivity 82
Recovery 82
Accuracy and precision 83
Stability 83
Results and Discussion 83
Mass Spectrometry/Chromatography 83
Internal Standard Selection 84
Selectivity 86
Recovery 86
Precision and Accuracy 87
Stability 88
7 SINGLE DOSE PHARMACOKINETICS OF INHALED MOMETASONE
FUROATE 89
Introduction 89
Materials and Methods 90
Subjects 90
Protocol 90
Bioanalytical Methods 91
Pharmacokinetic Data Analysis 91
Results and Discussion 94
Noncompartmental Analysis 94
Compartmental Analysis 97
8 SELECTIVITY OF MOMETASONE FUROATE AND OTHER INHALED
CORTICOSTEROIDS TO THE GLUCOCORTICOID RECEPTOR 101
Introduction 101
Materials and Methods 102
Chemicals 102
Glucocorticoid Receptor (GR) Binding Assay Experiments 102
Progesterone Receptor (PR) Binding Assay Experiments 103
Data Analysis 105
Molecular Dynamic Modeling 106
Results and Discussion 106
Vlll

CONCLUSIONS
117
APPENDIX NMR ANALYSIS OF MF AND DEGRADATION PRODUCTS 120
LIST OF REFERENCES 124
BIOGRAPHICAL SKETCH 138
IX

LIST OF TABLES
Table page
1-1. Cellular and transcriptional effects of corticosteroids 6
1-2. Pharmacokinetic and pharmacodynamic parameters of inhaled corticosteroids 17
2-1. Electrospray ionization mass spectral data of MF and its degradation products 35
2-2. Relative binding affinities (RBA) to the glucocorticoid receptor of rat lung tissue.. 37
4-1. Particle size distribution (in %) of MF and BUD micronized dry powders 56
5-1. Percentage of dose distributed into different tissues two hours after intravenous
administration of [1,2-3H]MF in male Sprague-Dawley rats 74
5-2. Average relative binding affinities (RBA) to the glucocorticoid receptor of rat lung
tissue 76
6-1. Intra-day and inter-day accuracy/precision for MF in human plasma 88
6-2. Stability (%) of MF in human plasma 88
7-1. Median (% coefficient of variation) pharmacokinetic parameters of MF obtained by
noncompartmental analysis following oral inhalation of 800 pg of MF by Asmanex
Twisthaler® dry powder device 95
7-2. Estimates of the pharmacokinetic parameters of MF after fitting using a one-
compartment body model with first order absorption 97
8-1. Relative receptor affinities to the progesterone receptor of sheep uterus tissue 107
8-2. Chemical structures of various corticosteroids and metabolites 111
A-l. 'H and l3C chemical shifts, characteristic couplings (Hz), 13C, ‘H long-range
correlations (HMBC) and 'h/H steric proximities NOE of compound MF 121
A-2. 'H and l3C chemical shifts, Characteristic couplings (Hz), I3C,'H and long-range
correlations (HMBC) of compound D2 122
x

LIST OF FIGURES
Figure page
1-1. Disposition of corticosteroids after inhalation 10
1-2. Molecular structure of mometasone furoate 12
2-1. A schematic degradation pathway for conversion of MF into its degradation
products 31
2-2. Representative chromatograms of drug free SLF and SLF incubated with MF 32
2-3. Graphical representation showing nonlinear curve fitting of the concentration-time
data of MF and its degradation products D1 and D2 33
2-4. The degradation profiles for MF in phosphate buffer 34
2-5. Competitive binding experiments to the glucocorticoid receptor 38
3-1. Representative chromatograms of drug free rat plasma and rat plasma incubated with
MF 45
3-2. Concentration-time profile of MF and its degradation products following incubation
of MF rat plasma and human plasma 46
3-3. Representative chromatograms for blank S9 fraction of rat lung and S9 fraction of rat
lung incubated with MF 48
3-4. Representative radiochemical elution profiles of [1,2-3H]-MF incubated with S9
fraction of rat lung 49
3-5. A schematic degradation pathway for conversion of MF into its degradation products
on incubation of MF in plasma 51
4-1. Dissolution profiles of micronized dry powders of MF and BUD 58
4-2. Dissolution profiles of micronized dry powders of MF and BUD 59
5-1. Representative radiochemical elution profiles of [1,2-3H]-MF incubated in S9
fraction rat liver 66
xi

67
5-2. Concentration-time profile of [1,2-3H]-MF and the metabolites formed after
incubation of 3H-MF in S9 fraction of rat liver
5-3. Representative chromatograms for blank S9 fraction of rat liver and S9 fraction of
rat liver incubated with MF 68
5-4. Representative radiochemical elution profiles of 3H-MF incubated in S9 fraction of
human liver at 37°C 70
•j
5-5. Concentration-time profile of [1,2- H]-MF and the metabolites formed on incubation
of MF in S9 fraction of human liver 71
5-6. Radiochemical elution profiles of intestinal contents two hours after intravenous
administration of 5 pCi [1,2-3H]-MF in male Sprague-Dawley rats 75
5-7. Competitive binding experiments to the glucocorticoid receptor 77
6-1. Full scan and daughter scan spectra of MF 85
6-2. Representative calibration curve of MF in human plasma 85
6-3. Chromatograms of blank human plasma for the I.S and MF channels 86
6-4. Chromatograms of human plasma spiked with 1000 pg/ml I.S and 15 pg/ml MF
channels 87
7-1. Mean (+ SD) plasma concentration-time profile of MF after oral inhalation of 800 pg
of MF by Asmanex Twisthaler® dry powder device 95
7-2. Representative fit using a one compartment body model for one subject following
oral inhalation of a 800 pg dose of MF 97
7-3. Absorption profile of MF after inhalation of a single dose of 800pg determined using
the Loo-Riegelman method 99
8-1. Competitive binding experiments to the progesterone receptors 108
8-2. Competitive binding experiments to the glucocorticoid receptors 109
8-3. Relationship between Log P value of different steroids to the observed steroid
selectivity 110
8-4. The structure of mometasone furoate superimposed over that of progesterone bound
to the ligand-binding domain of the human progesterone receptor 114
8-5. The structure of R-budesonide superimposed over that of progesterone bound to the
ligand-binding domain of the human progesterone receptor 115

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
PHARMACOKINETIC AND PHARMACODYNAMIC EVALUATION OF
MOMETASONE FUROATE
By
Srikumar Sahasranaman
August 2004
Chair: Guenther Hochhaus
Cochair: Hartmut Derendorf
Major Department: Pharmaceutics
Mometasone furoate (MF) is a corticosteroid that is being evaluated in the
treatment of mild-to-moderate persistent asthma. Clinical studies with orally inhaled MF
have shown that the systemic exposure and bioavailability (<1%) of MF are unusually
low in comparison to other inhaled corticosteroids. This study evaluated possible reasons
for the low systemic exposure of MF.
MF was found to be stable in the lung and blood, and this suggests that extra-
hepatic metabolism might not be a reason for the low systemic exposure of MF. MF
showed a slow in vitro rate of dissolution that indicates that MF could be removed by
mucociliary clearance mechanisms leading to the low systemic availability.
In vitro and in vivo studies showed that MF was efficiently metabolized by the
liver, and the 6(3-hydroxy-mometasone furoate metabolite showed strong binding to the
glucocorticoid receptor. MF was also found to be unstable in aqueous systems and one of
the degradation products showed significant activity to the glucocorticoid receptor. The

in vivo formation of these active products in humans needs to be evaluated and this is
important to assess the true pharmacological profile of MF.
A specific and sensitive HPLC-MS/MS assay was developed and was applied in a
clinical study to estimate the pharmacokinetic parameters of MF in asthmatic patients.
The systemic bioavailability of MF was found to be 3.2% in this study. This value was
assessed using robust methodology and is a more accurate estimate than the previously
reported value of <1%. This systemic availability along with the active metabolite could
be responsible for the systemic effects of MF. MF was also found to have a long mean
absorption time of 4.1 h that strongly suggests that MF could be removed from the lung
by mucociliary mechanisms.
It was of interest to study the glucocorticoid receptor selectivity of MF and other
corticosteroids as part of the pharmacodynamic evaluation of MF. The binding affinities
of MF and other corticosteroids to the glucocorticoid receptor and progesterone receptor
were assessed. MF showed the highest binding affinity to the progesterone receptor and
was the least selective among the glucocorticoids studied.
xiv

CHAPTER 1
INTRODUCTION
Asthma is a disease that afflicts 100 to 150 million people in the world and about
5% to 6% of the population in the United States (US) [1-3]. The prevalence of asthma in
the US has been increasing over the past decades and this disease has been reported to
cause more than 470,000 hospitalizations and 5000 deaths [2-5], Asthma also is a huge
economic burden, with a total cost of more than $ 11 billion being incurred because of this
disease in the US in 1998 [3].
Although there is no cure for asthma, advances in the understanding of the
pathophysiology of asthma have led to significant improvements in the management and
control of this disease [2], One of the most important developments has been the use of
inhaled corticosteroids (ICS) as the first-line therapy in patients with chronic asthma [2,
6]. Beclomethasone dipropionate (BDP) was developed in the early 1970s and was the
first ICS marketed for the treatment of asthma. Since then, other potent topical
corticosteroids have been developed and currently flunisolide (FLU), triamcinolone
acetonide (TA), budesonide (BUD) and fluticasone propionate (FP) along with BDP are
available in the US [2], The clinical efficacy and side effects associated with these drugs
have been widely documented. Mometasone furoate (MF) and ciclesonide (CIC) are
among the new ICS that are currently under evaluation for the treatment of asthma. In
this chapter, an overview of the pathophysiology of asthma, the mode of action of ICS
and the pharmacokinetic and pharmacodynamic issues related to MF have been
presented.
1

2
Asthma
Bronchial asthma is defined as a chronic inflammatory disease of the airways that
causes airway hyperresponsiveness and the appearance of variable and reversible airflow
obstruction [7, 8]. The inflammation is associated with hypersecretion of mucus, injury
to the epithelial cells, and deposition of fibrous material in the subepithelial basement
membrane. Asthma is characterized by the interaction between a multitude of cell types
that are involved in the pathogenesis of the disease. These interactions between mast
cells, eosinophils, epithelial cells, macrophages, and lymphocytes result in the release of
different mediators leading to bronchoconstriction, mucus hypersecretion, epithelial
damage, microvascular leakage and stimulation of neural reflex [7, 9].
An asthma attack is generally triggered upon the inhalation of an antigen and the
subsequent binding of the inhaled antigen to IgE antibodies on the mast cells in lungs
[10], The binding promotes the exocytosis of the mast cells leading to the release of
histamine and leukotrienes that may cause an acute episode of airway obstruction.
Histamine causes contraction of the smooth muscle cells of the bronchi that leads to
constriction of the lumen of the bronchi. The release of histamine and leukotrienes leads
to production of mucus and also the attraction and accumulation of inflammatory cells
like eosinophils [9], Eosinophils enhance the degranulation and release of granular
proteins, promote the formation of cytokines and are the main effector cells in the
inflammatory process. The major basic protein and other granular proteins contribute to
airway epithelial and tissue injury and airway hyperresponsiveness [10, 11]. The mast
cells also release proteases that are inflammatory enzymes, which exacerbate airway
inflammation and hyperresponsiveness [10]. Mast cells express and release cytokines that
lead to the infiltration of inflammatory cells into the airways. Several classes of

3
cytokines participate in allergic cell recruitment. These include specific endothelial
activators (IL-4 and IL-3), non-specific endothelial activators (TNF-a and IL-1),
miscellaneous activators (IL-5, granulocyte-macrophage colony-stimulating factor (GM-
CSF), and interferon gamma), and direct cell migrators like RANTES [10, 11].
Macrophages and activated lymphocytes are other cell types that contribute to airway
inflammation. Macrophages produce IL-1 that may prime eosinophils to respond to other
secretory stimuli [9]. The T helper lymphocyte, Th2, helps B lymphocyte cells make IgE
antibodies by synthesizing IL-4. Th2 also releases IL-5 that acts as a chemo-attractant for
eosinophils and other inflammatory cells.
Corticosteroids in Asthma Therapy
Inhaled corticosteroids represent the best available option in the treatment and
management of asthma because of their excellent therapeutic ratios [2], ICS are preferred
in the treatment of chronic asthma over other classes of medication like short and long
acting (32-adrenergic agonists (albuterol, salbutemol), anticholinergics (ipratropium
bromide) and other anti-inflammatory drugs like cromolyn sodium and nedocromil
sodium because of their ability to affect many of the inflammatory pathways involved in
asthma [12, 13]. ICS have been known to significantly improve lung functions and
reduce symptoms in comparison with newer therapeutic agents like leukotriene
antagonists [8]. Various dosing strategies have been used to maximize the therapeutic
effects of ICS at the pulmonary target site while minimizing the systemic side effects.
The National Institutes of Health and the National Heart, Lung and Blood Institute
(NHLBI), in their guidelines for asthma management and prevention, recommended
dosing strategies for inhaled steroids based on the severity of the disease determined

4
using lung function tests [2], This is an empirical approach and alternate rational
methods using pharmacokinetic/pharmacodynamic (PK/PD) modeling techniques have
been described to optimize the therapeutic ratios of treatment using ICS [14-18], In this
context, it is important to first understand the mode of action and the various
pharmacokinetic and pharmacodynamic concerns with ICS.
Mechanism of Action of Corticosteroids
Among the various therapies available, only corticosteroids are able to suppress
inflammation in the airways. Corticosteroids achieve this by targeting epithelial cells and
by suppressing the production of chemotactic mediators and adhesion molecules and also
by inhibiting the survival of inflammatory cells like mast cells, eosinophils, T
lymphocytes and dendritic cells [19]. This results in an inhibition of recruitment of
inflammatory cells into the airway. These cellular effects of corticosteroids are
summarized in Table 1-1.
Corticosteroids affect many of the inter-cellular interactions during the
pathogenesis of asthma that cause airway inflammation. Corticosteroids are lipophilic
molecules and diffuse easily across the cell membranes where they bind with high
affinity to the glucocorticoid receptor (GR) in the cytoplasm. The glucocorticoid
receptors (GR) are distributed throughout the body in many tissues and belong to the
steroid superfamily of cytosolic receptors [20]. The human GR is known to have two
isoforms: hGR-a and hGR-[3 [21]. Even though both isoforms are transcriptionally
active, only the hGRa isoform is activated by corticosteroids. The hGR(3 isoform
provides an antagonistic effect to the glucocorticoid-activated hGRa isoform [21]. In the
inactive state, the GR is bound by two molecules of 90 kD heat shock protein (hsp90) at

5
the C-terminal binding domain and one molecule of immunophilin p59 [22, 23]. This
dimeric complexation of the GR with hsp90 is required to maintain the C-terminal
domain of the GR in a favorable conformation for ligand binding [24]. Binding of
corticosteroids to the GR activates the receptor by dissociating hsp90 and the other heat
shock proteins and exposes a pair of DNA-binding “zinc fingers” [25, 26], This results in
a conformational change that allows the GR-ligand complex to either translocate into the
nucleus or to interact with transcription factors in the cytoplasm [27], Upon
translocation, the GR-ligand complex binds as a dimer to specific glucocorticoid response
elements (GREs) on the DNA and this leads to the modulation DNA transcription [28], It
has been believed that the binding of the GR-ligand complex to DNA results in either an
increase (transactivation) or decrease (transrepression) in the transcription of responsive
genes through the GREs depending on whether the GREs are positive (GRE+) or
negative (GRE-) [29-31]. However, recent studies indicate that negative GREs are
generally not a feature of the promoter region of the proinflammatory genes that are
repressed by corticosteroids [19].
The transrepression of the different genes responsible for encoding pro-
inflammatory proteins is thought to be responsible for the clinical anti-inflammatory
effects of corticosteroids. Corticosteroids repress the production of cytokines (IL-1, IL-2
IL-3,11-4, IL-5, IL-6, IL-11,11-13, GM-CSF, tumor necrosis factor-a (TNF-a)),
chemokines (IL-8, MCP-1, MCP-3, MCP-4, MIP-la, RANTES), mediator-synthesizing
enzymes (COX-2, cytoplasmic PLA2) and adhesion molecules (ICAM-1, VCAM-1, E-
selectin) [32-36].

6
Table 1-1. Cellular and transcriptional effects of corticosteroids (adapted from Barnes
and Adcock [19]).
Cellular effects
Cell Type
Cell
Effect
Inflammatory
Mast cells
Reduce numbers
Inflammatory
Eosinophils
Reduce numbers
Inflammatory
T-lymphocyte
Decrease cytokine production
Inflammatory
Macrophage
Decrease cytokine production
Inflammatory
Dendritic cell
Reduce numbers
Structural
Epithelial
Decrease cytokine production
Structural
Airway smooth
Decrease cytokine production
muscle
Increase [^-adrenergic receptor numbers
Structural
Endothelial
Decrease leakage
Structural
Mucus gland
Decrease mucus secretion
Effects on gene transcription
Decreased transcription
Cytokines
Chemokines
Adhesion molecules
Inflammatory receptors and enzymes
Peptides
Increased transcription
Annexin-1 (lipocortin-1, phospholipase A2 inhibitor)
IL-1 receptor antagonist
IL-1R2
IicBa (inhibitor of NF-kB)
P2-adrenergic receptor
Secretory leukocyte inhibitory protein
Clara cell protein (CC10, phospholipase Aj inhibitor)
Recent advances in the understanding of the mechanisms of corticosteroids have
shown that the activated glucocorticoid receptors cause deacetylation of histones and this
results in transrepression. Histones along with nucleosomes make up chromatin, and
alterations in the structure of chromatins are responsible for the regulation of gene
expression [19]. The closed conformation of the chromatin structure is associated with
suppression of gene expression and activated proinflammatory transcription factors such
as NF- kB cause the opening of the chromatin structure by acetylation of the core
histones [19]. The ability of corticosteroids to deacetylate these histones produces the

7
decrease in proinflammatory gene transcription at the therapeutically relevant low
concentrations of corticosteroids. Methylation, phosphorylation and ubiquitination, apart
from acetylation, have also been known to modify histones and these modifications may
regulate gene transcription. Corticosteroids may also inhibit or reverse these processes to
produce gene suppression.
In the case of genes encoding for proinflammatory proteins that lack GRE, the GR-
ligand complex modulates gene transcription through indirect protein-protein interactions
with transcription factors such as activating protein-1 (AP-1) and nuclear factor kB (NF-
kB) [37-40], These transcriptional factors induce many genes that encode
proinflammatory proteins like metalloproteinases and cytokines such as IL-2.
Corticosteroids may also produce nontranscriptional effects like inhibiting proteins
responsible for the stabilization of messenger RNAs of genes coding for GM-CSF [41].
Corticosteroids increase the expression of genes (transactivation) coding for anti¬
inflammatory proteins like annexin-1, IL-10, the inhibitor of NF-kP and IicB-a [19].
Corticosteroids also increase the production of lipocortin-1, a protein that inactivates
phospholipase A2 leading to the inhibition of proinflammatory lipid mediators like
leukotrienes, prostaglandins and the platelet activating factor [42], However, the
widespread anti-inflammatory actions of corticosteroids cannot be explained by
transactivation of limited numbers of anti-inflammatory genes [19]. The transactivation
of genes by corticosteroids is, however, believed to be responsible for producing the side
effects like growth retardation, bone and skin disorders that are associated with
corticosteroids [19]. Corticosteroids also enhance the transcription of the gene encoding
the P2-adrenergic receptor, thereby reversing or/and preventing the down-regulation

8
induced by long-term therapy with [fy-adrenergic agonists [43], This increase in P2-
adrenergic response has led to the development of combination therapy of corticosteroids
with long-acting P2-adrenergic agonists like salmeterol-fluticasone propionate (Advair
Diskus®) and formoterol-budesonide (Symbicort®) combinations [44],
The transactivation and transrepression effects of corticosteroids are summarized in
Table 1-1. Current research in the development of corticosteroids has shown increasing
attempts to identify corticosteroids with specific transrepression pharmacology. Such
steroids may be devoid of the side effects associated with transactivation. However, no
successful trials with these transrepression specific steroids have been reported in humans
yet.
Disposition of Inhaled Corticosteroids
The schematic representation of the disposition of corticosteroids after inhaled
administration is given in Figure 1-1. Upon inhalation of a glucocorticoid, only 10% to
60% of the administered dose is deposited in the lungs [13]. The majority of the dose
(40% to 90%) impacts on the oropharyngeal region and is swallowed. The swallowed
fraction of the dose is absorbed from the gastrointestinal tract and undergoes first pass
inactivation by the liver and the orally bioavailable fraction then enters the systemic
circulation. The currently marketed inhaled corticosteroids, except the prodrug BDP, are
metabolized by the liver into their inactive products [45]. The drug particles deposited in
the lungs dissolve according to the rate of dissolution of the drug. Only the dissolved
drug interacts with the glucocorticoid receptors (GR), as described in the previous
section, present in the lung to produce the anti-inflammatory effects. Following the
interaction with the GR, the drug is absorbed into the systemic circulation. The

9
absorption processes are rapid and it is believed that dissolution is the rate-limiting step
involved in pulmonary absorption of ICS [46]. The total systemic bioavailability of an
inhaled glucocorticoid is, therefore, the sum of the oral and pulmonary bioavailable
fractions. The non-protein bound, free drug in the systemic circulation is able to interact
with the systemic GR to produce the systemic side effects associated with corticosteroids
[47], The drug is eliminated from the systemic circulation mainly by hepatic clearance
mechanisms. Mucociliary clearance is an important mechanism that removes the
undissolved drug particles from the lung into the gastrointestinal tract [48]. Mucociliary
clearance acts as a primary defense mechanism that removes foreign particles and
aerosolized pollutants from the nose and the airways. This clearance mechanism is more
pronounced in the central airways of the lung compared to the peripheral airways and
plays an important role in reducing the pulmonary availability of highly lipophilic
corticosteroids that have a slow rate of dissolution.
The goal of treatment using ICS in asthma is to maximize the clinical anti¬
inflammatory effects in the lung while minimizing the systemic side effects. This can be
achieved by increasing the concentrations of the drug in the lung and decreasing the
levels in the systemic circulation and this is known as pulmonary targeting. Based on the
systemic disposition of ICS, computer simulations have been used to determine the
factors that are important in achieving good pulmonary targeting with ICS [49], These
factors are high pulmonary deposition, location of pulmonary deposition, optimal
pulmonary residence time, low oral bioavailability, high protein binding and high
systemic clearance. It is also important that the ICS bind specifically to the

10
glucocorticoid receptor as binding to the other members of the steroid family like the
progesterone receptors might result in undesired side effects.
40 - 90 % swallowec
Mouth and Pharynx
10 - 60 % Lungl | Mucociliary clearance
Deposition X
Gl tract
Lun
Absorption
from Gut
Pulmonary'"^ Absorption
Systemic
Circulatio
Liver
Orally absorbed
Fraction
Clearance
First-pass
Inactivation
Systemic
Side Effects
Figure 1-1. Disposition of corticosteroids after inhalation (adapted from Derendorf et al.
[50])
Clinical Effects and Systemic Effects of Inhaled Corticosteroids
The clinical anti-asthmatic effects of inhaled corticosteroids and the systemic side
effects associated with these drugs have been widely documented. Because of the
excellent anti-inflammatory properties of these drugs, treatments with ICS have been
shown to result in attenuation of symptoms and improved pulmonary function, reduction
in usage of supplemental P-adrenergic agonist and fewer exacerbations of asthma that
require the use of oral corticosteroids [51]. The assessment of lung function is normally

11
monitored using parameters such as peak expiratory flow (PEF) and forced expiratory
volume in one second (FEVi). Treatments with ICS also result in improvement in the
quality of life and reduction in hospitalizations in asthmatic patients. Studies have also
shown that ICS are useful in protecting against life-threatening and fatal asthma and there
is a possible reduction in the mortality rates in these cases [52, 53].
Even though most of the dose is swallowed upon inhalation and the ICS have low
oral bioavailability, these drugs are not without side effects as they are able to bind to
glucocorticoid receptors in the systemic circulation after pulmonary absorption. The
potential systemic side effects associated with ICS are suppression of growth in children,
bone demineralization and bone metabolic disorders, muscle atrophy, cataracts and
glaucoma [51]. Other rare adverse events that have been reported with ICS are dermal
thinning, psychosis, hypoglycemia, development of Cushingoid features and Addisonian
crisis. Corticosteroids may also produce topical side effects like dysphonia and oral
candidiasis upon inhalation as most of the dose is deposited in the oropharyngeal region.
However, these topical side effects can be minimized by using spacer devices and by
mouth rinsing after inhalation [54], The systemic side effects are commonly assessed by
monitoring the suppression of the hypothalamus-pituitary-adrenal (HPA) axis. The HPA
axis, which maintains homeostasis in both basal and stress-related states, is a sensitive
and convenient biomarker to detect the presence of exogenous corticosteroids [55, 56],
The administration of exogenous corticosteroids results in a negative feedback effect on
glucocorticoid receptors in the hypothalamus and anterior pituitary gland, resulting in the
suppression of corticotrophin and corticotrophin-releasing hormone, respectively. This
leads to a decrease in the secretion of the endogenous corticosteroid cortisol from the

12
adrenal cortex [57], The exogenous corticosteroids that are systemically available may
sometimes lead to prolonged adrenal suppression resulting in atrophy of the adrenal
cortex [56]. The presence of low endogenous cortisol levels may not be clinically
relevant as long as there is compensation from the additional glucocorticoid activity
because of the exogenous corticosteroids in the body. However, the presence of adrenal
cortical atrophy becomes clinically relevant if there is a sudden termination of exogenous
glucocorticoid therapy, or if there is unusual stress resulting in acute adrenal
insufficiency crisis [57]. Several PK/PD models have been reported to predict the
expected cortisol suppression after administration of inhaled corticosteroids [14, 58].
Pharmacokinetic and Pharmacodynamic Aspects of Mometasone Furoate
Mometasone furoate (MF, C27H30CI2O6, 9a,21-dichloro-l 1(3,17a-dihydroxy-16a-
methylpregna-l,4-diene-3,20-dione 17-(2-furoate), Figure 1-2) is a synthetic
glucocorticoid with anti-inflammatory activity. MF has been used in the treatment of
topical dermatological disorders [59]. MF has also been approved in the prophylaxis and
treatment of allergic rhinitis [60]. MF is currently available in the European Union and is
being evaluated in the United States as an oral inhalation powder in the treatment of
patients with mild-to-moderate persistent asthma [61-63],

13
MF is a potent glucocorticoid with a higher relative binding affinity (Table 1-2) to
the glucocorticoid receptor than the currently available inhaled corticosteroids [64],
However, higher receptor affinity does not necessarily translate into a higher activity with
MF and only indicates that lower doses of MF are required to produce the same degrees
of receptor occupancies. MF also shows a higher degree of plasma protein binding
(98%-99%) than the other ICS [65],
In vitro functional assays have shown that MF inhibits the various proinflammatory
processes in asthma such as the release of cytokines (IL-4, IL-5, interferon-y) by the T-
helper cells, adhesion molecule expression (VCAM-1) on human bronchial epithelial
cells, the proliferation of mononuclear cells, eosinophil survival and the release of
histamine and cysteinyl leukotriene by basophils [66-70]. The production of IL-1, IL-6
and TNF-a in murine myelomonocytic leukemia cells was also inhibited by MF in vitro
[71], MF has also been shown to transactivate GR-mediated transcription of the
chloramphenicol acetyltransferase reporter gene with greater potency than the other
inhaled corticosteroids in an in vitro study using kidney cells of African green monkeys
[72],
Clinical Efficacy of Mometasone Furoate
The use of MF in patients with mild-to-moderate persistent asthma results in a
significant improvement in lung function, reduction in symptoms and reduction in
bronchial hyper-responsiveness. Administration of 50 to 400 pg bid MF showed
significant improvement in the FEV i values after 6 days of treatment in a clinical study
based on allergen-induced decrease in FEVi [73].
In patients with mild-to-moderate asthma previously using short-acting inhaled 02-
adrenergic agonists, the use of MF administered as 200 pg bid, 400 pg once daily in the

14
morning or 200 pg once daily in the evening for 12 weeks showed significant
improvement in asthma symptoms and lung function [74, 75], Significant improvements
were seen in FEV \ and PEF values when compared to placebo in three randomized,
double blind, multi-center clinical trials. Significant improvement in the time to
worsening of asthma was also seen in patients using MF compared to those using
placebo. In moderate to severe asthmatic patients dependent on oral prednisolone,
treatment with 400 pg or 800 pg bid MF for twelve weeks improved asthma control,
quality of life, lung function and decreased dosage of the oral steroids [76]. Clinical
trials have also assessed MF to be equally or more efficacious than currently used inhaled
corticosteroids like fluticasone propionate (FP), budesonide (BUD) and beclomethasone
dipropionate (BDP) in patients with mild to severe persistent asthma [63, 77-80], Patients
with mild-to-moderate asthma showed similar improvement in lung functions determined
by FEV i and PEF, when treated with 100 or 200 pg bid MF or 200 pg bid of BDP over
12 weeks [77], MF 200 or 400 pg bid showed significant improvements in lung function
when compared to BUD 400 pg bid [78], There was also a significant improvement in
mean P2-adrenergic agonist usage and mean wheezing score during treatment with MF
compared to budesonide [78], Similar improvements in lung function and asthma
symptoms were seen with treatment using 200 pg bid MF and 250 pg bid fluticasone
propionate in patients with moderate persistent asthma [81].
The recommended starting dose of MF in adolescent and adult patients previously
on P2-adrenergic agonists or inhaled glucocorticoid therapy is 400 pg once daily
administered by a dry powder inhaler (Twisthalerâ„¢) [82], Once-daily administration of
MF has been shown to be more effective when administered in the evening than in the

15
morning [79], Mometasone furoate administered by dry powder inhaler has been shown
to be well tolerated. The most common adverse events associated with MF were oral
candidiasis, headache, pharyngitis and dysphonia [74, 75, 77, 78, 80], Clinical trials have
shown that MF <1600 pg/day is well tolerated up to one year and the incidence of
adverse events was similar with 100 to 400 pg bid MF, 200 pg bid BDP, 400 pg BUD or
250 pg bid FP [77, 78, 81].
Pharmacokinetic Properties of Mometasone Furoate
The pulmonary deposition of MF in asthmatic patients has been evaluated by
administering a 200 pg dose of technetium 99m ("mTc)-radiolabeled MF using a metered
dose inhaler [83]. Using gamma scintigraphy techniques, it was found that the mean
whole lung deposition was 13.9% while the mean deposition in the central, intermediate
and peripheral areas of the lung was 5.3%, 4.7% and 4.0% respectively [83], This pattern
of deposition is typical of inhaled drugs in patients with asthma [84]. However, the
deposition pattern of MF after administration by the marketed dry powder inhaler
formulation (Twisthaler®) has not yet been reported.
The pharmacokinetic (PK) data available, after oral inhalation of MF, is limited.
Affrime et al. reported the PK in healthy subjects after a single dose administration of
400 pg of MF given intravenously (IV), by metered dose inhaler (MDI) and by dry
powder inhaler (DPI) [62], Following the administration by MDI, MF was undetectable
in plasma with mean plasma concentrations being below the lower limit of quantification
(LLOQ, 50 pg/mL) at all time-points. After the DPI administration, plasma
concentrations of MF were also extremely low and close to the LLOQ. The mean
bioavailability of MF after DPI was estimated to be <1% (0.96%) [62]. The

16
pharmacokinetic parameters after the inhalation administration were unable to be
determined because of the low concentrations that were below the LLOQ. The systemic
clearance and volume of distribution of the terminal phase determined after the IV
administration were 54 L/h and 332 L respectively [62]. This systemic clearance of MF
is consistent with the high systemic clearance values seen with the other inhaled
corticosteroids. The pharmacokinetic parameters of MF and other commonly used
inhaled steroids are given in Table 1-2. Minimal systemic exposure to MF was also seen
after multiple-dose administration by DPI with mean Cmax values close to or below the
LOQ (50 pg/mL) after a 200 to 800 pg/day dose administered by DPI for 4 to 52 weeks
[63, 77], These initial studies on the PK of MF suggest that the systemic levels and
bioavailability of MF are extremely low after an oral inhalation when compared to other
inhaled corticosteroids (Table 1-2).
There is very limited information available on the metabolism of MF. An abstract
reported that MF undergoes extensive metabolism in vitro by liver hepatocytes and the
metabolites were tentatively identified [85]. Other studies have suggested that MF
undergoes hydroxylation at the 6-position to form 6|3-hydroxy-MF; hydrolysis of the
furoate ester and the substitution of the C-21 chlorine with a hydroxyl group [60, 63].
The above-mentioned routes of metabolism are tentative and there is no conclusive
evidence of a single major metabolite and none of the metabolites have been
unequivocally identified [61]. Two reports, by the same group, on the metabolism of MF
in liver and intestinal microsomes reported the formation of one metabolite (tentatively
identified as 6p-hydroxy-MF) while mentioning that other parallel and subsequent
metabolism pathways may be present [86, 87].

17
Table 1-2. Pharmacokinetic and pharmacodynamic parameters of inhaled corticosteroids.
Drug
BDP
17-BMP
FLU
TA
BUD
FP
MF
Fora! (%)
15-20
72a
7-20
23
6-1 lb
<1
< 1
Finh (%)
25(CFC)
27(CFC)
70 (HFA)
39
(MDI)
22-25
(MDI)
26 (MDI)
38 (TBH)
26 (MDI),
12 (DH)
17(DSK)
< 1
(MDI)
(DPI)
fu (%)
13
1.6
20
29
12
10
1-2
CL (L/h)
230
54c
58
37
84,
67 (22S),
117 (22R)
69
54
Vdss (L)
NA
84c
96
103
183,
245 (22S),
425 (22R)
356
NA
Vdnj-gn (L)
NA
NA
134
107
339
776
332
IV T'A (h)
0.1-0.5
0.6-1.7
1.6
2.0
2.8,
2.7 (22S),
2.7 (22R)
5-13
4.5
Inh T'A (h)
0.1
1.5-6.5
1.6
3.6
3.0
8-14
NA
MRT-IV
(h)
NA
1.6
1.7d
2.7
2.2
4.9
NA
MAT (h)
NA
NA
<1
2.9
0.3-2.6
5
NA
RRA
(Dexa=100)
NA
1022
190
233
935
1800
2900
References
[49, 88-
91]
[49, 88,
92, 93]
[49, 88,
94, 95]
[18, 49,
88, 96]
[49, 88, 97-
99]
[49, 88,
100-102]
[62, 64]
Abbreviations: BDP- Beclomethasone dipropionate; 17-BMP- Beclomethasone-17-monopropionate (active
metabolite of BDP); FLU-Flunisolide; TA- Triamcinolone acetonide; BUD- Budesonide; FP- Fluticasone
propionate; MF- Mometasone fiiroate; Foral- Oral bioavailability; Finh- Overall systemic bioavailability after
inhalation; fu- Fraction unbound; CL- Clearance; Vdss- Volume of distribution at steady state; Vdarea-
Volume of distribution of the terminal phase; IV T'A- Elimination half-life; Inh T!4- Terminal half-life after
inhalation; MRT-IV - Mean residence time after intravenous administration; MAT - Mean absorption time
after inhalation; RRA - Relative receptor affinity; Dexa - Dexamethasone; MDI- Metered dose inhaler;
DH- Diskhaler; DSK- Diskus; TBH- Turbohaler; CFC- Chlorofluorocarbon propellant based MDI; HFA-
Hydrofluoroalkane-134a propellant based MDI; NA- Not available;a -In rats;b- Radiolabeled budesonide;
c- Assuming complete conversion after intravenous administration of BDP; d- Calculated from Vdss and CL
after intravenous administration; 22S and 22R - Epimers of Budesonide.

18
Systemic Effects of Mometasone Furoate
Even though MF has minimal systemic exposure, clinical studies have shown that
MF produces considerable systemic effects as assessed by suppression of the
hypothalamus-pituitary-adrenal (HPA) axis after an oral inhalation. Mean serum cortisol
under the plasma concentration-time curve over 24 hours (AUCC24) was used as the
surrogate parameter to determine the HPA axis suppression in these studies. In a clinical
study involving 64 patients with mild-to-moderate asthma, 400 pg or 800 pg bid of MF
administered by a metered dose inhaler for 28 days produced a 10%-40% decrease the
AUCC24 compared to placebo (,P<0.05) [63], In comparison, 880 pg bid of fluticasone
propionate produced a 43%-56% suppression and an oral dose of 10 mg/day of
prednisolone caused a 64% to 72% suppression of AUCC24 compared with placebo
(P<0.01) [63, 103], Higher systemic levels of MF are observed when administered with
the dry powder inhaler than with the metered dose inhaler [62], Therefore, it is expected
that administration with the former formulation would result in greater cortisol
suppression. Unfortunately, there are no reports of direct comparisons between the two
formulations with respect to HPA axis suppression. Administration of 400 pg bid and
800 pg bid of MF by dry powder inhaler resulted in significant lowering of AUCC24 by
10% to 25% and 21% to 40% respectively compared to placebo treatment [63]. The
results from these studies have led to suggestions that the reported bioavailability of MF
is spurious or that MF has at least one active metabolite responsible for producing the
systemic effects [104-107],
Objectives of the Study
As mentioned earlier, the systemic exposure of mometasone furoate is extremely
low (systemic bioavailability <1%) when compared to other corticosteroids after oral

19
inhalation (Table 1-2). The deposition efficiency of MF has been reported to be
comparable to other ICS and therefore, the low systemic bioavailability cannot be a result
of poor deposition of MF into the lungs [62, 83]. Efficient hepatic metabolism might be
suggested as a reason for the low systemic levels of MF. However, hepatic metabolism
alone is insufficient to explain the unusually low systemic levels of MF compared to
other ICS as high clearances have also been reported for FP, BUD, triamcinolone
acetonide (TA) and flunisolide (FLU) (CL in Table 1-2). The systemic bioavailabilities
(Fjnh in Table 1-1) of these other steroids are much higher than the <1% estimate
proposed for MF. Since systemic clearances of MF and the other corticosteroids are
close to the hepatic blood flow rates, efficient hepatic metabolism alone does not explain
the difference between the bioavailability of MF and the other corticosteroids.
The other reasons that might explain the unusually low systemic exposure of MF
after an oral inhalation are:
1. MF is efficiently metabolized by the lung and therefore is not absorbed into the
systemic circulation leading to low concentrations in the blood.
2. MF is metabolized by the enzymes present in blood.
3. MF is a highly lipophilic drug [103] and could have a slow rate of dissolution, leading
to the drug being removed by mucociliary action out of the lung into the
gastrointestinal tract.
In the present study, these three reasons would be evaluated as possible factors
responsible for the low systemic exposure of MF.
In vitro metabolism of MF would be conducted in fresh rat plasma and in the S9
fraction of rat lung tissue and the metabolic profile of MF would be studied under
comparable conditions in human plasma and S9 fraction of human lung tissue. Stability
of MF in simulated lung fluid (SLF) would also be studied. The purpose for conducting

20
stability of MF in buffer systems is primarily to delineate degradation effect from
metabolism. This is to ensure that any change occurring in the parent structure (MF) is a
result of enzymatic action and not a result of degradation due the buffer system
employed.
Even though the systemic bioavailability of MF has been reported to be extremely
low, the drug shows considerable systemic effects as manifested by the suppression of the
hypothalamic-pituitary-adrenal axis [63], While the high potency of MF may be
suggested as a reason for the systemic effects in spite of the low availability, this is not
likely because MF also has high plasma protein binding (98%-99%) thereby reducing the
free levels available to produce the systemic effects. This contradiction could therefore
be possibly explained by the metabolism of MF into at least one active metabolite that
produces the systemic effects. To assess this hypothesis, this study also evaluates the
hepatic metabolism and the biological activities of the metabolites thus formed.
The validity of the reported <1% bioavailability estimate for MF has also been
questioned as the estimate was based on an assay of inadequate sensitivity (lower limit of
quantification, 50 pg/mL) and at a low dose (400 pg), which resulted in most of the
concentrations after oral inhalation being below the limit of quantification [106, 107],
The concentrations below the limit of quantification were then substituted as zero for area
under the curve calculations and this leads to an underestimation of the area under the
curve and the resulting systemic bioavailability. Therefore, another objective of this
study was to develop and validate a specific and sensitive analysis method using tandem
liquid chromatography-mass spectrometry (LC-MS/MS) for the quantification of MF in
human plasma. This assay would be used in a clinical study to determine the

21
pharmacokinetics of MF in patients with asthma after oral inhalation of a single dose of
800 pg of MF. It is expected that the use of a sensitive assay and administration of a
higher dose would enable the robust and accurate estimation of the systemic
bioavailability and other pharmacokinetic parameters of MF.
Austin et al. recently reported that MF and fluticasone propionate are agonists of
the progesterone receptor (PR) and weak antagonists of the androgen receptor [108],
Binding to the PR might be of clinical importance since inappropriate activation of the
progesterone receptor might result in dysregulation of the oestrus cycle and could lead to
a worsening of the symptoms of premenstrual tension. These effects may be exacerbated
in women with pre-existing difficulties. An excessive activation of PR will ultimately
have a contraceptive effect. Therefore, specificity to the glucocorticoid receptor (GR) is
an important requisite for the safety of inhaled corticosteroids. The specificity of MF and
other commonly inhaled corticosteroids to the GR will be assessed by comparing their
binding affinities to the GR and PR respectively.
Based on the above discussions, the overall objectives of the study are summarized
below:
1. Evaluate the stability of mometasone furoate in biological buffers and to characterize
the structure and the pharmacological properties of the degradation products.
2. Study the metabolism of mometasone furoate in rat and human lung, and plasma.
3. Assess the in vitro dissolution rate of mometasone furoate.
4. Study the hepatic metabolism of mometasone furoate and characterize the
pharmacological properties of the metabolites.
5. Develop and validate a specific and sensitive LC-MS/MS assay for the quantification
of mometasone furoate in human plasma.
6. Assess the pharmacokinetics of mometasone furoate after administration of a single
dose of 800 pg by oral inhalation in patients with asthma.

22
7. Investigate the specificity of mometasone furoate and other inhaled corticosteroids to
the glucocorticoid receptor.

CHAPTER 2
STABILITY OF MOMETASONE FUROATE IN AQUEOUS SYSTEMS
Introduction
There is very limited information available on the stability of MF in biological
tissues/fluids and aqueous systems. Teng and coworkers have characterized the
degradation of MF in simulated lung fluid (SLF) and have shown that MF is unstable in
SLF resulting in three degradation products [109]. They have also recently reported the
degradation of MF in aqueous systems and have hypothesized the structure of the
degradation products based on previous studies conducted with other corticosteroids
[110]. The present study aimed to clearly identify the structures of these degradation
products by MS and NMR analysis. It was of further interest to assess their potential
biological activity, by performing rat lung glucocorticoid receptor binding studies,
because of the possibility of these degradation products being formed in vivo.
Materials and Methods
Chemicals
Mometasone furoate (MF) was purchased from USP (Rockville, MD).
Dexamethasone was purchased from Sigma Chemicals Co (St Louis, MO). 3H-
Triamcinolone acetonide (specific activity 38 Ci/mmole) was purchased from Perkin
Elmer Life Sciences (Boston, MA). The degradation products of MF: D1 and D2 were
isolated and purified using HPLC and solid phase extraction in our laboratory. All other
chemicals and solvents were obtained from Sigma Chemicals Co. (St Louis, MO) and
Fisher Scientific Co. (Cincinnati, OH).
23

24
Stability of MF in Simulated Lung Fluid (SLF)
Composition of the simulated lung fluid buffer was adopted from the report of
Kalkwarf [111]. In brief, SLF comprised of MgCl2.6H20 (1 mM), NaCl (103 mM), KC1
(4 wM), Na2HP04.7H20 (1 mM), Na2SO4(0.5 mM), CaCl2.2H20 (2.5/wM),
NaH3C202.3H20 (7 mM), NaHC03 (31 mM) and Na3H3C60y.2H20 (0.33 mM) dissolved
in a liter of distilled water. The pH of the SLF buffer solution was adjusted to 7.4 using
dilute HC1 (20% v/v). 10 ml of the SLF was pre-incubated at 37°C for 10 min, and
spiked with MF (1 mg/mL stock solution) to achieve a concentration of 2.5 pg/mL.
Blank buffer was used in a control experiment. 500pL aliquot samples were withdrawn
at 0, 5, 10, 15, 30, 45 min and then at 1, 1.5, 2, 4, 6, 7, 24, 48 and 72 h into pre-chilled
tubes and the samples were diluted with 500pL of mobile phase (63:37% v/v MeOH-
H20). The samples were analyzed by HPLC-UV.
A LDC/Milton Roy CM4000 multiple solvent delivery system using a Milton Roy
SM 4000 programmable wavelength detector set at Xmax 254 nm, a CR-3A Chromatopac
integrator (Shimadzu Corporation, Japan) and an automatic injector (Perkin Elmer,
Boston, MA) was used as HPLC system. Chromatographic separation of the analytes
was achieved on Waters 5-pm symmetry RP-18 (150 x 4.6 mm i.d) column (Milford,
MA) preceded with a guard column (10 x 4.6 mm i.d, filled with reverse phase pre¬
column material) using a mobile phase of 63:37% v/v methanol-water at a flow rate of 1
ml.
Calibration curves based on MF peak areas over the concentration range of 0.125 to
5.0 pg/mL using unweighted least squares linear regression analysis resulted in r2 values
of at least 0.999. The limit of quantification of the assay was 0.125 pg/mL based on
good accuracy (> 90%) and precision (< 10% coefficient of variation).The good

25
reproducibility of the system allowed quantification without an internal standard as
potential internal standards either cross-eluted with degradation products or were unstable
in the buffer systems used.
Because of the lack suitable standards of D1 and D2, quantification of the
degradation products was based on the assumption that the molar absorptivities were
similar to that of MF. Results for MF and degradation products are presented in pM and
expressed in % of MF starting concentrations in graphical representations.
The concentration-time data obtained for MF and its degradation products were
fitted using SCIENTIST™ software (version 2.0, Micromath®, Salt Lake City, UT)
using the model shown in Figure 2-1. Alternative degradation pathways (broken arrows,
Figure 2-1) allowing first for the cyclization of MF followed by epoxide formation was
not indicated by the number of degradation products identified. The half-lives for the
different compounds were calculated using the equation below:
Ti/2 = ------, where kapp is the apparent degradation constant for that compound.
kapp
Effect of pH on Stability of MF in SLF
To assess the pH dependence, degradation process experiments were conducted in
50 mM K2HPO4 buffer at three different pH values (pH 7.0, 7.5 and 8.0). A fifty times
higher buffer strength (compared to SLF) of K2HPO4 was selected to improve the
buffering capacity. The pH of the buffer at the higher buffer strength did not change over
an incubation period of 24 h at 37°C. MF at a concentration of 2.5 pg/mL (4.8 pM) was
incubated at 37°C in a shaker bath. 200pL aliquot samples were withdrawn at 0, 5, 30,
min and then at 1,2, 4, 6, 8, and 10.6 h into pre-chilled tubes and the samples were

26
diluted with 200 pL of mobile phase (63:37 MeOH-I-hO). The samples were analyzed
using HPLC-UV as described above.
Isolation of Degradation Products of MF in SLF
For the purpose of isolation of degradation products, 4 vials containing 10 mL of
SLF buffer were spiked with MF (1 mg/mL stock solution) to give a concentration of 10
pg/mL. After incubation, the contents of the vials were pooled and 1 mL aliquots of the
spiked SLF were transferred to individual solid-phase extraction (SPE) cartridges and
extracted as reported previously [112]. The extracted and reconstituted samples were
injected onto the HPLC and the peaks corresponding to the various degradation products
were collected using a fraction collector (Gilson Model 203, Middleton, WI). Several
HPLC runs had to be performed to purify sufficient amount of material. The fractions
corresponding to each degradation product were pooled and extracted using the SPE
procedure described above. Purity (greater than 95%) and content of the isolated
degradation products were determined by HPLC. The isolated fractions were analyzed
by MS and NMR for structure determinations (see below). In addition, the activity of
these isolated degradation products was assessed in glucocorticoid receptor binding
experiments (see below).
Mass Spectrometry of MF and its Degradation Products
LC-MS analysis was used to identify the molecular mass of the degradation
products of MF. The chromatographic separation of the analytes was achieved on a
Waters 5-pm symmetry RP-18 (50 x 4.6mm i.d) column (Milford, MA) with an aqueous
mobile phase containing 70:30% v/v MeOH-H20 and flow rate of 0.3 ml/min. The LC
system was connected to a Micromass Quattro-LC-Z (Beverly, MA) triple quadrupole
mass spectrometer equipped with ES+ (electro-spray) ion source. The source

27
temperature was set to 120°C and the ES probe temperature was 400°C. Capillary and
cone voltages were set to 3kV and 25 V respectively. The samples were detected using a
full scan mode. The mass spectrometer was linked to a Perkin Elmer ISS-200 auto
sampler via a contact closure and the operation was controlled by computer software
MASSLYNX 3.1.
Electron ionization (El) mass spectra were taken on a Finnigan MAT 8430 mass
spectrometer under the following operating conditions: resolution, 1250; ion accelerating
voltage, 3 kV; electron energy, 70 eV; electron current, 500 pA, ion source temperature,
250 °C, evaporation temperature, 140 °C.
Structure Elucidation of Degradation Products Using NMR
NMR analysis was conducted at the IVAX Drug Research Institute, Budapest,
Hungary. The NMR spectra were recorded in CDCI3 at room temperature using Shigemi
sample tubes (180 pi) on Bruker Avance-500 spectrometer at 500.13/125.7 MHz. The
pulse programs were taken from Bruker software library.
Glucocorticoid Receptor Binding Assay Experiments in Rat Lung Cytosol
The various dilutions for MF (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0, 100 pM)
were prepared in methanol. Dilutions for degradation product D1 and dexamethasone
(0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1.0 pM) and degradation product D2 (0.001, 0.003,
0.01, 0.03, 0.1, 0.3, 1.0, 0.3 pM) were also prepared in methanol.
The animal protocol was approved by the local Institutional Animal Care and Use
Committee (IACUC) at the University of Florida, Gainesville, Florida. Sprague Dawley
rats (250 ± 25gms) were obtained from Harlan (Indianapolis, IN). The rats were
anesthetized using a cocktail mixture of ketamine, xylazine and acepromazine (3:3:1 v/v)

28
and were decapitated. The lungs were removed and homogenized in 8 volumes of ice-
cold incubation buffer (10 mM Tris/HCL, 10 mM sodium molybdate, 2 mM 1,4-
dithioerythritol). The homogenate was incubated with 5% w/v charcoal suspension (in
deionized water) for 10 min. The homogenate was then centrifuged for 20 min at
40,000X g in J2 rotor of Beckman centrifuge to obtain the cytosol. Fresh cytosol was
prepared and used for all the individual experiments.
10 nM of 3H-labeled triamcinolone acetonide (TA) solution was used as a tracer,
based on previous saturation binding experiments performed in our laboratory (data not
shown). 20 pL of the drug solution in methanol was added to pre-chilled tubes. Blank
methanol was used for the determination of total binding. Non-specific binding was
determined after addition of 20 pL of 100 pM unlabeled triamcinolone acetonide (10 pM
in the final incubation mixture). 20 pL of 100 nM3H-triamcinolone acetonide solution
(10 nM in final incubation mixture) was then added. 160 pL of the lung cytosol was
added and the tubes were vortexed and incubated at 4°C for 24 h. After the incubation,
200 pL of 5% w/v charcoal suspension (in water) was added to the tubes to remove the
excess unbound radioactivity. The tubes were vortexed and 300 pL of the supernatant
was transferred to the scintillation vials. 5 mL of the scintillation cocktail was added and
the scintillation vials were read in a liquid scintillation counter (Beckman Instruments LS
5000 TD, Palo Alto, CA). The stability of MF and the degradation products under the
incubation conditions (4°C), was demonstrated by HPLC after extraction of the
incubation mixture by solid phase extraction.
The data obtained were fitted by SCIENTISTâ„¢ (Micromath, Salt Lake City, UT)
using the following Emax model to obtain the estimates of Bmax and IC50.

29
CN
DPM = Bmax -Bmax h NS , where DPM represents the total tracer
IC50 +C
binding obtained at any given competitor concentration, NS represents non-specific
binding and N the Hill coefficient. Bmax is the specific binding by the ligand in the
absence of competitor.
The IC50 obtained for dexamethasone (ICso.dex) was used to calculate the relative
binding affinity (RBAtest) of the test compound form its IC50 value (ICso.test) as:
RB A =—-de-xl00
test rn
50,test
The stability of MF and its degradation products in the rat lung cytosol at 4°C was
checked by spiking the compounds separately in cytosol and incubating the samples for
24 h at 4°C. Blank cytosol was considered as control. One set of spiked cytosol was
analyzed at zero time and the other set was analyzed after 24 h (time period of
incubation).
Results and Discussion
Stability of MF in SLF
In agreement with findings of Teng and coworkers [109], incubation of MF (Co =
2.5 pg/ mL) in SLF at 37°C and subsequent HPLC analysis of the incubation mixture
suggests the formation of two major degradation products, named D1 and D2 (Figure 2-
2). The concentration-time profiles of MF, D1 and D2 (Figure 2-3) indicate that more
than 90% of MF undergoes conversion to either D1 and or D2 at the end of 4 h of
incubation with D1 being formed faster than D2. After 12 h, the majority of the starting
material entered the D2 pool with D1 being undetectable; therefore one can conclude that
D1 seems to be converted into D2. Consequently, it appears that MF is first converted

30
into Dl, which is subsequently converted into D2 (see Figure 2-3). Indeed, fitting of the
data to this model resulted in satisfying fits (Figure 2-3); small under or overestimations
might be related to the changes of the pH during incubation, which affected the
degradation rate constants (see below). The apparent degradation rate constant (ki = 0.52
h'1) for the conversion of MF to Dl was found to be nearly 4 times higher than the
degradation rate constant (k2 = 0.143 h'1) for the conversion of Dl to D2. The
degradation half-life for MF was found to be 1.3 h and the half-life of Dl was 4.8 h.
Thus, degradation profile shown in the upper part of Figure 2-1 (denoted by solid lines) is
supported by the data. This suggests that there are qualitative and quantitative
differences between this study and that conducted by Teng et al. who reported the
formation of another minor degradation product, D3 [109], However, D3 is not seen in
this study even though a more sensitive assay was used. The half-life of MF in SLF in
this study was found to be 3.5 times shorter compared to the half-life reported by Teng et
al. The concentration of D2 after 72 h in this study was approximately 80% as that of
parent MF compared to 30% by Teng et al. It might be speculated that differences in the
buffer composition, ionic strength or other factors might be the reason for the somewhat
different degradation pathways.
The rate of degradation of MF was found to vary as a function of pH of the buffer
solution. During incubation at 37°C, the starting pH of the SLF buffer solution was 7.4
that gradually became alkaline (pH 8.9) at the end of 72 h. The pH shift of the SLF
buffer solution was also monitored at ambient temperature (25°C) and it was observed
that the pH gradually changed from 7.4 to 7.9 in 4 h and was distinctly alkaline (pH 9.1)
at the end of 72 h. It is proposed that the buffer system gradually turns alkaline due to the

31
loss of acid (loss of CO2 from the buffer system). This could be due to the presence of
sodium bicarbonate in the buffer (~30 mM) and a low buffering capacity of phosphate
buffer (~1 mM) present in the SLF. However, the cause of the pH raise during incubation
is uncertain.
Figure 2-1. A schematic degradation pathway for conversion of MF into its degradation
products D1 and D2 on incubation of MF (Co = 2.5 pg mL'1) in SLF at 37°C
for 72 h. Broken arrows indicate the possible formation of structure E (D3),
which was not observed in this study. Structures A, B, D and E represent MF,
DI, D2 and D3 respectively. Structure C shows only D-ring of the steroid for
presenting the proposed reaction mechanism for the conversion of B to D. A
similar reaction mechanism would be possible for formation of structure E
from A.

32
(A)
Figure 2-2. Representative chromatograms of (a) drug free SLF at 24 h and (b) SLF
incubated with MF (C0 = 2.5 pg mL'1) for 24 h at 37 °C.

33
Figure 2-3. Graphical representation showing nonlinear curve fitting of the concentration¬
time data of MF and its degradation products D1 and D2 generated after
incubation of MF in SLF at 37°C for 72 h (N=2). The inset graph shows the
expanded time scale up to 12 h.
pH Effects on Stability of MF in SLF
In order to assess the pH dependence, degradation process experiments were
conducted in 50 mM K2HPO4 buffer at three different pH values (pH 7.0, 7.5 and 8.0,
Figure 2-4). The degradation rate of MF increased with increased pH. The degradation
rate constants were 0.03, 0.06, and 0.15 h'1 for pH 7.0, 7.5 and 8.0 respectively. The
results in this study are in agreement with the report by Teng et al. that was recently
published [110]. It may be noted that the degradation rate constant of MF (0.52 h'1) in
SLF was higher than the degradation rate constant observed in 50 mM K2HPO4 buffer at

34
pH 8.0. This suggests that ionic effects, because of the presence of salts in the
composition of SLF, might play a significant role in the degradation of MF.
Figure 2-4. The degradation profiles for MF (Co = 2.5 pg mL'1) in phosphate buffer at
pH 7.0, 7.5 and 8.0 at 37°C.
MS Analysis of MF and the Degradation Products
Based on the ESI+ mass spectra (Table 2-1), the molecular formulae of D1 and D2
agree with the general structures C27H29CIO6 and C27H27CIO5 respectively. The
molecular masses of D1 and D2 are in agreement with those reported by Teng et al.
[110]. The mass spectra data indicated a loss of HC1 for D1 when compared with MF.
Similarly, D2 differed from D1 by a loss of H2O. The presence of only one chlorine
atom in D1 and D2 was confirmed by the relative abundance of 37C1 isotope (Table 2) in

35
D1 and D2 (35%) compared to the relative abundance in MF (70%) that has two chlorine
atoms.
As electron ionization (El) mass spectra supply in general more structural
information, the El mass spectra of MF and D2 have also been taken. The El mass
spectrum of MF is dominated by the furoyl ion at m/z 95, while in the El mass spectrum
of D2 this peak is much less abundant and a very specific, chlorine-containing ion at m/z
211/213 gives rise to the base peak. The formation of this ion, which consists of the Cl 6-
C17 atoms of the steroid skeleton (with their substituents + 1 H) strongly support the
structure proposed here for D2.
Table 2-1. Electrospray ionization mass spectral data# of MF and its degradation
Compound
MF
D1
D2
Molecular formula
C27H3oC1206
c27h29cio6
C27H27C105
[MH]+
521(100)&
485(15)&
467 (60)&
[MH]+ (37C1 isotope)
523(70)
487 (5)
469 (20)
[M+Na]+
543 (30)$
507 (100)$
489 (35)$
[MH-H20]+
503 (10)$
-
-
[MH-HC1]+
485 (15)s
-
-
Others
Ur-. , ; ^ i
441 (5)
413 (15)
413(5)
413(10)
Listed are m/z. % relative abundance to nearest 5% is given in parenthesis.
1 Corresponding 37C1 isotope ion was also observed.
& This was determined to be the parent compound based on independent chemical
ionization mass spectrometry analysis (data not shown).
NMR Analysis of MF and the Degradation Products
The NMR analysis of MF and the degradation products were performed at the
IVAX Drug Research Institute, Hungary and the detailed report on the NMR analysis is
given in Appendix A.

36
Chemical Structures of the Degradation Products
As a result of the MS and NMR analysis, the structure of D2 is shown in Figure 2-
1. This structure of D2 is different from the structure proposed by Teng et al who
hypothesized a furoate-ester migration between Cl7 and C21 positions [110]. Even
though such ester migrations have been reported for other steroids like betamethasone 17-
valerate [113], beclomethasone monopropionate [114] and hydrocortisone butyrate [115],
this phenomenon cannot be expected to happen in steroids like MF that have a halogen
atom at C21 [59, 116], Based on these MS and NMR analyses, the proposed structure for
D2 in this study is accurate and unambiguous.
As mentioned earlier, D2 appears to be formed upon degradation of Dl. Based on
this hypothesis, the structure of D2 and the MS analysis of Dl, Dl is 9, 11-epoxide
mometasone furoate (Figure 2-1). The complete degradation pathway of MF in SLF is
summarized in Figure 2-1. The results of this study do not completely preclude the
formation of compound D3 (pathway shown by dashed lines in Figure 2-1) under
somewhat different experimental conditions even though the formation of this compound
is not seen in this study.
Glucocorticoid Receptor Binding of MF and its Degradation Products
The relative binding affinities of MF, mometasone and the isolated degradation
products Dl and D2 were determined in a competition binding experiment in rat lung
cytosol. Table 2-2 compares the average IC50 values and relative binding affinity (RBA)
values for dexamethasone, MF, Dl and D2. The data shows that MF is 29 times more
potent than dexamethasone. The binding affinity of Dl to the rat glucocorticoid receptor
was 4 times higher than dexamethasone. The IC50 value of the degradation product D2

37
could not be determined precisely as D2 did not show binding to the glucocorticoid
receptors in the concentration range studied.
Table 2-2. Relative binding affinities (RBA) to the glucocorticoid receptor of rat lung
tissue.
Compound
ICso(pM)
RBA
Dexamethasone
0.046 ±0.01
100
Mometasone Furoate
0.0016 ±0.0001
2938
Dl
0.011 ±0.0002
433
D2*
-0.290 ± 0.034
-15.8
* Indicates rough estimates of IC50 obtained by fixing the hill coefficient to 1.
Bledsoe et al. recently determined the crystal structure of the glucocorticoid
receptor ligand-binding domain in a ternary complex with dexamethasone [117]. The
glucocorticoid receptor ligand-binding domain has a unique side pocket that may account
for the selectivity of corticosteroids with substituents at the C-17a position [117, 118].
This may explain the strong binding of MF and Dl, which have a furoate moiety at the
Cl7a position, to the glucocorticoid receptor. However, the structural changes in D2,
with its condensation between the furoate ester carbonyl and the methylene group at C21
leading to the formation of the five-member ring, seem to interfere with the interaction of
the steroid with the receptor in this region of the receptor pocket resulting in a
significantly reduced binding affinity of D2.
The competition curves for dexamethasone, MF, Dl and D2 are shown in Figure 2-
5. In all binding experiments, non-linear curve fitting revealed slope factors (Hill
coefficient) for dexamethasone, MF and Dl were close to 1. The RBA value for MF in

38
this study is slightly higher but in agreement with earlier reported values [119]. 9,11-
epoxide-mometasone furoate (Dl) has a reduced binding affinity of 433, but this RBA
value is comparable to and even greater than many of the currently used inhaled
corticosteroids like flunisolide (RBA 180), triamcinolone acetonide (RBA 233) and
budesonide (RBA 935) [88], These results also indicate that the presence of an 11P-OH-
group is favorable but not absolutely necessary for receptor binding. The binding affinity
of 9,11-epoxide-MF compared to MF in this study to the receptor in rat lung is in
agreement with the values reported by Isogai et al for the glucocorticoid receptor of rat
skin [120].
Log of Competitor concentration (nM)
Figure 2-5. Competitive binding experiments to the glucocorticoid receptors in rat lung
cytosol. Nonlinear regression of non-transformed data was used for the
determination of IC50 values of dexamethasone, MF, degradation products Dl
and D2. An approximate IC50 value for D2 was determined to be 0.29 ± 0.034
pM (n=2). The estimated parameters for D2 were then used to predict bound
tracer at competitor concentrations higher than 0.3 pM. The hill coefficient
was fixed to 1.

39
The results from this show that MF is unstable in aqueous systems resulting in two
epoxidated degradation products. One of the degradation products shows a substantial
glucocorticoid receptor activity and is unstable, the other representing a new cyclized
stable structure whose pharmacological and toxicological properties have not been
described. These instabilities are not typically seen for corticosteroids that do not have
the specific features of MF (furoate group in Cl 7, Cl group at C21 and C9). Further
studies need to investigate whether these degradation products also occur in vivo or in
aqueous based MF formulations.

CHAPTER 3
IN VITRO EXTRA-HEPATIC METABOLISM OF MOMETASONE FUROATE
Introduction
The systemic levels and bioavailability (<1%) of mometasone furoate have been
reported to extremely low in comparison to other corticosteroids after an oral inhalation
[62]. Even though a major portion of the dose is swallowed upon oral inhalation [50],
efficient hepatic metabolism alone is insufficient to explain the low systemic exposure of
MF since the currently used corticosteroids also have high hepatic clearance values
approaching the liver blood flow. Efficient extra-hepatic metabolism may therefore, be
proposed as a hypothesis to explain the low systemic exposure of MF. This study
investigated the stability and metabolism of MF in the lung and in blood, the two major
organs apart from the liver that the drug encounters after an oral inhalation. In this study,
the metabolism was studied by using both non-labeled and tritium-labeled mometasone
furoate in rat and human lung S9 fractions. The use of radio-labeled drug enables the
detection of highly polar metabolites that might otherwise go undetected using HPLC-
UV because of co-eluting endogenous polar interferences present in the tissues.
Materials and Methods
Mometasone furoate (MF) was purchased from USP (Rockville, MD, USA) and
•j
[1,2- H]- mometasone furoate (specific activity: 0.56 Mbeq/mmole) was provided by
AstraZeneca (Lund, Sweden). All other chemicals and solvents were obtained from
Sigma Chemicals Co. (St Louis, MO) and Fisher Scientific Co. (Cincinnati, OH).
40

41
The use of male Sprague Dawley rats (Harlan, Indianapolis, IN) was approved by
the local Institutional Animal Care and Use Committee (IACUC) at the University of
Florida, Gainesville, Florida. Approval was obtained from the Institutional Review
Board, University of Florida for the use of human lung tissues. Human lung tissue
samples were obtained from persons aged between 18-60 years undergoing lung resection
surgery. Cigarette smoke has been known to induce metabolic enzymes like CYP1A1
and therefore, smokers were excluded in the study [121], Pregnant women were also
excluded for ethical reasons.
Stability of MF in Plasma
Male Sprague Dawley rats weighing 250±25g were euthanized to harvest fresh
blood. The blood was transferred to heparin sodium containing vacuutainers (Becton
Dickinson, 100 U.S.P. I.U.). The tubes were then centrifuged at 3500 rpm for 20 min to
obtain plasma. 4 ml of freshly collected rat plasma was pre-incubated at 37°C for 10 min
and then was spiked with MF (from a stock of 100 pg/mL) at a concentration of 3 pg/mL
(5.75 pM). Experiments were conducted in triplicates. 200 pL aliquots were removed at
regular intervals up to 72 h into pre-chilled tubes and the samples were precipitated with
600 pL of methanol. Blank rat plasma was used in a control experiment. Identical
stability studies were performed using fresh human plasma also. The samples were
stored at -20°C and were analyzed by HPLC within 48 h of storage.
The precipitated plasma samples were centrifuged at 10000 rpm for 5 min and 100
pL of the supernatant was injected directly for HPLC-UV analysis. A LDC/Milton Roy
CM4000 multiple solvent delivery system using a Milton Roy SM 4000 programmable
wavelength detector set at kmax 254 nm, a CR-3A Chromatopac integrator (Shimadzu
Corporation, Japan) and an automatic injector (Perkin Elmer, Boston, MA) was used as

42
the HPLC system. Chromatographic separation of the analytes was achieved on Waters
5-pm symmetry RP-18 (150 x 4.6 mm i.d) column (Milford, MA) preceded with a guard
column (10 x 4.6 mm i.d, filled with reverse phase pre-column material) using a mobile
phase of 65:35% v/v methanol-water at a flow rate of 1 ml.
Calibration curves based on MF peak areas over the concentration range of 0.125 to
5.0 pg/mL using unweighted least squares linear regression analysis resulted in r2 values
of at least 0.999. The limit of quantification of the assay was 0.125 pg/mL based on
good accuracy (> 90%) and precision (< 10% coefficient of variation). The good
reproducibility of the system allowed quantification without an internal standard as
potential internal standards cross-eluted with either the endogenous interferences or the
degradation products formed.
Because of the lack of suitable standards, quantification of the degradation products
was based on the assumption that their molar absorptivities were identical to that of MF.
Results for MF and degradation products expressed in % of MF starting concentrations in
the graphical representations.
Metabolism of MF in Lung
Preparation of homogenizing buffer (1.15% KC1 in 50 mM K2HPO4 pH 7.4)
870 mg of dipotassium hydrogen orthophosphate (K2HPO4) and 1.15 g of
potassium chloride (KC1) was dissolved in 100 ml of deionized water and the pH was
adjusted with 20% v/v orthophosphoric acid to 7.4. The buffer was chilled to 4°C before
use.
Preparation of NADPH generating system
NADPH generating system, which comprised of cofactors salts: NADP (2 mM),
glucose-6-phosphate (8 mM), nicotinamide (200 mM) and magnesium chloride (200

43
mM) was prepared in the homogenizing buffer. This cofactor solution was incubated at
37°C for 15 min to allow generation ofNADPH and then was used immediately.
Tissue metabolism using unlabeled MF.
S9 fractions of rat and human lung were used for studying the metabolism of MF.
Frozen human tissue samples were obtained from the Molecular Tissue Bank at the
University of Florida and the samples were pulverized before homogenizing. Rat tissues
were obtained by sacrificing the rats by decapitation and removing the lung tissue
immediately. The tissues were then rinsed in ice-cold homogenizing buffer, blotted dry
and weighed.
The lung tissues were homogenized in ice-cold homogenizing buffer to obtain 20
w/v of tissue. Lung tissue homogenate was then centrifuged at 4°C at lOOOOx g for 45
min to obtain the S-9 fraction for lung. S9 fractions were pre-incubated with equal
volumes of cofactor solution and incubated in a shaker bath to equilibrate the mixture to
37°C. Unlabeled MF was added to initiate the reaction and the final drug concentration
was 2.5 pg/mL (4.8pM) and final lung tissue concentration was 10% w/v. The lung S9
fractions were incubated at 37°C for up to 24 h under atmospheric air. Drug free S9
fraction of lung was used in a control experiment. Serial samples of250pL were
withdrawn at 0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 90, 120, 240 min and 24 h into pre¬
chilled tubes. The samples were precipitated with 250pL of acetonitrile and analyzed by
HPLC. The precipitated samples of lung homogenates were centrifuged at 10000 rpm for
5 min. 100 pL of the supernatant was injected directly into the column for HPLC
analysis. A mobile phase composition of 50:50% v/v of acetonitrile-water was used for
the analysis and the other chromatographic conditions for analysis of samples from S9
studies was identical to those mentioned earlier for analysis of the plasma samples.

44
Results for MF and metabolites are expressed in % of MF starting concentrations in
graphical representations. Semi logarithmic plots of % MF remaining versus time were
used to determine the apparent disappearance rate constant of MF. The disappearance
half-life was determined using the equation ti/2 = 0.693/kdiSS, where kdiss is the apparent
disappearance rate constant for that compound.
Metabolism in lung tissue using labeled [1,2-3H]-MF
An identical parallel incubation was performed simultaneously with 5 mL lung S9
fractions in which [1,2-3H]-MF (concentration <125 ng/mL) was added in addition to the
unlabeled MF (4.8 pM). These samples containing labeled MF were incubated as
mentioned in the previous section. The homogenizing buffer was also spiked with
identical concentrations of labeled and unlabelled MF and was used in a control
experiment. 250 pL aliquots were withdrawn at every 20 min for the first two hours and
finally at 24 h. The samples were withdrawn into pre-chilled tubes and were precipitated
with 250 pL of acetonitrile to terminate the reaction. The samples were immediately
analyzed using HPLC using the conditions mentioned for the analysis of unlabeled MF. 1
min fractions were collected using a fraction collector and transferred to individual
scintillation vials and counted using a liquid scintillation counter (Beckman Instruments
LS 5000 TD, Palo Alto, CA) for the presence of radioactivity. The total counts present
for MF at time zero were considered as 100%. The percentages of metabolites formed
were estimated as the ratio of dpm for metabolite to dpm of parent drug at time zero.
Results and Discussion
Stability in Plasma
The stability of MF was studied in fresh plasma harvested from rat blood.
Incubation of MF in rat plasma at 37°C for 72 h led to the formation of three degradation

45
products DI, D2 and D3 (Figure 3-1). However, MF was found to be stable in plasma
until 6 h of incubation. The formation of degradation products DI, D2 and D3 occurred
after 20 h of incubation (Figure 3-2). The decline of MF and the formation of DI, D2
and D3 are shown in Figure 3-2(a). The degradation half-life of MF as calculated form
the terminal phase of the log concentration-time curve was found to be 85 h.
A
B
MF
4
i IN.
. i • CO
. - in
a
tv
D1
1
* CO
3
D2 D3
44
HL
CO LO
»:o ez>
^ -cr¬
eo
cu
, I
1 1
i
0
Min
25 0
Min
25
Figure 3-1. Representative chromatograms of (a) drug free rat plasma at 48 h and (b) rat
plasma incubated with MF (Co = 3 pg/mL) until 48 h at 37°C.
MF was also found to be stable in human plasma until 6 h of incubation at 37°C.
However, prolonged incubation of MF in human plasma for 72 h led to the formation of
identical degradation products (DI, D2 and D3, Figure 3-2(b)) as observed in rat plasma.
The degradation half-life calculated for MF in human plasma was found to be 24 h which

46
was 3.5 times shorter compared to that in rat plasma and this might be attributed to
possible differences in protein binding of the compound between the two species.
0 8 16 24 32 40 48 56 64 72
Time (h)
(B)
Figure 3-2. Concentration-time profile of MF and its degradation products following
incubation of MF (Co = 6.2 pM) at 37°C in (a) rat plasma and (b) human plasma for 72 h
(n=3).

47
Using a mass spectrometry (positive electro-spray ionization) analysis method
reported in the previous chapter, the molecular masses ([MH+]) of MF, DI, D2 and D3
were determined to be 521, 485, 467 and 503 respectively. The structure elucidations of
these compounds are presented later in the chapter.
Stability in Lung
Incubation of MF in rat lung S9 fractions showed no metabolic conversion until 2
h. However, incubation of MF for 24 h resulted in a degradation product, D2 that was
found to be less polar than the parent drug itself (Figure 3-3). This degradation product
had identical retention times as the product D2 seen upon incubation of MF in plasma
and in the simulated lung fluid (chapter 2). Parallel control incubations of labeled MF in
the incubation buffer also showed formation of D2. This indicated that the formation of
D2 was a result of slow break down of MF in the homogenizing buffer and not a result of
metabolism in the lung. Incubations of labeled MF in the S9 fraction of lung were also
conducted, which also proved that MF was stable in lung fractions. The representative
radiochemical elution profiles for MF in rat lung incubations are shown in Figure 3-4.
Incubation of MF in human lung S9 fractions also indicated that MF was stable with no
metabolic conversion until 2 h of incubation. These results indicate that MF does not
undergo significant pulmonary metabolism.

48
Figure 3-3. Representative chromatograms for (a) blank S9 fraction of rat lung and (b) S9
fraction of rat lung incubated with MF (Co = 2.5pg mL'1) for 24 h at 37°C.
As the results of this study indicate, MF is stable in the plasma and lung S9
fractions up to 2 h. The degradation products (DI, D2 and D3 in plasma; D2 in lung) seen
upon prolonged incubation in plasma and lung appear to be a result of chemical
degradation and not metabolism, as these products (Figure 3-4) were observed in the
incubation buffer also. The degradation of MF in simulated lung fluid (an aqueous
system) and the determination of the structure and the pharmacological activity of the
two resulting degradation products have been reported in detail in chapter 2. Based on
the identical molecular masses and the retention times of the degradation products, it is
expected that the degradation products D1 and D2 observed in this study are identical to
the products reported earlier.

49
• . T
Figure 3-4. Representative radiochemical elution profiles of [1,2- H]-MF incubated with
S9 fraction of rat lung at 37°C (A) at time zero, (B) after 2 h at 37°C and (C)
after 24 h.
V

50
The structures of DI, D2 and D3 in plasma along with the proposed pathways of
degradation are provided in Figure 3-5. The conversion of D1 to D2 has been reported in
chapter 2. However, there is no conclusive evidence for the conversion of D3 to D2 in
this study and this pathway has been denoted by broken arrows in the figure. D1 has
been identified to be 9,11 -epoxide mometasone furoate and has strong glucocorticoid
receptor activity with RBA values of 433 while D2 is a condensed product of MF that
does not show significant binding to the glucocorticoid receptor (chapter 2). The
structure of D3 has been proposed based on the structures of D1 and D2 and the
molecular mass of D3. Based on the similarities in structure (condensed five-member
ring at position Cl7) between D2 and D3, it is expected that D3 would not show
significant binding to the glucocorticoid receptor either.

51
Figure 3-5. A schematic degradation pathway for conversion of MF into its degradation
products DI, D2 and D3 on incubation of MF (Co = 6.2 pM) in plasma at
37°C for 72 h. Structures A, B, D and E represent MF, DI, D2 and D3
respectively. Structure C shows only D-ring of the steroid for presenting the
proposed reaction mechanism for the conversion of B to D. A similar reaction
mechanism would be possible for formation of structure E from A. Broken
arrows indicate the possible formation of D2 from D3.
Even though degradation products of MF are observed in lung and plasma in vitro,
these products might not be seen in vivo since they are formed by relatively slow
processes and therefore might be superceded by the faster pulmonary absorption and
hepatic metabolic processes. The relative stability of MF in plasma and the lung rules out
the possibility of extra-hepatic metabolism in these organs being a cause for the low
systemic exposure of MF after an oral inhalation. Other reasons such as the removal of

52
slow-dissolving, highly lipophilic MF particles deposited in the lungs and airways by
mucociliary clearance mechanisms might be likely to explain the low levels.

CHAPTER 4
IN VITRO DISSOLUTION PROFILE OF MOMETASONE FURO ATE
Introduction
Extra-hepatic metabolism and clearance of MF by mucociliary mechanism are two
possible causes for the low systemic exposure of MF after an oral inhalation. Studies
described in the previous chapter indicate that extra-hepatic metabolism might not be the
reason for the low systemic exposure. Therefore, it is hypothesized that MF, being a
highly lipophilic drug [103], has a slow rate of dissolution leading to the drug being
removed by the mucociliary clearance mechanisms from of the lung resulting in the
unusually low systemic levels of MF seen after oral inhalation [62], To assess the effect
of the dissolution rate the absorption characteristics, the in vitro dissolution profile of dry
powder formulation of MF were compared to that of budesonide (BUD).
Pharmacokinetic studies in humans have shown that the major portion of the dose of
BUD is rapidly absorbed after oral inhalation [122]. If MF has a significantly slower rate
of dissolution than BUD, MF can be expected to undergo mucociliary clearance. The
dissolution studies were performed in a phosphate buffered medium with surfactant at
physiological temperature (37°C) and pH 7.4. Micronized powders of MF and BUD
were used in these studies. A 50 mM phosphate buffered medium was preferred over
simulated lung fluid in this study because of the increased stability of MF in the former
medium (chapter 2).
53

54
Materials and Methods
Chemicals
Budesonide was provided by AstraZeneca. Sodium dodecyl sulfate was obtained
from Fischer Chemicals (Fair Lawn, NJ) and lactose monohydrate NF extra-fine (particle
size 10-30 microns) was purchased from EM Industries (Hawthorne, NY). All other
chemicals and equipment have been described in method section in chapter 2.
Particle Size Distribution
The particle size of MF and BUD powders were checked using an Olympus BX60
optical microscope (Tokyo, Japan) equipped with SPOT RT digital camera and image
analysis software.
In Vitro Dissolution
In vitro dissolution of the dry powder formulations (500 mg of 2% drug in lactose)
was studied using a USP dissolution bath (VanKel Technology Group, Cary, NC) in 900
mL of pH 7.4 phosphate-buffered saline (50 mM) at 37°C. To ensure that the powders
dissolved completely (MF aqueous solubility 125 ng/mL vs. 140 pg/mL with surfactant),
2% sodium-dodecyl sulfate (SDS) was added to increase solubility and wetting and to
more closely mimic in vivo pulmonary surfactant levels [123], 1 mL samples were
removed and filtered through a 0.2 pM syringe filter at 0, 0.5, 1, 1.5, 2, 2.5, 5, 10, 15, 30,
45, 60 and 240 minutes and analyzed using HPLC. The concentrations of MF and BUD
were be determined by HPLC using the instruments described in the methods section in
chapter 2. For analysis of MF, BUD (2 pg/mL) was used as an internal standard and for
analysis of BUD; MF (1 pg/mL) was used as the internal standard. A mobile phase
composition of methanol-water (60:40, v/v) was used at a flow rate of 1 mL/min in the

55
analysis. The experiment was repeated using 0.5% SDS as surfactant while maintaining
all other conditions as described above.
Data Analysis
Half-life of dissolution in all cases, except the dissolution of MF in 0.5% SDS, was
calculated by non-linear curve fitting using SCIENTISTâ„¢ (Micromath, Salt Lake City,
UT) program and using the monoexponential formula:
Cumulative % Dissolved = 100 - (A*exp'at)
where a is the dissolution rate constant. Half-lives of dissolution (T5o%) were
calculated as 0.693/a.
The dissolution of MF in 0.5% SDS was characterized by a biphasic profile given
by the equation:
Cumulative % Dissolved = 100 - ((A*exp~at )+(B*exp~pt)),
where a and P are the rate constants for the initial rapid and latter slow phases of
dissolution. The corresponding half-lives for a and P were obtained by multiplying their
reciprocals with 0.693.
Differences in dissolution between drug formulations were tested for significance
using the FDA scale-up and postapproval changes (SUPAC) similarity test (f2):
f2 = 50 x LOG ([1+1/n S(Rt-Tt)2]'0 5 x 100)
where n=12 time points (0, 0.5, 1, 1.5, 2, 2.5, 5, 10, 15, 30, 45 and 60 minutes), Rt
is the reference % drug dissolved at time t and Tt is the test % drug dissolved at time t.
An f2 value between 50 and 100 suggests the two dissolution profiles are similar and an f2
value less than 50 indicates a lack of similarity [124],

56
Results and Discussion
Particle Size Distribution
The particle size distributions of MF and BUD micronized dry powders are given
in Table 4-1. The particle size distributions were comparable for the two compounds and
more than 88% of the particles had aerodynamic diameters in the range 1-5 pm. This is
in accordance with the particle size distribution of dry powder formulations of inhaled
corticosteroids, as particles with diameters 1 -5 pm have been known to cause maximum
pulmonary deposition [125], Particles with diameter greater than 5 pm impact on the
back of the throat after an oral inhalation while particles with diameter smaller than 1 pm
will be exhaled out.
<1 pm
1-5 pm
5-10 pm
>10 pm
MF
3.5
88.5
5.8
2.2
BUD
2.1
94.3
3.1
0.5
In Vitro Dissolution
Cumulative dissolution profiles for MF and BUD powders using 2% SDS as
surfactant are depicted in Figure 4-1. From the release profiles, it was observed that
steady state was reached (10 pg/ml) at 30 minutes. The dissolution half-life (T5o%) for
MF was calculated to be 4.2±0.3 min (n=6) and Tso% for BUD was 2.1±0.2 min (n=6).
Dissolution half-lives of release was significantly longer for MF when compared to BUD
and the dissolution profiles for the two drugs were not similar (f2=42). Previous studies
have shown that the dissolution half-lives was significantly longer for FP when compared
to BUD also under identical conditions (f2=34) [126],

57
The dissolution profiles of MF and BUD using 0.5% SDS as surfactant is shown in
Figure 4-2. MF exhibited a biphasic dissolution profile when lower concentration of the
surfactant was used. The T5o% for MF for the initial phase of dissolution was 3.5±0.2 min
while the Tso% for the latter phase of dissolution was 37.1±0.9 min. 66% of the total MF
powder dissolved at the faster rate while the remaining 34% showed the slower phase of
dissolution. The biphasic dissolution profile of MF could be because of different
polymorphs of MF in the powder studied. The overall dissolution of MF in the medium
with 0.5% SDS was slower than in the 2% SDS containing medium. BUD followed a
mono-phasic profile and there was no significant change (p>0.5) in the Tso% (1.9±0.3
min) of BUD compared to using 2% SDS. The dissolution profiles for MF and BUD
were not similar with an ii value of 28. The results clearly indicate that the concentration
of the surfactant used in the dissolution medium plays an important role in assessing the
dissolution profile of the compounds. While the use of surfactants has been
recommended in performing dissolution studies of water-insoluble compounds, there is
no recommendation on the exact concentration of the surfactant to be used [123]. The in
vitro model used in this study also utilizes a large volume of buffer and surfactant
compared to in vivo conditions. Considering the above-mentioned constraints, this
method is primarily used to qualitatively compare the dissolution rates of the
corticosteroids studied.

58
Time (min)
Figure 4-1. Dissolution profiles of micronized dry powders of MF and BUD in pH 7.4
PBS (50 mM, 2% SDS) at 37°C (n=6).

59
Figure 4-2. Dissolution profiles of micronized dry powders of MF and BUD in pH 7.4
PBS (50 mM, 0.5% SDS) at 37°C (n=6).
The lipophilic corticosteroids are more prone to mucociliary clearance because of
their slow dissolution rates. Removal of beclomethasone dipropionate from the lung by
mucociliary mechanisms has been suggested to explain the low systemic exposure of the
drug after administration by a chloro-fluoro-carbon pressurized MDI [127]. Mucociliary
clearance also appears to play a significant role in reducing the pulmonary uptake of
fluticasone propionate in patients with moderate to severe asthma [128].
The results of this study show that MF has slower in vitro rates of dissolution than
BUD. The in vitro dissolution rates of MF are comparable with that of FP [126] and

60
since mucociliary clearance has been proposed for FP, it is expected that MF would also
be cleared by mucociliary mechanisms resulting in lower systemic availability of MF.
This needs to be investigated by assessing the in vivo dissolution rate in terms of the
absorption profile and pulmonary residence times MF. A clinical study assessing the
absorption profile and the systemic availability of MF in asthmatic patients is presented
in chapter 7.

CHAPTER 5
HEPATIC METABOLISM AND CHARACTERIZATION OF METABOLITES OF
MOMETASONE FUROATE
Introduction
Although the systemic bioavailability of MF has been reported to be extremely low,
the drug shows considerable systemic effects as manifested by the suppression of the
hypothalamic-pituitary-adrenal axis [63]. This contradiction could possibly be explained
by the metabolism of MF into at least one active metabolite. To assess this hypothesis,
this section of the study evaluated the hepatic metabolism and the biological activities of
the metabolites thus formed. The pharmacological activity of the metabolites formed was
tested by assessing their binding to the rat lung glucocorticoid receptors. The evaluation
of the activity of the metabolites is important in estimating the correct pharmacological
profile of MF. The in vivo formation of these metabolites was also assessed by
performing disposition studies using radiolabeled MF in rats.
Materials and Methods
Chemicals
Mometasone furoate (MF) was purchased from USP (Rockville, MD, USA) and
[1,2-3H]- mometasone furoate (specific activity, 0.56 Mbeq/mmole) was provided by
AstraZeneca (Lund, Sweden). [l,2,4-3H]-dexamethasone (specific activity, 38
Ci/mmole) was purchased from Amersham Biosciences (Piscataway, NJ). All other
chemicals and solvents were obtained from Sigma Chemicals Co. (St Louis, MO) and
Fisher Scientific Co. (Cincinnati, OH). The use of Sprague Dawley rats (Harlan,
61

62
Indianapolis, IN) was approved by the local Institutional Animal Care and Use
Committee (IACUC) at the University of Florida, Gainesville, Florida. Approval was
obtained from the Institutional Review Board, University of Florida for the use of human
liver tissues. Human liver tissue samples were obtained from persons aged between 18-
60 years undergoing liver resection surgery. Cigarette smoke has been known to induce
metabolic enzymes like CYP1A1 and therefore, smokers were excluded in the study
[121]. Pregnant women were also excluded for ethical reasons.
In vitro Hepatic Metabolism of MF
The in vitro hepatic metabolism of MF was studied using the same protocol
described for lung metabolism in the methods section of chapter 3. Liver tissues of rat
and humans were processed as reported to obtain 40% w/v (20% w/v in final incubation
mixture) S9 fractions. The metabolism of MF was assessed using both unlabeled MF
(4.8 pM) and a mixture of labeled and unlabeled MF in rat and human lung S9 fractions.
The experiments were performed in triplicates. The sample collection and HPLC
analysis were performed as described in chapter 3.
In vivo Metabolism and distribution of MF
A total of 5 pCi of [1,2-3H]MF per 250 mg of bodyweight, in normal saline was
administered by injection into the tail vein of male Sprague-Dawley rats. The animals
were sacrificed by decapitation after two hours. The abdominal and thoracic cavities
were opened immediately and blood from the heart was withdrawn. Urine was collected
by puncturing the urinary bladder. The organs (Table 1) were dissected, rinsed with ice-
cold normal saline, blotted, weighed and then homogenized in methanol. 1 cm2 skin and
the vastus lateralis muscle from the right leg were resected and homogenized without
being rinsed. The contents of the stomach and the intestines (both small and large) were

63
flushed out of the organs before homogenization and collected. 30 mL of methanol was
used to homogenize the liver and 20 mL was used to homogenize the other tissues. 1 mL
of the homogenates was transferred to scintillation vials and 10 mL of the scintillation
cocktail was added. The scintillation vials were read in a liquid scintillation counter
(Beckman Instruments LS 5000 TD, Palo Alto, CA).
Glucocorticoid Receptor Binding Assay Experiments in Rat Lung Cytosol
As described earlier in the metabolism experiments, liver S9 fractions were spiked
only with unlabeled MF (4.8 pM) and incubated at 37°C in a shaker bath for 2 h. The 2 h
samples were analyzed by HPLC-UV analysis and the metabolites (MET1-MET5) were
isolated by collecting the fractions corresponding to the retention times of the
metabolites. The fraction corresponding to the elution of MF (denoted by MFfrac) in the
samples was also collected. These fractions were then evaporated under vacuum and
reconstituted in methanol. The concentrations of the MFfrac and the metabolites in the
fractions were calculated from % conversion of MF into the metabolites data obtained
from the previous section. The dilutions for MF (from a stock of 1 mg/mL), MFfrac and
the metabolites were prepared in methanol. Dexamethasone (DEXA) was used as a
reference compound for the receptor binding experiments. The concentration ranges for
the compounds in the study were 0.01 to 100 nM for MF, METI and MET3; 0.01 to 30
nM for MFfrac and MET2; 0.01 to 50 nM for MET4 and MET5 and 0.01 to 1000 nM for
DEXA.
A previously described method [129], with slight modifications, was used for
performing the receptor binding assays. Sprague Dawley rats were anesthetized using a
cocktail mixture of ketamine, xylazine and acepromazine (3:3:1 v/v) and were
decapitated. The lungs were removed and homogenized in 8 volumes of ice-cold

64
incubation buffer (10 mM Tris/HCL, 10 mM sodium molybdate, 2 mM 1,4-
dithioerythritol). The homogenate was incubated with 5% w/v charcoal suspension (in
deionized water) for 10 min. The homogenate was then centrifuged for 20 min at
40,000X g in J2 rotor of Beckman centrifuge to obtain the cytosol. Fresh cytosol was
prepared and used for all the individual experiments.
10 nM of 3H-labeled dexamethasone solution was used as a tracer, based on
previous saturation binding experiments performed in our laboratory (data not shown).
10 pL of the dilutions of the test compound in methanol was added to pre-chilled tubes.
10 pL of methanol instead of the test compound was used for the determination of total
binding. Non-specific binding was determined after addition of 10 pL of 100 pM
unlabeled DEXA (10 pM in the final incubation mixture). 10 pL of 100 nM 3H-
dexamethsone solution (10 nM in final incubation mixture) was then added. 80 pL of the
lung cytosol was added and the tubes were vortexed and incubated at 4°C for 24 h. After
the incubation, 100 pL of 5% w/v charcoal suspension (in water) was added to the tubes
to remove the excess unbound radioactivity. The tubes were vortexed and then
centrifuged and 150 pL of the supernatant was transferred to the scintillation vials. 5 mL
of the scintillation cocktail was added and the scintillation vials were read in the liquid
scintillation counter. Control experiments were also conducted in which receptor binding
assays were performed on fractions collected during HPLC-UV analysis of blank S9
fractions and on fractions collected during blank mobile phase runs.
The data obtained were fitted by SCIENTISTâ„¢ (Micromath, Salt Lake City, UT)
using the following Emax model to obtain the estimates of Bmax and IC50.
CN
DPM = B„„„ -B
max max
IC5oN+Cn
• + NS

65
where DPM represents the total tracer binding obtained at any given competitor
concentration, NS represents non-specific binding and N the Hill coefficient. Bmax is the
specific binding by the ligand in the absence of competitor.
The IC50 obtained for dexamethasone (ICso.dex) was used to calculate the relative
binding affinity (RBAtest) of the test compound form its IC50 value (ICso^est) as:
RBA„ =^5^x100
^50, test
Results and Discussion
Metabolism of MF in Rat Liver S9 Fractions
Incubation of a mixture of non-labeled and labeled MF with rat liver S9 fractions
over 1 h showed formation of five metabolites (Figure 5-1). The retention times for the
metabolites METI, MET2, MET3, MET4 and MET5 were between 1-2, 5-6, 7-8, 11-12
and 12-13 min respectively. A semi logarithmic plot of % dpm versus time displayed a
linear relationship with a correlation coefficient of 0.956 indicating first order elimination
kinetics for MF. The disappearance half-life of labeled MF was 18.5 min in rat liver S9
fractions. The concentration-time profiles of MF and its metabolites are shown in Figure
5-2. At the end of 1 h, METI was observed as the major conversion product with
MET/MF ratio of 32% and 7% of MF was converted to MET2. The sum of the MET/MF
ratios for metabolites 3, 4 and 5 were less than 10%. The control buffer sample did not
show any formation of polar degradation products indicating that the formation of
metabolites was a result of enzymatic activity present in the S9 fraction of liver sample
and not a result of degradation in the buffer.

66
A
Collection time interval (min)
B
Figure 5-1. Representative radiochemical elution profiles of (a) [1,2-3H]-MF incubated in
S9 fraction rat liver at time zero and (b) [1,2-3H]-MF incubated in S9 fraction
of rat liver for time 20 min at 37°C (bars in black and gray represent MF in S9
fraction of rat liver and homogenizing buffer respectively).

67
A
Time (min)
• T -1
Figure 5-2. Concentration-time profile of [1,2- H]-MF following incubation of [1,2- H]-
MF in S9 fraction of rat liver and (b) concentration-time profile of the
metabolites formed after incubation of 3H-MF in S9 fraction of rat liver at
37°C for 1 h.
The HPLC-UV profiles (Figure 5-3) observed after incubation of unlabeled-MF
with S9 fraction of rat liver indicated the formation of only one polar metabolite (MET2)
that eluted at 5.1 min. The disappearance half-life of unlabelled MF (16 min) as

68
determined by HPLC-UV analysis was found to be similar to the half-life of labeled MF
(18.5 min) that was estimated from the radiochemical elution profiles. The metabolite
MET 1 (retention time between 2-3) could not be viewed using a UV-VIS detector due to
the presence of endogenous interferences at the same retention time. MET3, MET4 and
MET5 could not be quantified using UV due to the very low concentrations of these
metabolites.
figure 5-3. Representative chromatograms for (a) blank S9 fraction of rat liver, (b) S9
fraction of rat liver incubated with MF (Co = 2.5pg/mL) until 0.25 h, (c) S9
fraction of rat liver incubated with MF until 1 h.

69
Metabolism of MF in Human Liver S9 Fractions
Incubation of a mixture of labeled and unlabelled MF in human liver S9 fractions
also showed formation of five metabolites (Figure 5-4). The retention times for these
metabolites were identical to the ones formed with rat liver S9 fractions. A semi
logarithmic plot of % dpm versus time displayed a linear relationship with a correlation
coefficient of 0.9584 and the disappearance half-life of 3H-MF in the S9 fraction was
found to be 0.76 h'1 with a corresponding half-life of 55 min. The concentration-time
profile for the disappearance of MF and appearance the metabolites are shown in Figure
5-5.
The HPLC-UV profiles observed after incubation of MF with S9 fraction of human
liver showed an endogenous tissue impurity eluting at around 5 min. Because of this
interference, MET2 could not be observed in human liver tissues. Hence, only the
decline of MF could be monitored under UV detection over time.

70
A
E
Q.
"D
30000
25000
20000
15000
10000
5000
CM CO
â– M-
lo
CO
r--
00
00
o
-r-
CM
CO
â– M-
uo
to
r--
oo
CO
o
Ó CM
CO
â– M-
ió
c¿
r¿-
OO
có
ó
-
CM
CO
ló
to
r-1
CO
CM

â–  Human liver 0 hr
â–¡ Buffer 0 hr
MF
Collection time interval (min)
B
12000.0
10000.0
8000.0
E
% 6000.0
4000.0
2000.0
0.0
MET1
â–  Human Liver 20 min
â–¡ Buffer 20 min
MET2 MEET3
n
MET4 MET5
MF
ó'4■ coO’-CMOO'i-iotDr--cocri
Collection time interval (min)
Figure 5-4. Representative radiochemical elution profiles of 3H-MF incubated in S9
fraction of human liver at 37°C (a) at time zero and (b) after 20 min (bars in
black and gray represent MF in S9 fraction of human liver and homogenizing
buffer respectively).

71
A
B
Time (min)
Figure 5-5. Concentration-time profile of [1,2-3H]-MF following incubation of [1,2-3H]-
MF in S9 fraction of human liver and (b) concentration-time profile of the
metabolites formed on incubation of MF in S9 fraction of human liver at 37°C
for 1 hr.

72
This study shows that MF is efficiently metabolized into at least five metabolites in
S9 fractions of both rat and human liver tissues. There were, however, quantitative
differences in the metabolites between the two species. The apparent half-life of MF in
the S9 fraction of human liver was found to be 3 times greater compared to that in rat.
METI, the most polar metabolite, was the major metabolite in rat liver fractions where as
both METI and MET2 were formed to an equal extent in human liver fractions. Such
species differences have been noted for other corticosteroids like budesonide also [130],
The molecular mass ([MH+]) of MET2 was estimated to be 538 using a mass
spectrometry method reported previously in chapter 2. Based on the molecular mass and
comparing the chromatographic elution profile of MET2 to putative metabolites [62],
MET2 is expected to be 6P-OH-MF. This is in agreement with other reports that have
hinted the formation of this metabolite [63, 85-87], Hydroxylation at the 6p-position is a
common route of metabolism among corticosteroids with budesonide, triamcinolone
acetonide and flunisolide getting metabolized into their respective 6p-hydroxy
derivatives [95, 130-133]. Using HPLC-UV detection in the present study, only the
decline of MF and the formation of MET2 could be viewed. This agrees with the report
by Teng et al., who monitored the formation of only one metabolite in human liver using
UV and concluded that other metabolites also should be formed to explain the loss of MF
[87], The use of the tritium-labeled drug in this study provided valuable information
about the formation of the other metabolites as well. METI is a metabolite that has not
been identified before and is not among the putative metabolites of MF that were
proposed by Affrime et al [62]. The formation of METI, however, is consistent with the
bioavailability study by the same group, who reported that majority of the radioactivity

73
eluting in the urine after administration of 3H-MF in humans eluted in the “very polar
region” [62]. However, no conclusive decision could be made about indications of
sequential metabolism in the present study. METI was extremely polar to be extracted
for further analysis, and therefore could not be identified. MET3, 4 and 5 were formed in
low amounts and could not be identified.
In vivo Metabolism and Disposition
The radioactivity (expressed as % of dose administered) in the tissues 2 h after an
intravenous injection of [1,2-3H]-MF in rats is given in Table 5-1. The data shows that
most of the radioactivity (—90%) is present in the stomach, intestines and the intestinal
contents. This strongly suggests that there is biliary excretion of MF and its metabolites.
Biliary excretion has also been reported for other corticosteroids like triamcinolone
acetonide and prednisolone [134-136]. The bioavailability study by Affrime et al in
humans after the inhalation of 3H-MF reported the presence of radioactivity associated
with metabolites in the feces also suggesting biliary excretion of the metabolites [62].
There was no significant concentration of MF into the other tissues. The amount of
radioactivity associated with the brain was negligible and the least among the organs
studied. This is consistent with results from other studies that show that the uptake of
corticosteroids into the brain is limited by the multidrug resistance (mdr) P-glycoprotein
(Pgp) efflux transporter at the blood-brain barrier [137, 138].
Radiochemical elution profiles of the intestinal contents show the formation of
three metabolites that have identical retention times as the metabolites METI, MET2 and
MET3 (Figure 5-6) that were seen upon incubation of MF with liver S9 fractions. There
was no radioactivity corresponding to MF and this is consistent with the efficient hepatic
metabolism of MF that was seen in vitro. Most of the radioactivity in the intestinal

74
contents was associated with METI (Figure 5-6) while MET2 and MET3 were minor
metabolites. MET4 and MET5 were either not formed or formed in extremely low
concentrations to be seen in the intestinal contents. The results of this study clearly
indicate that the MF is metabolized efficiently in vivo and the metabolites are
predominantly eliminated by biliary excretion into the intestinal tract.
Table 5-1. Percentage of dose distributed into different tissues two hours after
intravenous administration of [1,2-3H]MF in male Sprague-Dawley rats.
Tissue % of dose
Brain
0.05±0.01
Thymus
0.08±0.02
Heart
0.09±0.02
Spleen
0.24±0.16
Lung
0.32±0.01
Kidney
0.32±0.01
Stomach
1.3U0.39
Liver
2.0Ü1.36
Large intestine
2.47Ü.98
Small intestine
4.55±0.11
Intestinal contents
81.57±4.66
Muscle
0.20±0.05a
Fat
0.39±0.17a
Skin
0.05±0.02b
Plasma
0.26±0.08c
Urine
0.08±0.02c
a % of dose/gram tissue
b % of dose/cm2
c % of dose/ml

75
Collection time interval (min)
Figure 5-6. Radiochemical elution profiles of intestinal contents two hours after
intravenous administration of 5 pCi [1,2-3H]-MF in male Sprague-Dawley
rats.
Glucocorticoid Receptor Binding of MF and Metabolites
The relative binding affinities of DEXA, MF and the isolated metabolites were
determined in a competition binding experiment in rat lung cytosol. Table 5-2 gives the
relative binding affinities (RBA) values for DEXA, MF, MFfrac and the metabolites. The
competition curves for the compounds are shown in Figure 5-7. In all binding
experiments, non-linear curve fitting revealed slope factors (Hill coefficient) for all the
compounds were close to unity. The fractions collected during HPLC-UV analysis of
blank S9 fractions and during blank mobile phase runs did not show any binding to the

76
glucocorticoid receptor. The data shows that MF is 29 times more potent than
dexamethasone. The RBA of MFfracwas found to be 2700. The good agreement between
the RBA values of MF using a standard solution and MF collected from fractions (MFfrac)
indicates that the concentrations of the compounds in the fractions collected are accurate.
Table 5-2. Average relative binding affinities (RBA) to the glucocorticoid receptor of rat
lung tissue.
Compound
RBA
Dexamethasone
100
Mometasone furoate
2900
MFfrac
2700
METI
<25
MET2
700
MET3
25
MET4
<50
MET5
<50
The binding affinity of MET2 to the rat glucocorticoid receptor was 7 times higher
than dexamethasone. This RBA value of MET2 is greater than the RBA values of some
commonly used inhaled corticosteroids like flunisolide (RBA 180), triamcinolone
acetonide (RBA 233) and is comparable to budesonide (RBA 935) [88]. The strong
affinity of 6P-OH-MF is surprising because the 6P-hydroxy derivatives of other
corticosteroids do not show significant activity towards the glucocorticoid receptor. The
RBA values of 6p-OH-budesonide and 6p-OH-flunisolide have been reported to be 6 and
less than 1 respectively [119, 129]. The IC50 value of the metabolites METI, 4 and 5

77
could not be determined, as they did not show any binding to the glucocorticoid receptors
in the concentration range studied.
120 ,
Figure 5-7. Competitive binding experiments to the glucocorticoid receptor in rat lung
cytosol (n=2). Nonlinear regression of non-transformed data was used for the
determination of IC50 values of dexamethasone, MF, MFfrac, metabolites
MET2 and MET3.
Even though MF has minimal systemic exposure, MF produces significant systemic
effects after an oral inhalation. 800 pg BID of MF administered by a metered dose
inhaler for 28 days produced a 20%-30% suppression of the hypothalamic-pituitary-
adrenal axis (HPA) compared to placebo (PO.05) [63], In comparison, a 880 pg BID of
fluticasone propionate produced a 43%-56% suppression of HPA compared with placebo
(P<0.01) [63, 103]. The formation of the active metabolite, 6(3-OH-MF, might explain
the systemic effects of MF in spite of its low systemic exposure. Therefore, the

78
measurement of this metabolite would be critical in obtaining a true measure of the
systemic bioavailability of mometasone furoate. It would be of further interest also to
assess the plasma protein binding of the active metabolite to estimate the free levels of
the metabolite that would be able to interact with the systemic glucocorticoid receptors.

CHAPTER 6
A SENSITIVE LC-MS/MS METHOD FOR THE QUANTIFICATION OF
MOMETASONE FUROATE IN HUMAN PLASMA.
Introduction
Inhaled corticosteroids are now the drugs of choice in the treatment of chronic
asthma because of their distinct anti-inflammatory effects in the lung and minimal
systemic side effects. One of the goals of treatment with inhaled corticosteroids is to
reduce the levels of the drug in the blood to minimize the side effects. Therefore, the
quantification of these drugs in plasma demand a highly specific and sensitive assay.
HPLC-UV methods are not suitable because of their poor sensitivity [109]. Alternative
methods like radioimmunoassays (RIA) and HPLC/RIA have been used for the
quantification of inhaled corticosteroids in biological matrices. These methods are either
time-consuming or are limited by low selectivity [139, 140]. Liquid chromatography-
tandem mass spectrometry (LC-MS) techniques have become popular in the
determination of corticosteroids in biological matrices because of their increased
specificity and sensitivity [112, 141-145].
Affrime and coworkers estimated the pharmacokinetics of mometasone furoate
(MF) after an oral inhalation and plasma levels of MF were quantified using an LC-MS-
MS method with a lower limit of quantification of 50 pg/ml [62]. However, the details of
the assay are not available. It was found that the plasma levels of MF were undetectable
in most samples and there have been suggestions that a more sensitive assay is needed to
obtain a better estimate of the pharmacokinetic parameters of MF [106, 107], This paper
79

80
presents a specific and sensitive atmospheric pressure chemical ionization (APCI) based
LC-MS-MS assay using a multiple reaction monitoring technique to quantify MF in
human plasma after oral inhalation of therapeutic doses.
Materials and Methods
Chemicals and Reagents
Mometasone furoate was purchased from USP (Rockville, MD). 13C3-Fluticasone
propionate (l3C3-FP) was used as an internal standard (I.S.) and was provided by
GlaxoWellcome R&D, Ware, Hertz, UK. HPLC grade methanol, ethanol, ethyl acetate
and heptane were purchased from Fischer chemicals (Springfield, NJ, USA). The solid
phase LCis (3 ml) cartridges for sample extraction were obtained from Supelco
(Bellefonte, PA, USA). Drug-free human plasma was obtained from the Civitan regional
blood system (Gainesville, FL, USA).
Preparation of Calibration Standards and Quality Control Samples
A primary stock solution of MF was prepared by dissolving 20 mg of MF in 50 ml
of methanol and was stored at -20°C. The working solutions used in preparing quality
control samples (QC) and plasma calibration standards were 100 and 10 ng/ml of MF in a
mixture of methanol-water (85:15, v/v). The calibration standards (CC) ranged from 15 to
1000 pg/ml. Quality control (QC) samples in plasma (15, 30, 50, 80, 120 and 400 pg/ml)
were prepared from the stock solutions and were used against the independently prepared
plasma calibration standards. Two different series of stock solutions were prepared
separate weightings for the CCs and QCs. The I.S. stock solution was prepared by
dissolving 200 pg of C3-FP in 1 ml of methanol, and working solutions of 20 ng/ml
were prepared by diluting the stock solution with methanol.

81
Sample Processing
Plasma samples were thawed at room temperature and 50 pi of I.S. (corresponding
to approximately 1 ng/ml of 13C3-FP) working solution was added to 1 ml of plasma. The
compounds were then extracted using a previously reported method [112, 146], Briefly, 1
ml of 30% ethanol was added to 1 ml of the plasma sample and centrifuged. 2 ml of the
supernatant was then extracted using a 6 ml end-capped Cis cartridge and the analytes
were then eluted with 3 ml of a mixture of ethyl acetate-heptane (35:65, v/v). The residue
was then evaporated under vacuum and reconstituted in 100 pi of a mixture of methanol-
water (85:15, v/v) and a sample volume of 80 pi was injected into the HPLC-MS/MS
system.
HPLC-MS-MS Conditions
Isocratic high performance liquid chromatographic separations at ambient
temperature were achieved with a Waters 5-pm column (50mm x 4.6 mm i.d., Milford,
MA, USA) preceded by a Whatman 5-pm ODS Cig guard column cartridge (20mm x 2.0
mm i.d., Clifton, NJ, USA). The mobile phase consisted of methanol-water (85:15, v/v)
and was delivered at a flow rate of 1 ml/min using a LDC Analytical
constaMetric®35000 solvent delivery system (LDC/Milton Roy, Riviera Beach, FL,
USA). The analysis of MF was performed using a Micromass Quattro-LC-Z (Beverly,
MA, USA) triple quadrupole mass spectrometer equipped with an APCI ion source.
Negative APCI mode was chosen to acquire the mass spectra after tuning with MF.
Multiple reaction monitoring was used for the quantification. The source temperature and
the APCI probe temperature were set to 120°C and 500°C respectively. Argon was used
as the collision gas and the mass resolution was set to unit mass. The corona voltage for

82
both MF and I.S. transitions was set to 3.5 kV. The cone voltages for the MF and I.S.
transitions were set to 35 V and 20 V respectively. A dwell time of 0.7 s was used for
monitoring MF transition and dwell time of 0.1 s was used for monitoring the I.S
transition. The MS-MS signals were optimized by injecting 1 pg/ml solution of MF in
methanol at a flow rate of 100 pl/min using a Kd-Scientific® (Holliston, MA, USA)
infusion pump. The mass spectrometer was linked to a Perkin Elmer ISS 200 autosampler
by contact closure and was controlled by the software, Masslynx 3.1. Data analysis was
performed using the same software. Peak area ratios of MF to I.S. were plotted against
MF concentrations to obtain the calibration curves for MF. A weighted (1/x) linear
regression model with ten concentration points (including blank plasma) ranging from 15
to 1000 pg ml'1 in duplicates was used to plot the calibration curves.
Method Validation
Selectivity
Drug-free plasma from eight humans were extracted and screened for any potential
endogenous interference. Any apparent response from interference co-eluting at the
retention times of MF and the I.S. were compared to the response at the lower limits of
quantification for MF and to the response at the working concentration of the I.S.
respectively.
Recovery
The recovery of MF was determined at low (40 pg/ml), medium (80 pg/ml) and
high (400 pg/ml) concentrations by comparing the responses from plasma samples spike
prior to extraction with those from plasma samples extracted and spiked after extraction.
The recovery of the I.S., using an identical extraction method and analytical conditions,
has been reported earlier [112].

83
Accuracy and precision
Intra-day accuracy and precision were determined by analyzing the quality control
samples at concentrations of 15, 30, 50, 80, 120 and 400 pg/ml (n=6 at each
concentration) on the same day. Inter-day accuracy and precision were assessed by
repeating the experiment on three different days. Accuracy was expressed as the
percentage ratio of the measured concentration to the nominal concentration. Precision
was calculated as the coefficient of variation. The lower limit of quantification (LLOQ)
of the assay was defined as the lowest drug concentration that can be determined with an
accuracy of 80-120% and a precision <20% [147],
Stability
Freeze-thaw cycle stability was assessed by analyzing quality control samples at
concentrations of 50, 120 and 400 pg/ml in triplicate, following three cycles of freezing at
-80°C and thawing. Short-term temperature stability was evaluated for the quality control
samples at the same concentrations by thawing the samples and keeping them at room
temperature for 4 hours. Bench-top stability for 7 hours at the same concentrations was
determined after extraction and reconstitution. Stability was evaluated in terms of
accuracy, as the percentage ratio of measured concentration to the nominal concentration.
Results and Discussion
Mass Spectrometry/Chromatography
Preliminary tuning experiments were performed for MF in APCI and electrospray
ionization (ESI) modes. Negative APCI (APCF) was chosen because of better sensitivity
seen by the increased signal-to-noise ratio for the product ion peak. The full scan mass
spectra of the parent and daughter ions of MF in APCF under multiple reaction
monitoring mode are shown in Figure 6-1. The best sensitivity was observed with the

84
transition m/z 484.0 to m/z 389.2 and was chosen as the transition channel for MF. The
parent molecular ion {m/z 484.0) corresponds to a difference of 36 mass units from the
actual mass of MF {m/z 520.0) and this can be attributed to the loss of an HC1 molecule
from the structure of MF in the APCI source. The transition for l3C3-FP was selected as
m/z 503.3 to m/z 380.0 based on a previous report and the tuning conditions for this
compound has been described in the same report [112], Both MF and the I.S eluted at
0.97 min and the total analysis time for each sample was 4 min. The resulting calibration
curves for the validation were linear in the range 15-1000 pg/ml with correlation
coefficients (r2) >0.99 (Figure 6-2).
Internal Standard Selection
Isotope-labeled MF was unavailable for use as an internal standard. Therefore,
inhaled steroids structurally related to MF were screened and fluticasone propionate was
chosen as the I.S. because of similar lipophilicity values for MF and FP and identical
retention times for both compounds. Moreover, FP has excellent sensitivity under
negative ion mode APCI and shows good recovery with the extraction method used in the
assay [112], However, interference in the MF transition was seen when unlabeled
fluticasone propionate was used as the internal standard. No interferences in the MF
transition were observed with 13C3-FP and hence this compound was used as the internal
standard.

85
Figure 6-1. Full scan (top panel) and daughter scan (bottom panel) spectra of MF.
Compound 2 name: MF Method File: MF4-12-04
Coefficient of Determination 0.997646
Calibration curve: 0.121256 * x + 0.000341955
Response type: Internal Std ( Ref 1 ), Area * ( IS Cone. / IS Area )
Curve type. Linear, Origin: Include. Weighting. 1/x. Axis trans: None
Figure 6-2. Representative calibration curve of MF in human plasma.

86
Selectivity
No endogenous interference from human plasma was observed at the retention
times of MF and the I.S. (Figure 6-3). Figure 6-4 shows the chromatograms for extracted
plasma samples spiked with 15 pg/ml MF and I.S. (1 ng/ml of 13C3-FP).
Recovery
The recovery of MF (n=5) was for the low (40 pg/ml), medium (80 pg/ml) and high
(400 pg/ml) concentrations was 81.5±5.4, 74.5±3.9 and 73.7±5.8 respectively. The
recovery for the I.S. using an identical extraction method has been reported to be 82% for
a concentration of 100 pg/ml in an earlier report [112].

87
Precision and Accuracy
The intra and inter-day accuracy and precision data for the six QC samples of MF
used in the assay is listed in Table 6-1. The assay was reliable, consistent and showed
good accuracy (88.0-104.4% at LLOQ and 93.8-111.1% at other concentrations) and
precision (5.2-12.8 at LLOQ and 3.8-12.0 at other concentrations). 15 pg/ml was chosen
as the LLOQ for MF based on the accuracy (80-120%) and precision (<20%) on a day-to-
day basis [147]. The linear range for MF was 15-1000 pg/ml with correlation coefficients
(r2) >0.99.

88
Table 6-1. Intra-day and inter-day accuracy/precision for MF in human plasma (n=18).
15 pg/ml
30 pg/ml
50 pg/ml
80 pg/ml
120 pg/ml
400 pg/ml
Day 1
88.0/9.9
97.3/12.0
98.0/9.5
93.8/7.0
103.8/8.1
101.0/5.8
Day 2
104.4/5.2
93.9/9.6
98.8/7.2
104.3/4.4
106.5/5.0
105.8/7.1
Day 3
91.1/12.8
100.0/8.7
104.7/2.9
104.2/3.8
110.3/5.4
111.1/4.9
Inter-day
94.9/11.8
97.1/9.8
100.8/7.0
100.5/7.0
106.9/6.6
106.0/6.9
Stability
The data for the human plasma freeze-thaw cycle stability, short-term stability
under room temperature and bench-top stability at the low, medium and high
concentrations of MF is shown in Table 6-2. The results show that MF was stable under
the investigated conditions as the measured concentrations were within acceptable limits
(85 to 115% of the nominal concentrations).
Table 6-2. Stability3 (%) of MF in human plasma.
50 pg/ml
120 pg/ml
400 pg/ml
Three cycle freeze-thaw
100.0±7.2
101.9±7.5
94.2±5.2
Short term (4 h)
95.3±8.1
89.5±6.5
87.4±0.8
Bench top (6 h)
89.3±8.3
89.2±6.5
87.6±3.5
a Stability expressed as the % ratio of the measured concentration to the nominal
concentration (n=3).
A rapid, simple, sensitive and selective LC-(APCI')-MS-MS method was
developed and validated for the quantification of MF in human plasma samples. The
method is robust and linear over a wide range of concentrations. The assay has a limit of
quantification of 15 pg/ml, and is more sensitive than a previously reported competitive
enzyme immunoassay (LLOQ 50 pg/ml) and LC-MS-MS method (LLOQ 50 pg/ml) for
the quantification of MF in human plasma [62, 140], Considering all these attributes, this
assay is a significant improvement over existing assays for performing pharmacokinetic
studies following oral inhalation of therapeutic doses of MF.

CHAPTER 7
SINGLE DOSE PHARMACOKINETICS OF INHALED MOMETASONE FUROATE
Introduction
Mometasone furoate (MF) is an inhaled glucocorticoid characterized by high
lipophilicity, strong affinity to the glucocorticoid receptor, high protein binding and rapid
systemic clearance. These properties are important in achieving distinct anti¬
inflammatory effects in the lung while minimizing the systemic side effects.
Pharmacokinetic studies on MF after intravenous administration have shown that MF has
a rapid systemic clearance (54 L/h) and a large volume of distribution (332 L) [62].
However, pharmacokinetic investigations of MF after oral inhalation have been limited.
MF has been reported to have a systemic bioavailability of less than 1% after
administration by oral inhalation [62]. There has been a debate over the validity of this
claim as the bioavailability was estimated using an assay with insufficient sensitivity and
at doses that resulted in most of the concentrations being below the limit of quantification
of the assay [103, 106].
The development and validation of a specific and sensitive assay (lower limit of
quantification 15 pg/mL) for the quantification of MF in human plasma was described in
the previous chapter. This assay was used in the present study to determine the
pharmacokinetics of MF in patients with asthma after oral inhalation of a single dose of
800 pig of MF. This study was conducted as a part of a bigger project in the Nottingham
City Hospital, Nottingham, England that involved the assessment of the influence of lung
function on systemic availabilities of inhaled corticosteroids. Therefore, patients with
89

90
varying degree of lung functions were enrolled in the study and the systemic availabilities
of four inhaled corticosteroids- mometasone furoate, fluticasone propionate, budesonide
and beclomethasone dipropionate was assessed. The pharmacokinetics of MF alone are
presented in this report.
Materials and Methods
Subjects
Thirty non-smoking subjects with asthma were recruited between October 2003
and February 2004 from the volunteer database at the University of Nottingham,
England. Subjects were selected to provide a range in FEVi from 30% predicted
upwards and had to have stable asthma defined as no change in asthma symptoms or
treatment for at least two months. FEV i and forced vital capacity (FVC) were measured
with a dry bellows spirometer (Vitalograph, Buckingham, UK) as the higher of two
successive readings within 100ml. The enrolled subjects had a mean age of 56.6 (range
35-70) years, height of 167 (range 150-189) cm, weight of 78.9 (range 55.2-98.8) kg and
FEVi of 73.9 (range 36.4-137.7) mL. There were no restrictions based on treatment but
subjects were excluded if they had significant co-morbidity, were pregnant or lactating or
had a greater than 20 pack/year smoking history. All subjects gave written informed
consent to the study, which was approved by Nottingham City Hospital ethics committee.
Protocol
Subjects were screened at an initial visit to measure FEVi, assess suitability for the
study and to familiarize themselves with placebo dry powder inhalers. Subjects taking
fluticasone propionate, budesonide or mometasone furoate were changed to an equivalent
dose of beclomethasone dipropionate at least 4 days before the study day and were asked
to omit the beclomethasone dipropionate from the evening before the study day. Subjects

91
attended the department in the morning of the study, and after a ten minute rest, a venous
cannula was inserted, blood taken for baseline drug assay and FEVi measured on two
occasions 5 minutes apart. Subjects then inhaled 2 puffs from each of four inhalers in
random order with a mouth rinse after each drug. The drugs and doses inhaled were
beclomethasone dipropionate 800pg (Becodisks®, Allen & Hanburys), budesonide
800pg (Turbohaler®, Astra-Zeneca), fluticasone propionate lOOOpg (Accuhaler®, Glaxo-
Wellcome) and mometasone furoate 800pg (Asmanex Twisthaler®, Schering-Plough).
Venous blood samples were collected into heparinized tubes pre-dose (0 h) and at 0.08,
0.25, 0.5, 1,2, 3, 4, 6 and 8 h after dosing. Samples were centrifuged at 1500 rpm for 10
minutes and plasma samples were then frozen at -70°C. The order in which drugs were
given was randomized using random number tables in the pharmacy, Nottingham City
Hospital, Nottingham, England.
Bioanalytical Methods
The plasma concentrations of MF were quantified with a high performance liquid
chromatography/tandem mass spectrometry (LC/MS/MS) method using a Micromass
Quattro LC-Z triple quadrupole mass spectrometer (Beverly, MA), equipped with an
atmospheric pressure chemical ionization (APCI) source. The development and
validation of this method has been described in the previous chapter. The assay had a
lower limit of quantification of 15 pg/mL and the concentrations were measured over a
linear range of 15 to 1000 pg/mL.
Pharmacokinetic Data Analysis
Noncompartmental and compartmental pharmacokinetic data analysis was
performed using the WinNonlin® Professional Version 3.1 (Pharsight Corporation,
Mountain View, CA) software program. Concentrations below the limit of quantification

92
(15 pg/mL) were reported as missing and the missing values were extrapolated by
WinNonlin during noncompartmental analysis.
The following noncompartmental parameters were determined. The Cmax (peak
concentration) and Tmax (time of peak concentration) values were obtained by visual
inspection of the concentration-time profiles. The terminal half-life (ti/2) was calculated
as 0.693/k, where k is the elimination rate constant in the terminal phase, k was
determined based on visual inspection and subsequent linear regression of data points in
the terminal linear phase of a semi-log plot of concentration versus time. The area under
the plasma concentration-time curve (AUC) was calculated by the linear trapezoidal rule
in WinNonlin. The area under the curve up to infinity (AUCoo) was calculated by
extrapolating the last measurement point to infinity using the relationship CW/k, where
Ciast is the last measurable concentration above the limit of quantification (15 pg/mL).
The area under the first moment curve (AUMCoo) was calculated from C¡*t¡ versus t¡ pairs
using the trapezoidal rule. Extrapolation from the last measurement point (Qast) to infinity
was calculated as Qas^W/k + Qast/k2. The apparent clearance (CL/F) was calculated
using the relationship CL/F = D/AUCoo where D is the administered dose and F is the
overall systemic bioavailability. The apparent volume of distribution (Varea/F) was
calculated as Varea/F = CL/F/k.
The mean residence time after inhalation (MRT¡nh) was calculated by MRT¡nh =
AUMCoo/AUCoo- The mean absorption time (MAT) was calculated by the relationship
MAT = MRTjnh-MRTjv, where MRT¡V is the mean residence time after intravenous
administration (IV). The MRT¡V for MF was estimated to be 2.6 h by performing

93
noncompartmental analysis on the mean data read from a concentration-time plot in a
previously published report [62].
The overall systemic bioavailability of MF after oral inhalation (F) was calculated
as F,
inh
D,v • AUC, inh
Dinh -AUCwiv
where D,v and D¡nh are the doses after an intravenous administration and oral
inhalation respectively and AUCoo.iv and AUC^inh are the area under the curve after the
intravenous administration and oral inhalation respectively. Values for DIV (400 pg) and
AUCoojv (8.01 ng.h/mL) were taken from a previously published report [62],
Compartmental analysis was performed by using a one-compartment body model
with first order absorption to fit the plasma concentrations of MF. The model was
described by the equation:
c =. D F k0l
Vta-k.)
â– (<
e M -e~k(
")
where Ct is the concentration at time t, D is the dose, F is the systemic
bioavailability, Vc is the volume of distribution of the central compartment, koi is the
absorption rate constant and ke the elimination rate constant, ke was re-parameterized as
CL/Vc during fitting with WinNonlin. The goodness of fit was evaluated using the
Akaike information criteria (AIC).
The absorption profile and the absorption rate constant of MF were estimated by
Loo-Riegelman method [148] using Kinetica® Version 4.2 (InnaPhase Corporation,
Philadelphia, PA) software program. The pharmacokinetic parameters after intravenous
administration were obtained by fitting the mean concentration-time data read from a
previously published report to a two-compartment body model [62],

94
Results and Discussion
The pharmacokinetic analysis of MF after administration of a single dose of 800 pg
in patients with asthma is presented here. The concentration-time profile from one of the
subjects was unsuitable for performing pharmacokinetic analysis and the data from this
subject has not been included in the results. Therefore, the total number of subjects in the
analysis is 29.
Noncompartmental Analysis
Table 7-1 gives the summary of the results obtained from noncompartmental
analysis of a single dose of 800 pg MF following oral inhalation by Asmanex
Twisthaler® dry powder device. Peak concentrations of MF were observed at 1 hour
post dose and the median Cmax was 0.070 ng/mL (Figure 7-1). The median terminal half-
life of MF was 4.5 h and this is in good agreement with the half-life value of 4.45 h seen
after an intravenous administration of MF [62], The clearance (CL/F) estimate of MF
was 1573 L/h and the estimate of MRTmh was 6.7 h. The mean absorption time (MAT)
was estimated to be 4.1 h. The MRTIV of MF has not been reported in the literature and
MAT was estimated using the MRTiv (2.6 h) obtained from noncompartmental analysis
of mean concentration-time data taken from a graph in a published report [62], The
reliability of this estimate of MAT needs to be further assessed by obtaining the precise
MRTiv values from an intravenous study.

95
Table 7-1. Median (% coefficient of variation) pharmacokinetic parameters of MF
obtained by noncompartmental analysis following oral inhalation of 800 pg of
MF by Asmanex Twisthaler® dry powder device (n=29).
Parameter
Median (% CV)
Cmax (ng/mL)
0.070 (67)
Tmax * (h)
1.00 (0.08-4.00)
AUC» (ng.h/mL)
0.51 (73)
CL/F (L/h)
1573(59)
Varea/F (L)
9062 (94)
T i/2 (h)
4.5 (73)
MRTinh (h)
6.7 (70)
* - Tmax expressed in median (range).
0.15
Figure 7-1. Mean (+ SD) plasma concentration-time profile of MF after oral inhalation of
800 pg of MF by Asmanex Twisthaler® dry powder device (n=29).
The overall systemic bioavailability (F) of MF was estimated to be 3.2%. This
bioavailability is a result of absorption of the fraction of the dose that reaches the
intracellular steroid receptors in the lung. F was calculated using the AUC* estimates
from this study and the intravenous administration study by Affrime et al [62], The
pharmacokinetic parameters in the IV study were obtained from healthy subjects while
the parameters in this study were obtained from asthmatic patients. While this is not
expected to cause a difference in the basic pharmacokinetic parameters (CL and V)

96
between the two populations, as shown in the case of fluticasone propionate [128], the
systemic availability in healthy subjects is expected to be higher than the 3.2% seen in
patients. This is because inhaled corticosteroids are deposited centrally in the lungs of
patients as opposed to a more peripheral deposition in healthy subjects [149]. The
mucociliary clearance mechanisms are more pronounced in the central airways than in
the peripheral airways [48], This would lead to the mucociliary removal of slow-
dissolving highly lipophilic drugs like MF from the central airways in patients resulting
in lower systemic availability in asthmatic patients than in healthy subjects. Such
differences have been noted for the highly lipophilic steroid fluticasone propionate (FP),
with the systemic availability of FP being 10% in asthmatic patients compared to 21% in
healthy subjects [128]. Similarly, in a separate study, the systemic availability of FP was
about twice as high in healthy subjects versus patients [150].
The systemic bioavailability of MF (3.2% in patients) calculated in this study is a
more accurate estimate than the <1% value in healthy subjects reported in the literature
[62]. The <1% estimate was based on an assay of lower sensitivity (LLOQ 50 pg/mL)
and at a lower dose (400 pig), which resulted in most of the concentrations being below
the LLOQ. The concentrations below LLOQ were substituted as zero for AUC
calculations and this leads to an underestimation of the AUC and the resulting systemic
bioavailability. However, this study used a more sensitive assay (LLOQ 15 pg/mL) and a
higher dose (800 pg) to calculate the systemic bioavailability. In this study, the
concentrations below LLOQ were assumed missing and were extrapolated using the
WinNonlin® program while calculating the AUC. All these attributes make the systemic
bioavailability (3.2%) in the current study a more reliable estimate.

97
Compartmental Analysis
The one-compartment body model with first order absorption gave excellent fits (r2
> 0.94) for the observed concentrations of MF after administration of a single dose of 800
pg of MF. The one-compartment model also yielded better fits (lower AIC values) than a
two-compartment model. The pharmacokinetic parameters following fitting are
summarized in Table 7-2 and a representative fit for one of the subjects is shown in
Figure 7-2. The parameter estimates obtained after fitting were also consistent with the
results from the noncompartmental analysis.
Table 7-2. Estimates of the pharmacokinetic parameters of MF after fitting using a one-
compartment body model with first order absorption (n=29).
Parameter
Median estimate
CL/F (L/h)
1309
Vc/F (L)
12484
K0, (l/h)
18.0
Figure 7-2. Representative fit using a one compartment body model for one subject
following oral inhalation of a 800 pg dose of MF.

98
The estimate of koi from this study appears to be too large to represent the
absorption rate constant of MF. It is unlikely that a highly lipophilic glucocorticoid like
MF would be able to dissolve as rapidly as koi (half-life of absorption 2.3 min) suggests.
Moreover, the MAT of 4.1 h for MF also suggests a slow dissolution process. Therefore,
koi might actually represent a hybrid of distribution and elimination processes rather than
the absorption process. The sparseness and inadequate spacing of concentration-time
points, especially around Cmax (1.0 h), might be a reason for the inability of the software
program to distinguish between the absorption and distribution processes in this study.
Moreover, there is no literature information about the pharmacokinetic parameters after
intravenous administration and this makes it difficult to distinguish between the
microconstants involved. Similar flip-flop kinetics (distribution rate faster than
absorption rate) have been observed for fluticasone propionate and it was shown that an
early Tmax does not necessarily indicate a fast absorption process [151].
The absorption profile of MF (Figure 7-3) was determined using the Loo-
Riegelman method. The results show that there is a phase of rapid absorption initially
with more than 15% of the dose being absorbed in the first 5 min after inhalation. This
was followed by a slower absorption phase and 100% of the dose was absorbed 8 hours
after inhalation. This latter phase indicates a slow absorption process that is consistent
with the MAT of 4.1 h, the high lipophilicity and the slow in vitro dissolution
characteristics of MF (chapter 4). This biphasic absorption profile could be because of
the presence different polymorphs of MF in the inhalation powder and is consistent with
the biphasic dissolution observed in vitro (chapter 4). The absorption profile of MF was
obtained by fitting mean concentration-time data from healthy subjects in an IV study to

99
a two-compartment body model [62], Therefore, the absorption profile obtained might be
a rough estimate. However, it is unlikely that the overall pattern would be completely
different from the one seen in this study.
Figure 7-3. Absorption profile of MF after inhalation of a single dose of 800pg
determined using the Loo-Riegelman method. The profile is based on a two-
compartment disposition model for MF after intravenous administration.
The absorption profile of MF compares well with that of FP that has similar
lipophilicity. The MAT for FP has been reported to be 5 h [152] and FP has an
absorption half-life of 3.8 h [151]. The slow absorption processes of MF and FP make
them susceptible to be removed from the lung by mucociliary clearance mechanisms,
especially in patients with asthma in whom a more central deposition of inhaled
corticosteroids is seen [149].
This study illustrates the importance of using a sensitive analytical assay in
estimating the pharmacokinetic parameters of an inhaled glucocorticoid. The results

100
indicate that the systemic bioavailability of MF is greater than the <1% suggested earlier.
MF has a systemic bioavailability of 3.2% in asthmatic patients and this value is expected
to be higher in healthy subjects. This bioavailability of MF along with the fact that MF
has an active metabolite (6p-OH-mometasone furoate, chapter 5) is most likely to explain
the suppression of the hypothalamic-pituitary-adrenal axis seen after an oral inhalation of
MF [63]. This study also emphasizes the need for having prior information on
pharmacokinetic parameters from intravenous studies to enable precise estimation of the
absorption process and the pharmacokinetic parameters after oral inhalation. If such
prior information is not available, it is advisable to conduct parallel intravenous and
inhalation studies on the same subject population. Even though such parallel studies are
typically limited by regulatory constraints and formulation issues, they should be
conducted whenever possible for performing robust pharmacokinetic analysis.

CHAPTER 8
SELECTIVITY OF MOMETASONE FUROATE AND OTHER INHALED
CORTICOSTEROIDS TO THE GLUCOCORTICOID RECEPTOR
Introduction
Inhaled corticosteroids (ICS) have been used as the drugs of choice in the treatment
of asthma related symptoms for the past decade. The newer inhaled steroids show
increased potencies, indicated by pronounced receptor binding affinities, to the
glucocorticoid receptor (GR). The interaction of these synthetic ligands with the
systemic glucocorticoid receptors results in undesired systemic side effects. In addition
to these systemic side effects, other side effects could be result if the synthetic steroids
lacked selectivity towards the glucocorticoid receptor and bind to other members of the
steroid receptor family like the progesterone receptor (PR). As a matter of fact, some of
the endogenous corticosteroids like corticosterone and deoxycorticosterone have been
reported to bind to the progesterone receptor [153]. Similarly, the endogenous steroidal
ligands like progesterone have also been shown to non-selectively bind to the
glucocorticoid receptor [154], This cross reactivity of ligands between different nuclear
receptors is suggestive of a close homology in binding domain between the
glucocorticoid receptors (GR) and the other members of the steroid receptor family [22],
Recently, functional assays conducted by Austin and coworkers [108] have demonstrated
that mometasone furoate (MF) lacks glucocorticoid receptor selectivity, indicated by its
ability to bind non selectively to the PR. As potential systemic side effects could result
from the binding of these synthetic ligands to the PR, it was the aim of this study to
101

102
assess the selectivity of currently used corticosteroids towards the glucocorticoid
receptor, by comparing their relative receptor affinities to the GR and the PR.
Materials and Methods
Chemicals
Mometasone furoate (MF) and Mometasone (MO) were purchased from USP and
European Pharmacopoeia, France respectively. Fluticasone propionate (FP) and
budesonide (BUD) were provided by GlaxoWelcome (Research Triangle Park, NC) and
Sicor (Milan, Italy) respectively. Beclomethasone monopropionate was purchased from
European Directorate for Quality of Medicine (EDQM). Dexamethasone (DEX),
triamcinolone acetonide (TAA), beclomethasone (BECLO), betamethasone (BET) and
progesterone (PROG) were purchased from Sigma chemicals Co (St Louis, MO). H-
1 T
ORG-2058 (specific activity 36 Ci mmole') and H-TAA were purchased from Perkin
Elmer Life Sciences (Boston, MA). All other chemicals and solvents were obtained from
Sigma Chemicals Co. (St Louis, MO) and Fisher Scientific Co. (Cincinnati, OH).
Glucocorticoid Receptor (GR) Binding Assay Experiments
The various dilutions for DEX (0.001 - 1.0 pM), MF and MO (0.01 - 100 pM) were
prepared in methanol.
The animal protocol was approved by the local Institutional Animal Care and Use
Committee (IACUC) at the University of Florida, Gainesville, Florida. Sprague Dawley
rats (250 ± 25gms) were obtained from Harlan (Indianapolis, IN). The rats were
anesthetized using a cocktail mixture of ketamine, xylazine and acepromazine (3:3:1 v/v)
and were decapitated. The lungs were removed and homogenized in 8 volumes of ice-
cold incubation buffer (10 mM Tris/HCL, 10 mM sodium molybdate, 2 mM 1,4-
dithioerythritol). The homogenate was incubated with 5% w/v charcoal suspension (in

103
deionized water) for 10 min. The homogenate was then centrifuged for 20 min at
40,000X g in J2 rotor of Beckman centrifuge to obtain the cytosol. Fresh cytosol was
prepared and used for all the individual experiments.
10 nM of 3H-labeled triamcinolone acetonide (TAA) solution was used as a tracer,
based on previous saturation binding experiments performed in our laboratory (data not
shown). 20 pL of the drug solution in methanol was added to pre-chilled tubes. Blank
methanol was used for the determination of total binding. Non-specific binding was
determined after addition of 20 pL of 100 pM unlabeled TAA (10 pM in the final
incubation mixture). 20 pL of 100 nM3H-TAA solution (10 nM in final incubation
mixture) was then added. 160 pL of the lung cytosol was added and the tubes were
vortexed and incubated at 4°C for 24 h. After the incubation, 200 pL of 5% w/v charcoal
suspension (in water) was added to the tubes to remove the excess unbound radioactivity.
The tubes were vortexed and 300 pL of the supernatant was transferred to the
scintillation vials. 5 mL of the scintillation cocktail was added and the scintillation vials
were read in a liquid scintillation counter (Beckman Instruments LS 5000 TD, Palo Alto,
CA).
Progesterone Receptor (PR) Binding Assay Experiments
The various dilutions for PROG (0.01-100 pM), DEX (0.03 - 10000 pM), BET (0.3
-10000 pM), MF (0.01 - 100 pM), FP (0.01 - 100 pM), MO (0.01 - 100 pM), BMP (0.01
- 100 pM), BECLO (0.03 - 1000 pM), BUD (0.01 - 100 pM) and TAA (0.03 - 100 pM)
were prepared in methanol.
The animal protocol was approved by the local Institutional Animal Care and Use
Committee (IACUC) at the University of Florida, Gainesville, Florida. The personnel
handling the tissue were equipped with personal protective equipment (PPE) to prevent a

104
chance exposure to Coxiella burnetii virus normally present in the sheep. All steps till
the homogenization of the tissue was conducted under a hooded environment. The sheep
uterus tissue was obtained from pregnant sheep, which were sacrificed for another
experimental setup. The tissues were frozen in liquid nitrogen and were stored at -80°C
for not more than 4 days. During the day of the experiment the tissues were dipped in
liquid nitrogen for 1 min and then were pulverized to small pieces. The tissue was then
further homogenized after addition of 4 volumes of ice-cold incubation buffer (10 mM
Tris/HCL, 10 mM sodium molybdate, 2 mM 1,4-dithioerythritol) using a Bio
Homogenizer (5 sec, low speed, with 30 sec cool down period between each step). The
homogenate was incubated with 5% w/v charcoal suspension (in deionized water) for 10
minutes to remove the endogenous progestins. The homogenate was centrifuged for 1 hr
at 40,000 g in a Beckman high-speed centrifuge using a JA-20 fixed angle rotor. The
resulting aqueous supernatant (cytosol) was used on the same day of the experiment.
Fresh cytosol was prepared and used for all the individual experiments.
Portions of the cytosol (160 pi) were incubated with 20 pi of 20 nM of tritium
labeled [6,7-3H]ORG-2058 solution (final concentration in the incubation mixture: 2 nM)
and the same volume of varying concentrations of the competitor (prepared in incubation
buffer containing 50% ethanol). After 24 hrs incubation time, the unbound ligand was
removed by addition of 200 pi activated charcoal suspension (5% w/v in deionized
water). The mixture was incubated for 10 min at 0 - 4 °C, and then centrifuged for 5 min
at 10000 rpm (fisher scientific). The radioactivity in 300 pi of the supernatant was
determined using liquid scintillation counting (Beckman Instruments LS 5000 TD, Palo
Alto, CA). The non-specific binding was determined in presence of 2 x 10"' M unlabeled

105
PROG. The non-specific binding was always lesser than 15% of the total binding. All
determinations were performed in duplicate and the entire experiment was repeated on 3
different occasions.
Data Analysis
The IC50 values of the investigated steroids (competitor concentration necessary to
displace 50% of the specific H-ORG-2058 from the receptor site) and the slope factors
of the resulting competition curves were determined by non-linear curve fitting procedure
using Scientist . The data was fitted to the following Emax model to obtain the estimates
of Bmax srid ICso-
DPM =
Bmax - Bit
ic* +CN
+ NS
where, DPM represents the total tracer binding obtained at any given competitor
concentration. NS represents non-specific binding and N is the Hill coefficient. Bmax is
the specific binding by the ligand in the absence of competitor.
The resulting IC50 values were transformed into relative receptor affinities (RRA)
relative to progesterone (reference for PR assay) or dexamethasone (reference for GR
assay). RRArr and RRAgr describe the relationship between the IC50 values of the test
steroid to that of progesterone and dexamethasone respectively.
RRA
TEST
PR or GR
IC
REF
50
IC
TEST
50
x 100
The GR selectivity has been defined as
GR selectivity =
RRA
GR
RRA
PR

106
Molecular Dynamic Modeling
Structures 1A28 (for the human progesterone receptor complexed with
progesterone) [155], 1E3K (for the human progesterone receptor complexed with
metribolone) [156], and 1M2Z (for the human glucocorticoid receptor complexed with
dexamethasone) [117] were obtained from the Protein Data Bank, and are displayed
using DS ViewerPro 5.0 (Accelrys, Inc., San Diego, CA). Glucocorticoid structures
(beclomethasone monopropionate, fluticasone propionate, and mometasone furcate) were
individually fully geometry-optimized using AMI semi-empirical quantum chemical
calculations [157] in CAChe 5.0 (Fujitsu, Ltd., Chiba, Japan) and then superimposed with
the receptor-bound ligands by Discovery Studio’s (Accelrys, Inc., San Diego, CA)
Molecular Overlay algorithm within ViewerPro 5.0 using a targeted RMSD-minimizing
alignment on the ring-fusion atoms of the steroid structure (C5, C8, C9, C10, C14, and C13).
Results and Discussion
The relative receptor affinities (RRA) of MF, FP, BUD, BMP, BECLO, DEX,
TAA, BET, MO and PROG to the progesterone receptor were determined by competition
binding experiments in cytosol of sheep uterus. The RRA of MF, MO and DEX to the
glucocorticoid receptors was similarly determined by competition binding experiments in
rat lung cytosol. The RRA of FP, BUD, BMP, BECLO, BET and TAA to the GR were
obtained from previous experiments conducted in our group. The binding curves
resulting from a typical set of experiments conducted with sheep uterus and rat lung
cytosol are shown in Figures 8-1 and 8-2 respectively.
Table 8-1 compares the RRA values for PROG, MF, FP, BUD, BMP, BECLO,
DEX, TAA and MO to both the PR and the GR. Table 8-1 also shows the selectivity of
different corticosteroids. The high selectivity values range from 476-1375 for BET

107
(GR/PR ratio of 1375), BECLO (760) and DEX (476); moderate selectivity from 9-44 for
BMP (9), FP (12), TAA (18), MO (25), BUD (44); and a low selectivity for MF with
selectivity value of 1. Figure 8-3 shows the relationship between Log of selectivity and
Log P values with a correlation coefficient of (r2= 0.7499) for different steroids tested.
Table 8-1. Relative receptor affinities (RRA) to the progesterone receptor of sheep uterus
tissue.
Steroid
RRAgr
RRA PR
Selectivity
(RRAgr/RRApr)
DEX
100
0.21
476
BET
55*
0.04
1375
PROG
-
100
-
MF
2938
2580
1.1
FP
1800*
152
11.8
BMP
1022*
110
9.3
BECLO
76*
0.1
760
BUD
933*
21
44
TAA
233*
13
17.9
MO
88
3.5
25.1

108
Log of competitor concentration (pM)
Figure 8-1. Competitive binding experiments to the progesterone receptors in sheep
uterus cytosol. Nonlinear regression of non-transformed data was used for the
determination of IC50 values of (top) PROG, MF, MO, TAA, BMP, BUD, FP
and (bottom) PROG, MO, DEX and BECLO. The hill coefficient was fixed to
1.

109
Figure 8-2. Competitive binding experiments to the glucocorticoid receptors in rat lung
cytosol. Nonlinear regression of non-transformed data was used for the
determination of IC50 values of DEX, MF and MO. The hill coefficient was
fixed to 1.
As mentioned earlier in the text, there are a few endogenous corticosteroids that
have been reported to be nonselective towards the GR by the fact that they have the
ability to bind to the progesterone receptor. The cross reactivity of ligands between GR
and PR might be of greater concern with synthetic steroids often used in the
glucocorticoid treatment, where the associated systemic side effects are very common.
Raynaud and coworkers have previously shown that synthetic corticosteroids like TAA
and fluocinolone acetonide have the ability to bind to the progesterone receptors
indicating low GR selectivity [153, 158]. Recently, cell based functional assays
conducted by Austin and coworkers have also shown that MF exhibits a potent agonist

110
activity towards the progesterone receptor, indicating low GR selectivity [108]. As a
further attempt to evaluate the selectivity of other clinically proven inhaled
corticosteroids, competitive receptor binding assays were performed both at the
progesterone and the glucocorticoid receptors in this study. This study also analyzed
some of the structure affinity relationships, by relating the influence of functional groups
(Table 8-2) to the observed differences in selectivities among steroids.
Figure 8-3. Relationship between Log P value of different steroids to the observed steroid
selectivity. The calculated Log P values are the averages of the computed
values: CLOGP (from ChemDraw Ultra 7.0), ACD/LogP and QlogP [159].
The solid line represents the line of best fit (linear regression) through all data
points. The Log P correlates to the selectivity with a correlation coefficient
(r2) of 0.7499.

Ill
Table 8-2. Chemical structures of various corticosteroids and metabolites
(a)
r5-
ch3
o^r2 (b)
ch3 2
"Ji 1111R
H > CH3
r3 H F
r Cr >
R4 Í
VR2 R
^ 0
H y'
H
4
Steroid
A1
A4
aR
aRl
R2
aR3
R4
(3R5
RBApk
DEX
+
+
OH
ch3
CH2OH
F
H
OH
0.21
BET
+
+
OH
PCH3
CH2OH
F
H
OH
0.04
PROG
-
+
H
H
ch3
H
H
H
100
MF
+
+
OCOR’*
ch3
CH2C1
Cl
H
OH
2580
MO
+
+
OH
ch3
CH2C1
Cl
H
OH
3.5
FP
+
+
OCOCH2CH3
ch3
SCH2F
F
aF
OH
152
BMP
+
+
OCOCH2CH3
(3CH3
CH2OH
Cl
H
OH
110
BECLO
+
+
OH
(3CH3
CH2OH
Cl
H
OH
0.1
BUDb
+
+
CH2CH2CH3
H
CH2OH
H
H
OH
21
TAAb
+
+
ch3
ch3
CH2OH
F
H
OH
13
* where R' =
It has been previously been established that the binding forces that are responsible
for interaction of steroidal ligands with the GR are hydrophobic in nature and there exists
a good correlation between the lipophilicity of the ligand and the GR relative binding
affinities [88], Interestingly, our data (Figure 8-3) showed that lipophilicity of the steroid
could be considered as a predictor of GR selectivity within these structures, with lower
selectivity associated with higher Log P values of the steroid.
The data obtained in this study showed that some of the tested inhaled
corticosteroids were found to display a lack in GR selectivity due to their ability to bind
to the progesterone receptor. McGuire et al have shown that the presence of free OH
group at Cl 7a position of the steroid dramatically reduces the ability of the steroid to

112
bind to the progesterone receptor compared to the corresponding substituted esters at
Cl7a [160]. Similarly, in this study, experiments with C17a-OH containing
corticosteroids like DEX, BET, MO and BECLO also showed decreased affinities
towards the progesterone receptor. Esterification of the Cl 7a OH group in BECLO and
MO significantly increased the PR binding affinities and contributed to an overall
decrease in the GR selectivity. GR selectivity was much lower when furoate ester was
present at the Cl7a position compared to the propionate (as in BMP or FP) and a
definitive explanation for this behavior is not very evident from the current experiments.
Nevertheless, a greater lipophilicity of the furoate substitution compared to the
propionate might favor greater Van der Waals interactions with surrounding hydrophobic
residues (e.g., Leu , Leu , Phe , Leu , and Tyr ) resulting in stronger binding.
There is sufficient evidence from literature that suggests that there exists an
additional binding pocket in the glucocorticoid receptor, which has the ability to
accommodate larger Cl 7a substituents of corticosteroids [117]. However, this binding
pocket is absent in progesterone, androgen and the estrogen receptors. Surprisingly, in
spite this absence, several steroids like (MF, BMP, FP) having large substitution at the
C17a showed significant increase in the PR affinity and consequently a decrease in the
GR selectivity. Figure 8-4 indicates the Cl7a side chain of mometasone furoate can still
easily fit within the ligand-binding domain of the progesterone receptor where it is
surrounded by hydrophobic residues (e.g., Leu715, Leu718, Phe794, Leu 191, and Tyr890).
The AMI-optimized structure of mometasone furoate was superimposed on the receptor-
bound conformation of the original progesterone ligand as determined in the crystal
structure [155]. In the superimposed AMl-optimized conformation, this side-chain

113
encounters only some minimal structural hindrance from these residues, but it could
easily rearrange in a more convenient conformation as there is sufficient space and the
receptor itself is also flexible to a certain extent [161]. The Van der Waal interactions
between the lipophilic Cl7a ester substitution (as in MF and BMP) and the surrounding
hydrophobic residues may result in sufficient favorable interactions to justify an
increased binding affinity towards the progesterone receptor. Similar considerations
could apply to FP vs. fluticasone.
The superimposition of BUD over the PR also shows (Figure 5) sufficient space
within the receptor to accommodate the side chains. However, C16a-0 and C21-OH
groups experience hindrance from the surrounding hydrophobic residues Ph905 and
Leu797 respectively. These hindrances might restrict the C21 and Cl6a side chains in
BUD to interact freely with the progesterone receptor, thus worsening the overall
receptor fit of BUD. This hypothesis could be explained by our experimental observation
that showed increased GR selectivity for BUD compared to MF.
Greater GR selectivity of BECLO compared to MO could be due to the differences
in substituent at C21 and p orientation of C16 methyl in BECLO. To further investigate
the effect of orientation of CI6-CH3 group on GR selectivity, competition assays were
conducted with DEX and its epimer betamethasone (BET) at the progesterone receptor.
Selectivity is a ratio of relative affinities at two different receptor types (GR and PR), and
therefore a change in the PR affinity would then reflect a corresponding change in the
overall selectivity. Results indicated that presence of P-CH3 group as in BET increased
GR selectivity over DEX. The difference in the orientation of Cl 6 methyl between DEX
and BET indicates that a methyl in the P plane at C16 position of the steroid might

114
sterically hinder favorable Van der Waals interaction with the surrounding hydrophobic
residues at the PR. This could be a reason why BECLO showed greater selectivity than
MO. The affect of (3-alkyl substitution on the relative receptor affinity of steroids at the
PR as observed from our experiments was in agreement with the previous observations
by McGuire et al [160],
Figure 8-4. The structure of mometasone furoate (shown as a darker ball-and-stick
structure) superimposed over that of progesterone bound to the ligand-binding
domain of the human progesterone receptor. Note that there is sufficient space
to accommodate the 17a side chain, which is surrounded by hydrophobic
residues (Leu715, Leu718, Phe794, Leu 797, and Tyr890; also shown as ball-and-
stick structures) that can provide favorable van der Waals interactions. Amino
acid residues in front of the ligand are shown only as line structures to not
hinder the view.

115
Figure 8-5. The structure of R-budesonide (shown as a darker stick structure)
superimposed over that of progesterone bound to the ligand-binding domain
of the human progesterone receptor. Note the C16a-0 in BUD “hits” a
hydrophobic Leu797 and the C21-OH “hits” a hydrophobic Ph905 residue of
the PR. The purple dotted lines indicate the structure bump monitors. The
structures of surrounding hydrophobic residues (Leu715, Leu718, Phe794, Leu 797
and Tyr890) are also shown as ball-and-stick structures that can provide
favorable van der Waals interactions. Amino acid residues in front of the
ligand are shown only as line structures to not hinder the view.
The results indicate that the synthetic glucocorticoid MF is the least specific
followed by BMP, FP, TAA, MO and BUD as steroids with moderate selectivity. The
extra binding pocket present in the GR to accommodate bulky substituents in the Cl7a
position is absent in the PR. However, superimposed AMI geometry optimized structure
of steroid ligands at the PR indicates that there could be an extra space around the ligand
at the PR than previously thought and nonspecific Van der Waals interaction could be the
main determinants of ligand binding. The systemic side effects associated with high
systemic levels of steroids are the major concern during corticosteroid therapy. Steroids

116
like MF that show sufficient cross reactivity with progesterone receptors [108] could
aggravate these systemic side effects in case of a severe systemic spill-over. However, in
view of the observed differences in selectivities, it would be interesting to further
investigate whether the systemic drug concentrations achieved in patients would be
sufficient enough to cause clinical concerns.

CHAPTER 9
CONCLUSIONS
Inhaled corticosteroids are now the first line of therapy in the control and
management of asthma because of their excellent therapeutic ratios. Mometasone furoate
is a glucocorticoid that is being currently evaluated in the treatment of mild-to-moderate
persistent asthma. The systemic exposure and bioavailability (<1%) of MF has been
reported to be extremely low in comparison with other inhaled corticosteroids. The
major focus of this dissertation was to evaluate the extra-hepatic metabolism and
mucociliary clearance of MF as possible reasons for the low systemic bioavailability of
MF. MF was found to be relatively stable in the lung and blood and therefore, extra-
hepatic metabolism might not be a reason for the low systemic exposure of MF. It was
found during these experiments that MF was unstable in aqueous systems and one of the
resulting degradation products showed significant activity to the glucocorticoid receptor.
The in vivo formation of this degradation product needs to be investigated. MF has a
slow in vitro rate of dissolution that suggests that MF could be removed by mucociliary
clearance mechanisms leading to the unusually low systemic exposure.
In vitro assessment of the hepatic metabolism showed that MF is efficiently
metabolized into at least five metabolites and one of the metabolites, 6P-OH-mometasone
furoate showed strong binding affinity to the glucocorticoid receptor. In vivo disposition
studies in rats also showed the formation of this metabolite and there was a strong
indication of biliary elimination of the metabolites. The plasma protein binding and the
in vivo formation of this metabolite in humans need to be investigated. The measurement
117

118
of the active metabolite would be important in estimating the true pharmacological
profile of MF.
It has been speculated that the reported low bioavailability estimate of MF might
not be accurate because of the use of an insensitive assay method and biased
methodology in calculating this estimate. To evaluate this hypothesis, a simple, specific
and sensitive LC-(APCT)-MS/MS assay method was developed in this study for
quantifying MF in human plasma samples. Validation results have shown that this
method is robust and more sensitive than current methods and is ideally suited for the
pharmacokinetic analysis of MF. This assay was used to assess the pharmacokinetics of
MF after oral inhalation of a single dose of 800 pg in patients with asthma in a clinical
study. The systemic bioavailability of MF was estimated to be 3.2%. This bioavailability
estimate of MF is more accurate than the <1% estimate proposed earlier because of the
use of the more sensitive assay and a higher dose in this study. However, this new
bioavailability estimate (3.2%) of MF is still lower than the bioavailability of other
inhaled corticosteroids. The slow in vitro dissolution of MF and the long mean
absorption time of MF strongly suggest that MF could be removed by mucociliary
clearance mechanisms and this could explain the low systemic availability of MF. This
bioavailability of MF and the formation of the active metabolite could explain the
considerable systemic effects (HPA axis suppression) seen upon oral inhalation of MF.
Selectivity studies show that MF has a strong binding affinity to the progesterone
receptor and MF is the least selective among inhaled corticosteroids towards the
glucocorticoid receptor. However, the clinical side effects that may be caused by MF
because of the binding to the progesterone receptor need to be assessed. This study also

119
showed that the selectivity of steroids was negatively correlated with the lipophilicity of
these drugs. Therefore, lipophilicity could be used as a good predictor of the selectivity
of corticosteroids.

APPENDIX
NMR ANALYSIS OF MF AND DEGRADATION PRODUCTS
The NMR analysis of MF and the degradation products were performed at the
I VAX Drug Research Institute, Hungary. The structure determination of MF was based
on the NMR spectral assignments, confirmed by DEPT, two-dimensional 'h/H-COSY,
’H,i3C-HSQC, 'H,i3C-HMBC and one-dimensional selective NOESY experiments. The
113 *131
H and C chemical shifts, characteristic proton-proton couplings (Hz), C, H long-
range correlations (HMBC) and ’H.'H steric proximities (NOE) are summarized in Table
A-l.
The 'H^H-COSY spectrum gave the geminal and vicinal proton-proton
connectivities and the 'H,I3C-HSQC provided the chemical shifts of the one-bonded
coupled 13C-'H nuclei. The ‘H,13C-HMBC experiment was used for the assignment of the
quaternary carbons since the cross peaks revealed the two- and three-bond correlations
between protons and carbons. The stereochemistry of the steroid skeleton and the
substituents, the determination of the a- and P-positions of the protons were achieved on
the basis of the one-dimensional NOESY experiments irradiating the Me-16, Me-18 and
Me-19 protons. These experiments marked out the protons that are located in a distance
less than 5 Á from the irradiated protons.
120

121
Table A-l. 'H and l3C chemical shifts, characteristic couplings (Hz), l3C, long-range
correlations (HMBC) and 'H,‘H steric proximities NOE of compound MF.
5‘H
8I3C
HMBC (13C
partner)
NOE
1
7.18 d (10.0)
151.5
3, 5
2
6.37 dd (10.0)
129.8
10
3
-
186.3
4
6.13 dd (2.0, 1.5)
125.3
1,6, 10
5
-
165.2
6
P
2.66 m
30.6
4,5
a
2.43 ddd (14.3, 4.3,
4,5, 8, 10
~1)
7
p/a
1.81 m
27.2
8
2.64 m
34.5
9
-
82.8
10
-
49.8
11
a
4.64 dd (3.6, 2.8)
75.2
HO-11
P
1.76 d (2.8)
-
12
P
1.72 dd (14.2, 2.5)
36.7
9, 13, 14, 17
a
2.94 dd (14.2, 3.6)
13
-
48.9
14
a
2.66 m
43.8
15
P
1.92 m
32.9
a
1.33 ddd (11.3, 7.6,
3.5)
16
P
3.47 m
35.8
Me-16
a
0.99 (7.1)
16.7
15, 16, 17
15a,16
17
-
97.4
18
a
1.15 s
17.7
12, 13, 14, 17
8, HO-11, 120, 15P,
16,21
19
a
1.68s
24.5
1,5,9, 10
1,6P,8
20
-
196.8
21
4.14s
44.7
20
ooo
-
158.1
f2
-
143.0
f3
7.25 dd (3.6, 0.7)
119.6
f4
6.54 dd (3.6, 1.7)
112.3
F5
7.63 dd (1.7, 0.7)
147.7
f2, f3

122
Table A-2. ’H and l3C chemical shifts, Characteristic couplings (Hz), 13C,'H and long-
range correlations (HMBC) of compound D2.
6'H
5I3C
HMBC (l3C
partner)
1
6.57 d (10.4)
151.9
3,5
2
6.18 dd (10.4, 1.8)—
128.1
10
3
-
186.0
4
6.20 d (1.8)
125.1
1,6, 10
5
-
164.7
6
P
2.71 dddd (15.5, 11.3,
29.3
4,5
5.5, 1.7)
a
2.53 dt (15.5, 5.3)
4,5,8, 10
7
P
2.41 m
30.5
a
1.53 m
8
P
2.36 m
34.2
9
-
66.1
10
-
44.1
11
a
3.13 t (2.3)
62.4
12
P
1.71 d (2.3)
30.4
9, 13, 14, 17
a
1.71 d (2.3)
13
49.7
14
a
1.89 m
50.0
15
P
34.2
16
P
2.87 m
39.5
Me-16
a
0.88 d (7.1)
13.9
15, 16, 17
17
-
100.1
18
P
1.27 s
15.8
12,13,14,17
19
P
1.46 s
23.7
1,5,9, 10
20
-
194.6
21
-
106.7
20
oc=o
-
166.8
£2
-
142.8
fi
7.50 dd (3.8,0.7)
118.8
f4
6.68 dd (3.8, 1.8)
112.9
f5
7.61 dd (1.8, 0.7)
147.0
f2,f3
NMR analysis on D1 could not be performed satisfactorily since the quantity of D1
generated was insufficient because of its inherent instability. Structural determination of
D2 was based on NMR spectral assignments which were confirmed by two-dimensional

123
’H,i3C-HSQC, 'H,13C-HMBC and one-dimensional selective NOSEY experiments. The
'H and 13C chemical shifts, characteristic proton-proton couplings (Hz), 13C,'H long-
range correlations (HMBC) and 'H,'H steric proximities (NOE) are summarized in Table
A-2.
The 'H,13C-HSQC provided the chemical shifts of the one-bonded 13C-*H nuclei.
The 'H,i3C-HMBC experiment was used for the assignment of the quaternary carbons
since the cross peaks revealed the two- and three- bond correlations between protons and
carbons. The stereochemistry of the steroid skeleton and the substituents, the
determination of the a- and P-positions of the protons were achieved on the basis of their
coupling pattern and utilizing the results of the one-dimensional NOSEY experiment
irradiating the Me-18 protons. This measurement gave well-defined NOE responses on
Hp-16, H-8, H2-I2 protons, and a rather small one on Hp-15.
The 13C spectrum shows the absence of the C2I-H2 methylene group and the
corresponding carbon signal appeared at 5106.7. This clearly indicates that condensation
took place between the ester carbonyl and the above-mentioned methylene resulting in a
new, five-member spiro-ring at position Cl 7. The very high value of the one-bond
coupling *J(H-11, C-l 1 )= 175 Hz, unambiguously prove the presence of an epoxide group
in the “C” ring.

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BIOGRAPHICAL SKETCH
Srikumar Sahasranaman was bom on June 30, 1976, to Srimati Jayalakshmy
Sahasranaman and Shri Sahasranaman Ramaswami in West Pallassana, Kerala, India. He
graduated from St. Johns Junior College, Chennai, India, in 1993. Srikumar entered the
Bachelor of Pharmacy program at the Birla Institute of Technology and Science, Pilani,
India, in 1993 and graduated with honors in 1997. He then joined the graduate school in
National University of Singapore and completed his master’s in pharmacy in 2000. In
August 2000, Srikumar entered the Ph.D. program at the Department of Pharmaceutics,
College of Pharmacy, University of Florida, and worked under the guidance of Dr.
Guenther Hochhaus. Srikumar received his Doctor of Philosophy degree in
pharmaceutics in August 2004.
138

I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy. , .
Guenther Hochhaus, Chair
Professor of Pharmaceutics
I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.
Hartmut Derendorf
Professor of Pharmaceutics
I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.
Associate Professor of Pharmaceutics
I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosopl;
Scott Powers
Professor of Exercise and Sport Sciences
This dissertation was submitted to the Graduate Faculty of the College of Pharmacy
and to the Graduate School and was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy. ,
August 2004 ^£>-
Dean, College of Ph¡

UNIVERSITY OF FLORIDA
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