Pharmacokinetic and pharmacodynamic evaluation of mometasone furoate

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Pharmacokinetic and pharmacodynamic evaluation of mometasone furoate
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Thesis (Ph. D.)--University of Florida, 2004.
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Includes bibliographical references.
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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
































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. I 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.








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.















TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ........................................................................................... iv

LIST O F TA B LES ........................................................................................................ x

LIST O F FIG U RES ..................................................................................................... xi

A B ST R A C T ....................................................................................................... .............. xiii

CHAPTER

1 IN TRO D U CTIO N ....................................................................................... ......... 1

A sthm a .......... ..... ........ .... ............................. .......... ........................................ 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
O objectives of the Study ..................................................................................... ... 18

2 STABILITY OF MOMETASONE FUROATE IN AQUEOUS SYSTEMS......... 23

Introduction ................. ................................................................................ 23
Materials and Methods .................................................................................... 23
C hem icals .. .... ... .... .......................................... ................................. ..... 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 D iscussion........................................................................................ 29
Stability of M F in SLF.............................................................................. 29
pH Effects on Stability of MF in SLF...................................... ........... ... 33
MS Analysis of MF and the Degradation Products................................... 34








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
M materials and M ethods......................................................................................... 40
Stability of M F in Plasm a............................................................................ 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
R results and D discussion ......................................................................................... 44
Stability in Plasm a................................................................................. ... 44
Stability in Lung............. ................................... ............................ 47

4 IN VITRO DISSOLUTION PROFILE OF MOMETASONE FUROATE............. 53

Introduction...................................................................................... .... ......... 53
M materials and M ethods ................................................................. ...................... 54
Chem icals.................... ....................................................................... 54
Particle Size Distribution................................................. ................. 54
In Vitro Dissolution................................................... .......................... 54
D ata A nalysis.................................................................... ................. 55
Results and Discussion..................................................................................... 56
Particle Size D istribution........................................... ............................. 56
In Vitro Dissolution...... ..................................................................... 56

5 HEPATIC METABOLISM AND CHARACTERIZATION OF METABOLITES
OF MOMETASONE FUROATE ...................................................................... ... 61

Introduction ................................................................... ................................ 6 1
Materials and Methods .................................................... ...................... 61
C hem icals.................................................................................. ... 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









6 A SENSITIVE LC-MS/MS METHOD FOR THE QUANTIFICATION OF
MOMETASONE FUROATE IN HUMAN PLASMA........................................... 79

Introduction........................................................................................ 79
Materials and Methods .............................................................. ...... 80
Chem icals and Reagents ........................................................................... ... 80
Preparation of Calibration Standards and Quality Control Samples ............. 80
Sam ple Processing ............. ............................................................. .. ..... 81
H PLC-M S-M S Conditions ............................................... .......................... 81
M ethod V alidation.. .. .................................... .......................... ........ 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
FU R O A T E ............................................................................................................... 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
Com partm ental A nalysis............................................ ....... .......... ............. 97

8 SELECTIVITY OF MOMETASONE FUROATE AND OTHER INHALED
CORTICOSTEROIDS TO THE GLUCOCORTICOID RECEPTOR............... 101

Introduction........ .......................................................... ......................... 101
M materials and M methods ............................................................... .......... ........... 102
Chem icals................................................................................ ... 102
Glucocorticoid Receptor (GR) Binding Assay Experiments.................... 102
Progesterone Receptor (PR) Binding Assay Experiments......................... 103
D ata A nalysis...................................................................................... 105
Molecular Dynamic Modeling...................................... ...... 106
R results and D discussion ......................................... ........................................... 106









CO N C LU SIO N S....................................................................................................... 117

APPENDIX NMR ANALYSIS OF MF AND DEGRADATION PRODUCTS ........ 120

LIST O F REFEREN CES ................................................................................................ 124

BIOGRAPHICAL SKETCH .................................................................................... 138














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 Pig 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-1. 'H and '3C 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 13C chemical shifts, Characteristic couplings (Hz), '3C,'H and long-range
correlations (HMBC) of compound D2............................................................... 122














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
produ cts.................................................................. ............................................ 3 1

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 M F. .............................................. ........................ 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








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................................................ 67

5-3. Representative chromatograms for blank S9 fraction of rat liver and S9 fraction of
rat liver incubated with M F. ................................... ....................................... 68

5-4. Representative radiochemical elution profiles of 3H-MF incubated in S9 fraction of
hum an liver at 370C ........................................................................................... 70

5-5. Concentration-time profile of [1,2-3H]-MF and the metabolites formed on incubation
of M F in S9 fraction of human liver................................................. ............. 71

5-6. Radiochemical elution profiles of intestinal contents two hours after intravenous
administration of 5 tCi [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
ch ann els................................................................................................................... 87

7-1. Mean (+ SD) plasma concentration-time profile of MF after oral inhalation of 800 Lg
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 utg dose of M F............................................ ............ ... 97

7-3. Absorption profile of MF after inhalation of a single dose of 800ptg 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 613-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
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.













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.








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








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

secretary 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 p2-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








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-p [21]. Even though both isoforms are transcriptionally

active, only the hGRa isoform is activated by corticosteroids. The hGRP 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








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, I1-13, 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].









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 32-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)
02-adrenergic receptor
Secretory leukocyte inhibitory protein
Clara cell protein (CC10, phospholipase A2 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








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 ofNF-KP and IKB-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








induced by long-term therapy with P2-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








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








glucocorticoid receptor as binding to the other members of the steroid family like the

progesterone receptors might result in undesired side effects.





40 90 % swallow. 9 :: Mouth and Pharynx


10- 60 %Lung ,, clearance
Deposition 4

GI tract Lunt oa Absorption
S~gtoJ^ Gl trct fifi|^ ^^^

V>t: ^ ^


First-p
Inactiv


Orally absorbed I
Fraction Clearance

Systemic
>ass Side Effects
ation


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








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








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, C27H30C1206, 9a,21-dichloro- 11P3,17a-dihydroxy-16a-

methylpregna-1,4-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].


Figure 1-2.








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 gg bid MF showed

significant improvement in the FEVI 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 uig bid, 400 ug once daily in the








morning or 200 lg once daily in the evening for 12 weeks showed significant

improvement in asthma symptoms and lung function [74, 75]. Significant improvements

were seen in FEVI 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 jlg or 800 lag 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 FEVI and PEF, when treated with 100 or 200 pg bid MF or 200 jlg bid of BDP over

12 weeks [77]. MF 200 or 400 pLg bid showed significant improvements in lung function

when compared to BUD 400 lg bid [78]. There was also a significant improvement in

mean 32-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 jLg bid MF and 250 Lg bid fluticasone

propionate in patients with moderate persistent asthma [81].

The recommended starting dose of MF in adolescent and adult patients previously

on 32-adrenergic agonists or inhaled glucocorticoid therapy is 400 Pg once daily

administered by a dry powder inhaler (TwisthalerTM) [82]. Once-daily administration of

MF has been shown to be more effective when administered in the evening than in the








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 lig/day is well tolerated up to one year and the incidence of

adverse events was similar with 100 to 400 gg bid MF, 200 plg bid BDP, 400 Pg BUD or

250 jg 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 (99mTc)-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 ug 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








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 jig/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 60-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 60-hydroxy-MF) while mentioning that other parallel and subsequent

metabolism pathways may be present [86, 87].









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 6-11 1 <1


Fi (%)


25 (CFC) 27 (CFC) 39 22-25 26 (MDI)
70 (HFA) (MDI) (MDI) 38 (TBH)

13 1.6 20 29 12


26 (MDI),
12 (DH)
17 (DSK)
10


<1
(MDI)
(DPI)
1-2


CL (L/h) 230



Vds (L) NA



Vdarea (L) NA


58 37 84,
67 (22S),
117 (22R)


96 103


183,
245 (22S),
425 (22R)


107 339


IV T/ (h) 0.1-0.5 0.6-1.7 1.6 2.0 2.8,
2.7 (22S),
2.7 (22R)
Inh T2 (h) 0.1 1.5-6.5 1.6 3.6 3.0


MRT-IV
(h)
MAT (h)

RRA
(Dexa=100)


2.7 2.2


<1 2.9


1022


0.3-2.6


233 935


69



356



776


5-13


8-14

4.9

5

1800


References [49, 88- [49, 88, [49, 88, [18, 49, [49, 88, 97- [49, 88,
91] 92,93] 94,95] 88,96] 99] 100-102]


4.5


NA

NA

NA

2900


[62, 64]


Abbreviations: BDP- Beclomethasone dipropionate; 17-BMP- Beclomethasone- 7-monopropionate (active
metabolite of BDP); FLU-Flunisolide; TA- Triamcinolone acetonide; BUD- Budesonide; FP- Fluticasone
propionate; MF- Mometasone furoate; Fora,- Oral bioavailability; Fnh- Overall systemic bioavailability after
inhalation; fu- Fraction unbound; CL- Clearance; Vd,,- Volume of distribution at steady state; Vdaea-
Volume of distribution of the terminal phase; IV TV2- Elimination half-life; Inh TV2- 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 Vds, and CL
after intravenous administration; 22S and 22R Epimers of Budesonide.








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 lIg or 800 lg 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 lig 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 jlg bid and

800 ig 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








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

(Finh 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








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 [ig), 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








pharmacokinetics of MF in patients with asthma after oral inhalation of a single dose of

800 utg 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 tg 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 ofMF 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: Dl 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).








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), NaC1 (103 mM), KCI

(4 mM), Na2HP04.7H20 (1 mM), Na2SO4 (0.5 mM), CaCl2.2H20 (2.5mM),

NaH3C202.3H20 (7 mM), NaHCO3 (31 mM) and Na3H3C607.2H20 (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 370C 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. 500p1L 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 500[L 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 kmax 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 ug/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 ofvariation).The good








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 Dl 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 SCIENTISTT 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:

0.693
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 K2HP04 buffer at three different pH values (pH 7.0, 7.5 and 8.0). A fifty times

higher buffer strength (compared to SLF) of K2HP04 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 370C. MF at a concentration of 2.5 Pg/mL (4.8 JiM) was

incubated at 370C 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








diluted with 200 pL of mobile phase (63:37 MeOH-H20). 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

jig/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-gtm 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








temperature was set to 1200C and the ES probe temperature was 4000C. 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 CDC13 at room temperature using Shigemi

sample tubes (180 [l) 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 iM)

were prepared in methanol. Dilutions for degradation product Dl and dexamethasone

(0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1.0 gM) and degradation product D2 (0.001, 0.003,

0.01, 0.03, 0.1, 0.3, 1.0, 0.3 gM) 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)








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 iL 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 piL 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 40C for 24 h. After the incubation,

200 uL 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 piL 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 (40C), was demonstrated by HPLC after extraction of the

incubation mixture by solid phase extraction.

The data obtained were fitted by SCIENTISTTM (Micromath, Salt Lake City, UT)

using the following Emax model to obtain the estimates of Bmax and ICso.








CN
DPM = Bm -B -, + NS where DPM represents the total tracer
IC50 +CN

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 (IC50,dex) was used to calculate the relative

binding affinity (RBAtest) of the test compound form its IC50 value (ICs5,test) as:

IC50dex
RBA test 50,dex x100
IC 50,test

The stability of MF and its degradation products in the rat lung cytosol at 40C 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 tg/ mL) in SLF at 370C and subsequent HPLC analysis of the incubation mixture

suggests the formation of two major degradation products, named Dl and D2 (Figure 2-

2). The concentration-time profiles ofMF, Dl and D2 (Figure 2-3) indicate that more

than 90% of MF undergoes conversion to either Dl and or D2 at the end of 4 h of

incubation with Dl being formed faster than D2. After 12 h, the majority of the starting

material entered the D2 pool with Dl being undetectable; therefore one can conclude that

Dl seems to be converted into D2. Consequently, it appears that MF is first converted








into D1, 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 (kl = 0.52

h-') for the conversion of MF to D1 was found to be nearly 4 times higher than the

degradation rate constant (k2 = 0.143 h-') for the conversion of D1 to D2. The

degradation half-life for MF was found to be 1.3 h and the half-life of D 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 370C, 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









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.



NulohlcatcI i Bs,-


Figure 2-1. A schematic degradation pathway for conversion of MF into its degradation
products Dl and D2 on incubation of MF (Co = 2.5 ig mL-1) in SLF at 370C
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,
D1, 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.


- HCI


. -HCI


Nucleophilic attack


B P Base, -H












(A) (B)









D2


D1
MF I








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



































Figure 2-3. Graphical representation showing nonlinear curve fitting of the concentration-
time data of MF and its degradation products Dl and D2 generated after
incubation of MF in SLF at 370C 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-') in

SLF was higher than the degradation rate constant observed in 50 mM K2HPO4 buffer at


100

MF
S 80 T A D1
.. U D 2



40 l i
E
20 P


0 15 30 45 60 75
Time (hr)










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 tg mL-') in phosphate buffer at
pH 7.0, 7.5 and 8.0 at 370C.




MS Analysis of MF and the Degradation Products

Based on the ESI+ mass spectra (Table 2-1), the molecular formulae ofDl and D2

agree with the general structures C27H29C106 and C27H27CO05 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 HCl for Dl when compared with MF.

Similarly, D2 differed from Dl by a loss of H20. The presence of only one chlorine

atom in Dl and D2 was confirmed by the relative abundance of 37C1 isotope (Table 2) in


2.5
u-


L 2.0
uD


E
I 1.5

0
-J


0 2 4 6
Time (hr)


8 10 12








Dl 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 C16-

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
products.
Compound MF D1 D2
Molecular formula C27H30C1206 C27H29C106 C27H27C1O5
[MH]+ 521 (100) 485 (15)' 467 (60)a
[MH]+ (37C1 isotope) 523 (70) 487 (5) 469 (20)
[M+Na]f 543 (30)$ 507 (100)$ 489 (35)$
[MH-H20] 503 (10) -
[MH-HCI]+ 485 (15) -
Others 441 (5) 413 (5) 413 (10)
413 (15)
Listed are m/z. % relative abundance to nearest 5% is given in parenthesis.
SCorresponding 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.








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 C17 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 D1. Based on

this hypothesis, the structure of D2 and the MS analysis of Dl, D1 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 D to the rat glucocorticoid receptor

was 4 times higher than dexamethasone. The IC50 value of the degradation product D2








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 IC50 (pM) RBA
Dexamethasone 0.046 0.01 100

Mometasone Furoate 0.0016 0.0001 2938

D1 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-17oa position [117, 118].

This may explain the strong binding of MF and D1, which have a furoate moiety at the

C17a 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, D1 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 D1 were close to 1. The RBA value for MF in









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 1 I3-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].







120
SDEX (obs)
DEX (pred)
100 L T m MF(obs)
S- MF (pred)
o A D1 (obs)
.- 80- D \ (pred)
2 v\y D2 (obs)
C D2 (prod)
0 8 60

S 40

20

0
10-2 10-1 100 101 102 103 104 105
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
tM (n=2). The estimated parameters for D2 were then used to predict bound
tracer at competitor concentrations higher than 0.3 tM. The hill coefficient
was fixed to 1.








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 ofMF (furoate group in C17, 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%) ofmometasone 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 interference present in the tissues.

Materials and Methods

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). All other chemicals and solvents were obtained from

Sigma Chemicals Co. (St Louis, MO) and Fisher Scientific Co. (Cincinnati, OH).








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 CYPlAI

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 370C for 10 min

and then was spiked with MF (from a stock of 100 pg/mL) at a concentration of 3 tg/mL

(5.75 tM). Experiments were conducted in triplicates. 200 iL aliquots were removed at

regular intervals up to 72 h into pre-chilled tubes and the samples were precipitated with

600 gL 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 -200C and were analyzed by HPLC within 48 h of storage.

The precipitated plasma samples were centrifuged at 10000 rpm for 5 min and 100

tL 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 Amax 254 nm, a CR-3A Chromatopac integrator (Shimadzu

Corporation, Japan) and an automatic injector (Perkin Elmer, Boston, MA) was used as








the HPLC system. Chromatographic separation of the analytes was achieved on Waters

5-gm 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 ug/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 ug/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 interference 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% KCI in 50 mM K2HP04 pH 7.4)

870 mg of dipotassium hydrogen orthophosphate (K2HPO4) and 1.15 g of

potassium chloride (KCI) 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 40C 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








mM) was prepared in the homogenizing buffer. This cofactor solution was incubated at

370C for 15 min to allow generation of NADPH 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 10000x 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 lg/mL (4.8tM) and final lung tissue concentration was 10% w/v. The lung S9

fractions were incubated at 370C for up to 24 h under atmospheric air. Drug free S9

fraction of lung was used in a control experiment. Serial samples of 250tL 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 250IL of acetonitrile and analyzed by

HPLC. The precipitated samples of lung homogenates were centrifuged at 10000 rpm for

5 min. 100 [tL 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.








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 tM). 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 370C for 72 h led to the formation of three degradation








products Dl, 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 Dl, D2 and D3 occurred

after 20 h of incubation (Figure 3-2). The decline of MF and the formation of D1, 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

















0 Min 25 0
0Min 25 0


*1 f


D2D3

-. 0A


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 [tg/mL) until 48 h at 370C.


MF was also found to be stable in human plasma until 6 h of incubation at 370C.

However, prolonged incubation of MF in human plasma for 72 h led to the formation of

identical degradation products (D1, 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








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.


Figure 3-2. Concentration-time profile of MF and its degradation products following
incubation of MF (Co = 6.2 tM) at 370C in (a) rat plasma and (b) human plasma for 72 h
(n=3).








Using a mass spectrometry (positive electro-spray ionization) analysis method

reported in the previous chapter, the molecular masses ([MH+]) of MF, D1, 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.
























Figure 3-3. Representative chromatograms for (a) blank S9 fraction of rat lung and (b) S9
fraction of rat lung incubated with MF (Co = 2.5utg mL'') 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 (Dl, 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 Dl and D2 observed in this study are identical to

the products reported earlier.



















































Figure 3-4. Representative radiochemical elution profiles of [1,2-3H]-MF incubated with
S9 fraction of rat lung at 370C (A) at time zero, (B) after 2 h at 370C and (C)
after 24 h.








The structures of Dl, D2 and D3 in plasma along with the proposed pathways of

degradation are provided in Figure 3-5. The conversion of Dl 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. Dl 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 Dl and D2 and the

molecular mass ofD3. Based on the similarities in structure (condensed five-member

ring at position C17) between D2 and D3, it is expected that D3 would not show

significant binding to the glucocorticoid receptor either.










Nucleophilic r_ .


Figure 3-5. A schematic degradation pathway for conversion of MF into its degradation
products D1, D2 and D3 on incubation of MF (Co = 6.2 pM) in plasma at
370C for 72 h. Structures A, B, D and E represent MF, D1, 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


/H20


"*..... -HCI
''' '






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 FUROATE

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 (370C) 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).








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 ofpH 7.4 phosphate-buffered saline (50 mM) at 370C. 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 gg/mL) was used as the internal standard. A mobile phase

composition ofmethanol-water (60:40, v/v) was used at a flow rate of 1 mL/min in the








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 SCIENTISTTM (Micromath, Salt Lake City,

UT) program and using the monoexponential formula:

Cumulative % Dissolved = 100 (A*expat)

where a is the dissolution rate constant. Half-lives of dissolution (T50%) 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"a )+(B*exp-t)),

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 E(Rt-Tt)2]05 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].








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 g.m. This is

in accordance with the particle size distribution of dry powder formulations of inhaled

corticosteroids, as particles with diameters 1-5 ptm 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 m

will be exhaled out.



Table 4-1. Particle size distribution (in %) of MF and BUD micronized dry powders.
<1 CPm 1-5 Cpm 5-10 gm >10 tm
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 tg/ml) at 30 minutes. The dissolution half-life (T50%) for

MF was calculated to be 4.20.3 min (n=6) and T50% 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].








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 T50% for MF for the initial phase of dissolution was 3.50.2 min

while the T50% 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 T50% (1.90.3

min) of BUD compared to using 2% SDS. The dissolution profiles for MF and BUD

were not similar with an f2 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.















120


100 -


7 80

:5 MF
60 BUD


40
o 40
E

20


0
0 20 40 60

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 370C (n=6).








































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


120


100


880


S60- MF
0 BUD

40


20

0
0 20 40 60
Time (h)






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). [1 ,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,








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 CYP1Al 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 g.M) 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 uiCi 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 rejected and homogenized without

being rinsed. The contents of the stomach and the intestines (both small and large) were








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 gM) and incubated at 370C in a shaker bath for 2 h. The 2 h

samples were analyzed by HPLC-UV analysis and the metabolites (METI-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








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 jL of the dilutions of the test compound in methanol was added to pre-chilled tubes.

10 uL of methanol instead of the test compound was used for the determination of total

binding. Non-specific binding was determined after addition of 10 tL 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 [iL 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 SCIENTISTTM (Micromath, Salt Lake City, UT)

using the following Emax model to obtain the estimates of Bmx and IC5o.

CN
DPM= Bmx -Bm. + NS
ICo50 +CN








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,test) as:


RBA.tes I 50,dex 100
IC50,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 MET1, 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, MET1 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.

















* Rat Liver 0 hr
MBuffer0 hr


25000

20000

S15000

10000


4-I MF


5000


0 Collection time interval (m

Collection time interval (min)


M Rat Liver 20 min
SBuffer 20 min


MET2

SMET3

I


MET4 MET
MET5


o cM C D I '? T T C V
0M 0 r- (M C') i 10 CD C OD CD

Collection time interval (min)




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 370C (bars in black and gray represent MF in S9
fraction of rat liver and homogenizing buffer respectively).


30000


7500


6000


4500


3000


1500


0













A

100
'H-MF
80
u-



2











35
Io



















SH-MET2
S 40


20H-M

















0 -------H-MET-
0
0 10 20 30 40 50 60
Time (min)


B
35

30


LL- 'H-MET2
c 20 3 'H-MET4
U 'H-MET5
0-
n 15
C5

10
,__..-.-----A- ^---)---
/ --A"" 3


0 10 20 30 40 50 60
Time (min)


Figure 5-2. Concentration-time profile of [1,2-3H]-MF following incubation of [1,2-3H]-
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
370C 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








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 interference at the same retention time. MET3, MET4 and

MET5 could not be quantified using UV due to the very low concentrations of these

metabolites.


. Representative chromatograms for (a) blank S9 fraction of rat live
fraction of rat liver incubated with MF (Co = 2.5pg/mL) until 0.25
fraction of rat liver incubated with MF until 1 h.









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-' 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.












30000

25000


20000

15000


10000

5000

0
0 ------
0 C\5


12000.0

10000.0

8000.0

6000.0


* Human liver 0 hr

E Buffer 0 hr


..........--...........---.--- |....Ib.
c^) t (o T- o... T ............
0 0 <,J IO (O -- Co 0C


Collection time interval (min)


4000.0 i. '.IJ MET4 MET5

2000.0 I |4

0 .0 ... ._ r I _I '
0.0 ,

SY- ? Collection time interval (m C in)
Collection time interval (min)


Figure 5-4. Representative radiochemical elution profiles of 3H-MF incubated in S9
fraction of human liver at 370C (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).


-IMF



























0 10 20 30 40
Time (min)


50 60 70


0 10 20 30 40 50 60
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 370C
for 1 hr.








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.

MET1, the most polar metabolite, was the major metabolite in rat liver fractions where as

both MET1 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 63-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. MET1 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 MET1, however, is consistent with the

bioavailability study by the same group, who reported that majority of the radioactivity








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. MET1 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 MET1, 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








contents was associated with MET1 (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 0.090.02
Spleen 0.240.16
Lung 0.32+0.01
Kidney 0.320.01
Stomach 1.31+0.39
Liver 2.011.36
Large intestine 2.471.98
Small intestine 4.550.11
Intestinal contents 81.574.66
Muscle 0.20+0.05a
Fat 0.390.17a
Skin 0.050.02b
Plasma 0.260.08c
Urine 0.08+0.02c
a % of dose/gram tissue
b % of dose/cm2
c % of dose/ml














25000
MET1


20000



15000
E
"-o
10000 -

MET2 MET3
5000 -




FJ,? I 0 C1M cI L n CO r- W 0. .
] i i- i -

Collection time interval (min)



Figure 5-6. Radiochemical elution profiles of intestinal contents two hours after
intravenous administration of 5 iCi [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


I








glucocorticoid receptor. The data shows that MF is 29 times more potent than

dexamethasone. The RBA of MFfra was 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
MET1 <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










could not be determined, as they did not show any binding to the glucocorticoid receptors

in the concentration range studied.








120

2 100
DEXA
MF
S80 MFfrac
S\ MET2
Z;o MET3
C
0 60

40
I-

20


0.01 0.1 1 10 100 1000
Concentration (nM)



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 (P<0.05) [63]. In comparison, a 880 ug 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, 6p-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








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). '3C3-Fluticasone

propionate (13C3-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 LC18 (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 -200C. 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 lg of '3C3-FP in 1 ml of methanol, and working solutions of 20 ng/ml

were prepared by diluting the stock solution with methanol.








Sample Processing

Plasma samples were thawed at room temperature and 50 il 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 C18 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 p.1 of a mixture of methanol-

water (85:15, v/v) and a sample volume of 80 gl 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-jim column (50mm x 4.6 mm i.d., Milford,

MA, USA) preceded by a Whatman 5-p.m ODS C18 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 1200C and 5000C respectively. Argon was used

as the collision gas and the mass resolution was set to unit mass. The corona voltage for








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 gl/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 mlF' 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].








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

-800C 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 (APCI) 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 APCI" under multiple reaction

monitoring mode are shown in Figure 6-1. The best sensitivity was observed with the








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 HCI molecule

from the structure of MF in the APCI source. The transition for 13C3-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 interference in the MF

transition were observed with '3C3-FP and hence this compound was used as the internal

standard.












TUNE-MF-3-5-01-5 8 (0.161)
100-1


408-410 432433 44744?449 463469 476
... h ............ .I .h I I. .


Scan AP-
520 3.44e5


486
/

487
488 50 507


523
524

1-525


410 420 430 440 450 460 470 480 490 500 510 520 530


TUNE-MF-3-5-01-7 1 (0.042)
100-


536 552 555 5679 585"7 597
/ Afi III l in i iiif i| iiiirlii i / m/z
540 550 560 570 580 590 600


Daughters of 484AP-
4848.50e4


389




307 31324 333339345 360 1 371 374 387 390


404 430
.-4 /6 4 25\


448

434 447 46 ,470
S\ 4 / 466 469470


300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480


Figure 6-1. Full scan (top panel) and daughter scan (bottom panel) spectra of MF.


0.0 0.1 0.2 0.3 0.4 0:5


0.7 0.


Y Tmm~mrrr lr~r~~n ~ ~ --""""- """" -C


I


ill


II-LYI


-i_


,,,mt


i1 ....


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. Aids trans: None
0.1251 X


Figure 6-2. Representative calibration curve of MF in human plasma.










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 3C3-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 81.55.4, 74.53.9 and 73.75.8 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].




04-21-04-03 Sm (Mn. 1x2) MRM of 3 Channels AP-
1 503.27 > 380
1717
Area




04-21-04-03 Sm (Mn, 1x2) MRM of 3 Channels AP-
484 > 389.21
10 506
Area



----- Time
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75

Figure 6-3. Chromatograms of blank human plasma for the I.S (3C3-FP, top panel) and
MF (bottom panel) channels.