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In Vitro Interaction of Corticosteroids with P-Glycoprotein and Cytokines

Permanent Link: http://ufdc.ufl.edu/UFE0024063/00001

Material Information

Title: In Vitro Interaction of Corticosteroids with P-Glycoprotein and Cytokines
Physical Description: 1 online resource (52 p.)
Language: english
Creator: Wang, Nasha
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: budesonide, calu, corticosteroids, cytokines, fluticasone, mometasone, pglycoprotein
Pharmacy -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, M.S.P.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Asthma is a chronic lung disease which can inflame and narrow the airways. Current trends indicate that the prevalence rates for asthma have been increased more than double from 1980 to 2003. Inhaled corticosteroids are the most potent anti-inflammatory agents and offer advantages of fast, effective control in asthma therapy. P-glycoprotein (Pgp) functions as a transmembrane drug transport mechanism that can actively pump out many currently prescribed medicals. In vitro transport studies were conducted to understand how Pgp may affect corticosteroids budesonide (BUD) and fluticasone propionate (FP) transportation in Calu-3 cell model. The results showed that BUD and FP are substrates of Pgp and inhibition of Pgp increased and decreased the concentrations of BUD and FP in acceptor chamber on apical to basolateral direction and basolateral to apical direction respectively. Cytokines are reported as substrates of Pgp. To understand if corticosteroids BUD, FP, and mometasone furoate (MF) could affect cytokine release by Pgp, in vitro cytokine release studies were preformed with interleukine (IL)-4, IL-5, and IL-13 in peripheral blood mononuclear cell (PBMC) model. The results showed that there is competition between corticosteroids and cytokines on transport by Pgp and different concentrations of BUD, FP and MF could significantly reduced three cytokines release out of PBMC cells.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Nasha Wang.
Thesis: Thesis (M.S.P.)--University of Florida, 2008.
Local: Adviser: Hochhaus, Guenther.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0024063:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024063/00001

Material Information

Title: In Vitro Interaction of Corticosteroids with P-Glycoprotein and Cytokines
Physical Description: 1 online resource (52 p.)
Language: english
Creator: Wang, Nasha
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: budesonide, calu, corticosteroids, cytokines, fluticasone, mometasone, pglycoprotein
Pharmacy -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, M.S.P.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Asthma is a chronic lung disease which can inflame and narrow the airways. Current trends indicate that the prevalence rates for asthma have been increased more than double from 1980 to 2003. Inhaled corticosteroids are the most potent anti-inflammatory agents and offer advantages of fast, effective control in asthma therapy. P-glycoprotein (Pgp) functions as a transmembrane drug transport mechanism that can actively pump out many currently prescribed medicals. In vitro transport studies were conducted to understand how Pgp may affect corticosteroids budesonide (BUD) and fluticasone propionate (FP) transportation in Calu-3 cell model. The results showed that BUD and FP are substrates of Pgp and inhibition of Pgp increased and decreased the concentrations of BUD and FP in acceptor chamber on apical to basolateral direction and basolateral to apical direction respectively. Cytokines are reported as substrates of Pgp. To understand if corticosteroids BUD, FP, and mometasone furoate (MF) could affect cytokine release by Pgp, in vitro cytokine release studies were preformed with interleukine (IL)-4, IL-5, and IL-13 in peripheral blood mononuclear cell (PBMC) model. The results showed that there is competition between corticosteroids and cytokines on transport by Pgp and different concentrations of BUD, FP and MF could significantly reduced three cytokines release out of PBMC cells.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Nasha Wang.
Thesis: Thesis (M.S.P.)--University of Florida, 2008.
Local: Adviser: Hochhaus, Guenther.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0024063:00001


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1 IN VITRO INTERACTION OF CORTICOS TEROIDS WITH P-GLYCOPROTEIN AND CYTOKINES By NASHA WANG A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

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2 2008 Nasha Wang

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3 To my parents, husband and daughter

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4 ACKNOWLEDGMENTS I wish to express m y gratitude to my superv isor, Dr. Guenther Hochhaus, for giving me the opportunity to work with him. I thank him for his guidance, support and encouragement. I thank Dr. Hartmut Derendorf and Dr. Jeffr ey Hughes for serving on my supervisory committee. I thank Dr. Shihong Song and Dr. Veroni ka Butterweck for allowing me to use their laboratory facilities. I take this opportunity to express my gratitude to Yufei Tang for her invaluable technical assistance. I would also like to thank my group members (Kai, Elanor, Keerti, Navin, Gina, Wan, and Buja) and other graduate students and post-docs in the department for their valuable support and friendship. Finally, I would like to thank my parents for th eir support and encouragement. None of this work would have been possible without them. I also express my heartfelt gratitude to my husband and daughter, Yi and Eileen, for thei r support, understanding, and encouragement.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF TABLES ...........................................................................................................................7LIST OF FIGURES .........................................................................................................................8LIST OF ABBREVIATIONS ......................................................................................................... .9ABSTRACT ...................................................................................................................... .............11CHAPTER 1 INTRODUCTION ................................................................................................................ ..12Asthma ........................................................................................................................ ............12Mechanism of Action of Corticosteroids in Asthma ..............................................................13Molecular Mechanism .....................................................................................................13Cellular Effects ................................................................................................................13Effects on Inflammation ..................................................................................................14Effects on Airway Hyperresponsiveness .........................................................................14Function of P-glycoprot ein on Cell Membrane ......................................................................14Function of P-glycoprotein in Human Body ..........................................................................15Cytokines and Their Roles in Human Body and Asthma .......................................................16Cytokines ..................................................................................................................... ....16Functions of Cytokines in Human Body .........................................................................16Cytokines in Asthma .......................................................................................................17Hypothesis and Objectives of Study .......................................................................................18Specific Aim 1 .................................................................................................................18Specific Aim 2 .................................................................................................................182 IN VITRO CORTICOSTEROI DS TRANSPORT STUDY...................................................23Introduction .................................................................................................................. ...........23Materials and Methods ...........................................................................................................24Calu-3 Cell Culture Method ............................................................................................24Transport Experiment Method ........................................................................................25Data Analysis ...................................................................................................................26Statistical Analysis .......................................................................................................... 27Results and Discussion ........................................................................................................ ...27Conclusion .................................................................................................................... ..........27

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6 3 IN VITRO EFFECT OF CORTIC OSTEROIDS ON CYTOKI E RELEASE STUDY ..........35Introduction .................................................................................................................. ...........35Materials and Methods ...........................................................................................................35PBMC Separation and Stimulation Method ....................................................................35Cytokine Release Experiment .........................................................................................36Statistical Analysis .......................................................................................................... 36Results and Discussion ........................................................................................................ ...36Conclusion .................................................................................................................... ..........374 CONCLUSIONS ................................................................................................................. ...48LIST OF REFERENCES ...............................................................................................................49BIOGRAPHICAL SKETCH .........................................................................................................52

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7 LIST OF TABLES Table page 2-1 Results of BUD transport study in Calu-3 cell model....................................................... 312-2 Results of FP transport study in Calu-3 cell model........................................................... 333-1 Results of IL-4, IL-5 and IL-13 release studies after treatments with BUD, FP, and MF at concentration 0.01M, 0.1M, and 1M................................................................47

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8 LIST OF FIGURES Figure page 1-1 Normal versus asthmatic bronchiole in huma n lung.......................................................... 19 1-2 Structures of inhaled corticos teroids and 2-adrenergic agonist........................................ 20 1-3 Mechanism of P-glycoprotein (Pgp) as a transmembrane drug efflux pump .................... 21 1-4 A central role for inflammatory Th2 cells in asthma......................................................... 22 2-1 A 6-well Transwell plate used in transport study............................................................ 29 2-2 Representation of Calu-3 m onolayer in polarized Transwell system.............................. 30 2-3 Percentage of BUD appear ed in the acceptor co mpartment at different time points......... 32 2-4 Percentage of FP appeared in the acceptor comp artment at different time points............. 34 3-1 Box plots of IL-4 release results after 24 h treatment with BUD...................................... 38 3-2 Box plots of IL-4 release results after 24 h treatment with FP.......................................... 39 3-3 Box plots of IL-4 release re sults after 24 h treatment with MF ........................................ 40 3-4 Box plots of IL-5 release results after 24 h treatment with BUD...................................... 41 3-5 Box plots of IL-5 release results after 24 h treatment with FP.......................................... 42 3-6 Box plots of IL-5 release results after 24 h treatment with MF......................................... 43 3-7 Box plots of IL-13 release resu lts after 24 h treatment with BUD .................................... 44 3-8 Box plots of IL-13 release resu lts after 24 h treatment with FP ........................................ 45 3-9 Box plots of IL-13 release resu lts after 24 h treatment with MF ....................................... 46

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9 LIST OF ABBREVIATIONS ABC Adenosine triphosphate binding cassette ATP Adenosine triphosphate BUD Budesonide Da Dalton DMEM Dulbeccos modified eagles m edium EGF Epidermal growth factor ELISA Enzyme-linked immunosorbent assay FCS Fetal calf serum FGF Fibroblast growth factor FP Fluticasone propionate GR Glucocorticord receptor GRE Glucocorticoid response element HBSS Hanks balanced salt solution HEPES N-2-hydroxyethyl-piper azine-N-2-ehtanesulfonic ICAM Intercellular adhesion molecule IgE Immunoglobulin E IGF Insulin-like growth factor IL Interleukin INF Interferon MCP Monocyte chemoattractant protein MDR Multidrug resistance MF Mometasone furoate MRP Multidrug resistance related protein MXR/BCRP Mitoxantrone resistance/br est cancer resistance protein

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10 Papp Coefficient of permeability PBMC Peripheral blood mononuclear cell PBS Phosphate buffered saline PHA Phytohaemagglutinin PDR Permeability directional ratio PN Passage number TEER Transepithelial electrical resistance TGF Transforming growth factor TNF Tumor necrosis factor VLA Very late antigen

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11 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science IN VITRO INTERACTION OF CORTICOS TEROIDS WITH P-GLYCOPROTEIN AND CYTOKINES By Nasha Wang December 2008 Chair: Guenther Hochhaus Major: Pharmaceutical Sciences Asthma is a chronic lung disease which can inflame and narrow the airways. Current trends indicate that the preval ence rates for asthma have been increased more than double from 1980 to 2003. Inhaled corticosteroids are the most potent anti-inflammatory agents and offer advantages of fast, effective control in asthma therapy. P-glycoprotein (Pgp) functions as a transmembrane drug transport mechanism that can actively pump out many cu rrently prescribed medicals. In v itro transport studies were conducted to understand how Pgp may affect corticostero ids budesonide (BUD) and fluticasone propionate (FP) transportation in Calu-3 cell model. The results showed that BUD and FP are substrates of Pgp and inhibition of Pgp increased and decreased the concentrations of BUD and FP in acceptor chamber on apical to basolateral direction and basolateral to apical direction respectively. Cytokines are reported as substrates of Pgp. To understand if corticosteroids BUD, FP, and mometasone furoate (MF) could affect cytokine re lease by Pgp, in vitro cytokine release studies were preformed with interleukine (IL)-4, IL5, and IL-13 in peripheral blood mononuclear cell (PBMC) model. The results showed that ther e is competition between corticosteroids and cytokines on transport by Pgp and different concentrations of BUD, FP and MF could significantly reduced three cytoki nes release out of PBMC cells.

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12 CHAPTER 1 INTRODUCTION Asthma The word asthma originates from an anci ent Greek word meaning pain. When a person inhales, the air travels thr ough lung and flows through progressively narrower airways called bronchioles. Asthma is a chronic, inflammatory disease that causes swelling and narrowing of the bronchioles or airways. Figure 1-1 shows th e normal bronchiole and asthmatic bronchiole in human lung. The narrowing of the airways is usually either totally or part ially reversible with asthma therapy. The symptoms of asthma range from minor wheezing, difficulty breathing to life-threatening asthma attacks. Most asthma patients have wheezing attacks separated by symptom-free periods. Some patients have long-term shortness of breath with episodes of increased shortness of breath. Some patients may have a cough as the main symptom. Asthma attacks can last minutes to days and can beco me dangerous if the airflow becomes severely restricted (1). Asthma affects about 300 million people worldwide, a total that is expected to rise to about 400 million over the next 15-20 years (2). In sens itive individuals, asthma symptoms can be brought by a variety of triggers, such as, allergen s (pollen, mode or dust mites), stress, weather change, or foods. Because asthma is a chronic di sease, it requires continuous management and appropriate treatment. Treatment of asthma involves avoiding know n triggers and take one or more asthma medications. Asthma therapy varies from different patients. Most patients with persistent asthma take a combination of l ong-term control medications and quick-relief medications. Long-term control medications are us ed on a regular basis to prevent attacks and quick-relief medications are used to relieve symptoms during an attack. Inhaled corticosteroids are the most effective anti-inflammatory ag ents for chronic persistent asthma (3). 2-adrenergic

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13 agonists are also used in asthma therapy as br onchodilators, which open up the bronchial tubes so that more air can move through. Figure 1-2 show s the structures of inhaled corticosteroids and 2-adrenergic agonists. Most pati ents with asthma respond well to current treatments; however, 5-10% of patients have severe disease that of ten fails to respond to conventional therapy. Mechanism of Action of Corticosteroids in Asthma Initial approach to trea t asthma emphasi zes the relief of bronchoconstriction with bronchodilators, particularly 2-adrenergic agonists, but the disc overy of airway inflammation as an important pathophysiological component of asthma has led to the use of inhaled corticosteroids as the mainstay of asthma therap y (4). All levels of pe rsistent asthma require daily inhaled corticosteroids as anti-inflammatory treatment, becau se they are the safest, most effective treatment for persistent asthma. The ex act mechanism of action of corticosteroids in airway anti-inflammation is not fully known. However the effect is believed to occur through some different pathways (5). Molecular Mechanism Inhaled corticosteroids are hi ghly lipophilic. They can rapi dly diffuse across airway cell me mbranes and then bind to glucocorticoid receptor (GR) in the cytoplasm. The GRcorticosteroid complexes then move quickly into the nucleus. In the nu leus, GR-corticosteroid complexes bind to DNA at specific sequences in the promoter region of corticosteroidresponsive genes known as glucocorticoid response element (GRE), leading to changes in gene transcription (6). These changes include both anti-inflammatory gene activation and inflammatory gene suppression. Cellular Effects Corticosteroids ma y have direct inhibitory effects on many of the cells involved in airway inflammation in asthma, including macrophage s, T-lymphocytes, eosinophils, and airway

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14 epithelial cells (7). In addition to their suppressive effects on inflammatory cells, corticosteroids may also inhibit plasma exudation (8) and mucus secretion (9) in inflammatory airways (4). Effects on Inflammation In patients treated with inhale d corticosteroids for one to th ree months there is a m arked reduction in the numbers of mast cells, macr ophages, T-lymphocytes and eosinophils in the bronchial epithelium and submucosa (10-13). Furt hermore, corticosteroids reverse the shedding of epithelial cells and the gobletcell hyperplasia characteristically seen in biopsy specimens of bronchial epithelium from patients with asthma (4, 10). Effects on Airway Hyperresponsiveness By reducing airway inflammation, inhaled co rticosteroids consiste ntly lessen airway hyperresonsiveness (14). Long-term treatm ent wi th inhaled corticosteroids reduces airway responsiveness to histamine, cholinergic agonists, and allergens. Such treatment similarly lowers responsiveness to exercise, fog, cold air, bradykinin, adenosine, and irri tants such as sulfur dioxide and metabisulfites. The reduction in airway hyperresponsiveness may not be maximal until treatment has been given for severa l months. Although the treatment suppresses inflammation, it may be unable to reverse the pers istent structural changes that underlie the disease (4). Function of P-glycoprotein on Cell Membrane P-glycoprotein (Permeability-glycoprotein, P gp) is N-glycosylated 170 kilodalton (kDa) protein of about 1280 amino acids. Pgp belongs to the subfam ily B of the adenosine triphosphate (ATP) binding cassette (ABC) superf amily of transporter proteins and has an important role in multidrug resistance (MDR). ABC transporters are ubiquitous with over 300 family members identified in all known organisms from bacteria to mammals. They are involved in transport of a great variety of substrates including sugars, am ino acids, steroids, cholesterol, phospholipids,

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15 peptides, proteins, toxins, antibiotics and xenobio tics (15). In addition, some of corticosteroids, such as flunisolide, dexomethasone, methotrexate (16, 17) have been shown as substrates of Pgp. Besides Pgp, other members include multidr ug resistance related protein (MRP1-MRP7), mitoxantrone resistance/brest cancer resistance protein (MXR/ BCRP) and the cystic fibrosis transmembrane regulator (18). Many members of ABC superfamily are involved in MDR, which has become a major impediment to the success of cancer chemotherapy. The 170-kDa Pgp has an overall molecular ar chitecture similar to that of all ABC transporters. The Pgp molecule is composed of two halves, each consisting of transmembrane helices and the cytoplasmic ATP-binding domain. Th e two halves of Pgp are joined by a single polypeptide chain (19). The compounds transported by Pgp have diverse chem ical structures and it is difficult to define common properties of a typical substrate. The only features common to Pgp substrates appear to be that they are all hydrophobic a nd contain spatially separated hydrophilic and hydrophobic moieties, with a molecular mass of 300-2000 Da (20,21). Pgp substrates include many medications, such as steroids, antibiotics, anticancers, human immunodeficiency virus (HIV) proteases inhibito rs, opioids, cardiovasculars. (22). There are different models proposing the mechanism of tr ansportation by Pgp. All m odels agree that Pgp uses ATP hydrolysis energy to transport its substrates outside the cell. But the precise mechanism of transportation is still not well unde rstood. Figure 1-3 shows P-glycoprotein as a transmembrane drug efflux pump, which recognizes its substrates in cytoplasm or inside the membrane and transport them into the extracellu lar medium using the energy of ATP hydrolysis. Function of P-glycoprotein in Human Body In human body, Pgp is highly expressed in intes tine, adrenal, pregnant uterus and placenta. Significant levels of Pgp are further found in brai n, spinal cord, liver, kidney, heart, testes, lung and spleen. Also Pgp is highly ex pressed at blood-tissue barriers and the brain and spinal cord.

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16 The characterizations, together with distributi on of Pgp, suggest that the function of Pgp in human body is to prevent drug en tering into the central nervou s system, limit absorption and distribution of exogenous toxins and increase excretion of thes e xenobiotics or metabolites. Pgp was also suggested to actively translocate chol esterol to the outer cell membrane. Therefore, exist of Pgp in human body can protect the body from harmful substances and affect pharmacokinetics (i.e., absorption, distribution, meta bolism, and excretion of corticosteroids in asthma therapy). Cytokines and Their Roles in Human Body and Asthma Cytokines Cytokines are ma ny kinds of small peptides re leased from inflammatory tissue, connective tissue and immune system cells; they medi ate and regulate immunity, inflammation, and hematopoiesis, i.e. the formation of blood cells, in the body. Cytokines used to have different names depending either on their origin, such as lymphokines (produced by lymphocytes), monokines (produced by monocytes) or on thei r activity: chemokines, interleukins (IL), interferon (INF), monocyte chemoa ttractant protein (MCP), transforming growth factor (TGF), tumor necrosis factor (TNF), epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF). The same cytokine can be secret ed by different kinds of cells, hand even that the same cytokine can present different functions. Functions of Cytokines in Human Body Cytokines usually have an e ffect on closely adjacent cells (paracrine) predominantly, although they m ay also act at a distance (endocrine) or may have effects on the cell of origin (autocrine). Cytokines bind the target cell on a specific membra ne receptor (cytokine receptor) and then cascades of intracellular signaling alter cell functions. Th e effect of a certain cytokine on a given cell depends on the ki nd of cytokine, its extracellular abundance, the presence and

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17 abundance of the complementary receptor on the cell membrane. Generally, cytokines are involved in cell-to-cell interactions/communications and critical to induct the immune response and involved in anti-inflammatory process in human body. Cytokines in Asthma Because of the overlapping and abundance propertie s of cytokines, it is h ard to clearly classify cytokines which are potentially involve d in asthma. However, considering about the abnormalities of asthma, cyt okines can be generally grouped as follows (23): (1) Lymphokines: IL-2, IL-3, IL-4, IL -5, IL-13, IL-15, IL-16, IL-17. (2) Pro-inflammatory cytokines: IL-1, TNF, IL-6, IL-11, GM-CSF, SCF. (3) Anti-inflammatory cytokines: IL-10, IL-1 IFN, IL-12, IL-18. (4) Chemokines: MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, IL-8. (5) Growth factors: TGF, FGF, EGF, IGF. Therefore, many cytokines are involved in the development of the atopic state and of the chronic inflammatory processes of asthma, cont ributing to the release of mediators such as histamine. The potential role of each cytokine can be indentified from its expression in asthmatic airways, from studies in transgenic or knockout mice or from studies involving the use of inhibitors of synthesis or antibodies or blockers at the receptor level (23). But remember, the fact that cytokines work as a networ k should not be underestimated. Asthma is characterized by the infiltration of T helper 2 (Th2)-type cells, eosinophils, and mast cells to the airway wall. Picture 1-4 show s a central role for inflammatory Th2 cells in asthma. The Th2-cell-asscociated cytokines interleukin-4 (IL-4), IL-5, IL-9, IL-13 and tumor necrosis factor (TNF) have an important role in the pathogenesis of allergic asthma, as shown in their reduction of most of the salient features of asthma, su ch as goblet-cell hyperplasia, airwaywall remodeling and bronchial hyper-reactivity. T h2-cell-associated cytokines are known to induce changes in blood vessels th at lead to the upregulation of intercellular adhesion molecule 1 (ICAM1) and vascular cell-adhesion molecule 1 (VCAM1). This lead s to the recruitment of very

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18 late antigen 4 (VLA4)-expressing eosinophils into the airway wa ll. These factors also induce promoting immunoglobulin class switching to the immunoglobulin E (IgE) heavy chain, allowing for the production of IgE by B cells, a feat ure of allergic asthma In addition, Th2-type cytokines lead to stimulation of the epithelial-me senchymal tropic unit, thus stimulating collagen deposition (24). Therefore, cytokines play an inte gral role in the coordi nation and persistent of the inflammatory process in the chronic inflammation of the airways in asthma. Hypothesis and Objectives of Study Specific Aim 1: Perform in vitro transport studies of corticosteroids BUD and FP using Calu-3 cell line. Some of the corticosteroids have been reported as a substrate of Pgp (16). We hypothesize that there is interaction betw een Pgp and corticosteroids budesonide (BUD) and fluticasone propionate (FP) and will affect pharmac okinetics of corticos teroids in human body. Specific Aim 2: Conduct in vitro cytokine release studies of IL-4, IL-5, and IL-13 with the existence of BUD, FP using peri pheral blood mononuclear cells (PBMC). Several studies have subsequently suggested th at cytokines can be tr ansported out of cell membranes by Pgp (3). Since both cytokines and co rticosteroids could be substrates of Pgp. We hypothesize that corticos teroids will affect the tran sportation of cytokines by Pgp.

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19 Figure 1-1. Normal versus asth matic bronchiole in human lung Trachea Lung Asthmatic Bronchiole Healthy Bronchiole

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20 Figure 1-2. Structures of i nhaled corticosteroids and 2-adrenergic agonist. A) Structure of 2adrenergic agonists, Albuterol. B) Structur e of inhaled corticoste roid, Budesonide. C) Structure of inhaled corticoste roid, Fluticasone. D) Structur e of inhaled corticostetoid, Mometasone furote. A B C D

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21 Figure 1-3. Mechanism of P-glycoprotein (Pgp) as a transmembrane drug efflux pump Transmembrane domain Homologous half Plasma membrane Drug efflux Corticosteorids or other Pgp substrates ATP-binding domain

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22 Figure 1-4. A central role for inflammatory Th2 cells in asthma. Reprint with permission from Hammad H, Lambrecht B.N. Dendritic cell s and epithelial cells: linking innate and adaptive immunity in asthma. Natu re Reviews Immunology 8(2008) 193-204.

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23 CHAPTER 2 IN VITRO CORTICOSTEROIDS TRANSPORT STUDY Introduction Inhaled corticosteroids a re used in the treatm ent of asthma and are of significant benefit because they are delivered direc tly to the site action, the lung. However, after inhalation, some portion of the corticosteroid dosag e is deposited in the oral cav ity, swallowed, and then available for systemic absorption from the gastrointestin al tract, resulting in unwanted systemic side effects. The aim of inhaled corticosteroids is to increase desired local drug concentrations and decrease unwanted drug systemic bioavailability. Since Pgp is expressed on bronchial epithelial cells, it will sure affect local absorption of cortic osteroids in human lung. In order to identify the effect of Pgp on corticosteroids absorption in lung, we perform co rticosteroid transport study in vitro using Calu-3 cell line. The aim of this st udy was to investigate the role of Pgp in the transport of the inhaled corticosteroids, bude sonide and fluticasone, across Calu-3 cells. The literature cites several cell lines that are currently being used as in vitro models to investigate pulmonary drug absorption. Among thes e are a Type II-like pu lmonary epithelial cell line, A549, and the bronchiole cell lines HBE4/E6/E7 and Calu-3 cell line. The v-3 cell line, derived from human bronchial epit helium, is believed to have ser ous cell properties (24). This cell line has been extensively researched due to its ability to form tight monolayers in culture and its ability to express Pgp (25). In this transport study, we have c hosen the Calu-3 cell line as a tool to identify the mechanism of corticosteroids transepithelial transport in the airways in vitro. Calu-3 cells were reconstitute d as functional epithelial monolayers on permeable filter support in a 6-well Transwell plate. Figure 2-1 is a picture of a 6-well Transwell plate. Figure 2-2 represents Calu-3 monolayer in Transwell system.

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24 Materials and Methods Calu-3 Cell Culture Method Calu-3 cells, a human mucusproducing, subm ucosal gland carcinom a bcell line, were obtained from the American Type Culture Collection (HTP-55; ATCC, Manassas, USA). Calu-3 cells were cultured in Dulbeccos Modified Eagles medium (DMEM) (Invitrogen, 12430-054) supplemented with 1% (v/v) nonessential ami no acids (Invitrogen, 11140-050), 20% (v/v) fetal calf serum (FCS, Fisher MTT35011CV), and 1% (v/v ) antibiotic solution containing penicillin and streptomycin (Invitrogen, 15140-122). Calu-3 cells were maintained in a culture flask at 37C, 95% rela tive humidity and 5% CO2, and passaged at approximately 90% confluence, as determined by light microscopy. All transport experiments were performed with Ca lu-3 cells within passage number (PN) 29-45. For transport experiments, Calu-3 cells we re grown in six-well plates on Transwell filters with a pore size of 0.4 m permeable support and a surface area of 4.5 cm2 (Corning Incorporated, 34606005). Before seed ing the cells, the filter membranes were coated with 660 L of a collagen solution (30 g/mL in phosphate -buffered saline, INAMED 5409) and air dried under sterile conditions. In each Transwell plate, the first well of th e six well was bland without any cells. Cells were seeded at an initial density of 2.55 cells/cm2. The culture medium was replaced every other day for a total of 18-21 days in order to have a stable expression of Pgp in cell monolayer. At the first day, the culture medi um was added into both apical and basolateral sides of the Transwell well. After 1 day in culture, the cultu re medium on the apical side of the cell monolayer was removed, and cells were grown at an air interface, i. e. the culture medium was added only into the basola teral side of the Transwell well. Interface culture conditions were shown to induce ciliogenesis and the production of mucu s by Calu-3 cells (26).

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25 Transport Experiment Method There were two study groups in each corticos tero id transport experiment. One group was the control group which Calu-3 cell monolayer wa s treated with only cort icosteroid budesonide (BUD, Sigma-Aldrich, US) or flut icasone propionate (FP, Sigma-Aldrich, US). The other group was the experimental group with Calu-3 cell m onolayer was treated with both corticosteroid BUD or FP and the Pgp inhibitor vera pamil (100 M, Sigma-Aldrich, US). Before and also after the transport experime nts, the tightness of the cell monolayers was determined by means of the transepithelial elec trical resistance (TEER) values using EndOhm Voltohmmeter equipped with a STX-2 chopstick electrode (World Precision Instruments Inc., Sarasota, FL, USA). To make sure the tightne ss of the cell monolayer, he difference of TEER values between the blank and cell monolayer membrane needs to be bigger than 300 cm2. Also, when the experiment finished, the TEER va lue of each cell monolayer membrane needs to be larger than 300 cm2. The transport experiments were performed in transport medium (HBSS/HEPES) consisting of HBSS (Hanks balanced salt solution; Sigm a-Aldrich, US) supplemented with 30 mM HEPES (N-2-hydroxyethyl-piperazi ne-N-2-ehtanesulfonic acid, final pH 7.4; Fisher Scientific, US). Before each experiment, the cells were washed tree times with HBSS/HEPES to remove growth medium. Cells were then allowed to equilibrate by incubation with HBSS/HEPES for 1 h at 37C, 95% relative humidity and 5% CO2 incubator. The composition of the buffers in the apical and basolateral compartments was identical in all experiments. In both BUD and FP transport studies, we tested two directions of corticosteroid transport: from apical to basolateral side (A B) and basolateral to apical side (B A). Transport study st arted by placing 2.0 mL HBSS/HEPES containing 30 m BUD or 5 m FP in control group and 30 m BUD or 5 m FP with 100 M Pgp inhibitor vera pamil in either apical side or basolateral side (donor

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26 compartment). Then we added 2.0 mL HBSS/HEPES in basolateral side or apical side (acceptor compartment). BUD or FP used in this study was the mixture of 2% of 50 Ci 3H-BUD or 3H-FP with 98% of 30 m BUD or 5 m FP in transpor t buffer. All radioactiv e corticosteorids were received from AstraZeneca, Sweden as a gift. The appearance of the corticosteroids was followed in time by removing 200 L liquid from the acceptor compartment. Then add 200 L fresh HBSS/HEPES to maintain a constant volu me. In BUD transport experiment, 200 L liquid was sampled at time 0, 30, 45, 60, 90, 120, 150, 180, 240, 300, 360 min. In FP transport experiment, 200 L liquid was sampled at time 0, 5, 10, 15, 30, 45, 60, 90, 120, 180, 240 min. All solutions used were preheate d to 37C. Then, we quantified the percentage of BUD or FP concentration appeared in acceptor compartment co mpared to the initial concentration added in donor compartment. All samples were read by scintillation counter Beckman LS6500. All procedures in this transport study were perfor med under sterile environment. All experiments were done in triplicate. Data Analysis The value of coefficient of permeability (Papp) was calculated by following equation: Papp= Q/( TAC0) where Q/ T is the amount of drug (ng/min) appearing in the acceptor compartment as a function of time obtained from the slope of the li near portion of the amou nt transported vs time plot, C0 is the initial con centration of corticosteroid in donor compartment (ng/mL), and A is the surface area of the permeable membrane in cm2 (27,28). To determine the affinity of corticosteroids to Pgp, we calculated the permeability directional ratio (PDR) by following equation: PDR = Papp(B A)/Papp(A B)

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27 If the ration of these two coeffici ent of permeability values is la rger than 1, it indicates that the compound is a substrate of Pgp. Statistical Analysis Data from individual transpor t experim ent were analyzed using an unpaired Students t -test (2-tailed) in Microsoft Excel 2007. P values smaller that 0.05 were considered to indicate statistical differences. Results and Discussion The PDR value of BUD and FP transport st udy results were 2.02 and 1.06 respectively. The results show that both BUD and FP are s ubstrates of Pgp. Fi gure 2-3 and figure 2-4 represent the percentage of budesonide (BUD) and fluticas one propionate (FP) appeared in accepto r compartment at different time points. Students t -test (2-tailed) P values of two transport direction results for BUD and FP are a ll less than 0.05. The resu lts show significant differences between control gr oup (without Pgp) and experimental group (with Pgp) in both BUD and FP experiments. And the difference be tween control group and experimental group in A B direction is smaller than that between two groups in B A direction. Conclusion TEER values rema ined almost unchanged during transport studies. This demonstrates no adverse alteration of the monolayers occurred. The PDR values suggest that budesonide (BUD) and fluticasone propionate (FP) are actively transported across Calu-3 cell monolayers by efflux pump P-glycoprotein (Pgp). The in hibition of Pgp increased the concentration of BUD and FP in acceptor compartment on A B direction. Also, the inhibi tion of Pgp decreased the concentration of BUD and FP in acceptor compartment on B A direction. The results suggested that BUD and FP were substrates of Pgp and th e transportation of BUD and FP by Pgp was more pronounced from basolateral side to apical side in both A B and B A directions. This

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28 suggested that the major amount of Pgp might express on the apical side of the cell membrane in Calu-3 cells which were attached on the permeable membrane.

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29 Figure 2-1. A 6-well Transwell plate used in transport study 6-well Transwell p late Transwell insert Transwell well

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30 Figure 2-2. Representation of Calu-3 monolayer in polarized Transwell system

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31 Table 2-1. Results of BUD transport study in Calu-3 cell model Time (min) % of BUD presence in acceptor compartment s.d. BUD_a BUD_b BUD_av BUD_bv 30 0.77.09 1.50.20 1.21.12 1.39.30 45 1.03.22 2.15.18 1.38.28 1.57.13 60 1.22.07 2.44.21 1.48.15 1.83.49 90 1.39.08 2.87.56 1.74.40 2.31.77 120 1.52.34 3.14.26 1.79.51 2.37.58 150 1.97.26 3.46.18 2.02.04 2.62.16 180 2.15.34 3.67.59 2.18.20 2.95.48 240 2.21.32 3.77.62 2.36.44 3.08.42 300 2.17.17 3.68.25 2.12.29 2.99.49 360 2.07.31 3.83.25 2.03.66 3.08.36

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32 Figure 2-3. Percentage of BUD appeared in the acceptor compar tment at different time points BUD_a BUD_b BUD_av BUD_bv

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33 Table 2-2. Results of FP transport study in Calu-3 cell model Time (min) % of FP presence in acceptor compartment s.d. FP_a FP_b FP_av FP_bv 5 3.72.41 5.72.59 4.41.44 5.29.81 10 4.6.58 7.15.24 5.58.42 6.341.51 15 5.77.44 8.19.13 6.36.43 7.23.30 30 7.57.85 9.44.80 8.10.47 8.91.58 45 7.98.44 9.84.11 8.68.53 9.35.93 60 8.15.56 10.24.67 8.80.65 9.47.29 90 8.06.24 10.57.95 9.63.78 8.88.24 120 8.37.85 10.24.47 8.62.62 9.91.46 180 8.41.07 10.02.23 8.68.67 9.36.70 240 8.14.18 9.97.76 8.65.89 9.08.44

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34 Figure 2-4. Percentage of FP app eared in the acceptor compartment at different time points FP_a FP_b FP_av FP_bv

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35 CHAPTER 3 IN VITRO EFFECT OF CORTICOSTEROIDS ON CYTOKIE RELEASE STUDY Introduction Cytokines are produced by ma ny different types of cells which are involved in cell interactions acting through cytokine receptors on cell membrane. Lymphokines are a subset of cytokines that are produced by lymphocyte. Lym phokines have important ro les in asthma. They can induce the attraction of other immune cells, such as macrophages and other lymphocytes to an infected site and their subsequent activation prepare them to attack the invaders. They encourage cell growth, promote cell activation, direct cellular tra ffic, destroy target cells, and incite macrophages. The lymphokines, which are mo st important in asthma, are IL-4, IL-5, and IL-13. In our 2nd specific aim, we tested the release of IL-4, IL-5, and IL-13 from peripheral blood mononuclear cell (PBMC). PBMC is human blood cell line which has a round nucleus, such as a lymphocyte and monocyte. PBMC cells are a critical component in the immune system to fight infection and adapt to in truders. In this cytokine releas e study, we tested the effect of corticosteroids BUD, FP, and MF on the re lease of lymphocyte IL-4, IL-5, and IL-13. Materials and Methods PBMC Separation and Stimulation Method In cytokine release study, PBMC cells were isolated from buffy coats (L ife South Community Blood Center, Florida, US) of 8 healthy subjects using LymphoprepTM (Axis-Shield PoC AS, Oslo, Norway). The isol ation procedure is as follow: 1. Dilute 5 mL buffy coat with 5 mL phos phate buffered saline (PBS, Fisher) (without Ca2+ and Mg2+) at 18-25C. 2. Slowly add the diluted buffy coat on top of 5 mL of LymphoprepTM using sterile fine tip pipet. 3. Centrifuge at 400 g (1900 rpm) for 20 mi n at 20C. Allow to decelerate without brakes.

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36 4. Carefully recover PBMC cells from the plas ma/Lymphoprep interface using sterile fine tip pipet without remove the supernatant and transfer the cells to a 50 mL centrifuge tube. 5. Wash PBMC cells once with PBS by centrifugation at 400 g for 8 min at 2-8C. 6. Wash PBMC cells twice with PBS by centrifugation at 225 g for 8 min at 2-8C. 7. Resuspend PBMC in 10 mL CDME M (DMEM with 0.05 M Hepes (4-(2hydroxyethyl)-1-piperazineethane sulfonic acid, Fisher), 0.5 % FCS, 2 mM glutamine (Invitrogen)). 8. Aliquots of 10 mL are seeded in to plastic petri dishes (Fisher) and incubated for 1 h at 37C in a 5% CO2 atmosphere. 9. Adherent cells are recovered by gently sc raping with a rubber police man (Corning) and resuspended in CDMEM (36 cells/mL). 10. Viability is assessed by trypan blue dye (Fisher) exclusion. PBMC cells were allowed to grow overnight. In order to secret cy tokines, PBMC were then stimulated with 5 g/mL phytohaemagglutinin (PHA, Fisher Scientific, US) and incubated for 24 h at 37C in a 5% CO2, in RPMI 1640 (Invitrogen) medium. Cytokine Release Experiment PHA-stimul ated PBMC are incubated with blank CDMEM, 0.01, 0.1, and 1 M BUD, or FP, or mometasone furoate (MF) in CDMEM for 24 h. Then PBMC were centrifuge after incubated at 350 g for 20 min. IL-4, IL-5, and IL -13 in supernatant were quantified by human IL-4, IL-5, and IL-13 Enzyme-Linked ImmunoSorbe nt Assay (ELISA) kits (eBioscience Inc., CA, US) respectively. Statistical Analysis All data are expressed as m ean values s.d. After data anal ysis in R program, distribution of cytokine concentration was different from normal distribution (Shapiro-Wilk normality test and Kolmogorove-Smirnov test). Therefore, Wilc oxons test were applied to compare all three concentrations of three corticostero ids with control groups in R program. Results and Discussion Figures 3-1 to 3-9 show the box plots of IL4, IL-5, and IL-13 release concentrations in PHA-stimul ated PBMC cell supernatant afte r 24 h treatment with BUD, FP, and MF

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37 respectively. Table 3-1 shows the effect of different concentrati on of BUD, FP, and MF on IL-4, IL-5, and IL-13 release study in PHA-stimulated PBMC cells. The treatment of FP at three concentrations for 24 h resulted in a significant reduction in IL-4, IL-5, and IL-13 concentrations in the PBMC culture supernatants Also treatments with all thr ee concentrations of BUD and MF for 24 h had a significant reduction effect in IL-5 concentration in cell supernatants. However, it seems that only 24 h treatment of higher concen trations of BUD and MF significantly decreasing the release of IL-4 and IL-13 out of cell by Pgp. Conclusion We investigated the hypothesis that corticosteroids have e ffect on the transportation of cytokines by Pgp. We tested the effect of BUD, FP, and MF at 3 concentrations 0.01M, 0.1M, and 1M on out-of-cell transport of cytokines IL -4, IL-5 and IL-13 in PHA-stimulated PBMC cell supernatant. Results of this study suggest that the transport of three cytokines IL-4, IL-5 and IL-13 by Pgp are inhibited by corticosteroids BUD, FP, and MF at different concentration levels. Therefore, there is interaction between cortic osteroids and cytokines BUD, FP, and MF on the transport by membrane transporte r Pgp. With increasing concentration of corticosteroids, there was more induction in concentration of cytokine s which were transported out of cells by Pgp. The competition of corticosteroids with cytokines decreased the out-of-cell release of IL-4, IL-5, and IL-13 by Pgp in PHA-stimulated PBMC cells. In addition, the results of cytokine releases study suggested that MF is also a substrate of Pgp.

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38 Figure 3-1. Box plots of IL-4 release results after 24 h treatment with BUD. Treatment with 1M BUD for 24 h significantly decrease th e IL-4 concentration from 46 pg/mL to 20 pg/mL in PHA-stimulated PBMC cell supernatant. ** P < 0.001 Initial Concentration of BUD in Donor Compartment 50 b lank 0.01 M 0.1 M 1 M** IL-4 (pg /mL ) 60 40 30 20 10

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39 Figure 3-2. Box plots of IL-4 release results after 24 h treatment with FP. Treatment with 0.01 M, 0.1 M, and 1 M FP for 24 h significantly decrease the IL-4 concentrations in PHA-stimulated PBMC cell supernatant. P < 0.05. ** P < 0.001. Initial Concentration of FP in Donor Compartment 50 b lank 0.01 M* 0.1 M** 1 M** IL-4 (pg /mL ) 60 40 30 20 10

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40 Figure 3-3. Box plots of IL-4 release results after 24 h treatment with MF. Treatment with 0.1 M, and 1 M MF for 24 h significantly decrease the IL-4 concentrations in PHAstimulated PBMC cell supernatant. P < 0.05 Initial Concentration of MF in Donor Compartment b lank 0.01 M 0.1 M* 1 M* 50 IL-4 (pg /mL ) 60 40 30 20

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41 Figure 3-4. Box plots of IL-5 release results after 24 h treatment with BUD. Treatment with 0.01 M, 0.1 M, and 1 M BUD for 24 h significan tly decrease the IL-5 concentrations in PHA-stimulated PBMC cell supernatant. P < 0.05 ** P < 0.001 Initial Concentration of BUD in Donor Compartment b lank 0.01 M* 0.1 M** 1 M** 80 IL-5 (pg /mL ) 100 60 40 20

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42 Figure 3-5. Box plots of IL-5 release results after 24 h treatment with FP. Treatment with 0.01 M, 0.1 M, and 1 M FP for 24 h significantly decrease the IL-5 concentrations in PHA-stimulated PBMC cell supernatant. P < 0.05 ** P < 0.001 Initial Concentration of FP in Donor Compartment b lank 0.01 M* 0.1 M* 1 M** 80 IL-5 (pg /mL ) 100 60 40 20

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43 Figure 3-6. Box plots of IL-5 release results after 24 h treatment with MF. Treatment with 0.01 M, 0.1 M, and 1 M MF for 24 h significantly decrease the IL-13 concentrations in PHA-stimulated PBMC cell supernatant. P < 0.05 ** P < 0.001 Initial Concentration of MF in Donor Compartment b lank 0.01 M* 0.1 M* 1 M** 80 IL-5 (pg /mL ) 100 60 40 20

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44 Figure 3-7. Box plots of IL-13 release results after 24 h treatment with BUD. Treatment with,0.1 M, and 1 M BUD for 24 h significantly de crease the IL-13 concentrations in PHAstimulated cell supernatant. P < 0.05 ** P < 0.001 Initial Concentration of BUD in Donor Compartment b lank 0.01 M 0.1 M* 1 M** 100 IL-13 (pg /mL ) 150 50

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45 Figure 3-8. Box plots of IL-13 release results after 24 h treatment with FP. Treatment with, 0.1 M, and 1 M FP for 24 h significantly decr ease the IL-13 concentrations in PHAstimulated PBMC cell supernatant. P < 0.05 Initial Concentration of FP in Donor Compartment b lank 0.01 M* 0.1 M* 1 M* 100 IL-13 (pg /mL ) 150 50

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46 Figure 3-9. Box plots of IL-13 re lease results after 24 h treatment with MF. Treatment with, 0.1 M, and 1 M MF for 24 h significantly decrease the IL-13 concentrations in PHAstimulated PBMC cell supernatant. P < 0.05 Initial Concentration of MF in Donor Compartment b lank 0.01 M 0.1 M* 1 M* 100 IL-13 (pg/mL) 140 50 120 160

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47 Table 3-1. Results of IL-4, IL-5 and IL-13 releas e studies after treatments with BUD, FP, and MF at concentration 0.01M, 0.1M, a nd 1M. Values are means s.d. P <0.05, **P <0.001 vs control after Shapiro-Wilk normality test, Kolmogorov-Smirnov test and Wilcoxons test. Treatment IL-4 (pg/mL) IL-5 (pg/mL) IL-13 (pg/mL) Control 46.24.23 80.08.67 140.01.58 BUD 0.01M 36.16.07 59.73.28* 114.80.05 0.1M 30.75.72 41.95.92** 103.80.51* 1M 19.36.09** 26.95.87** 69.02.85** FP 0.01M 35.04.12* 54.57.81* 110.23.64* 0.1M 29.79.24** 38.81.80* 100.67.80* 1M 23.24.21** 23.27.40** 83.16.22* MF 0.01M 36.87.25 56.76.65* 124.04.66 0.1M 33.32.12* 41.40.13* 110.57.16* 1M 27.52.67* 28.22.04** 94.30.71*

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48 CHAPTER 4 CONCLUSIONS One important advantage of Inhaled corticosteroid s in asthm a therapy is that a considerable part of the drug can be delivered directly into the lung right afte r inhalation. In order to quickly turn on the effect, it is critic al that corticosteroid can tr avel through lung epithelial cell membrane and reach the nucleus. Pgp on epithel ial cell membrane fbunctions as membranelocalized drug transporter and can actively trans port its substrates out of cell. However, the existence of Pgp may reduce the corticosteroids concentration and effect inside the cells. In the first in vitro trans port study with Calu-3 cell model, the results showed that budesonide and fluticasone propionat e were substrates of Pgp with both PDR value larger than 1. With inhibition of Pgp, the concentration of budesonide and fluticasone propionate in acceptor compartment increased in A B direction and decreased in B A direction. It suggested that Ppg were expressed more on the apical side cell memb rane in Calu-3 cells and the basolateral side membrane were tightly attached on the pe rmeable support membrane in the transwell insert. In the second in vitro cytoki ne release study with stimulated-PBMC cell model, the results suggested that certain concentration of budesonide, fluticas one propionate, and mometasone furoate can interact with cyt okines IL-4, IL-5, and il-13 on th e transport by Pgp and decrease three cytokines out-of-cell-release in stimulatedPBMC cell line. Th is suggested that one effect of corticosteroids on anti-inflammation might depend on the decrea se of cytokine release from lymcytes. Future studies need to address the in teraction between cortic osteroids and cytokines with Pgp in vivo using appropriate animal models.

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49 LIST OF REFERENCES 1. Adcock P.M., Caramori G., Chung P.K.F., Ne w Targets for drug developm ent in asthma. Lancet. 372: 1073-1087, 2008. 2. Barnes P.J., Corticosteroids: The drugs to beat. Eur. J. Pharmacol. 533: 2-14, 2006. 3. Barnes P.J., Inhaled glucocorticoids fo r asthma. New Eng. J. Med. 332: 868-875, 1995. 4. Fong E.W., Levin R.H., Inhaled Corticostero ids for Asthma. Pedia. Rev. 28: 30-35, 2007. 5. Barnes P.J., Adcock I., Anti-inflammatory actions of steroids: molecular mechanisms. Trends Pharmacol. Sci. 14: 436-441 1993. 6. Schleimer R.P., Effects of glucocorticoids on inflammatory cells relevant to their therapeutic applications in asthma Am. Rev. Respir. Dis. 141: 59-69, 1990. 7. Boschetto P., Rogers D.F., Fabbri L.M., Barnes P.J., Corticosteroid inhibition of airway microvascular leakage. Am. Re v. Respir. Dis. 143: 605-609, 1991. 8. Shimura S., Sasaki T., Ikeda K., Yamauchi K, Sasaki H., Takishima T., Direct inhibitory action of glucocorticoid on gl ycoconjugate secretion from airway submucosal glands. Am. Rev. Respir. Dis. 141: 1044-1049, 1990. 9. Laitinen L.A., Haahtela T., A comparative study of the effects of an inhaled corticosteroid, budesonide, and of a 2-agonist, terbutaline, on airway inflammation in newly diagnosed asthma: a randomized, double-b lind, parallel-group controlled trial. J. Allergy Clin. Immunol. 90: 32-42, 1992. 10. Djukanovic R., Wilson J.W., Britten K.M., Wilson S.J., Walls A.F., Roche W.R., Howarth P.H., Holgate S.T., Effect of an i nhaled corticosteroid on airway inflammation and symptoms in asthma. Am. Rev. Respir. Dis. 145: 669-674,1992. 11. Jeffery P.K., Godfrey R.W., Adelroth E., Ne lson F., Rogers A., Johansson S.A., Effects of treatment on airway inflammation and th ickening of basement membrane reticular collagen in asthma: a quantitative light a nd electron microscopic study. Am. Rev. Respir. Dis. 145: 890-899, 1992. 12. Burke C., Power C.K., Norris A., Condez A., Schmekel B., Poulter L.W., Lung function and immunopathological changes after inhale d corticosteroid therapy in asthma. Eur. Rspir. J. 5: 73-79, 1992. 13. Barnes P.J., Effect of corticosteroids on airway hyperresponsiveness. Am. Rev. Respir. Dis. 141: 70-76, 1990. 14. Higgins C.F., ABC transporters: from micr oorganisms to man. Ann. Re.v Cell. Biol. 8: 67-113, 1992.

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50 15. Pawlik A., Ba kiewicz-Masiuk M., Machali ski B., Safranow K., and Gawro skaSzklarz B., Involvement of Pglycoprotein in the release of cytokines from peripheral blood mononuclear cells treate d with methotrexate and dexamethasone. J. Pharm. Pharmacol. 57: 1421-1425, 2005. 16. Florea B.I., Sandt C.J., Schrier S.M., Kooi man K., Deryckere K., Boer A.G., Junginger H.E., Borchard G., Evidence of P-glycoprotein me diated apical to basolateral transport of flunisolide in human broncho-tracheal epitheli al cells (Calu-3). Br J. Pharmacol.134: 1555-1563, 2001. 17. Dean M., Allikmets R., Complete characterization of human ABC gene family. J. Bioen. Biomem. 33(2001) 475-479 18. Borowski E., Bontemps-Gracz M.M., Piwkowska A., Strategies for overcoming ABCtransporters-mediated multidrug resistance (MDR ) of tumor cells. Acta Biochim. Pol. 52: 609-627, 2005. 19. Ford J.M., Hait W.N., Pharmacology of drugs that alter multidrug resistance in cancer. Pharmacol. Rev. 42: 155-199, 1990. 20. Sharom F.J., Liu R., Romsicki Y., Lu P., Insights into the stru cture and substrate interactions of the P-glycoprotein multidrug transporter from spectroscopic studies. Biochim. Biophys. Acta 1461: 327-345, 1999. 21. Petrovic V., Teng S., Piquette -Miller M., Regulation of drug transporters: during infection and inflammation. Mol. Interv. 7: 99-111, 2007. 22. Hammad H., Lambrecht B.N., Dendritic cells and epithelial cells: linking innate and adaptive immunity in asthma. Nat. Rev. Immunol. 8: 193-204,2008. 23. Neyfakh A.A., Serpinskaya A.S., Chervonsky A.V., Apasov S.G., Kazarov A.R., Multidrug-resistance phenotype of a subpopulation of T-lymphocytes without drug selection. Exp. Cell Res. 185: 2735-2739,1989. 24. Foster K.A., Avery M.L., Yazdanian M., Audus K.L., Characterization of the Calu-3 cell line as a tool to screen pulmonary drug delivery. Int. J. of Pharm. 208: 1-11, 2000. 25. Hamilton K.L., Bachstrom G., Yazdanian M. A., Audus K.L., P-glycoprotein efflux pump expression and activity in Calu-3 ce lls. J. Pharm. Sci. 90: 647-658, 2001. 26. Meaney C., Florea B.I., Borchard G., Junginger H.E., Characterization of a human submucosal gland cell line (Calu-3) as an in vitro model of the airw ay epithelium. Proc. Int. Symp. Control Rel. Bioact. Master. 26: 198-199, 1999. 27. Xu J., Go M.L., Lim L.Y., Modulation of digoxin transport across Caco-2 cell monolayers by citrus fruit juices: lime, lem on, grapefruit, and pummel. Pharm. Res. 20: 169-176, 2003.

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51 28. Honda Y., Ushigome F., Koyabu N., Morimoto S., Shoyama Y., Uchiumi T., Kuwano M., Ohtani H., Sawada T., Effects of grap efruit juice and orange juice components on Pglycoproteinand MRP2-mediated drug e fflux. Br. J. Pharmacol. 143: 856-864, 2004.

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52 BIOGRAPHICAL SKETCH Nasha Wa ng was born in Harbin, Heilongjiang, China. She obtained her Bachelor of Pharmacy degree from China Pharmaceutical Un iversity in 2001. She obtained her Master of Medicinal Chemistry from Peking Union Medi cal College in 2004. She was admitted to the graduate program at the College of Pharmacy, University of Florida in 2004 and joined in Department of Pharmaceutics in 2005. She comple ted her Master of Science in Pharmacy in December 2008 under the supervision of Dr. Guenther Hochhaus.