Biopharmaceutical aspects of corticosteroid therapy in preterm infants

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Table of Contents
    Title Page
        Page i
    Dedication
        Page ii
    Acknowledgement
        Page iii
    Table of Contents
        Page iv
        Page v
    Abstract
        Page vi
        Page vii
    Chapter 1. Background
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
    Chapter 2. Role of P-glycoprotein transporters in modulating the brain permeability of inhaled corticosteroids
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
    Chapter 3. Assessment of pulmonary targeting and brain permeability of triamcinolone acetonide phosphate, an inhaled steroid, in neonatal rats using ex vivo receptor binding assay
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
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        Page 43
        Page 44
        Page 45
        Page 46
        Page 47
        Page 48
        Page 49
    Chapter 4. Pulmonary targeting of sustained release formulation of budesonide in neonatal rats
        Page 50
        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
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        Page 60
        Page 61
    Chapter 5. Conclusions
        Page 62
        Page 63
        Page 64
    List of references
        Page 65
        Page 66
        Page 67
        Page 68
        Page 69
        Page 70
        Page 71
        Page 72
        Page 73
        Page 74
    Biographical sketch
        Page 75
        Page 76
        Page 77
        Page 78
Full Text










BIOPHARMACEUTICAL ASPECTS OF CORTICOSTEROID THERAPY IN
PRETERM INFANTS














By

VIKRAM ARYA


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


2003
































This thesis is dedicated to my family for their constant love, encouragement and support
of my goals.














ACKNOWLEDGMENTS

I would like to express my sincere gratitude to Dr. Guenther Hochhaus, my mentor and

advisor whose constant encouragement, endless patience and unfaltering support was

instrumental in the successful completion of this dissertation. I would also like to thank the

members of my supervisory committee (Drs. Hartmut Derendorf, Jeffrey Hughes and S.

Khan) who individually, and as a group, provided excellent input and guidance during the

course of my doctoral research.

I would also like to sincerely thank my friends in the lab (Boglarka, Intira, Kai,

Manish, Sriks, Yaning) for their willingness to share their academic and personal

experiences. I would also thank the secretaries of the department for their technical support.

I would like to thank my brother, Dr. Vivek Arya for his constant support and for

always believing in me. I would not have even able to begin, much less complete, this great

academic endeavor without his help.

I also wish to acknowledge my American "family" for making me feel at home away

from home, providing insight into American culture, and sharing all the fun times.

Finally, I wish to extend my deepest gratitude to my parents for their constant love and

encouragement. There is nothing without their presence.














TABLE OF CONTENTS
Page

ACKNOW LEDGMENTS ................................................................................................. iii

ABSTRACT....................................................................................................................... vi

CHAPTER

1. BACKGROUND........................................................................................................ 1

Introduction............................................................................................................... 1
Pathogenesis of Chronic Lung Disease...................................................................... 3
Corticosteroids in Chronic Lung Disease................................................................... 5
Adverse Effects of Corticosteroids in Preterm Infants............................................... 7
P-glycoprotein Transporters and Blood Brain Barrier............................................. 10
Inhaled Corticosteroids In Chronic Lung Disease ................................................... 13
Strategies for Improving Pulmonary Selectivity...................................................... 17
Sustained Release Drug Delivery Systems .............................................................. 20
Liposomes..................................................................................................... 20
Microencapsulation....................................................................................... 22
Microspheres................................................................................................. 22
Objectives ............................................................................................................... 24

2. ROLE OF P-GLYCOPROTEIN TRANSPORTERS IN MODULATING THE BRAIN
PERMEABILITY OF INHALED CORTICOSTEROIDS...................................... 25

Introduction ... ......................................................................................................... 25
Hypothesis............................................................................................................... 26
Materials and Methods............................................................................................. 26
Preparation of Drug and Radiolabelled Solutions ........................................ 27
Animal Procedures........................................................................................ 27
Ex Vivo Receptor Binding Assay ................................................................. 28
Results ......... ......................................................................................................... 29
Discussion .... ............................................................................................................ 31
Conclusions ... ......................................................................................................... 33

3. ASSESSMENT OF PULMONARY TARGETING AND BRAIN PERMEABILITY OF
TRIAMCINOLONE ACETONIDE PHOSPHATE, AN INHALED STEROID, IN
NEONATAL RATS USING EX VIVO RECEPTOR BINDING ASSAY ..............34

Introduction ... ................................................ ......................................................... 34














Hypothesis..... .....................................................................................................35
M materials and M ethods............................................................................................. 35
Preparation of TAP and Radiolabelled Solution........................................... 35
Animal Procedures................................................................... ..................36
Results ......... ......................................................................................................... 38
Discussion .............................................................................................................. 44
Conclusion ............................................................................................................... 48


4. PULMONARY TARGETING OF SUSTAINED RELEASE FORMULATION OF
BUDESONIDE IN NEONATAL RATS ................................................................. 50

Introduction .................................................... ..................................... ...................50
Hypothesis ............................................................................................................... 51
M materials and M ethods.............................................................................................51
Preparation of Uncoated/PLA coated Budesonide Suspensions and
Radiolabelled Solutions................................................................................ 52
Coating Procedure......................................................................................52
Animal Procedures .....................................................................................52
Results ................................................................................................................... 55
Discussion....................................... .................... ......................58
Conclusion ............................................................................................................... 61

CONCLUSIONS................................................................................................................ 62

LIST OF REFERENCES................................................................................................... 65

BIOGRAPHICAL SKETCH ............................................................................................. 75








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

BIOPHARMACEUTICAL ASPECTS OF CORTICOSTEROID THERAPY IN
PRETERM INFANTS

By

Vikram Arya

December 2003

Chair: Guenther Hochhaus
Major Department: Pharmaceutics

Premeture birth is a major cause of infant mortality in the United States. The

immaturity of the vital organs such as lungs necessitates the use of artificial respiratory

support. The ensuing pulmonary damage predisposes the preterm infant to a wide array

of medical complications such as chronic lung disease (CLD). The benefits of using

systemic corticosteroids for the treatment/prevention of CLD in preterm infants are well

documented. However, the concomitant observance of neurotoxic adverse effects in

premature infants (and absent in adults), after systemic corticosteroid administration,

have led to exploration of alternate routes of corticosteroid delivery. The administration

of corticosteroids through the inhalation route has met with limited success, partly due to

the rapid absorption of the corticosteroid from the lungs into the systemic circulation

leading to loss of pulmonary targeting. Computer simulations have reiterated the

importance of optimizing the drug release rate for improving pulmonary targeting. The

overall objective was to study the biopharmaceutical factors such as brain permeability

and pulmonary residence time that modulate the disposition of inhaled corticosteroids in

preterm infants.








The role of p-glycoprotein transporters in modulating the brain permeability of

inhaled corticosteroids was evaluated by assessing the brain and liver receptor occupancy

in wild type and mdrl a mice after intravenous administration of TAP.

Ex vivo receptor binding assay was used for assessing pulmonary and systemic

corticosteroid exposure in neonatal rats after intratratracheal administration of

triamcinolone acetonide phosphate (TAP) solution. To gain more insight into pulmonary

residence time and pulmonary targeting, the neonatal rat model was used to determine the

pulmonary targeting of poly (1-lactic acid) (PLA) coated budesonide.

Mdrl a mice showed significantly higher brain receptor occupancy than wild type

mice, which suggests the pivotal role played by p-gp in modulating the brain permeability

of corticosteroids. We did not observe pulmonary targeting after intratracheal

administration of TAP. However, we observed significant brain receptor occupancy in

neonatal rats that was in sharp contrast to minimal brain receptor occupancy in adult rats.

Polymeric coated budesonide significantly higher pulmonary targeting as compared to

uncoated budesonide.

Overall, the results underscore the urgent need to develop pulmonary targeted

sustained-release delivery systems for corticosteroids in preterm infants. This will

potentially result in an improved benefit-to-risk ratio of inhaled corticosteroid therapy for

CLD.













CHAPTER
BACKGROUND

Introduction

Preterm birth, observed in 7-10 % of all pregnancies in the United States, continues

to be a major cause of infant morbidity and mortality (1). Medical complications arising

due to prematurity result in significant health care costs (estimated to be $10 billion in the

US annually), frequent hospitalizations and great emotional burden for the family.

The normal gestational age (number of completed weeks of pregnancy from the last

menstrual period) of a full term baby is 40 weeks. Preterm (or premature) babies are

born before 37 weeks of completed gestation. Although some preterm births are elective,

a variety of factors such as previous preterm birth, uterine or cervical abnormalities, use

of illicit drugs and low socio-economic status increase the risk of women delivering

preterm.

Because of immature birth, the vital organs of the preterm infant such as the lungs

and brain are not fully developed and are incapable of performing the vital functions

required for healthy survival. Bolt et al. (2) reviewed lung development in premature

infants. The human lung development can be classified into five distinct phases

embryonic, pseudoglandular, canilicular, and saccular: and the alveolar phase (that

continues after birth). These phases encompass the various stages of the pulmonary

development process and are operative at distinct phases of gestation (e.g., the embryonic

phase lasts until the 6th week of gestation and involves the formation of

bronchopulmonary segments). This suggests that the gestational age of the preterm infant










at the time of birth governs the degree of pulmonary immaturity. Fig 1-1 illustrates the


pulmonary development as a function of gestational age.

0-
Embryonic Formation of Bronchopulmonary
) Phase segments
-. 5-- (0-6 weeks) sge
g 5


> Pseudo-
10 -- glandular Airway division completed
Phase Development of smooth muscles
S(7-16 Formation of cilia and cartilage
1-" weeks)
15--



Canalicular Airway branching completed
20 Phase Type I and Type II pneumocytes
(16-26 formed
weeks) SURFACTANT PRODUCTION
STARTS
Very Premature birth -
(22-26 weeks) 25 -



30 Saccular
Phase Thinning of airspace walls
Premature birth (26-40 Increased formation of pulmonary
(26-37 weeks) weeks and alveoli
35 -- continues Increased development of Type I and
after birth) Type H pneumocytes



Term Birth 4-- 40 -


Post-term birth
45 --


Fig 1-1: Different Stages of Pulmonary Development as a Function of
Gestational Age.

The immaturity of the lung necessitates the use of mechanical ventilators to provide

artificial respiratory support to the preterm infant. This use of mechanical ventilation

leads to significant damage of an already fragile immature lung. Clark et al. (3) have


shown that the mechanical damage caused to the immature lungs by mechanical








ventilators leads to fluid and protein leak in the airways, inhibition of surfactant

production and increase in pulmonary inflammation. This pulmonary damage

predisposes the preterm infant to a wide array of pulmonary complications such as apnea

(interruption in breathing), respiratory distress syndrome (pulmonary complication due to

insufficient surfactant production) and chronic lung disease (CLD). In addition to the

pulmonary complications, the preterm infant also suffers from other physiological

complications of premature birth. These include intraventricular hemorrhage (bleeding in

the brain which eventually fills up the ventricles leading to brain damage); patent ductus

arteriosus (failure of closure of ductus arteriosus leading to heart failure and lack of

oxygen to the heart); and retinopathy of prematurity (abnormal growth of blood vessels in

the eyes leading to scar formation that can damage the retina).

Pathogenesis of Chronic Lung Disease

Despite significant advances in perinatal and neonatal care, CLD (also known as

bronchopulmonary dysplasia) persists as one of the major complications in premature

infants who require prolonged mechanical ventilation. Northway et al. (4) have described

the occurrence of bronchopulmonary dysplasia as a result of prolong mechanical

ventilation. The clinical definition of CLD varies among different healthcare settings.

However, the two most commonly accepted definitions in neonatal intensive care units

(NICU) are 1) mechanical ventilation and dependence on supplemental oxygen at 28 days

postnatal age and 2) the same features at 36 weeks postmenstrual age. The incidence of

chronic lung disease among ventilated infants is estimated to be between 4 and 40 %

depending on the gestational age; but the highest incidence (in excess of 70 %) occurs in

infants weighing less than 1000 g at birth (5). Moreover, the increasing survival of very








immature infants due to significant advancements in neonatal care made in recent years

has dramatically increased the number of infants at a risk for developing CLD (6).

An increasing body of evidence suggests that exposure to mechanical ventilation

triggers a cascade of inflammatory responses that play a key role in the pathogenesis of

CLD in preterm infants (7). A number of factors such as barotraumas induced by

mechanical ventilation and production of oxygen-derived free radicals result in the

release of pro-inflammatory cytokines such as tumor necrosis factor-a (TNF- a),

interlukin 6 (IL-6) and interlukin 8 (IL-8). Dooy et al. (8) have shown that lung damage

in premature infants may be caused by the failure to downregulate this inflammatory

response. Consequently, the discordance between high concentrations of pro-

inflammatory mediators and the inability of the premature infant to generate a sufficient

anti-inflammatory response makes the premature infant very susceptible to the

development of CLD.

The impact of CLD on both mortality and morbidity has made it imperative to

develop and implement treatment strategies aimed at preventing/treating CLD. The

recognition of a strong correlation between pulmonary inflammation and the

development of CLD has resulted in clinical intervention with anti-inflammatory agents.

The rationale for using these agents is the modulation of the inflammatory process in the

lung thereby reducing the incidence or severity of CLD. Currently, systemic

corticosteroids, because of their strong anti-inflammatory properties, appear to be suitable

therapeutic agents for the treatment/prevention of CLD. Fig 1-2 shows the various risk

factors responsible for preterm birth and eventual development of CLD; and the








beneficial and adverse effects of using systemic corticosteroids, the most widely accepted

clinical intervention in CLD.


Fig 1-2: Schematic Representation of Development and Treatment of CLD.

Corticosteroids in Chronic Lung Disease

As previously noted, the scientific rationale for using systemic corticosteroid

therapy is the reduction in pulmonary inflammation which is considered to play a pivotal

role in the onset of CLD (7). Corticosteroids reduce the polymorphonuclear induction in

the cells, reduce the production of pro-inflammatory cytokines such as leukotrienes and

TNF and induce the closure of patent ductus arteriosus (9). Corticosteroids also enhance

the production of surfactant and antioxidant enzymes, decrease bronchospasm,

pulmonary and bronchial edema thereby improving the pulmonary compliance in preterm








infants (10, 11). These beneficial effects of corticosteroids facilitate the faster weaning

ofpreterm infants from mechanical ventilators and reduce the duration of supplemental

oxygen, factors that are highly implicated in the development of CLD.

Liggins and Howie introduced the concept of using antenatal (steroids administered

to the mother at the risk of delivering preterm) corticosteroids for the enhancement of

fetal lung maturation (12). They showed that the administration of antenatal

corticosteroids to enhance fetal lung maturation resulted in a significant reduction in the

incidence of respiratory distress syndrome (RDS) in preterm infants. The landmark study

by Liggins and Howie paved the way for a plethora of randomized clinical trials (RCT)

that investigated the efficacy of antenatal and postnatal corticosteroids to reduce/prevent

the occurrence ofCLD. Mammel et al. (13) and Schick et al. (14) showed short-term

improvement in pulmonary function and faster weaning from the mechanical ventilator in

preterm infants treated with dexamethasone, a potent corticosteroid. Avery et al. (15)

showed that in infants treated with dexamethasone, there was significant facilitation in

weaning from mechanical ventilators, however, there were no significant differences in

the length of hospital stay. Halliday et al. (16) showed the beneficial effects of

corticosteroids on lung function leading to earlier extubation of premature infants. Yeh et

al. (17) reported that early (< 12 h) postnatal dexamethasone therapy facilitated removal

of the endotracheal tube and minimized lung injury in premature infants with severe

RDS. Canterino et al. (18) evaluated the effect of antenatal steroid treatment on the

development of neonatal periventricular leukomalacia. It was shown that antenatal

steroid treatment led to over 50 % decrease in the incidence ofperiventricular

leukomalacia in preterm neonates.








The National Institute of Health (NIH) issued a consensus statement in the spring

of 1994 on the multiple benefits of administering a single dose of antenatal steroids for

fetal maturation (19). The panel concluded that administration of antenatal

corticosteroids to pregnant women at a risk of preterm delivery reduces the incidence of

RDS and neonatal mortality. Evidence in the literature was sufficient to advocate the use

of antenatal corticosteroids (dexamethasone/betamethasone) up to 7 days before delivery.

However, the continuation of corticosteroid therapy beyond 7 days and the

advantages/disadvantages of multiple administration of systemic corticosteroids were

topics that warranted further research. In the spring of 2000, the NIH again issued

clinical recommendations regarding antenatal corticosteroid therapy that entailed giving a

single dose of corticosteroids to all pregnant women at 24-34 weeks of gestation who are

at a risk of preterm delivery within 7 days (20). However, the report concluded with a

cautionary note: "Because of insufficient scientific data from randomized clinical trials

regarding the efficacy and safety of repeated courses of corticosteroids, such therapy

should not be used routinely. In general, it should be reserved for patients enrolled in

randomized controlled trials" (20).

Adverse Effects of Corticosteroids in Preterm Infants

In addition to recognizing the beneficial effects of corticosteroids, the NIH

consensus statement also noted the occurrence of serious adverse effects of using

systemic corticosteroids in preterm infants. This occurrence of adverse effects after

systemic corticosteroid therapy had been shown as early as 1972 by Baden et al. (21) who

studied the effect of two doses of hydrocortisone on the incidence of RDS. Follow up

studies of surviving premature infants from this trial revealed increased risk of

intraventricular hemorrhage (22). Ewerbech and Helwig also reported an increased risk








of intraventricular hemorrhage after using prednisolone in 10 premature infants with

severe RDS (23). Fitzhardinge and co-workers (24) followed the trial conducted by

Baden et al. (21) and showed that infants who received systemic corticosteroid therapy

had increased incidences of neurological complications and lowered motor development.

Nevertheless, the attainment of immediate beneficial effects in premature infants after

systemic corticosteroid administration led to its unfortunate and indiscriminate use in the

1980s and 1990s despite the early alarming indications of adverse effects and without

sufficient establishment of the benefit/risk ratio.

The last two decades have witnessed an alarming increase in the nature and degree

of clinical complications observed in preterm infants who are treated with systemic

corticosteroids. The degree of adverse effects is highly dependent on the gestational age

of the preterm infant (which determines the degree of prematurity), and the degree of

development of vital organs and drug transport systems. Many studies have shown the

adverse effects after single and multiple doses of systemic corticosteroids. Table 1-1

shows the adverse effects associated with different vital organs of the body (25)..

Table 1-1: Adverse Effects of Postnatal Corticosteroid Treatment.

Region of the Body Adverse Effects
Central nervous system Motor developmental retardation
Atrophy of the dendrites
Cerebral palsy
Cardiovascular Hypertension
Cardiac hypertrophy
Sustained bradycardia
Metabolic and endocrine Somatic growth failure
Hyperglycemia
Proteolysis
Respiratory Pneumothorax








Yeh et al. (26) studied the outcome at 2 year corrected age of infants who

participated in a controlled trial of early (< 12 h) dexamethasone therapy for prevention

of chronic lung disease. Results of the study advised against the use of corticosteroids

because of its adverse effects on neuromotor function and somatic growth. In addition,

the preterm infants also showed transient albeit significant adverse effects such as

hyperglycemia, hypertension. Papile et al. (27) conducted a randomized clinical trial to

determine the efficacy of early vs late dexamethasone therapy in infants at a risk of CLD

and reported a decrease in head growth in infants who were receiving dexamethasone.

Stark et al. (28) studied the adverse effects of early dexamethasone treatment in

extremely low-birth weight (501-1000 g) infants who received mechanical ventilation

within 12 h after birth and were randomized to receive either placebo or dexamethasone.

Results of the study showed that treatment with dexamethasone was associated with

gastrointestinal perforation and decreased growth. In addition, alarming reports in the

literature document the termination of clinical trials involving postnatal corticosteroids

because of short-term adverse events, including gastrointestinal hemorrhage and

intestinal perforation requiring surgery (29). Murphy et al. (30) reported impaired

cerebral gray matter growth after treatment of premature infants with dexamethasone.

Israel et al. (31) showed in a retrospective study that prolonged treatment of premature

infants suffering from chronic lung disease with dexamethasone was associated with

hypertrophic cardiomyopathy.

Adverse effects of corticosteroids on the brain have also led to long-term

neurological complications. This concern has been amplified by two studies that show

significantly more infants with cerebral palsy (32) and reduced neuromotor function (26)








in corticosteroid-treated groups. In addition to the brain-related adverse effects, a variety

of adverse effects such as adrenal suppression, immune suppression, bradycardia, weight

loss and hyperglycemia have been reported (9). All this information gleaned from a wide

variety of scientific literature strongly suggests the adverse effects observed in preterm

infants after antenatal and postnatal systemic corticosteroid use. Consequently, the

observance of short- and long- term adverse effects, especially on the brain, has

simmered the enthusiasm for using corticosteroids systemically for the treatment and

prevention of CLD in preterm infants.

P-glycoprotein Transporters and Blood Brain Barrier

As previously mentioned, the systemic administration of corticosteroids results in a

variety ofneurotoxic adverse effects in preterm infants. This can be due to the enhanced

permeability of the corticosteroid across an immature blood brain barrier. The

immaturity of the blood brain barrier also leads to incomplete development of efflux

systems such as P-glycoprotein transporters.

P-glycoprotein (P-gp, MW 170 KDa) is a 128 amino acid transmembrane

glycoprotein and belongs to the family of ATP binding cassette (ABC) transporter

proteins. It is highly concentrated on the apical membrane of the endothelial cells of the

brain capillaries. It was originally identified because of it's ability to confer multi drug

resistance (development of resistance by cancerous cells against a variety of drugs) in

mammalian tumor cells (33). The efflux transporters present on the blood brain barrier

actively extrude a wide variety of structurally unrelated substrates such as ivermectin,

dexamethasone, vinblastin, digoxin, loperimide, domperideone, phenytoin, and

cyclosporine A (34, 35). Fig 1-3 schematically represents the blood brain barrier and

selected transport mechanisms.











brain tight
extracellularr fluid)A functions


endothelium V




c "^ .1
I~~~ is flB J^

0. E E.
W0
Fig 1-3: Schematic representation of the blood brain barrier and selected transport
mechanisms. The arrows indicate the direction of transport (taken from (36)).


The clinical implications of poor development of the p-glycoprotein transporters

(due to an immature blood brain barrier) have been previously shown by a number of

research groups. Smit et al. (37) have shown that the absence or pharmacological

blocking of placental p-gp profoundly increases fetal drug exposure. Lankas et al. (38)

have shown that the placental mdrla p-gp in mice is present in the fetus derived epithelial

cells and constitutes a barrier between the fetal and maternal blood circulation. Kalken et

al. (39) have studied the expression of p-gp transporters in human tissues at different

developmental stages using immunohistochemistry. They did not observe any staining of

the embryonic and fetal brain cells upto 28 weeks of gestation. This strongly indicates

the absence/poor development of p-gp transporters on the blood brain barrier in preterm

infants.

In sharp contrast, the permeability of systemically administered corticosteroids

across the blood brain barrier in adults is severely restricted. Dekloet et al. (40) have









shown that the permeability of dexamethasone, a systemic corticosteroid, is restricted

across the blood brain barrier of adult rats due to active efflux by the p-gp pump. In

addition, Talton et al.(41) and Wang et al. (42), using ex vivo receptor binding assay,

have evaluated the brain receptor occupancy after intravenous administration of two

widely used inhaled corticosteroids, fluticasone propionate (FP) and beclomethasone

monopropionate (BMP). Fig 1-4 shows the plot of percent free receptors as a function of

time after intravenous administration of FP and BMP.




150-
L.
0

A 100-

A BRAIN
50- --KIDNEY
LL 50-

I~0

0 2 4 6 8 10 12 14
TIME (hrs)


Fig 1-4: Brain and kidney receptor occupancy in rats after intravenous
administration (100 fig/kg) of (A) fluticasone propionate (B) beclomethasone
monopropionate. Data taken from references (41) and (42) respectively.












150-
1A
'0
4) 100- 1
B 1
- BRAIN
U.5- -50- KIDNEY

I I I I I+^----
0- -- ----------- -
0 2 4 6 8 10 12 14
Time (hrs)


Fig 1-4: Continued

The results clearly show that the permeability of inhaled corticosteroids is severely

restricted in adult rats (as indicated by minimal brain receptor occupancy). Chapter 2

provides a detailed evaluation of the role played by p-gp transporters in modulating the

brain permeability of inhaled corticosteroids.

Inhaled Corticosteroids In Chronic Lung Disease

Systemic corticosteroids have established profiles of beneficial effects and adverse

effects. This leads to the rational question that "how can treatment strategies with

systemic corticosteroids be optimized with respect to dose administered, timing of

intervention with corticosteroids after birth or perhaps by changing the route of drug

administration so that the beneficial effects of the corticosteroids can be maximized and

the adverse effects (from high systemic exposure) can be minimized?"

The review of literature shows that a consensus is lacking on the dose that can be

used for the treatment or prevention of CLD. Yeh et al. (17) used 1 mg/kg/day of

dexamethasone for 3 days and then tapered the dose for 12 days. Stark et al. (43) used








dexamethasone within 24 h after birth (0.15 mg/kg/day) for 3 days and tapered it off over

7 days. 0' Shea and colleagues (32) used 0.5 mg/kg/day of dexamethasone and tapered

the dose over 42 days. The consensus on optimal dosing schedule is also lacking. Cole

and Fiascone (44) have shown that early use (< 2 weeks age) of systemic steroids leads to

reduction in CLD and mortality. They also showed that very early use (< 3 days of age)

elevates the risk of gastrointestinal complications. However, both of the schedules had

adverse effects.

An important parameter that can be modulated to increase beneficial effects and

decrease systemic exposure to corticosteroids is the route of drug administration.

Delivery of corticosteroids through the inhalation route is a plausible alternative. Major

advantages of delivering drugs through the inhalation route include direct delivery of the

drug to the site of inflammation (i.e., the lungs), rapid onset of action, lower doses needed

for effective therapy leading to less spill-over into the systemic circulation and

accessibility to systemic circulation without traversing the liver (particularly suitable for

drugs that are systemically active but show a high first pass effect after oral

administration) by absorption across the pulmonary epithelium. The advantages of

delivering corticosteroids through the inhalation route for treating pulmonary

inflammatory disorders such as asthma have been clearly established. As the use of

corticosteroids to counteract the inflammatory reaction in the lung is the common

denominator between asthma and CLD, it can be expected that administering

corticosteroids through the pulmonary route to premature infants suffering from CLD

will result in a higher benefit/risk ratio.








A number of research groups have investigated the benefits of delivering a variety

ofcorticosteroids to premature infants through the pulmonary route. Amon et al. (45)

studied the clinical efficacy ofbudesonide (600 gg twice daily) vs placebo administered

by metered dose inhaler and spacer directly into the endotracheal tube of intubated

infants. Results showed a significant reduction in the need for mechanical ventilation in

the budesonide-treated group without concurrent adverse effects. Jonsson et al. (46)

showed that budesonide aerosol delivered through a dosimetric jet nebulizer decreased

the requirement for mechanical ventilation without significant adverse effects in

premature infants who were at a high risk for developing CLD.

On the other hand, a number of studies have shown the limited effectiveness of

inhaled glucocorticoid therapy in premature infants suffering from CLD. Groneck et al.

(47) did not observe any reduction in tracheal inflammatory markers after 10 days of

inhaled beclomethasone therapy (500 gg tid) initiated on day 3 of life in ventilated

infants compared to rapid reduction in tracheal inflammatory markers after 3 days of

systemic dexamethasone therapy (0.5 mg/kg/day). Dimitriou et al. (48) investigated the

degree and onset of the clinical response and adverse effects observed after a 10 day

course of either systemically administered dexamethasone (0.5 mg/kg/day) or nebulized

budesonide (100 jg qid) in a randomized trial of 40 preterm infants who required

mechanical ventilation after 5 days or supplemental oxygen for at least 14 days. Results

indicated a greater and faster onset of action after systemic administration of

dexamethasone. Inwald et al. (49) have previously shown elevated levels of chemokines

in the broncho-alveolar lavage (BAL) fluid of infants treated for respiratory distress

syndrome (RDS). To study the effect of inhaled budesonide in reducing the levels of








chemokines, Inwald et al. (50) measured the levels of chemokines in 12 preterm infants

who were ventilated for RDS. No significant changes in the levels ofchemokines were

found in the inhaled budesonide group. Cole et al. (51) conducted a multicenter trial to

determine if inhaled beclomethasone dipropionate in premature infants (< 33 weeks of

gestation) would reduce the frequency ofbronchopulmonary dysplasia (BPD). Results

showed a similar frequency of BPD in the beclomethasone and placebo treated groups;

however, fewer infants in the inhaled beclomethasone therapy group required additional

systemic corticosteroids or mechanical ventilation.

One should be very cautious in interpreting the limited success of inhalation

therapy in premature infants. Results should be analyzed in light of the various

complexities associated with delivering aerosolized medication to preterm infants. The

clinical efficacy of aerosolized corticosteroids for treatment of pulmonary disorders such

as CLD is contingent on stringent control of a variety of factors. These factors include

amount of dose deposited in the lungs, particle size and regional distribution of the

deposited dose in the lung, and device used to deliver the dose. In addition to these

factors, patient-related factors such as degree of lung development influence the clinical

efficacy of inhaled formulations. Further, the highly lipophilic nature of the

corticosteroids in conjugation with high absorptive surface provided by the pulmonary

epithelium results in rapid absorption of most commercially available glucocorticoids

such as flunisolide, triamcinolone acetonide, beclomethasone dipropionate and

budesonide (Fig 1-5). This rapid absorption from the lungs into the systemic circulation

results in low pulmonary residence time (the time for which the drug stays in the lung









before being absorbed into the systemic circulation) leading to very low

pharmacologically active pulmonary corticosteroid concentrations.

CH2OH f H2OH
I c=O
C--O CHs HOCH
CH3 / 3HO CH HCHzCH2CH3
HO 0Ho C -0.
CH 6 3 HH3




Triamcinolone acetonide Budesonide O
II
CH20H H 2C-O-C-C-CH3
I H 2
C--O CH3 H CH31 11
C-3U / HO. ;,.s -O-C-C--CH3
HOH 0-O -- CC H / C H2

C1 H
0 O~0

F
Flunisolide Beclomethasone dipropionate
Flunisolide

Fig 1-5: Chemical Structures of Some Commonly Used Inhaled Corticosteroids.

Hochhaus et al. (52), through a series of simulations, have shown that inhaling a

glucocorticoid solution does not necessarily result in pulmonary targeting because a

solution is rapidly absorbed from the lung into systemic circulation. This leads to

adverse systemic effects and a lower benefit-to-risk ratio. Hence, alternative strategies

for drug delivery are urgently required that will increase the pulmonary selectivity of the

drug. This will help in achieving increased pulmonary targeting and reduction in

undesired systemic effects thereby leading to a higher benefit-to-risk ratio.

Strategies for Improving Pulmonary Selectivity

The ultimate goal of achieving pulmonary selectivity is a reduction in dose required

to produce the desired beneficial effects with concomitant reduction in adverse effects.








For all forms of pulmonary administration, only a small portion of the drug is delivered to

the lungs whereas the major part of the drug is deposited in the oropharynx and

consequently swallowed. The portion of the drug reaching the lungs is either rapidly

absorbed into systemic circulation or removed from the upper portions of the airways by

mucociliary transporters. The swallowed portion of the drug, depending on the oral

bioavailability, enters the systemic circulation where it can show systemic adverse

effects. Hence, the efficient removal of this systemically available drug (which is a

combination of drug coming from the lungs and the drug that is orally absorbed) is

pivotal for achieving pulmonary selectivity. It has been shown that a variety of local and

systemic factors are involved in achieving pulmonary selectivity (53). Table 1-2 lists the

factors important for achieving pulmonary targeting

Table 1-2: Factors for Achieving Pulmonary Targeting.

Pulmonary Components Systemic Components

Efficiency of pulmonary deposition Oral bioavailability
Pulmonary residence time Clearance
Pulmonary absorption rate Volume of distribution
Pharmacodynamic drug
characteristics in the lung

As indicated in the table, one of the factors that plays a key role in determining

pulmonary selectivity is the pulmonary residence time. A variety of approaches can be

adopted to increase the pulmonary residence time of the drug. These approaches include:

1) slow dissolution rate of the drug particles, 2) corticosteroid esterification (in case of

budesonide) and 3) use of slow release systems such as liposomes and nano-coatings.

The data available for inhaled corticosteroids suggests that drugs with slower

dissolution rate such as fluticasone propionate show higher pulmonary targeting (54). In








addition, using an animal model for pulmonary targeting, it was shown that the degree of

pulmonary targeting of intratracheally administered TA increased from solutions to

micronized particles to crystal suspensions (55, 56).

Recent biochemical studies have shown that budesonide, a widely used inhaled

corticoseroid, is intracellularly esterified (57, 58). These esters are unable to traverse the

pulmonary membranes and are trapped as inactive "pro-drugs". The esters are eventually

cleaved by esterases present in the lung thereby releasing the active drug. Although this

is a novel mechanism to increase the pulmonary residence time, more studies are required

to determine whether the drug being "trapped" as ester represents a clinically relevant

portion of the dose.

The use of sustained release systems has gained widespread attention during the

last two decades. Better control over the rate of drug release, less frequent drug

administration, improvement in patient compliance and reduction in the fluctuation of

plasma levels are some of the factors that have led to the successful adoption of

sustained-release drug-delivery systems in a variety of therapeutic areas (such as

ophthalmic, transdermal etc.). In addition to the advantages previously mentioned, a

major advantage of sustained-release formulation is retention of the drug in the local area

for a longer period of time. This leads to an increase in the local effects and significant

reduction of systemic exposure. In contrast, conventional dosage forms provide

immediate release of the drug that necessitates frequent dosing for maintenance of

therapeutic levels.

As previously noted, the enthusiasm of using systemic corticosteroids in preterm

infants suffering from chronic lung disease has simmered due to high incidence of








systemic adverse effects. Hence, the pulmonary delivery of sustained release

formulations of inhaled corticosteroids are expected to exhibit higher pulmonary

residence time thereby leading to significant improvement in pulmonary selectivity.

Appropriate modifications of the drug/delivery system can potentially result in a wide

spectrum of sustained release formulations that markedly differ in their pharmacokinetic/

pharmacodynamic properties when compared with the conventional form of the drug.

Sustained Release Drug Delivery Systems

Liposomal and microencapsulated (polymer coated) formulations such as

microspheres have gained widespread attention for their ability to provide sustained

release of the encapsulated drug. Next is a brief description, including pharmaceutical

applications and potential limitations.

Liposomes

Liposomes have evolved into a major class of drug-delivery systems since their

discovery by Bangham et al. (59). Liposomes are microscopic vesicles composed of

multilamellar phospholipid bilayers alternating with hydrophilic compartments. The

drug, depending on its physicochemical characteristics, is either incorporated in the

aqueous or the lipid bilayer. The size (diameter) of the liposomal formulation varies

from 20 nm to 20 grm. The ability of the liposomes to modulate the pharmacokinetics

and biodistribution of the encapsulated drug has provided impetus for their use in a

number of medical complications (such as cancer, fungal infections etc.). A number of

commercially available liposome based products such as DoxilTM (doxorubicin) and

ambisomeTM (amphotericin B) have obtained FDA approval and are being routinely used

(60).








The advantages of using liposomes in inhalation therapy have been well

documented (61). In addition to acting as a drug reservoir in the lungs, liposomes also

facilitate the achievement of high concentrations of the drug in the infected macrophages.

The similarity between the phospholipids used for preparation of liposomes and the

naturally occurring phospholipids (which form the surfactant system in the lungs)

minimizes the incidence of toxicity. Optimally designed liposome-based drug-delivery

systems can potentially prolong the pulmonary residence time of the drug and lead to

significant decrease in systemic exposure.

Rothi et al. (62) and Hochhaus et al. (63) have explored the use of triamcinolone

acetonide phosphate (TAP) as pulmonary targeted drug-delivery systems. They showed

that intratracheal administration of liposome encapsulated TAP provided sustained

receptor occupancy and improved pulmonary targeting in comparison to TAP solution.

Other groups have also reiterated the beneficial effects of encapsulating drugs in

liposome to increase the pulmonary retention time. For example, Brattsand and co-

workers (64) demonstrated that budesonide palmitate liposomes, but not budesonide,

showed improved pulmonary targeting in a rat alveolar model of pulmonary

inflammation. Although the various formulation parameters such as choice of lipids,

incorporation of cholesterol (for decreasing the permeability of the bilayer to avoid

leakage etc.) influence the liposomal characteristics such as size, encapsulation

efficiency, the route of administration of the liposomal formulation ultimately determines

the PK/PD profile of the entrapped drug.

The liposomal encapsulated formulations also have potential limitations such as

low shelf life stability, leakage of the encapsulated drug, inability to permeate capillary








endothelial cells in the intact form, and low encapsulation efficiencies with hydrophilic

drugs.

Microencapsulation

Microencapsulation is a technique of applying thin coatings to small solid particles.

The basic parameters that need to be taken into account while designing

microencapsulated formulations include the core (i.e., the active drug), the coating

material (which to a large extent governs the physical and chemical properties of the

microencapsulated formulation), and the method used to microencapsulate the drug.

Flexibility in the choice of core material (solid particles or dispersed material) has

significantly contributed toward improving formulation acceptability (e.g., taste masking,

in the case of acetaminophen; reduction of gastric irritation from potassium chloride; and

stability toward oxidation for vitamin A palmitate) (65). The choice of the coating

material is, in part, contingent on the nature of the drug to be encapsulated, as the coating

material should be nonreactive and compatible with the active drug. In addition, the use

of biodegradable polymers such as poly (1-lactic acid (PLA) and poly (lactic-co-

glycolic acid) (PLGA) as coating material has also gained popularity because of the easy

bio-degradation of these polymers by in vivo enzymatic hydrolysis.

Microspheres

Microspeheres have gained widespread importance as pulmonary drug delivery

systems due to several advantages such as higher shelf life and longer in vivo retention of

the drug as compared to liposomes. Respirable PLGA micropsheres of rifampin have

been shown to reduce the incidence of inflammation and lung damage in a guinea pig

lung infection model(66). Kawashima et al. (67)have shown the utility of pulmonary

delivered insulin with nubulized PLGA microspheres to prolong the hypoglycemic effect.








PLGA microspheres of isoproterenol have been shown to reduce bronchoconsriction(68).

The coating of biodegradable polymers are mainly applied using spray drying technology

which significantly increases the polymer load. This has led to development of alternate

methods of coating the drug, which can reduce the polymer load on the drug particles.

One way to coat the drug is by using pulse laser deposition (PLD), a novel laser

based technique. The method essentially involves the deposition of ultra thin coatings

(10-1000 nanometers) of biodegradable polymers on the drug particles that are typically

in the size range of 1-5 tm. This results in an extremely low polymer load (generally

less than 1% by mass) (69). Fig 1-6 provides a schematic diagram of the PLD set up

which is used to deposit polymeric coatings on drug particles.


polymer target pulsed laser beam





plume 1.



fluid ization cor p
system core particles




vacuum chamber
Fig 1-6: Schematic Diagram of the PLD set up (taken from reference (41).

Briefly, the coating procedure consists of a biodegradable polymer target and a

fluidized bed of drug particles. The laser beam enters the vacuum chamber and ablates

the polymer target that forms the plume. The plume is consequently deposited on the








fluidized drug particles. Various factors such as choice of the polymer, coating time can

be optimized to obtain sustained release formulations. The microencapsulated

formulation thus obtained is expected to sustain the release of the drug powder, thereby

leading to higher pulmonary residence time and improved pulmonary targeting.

The in vivo efficacy of microencapsulated (using PLD) corticosteroids has been

shown in adult rats by Talton et al. (41). It was shown that microencapsulated

budesonide dry powder exhibited slower pulmonary absorption and significant increase

in pulmonary targeting as compared to the free powders of budesonide in adult rats.

Assuming the applicability of these results to the neonatal rat model, higher pulmonary

targeting can be expected after pulmonary delivery of microencapsulated corticosteroids.

Objectives

The following specific aims were tested:

To study the role played by p-gp transporters in modulating the brain
permeability of inhaled corticosteriods in mice.

To determine the pulmonary targeting and investigate the potential reasons for
differences in brain receptor occupancies between neonatal and adult rats after
intratracheal instillation of triamcinolone acetonide phosphate (TAP) solution.

To determine whether intratracheal instillation of poly (1-lactic acid) (PLA)
encapsulated budesonide demonstrates pulmonary targeting in the neonatal rat
model.














CHAPTER 2
ROLE OF P-GLYCOPROTEIN TRANSPORTERS IN MODULATING THE BRAIN
PERMEABILITY OF INHALED CORTICOSTEROIDS

Introduction

The blood brain barrier (BBB) restricts the entry of a variety of therapeutically

active agents from the systemic circulation into the central nervous system (CNS). The

endothelial cells of the brain capillaries, connected via tight junctions, form a physical

barrier and limit the penetration ofhydrophilic substrates. In addition, the efflux

transporters present on the BBB actively extrude a wide variety of structurally unrelated

substrates such as ivermectin, vinblastin, digoxin, loperimide, domperideone, phenytoin,

and cyclosporine A (34, 35). This active extrusion by the efflux pumps has severely

limited the clinical efficacy of therapeutic moieties used for treating brain cancer (70) and

HIV infections in the brain (71). As previously mentioned, P-glycoprotein (P-gp) plays a

very critical role in regulating the movement of xenobiotics across the blood brain

barrier.

The availability of knockout mice has proven to be a major tool to investigate the

role of p-glycoprotein transporters in modulating the permeability of drugs across the

BBB (72). Using this model, Schinkel et al. (72) have shown that the levels of

ivermectin in the brain ofmdrla (-/-) increased about 90 fold as compared to wild type

mice. Mayer et al. (73) showed that digoxin accumulated in the brain ofmdrla (-/-)

mice, which was in sharp contrast to very low levels in wild type mice. Schinkel et al.

(35) showed a sevenfold and fourfold increase in the levels of loperamide and









ondansetron respectively in mdrla (-/-) than wild type mice. These results clearly

demonstrate the pivotal role played by P-gp in modulating the permeability of drugs

across the blood brain barrier and show that the presence or absence of p-gp on the blood

brain barrier can either restrict the permeability or lead to significantly elevated levels of

the P-gp substrates.

The primary focus of our work was to evaluate whether p-gp transporters influence

the permeability of triamcinolone acetonide phosphate (TAP), one of the clinically

relevant inhaled glucocorticoid. A previously developed (55) ex vivo receptor binding

assay was used to monitor the free cytosolic receptors in the brain and liver of wild type

and knockout mice after intravenous administration of 100 tg/kg TAP. This assay was

used because it is a surrogate marker of pharmacologically relevant free drug

concentrations in different tissues.

Hypothesis

We expect to see significantly higher brain receptor occupancy in mdr l a mice due

to absence ofp-gp transporters. To test our hypothesis, the brain and liver receptor

occupancies were monitored in wild type and mdrl a mice after intravenous

administration of TAP (100 tg/ kg).

Materials and Methods

Triamcinolone acetonide phosphate solution (TAP) (54.4 mg/mL) was obtained

from Bristol Myers Squibb (BMS), Munich, Germany. ({6,7- 3H} triamcinolone

acetonide, 38 Ci/mmol) was obtained from New England Nuclear (Wilmington, DE). All

other unlabelled chemicals were obtained from Sigma (St. Louis, MO) or equivalent

sources.








Preparation of Drug and Radiolabelled Solutions

TAP solution (54.4 mg/mL) was diluted with PBS to obtain a final concentration of 50

[tg/mL. Suitable volume of this solution (equivalent to 100 tg/ kg) was injected into the

mice through the tail vein. 20 nM 3H labeled TA (prepared in the incubation buffer) and

a mixture of 20 nM 3H labeled TA and 20 tM unlabelled TA was used to determine the

total and non- specific binding respectively.

Animal Procedures

All animal procedures were approved by the Institutional animal care and use

(IACUC), University of Florida, an Association for the Assessment and Accreditation of

Laboratory Animal Care (AAALAC) approved facility. Wild type mice and mdria

knockout mice (30 + 5 g) were obtained from Taconic (Germantown, NY) and were

housed in sterile pathogen free (SPF) environment. The animals were housed in the

operating room 12 h before the experiment to accustom them to the new environment.

On the day of the experiment, the mice were gently handled (to produce minimum stress)

and weighed. The mice were anesthetized with an anesthetic mixture (1.5 ml of 10 % v/v

ketamine, 1.5 ml of 2 % v/v xylazine and 0.5 ml of I % v/v acepromazine) at the dose 1

ml/kg. The depth of anesthesia was checked using tail pinch or pedal withdrawal reflex.

Once the mice were under complete anesthesia, either 100-125 iL of glucocorticoid

(TAP) solution or saline (for placebo) was slowly injected into the tail vein using a

tuberculin syringe with a 27-guage needle. The mice were decapitated at 1, 2.5 and 6

hours after tail vein injection of the glucocorticoid drug solution. The brain and liver

were removed and immediately processed for receptor binding studies.








Ex Vivo Receptor Binding Assay

A previously developed ex-vivo receptor binding assay was used (63). Immediately

after decapitation, the brain and liver were rejected and placed on ice. The weighed

tissue was added to 10 times (for liver) and 4 times (for brain) organ weight of ice-cold

incubation buffer (10 mM Tris/HCl, 10 mM sodium molybdate, 2 mM 1,4-

dithioerythritol). 2 mL of the homogenate was incubated with 5 % charcoal (in distilled

water) for 10 minutes to remove endogenous corticosteroids. The homogenate was

centrifuged for 20 min at 20,000 g at 4 C in a Beckman centrifuge equipped with a JA-

21 rotor to obtain a clear supernatant. Since the amount of cytosol obtained from various

tissues of mice was very less, for all mice experiments, the volume ofcytosol used, the

volume of tracer added, the volume of charcoal added to remove excess radioactivity and

the supernatant collected for reading in the scintillation counter were reduced to half of

the volumes used for the rat experiments.

Aliquots of the supernatant (75 QL) were added to pre-chilled microcentrifuge

tubes containing 25 tL of 20 nM 3H labeled TA or a mixture of 20 nM 3H labeled TA

and 20 tM of unlabelled TA to determine the total binding and the non-specific binding

respectively. The microcentrifuge tubes were vortexed and incubated at 4 C for 18 h.

After the incubation, 100 tL of activated charcoal (5 % in water) was added to the

microcentrifuge tubes to remove excess radioactivity. The microcentrifuge tubes were

vortexed, centrifuged for 5 minutes and 125 .tL of supernatant was removed and added to

the scintillation vial. 2.5 mL of the scintillation cocktail (CytoscintTM, ICN Biomed,

Costa Mesa, CA) was added and the scintillation vials were read in a scintillation counter

(Beckman, LS 5000 TD, Palo Alto, CA) to obtain the radioactive counts (measured in

disintegrations per minute (dpm's)) in different tissues.








For a given tissue (liver or brain), the radioactivity counts corresponded to the total

binding (specific + non specific) of the tracer. The dpm's corresponding to the non-

specific binding (obtained by incubating the cytosol with a high concentration of a

mixture of 20 nM 3H labeled TA and 20 tM unlabelled TA for mice experiments,

removing excess radioactivity and determining the radioactive counts in the supernatant)

was subtracted from the total binding to obtain estimates of the specific binding. The

specific binding obtained in the rats administered saline (placebo) corresponded to 100 %

free receptors. The % free receptors present in the brain or liver was calculated as

% free receptors in a tissue= specific binding in a tissue of rat administered TAP *100
specific binding in a tissue of rat administered saline

For each tissue, the cumulative AUCO-6h calculated from the % free receptors vs

time profile was subtracted from 600 (each hour of the experiment corresponds to 100 %

free receptors) to obtain the cumulative AUCO-6h for % bound receptors. The average

receptor occupancies (AUC) in the brain and liver for wild type and mdrl a mice were

obtained by dividing the cumulative receptor occupancy (AUCo-6h) by 6h (the duration of

experiment). The average receptor occupancies observed in each tissue was compared

using a student t test.

Results

Fig 2-1 and 2-2 show the % free receptors vs time profiles in the liver and brain

respectively, after intravenous administration (100 jtg/kg) of TAP to wild type and

knockout mice. Table 2-1 shows the average AUC estimates obtained in the brain and

liver. Intravenous administration of TAP resulted in similar average hepatic AUC in

mdrla and wild type mice (37.8 % vs 34.9 %) (p>0.05). However, the average brain









AUC in mdrl a deficient mice was significantly higher in knockout mice than wild type

mice (47.5 % vs 11.5 %) (p

-- WLD TYPE
-'- KNOCK OUT


6 7


Time (hrs)


Fig 2-1: Liver Receptor Occupancy in wild type and mdrla (-/-) mice after
intravenous administration (100 utg/kg) of triamcinolone acetonide
phosphate.



200-


LO150-
M-a-WLD TYPE MICE
1i 00- -A- KNOCK OUT MICE
\\'
IL \ -
os50- -- '
50

0 1 2 3 4 5 6 7
Tinme (hrs)

Fig 2-2: Brain Receptor Occupancy in wild type and mdrla (-/-) mice after
intravenous administration (100 gg/kg) of triamcinolone acetonide
phosphate.












Table 2-1: Average AUC's in the brain and liver of wild type and mdrla mice after
intravenous administration of TAP.


Dose Average AUC (%)
~________(jig/kg)
Brain Liver
Wild type mice 100 11.5 34.9
mdrla mice 100 47.5 37.8


Discussion

The blood -brain barrier regulates the composition of extra cellular fluid and

protects the brain against changes in the systemic circulation (74). The permeability

across the blood brain barrier increases with increasing lipophilicity but decreases again

when a maximum lipophilicity is achieved (75). However, the CNS permeability of some

lipophilic substances such as vinblastin (Log P= 1.7), vincristine (Log P=2.1) is very

limited. This can be explained on the basis of the presence of efflux mechanisms which

actively efflux the drugs from CNS into the systemic circulation. P-gp is one of the

efflux transporters that plays a critical role in modulating the permeability of xenobiotics

across the blood brain barrier.

Although corticosteroids are lipophilic and are expected to easily cross the blood

brain barrier, the limited amount of available literature clearly shows that penetration of a

number of systemically used glucocorticoids such as dexamethasone and prednisolone is

modulated by p-gp (76-78). In addition, Talton et al. (41) and Wang et al. (42) have used

a previously developed ex vivo receptor binding assay (55) to monitor the glucocorticoid

receptor occupancy in the brain and kidney after intravenous administration of a majority

of clinically relevant inhaled glucocorticoids such as budesonide, fluticasone propioinate,








beclomethasone dipropionate, beclomethasone monopropionate and triamcinolone

acetonide. The results from these studies have shown minimal brain receptor occupancy

after intravenous/intratracheal administration of inhaled corticosteroids thereby

suggesting the the involvement of efflux mechanisms. To establish a clear link between

the minimal receptor occupancy in the brain and the active efflux by the p-gp pump, the

brain and liver receptor occupancy was monitored in wild type and mdrl a adult mice

after intravenous administration of TAP. The average AUC estimates calculated from the

% free receptors vs time profiles clearly show significantly higher brain receptor

occupancy of TAP in knockout mice. This strongly indicates the involvement ofp-

glycoprotein transporters in the active efflux of corticosteroids from the brain thereby

modulating the pharmacologically active concentrations in the brain. The hepatic

receptor occupancies were similar for wild type and knock out mice.

Although similar results have been shown by De Kloet et al. (40) for systemic

corticosteroids, the assay methodology used and the nature (inhaled vs systemic) of

corticosteroid used in our study were different. De Kloet et al. measured the total

concentrations of subcutaneously administered 3H dexamethasone in mdrla (-/-) and

mdrl a (+/+) mice, whereas we used ex vivo receptor binding assays (surrogate marker of

free levels) to assess the corticosteroid receptor occupancies in the brain.

The incidence of drug-drug interactions due to modulation of brain permeability of

p-gp substrates have been widely reported (79-81). These and similar studies shed light

on the changes in disposition of p-gp substrates when co-administered with p-gp

modulators. The results of our study suggest that the brain permeability of inhaled

glucocorticoids is also modulated by p-gp transporters. Consequently, concomitant








administration of inhaled glucocorticoids and p-gp inducers/inhibitors such as quinidine

and verapamil can potentially lead to clinically relevant drug-drug interactions.

In conclusion, the results of our study strongly suggest the critical role played by p-

gp in modulating the permeability of inhaled glucocorticoids. The understanding of the

important role played by p-gp transporters in modulating the permeability of drugs across

the blood brain barrier will significantly contribute towards development of effective

medications for CNS related disorders.

Conclusions

We observed significantly higher brain receptor occupancy in knockout

mice than wild type after intravenous administration of TAP. This suggest

the extrusion of inhaled corticosteroids by the p-gp transporters thereby

preventing brain receptor occupancy.

These results in conjugation with the minimal brain receptor brain

occupancy observed after intravenous administration suggest the pivotal

role played by p-gp transporters in reducing pharmacologically relevant free

levels of inhaled corticosteroids.

The involvement of active transport mechanisms in modulating the brain

uptake of inhaled corticosteroids argue for the possibility of drug-drug

interactions.














CHAPTER 3
ASSESSMENT OF PULMONARY TARGETING AND BRAIN PERMEABILITY OF
TRIAMCINOLONE ACETONIDE PHOSPHATE, AN INHALED STEROID, IN
NEONATAL RATS USING EX VIVO RECEPTOR BINDING ASSAY

Introduction

Inhaled corticosteroids are highly lipophilic moieties and are rapidly absorbed

across the pulmonary epithelium into the systemic circulation (82, 83). This rapid

absorption of the corticosteroids into the systemic circulation may explain the high

incidence of adverse effects observed in preterm infants after systemic corticosteroid

administration. It has been previously shown that the extent of the pharmacological

effects (or side effects) of the glucocorticoid is directly related to the fraction of receptors

occupied (84). Hence, tracking corticosteroid receptor occupancy in the local (lungs) and

systemic (brain, liver) organs can potentially provide a reasonably accurate assessment of

the beneficial effects/side effects of inhaled corticosteroids.

As previously mentioned, the placental p-gp transporters play a very critical role

role in protecting the developing fetus against maternal xenobiotic exposure.

Consequently, the absence or pharmacological blocking of these transporters results in

increased fetal exposure(37, 38).

We used a previously developed ex vivo receptor binding assay to simultaneously

assess the fraction of receptors occupied in the local (lung) and systemic (liver, brain)

organs after intratracheal administration of different doses of triamcinolone acetonide

phosphate (TAP). The validity of such a model has been previously established in adult

rats by Hochhaus et al. (55) for assessing the pulmonary targeting observed after








intratracheal instillation of TAP solution and liposomal encapsulated TAP. The same

model has been utilized for determining the degree of pulmonary targeting in neonatal

rats.

Hypothesis

We expect to see similar pulmonary and hepatic receptor occupancies after

intratracheal administration of various doses of TAP. Further, we expect to see

significant brain receptor occupancy at the higher doses (25 and 50 utg/kg) of TAP. To

test our hypothesis, the local (lung) and systemic (liver and brain) receptor occupancies

were monitored in neonatal (10-11 days old) rats after intratracheal instillation of TAP at

at different doses (2.5, 25 and 50 ug/kg).

Materials and Methods

Triamcinolone acetonide phosphate solution (TAP) (54.4 mg/mL) was obtained

from Bristol Myers Squibb (BMS), Munich, Germany. Phosphate buffered saline (pH

7.4) was obtained from Cellegro (Mediatech, Hemdon, VA). ({6,7- 3H} triamcinolone

acetonide, 38 Ci/mmol) was obtained from New England Nuclear (Wilmington, DE). All

other chemicals were obtained from Sigma (St. Louis, MO) or equivalent sources.

Preparation of TAP and Radiolabelled Solution

TAP solution (54.4 mg/mL) was suitably diluted with PBS to obtain 50 jig/mL of

working stock solution. Suitable volumes of the working stock solution were

intratracheally administered at doses of 2.5, 25 and 50 jig/kg TAP.

10 nM 3H labeled triamcinolone acetonide (TA), prepared in incubation buffer

(mixture of 10 mM Tris/HCl and 10 mM sodium molybdate in cold water) was used as

tracer solution. A mixture of 10 nM 3H labeled TA and 10 gM unlabelled TA, prepared

in incubation buffer, was used to estimate the non-specific binding.








Animal Procedures

All animal procedures were approved by the institutional animal care and use

committee, (IACUC), University of Florida, an Association for the Assessment and

Accreditation of Laboratory Animal Care (AAALAC) approved facility. Neonatal rats

(10-11 days old) were obtained from Harlan (Indianapolis, Indiana). The rats were

anesthetized with an anesthetic mixture (1.5 ml of 10 % ketamine, 1.5 ml of 2 % xylazine

and 0.5 ml of 1 % acepromazine) at the dose of 1 ml/kg. The skin on the neck was

shaved and the area was cleaned with betadine solution. A 1-cm incision was made in

the skin with a sterile scalpel blade to expose the underlying musculature. The muscles

were gently teased apart with a sterile curved hemostat to expose the trachea. Silk suture

was passed under the trachea for further manipulation. An incision was made between a

pair of tracheal rings and either TAP solution (2.5, 25 or 50 ig/kg) or saline (to placebo

rats) was administered. Following surgery, animals were placed on a fresh drape

overlying a heating pad. The neonatal rats were kept warm with the aid of a heating pad

and overhead light and the body temperature was monitored via a mouse rectal probe

connected to a microprobe thermometer. The neonatal rats were decapitated at various

time points (1, 2.5, 4 and 6 h) and the lungs, liver and brain were removed. The weighed

tissue was added to 4 times (for lungs and brain) and 10 times (for liver) organ weight of

ice cold incubation buffer (10 mM Tris/HCl, 10 mM sodium molybdate, 2 mM 1,4-

dithioerythritol). The homogenate was incubated with 5 % charcoal (in distilled water)

for 10 minutes to remove endogenous corticosteroids. The homogenate was centrifuged

for 20 min at 20,000 X g at 4 C in a Beckman centrifuge equipped with a JA-21 rotor to

obtain a clear supernatant. Aliquots of the supernatant (150 gL) were added to pre-

chilled microcentrifuge tubes containing 50 uL of either 10 nM 3H labeled TA for








determining the total binding or a mixture of 10 nM 3H labeled TA and 10 tM of

unlabelled TA for determining the non-specific binding. The microcentrifuge tubes were

vortexed and incubated at 4 C for 18 h.

After the incubation, 200 jiL of activated charcoal (5 % in water) was added to the

microcentrifuge tubes to remove excess radioactivity. The microcentrifuge tubes were

vortexed, centrifuged for 5 minutes and 300 U.L of the supernatant was removed and

added to the scintillation vial. 5 mL of the scintillation cocktail (CytoscintTM, ICN

Biomed, Costa Mesa, CA) was added and the scintillation vials were read in a

scintillation counter (Beckman, LS 5000TD, Palo Alto, CA) to obtain the radioactive

counts (measured in disintegrations per minute (dpm's)) in different tissues.

For a given tissue (lung, liver or brain), the radioactivity counts (measured in

dpm's) correspond to the total binding (specific+ non specific) of the tracer. The dpm's

corresponding to the non-specific binding (obtained by incubating the cytosol with a high

concentration of unlabelled TA, removing excess radioactivity and determining the

radioactive counts in the supernatant) was subtracted from the total binding to obtain

estimates of the specific binding. The specific binding obtained in the rats administered

saline (placebo) corresponded to 100 % free receptors. The % free receptors present in

the lung, liver or brain was calculated as

% free receptors in a tissue= specific binding in a tissue of rat ad ministered TAP *. 100
specific binding in a tissue of rat administered saline

For each tissue, the cumulative AUCO-6h calculated from the % free receptors vs

time profile was subtracted from 600 (each hour of the experiment corresponds to 100 %

free receptors) to obtain the cumulative AUCo-6h for % bound receptors. The average

receptor occupancies (AUC) in the lung, liver and brain were obtained by dividing the








cumulative receptor occupancy (AUCO-6h) by 6h (the duration of experiment). The

differences in pulmonary and hepatic receptor occupancies (AUC lung-AUC liver) after

different doses were compared using student t test.

Results

Fig 3-1 (A-C), 3-2 (A-C) and 3-3 (A-C) show the plots of% free corticosteroid

receptors in the lung, liver and brain of neonatal rats as a function of time after

intratracheal administration of different (2.5, 25 and 50 ug/kg) doses of TAP. Fig 3-4

shows the plot of% free receptors as a function of time after intratracheal instillation of

100 tg/ kg of TA. Table 3-1 gives the average area under the curve (AUC) estimates

obtained from the plots of% free receptors vs time.

After intratracheal administration of 2.5 jg/kg TAP to neonatal rats, the average

lung, liver and brain receptor occupancies were 18.3 4.5 %, 17.4 13.5 %, -14.7 +

11.9%. After intratracheal administration of 25 jg/kg TAP, the average lung, liver and

brain receptor occupancies were 36.3 12.6 %, 45.3 7.8 % and 45.7 9.7 respectively.

However,.after administration of 50 jg/kg TAP, the lung, liver and brain receptor

occupancies were 59.9 9.4. %, 50.8 13.0, 47.0 + 10 respectively. As shown the table,

the average AUC estimates in the lung and liver were similar after intratracheal

instillation of various doses of TAP. However, the average AUC estimates in the brain

were significantly higher at 25 and 50 jtg/kg as compared to the lowest dose (2.5 jig/kg).





















U_
0
o* 100*
M
o
0
CD
1 0
Il 50


(A
0



I 100-
s10
loo
0
L-
U.
S50O


--Lung
Liver


0 2 4 6

Time (hrs)


-- Lung
-*- Brain


0 2 4 6

Time (hrs)


Fig 3-1: Percent free receptors vs time profiles in (A) lung vs liver (B) lung vs
Brain and (C) brain vs liver after intratracheal instillation of 2.5 gg/kg of TAP
in neonatal rats.





















--- Liver
-- Brain


0 2 4 6
Time (hrs)


Fig 3-1: Continued


W
L.
0
4)
0
1-
8
0:
5


-u-Lung
-*-- Liver


0 2 4 6 8
Time (hrs)
Fig 3-2: Percent free receptors vs time profiles in (A) lung vs liver (B) lung vs
brain and (C ) brain vs liver after intratracheal instillation of 25 tg/kg of TAP in
neonatal rats.














-- Lung
-- Brain


0 2


Time (hrs)


150-


1004

50-


-4


--- Liver
--- Brain


Time (hrs)


Fig 3-2: Continued


I :












































L
0



o
CL
J.

0
ul
a0


--Lung
-*-- Liver


0 2 4 6
Time (hrs)


--Lung
SBrain


0 2 4 6 8
Time (hrs)

Fig 3-3: Percent free receptors vs time profiles in (A) lung vs liver (B) lung vs brain
and (C) brain vs liver after intratracheal instillation of 50 tg/kg of TAP in
neonatal rats.























Liver
-- Brain


0 2 4 6 8
Time (hrs)


Fig 3-3: Continued


SLiver
-U-Brain


Time (hrs)
Fig 3-4: Percent free receptors vs time profiles in the brain and liver of adult rats
after intratracheal instillation of 100 gg/kg TA (data taken from reference(41)).


U)
L-
'0
100



U. 50

;5O


150
?A
U)
L-
0
OL
o
I,


. 50.








Table 3-1: Average AUC estimates in the lung, liver and brain after intratracheal
administration of 2.5, 25 and 50 tg/kg of TAP to neonatal rats and 100 jig/kg
to adult rats.
Average AUC (%)
Dose N Lung Liver Brain
(uig/kg)_________________
2.5 4 18.3 4.5 17.4 13.5 -14.7 11.9
25 2 36.3 12.6 45.3 7.8 45.7 9.7
50 4 59.9 9.4 50.8 13.0 47. 9.6
100* 3 63.1 8 11.3 6.7
* 100 jtg/kg of TA was administered intratracheally to adult rats, data from reference (41)

Discussion

A previously developed model by Hochhaus et al. (55) was used to simultaneously

assess the fraction of receptors occupied in the local (lung) and systemic (liver, brain)

organs after different doses of TAP in neonatal rats. The assay is a radioligand binding

assay which is used to monitor the decrease in % free receptors (increase in receptor

occupancy) as a function of time. Using this functional assay, we could determine the

receptor occupancy of TAP in different organs (lung, liver and brain) as a function of

time. TAP is a prodrug of triamcinolone acetonide (TA) and is efficiently metabolized to

TA (85).

We did not see appreciable pulmonary targeting after administering TAP solution

at the different doses (p > 0.05). This can be attributed to the rapid absorption of the TAP

solution from the lungs into the systemic circulation resulting in similar pulmonary and

hepatic receptor occupancies. In fact, Hochhaus et al. (52), through a series of computer

simulations, have shown that the rapid removal of the dissolved drug from the lungs and

its absorption into the systemic circulation results in similar pulmonary and systemic drug

levels leading to negligible pulmonary targeting. The results obtained from the computer

simulations have been experimentally corroborated by Talton et al. (41) and Suarez et al.








(63) for a wide variety of inhaled corticosteroids. In addition, we did not observe a clear

dose response relationship between different doses of TAP administered and the AUC's

obtained in the lung and liver. This can be due to saturation of the corticosteroid

receptors in the lung and liver at the different doses used. The log-linear nature of the

dose response relationship can explain the non-linearity observed between the doses of

TAP administered and the response (average AUC's) obtained in the different organs of

neonatal rats.

We used 10-11 days old rats as the neonatal rat model in our study. The pattern of

brain development is highly species specific. In mammals such as guinea pig and

primates, the majority of neurodevelopmental processes are completed in utero (86, 87).

However, in animals which give birth to immature young ones such as rats and mice, the

major portion ofneurodevelopment takes place after birth (88). Consequently, the

neonatal rat model used in our study represents a valid model to assess the

pharmacologically relevant concentrations of the corticosteroid in the brain that might be

linked to the neurotoxic adverse effects of corticosteroids in preterm infants.

An interesting and important observation from the dose response studies was that

TAP showed receptor occupancy in the brain of neonatal rats. As previously noted,

similar receptor binding studies performed in adult rats by Saurez et al. (89), Talton et al.

(41) and Wang et al (42) after have shown the absence of brain receptor occupancy in

adult rats irrespective of the corticosteroid used (budesonide, fluticasone propionate,

triamcinolone acetonide) and the route of administration (intravenous, intratracheal).

The lower brain receptor occupancy observed in adult rats after intratracheal

instillation of TA powder can be due to the higher hepatic clearance of the corticosteroid








from the systemic circulation. The efficient removal of the drug from the systemic

circulation in adult rats (due to well developed hepatic system) can result in less drug

available for entering the brain. On the other hand, the incomplete development of

hepatic metabolic pathways in neonatal rats can lead to higher systemic levels and

consequently higher availability of the drug to enter the brain. However, the ex vivo

receptor binding assay used simultaneously tracks the receptor occupancy in the local

(brain) and systemic (liver) organs. Our results show pronounced liver receptor

occupancy in adult rats that seem to suggest that higher clearance in adult rats cannot

explain the differences in brain receptor occupancy between neonatal and adult rats.

A potential reason for differences in brain receptor occupancies between neonatal

rats and adult rats can probably be due to the lack of a functional blood brain barrier in

neonatal rats. In addition, the higher brain receptor occupancy in neonatal rats suggests

the absence of a fully matured blood brain barrier. This absence of a fully matured blood

brain barrier can be one of the likely explanations for the neurotoxic adverse effects

observed in preterm infants after systemic corticosteroid administration.

Another reason for observing higher brain receptor occupancy in neonatal rats can

be the absence of fully functional p-gp transporters (due to immaturity of the blood brain

barrier). As described in chapter 2 the results from studies with wild type and mdrl a (-

/-) mice have explicitly shown that after intravenous administration of TAP (100 jg/kg)

to knockout mice and wild type mice, there is a significantly higher brain receptor

occupancy in knockout mice as compared to wild type mice (90). Matsuoka et al. (91)

have studied the expression of p-gp transporters in the brain of rats as a function of

gestational age. It was shown that p-gp was undetectable until postnatal day 7, after








which the p-gp expression showed a steady increase to reach a plateau at day 20 with

about 25 % development at day 10. Since the neonatal rats used in our study were 10-11

days old, there is a strong possibility that the enhanced permeability of TAP in the brain

of neonatal rats (resulting in significantly higher brain receptor occupancy) was a

consequence of incomplete development of the p-gp transporters (due to immaturity of

the blood brain barrier). The presence, albeit insignifant, of p-gp transporters in 10 day

old rats can probably explain the absence of brain receptor occupancy (due to active

extrusion of TAP by the p-gp transporters) at the lowest dose used in our study (2.5

tg/kg). Further, the negative brain receptor occupancy observed at the lowest dose was

most likely due to the inherent variability of the assay used. Although there was

significant brain receptor occupancy at higher doses (25 and 50 ig/kg) as compared to

the lowest dose, the absence of a linear relationship between the higher doses of TAP

administered and the brain receptor occupancy can be attributed to saturation of p-gp

transporters resulting in reduced efflux of TAP. This further indicates the critical role

played by p-gp in modulating the permeability of corticosteroids across the blood brain

barrier.

The presence of fully functional p-gp transporters (due to fully developed blood

brain barrier) in adult rats can possibly explain the minimal brain receptor occupancy

observed after intratracheal insitllation of TA (fig 3-4). This active efflux by p-gp results

in very low pharmacologically relevant brain concentrations. In addition, similar results

have been reported by deKloet et al. (40) who have shown that dexamethasone poorly

penetrates the brain of adult rats. Although our results, together with the results of

Matsuoka et al. implicate the poor development of p-gp transporters on the blood brain








barrier as one of the major factors for increased permeability of TAP in neonatal rats,

more conclusive biochemical studies need to be performed to study the expression ofp-

gp transporters in preterm infants. This would lead to a significant understanding of the

role played by p-gp transporters in modulating the permeability of corticosteroids in

humans. The information on the expression of p-gp transporters as a function of

gestational age can be utilized for making detailed dosing recommendations for

antenatal/postnatal corticosteroid therapy in preterm infants suffering from CLD.

Conclusion

A previously developed ex vivo receptor binding method was successfully adapted

in neonatal (10-11 days old) rats to simultaneously monitor the glucocorticoid

receptor occupancy in the local (lung) and systemic (liver, brain) organs after

intratracheal instillation of different doses of TAP.

We did not observe a clear dose response relationship between different doses of

TAP used and the average AUC estimates obtained in the lung, liver and brain.

This can probably be due to saturation of glucocorticoid receptors at the various

doses used and the log linear relationship between dose and response.

The results show significantly higher brain receptor occupancy at higher doses in

neonatal rats than adult rats thereby suggesting the lack of a functional blood

brain barrier in preterm infants. This is in close agreement with the observance of

adverse effects in preterm infants after systemic corticosteroid administration.

We did not observe brain receptor occupancy in adult rats after intratracheal

instillation of TA. This can be attributed to the active efflux of TA by p-gp

transporters.





49


* The results from our study underscore the important and urgent need to develop

targeted delivery systems to the lungs for administering inhaled corticosteroids to

preterm infants. This will greatly assist in increasing the local effect of steroid in

the lungs and reduce the systemic spill over thereby increasing the benefit to risk

ratio.














CHAPTER 4
PULMONARY TARGETING OF SUSTAINED RELEASE FORMULATION OF
BUDESONIDE IN NEONATAL RATS

Introduction

The delivery of corticosteroids through the inhalation route for the

treatment/prevention of chronic lung disease has gained attention in recent years.

However, as previously noted, a number of studies have also shown the limited

effectiveness of inhaled glucocorticoid therapy in premature infants suffering from CLD

(47,48).

The limited success of inhaled corticosteroid therapy in preterm infants can be, in

part, explained on the basis of rapid absorption of lipophilic corticosteroids across the

high absorptive surface provided by the pulmonary epithelium (83). This rapid

absorption from the lungs into the systemic circulation results in very low

pharmacologically active pulmonary drug concentrations and low pulmonary residence

time (the time for which the drug stays in the lung before being absorbed into the

systemic circulation). This leads to adverse systemic effects and a lower benefit to risk

ratio. Hence, alternative strategies for drug delivery are urgently required which will

increase the pulmonary residence time of the drug thereby increasing the desired local

effects with concomitant reduction in systemic exposure.

Recently, the use of pulse laser deposition (PLD) technique to coat drug particles

with nano thin films (thereby significantly reducing the polymer load) of biodegradable

polymers such as poly (1-lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) has








gained widespread attention (92, 93). The polymer coated formulation thus obtained is

expected to sustain the release of the drug powder thereby leading to higher pulmonary

residence time and improved pulmonary targeting. Talton et al. (93) have performed in

vitro (using dissolution tests) and in vivo (using ex vivo receptor binding assay)

characterization of PLGA coated budesonide and PLA coated triamcinolone acetonide

dry powders. They showed that the half-life of release (ts50 %) of polymer coated

budesonide was significantly higher as compared to uncoated budesonide (60 1.6 min

vs 1.2 min). Using a previously developed ex vivo receptor binding assay (55), it was

shown that the alteration in dissolution behavior of the coated budesonide translated into

significant improvement in pulmonary targeting.

Hypothesis

We hypothesize that the pulmonary instillation of PLA coated budesonide in

neonatal (10-11 days old) rats will also result in sustained lung receptor occupancy and a

higher degree of pulmonary targeting as compared to uncoated budesonide. To test our

hypothesis, ex vivo receptor binding assays were performed in neonatal (10-11 days old)

to track the % free receptors in the lung, liver and brain after intratracheal administration

of uncoated/polymer coated budesonide. The average receptor occupancies (AUC 0-6 h /

6) in the lung, liver and brain and the pulmonary targeting (defined as AUC iung/AUC liver)

were obtained from the % free receptors vs time profiles.

Materials and Methods

Micronized BUD was obtained from Astra Zeneca Pharmaceuticals (Wilmington,

DE). Extra fine lactose monohydrate was donated from EM industries (Hawthrone, NY).

Phosphate buffered saline (PBS) (pH 7.4) was obtained from Cellegro (Mediatech,

Hemdon, VA). ({6,7- 3H} dexamethasone, 35-40 Ci/mmol) was obtained from New








England Nuclear (Wilmington, DE). All other chemicals were obtained from Sigma (St.

Louis, MO) or equivalent sources.

Preparation of Uncoated/PLA coated Budesonide Suspensions and Radiolabelled
Solutions

0.4 % of the uncoated/ PLA coated budesonide powders were prepared in extrafine

lactose. Approximately 6.25 mg of the powders (equivalent to 25 gg of the free drug)

were weighed in a 1.5 ml tubes. 300 jtl of the PBS was added prior the administration.

This suspension was intratracheally administered (50 gig/kg) to the neonatal rats.

25 nM 3H labeled dexamethasone was prepared in incubation buffer (mixture of 10

mM Tris/HCl and 10 mM sodium molybdate in cold water) and used as tracer solution.

A mixture of 25 nM 3H labeled dexamethasone and 25 p.M unlabelled dexamethasone,

prepared in incubation buffer, was used to estimate the non-specific binding.

Coating Procedure

The PLA polymer target was prepared in a Carver Press(Wabash, IN). One gram

of polymer was weighed, transferred into a 1 inch x 0.25 inch circular mold and pressed

with 2500 psi at 100 C for 10 min. A pulsed excimer laser using Krypton Fluoride

source (.=248 rnm) was used to ablate the polymer in a vacuum chamber. A 5 Hz laser

frequency was used to perform the ablation. The polymer was ablated onto 100 mg of

fluidized micronized BUD within the same chamber for 1 h. The coating procedure was

performed by personnel at the Engineering Research Center (ERC), University of

Florida.

Animal Procedures

All animal procedures were approved by the institutional animal care and use

committee, (IACUC), University of Florida, an Association for the Assessment and








Accreditation of Laboratory Animal Care (AAALAC) approved facility. Neonatal rats

(20 + 5 g) were obtained from Harlan (Indianapolis, Indiana). The neonatal rats were

anesthetized with an anesthetic mixture (1.5 ml of 10 % v/v ketamine, 1.5 ml of 2 % v/v

xylazine and 0.5 ml of 1 % v/v acepromazine) at the dose of 1 ml/kg. The skin on the

neck was shaved and the area cleaned with betadine solution. A 1-cm incision was made

in the skin with a sterile scalpel blade to expose the underlying musculature. The

muscles were gently teased apart with a sterile curved hemostat to expose the trachea.

An incision was made between a pair of tracheal rings and uncoated/coated budesonide

(50 jg/kg) suspension was intratracheally administered. The placebo rats were

administered saline. Following surgery, the rats were placed on a fresh drape overlying a

heating pad and were kept warm with the aid of a heating pad. The rats were decapitated

at 1, 2.5, and 6 h and the lung, liver and brain were removed. The weighed tissue was

added to 10 times (for liver) and 4 times (for lung and brain) organ weight of ice-cold

incubation buffer (10 mM Tris/HCl, 10 mM sodium molybdate, 2 mM 1,4-

dithioerythritol). The homogenate was incubated with 5 % charcoal (in distilled water)

for 10 minutes to remove endogenous corticosteroids. Aliquots of the supernatant (150

gL) were added to pre chilled microcentrifuge tubes containing 50 jiL of either 25 nM 3H

labeled dexamethasone for determining the total binding or a mixture of 25 nM 3H

labeled dexamethasone and 25 ptM of unlabelled dexamethasone for determining the non-

specific binding. The microcentrifuge tubes were vortexed and incubated at 4 0 C for 18

h.

After the incubation, 200 uL of activated charcoal (5 % in water) was added to the

microcentrifuge tubes to remove excess radioactivity. The microcentrifuge tubes were








vortexed, centrifuged for 5 minutes and 300 g.L of the supernatant was removed and

added to the scintillation vial. 5 mL of the scintillation cocktail (CytoscintTM, ICN

Biomed, Costa Mesa, CA) was added and the scintillation vials were read in a

scintillation counter (Beckman, LS 5000TD, Palo Alto, CA) to obtain the radioactive

counts (measured in disintegration per minute (dpm's)) in various tissues.

For a given tissue (lung, liver or brain), the radioactivity counts (measured in

dpm's) corresponded to the total binding (specific + non specific) of the tracer. The

dpm's corresponding to the non-specific binding (obtained from incubating the cytosol

with a mixture of 25 nM 3H labeled dexamethasone and 25 tM unlabelled

dexamethasone, removing excess radioactivity and determining the radioactive counts in

the supernatant) was subtracted from the total binding to obtain estimates of the specific

binding. The specific binding obtained in the rats administered saline (placebo)

corresponded to 100 % free receptors. The % free receptors present in the brain or liver

was calculated as

% free receptors in a tissue= specific binding in a tissueofrat administered TAP *100
specific binding in a tissue of rat administered saline

For each tissue, the cumulative AUCO-6h calculated from the % free receptors vs

time profile was subtracted from 600 (each hour of the experiment corresponds to 100 %

free receptors) to obtain the cumulative AUCO-6h for % bound receptors. The average

receptor occupancies (AUC) in the brain and liver for wild type and mdrl a mice were

obtained by dividing the cumulative receptor occupancy (AUCo-6h) by 6h (the duration of

experiment). The differences in pulmonary and hepatic receptor occupancies (AUC iung-

AUC liver) after different doses were compared using student t test.








Results

Fig 4-1 (A-B) shows the % free receptors as a function of time in the lung and liver of

neonatal rats after administration of uncoated and polymer coated budesonide

respectively. Fig 4-2 (A-B) shows the % free receptors as a function of time in the lung

and brain of neonatal rats after administration of uncoated and polymer coated

budesonide respectively. Table 4-1 shows the pulmonary targeting and the average

receptor occupancy estimates obtained in the lung, liver and brain of neonatal rats after

intratracheal administration of uncoated/coated budesonide. The average receptor

occupancy in the lung, liver and brain after intratracheal administration of micronized

uncoated budesonide were 58.4 12.9 %, 56.4 6.8 % and 38.3 6.7 %. However, after

administration of PLA coated budesonide, the average AUC estimates in the lung, liver

brain were 75.8 3.7 %, 46.6 + 14.5 % and 29 7 %. The average receptor occupancies

in the lung and liver after administration of uncoated budesonide were similar (p>0.5).

However, the average lung and liver receptor occupancies after administration of PLA

coated budesonide were significantly different (p < 0.05). The pulmonary targeting

(AUCiung/AUCiver) after intratracheal administration of uncoated budesonide was 1.03

0.13 and 1.72 0.46 respectively.













(0

u
o










LL
O
U,








I-
0








1



U.
e








I.
0
0.


-u--Lung
-*- Liver























-- Lung
-*-Liver


5 6 7


Fig 4-1: Percent free receptors vs time in the lung and liver of neonatal rats after
intratracheal instillation of (A) micronized uncoated budesonide and (B)
PLA coated budesonide (50 gtg/kg).


3 4 5 6 7
TIME (hrs)


0 1 2 3 4
TIME (hrs)










150-
UA
0
100
A I
S
Ix
u. 50-


-*-Brain
=Lung


6 7


Time (hrs)


75-1 ---Brain
"I -u- Lung

0
u. 50- \ ~f ^"
25-
0 I I I I

0 1 2 3 4 5 6 7
Time (hrs)
Fig 4-2: Percent free receptors vs time in the lung and brain of neonatal rats after
intratracheal instillation of (A) micronized uncoated budesonide and (B)
PLA coated budesonide (50 tg/kg).








Table 4-1: Average AUC's (n=3) in the lung, liver and brain and pulmonary targeting
(PT) in neonatal rats after intratracheal administration (50 ig/kg) of uncoated
budesonide and PLA coated budesonide.

Average AUC ( PT (AUCiung/
Formulation Dose (gg /kg) Lung Liver Brain AUCiiver)
Uncoated 50 58.4 12.9 56.4 6.8 38.3 6.7 1.03 0.13
Budesonide____________
PLA Coated 50 75.8 3.7 46.6 14.5 29 7 1.72 0.46
Budesonide_____________________________________

Discussion

The enthusiasm of using inhaled corticosteroid therapy in preterm infants for the

treatment/prevention of chronic lung disease has simmered due to the observance of

extrapulmonary adverse effects. Hence, treatment strategies need to be developed which

can improve the clinical effectiveness (topical efficacy:systemic activity) of inhaled

corticosteroids.

The last few years have witnessed the development of inhaled corticosteroids such

as budesonide and fluticasone propionate based on optimized pharmacokinetic properties.

Ideally, an inhaled corticosteroid should produce therapeutic effect at the pulmonary site,

should have minimum oral bioavailability and should be rapidly cleared once it is

absorbed into the systemic circulation. In addition to all these factors, another parameter

that is responsible for achieving improvement in pulmonary selectivity is the pulmonary

residence time.

The pulmonary residence time is governed by a combination of factors such as the

release rate of the drug (from the powder/delivery system), rate of absorption into

systemic circulation and the pulmonary clearance of the drug by the mucociliary

transporters. Computer simulations have shown that rapid release of the drug (incase of

solutions) results in fast absorption leading to similar pulmonary and systemic drug levels








and loss of pulmonary targeting (expressed as the difference between pulmonary and

systemic receptor occupancies) (52). As the dissolution rate (release rate) is decreased,

the pulmonary targeting increases and reaches a maximum at an "optimal" dissolution

rate. Further reduction is dissolution rate leads to pulmonary clearance of a major portion

of the drug via the mucociliary transporter before the drug can show its therapeutic effect.

Hence, a sustained release system optimized for the release rate can potentially lead to

pronounced pulmonary selectivity.

As previously noted, a variety of approaches such as slowly dissolving drug

particles, intracellular formation of esters (incase ofbudesonide) and slow release

systems such as liposomes and microspheres can be employed to increase the pulmonary

residence time. The in vivo utility of slow release systems to increase the pulmonary

residence time has also been emphasized using animal models. Gonzales rothi et al. (62)

have shown that pulmonary instillation of liposomal encapsulated triamcinolone

acetonide phosophate (TAP) resulted in increased pulmonary residence time thereby

leading to improved pulmonary targeting. Similarly, Brattsand et al. (64) have reported

increase in pulmonary selectivity with budesonide palmitate liposomes. However,

limitations such as leakage of encapsulated material and low encapsulation efficiencies

have limited the use of liposomes as model systems for demonstrating pulmonary

targeting. In order to overcome these formulation related limitations, we used the PLD

method for preparing polymer coated sustained release formulation ofbudesonide.

As budesonide has been shown to rapidly dissolve in the lungs of rats (94) and

humans (95), the pulmonary administration of budesonide by a sustained release delivery








system is expected to increase the pulmonary residence time and improve pulmonary

targeting.

We compared the receptor occupancy in the lung, liver and brain of neonatal (10-11

days old) rats after intratracehal administration of PLA coated/uncoated formulations of

budesonide using an ex vivo receptor binding assay. The assay is a surrogate marker for

pharmacologically active free drug concentrations in various tissues, hence determining

the receptor occupancy in various tissues will help in the indirect assessment of local

(lungs) and systemic (liver and brain) corticosteroid exposure.

We did not observe significant pulmonary targeting after intratracheal

administration of uncoated budesonide that can be attributed to the rapid absorption of

budesonide from the lung into the systemic circulation leading to similar local and

systemic exposure. However, we observed significant brain receptor occupancy after

intratracheal administration of uncoated budesonide. These results are in good agreement

with the our previous results (chapter 3) where we observed significant brain receptor

occupancy in neonatal rats after intratracheal instillation of triamcinolone acetonide

phosphate, an inhaled corticosteroid (96). Further, the incomplete development of the p-

gp transporters (due to an immature blood brain barrier) can, in part, explain the

observance of significant brain receptor occupancy. Matsuoka et al. (91) have shown that

that the development ofp-gp transporters in rats starts at day 7 and steadily increases to

reach a plateau at day 20 (with about 25 % developed at day 10). As the rats used in our

study were 10-11 days old, the significant brain receptor occupancy can probably be due

to poor development of the p-gp transporters (due to an immature blood brain barrier).








We observed significant pulmonary targeting after intratracheal administration of

PLA coated budesonide. This can probably be explained on the basis of an increase in

the pulmonary residence time of the polymer-coated formulation leading to sustained

receptor occupancy. Although the brain receptor occupancy after intratracheal

administration of polymer-coated budesonide was lower as compared to uncoated

budesonide, the results were not significantly different to make any conclusions regarding

the differences in brain exposure. However, sustained receptor occupancy (resulting in

higher pulmonary targeting) observed after intratracheal administration of polymer-

coated budesonide can lead to reduction in the dose of corticosteroid administered. This

will potentially result in the reduction of systemic exposure.

Conclusion

The results from our study show that the pulmonary targeting in neonatal

rats was significant improved by using polymer-coated slow release

formulation of budesonide.

The significant improvement in pulmonary targeting potentially allows for a

reduction in dose administered thereby leading to reduction in systemic

adverse effects.

The efficacy (by increase in pulmonary residence time) and safety (by

administering lower doses of the glucocorticoids resulting in less "spill

over" into systemic circulation) of inhaled glucocorticoids can be improved

by use of optimally designed slow release formulations.














CHAPTER 5
CONCLUSIONS


Systemic corticosteroids are widely used for the treatment/prevention of chronic

lung disease (CLD). Although systemic corticosteroids have shown beneficial effects,

the concomitant adverse effects have simmered the enthusiasm for using them. The last

few years have witnessed the use of inhaled steroids for the prevention/treatment of CLD.

However, inhaled corticosteroid therapy has met with limited success, partly due to high

lipophilicity of commercially available corticosteroids resulting in rapid absorption of the

corticosteroid from the lungs into systemic circulation. This rapid absorption leads to

significant reduction in the pulmonary residence time and consequently, a loss of

pulmonary targeting.

The overall objective of this thesis was to study the biopharmaceutical factors that

modulate the disposition of inhaled corticosteroids in preterm infants. In addition, the

usefulness ofmicroencapsulated corticosteroid formulations for increasing the pulmonary

targeting was evaluated.

In the first set of experiments, we investigated the role played by p-gp transporters

in modulating the brain permeability of inhaled corticosteroids in mice. The brain and

liver receptor occupancies were determined in wild type and mdrl a mice after

intravenous administration of TAP. The results showed significantly higher brain

receptor occupancy in mdrl a mice that underscores the critical role played by p-gp

transporters in modulating the brain permeability of inhaled steroids. Previous studies








performed in our laboratory have shown minimal brain receptor occupancy in adult rats.

Hence, our results, taken in conjugation with previous studies, suggest that p-gp

transporters modulate the brain permeability of all clinically relevant inhaled

corticosteroids.

The next set of experiments involved the simultaneous monitoring of corticosteroid

receptor occupancy in the local (lung) and systemic (liver, brain) organs of neonatal rats

after intratracheal instillation of different doses of triamcinolone acetonide phosphate

(TAP). This was done primarily to determine if pulmonary instillation of TAP

demonstrates pulmonary targeting in neonatal rats. As expected, we observed similar

pulmonary and hepatic receptor occupancies (no pulmonary targeting) that can be

attributed to the rapid absorption of TAP into systemic circulation. However, an

interesting and important observation from these experiments was that TAP showed brain

receptor occupancy in the neonatal rats. This was in sharp contrast to minimal brain

receptor occupancy observed in adult rats observed in previous similar studies performed

in our laboratory. This higher brain receptor occupancy in neonatal rats (and absent in

adult rats) can probably be explained on the basis of an immature blood brain barrier in

neonatal rats. In addition, the poor development of p-gp transporters (due to an immature

blood brain barrier) in neonatal rats can also, in part, explain the increased permeability

of the corticosteroid.

In our last set of experiments, we evaluated the use of a novel sustained release

drug-delivery system for improving the pulmonary targeting ofbudesonide, a widely

used inhaled corticosteroid. Poly (1-lactic acid) coated budesonide and uncoated

budesonide was intratracheally administered to neonatal rats and the degree of local








(lungs) and systemic (liver, brain) corticosteroid receptor occupancies were determined

using ex vivo receptor binding assay. The results showed significantly higher pulmonary

targeting after intratracheal administration of polymer-coated budesonide as compared to

uncoated budesonide. The significant differences in pulmonary targeting can potentially

help to reduce the amount of dose. This can lead to reduction in systemic adverse effects

observed after corticosteroid administration thereby improving the benefit to risk ratio.

Overall, the results from our study underscore the important and urgent need to

develop targeted delivery systems to the lungs for administering inhaled corticosteroids

to preterm infants. This will greatly assist in increasing the local effect of steroid in the

lungs and reduce the systemic "spill over" thereby increasing the benefit to risk ratio.














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BIOGRAPHICAL SKETCH

Vikram Arya was born on August 26, 1974 in Delhi, India. He completed his BS

in pharmaceutical sciences from Birla Institute of Technology (BIT), Ranchi, India. He

joined the doctoral program in the Pharmaceutics Department at the University of Florida

in Fall 1998. During the course of doctoral studies, he did his summer internship at

Aventis Pharmaceuticals in Bridgewater, New Jersey. He worked under the supervision

of Dr. Guenther Hochhaus, Professor of Pharmaceutics. Vikram Arya earned his PhD in

December 2003.













I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.


Guenther Hochhaus, Chair
Professor of Pharmaceutics

I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.


Hartmut Derendorf
Professor of Pharmaceutics

I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.


jiTteo/ gnes'
Associate Professor of Pharmaceutics

I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.


Saeed Khan
Professor of Pathology, Immunology and
Laboratory Medicine











This dissertation was submitted to the Graduate Faculty of the College of
Agricultural and Life Sciences and to the Graduate School and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
December, 2003 //Jf//o Jo/r


Dean, Graduate School





















































UNIVERSITY OF FLORIDA
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