Pulmonary targeting of inhaled glucocorticoid dry powders

MISSING IMAGE

Material Information

Title:
Pulmonary targeting of inhaled glucocorticoid dry powders
Physical Description:
x, 136 leaves : ill. ; 29 cm.
Language:
English
Creator:
Talton, James David, 1971-
Publication Date:

Subjects

Subjects / Keywords:
Glucocorticoids -- pharmacokinetics   ( mesh )
Glucocorticoids -- metabolism   ( mesh )
Triamcinolone Acetonide -- pharmacokinetics   ( mesh )
Triamcinolone Acetonide -- metabolism   ( mesh )
Budesonide -- pharmacokinetics   ( mesh )
Budesonide -- metabolism   ( mesh )
Androstadienes -- pharmacokinetics   ( mesh )
Androstadienes -- metabolism   ( mesh )
Department of Pharmaceutics thesis Ph.D   ( mesh )
Dissertations, Academic -- College of Pharmacy -- Department of Pharmaceutics -- UF   ( mesh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1999.
Bibliography:
Includes bibliographical references (leaves 120-135).
Additional Physical Form:
Also available online.
Statement of Responsibility:
by James David Talton.
General Note:
Typescript.
General Note:
Vita.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
oclc - 51625251
System ID:
AA00025279:00001

Full Text














PULMONARY TARGETING OF INHALED GLUCOCORTICOID
DRY POWDERS










By

JAMES DAVID TALTON


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


1999























Copyright 1999

by

JAMES DAVID TALTON













ACKNOWLEDGEMENTS


I would like to extend my appreciation to many people who have

helped me through the years. I would first like to thank my advisor, Dr. Guenther

Hochhaus, for his support and patience over the last four years. I would like to

thank the members of my supervisory committee, Dr. Hartmut Derendorf, Dr.

Jeffrey Hughes, and Dr. Gayle Brazeau, for their leadership and friendship. I

would especially like to acknowledge my external committee member, Dr.

Christopher Batich, for his guidance over the last seven years throughout my

undergraduate and graduate research.

I would like to thank the graduate students, staff, and professors in

Materials Science and Engineering. Special thanks to: James Fitz-Gerald,

James Marotta, Bob Hadba, Chris Widenhouse, Drew Amery, James Kirk,

Kaustubh Rau, Paul Martin, Brent Gila, Sean Donovan, Mike Ollinger, Dr. Rajiv

Singh, Dr. Eugene Goldberg, Dr. Anthony Brennan, and many others.

I would also like to thank the graduate students, staff, and

professors in Pharmaceutics and the College of Pharmacy. Special thanks to:

Sandra Suarez, Suliman AI-Fayoumi, Earvin Liang, Jeff Stark, Scott Poxen,

Ampara de la Pena, Sriram Krishnasmami, Fuxing Tang, Brett Houk, Intira

Coowanitwong, Vikram Arya, Pretti Ajmani, Yufei Tang, Marge Rigby, Pat Khan,








Jim Ketcham, Vada Taylor, and the many exchange students who visited the lab

over the years.

In addition I would also like to thank several people who have

donated their expertise and equipment to assist me in my research. I would like

to thank Dr. Greg Schultz and the Center for Wound Healing for first getting me

interested in drug delivery. I would also like to thank the staff of the Major

Analytical Instrumentation Center and the Engineering Research Center for

Particle Science and Technology for their assistance in sample characterization.

Special thanks to Dr. Sue Way and Dr. Shirlynn Chen at Boehringer Ingelheim

for their help during my summer internship.

Finally, many thanks to some special family and friends that have

been supportive over the years. Special thanks to my mother, father, brother,

sister, and family for their love and support during my extended years of

education.













TABLE OF CONTENTS
page

ACKNOW LEDGEM ENTS ..................................................................................... iii

KEY TO ABBREVIATIONS................................................................................. viii

ABSTRACT.......................................................................................................... ix

CHAPTERS

1. BACKG ROUND ......................................................................................... 1

Introduction ............................................................................................... 1
Glucocorticoids in Asthm a Therapy............................................................ 1
Glucocorticoid M ode of Action......................................................... 4
Asthm a .............................................................................................. 6
Therapy of Asthm a .......................................................................... 8
Physical Factors of Inhaled Glucocorticoid Therapy ................................ 10
Inhalers .................................................................... ...................... 10
Structure and Function of the Respiratory Tract............................ 12
Mechanisms of Drug Deposition.................................................... 14
Particle Characteristics.................................................................. 14
Pharm acokinetic / Pharm acodynam ic Parameters................................... 15
Oral Bioavailability ......................................................................... 16
Pulm onary Absorption .................................................................. 16
Distribution .................................................................................... 17
Metabolism .................................................................................... 19
Clearance ...................................................................................... 20
Receptor Binding Affinity (RBA) .................................................... 20
Pharm acodynam ic Effects............................................................. 21
Com prison of Inhaled Glucocorticoids.................................................... 22
Triam cinolone Acetonide (TA) ....................................................... 23
Budesonide (BUD)......................................................................... 26
Fluticasone Propionate (FP).......................................................... 27
Controlled Release...................................................................... ......... 28
Liposomes ..................................................................................... 29
Biodegradable M icrospheres......................................................... 30
M icroencapsulation ....................................................................... 31
Objectives ................................................................................................ 33








2. CHARACTERIZATION, IN VITRO DISSOLUTION, AND IN VIVO
PLASMA CONCENTRATIONS IN RATS OF TRIAMCINOLONE
ACETONIDE, BUDESONIDE, AND FLUTICASONE PROPIONATE DRY
P O W D E R S ............................................................................................... 35

Introd uctio n .............................................................................................. 3 5
H ypothe sis ............................................................................................... 3 7
Materials and Methods............................................................................. 37
C he m ica ls ...................................................................................... 37
S E M A na lysis ................................................................................ 37
In Vitro D issolution ........................................................................ 38
IT Administration in Rats ............................................................... 41
IV Administration in Rats ............................................................... 42
Solid Phase Extraction of Plasma Samples................................... 42
LC/MS/MS of Extracted Plasma Samples ..................................... 43
Non-Compartmental Pharmacokinetic Analysis ............................ 44
R e s u lts ..................................................................................................... 4 7
S E M A analysis ............................................. ................................... 47
In V itro D issolution ........................................................................ 47
Pharmacokinetics of TA in Rats .................................................... 50
Pharmacokinetics of BUD in Rats ................................................. 50
Pharmacokinetics of FP in Rats .................................................... 51
D iscussio n ................................................................................................ 56
C o nclusio ns........................................................... ................................... 59

3. ASSESSMENT OF PULMONARY TARGETING OF TRIAMCINOLONE
ACETONIDE, BUDESONIDE, AND FLUTICASONE PROPIONATE
USING AN EX VIVO RECEPTOR BINDING ASSAY............................... 61

Intro d u ctio n .............................................................................................. 6 1
H y pothe sis ............................................................................................... 6 2
Materials and Methods............................................................................. 62
C he m ica ls ...................................................................................... 6 2
IT Administration in Rats ............................................................... 63
IV Administration in Rats ............................................................... 64
Ex Vivo Receptor Binding Assay................................................... 65
Non-Compartmental Analysis........................................................ 66
R e s u lts ..................................................................................................... 6 8
Receptor-Binding of TA after IT Administration in Rats................. 68
Receptor-Binding of TA after IV Administration in Rats................. 68
Receptor-Binding of BUD after IT Administration in Rats..............69
Receptor-Binding of BUD after IV Administration in Rats .............. 69
Receptor-Binding of FP after IT Administration in Rats................. 69
Receptor-Binding of FP after IV Administration in Rats................. 70
Comparison of Pulmonary Targeting between Drug Formulations 70
D is c u s s io n ......................................................................... ...................... 7 0








C o nclusio ns.......................................................... ................................... 84

4. CHARACTERIZATION, IN VITRO DISSOLUTION, AND IN VIVO
PLASMA CONCENTRATIONS AND PULMONARY TARGETING IN
RATS OF A NOVEL SUSTAINED-RELEASE FORMULATION OF
MICROENCAPSULATED BUDESONIDE................................................ 85

Intro d u ctio n .............................................................................................. 8 5
H y pothe sis ............................................................................................... 87
Materials and Methods............................................................................. 87
C he m ica ls ...................................................................................... 87
Pulsed Laser Deposition (PLD) Setup........................................... 88
SEM Analysis ................................................................................ 88
NMR Analysis ................................................................................ 90
FTIR Analysis ................................................................................ 90
In Vitro Dissolution ........................................................................ 90
IT Administration in Rats ............................................................... 92
Solid Phase Extraction of Plasma Samples................................... 94
LC/MS/MS of Extracted Plasma Samples..................................... 94
Ex Vivo Receptor Binding Assay................................................... 95
Non-Compartmental Pharmacokinetic Analysis ............................ 97
R e s u lts ................................................................................................... 10 0
S E M A na lysis .............................................................................. 10 0
N M R A na lysis .............................................................................. 10 0
FT IR a na lysis ............................................................................. 10 0
In Vitro Dissolution ...................................................................... 105
Pharmacokinetics of Coated Budesonide (BUD25) in Rats....... 105
Receptor-Binding of Coated Budesonide (BUD25) in Rats ....... 109
Comparison of Pulmonary Targeting between Drug Formulations1 09
D isc uss io n .............................................................................................. 1 13
C o n c lu s io n s ............................................................................................ 1 17

5. CONCLUSIONS..................................................................................... 118

R E F E R E N C E S ................................................................................................. 12 0

BIOGRAPHICAL SKETCH ............................................................................... 136











vii














KEY TO ABBREVIATIONS


BDP
BUD
Cl
DEX
DPI
F
FP
fu
GR
GRE
IT
IV
LC/MS/MS
MAT
MDI
MET
MRT
PGA
PLA
PLGA
RBA
SPE
T1/2
T50%
TA
TAP
TFA
Vd


Beclomethasone dipropionate
Budesonide
Clearance
Dexamethasone
Dry-powder inhaler
Bioavailability
Fluticasone propionate
fraction unbound
Glucocorticoid receptor
Glucocorticoid response element
Intratracheal
Intravenous
Liquid chromatography with double mass spectrometer
Mean absorption time
Metered-dose inhaler
Mean effect time
Mean residence time
Poly(glycolic acid)
Poly(lactic acid)
Poly(Iactic-co-glycolic acid)
Receptor binding affinity
Solid phase extraction
Half-life
Dissolution half-life
Triamcinolone acetonide
Triamcinolone acetonide phosphate
Tri-fluoroacetic acid
Volume of distribution













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

PULMONARY TARGETING OF INHALED GLUCOCORTICOID
DRY POWDERS

By

James David Talton

August 1999

Chairman: GQnther Hochhaus
Major Department: Pharmaceutics

Although new inhaled glucocorticoids introduced for the treatment

of asthma in the last decade have been shown to have lower oral bioavailability

and higher systemic clearance, systemic side effects are still observed. Although

the efficacy of inhaled glucocorticoids is well established, it is difficult to assess

their pulmonary targeting (local vs. systemic effects) in humans, mainly because

of the lack of appropriate surrogate markers for pulmonary effects. Computer

simulations have shown that, based on receptor occupancies in individual

organs, pulmonary targeting can be improved by optimization of drug release

rate. In addition, it has previously been shown using liposomes that pulmonary

targeting is improved by increasing the pulmonary residence time, but the

industrial application of liposome formulations are limited.

The overall objective was to compare the dissolution rates in vitro,

and pharmacokinetics and pulmonary targeting in vivo of three currently available








inhaled glucocorticoid dry powders (triamcinolone acetonide, budesonide, and

fluticasone propionate) and one sustained release formulation (micro-

encapsulated budesonide). First, dissolution of fluticasone propionate was

shown to be slower than triamcinolone acetonide and budesonide in vitro. In vivo

experiments in rats showed comparable fast absorption times for budesonide

and triamcinolone acetonide and slower absorption for fluticasone propionate.

Next, comparison of the pulmonary targeting using an ex vivo receptor-binding

assay showed fluticasone propionate displayed the highest level of pulmonary

targeting followed by triamcinolone acetonide and budesonide. Then, to further

evaluate the relationship of dissolution rate on pulmonary targeting, polymeric

microencapsulated budesonide was investigated. Although SEM analysis

showed that polymer coatings did not substantially increase particle size, the in

vitro dissolution half-life increased ten-fold. The pharmacokinetic profile in vivo

showed a slower absorption rate for coated budesonide than free dry powder

formulations. Finally, ex vivo receptor binding of coated budesonide showed a

statistically significant increase in pulmonary targeting when compared to free

powders of budesonide and fluticasone propionate. This method of extending

the release-rate of the encapsulated material using polymeric-coated particles

has promising industrial applications, and further emphasizes the relationship

between increasing pulmonary residence time to improve pulmonary targeting.













CHAPTER 1
BACKGROUND



Introduction


To clearly understand the effects of inhaled glucocorticoids used for

asthma therapy, the pharmacokinetic and pharmacodynamic factors involved

must be evaluated. In this background section the current literature on asthma

therapy as it pertains to glucocorticoids, the physical factors of aerosol delivery,

and the pharmacokinetic / pharmacodynamic factors are discussed. Also,

descriptions of the three currently available glucocorticoids that are evaluated in

this thesis are given, namely triamcinolone acetonide (TA), budesonide (BUD),

and fluticasone propionate (FP). Finally, a background of the different sustained

release mechanisms for pulmonary drug delivery is reviewed.
(


Glucocorticoids in Asthma Therapy


Glucocorticoids are used as the first-line treatment in asthma

therapy [1]. Systemic administration of glucocorticoids is only recommended if

the disease can not be controlled with local therapy. Thus, inhalation therapy is

generally applied to achieve high local activity with reduced systemic side effects.

There are currently several inhaled glucocorticoids on the market, such as








beclomethasone dipropionate, triamcinolone acetonide, flunisolide, budesonide,

and fluticasone propionate.

While there have been numerous review articles describing the

clinical efficacy of inhaled glucocorticoids in asthma therapy [2-9], detailed

reviews on the underlying pharmacokinetic behaviour and local vs. systemic

effects of these drugs are rare. More studies regarding the pharmacokinetic

properties of inhaled glucocorticoids and their relationship to the degree of

pulmonary targeting or selectivity would be beneficial [4, 10-13].

A pharmacokinetic multi-compartment model is shown in Figure 1.1

(adapted from [14]) that illustrates several of the factors involved in pulmonary

drug delivery that will be reviewed in this chapter. Briefly, the aerosol dose is

inhaled through the mouth and a portion is deposited in the oropharynx and

swallowed (swallowed fraction) or is deposited in the lungs (inhaled fraction).

The swallowed fraction, including a portion that is deposited in the upper lung

that is subjected to mucociliary transport, is then available to be absorbed in the

GI and is subject to the first pass effect in the liver before passing into the

systemic compartment (according to its oral bioavailability). The drug particles

deposited in the lungs will dissolve according to its dissolution rate kdiss and will

be available for absorption into the lung tissue (which is generally fast for

glucocorticoids). Absorbed drug in the lungs, Clung, may then be bound to the

pulmonary glucocorticoid receptors (according to the EC5o) and then absorbed

systemically according to its absorption rate kabs (also generally fast). A portion

of drug that enters the systemic compartment binds to tissues according to the








volume of distribution and the plasma protein binding. Non-protein bound drug in

the plasma is then available to be bound to systemic glucocorticoid receptors

(according to the EC50). Elimination of drug from the systemic compartment

occurs via clearance mainly in the liver. From this model the physical and

pharmacokinetic parameters of inhaled glucocorticoids that improve the local vs.

systemic effect ratio, or the pulmonary targeting, are a high pulmonary deposition

ratio, low oral bioavailability (F), high volume of distribution (Vd), high clearance

(Cl), and most importantly a slow pulmonary dissolution or absorption rate (see

Table 1.1). These pharmacokinetic factors in humans will be discussed

individually in this background chapter.





Table 1.1: Factors important for pulmonary targeting.

Pulmonary components Systemic components


Pulmonary deposition efficiency
Location of pulmonary deposition
Pulmonary residence time (dissolution
rate and other factors)
Pulmonary absorption rate
Pharmacodynamic drug characteristics
in the lung


Oral bioavailability
Oral deposition efficiency
Clearance
Volume of distribution
Tissue distribution pattern
Pharmacodynamic drug characteristics
in systemic tissues

















Swallowed Fraction '/ S.Inhaled Fraction

Drug Powder
I~ru'Drug-
G I






Absorption Systemic a etsma Bmdiding, %
| L Free EC-0
^Kabs, ~Drug. .--------
First Dg at systemic receptor
Pass, f


Elimination
tCI,Vd i f


Figure 1.1: Pharmacokinetic multi-compartmental model of inhaled drug
delivery.




Glucocorticoid Mode of Action

The glucocorticoid receptor (GR) belongs to the steroid superfamily

of cytosolic receptors [15]. In its inactive state the GR is bound by two molecules

of 90 kD heat shock protein (HSP-90) and one molecule of the immunophilin p-

59, covering the DNA binding region of the receptor [16, 17]. Glucocorticoids








exert their action directly by binding to the intracellular GR and translocating to

the nucleus, binding to specific glucocorticoid response elements (GRE) and

modulating DNA transcription through several mechanisms (see Figure 1.2,

adapted from [18]). Two isoforms of the human GR, hGR-oc and hGR-p, have

been identified recently [19], with only the hGRa isoform activated by

glucocorticoids. Both isoforms are transcriptionally active, though, with the hGRP3

isoform providing an antagonistic effect to the glucocorticoid-activated hGRa

isoform [19].

When the glucocorticoid receptor is activated, it dissociates from

the complex with HSP-90 and p-59, exposing a pair of DNA-binding zinc fingers

[20]. Two activated GR's must be present to form a homodimer which is then

able to interact with specific DNA GREs. Binding of the liganded GR to DNA

results in either induction or repression of responsive genes through these

GRE's, such as increased mRNA expression for tyrosine aminotransferase (TAT)

in the liver [21].

Recent studies have found that the bound GR also modulates gene

transcription through indirect protein-protein interactions with transcription factors

such as AP-1 (activating protein-I) and NF-Kf3 (nuclear factor K3), which induce

many of the inflammatory genes that are abnormally expressed in asthma [22].

These recently discovered indirect pathways are extremely important because

they provide a crosstalk between glucocorticoids on one hand, and polypeptide

hormones and cytokines on the other. Some of the genes regulated by this

pathway include metalloproteinases, such as stromelysin and collagenase, and








many cytokines, such as IL-2 [22]. The repression of these genes is likely to

underlie the anti-inflammatory and immunosuppressive effects of glucocorticoid

therapy.

Glucocorticoid receptors are distributed throughout the body in

many tissues and are essentially the same in humans and other species [8].

Independent of the binding mechanism, the degree of effects and side effects

from a glucocorticoid dose is directly related to the number of activated receptors

in the cells of a tissue [21]. This suggests that, for both local effects and

systemic side effects, the receptor occupancies in local and systemic organs are

highly suitable for describing the activity of a glucocorticoid [21]. Although, more

potent glucocorticoids with high receptor binding affinities (such as budesonide,

beclomethasone monopropionate, and fluticasone propionate) have recently

been developed, they are not necessarily more efficacious. More potent

glucocorticoids with lower EC50 values (effective concentration which binds 50%

of the receptors) have corresponding increasing receptor binding affinities (RBA)

for systemic glucocorticoid receptors as well in the lung [8]. The relationship of a

high receptor binding affinity increasing the extent of local and systemic effects

has been demonstrated previously using computer simulations [14].

Asthma

Asthma is an inflammatory disease of the airways associated with

mucosecretion, hyperreactivity, epithelial cell injury, and deposition of fibrous

material in the subepithelial basement membrane. The physiological changes









Glucocortlcold


Figure 1.2 Mechanism of glucocorticoid binding to cytosolic glucocorticoid
receptor (GR) and translocation to nucleus (adapted from [18]).




that occur in asthma include an increase in the non-specific reactivity of the

airways to a variety of bronchospastic agents including histamine, metacholine,

adenosine, and leukotrienes [23]. It is unknown if damage in the airway

epithelium occurs prior to the inflammatory reaction or as a result of released

mediators from inflammatory and mast cells. The interactions between the

epithelial cytoskeleton and its transmembrane receptors and the epithelial

basement membrane and stroma may play an important role in the pathogenesis

of asthma [24].








Many cells are involved in asthma including eosinophils,

macrophages, mast cells, epithelial cells, and lymphocytes (see Figure 1.3

adapted from [25]). The interactions of these cells result in the release of

different mediators leading to bronchoconstriction, microvascular leakage, mucus

hypersecretion, epithelial damage, and stimulation of neural reflex [26]. For

example, macrophages produce IL-1 which may prime eosinophils to respond to

other secretary stimuli [27]. It has been suggested that the immunosuppressive

effects of glucocorticoids can also be mediated via inhibition of NF-KP through

induced synthesis of the NF-KP inhibitory protein IKP3-9 [22]. Similar to this

mechanism, glucocorticoids inhibit the transcription of a number of cytokines and

chemokines relevant to the pathogenesis of asthma [28].

Cytokine generation in the airways triggers in part infiltration of

inflammatory cells into the airways. There are several classes of cytokines

involved in allergic cell recruitment including non-specific endothelial activators

(TNF-a and IL-1), specific endothelial activators (IL-4 and IL-3), miscellaneous

activators (IL-3, IL-5, granulocyte-macrophage colony-stimulating factor (GM-

CSF), and interferon gamma), and direct cell migrators (RANTES) [29]. The

release of these cytokines has been shown to be modulated by glucocorticoid

therapy, thus minimizing mediator release and inflammatory cell migration [30].

Therapy of Asthma

The overall management of asthma involves immediate relief from

bronchconstriction (for example with P-agonists) and the control of the













Macrophage : Mast Cell

T-lymphocyte / Neutrophil


.'. 1^0t Eosinophil


Globet cell Epithelial shedding

Plasma leak Sensory nerve

F r 3 t ee iBronchoconstriction




Figure 1.3 Antigen effect on pulmonary cells associated with asthma [25].


inflammatory response (for example with glucocorticoids) [31]. Controlling the

allergic response to the local environment, due to smoking and allergens found at

home or in the workplace, is the first priority in minimizing asthma hyper-

responsivity [31]. Drug therapy for asthma includes five classes of medications:

anticholinergics, beta-adrenergic agonists, theophylline, sodium cromolyn

glycate, and glucocorticoids [24]. Many controlled trials have now established

that inhaled glucocorticoids are the most effective medications because of their








effects at the cellular levels to attenuate the late response and reduce the overall

need for beta-agonists [32, 33].



Physical Factors of Inhaled Glucocorticoid Therapy


Localized delivery of drugs to the lungs has become an increasingly

important therapeutic method for treating a variety of diseases including asthma,

bronchitis, and cystic fibrosis [34]. The theory behind inhalation therapy for

localized delivery is that only relatively small doses are required for effective

therapy, reducing exposure of drug to the systemic circulation, and potentially

minimizing systemic side-effects [35]. These low dosage regimens may provide

considerable cost savings, especially with expensive therapeutic agents.

Localized drug delivery directly to the lung should, in theory, maximize

therapeutic effect while minimizing unwanted side effects. As mentioned earlier,

these advantages are the basis of inhalation therapy of glucocortioids.

Inhalers

There are a variety of inhalers on the market today with different

methods of delivering aerosolized drug to the lung, including metered-dose

inhalers (MDI's), dry-powder inhalers (DPI's), and nebulizers. The pulmonary

deposition efficiency of the inhaled drugs, or the ratio of dose effectively

deposited in the lungs to the dose entering the mouth, can be either determined

by gamma scintigraphy or pharmacokinetic techniques [36-38]. The deposition

efficiency depends on the physical characteristics of the liquid aerosol or dry








powder particles, the velocity of the aerosolized particles, and breathe

coordination [39, 40].

Pulmonary deposition also depends on the nature of the delivery

device used. Pulmonary deposition of conventional metered-dose inhalers

(MDIs) depends on physicochemical characteristics of the device, such as

aerosol size, density, and velocity, as well as by the coordination of inhalation

[37]. Pulmonary deposition of MDIs has been generally shown to be in the range

of 10-20% [41-43] with significant oropharyngeal deposition because of the

exiting aerosol velocity [44]. Moderate increases in pulmonary deposition

efficiency of metered dose inhalers has been achieved with the use of spacer

devices that slow aerosol velocity [41, 45-49]. Aerosol deposition in the human

lung has been also optimized by actuating the aerosol in coordination with breath

rate following administration from a microprocessor-controlled pressurized

metered-dose inhaler [50, 51]. Improvement in newer MDIs with increases in

deposition efficiencies of up to 40% have been reported [52, 53].

Because of the FDA limiting the use of CFC's earlier this decade,

dry-powder inhalers have gained much attention. Dry-powder formulations have

been characterized by increases in stability, more uniform particle size

distributions, and less impaction in the oropharyngeal because the aerosol is not

pressurized [54]. Early reports on DPI's suggested similar deposition efficiencies

to MDI's (10-20%). Newer dry-powder inhalers have shown high pulmonary

deposition efficiencies [55, 56], but show high variabilities across different studies

[57]. For DPI's, the deposition efficiency depends on the inspiratory flow of the








patient [58] and dry powder inhalation devices might be unsuitable for patients

with very low inspiratory flow.

Common nebulizers use ultrasonic or jet aerosolization of solutions,

and although bulky are common in home therapy. Nebulizer efficiency has been

tested by Hardy [59] and found to be dependent on the brand and system of

nebulizer employed with pulmonary deposition ranging from 2 to 20%. Spacer

use generally increases efficiency of jet nebulizers [60]. Newer studies by the

same group showed, in general, a higher deposition of ultrasonic nebulizers

when compared with jet nebulizers [60]. The use of nebulizers has been

recommended for patients with severe bronchoconstriction [61] and in chronic

asthma in adults [62], but depends on the aerosol characteristics and output [63].

Generally, the pulmonary selectivity of an inhaled glucocorticoid will

increase with increased pulmonary deposition. A high pulmonary deposition will

therefore be advantageous. However, the benefits of improved pulmonary

deposition are more important for substances with distinct oral bioavailability [14,

64].

Structure and Function of the Respiratory Tract

The respiratory tract can be divided into upper airways, including

the mouth, trachea, and larynx, and lower airways, including the bronchi,

bronchioles, and alveoli [65]. The lower airways resemble a series of tubes

undergoing regular dichotomous branching. Further branching from the bronchi

to the alveoli reduces the diameter of the tubes, with marked increases in the

surface area of the airways, which allows ease of gas exchange [66]. The lower








airways can be divided into two physiological zones: central and peripheral. The

central zone consists of the larger tubes responsible for the bulk movement of air

and blood. In the central airways, airflow is rapid and turbulent and less gas

exchange occurs. The epithelial layers of the trachea and main bronchi are

made up of several cell types including ciliated, basal, and goblet. A large

number of mucous- and serum-producing glands are located in the submucosa

[66].

The peripheral section of the human lung consists of smaller

bronchioles and the alveoli. Each bronchiole ends in many acini, which consists

of the alveolar ducts, alveolar sacs, and alveoli. The primary cells of the

epithelium in the acini are the type I pneumocytes, which cover 90% of the entire

alveolar surface, and type II pneumocytes, which are more numerous but have a

smaller total volume and are responsible for the storage and secretion of lung

surfactant. Other less prevalent cell types include type III pneumocytes and

alveolar macrophages. The alveolar blood barrier consists of a single epithelial

cell, a basement membrane, and a single endothelial cell. While this

morphological arrangement readily facilitates the exchange of gases and small

molecules, it can still represent a major barrier to large molecules [67]. Once

dissolved, though, absorption of lipophilic and low molecular weight drugs such

as glucocorticoids is fast because the lung tissue is so highly vascularized,

similar to the gastrointestinal (GI) tract. Unlike oral delivery of drugs through the

GI, inhaled drugs are not subject to changes in pH or first-pass metabolism [34].








Mechanisms of Druc Deposition

As discussed above, drugs for inhalation therapy are administered

in aerosol form. From the inhaler, the aerosol is characterized as a suspension

of liquid or solid particles dispersed in a gas phase. The main physical

parameter for high therapeutic efficacy is the ability of the aerosolized drug to

reach the airways of the lungs. The physiology of the human respiratory tract,

though, has evolved to prevent the entry of particulate matter. Upon actuation of

the inhaler, the deposition efficiency is determined by the ratio of drug that exits

the inhaler and the amount that is absorbed through the lung, with the rest either

swallowed or redistributed by the mucociliary transport system in the upper

bronchi [68].

Particle Characteristics

The particle's geometric and aerodynamic diameter are critical

parameters affecting their flow and site of deposition, since these parameters

determine the aerodynamic and gravitational mechanisms that affect the extent

of penetration into the lungs. Aerosol particle size is expressed in terms of

aerodynamic diameter (Dae), which is defined as the diameter of a similar

spherical particle having unit density (1.0 g/cc) that has the same flow velocity

[68]. Thus, larger particles with lower densities will have similar aerodynamic

diameters and may yield similar deposition profiles to smaller particles at

standard densities [69]. Byron [70] showed that maximum pulmonary deposition

occurs with particles between 1 to 5 microns, with particles larger than 5 microns

gaining sufficient momentum to impact in the back of the throat, and particles








smaller than 5 microns being breathed back out. Overall the physical factors for

maximum aerosol deposition in the lung include (1) aerosol Dae < 5 microns to

minimize oropharyngeal deposition, (2) aerosol Dae > 1 micron, and (3) slow,

steady inhalation and a period of breath holding on completion of inhalation to

minimize exhalation of the drug.



Pharmacokinetic / Pharmacodynamic Parameters


It is important to understand the underlying pharmacokinetic factors

of the inhaled glucocorticoids (deposition ratio, oral bioavailability, absorption,

distribution, and clearance) that lead to the local and systemic effects for

improving the overall clinical efficacy [4, 10-13]. A pharmacokinetic

compartmental model after inhalation is shown in Figure 1.1 and pharmacokinetic

parameters for three of the five currently available inhaled glucocorticoids

(dexamethasone shown for comparison only) listed in Table 1.2.



Table 1.2: Pharmacokinetic parameters of inhaled glucocorticoids.____
Drug Abbr RBA fu CL Vdss ti/2 Forai
(%) (L/h) (L) (h) (%)

Dexamethasone DEX 100 32 73 17 77 106 77 4.6 77 83 82
Triamcinolone TA 361 72 23 74 37 79 103 79 2.0 79 23 79
Acetonide
Budesonide BUD 935 72 12 75 84 75 183 75 2.8 75 11 75
Fluticasone FP 1800 72 10 76 69 78 318 78 6.0 78 <1 81
Propionate








Oral Bioavailability

Glucocorticoids are inhaled to achieve localized pulmonary effects,

but unfortunately much of the inhaled dose is swallowed [14]. Considering the

high degree of orally-impacted and swallowed drug (commonly > 50% of the

dose), a significant amount of the dose is available for oral absorption. This part

of the dose will reduce the pulmonary selectivity of the drug, if orally bioavailable.

Thus, the optimal inhaled glucocorticoid should consequently show minimal oral

bioavailability. The hepatic clearances of most of the inhaled glucocorticoids are

close to the liver blood flow of 90 L/hr, resulting in a high first-pass effect. In

particular, fluticasone propionate [82, 83] possesses an extremely low oral

bioavailability of close to 0%. It has be shown for fluticasone propionate that high

first pass effect and poor absorption from the gastrointestinal tract are

responsible for its low oral bioavailability [82, 83]. Slightly higher oral

bioavailabilities have been reported for budesonide (11%, [74]) and triamcinolone

acetonide (23%, [84]).

Pulmonary Absorption

A slow pulmonary absorption has been classified as a key

component for successful targeting of the inhaled glucocorticoids [14]. It is now

recognized that an increased pulmonary mean residence time of an inhaled

glucocorticoid is not only beneficial for a prolonged activity but also for increased

pulmonary targeting [14, 85]. However, obtaining a distinct pulmonary residence

time is difficult because of the high surface area and high blood flow.








Comparison of plasma concentrations after inhalation with those

obtained after intravenous administration allows the calculation of the mean

absorption times. Detailed studies on the pulmonary absorption rate of

glucocorticoids have been performed in studies by Schlagel and co-workers [86,

87]. These studies suggest that absorption of aerosolized glucocorticoid

solutions from the lung are generally fast, with half-lives of absorption for cortisol,

cortisone and dexamethasone of 1.0 to 1.7 minutes [87]. Studies in humans

suggest that inhaled glucocorticoids such as flunisolide [88] and budesonide

(presumably reaching the lung as solid particles) are also absorbed relatively

fast, with maximum plasma concentration times (tmax) observed after 0.2 [88] and

0.5 hours [55], respectively. Triamcinolone acetonide [89] and fluticasone

propionate [90, 91] are absorbed more slowly, though, with tmax values of 2.1

hours for triamcinolone acetonide [78] and 1.0 hour for fluticasone propionate

[92, 93].

Distribution

The volume of distribution (Vd) is the main pharmacokinetic

parameter that describes the plasma protein binding and tissue distribution

proportional to the free plasma levels of drug. Although a lower Vd would result

in a decrease in half-life of drug (clearance staying the same), a lower Vd only

slightly decreases the cumulative systemic side effects. Computer simulations

have shown that a short half-life from a smaller volume of distribution is not as

substantial a pharmacokinetic factor for improving pulmonary targeting as the

clearance [14].








Glucocorticoids are distributed throughout the body extensively

because of their high lipophilicity and low molecular weight. Tissue distribution of

glucocorticoids is, however, affected by specific glucocorticoid transport systems.

Membrane-binding components for cortisol which involve the uptake of

corticosteroids into thymocytes [94] and liver cells [95, 96] have been identified.

Increased penetration of dexamethasone into the brain has been observed in

multi-drug resistance (mdrlA) P-glycoprotein knockout mice when compared to

normal mice [97, 98], suggesting that glucocorticoids are substrates for p-

glycoprotein efflux.

Unlike the endogenous glucocorticoid cortisol, the currently

available inhaled glucocorticoids do not bind significantly to the plasma cortisol

transporter transcortin [99]. Rather, o-1 acid glycoprotein [100] and albumin

represent the most important plasma protein binding components. Plasma

protein binding (see Table 1.2) of the inhaled glucocorticoids ranges from 70-90

% and is roughly related to the lipophilicity of the glucocorticoid. Increases in

non-specific binding to tissues also increases with lipophilicity, resulting in

increased volume of distributions. The volume of distribution in humans

increases such that triamcinolone acetonide < budesonide < fluticasone

propionate (see Table 1.2). Although suggested by some investigators [101],

increased overall tissue binding does not necessarily increase lung selectivity

after inhalation because only free drug concentration and not bound drug is

relevant for inducing pharmacological effects.








Interestingly, reversible esterfication of 21-alcohols, such as

budesonide, has recently been suggested to increase the pulmonary residence

time [102]. Since the fatty acid conjugates are retained intracellularly for a longer

time than unchanged budesonide, the duration of tissue exposure to free

budesonide depends partly on the rate of lipase-catalyzed hydrolysis of the

conjugates [102]. To sustain therapeutic levels, though, a significant portion of

the drug would need to be esterified for it to act as an effective depot over time.

Metabolism

Most glucocorticoids are stable in the lung and most other tissues,

such as triamcinolone acetonide [103], budesonide [104], and fluticasone

propionate [105], with metabolism occurring mainly in the liver by oxidative

processes. Relevant oxidative processes include 6-11-hydroxylation (flunisolide,

budesonide, triamcinolone acetonide, fluticasone propionate), 11-oxidation, 6-

ketoformation (flunisolide), B-ring dehydrogenation (budesonide, triamcinolone

acetonide, flunisolide), 21-oxidation (triamcinlolone acetonide), acetal oxidation

(budesonide [106]), A-ring reduction (budesonide), 1711-side chain cleavage

(dexamethasone) and thio-ester cleavage (fluticasone propionate) [107].

Deactivation by cytochrome P450 by hepatic cells and subsequent fast

elimination for most of the inhaled glucocorticoids has been reported [85, 104,

107]. Lipophilicity of the glucocorticoid has been shown to enhance the

metabolic activity with these enzymes with very lipophilic glucocorticoids showing

increased intrinsic clearance (such as fluticasone propionate, [85]).








Clearance

A primary requirement for increased pulmonary selectivity is a high

systemic clearance [14, 85]. Figure 1.1 illustrates that high clearance from the

systemic compartment will improve pulmonary selectivity by lowering systemic

exposure (expressed as the ratio of pulmonary to systemic receptor

occupancies). As most of the currently available inhaled glucocorticoids are

being inactivated through hepatic metabolism, most inhaled glucocorticoids show

systemic clearance values close to the liver blood flow (see Table 1.2) of 90 L/hr.

One significant way of improving pulmonary selectivity is to further increase

systemic clearance through other mechanisms such as extrahepatic inactivation

mechanisms. However, to be a successful inhaled glucocorticoid, such

derivatives need to show sufficient pulmonary activity.

Receptor Binding Affinity (RBA)

Receptor binding affinities (RBA) of inhaled glucocorticoids to the

glucocorticoid receptor vary significantly (see Table 1.2). Structure-affinity

relationships for a variety of synthetic glucocorticoids have shown that the

presence of a 1,2 double bond, the existence of a 6 and 9 fluoro or methyl group,

and the introduction of lipophilic residues in the 16 and 17 position increase

affinity [108-112]. Changes in position 16 and 17 consequently led to the design

of more potent inhaled glucocorticoids, such as beclomethasone monopropionate

(active species of beclomethasone dipropionate), triamcinolone acetonide,

flunisolide, and budesonide, and fluticasone propionate. Glucocorticoids with

intrinsic activities 20 times more pronounced than that of dexamethasone have








been described [113]. Interestingly, modifications on the 171 side chain by

adding a thioester of cortienic acid for fluticasone propionate retained

glucocorticoid receptor binding affinity [110, 114-116], while introducing a

predictable metabolic inactivation group [4]. In contrast, traditional 21-ester of

glucocorticoids (such as beclomethasone dipropionate) do not bind to the

receptor. As stated earlier, all of the relevant inhaled glucocorticoids bind to the

receptor with different potencies.

Pharmacodynamic Effects

A significant body of literature suggests that the receptor binding

affinity of a glucocorticoid correlates with its effect at the site of action [14, 21,

117]. Consequently, good correlation's between the receptor binding affinity and

the activity in systems not affected by pharmacokinetic properties have been

found for a number of pharmacological parameters [118] including topical anti-

inflammatory properties [15, 119, 120], activity in skin blanching [116], and

modulation of the activity of enzymatic systems such as tyrosine

aminotransferase [121]. These in vitro assays are useful in providing rankings of

relative potencies but, as described previously, many pharmacokinetic factors

are involved in obtaining a high local vs. systemic effect.

Studies comparing the receptor binding affinity have failed to

compare the clinical efficacies, defined as ratio of dose of drug to therapeutic

effect, of the different inhaled glucocorticoids through indirect measurements of

asthma severity including full expiratory volume (FEV), peak expiratory flow

(PEF), and nonspecific bronchial hyper-responsiveness [8]. Unfortunately, there








is no direct measure of airway inflammation, though, and these measurements

are only secondary markers from the pharmacological effect of the inhaled

glucocorticoids and show great variability between studies. In addition, surrogate

markers of systemic effects in humans such as cortisol suppression and

immunosuppression show correlation's to potencies when given in equimolar

doses, but little information on pulmonary efficacy [8].

Overall, desired pulmonary effects and deleterious systemic side

effects are non-dissociable, with pharmacokinetic properties rather than

pharmacodynamic properties important for describing pulmonary selectivity [8].

In fact, theoretical simulations showed that differences in receptor affinity can be

overcome by selecting the appropriate dose, assuming larger doses can be

inhaled [14]. Thus the pharmacokinetic factors involved in improving the local /

systemic effects in pulmonary drug delivery include a high deposition ratio

(inhaled fraction / swallowed fraction), low oral bioavailability (F), slow dissolution

or absorption rate (kdiss or kabs), high volume of distribution (Vd), and a high

clearance rate (Cl).



Comparison of Inhaled Glucocorticoids


Currently available inhaled glucocorticoids are based on the 21

carbon atom cortisol structure with four rings, three six-carbon rings and a five-

carbon ring. The synthetic anti-inflammatory glucocorticoids are characterized by

lipophilic moieties in the 16 and 17 position; CH3, F or Cl moieties in the 6 and 9

positions; and/or double bound carbons in the 1,2 position. Other essential








features include a ketone oxygen at the 3 position, an unsaturated bond between

the 4,5 carbons, a hydroxyl group at the 11 position, and a ketone oxygen at the

20 position. By modifying the basic structure of glucocorticoids, it is possible to

alter the affinity for the glucocorticoid receptor (GR) and plasma protein binding,

modulate the metabolism pathway (oxidation or hydrolytic), and the tissue

binding and clearance [122].

Adequate characterization of the overall pharmacokinetic drug

properties is a necessary prerequisite for comparing the pulmonary targeting.

The time course of the pharmacological response is determined by both the

concentration and time of free drug at the receptor site. Therefore to assess the

systemic exposure of the drug, it is important to observe the glucocorticoid

concentration vs. time profile in the systemic compartment by monitoring the

plasma levels. This section summarizes the important pharmacokinetic

parameters of three commercially available inhaled glucocorticoids, triamcinolone

acetonide (TA), budesonide (BUD), and fluticasone propionate (FP) (see

structures in Figure 1.4).

Triamcinolone Acetonide (TA)

Triamcinolone acetonide (TA) entered the asthma market as the

Azmacort MDI by Rhone-Poulenc in 1992. Doses of 200 to 400 mcg/day (100

mcg/puff) at 2 to 4 times daily were recently shown to have comparable

therapeutic effect in forced expiratory volume [123]. The pulmonary deposition

ratio from Azmacort MDI with spacer has been reported to be approximately 22%

[73]. First-pass metabolism in the liver to less active metabolites accounts for the









Triamcinolone Acetonide
CH2OH

CO

I






HO CH3 ,C(CH3)2CH2CH3
HO, -0
CH3





0



Fluticasone Propionateide

CH2FH
I
Lou







CO
HCH3 | I-OCOCH2CH2CH3


CH3








F








Figure 1.4: Triamcinolone acetonide (TA), budesonide (BUD), and fluticasone
propionate (FP) molecular structures.
SCH2F


HOCH I\|>-OCOCH12CH3
HO1' I 1--CH3






F





Figure 1.4: Triamcinolone acetonide (TA), budesonide (BUD), and fluticasone
propionate (FP) molecular structures.








reduced oral bioavailability of 20 to 25% [78]. Absorption of TA suspension in the

lungs has been measured to be approximately 2 hours by the difference in half-

lives of intravenous (1.4 to 2.0 hours) versus inhaled (3.6 hours) doses [73, 124].

Triamcinolone acetonide (TA), along with flunisolide, belongs to the

second generation of glucocorticoids that show an increased topical potency

compared to dexamethasone due to increased receptor binding affinity (RBA =

361) [71]. Plasma protein binding for TA, similar to the other inhaled

glucocorticoids, has been reported at 71% [84]. TA has a volume of distribution

of 100 to 150 L and has a mean residence time of 2.7 hours after intravenous

administration [84, 89, 124]. Clearance of TA is 37.3 L/hr and the major

metabolite of TA is 6-hydroxytriamcinolone acetonide, whereas triamcinolone

(TC) is only a minor metabolite [89, 124].

Triamcinolone acetonide phosphate (TAP), a water-soluble prodrug

which is rapidly metabolized to TA, has been used for IV administration in

humans [124]. TAP, which shows dose-dependent kinetics, has a plasma half-

life of 3 to 4 minutes and releases active TA immediately. No unchanged ester is

found in urine after IV administration, indicating a complete conversion of TAP

prodrug to TA. In addition, the total body clearance of TAP exceeds the hepatic

blood flow, indicating a large contribution of extrahepatic metabolism due to

hydrolysis in the plasma [124]. Previously, our group has shown that pulmonary

administration of TAP in a sustained-release liposome formulation resulted in a

higher pulmonary residence time, a prolonged pulmonary effect, and a higher

lung to systemic drug ratio [125].








Budesonide (BUD)

Budesonide recently entered the US market as Pulmicort

Turbohaler (Astra USA) as the first inhaled glucocorticoid dry-powder system.

Prescribed doses of 400 to 1,600 mcg per day have been reported [123], with a

pulmonary deposition ratio reported of 32% (16 to 59%) for the DPI and 15% (3

to 47%) for the MDI sold in Europe [126]. About 89% of an oral dose of

budesonide undergoes first-pass metabolism resulting in an oral bioavailability of

11% [55].

Budesonide has a higher receptor binding affinity (RBA=935) than

TA and a higher protein binding (88%) [55]. Its volume of distribution at steady

state is 183 L, indicating high tissue affinity. Budesonide is a drug with a very

high hepatic extraction ratio and a high clearance (84 L/h) close to hepatic blood

flow. The plasma half-life of budesonide is 2.8 hours and is approximately the

same after intravenous and inhalation administration, reflecting a fast rate of

dissolution and absorption in the lung [74]. Similarly, Thorsson at al. [55]

reported a Cmax of 3.5 nmol/L at 0.3 hour after inhalation via Turbohaler and a

Cmax of 2.3 nmol/L at 0.5 h after inhalation via MDI, indicating dissolution of the

dry powder is not rate-limiting.

Budesonide has been shown to have fast dissolution rate in the

lung of rats [127] and humans [74]. Thus, decreasing its pulmonary release by

encapsulation in microspheres or liposomes is expected to improve the lung

selectivity. The lung absorption rate of micronized budesonide in suspension was

compared with that of budesonide in solution using isolated perfused rat lungs








[128] with only a marginal difference in lung absorption rate. However, when

budesonide 21-palmitate was incorporated into liposomes budesonide showed

prolonged retention time (half-life = 6 hr) after intratracheal administration [85].

However, some studies give evidence that a portion of the budesonide dose is

retained in lung tissue longer than other steroids because it forms conjugates

with long-chain fatty acids (mostly oleic acid) within cells [102]. Such conjugation

does not appear to occur with beclomethasone dipropionate, fluticasone

propionate or other inhaled glucocorticoids. Budesonide fatty acid conjugates act

as an intracellular store of inactive drug since only free budesonide binds to the

glucocorticoid receptor. Currently, this depot effect has not been directly

correlated to an increase in the therapeutic effect.

Fluticasone Propionate (FP)

Fluticasone propionate (FP) is commercially available as Flovent

MDI (Glaxo-Wellcome) and the Diskhaler DPI (Glaxo-Wellcome). Doses of 100-

200 mcg/day for children, 200-500 mcg/day for adults with mild asthma, 500-

1000 mcg/day for adults with moderate asthma, and 1000-2000 mcg/day for

adults with severe asthma are recommended [129]. Following inhalation, 26% of

the dose from MDI or 15% of the dose from DPI is deposited in the lung [90],

while the majority impacts on the oropharyngeal region and is swallowed.

Fluticasone propionate undergoes extensive first-pass metabolism, resulting in a

oral bioavailability of less than 1%, and an overall bioavailability after inhalation

of 10-15% [83, 130]. Absorption of the lipophilic fluticasone molecule is slow








(MAT of 4.9 hours), leading to prolonged retention in the lungs and lower peak

plasma concentrations [131].

Fluticasone propionate has a high RBA of 1,800 and a high plasma

protein binding of 90% [Meibohm, 1998] compared to TA and BUD. The volume

of distribution of fluticasone propionate at steady state (Vdss) is 318 L, which is in

agreement with the high lipophilicity of the molecule [79]. Rapid hepatic

clearance of 66 L/hr minimizes systemic side, with almost 87-100% of the drug

excreted in the feces, and 3-40% as the inactive 17-carboxylic acid [4].

After IV administration, FP follows a three-compartment body

model with its terminal half-life ranging between 7.7 and 8.3 hrs [79]. Absorption

of FP in humans is slower than that of TA and BUD and is the overall rate-limiting

step in the lungs, and as a result terminal half-life values of 10 hours have been

reported after inhalation [93]. In a recent study it was shown that the t% is dose-

dependent and ranged between 5.2 and 7.4 hours with a mean of 6.0 + 0.7 hours

[90]. The reported value for the mean residence time of FP after inhaled

administration, calculated as the area under the first moment curve (AUMC)

divided by AUC, averaged 9.1 1.1 hours (ranging from 7.8 to 11 hours [90]).

The mean absorption time after inhalation of FP was found to range from 3.6 to

6.8 hours with a mean of approximately 5.0 hours [90].



Controlled Release


It has been demonstrated that encapsulation of glucocorticoids into

liposomes can lead to the enhancement of therapeutic efficacy, with a reduction








in their toxicity and prolongation of their therapeutic effect [85, 125]. Other

methods of obtaining controlled release in the lungs, such as polymeric

microspheres and microencapsulation techniques [34], are described in this

section.

Liposomes

In the last two decades, it has been demonstrated that

encapsulation of drugs into liposomes can lead to the enhancement of

therapeutic efficacy, reduction of their toxicity, and prolongation of their

therapeutic effect [125]. Liposomes have similar components to natural

membranes, composed of phospholipid bilayers entrapping hydrophilic or

hydrophobic materials between their aqueous or lipophilic membranes. Through

variation in size, lipid composition, charge, number of bilayers and surface

characteristics, the release-rate of drug from liposomes and therefore the

pharmacokinetics of the encapsulated drug can be modified [67].

Reports of pulmonary delivery of liposomal encapsulated-

glucocorticoids are rather scarce. Some glucocorticoids show low encapsulation

efficiencies, though, and tend to escape very easily from liposomes, as has been

demonstrated for hydrocortisone [132] and for triamcinolone acetonide [133]. In

order to overcome this problem different derivatives of glucocorticoids have been

used [85, 134, 135]. Encapsulation of triamcinolone acetonide-21-palmitate was

shown to have increased liposomal retention of 85%, compared to 5% for the

parent drug [134]. Brattsand [85] demonstrated that budesonide 21-palmitate

incorporated into liposomes showed prolonged residence time in the lungs of rats








(ti/2 = 6 hours) compared to free budesonide (t1/2 < 2 hours) after intratracheal

administration. Recently, pulmonary targeting was enhanced in rats by

incorporating triamcinolone acetonide phosphate (TAP) into multi-lamellar

liposomes [125]. Overall, these studies suggest that slow release from

liposomes induces increased pulmonary targeting, although difficulties in the

scale-up manufacturing of liposomes has limited their industrial use [34].

Biodegradable Microspheres

Biodegradable polymers are being used in a large number of

biomedical applications such as resorbable sutures, internal fixation devices,

degradable scaffolds for tissue regeneration, and matrices for drug delivery. The

biocompatibility of these polymers has been reviewed [136]. A variety of

synthetic and natural polymers have been found to exhibit minimal inflammatory

response in various implantation sites [34].

The advantages of microspheres over liposomes include greater

range of sizes, higher stability and shelf life, and longer retention in vivo (up to 6

months) [34]. Biodegradation is associated with materials that can be broken

down by natural means such as enzymatic or hydrolytic degradation [137].

Biodegradation of poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and their

copolymers poly(lactic-co-glycolic acid) (PLGA) yield the natural metabolic

products lactic acid and glycolic acid, which are incorporated into the tricarboxylic

acid cycle and excreted [69].

Although several reports of inhaled microsphere preparations have

shown improvements in targeted and sustained drug release, there have been no








reports on glucocorticoid microspheres. PLGA microspheres of isoproterenol, a

beta-agonist bronchodilator, intratracheally administered in rats was shown to

ameliorate bronchconstriction for 12 hours in contrast to 30 minutes after free

isopreterenol administration [138]. Preparations of large, porous particles of

PLGA encapsulated testosterone and insulin by double-emulsion solvent

evaporation showed effects up to 96 hours while improving deposition [69].

Sustained release of 2% rifampicin from PLGA microspheres from 3 to 7 days in

guinea pigs has been shown reduce mycobacterium infection in macrophages

[139]. Unfortunately, low encapsulation efficiencies (<40%) and concerns of

accumulation of slowly degrading polymers in the lungs for long-term use have

limited the therapeutic application of polymeric pulmonary sustained release

systems.

Microencapsulation

The area of microencapsulation is relatively new, previously limited

to solvent evaporation techniques [140-143]. Currently there are several different

ways of applying coatings to particles in industry, mainly through spray-coating

technologies [143]. Pranlukast, a luekotriene inhibitor, encapsulated with

hydroxypropylmethylcellulose (HPMC) nanospheres were prepared by spray

drying showed an improvement in inhalation efficiency but did not show a

significant difference in the dissolution rate [144]. The disadvantages applying

micron-thick coatings for sustained-release (10-100 microns thick) [145] is that

large quantities of solvents that must be dried under strong venting and that an

increase in particle size reduces the inhalation efficiency [34].








A novel coating method has recently been developed using rapid

thermal evaporation from a pulsed eximer laser to coat solid materials onto

particles [146]. The technique can deposit nanometric coatings (10-1000

nanometers) on core particles ranging from 500 nm to 1000 pm [146]. Through

this method, the coating material is generally less than 1% by mass, and coating

times are under one hour without the need for drying solvents.

This variation of pulsed laser deposition (PLD) uses high-energy

pulses of ultraviolet light to deposit solid coating materials onto particles.

Previously there has been a significant emphasis given to control of the particle

characteristics (shape, size, surface chemistry, adsorption, etc.), but little

attention has been on designing the desirable properties at the particulate

surface, which can ultimately lead to enhanced properties of the product [146].

By attaching atomic to nano-sized organic or inorganic, multi-elemental particles

either in discrete or continuous form onto the surface of the core particles, that is,

nano-functionalization of the particulate surface, materials and products with

significantly enhanced properties can be obtained.

Only limited reports have used pulsed laser deposition to deposit

polymeric nano-particle coatings on flat surfaces [147-150], and none have

reported coatings on particles. Through this coating method the coating material

is generally less than 1% by mass, and coating times are under one hour without

the need for drying solvents. This method has a wide variety of pharmaceutical

applications ranging from coatings to improve agglomeration and flowability,








stability, cell uptake and interactions, as well as controlling the release rate of the

drug.



Objectives


Many clinical trials have been performed comparing efficacy of the

commercially available glucocorticoids. Unfortunately establishing dose-

response studies for comparing clinical effectiveness of the inhaled

glucocorticoids has been difficult because of the lack surrogate markers. While

glucocorticoids exert many different effects in tissues, the extent of binding to the

cytosolic receptors (receptor occupancy) in each organ is presented as a

'universal' surrogate marker from which other local antiinflammatory effects and

systemic side effects can be correlated to.

The first objective is to compare the particle size, in vitro

dissolution, and in vivo absorption rate in rats of three inhaled glucocorticoid dry

powders, triamcinolone acetonide, budesonide, and fluticasone propionate. As

stated above, by comparing the plasma concentrations vs. time the absorption

rate can be observed. Then, the relationship of dissolution and absorption rate

will be further investigated by comparing the pulmonary targeting in one local

(lung) and four systemic (liver, kidney, spleen, and brain) organs of the same

three glucocorticoids. For both sets of experiments dry powder formulations will

be delivered intratracheally and drug solutions will be delivered intravenously to

compare differences in absorption and pulmonary targeting. Then, to further

evaluate the dissolution rate and its effect on the absorption rate and pulmonary





34

targeting, the dissolution rate in vitro, absorption rate, and pulmonary targeting in

vivo of coated budesonide particles will be evaluated.













CHAPTER 2
CHARACTERIZATION, IN VITRO DISSOLUTION, AND IN VIVO PLASMA
CONCENTRATIONS IN RATS OF TRIAMCINOLONE ACETONIDE,
BUDESONIDE, AND FLUTICASONE PROPIONATE DRY POWDERS



Introduction


In the past three years there have been two new inhaled

glucocorticoids introduced to the U.S., namely fluticasone propionate

(GlaxoWellcome) in 1997 and budesonide (Astra) in 1998. These drugs show

increased receptor binding affinity, total clearance values approaching the liver

blood flow, and as a result low oral bioavailability [74, 129]. Unfortunately,

comparing the clinical efficacy and pulmonary targeting of these drugs to

traditional inhaled glucocorticoids such as triamcinolone acetonide has been

difficult because of the lack of surrogate markers in humans [8].

As discussed previously, our group developed an ex vivo receptor

binding assay in rats that links a longer release rate of liposomal encapsulated of

triamcinolone acetonide phosphate (compared to free drug solutions) with an

increase in pulmonary targeting [151]. This relationship is now further evaluated

by investigating the in vitro dissolution rates and the in vivo absorption rates of

three different inhaled glucocorticoid dry powders, triamcinolone acetonide,

budesonide and fluticasone propionate. Comparisons of the dissolution and








absorption rates observed in this chapter can then be further analyzed by

measuring the pulmonary targeting in Chapter 3.

Micronized powders of triamcinolone acetonide, budesonide, and

fluticasone propionate were used for these studies. Scanning electron

microscopy was performed to ensure particle sizes were homogenous. Then, to

evaluate the effect of dissolution rate on the pharmacokinetics, the in vitro

dissolution of dry powder formulations was performed in a phosphate buffer

solution with surfactant at physiological temperature and pH. Because this in

vitro model utilizes a large volume of buffer and surfactant compared to in vivo

conditions, this method is used primarily to observe the trend of dissolution rates

of the three glucocorticoids.

To analyze the pharmacokinetics of triamcinolone acetonide,

budesonide, and fluticasone propionate in rats, plasma concentrations were

analyzed after administering equivalent doses in vivo in rats. The plasma

concentrations were measured by extracting drug from the plasma samples

using solid phase extraction (SPE) and injecting into a high-performance liquid

chromatography with double mass-spectrometer (LC/MS/MS) system. Overall,

the objective of the present study was to compare the particle size, dissolution

rates in vitro, and pharmacokinetic profiles of triamcinolone acetonide,

budesonide, and fluticasone propionate following intratracheal and intravenous

administration in vivo in rats at equal doses (100 ag/kg rat).








Hypothesis


Glucocorticoid powders of similar particle size with a slower

dissolution rate in vitro should display a slower absorption rate in vivo.



Materials and Methods


Chemicals

Analytical grade chemicals were obtained from Sigma Chemical

Co. (St. Louis, MO.). Micronized triamcinolone acetonide was obtained from

Sigma. Micronized budesonide was obtained from Sicor (Milan, Italy).

Micronized fluticasone propionate was obtained from Glaxo-Wellcome (Research

Triangle Park, NC). Micronized fluticasone propionate internal standard, 13C3-FP

(I.S.), was kindly provided by Glaxo Group Research (Herts, UK). Lactose

monohydrate NF extra-fine (particle size 10-30 microns) was obtained from EM

Industries (Hawthorne, NY). All other reagents were of analytical grade.

SEM Analysis

Micronized dry powders were analyzed using a Joel model 6400

EDS scanning electron microscope (SEM) to obtain information on the size,

shape, and surface morphology. Micrographs of powder samples were prepared

by applying a thin layer of carbon paint onto a graphite sample mount and

spreading approximately one milligram on top. Sample mounts then underwent a

carbon evaporation process prior to characterization. Characterization was








performed at 1-2 kEV in vacuum. Magnification of representative micrographs

was x 5,500.

In Vitro Dissolution

In vitro dissolution of dry powder formulations (500 mg of 2% drug

in lactose) was tested using a USP dissolution bath (VanKel Technology Group,

Cary, NC) in 900 ml of pH 7.4 phosphate-buffered saline (50 mM) at 37C. In

order to assure that the powders dissolved completely (FP aqueous sol. 100

ng/ml vs. 90 jg/ml with surfactant), 2% sodium-dodecyl sulfate was added to

increase solubility and wetting and to more closely mimic pulmonary surfactant

levels in vivo [152]. After drug was added one-milliliter samples were removed

and filtered through a 0.2 im syringe filter at 0, 0.5, 1, 1.5, 2, 2.5, 5, 10, 15, 30,

45, 60, and 600 minutes and analyzed using HPLC. Steady-state was reached

(10 Vig/ml) at 60 minutes (observed from release profiles) and replaced as 100%

dissolved.

Glucocorticoid concentrations in solution were analyzed using an

HPLC setup consisting of a Perkin Elmer series 3B pump with a flow rate of 1.0

ml/min and a Zorbax C-18 (150 x 4.6 mm) column connected to a Perkin Elmer

ISS 100 autoinjector. The detector was a Milton Roy SM-4000 attached to a

Hewlett Packard HP-3394A integrator. The eluent was monitored at 254 nm.

The mobile phase consisted of a mixture of 40%:60% v/v acetonitrile : 0.03%

triflouroacetic acid buffer for TA and 50%:50% v/v acetonitrile : 0.03%

triflouroacetic acid buffer for BUD and FP (see Figure 2.1). Samples were

injected after standards (5 standards of 1, 5, 10, 50, and 100 jig/ml) for each data








set. Slopes from the calibration curve from standards were used to calculate the

sample concentration for each data set (r2>0.99).

Half-life of dissolution was calculated by curve fitting using

SCIENTIST program and using the monoexponential formula:



Cumulative % Dissolved = 100 [ A exp 't ] (eq. 2.1)



to obtain the dissolution rate constant (a). Half-lives of dissolution (T50o/o) were

calculated as 0.693/a.

Differences in dissolution between drug formulations were tested

for significance using the FDA scale-up and postapproval changes (SUPAC)

similarity test (f2):



f2= 50 x LOG {[1+1/n X(Rt-Tt)2]-05 x 100} (eq. 2.2)



where n=13 time points (0, 0.5, 1, 1.5, 2, 2.5, 5, 10, 15, 30, 45, 60, and 600

minutes), Rt = the reference % drug dissolved at time t, and Tt. = the test % drug

dissolved at time t. An f2 value between 50 and 100 suggests the two dissolution

profiles are similar and an f2 value less than 50 indicates a lack of similarity [153].





40

Triamcinolone Acetonide


-I


4.23


Budesonide


Fluticasone Propionate


3.20
















7.64


Figure 2.1: HPLC chromatagrams of 100 ig/ml standards of: A) TA, B) BUD,
and C) FP


f~^ pop








IT Administration in Rats

The Animal Care Committee of the University of Florida, an

AAALAC approved facility, approved all animal procedures. Specific-pathogen-

free male F-344 rats, weighing approximately 220 to 250 grams, were housed in

a 12 hr light/dark cycle / constant temperature environment. Animals were

allowed free access to water and rat chow, but were food-fasted overnight prior

to each experiment.

On the day of the experiment rats were handled gently to produce

minimum stress. Once weighed, the rats were placed in a rat holder for

intraperitoneal administration of the anesthetic (fresh preparation of the

combination of 1.5 ml of ketamine 10%, 1.5 ml of xylazine 2% and 0.5 ml of

acepromazine 1%) at the dose of 1 ml/kg. The depth of anesthesia was checked

by tail pinch or pedal withdrawal reflex.

The neck of the completely anesthetized animal was shaved and

aseptically cleaned with isopropyl alcohol 70%. A one-centimeter midline vertical

incision was made originating above the sternal notch. The neck muscles and

glands were carefully dissected midline, until the trachea was exposed and a

tracheotomy was performed between the third and fourth tracheal rings. One

inch of a 14 gauge Novalon catheter sheath attached to a delivery device for

intratracheal (IT) administration of dry powders (Penn-Century, Philadelphia, PA),

was introduced into the trachea. A mixture of 5 0.5 mg (calibrated with lactose,

n=16) of extra fine monohydrate lactose and TA, BUD, or FP (0.4%, 100 jig/kg

dose) was placed in the chamber of the device and instilled in the lungs with








insufflation of 3 ml of air. A sham rat was included in each set of experiments

that received 5 mg of the vehicle (lactose). Animals (one animal per time point)

including sham rat, were decapitated at 0.5, 1, 2, 4, 6, or 12 hours after

instillation. Plasma was snap frozen and stored at -70 C until processing.

Organs were processed as discussed in Chapter 3 for receptor binding. Plasma

samples for three to five independent experiments using TA, BUD, and FP were

performed for a given time point after IT administration.

IV Administration in Rats

For the intravenous (IV) injection of budesonide, animals were

anesthetized as described above and the intracardial approach was used. For

this approach, IV solution was injected after needle placement in the heart and

verified by aspiration of blood. Either 1001 of IV solution (200[g/ml drug) or

100[1 of the vehicle (for the sham rat) was injected using a tuberculin syringe

with a 27-gauge needle. Animals (one animal per time point) including sham rat,

were decapitated at , 1, 2, 4, 6, or 12 hours after instillation. Plasma was snap

frozen and stored at -70C until processing. Organs were processed as

discussed in Chapter 3 for receptor binding. Plasma samples for three

independent experiments for BUD were performed for a given time point after IV

administration.

Solid Phase Extraction of Plasma Samples

Frozen plasma samples were thawed at room temperature. A

volume of 200 pil plasma was added to 1.8 ml of 10% ethanol with 10 ng/ml final

concentration of internal standard (13C3-FP for FP analysis, BUD for TA analysis,








and TA for BUD analysis). Recovery from the extraction technique was

measured by spiking blank plasma with 10 ng/ml final concentration of TA, BUD,

and FP standard for each sample set. Solid phase extraction (SPE) of

compounds was performed as previously described with modifications [154][Li,

1997 #8]. Briefly, samples with 10% ethanol were refrigerated for 15 minutes to

precipitate proteins and centrifuged at 4000 rpm. Supernatants were then

extracted using ethanol-activated 6 ml endcapped C-18 cartridges (Supelco).

The analytes were eluted with 2 x 1 ml of 60% ethyl acetate in heptane. The

residue was evaporated under vacuum and reconstituted in 100 pI of methanol-

water (80:20, v/v) mobile phase. A total sample volume of 80 Il was injected into

the LC/MS/MS system.

LC/MS/MS of Extracted Plasma Samples

The analysis of TA, BUD, and FP was performed as described

previously for FP with modifications [155]. A Micromass Quattro-LC-Z (Beverly,

MA) triple quadrupole mass spectrometer equipped with an atmospheric

pressure chemical ionization (APCI) ion source was used for analysis. The APCI

probe temperature was set to 500C and corona and cone voltages were set at

optimum conditions after tuning. The MS/MS signals were optimized by injecting

a 1 ptg/ml standard in methanol at a flow-rate of 100 pl/min using a Kd-Scientific

infusion pump. At low collision energy, mass analysis was collected through the

second quadrople for 432.0>432.0 for TA, 428.0>428.0 for BUD, and

500.0>500.0 for FP. Argon was used as the collision gas at 1.5 x 10-3 mBar.

The mass spectrometer was linked to a Perkin Elmer ISS-200 autosampler via








contact closure and the operation was controlled by computer software,

Masslynx 3.1. The mobile phase was a mixture of methanol-water (80:20, v/v)

delivered at a flow-rate of 1.2 ml/min by a LDC/ Milton Roy CM4000 multiple-

solvent pump. Chromatographic separations were achieved using a Waters 5-pt

m ODS2 (4.6 x 50 mm) column (Milford, MA) preceded by a Whatman 5-ptm ODS

C-18 guard column cartridge (Clifton, NJ). Data analysis was performed using

Masslynx software. Samples were injected with calibration curves (4 standards

of 1, 10, 20, and 100 ng/ml) for each data set (see Figure 2.2). Slopes from

concentration vs. peak area profiles were used for each data set (r2>0.97) and

plasma concentrations calculated by multiplying by (Area / Slope) 2 / Recovery.

Recovery percentages, calculated by comparing extracted plasma samples

spiked with 10 ng/ml final concentration of TA, BUD, and FP to standards, were

calculated as 56.3 5.6% for TA (n=3), 64.1 3.2% for BUD (n=12), and 86.9 +

3.9% for FP (n=3).

Non-Compartmental Pharmacokinetic Analysis

The non-compartmental analysis for plasma concentrations over

time was performed using standard techniques [156]. The time to peak tmax (at

Cmax) and Cmax was determined as the highest plasma concentration from the

average plasma concentration vs. time profile.

Mean residence time (MRT) for plasma data was calculated from

the relationship:


MRToo =AUMCo0. / AUC0,o(


(eq. 2.3)











PG c073 .
TA A
j/ \





7 ~ / ... __-___--- _
(PC- .10 '

BUD / \




0- -20 6AO O n 0 S l0 1.20 \ T40 16


1.27
13C FP


FP


1.00 1-.. 1,SO 1.75


2.0( 3.2-< ,>M


2.7G 2.0 3.o S1 SO 3.75


Figure 2.2:


LC/MS/MS chromatograms of TA (432>432), BUD (429>429),
13C FP (503>503), FP (500>500)


0.2S 0-&0 16.T


Tirw








where AUMCO-,0 is the area under the first moment curve, and AUC0o- is the

area under the curve. The area under the curve (AUCo0.) was calculated by the

trapezoidal rule from the C versus time curve (AUCo-,12) and extrapolation to

infinity (AUC12_o) calculated by adding the ratio of the last measured C12 and the

first-order rate constant of the terminal phase (ke, exponential regression from 4

to 12 hours, r2>0.97). The AUC after IV administration was calculated by

interpolating to from the slope of the first 3 time points (0.5 to 2 hours). The area

under the first moment curve (AUMCo0_o) was calculated from the C t versus

time curve (AUMCo-12) and extrapolation to infinity (AUMC12-_o) calculated by

adding the ratio of the last measured C12 t and the squared of the rate constant

of elimination (C12*t/ke + C12/ke2).


Differences in AUC's between IV and IT administration were tested

for significance using unpaired Student's T-test. Pulmonary bioavailability for

clearance determinations (CI/F) was approximated as 1.0 because the total dose

was delivered directly to the lungs. The total body clearance (CI/F) was

calculated as dose divided by AUC0ooo divided by bioavailability (Dose/AUC).

Mean absorption times for TA and BUD plasma data were

calculated from the relationship:



MAT = MRTIT MRTiv (eq. 2.3)



For TA IV, the plasma concentration profile for TAP-sol was used from a previous

study [151] based on the assumption that TAP conversion time to TA is negligible








[124] and glucocorticoids in solution are rapidly absorbed into the systemic

circulation upon intratracheal instillation [157].



Results


SEM Analysis

SEM photomicrographs of micronized dry powders are depicted in

Figure 2.3. SEM analysis revealed little difference in particle size and

morphology between TA, BUD, and FP from different sources. TA powders

displayed some spherical particulates > 3 microns, but were otherwise

homogenous. BUD and FP powders were slightly agglomerated but particle

sizes were more monodisperse. A quantitative analysis of TA, BUD, and FP

particles was not performed, but particle sizes ranged generally from 1 to 3

microns.

In Vitro Dissolution

Cumulative dissolution profiles for TA, BUD, and FP powders are

depicted in Figure 2.4. Fitted monoexponential curves showed some divergence

from 5 to 10 minutes, but resulting fits showed good correlation (r2>0.99 for all

three). Dissolution half-lives of release (tso50% ) were not statistically different for

TA and BUD (f2=74), calculated as 1.1 0.1 minutes (n=3) and 1.2 0.1 minutes

(n=6), respectively. Dissolution half-life for FP (n=3) was significantly longer than

TA (f2=33) and BUD (f2=34) at 4.0 + 0.4 minutes.










A











B













C













Figure 2.3: SEM micrographs of: A) micronized TA powder, B) micronized BUD
powder, and C) micronized FP powder. Resolution 5,500 times
magnification.














120


100


80


60


40


20


0


0 10 20 30 40 50 6C


Time (min)


Figure 2.4:


Dissolution of triamcinolone acetonide N (TA), budesonide *
(BUD), and fluticasone propionate A (FP) dry powders in pH 7.4
PBS (50 mM, 2.0 % SDS) at 37C (n=3).








Pharmacokinetics of TA in Rats

The average triamcinolone acetonide (TA) plasma concentrations

after IT administration of TAP-sol (n=3) and TA dry powder (n=3) are shown in

Figure 2.5. The pharmacokinetic parameters resulting from the analysis of

plasma data are listed in Table 2.1. The plasma concentrations of triamcinolone

acetonide after intratracheal delivery of triamcinolone acetonide phosphate

solution (TAP-sol) in rats was previously investigated [151]. TAP-sol was

compared to TA dry powder in place of IV data because it has been shown that

metabolism of TAP is fast in vivo [124] and absorption of glucocorticoids in

solution after IT administration occurs relatively unhindered [87].

The maximum TA plasma concentration of 62 ng/ml was observed

after the first time point of 1.0 hour for TAP-sol and 200 ng/ml after the first time

point of 0.5 hour for TA dry powder. The terminal slope (ke) was 0.6 hr'1 for TAP-

sol and 0.35 hr1 (r2=0.99) for TA dry powder. The AUCo0, for the drug plasma

concentration-time profiles was 179 ng/ml*hr for TAP-sol and 234 15.9

ng/ml*hr. The clearance (CI/F) was calculated as 112 ml/hr for TAP-sol and 85.5

ml/hr for TA dry powder. The mean residence time (MRT) was calculated as 1.8

hours for TAP-sol and 2.0 for TA dry powder, with a resulting mean absorption

time (MAT) of 0.2 hours for TA dry powder.

Pharmacokinetics of BUD in Rats

The average budesonide (BUD) plasma concentrations after IV

administration (n=3) and IT administration (n=5) are shown in Figure 2.6. The








pharmacokinetic parameters resulting from the analysis of plasma data are listed

in Table 2.1.

The maximum BUD plasma concentration of 29.3 ng/ml was

observed after the first time point of 0.5 hour after IV administration and 48.1

ng/ml after the first time point of 0.5 hour for IT administration. The terminal

slope (ke) was 0.26 hr1 (r2=0.99) after IV administration and 0.35 hr-1 (r2=0.99)

after IT administration. The AUCo0co was 60.6 + 26.9 ng/ml*hr after IV

administration compared to 85.3 41.2 ng/ml*hr after IT administration, and were

not statistically different (p>0.2). The clearance (CI/F) was calculated as 330

ml/hr after IV administration and 234 ml/hr after IT administration. The mean

residence time (MRT) was calculated as 2.4 hours after IV administration and 2.7

after IT administration, with a resulting mean absorption time (MAT) of 0.3 hours

after IT administration.

Pharmacokinetics of FP in Rats

The average fluticasone propionate (FP) plasma concentrations

after IT administration (n=3) are shown in Figure 2.7. The pharmacokinetic

parameters resulting from the analysis of plasma data are listed in Table 2.1.

The maximum FP plasma concentration of 15.2 ng/ml was

observed after 1.0 hour after IT administration. The terminal slope (ke) was 0.22

hr1 (r2=0.98) after IT administration. The AUCo0_o for the drug plasma

concentration-time profile was 92.0 ng/ml*hr after IT administration. The

clearance (CI/F) was calculated as 217 ml/hr after IT administration. The mean

residence time (MRT) was calculated as 5.6 hours after IT administration.


















Table 2.1 Pharmacokinetic parameters for BUD TA, BUD, and FP.
TAP-sol* TA IT BUD IV BUD IT FP IT

Cmax (ng/ml) 62 200 29.3 48.1 15.2

tmax(hr) 1.0 0.5 0.5 0.5 1.0

ke (hr1) 0.6 0.35 0.33 0.26 0.22

AUC (ng/ml*hr) 179 234 + 60.6 + 85.3 + 92.0
15.9 26.9 41.2 54.5

CI/F (ml/hr) 112 85.5 330 234 217

MRT(hr) 1.8 2.0 2.4 2.7 5.6

MAT (hr) 0.2 0.3


* previously reported from [151].















STAP-sol (=_3)*
STA IT (n=3)


0 5 10 15


Time (hrs)


Figure 2.5.


Average plasma concentrations (mean + SD) of TAP-sol (n=3*) and
TA dry powder (n=3) after intratracheal administration.


* previously reported from [151].


1000.000


100.000



10.000



1.000


0.100















--BUD IV (n=3)
-u--BUD IT (n=5)


0 5 10


Time (hrs)


Figure 2.6.


Average plasma concentrations (mean + SD) of BUD after
intravenous (n=3) and intratracheal administration (n=5).


1000.00


100.00



10.00



1.00


0.10














--TA IT (n=3)
--BUD IT (n=5)
---FP IT(n=3)


0 5 10 15


Time (hrs)


Figure 2.7.


Average plasma concentrations (mean + SD) of TA (n=3), BUD
(n=5), and FP (n=3) after intratracheal administration.


1000.00


100.00



10.00



1.00



0.10








Discussion


In the present study we investigated the particle size and

morphology, in vitro dissolution, and in vivo systemic plasma concentrations of

triamcinolone acetonide (TA), budesonide (BUD), and fluticasone propionate

(FP) after intratracheal (IT) and intravenous (IV) administration. Unfortunately

budesonide was the only drug that plasma samples were obtained for after

intravenous administration, but aided in comparing absorption of free budesonide

powders to the coated powders in Chapter 4. Triamcinolone acetonide

phosphate solution delivered intratracheally previously analyzed [151] was

substituted for TA IV because, as stated before, absorption of glucocorticoids in

solution after IT administration occurs relatively unhindered [87].

The comparison of particle size and morphology using SEM was

qualitative rather than quantitative, but verification of particle sizes was

necessary to ensure that they were homogenous. Mean particle size was

estimated as 1 to 3 microns for all three drugs, which is similar to the particle size

used in dry-powder formulations in humans.

Comparison of the dissolution rates of all three powders showed FP

had the slowest dissolution half-life of approximately four minutes, with TA and

BUD dissolution half-lives close to a minute. While the USP dissolution setup is

typically used for monitoring tablet disintegration, this model needs improvement

to more closely mimic pulmonary conditions. SDS was used as a surfactant, but

the use of human lung surfactant has been studied visually in vitro on glass








slides for the inhaled glucocorticoids and produced a similar order in dissolution

times comparing BUD (6 minutes) and FP (< 8 hours) [12].

The plasma concentrations of TA after IT administration of TAP

solution and TA dry powder were similar at the observed time points from 1 to 6

hours, which indicates that relative bioavailabiltities were similar. Plasma

concentrations of BUD after intravenous and intratracheal administration were

also similar, which indicates that relative bioavailabilities of BUD were also

similar. The AUC for intravenous administration of BUD of 60.6 ng/ml*hr also

agrees well with previously reported studies in rats by Chanoine [127] of IV

administration of BUD (68 ng/ml*hr). Chanoine also found similar fast absorption

of BUD after intratracheal administration with <10% remaining in the lung after

0.5 hour [127]. Plasma concentrations of FP appeared lower after IT

administration than TA and BUD, but with a flatter terminal slope.

Compartmental analysis was not performed because of the lack of early time

points, where a clearer absorption profile should be observable if sampled more

frequently. This data was necessary for comparison, though, to the coated

particles in Chapter 4.

Comparison of the clearance values (CI/F) at equal doses (100

pLg/kg) were calculated as TA the lowest (85.5-112 ml/hr), FP in the middle (217

ml/hr), and BUD slightly higher (234-433 ml/hr). This trend (TA
similar to the order of clearance values in humans, is lower than a reported liver

blood flow in rats of similar size (250 grams) of 828 ml/hr [158]. The calculated

clearance of budesonide in rats was comparable to a previously reported value of








375 ml/hr found by Chanoine after IV administration [127]. The order of

calculated clearance values in rats is similar to the trend of clearances in humans

of TA being the lowest (37.3 L/hr, [78]), FP in the middle (66 L/hr, [79]), and BUD

being the highest (84 L/hr, [74]). Although these values in humans are closer to

the liver blood flow of 90 L/hr [158] possibly due to differences in liver enzyme

activity or size scaling, the relative rankings are similar.

The mean absorption time (MAT) of TA dry powder was relatively

short (MAT=0.2 hour), calculated by the small difference in mean residence times

after IT administration of TAP-solution (MRT=1.8 hours) and dry-powder

formulation (MRT=2.0 hours). This agrees well with the trend of fast dissolution

in vitro but not with reported mean absorption time values in humans of 2.9 hours

[129], which may be due a slower dissolving formulation in the MDI. Also, the

mean absorption time of BUD powders was short (MAT=0.3 hour), calculated by

the small difference in mean residence times after IV (MRT=2.4 hours) and IT

(MRT=2.7 hours) administration. This agrees well with the fast in vitro dissolution

and with reported values of fast in vivo absorption rates in rats [127] and in

humans [129]. Although IV data for FP in rats was not available, the flatter

plasma concentration profile of FP showed a later tmax at 1.0 hour and the highest

MRT of 5.6 hours. This suggests that absorption of FP is slower than TA and

BUD, which may be linked to slower dissolution similar to observed in vitro

results. This also agrees well with the reported tmax of FP after inhalation of

different doses in humans of 1.0 hour and a high MRT of 9.0 hours [90].








In conclusion, the in vitro dissolution of TA and BUD powders was

faster when compared to FP powders, which correlated to a faster absorption

time for TA and BUD than FP in vivo. Overall, the rankings of calculated

absorption rates and clearance values for TA, BUD, and FP suggests that similar

trends in rankings of absorption and clearance exist between these studies in

rats and in published human studies. As previously shown in computer

simulations, theoretically glucocorticoids with fast dissolution rates, such as TA

and BUD, rapidly enter pulmonary cells but are removed immediately to the

systemic circulation and low pulmonary targeting is observed. On the other

hand, glucocorticoids with slower dissolution rates, such as FP, obtain higher

targeting because drug concentrations in the lung are higher than systemic levels

over an extended period of time [14]. To experimentally compare drugs with

different dissolution rates and the level of pulmonary targeting the same three

drugs (TA, BUD, and FP) will be compared using an ex vivo receptor binding

assay in rats in Chapter 3.



Conclusions


Using SEM analysis, the particle sizes of micronized TA, BUD, and

FP powders appeared to be similar (1-3 microns).

The dissolution of TA and BUD in vitro was fast (t50% < 2 min.), with

FP dissolution being slower (t50% = 4 min.).

Calculated clearance values for TA, BUD, and FP in rats showed a

similar order to calculated values in human studies.





60

* Absorption in vivo of TA and BUD appeared faster and FP, showing

a similar trend to dissolution results.













CHAPTER 3
ASSESSMENT OF PULMONARY TARGETING OF TRIAMCINOLONE
ACETONIDE, BUDESONIDE, AND FLUTICASONE PROPIONATE USING AN
EX VIVO RECEPTOR BINDING ASSAY



Introduction


Computer simulations have shown that a higher pulmonary

selectivity can be achieved at slower dissolution or release rates, therefore

increasing the pulmonary residence time [14]. In addition, the limited data

available for currently available inhaled glucocorticoids suggests that drugs with

slower dissolution rates, such as fluticasone propionate, have higher pulmonary

targeting [159]. Based on these findings it is predicted that drugs with slower

dissolution and pulmonary absorption will have increased pulmonary targeting,

and conversely that drugs with fast dissolution and absorption rates will show

similar profiles in receptor occupancies in local and systemic organs.

The proposed method of investigating this hypothesis was to

evaluate the pulmonary targeting (PT) in vivo of triamcinolone acetonide,

budesonide, and fluticasone propionate administered intratracheally in dry

powder lactose formulations and intravenously in IV solutions. These in vivo

studies will utilize an ex vivo animal model previously described [160].

Monitoring the receptor binding in local and systemic organs should provide a

more complete profile of the pulmonary targeting. Intratracheal (IT)








administration to the site of action will be compared to intravenous (IV)

administration to observe differences in pulmonary targeting after pulmonary

absorption. Pulmonary targeting of intravenous administration of TA was

previously investigated only in the lung vs. liver receptor binding profiles, which

are used for comparison [160]. While these and other studies involving liposomal

delivery monitored only receptor binding in the liver to assess the extent of

systemic effects, kidney, spleen, and brain were included in this study as

systemic organs to further investigate the distribution of the drug throughout the

body. Overall, the objective of this section is to compare the pulmonary targeting

of TA, BUD, and FP in rats by monitoring the decrease in % free receptors

(receptor occupancy) in one local and four systemic organs.



Hypothesis


Glucocortioid powders with slower dissolution in vitro should display

higher pulmonary targeting in vivo.



Materials and Methods


Chemicals

Analytical grade chemicals were obtained from Sigma Chemical

Co. (St. Louis, MO). Micronized triamcinolone acetonide was obtained from

Sigma. Micronized budesonide was obtained from Sicor (Milan, Italy).

Micronized fluticasone propionate was obtained from Glaxo-Wellcome (Research








Triangle Park, North Carolina). Lactose monohydrate NF extra fine (particle size

10-30 microns) was obtained from EM industries (Hawthorne, NY). Radiolabeled

triamcinolone acetonide ({6,7-3H} TA, 35.4 Ci/mmol) was purchased from DuPont

NEN Research Products (Boston, MA). All other reagents were of analytical

grade.

IT Administration in Rats

Intravenous solutions were prepared by dissolving the

glucocorticoids in a mixture of PEG 300 and saline (2:1 v/v) to obtain a final

concentration of 200 ptg/ml. Intratracheal dry powders were mixed by

geometrically diluting glucocorticoid powders with lactose to obtain a

concentration of 4 ptg / mg lactose.

The Animal Care Committee of the University of Florida, an

AAALAC approved facility, approved all animal procedures. Specific-pathogen-

free male F-344 rats, weighing approximately 220 to 250 grams, were housed in

a 12 hr light/dark cycle / constant temperature environment. Animals were

allowed free access to water and rat chow, but were food-fasted overnight prior

to each experiment.

The animals were housed in the operating room 12 hours before

the experiment to get them accustomed to the new environment. The day of the

experiment the rats were handled gently to produce minimum stress. Once

weighed, the rats were placed in a rat holder for intraperitoneal administration of

the anesthetic (fresh preparation of the combination of 1.5 ml of ketamine 10%,








1.5 ml of xylazine 2% and 0.5 ml of acepromazine 1%) at the dose of 1 ml/kg.

The depth of anesthesia was checked by tail pinch or pedal withdrawal reflex.

The neck of the completely anesthetized animal was shaved and

aseptically cleaned with isopropyl alcohol 70%. A one-centimeter midline vertical

incision was made originating above the sternal notch. The neck muscles and

glands were carefully dissected midline, until the trachea was exposed and a

tracheotomy was performed between the third and fourth tracheal rings. One

inch of a 14 gauge Novalon catheter sheath attached to a delivery device for

intratracheal administration of dry powders (Penn-Century, Philadelphia, PA),

was introduced into the trachea. A mixture of 5 0.5 mg (calibrated with lactose,

n=16) of extra fine monohydrate lactose and TA, BUD, or FP (0.4%, 100 ag/kg

dose) was placed in the chamber of the device and instilled in the lungs with

insufflation of 3 ml of air. A sham rat was included in each set of experiments

that received 5 mg of the vehicle (lactose). Animals (one animal per time point)

including sham rat, were decapitated at 0.5, 1, 2, 4, 6, or 12 hours after

instillation. Lung, liver, kidney, spleen and brain were immediately processed for

receptor binding studies. A total of six to nine independent experiments for TA,

BUD, and FP, respectively, were performed for a given time point after IT

administration.

IV Administration in Rats

For the intravenous (IV) injection of budesonide, animals were

anesthetized as described above and the intracardial approach was used. For

this approach, IV solution was injected after placement in the heart and verified








by aspiration of blood. Either 100 pl of IV solution (200 tg/ml drug) or 100l of

the vehicle (for the sham rat) was slowly injected using a tuberculin syringe with

a 27-gauge needle. Animals (one animal per time point) including sham rat,

were decapitated at , 1, 2, 4, 6, or 12 hours after instillation. Lung, liver,

kidney, spleen and brain were immediately processed for receptor binding

studies. A total of three to six independent experiments for TA (previously

reported [160]), BUD, FP were performed for a given time point after IV

administration.

Ex Vivo Receptor Binding Assay

An ex vivo receptor binding assay previously described [125] was

employed with minor modifications. Briefly, immediately after decapitation, the

lungs (without trachea), a lobe of the liver, kidney, spleen, and brain were

rejected and placed on ice. The weighed tissue was added to 10 times (for liver

and spleen) and 4 times (lung, kidney, and brain) organ weight of ice-cooled

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

dithiothreitol). The mixture was then homogenized in a Virtis 45 homogenizer at

40% of full speed, for three periods of 5 seconds each with a 30-second cooling

period between each step. One-tenth volume of 5% activated charcoal (prepared

in incubation buffer) was added to the homogenate and mixed. After 5 minutes, the

suspension was centrifuged at 4C at 50,000g for 10 min. in a Beckman centrifuge

equipped with a JA-21 rotor (Beckman instruments, Palo Alto, CA) to obtain a clear

supernatant. Aliquots of the supernatant (150 p1) were transferred into

microcentrifuge tubes that contained 25 1\ of 3H-triamcinolone acetonide in








incubation buffer (final concentration 10 nM) and 25 [1 of incubation buffer or 25 p1

of unlabeled TA (10 jM) to determine total and non-specific binding respectively.

Aliquots of 150 p1 of the resultant cytosol preparations were transferred into

microcentrifuge tubes which contained 25 p1 of 3H-triamcinolone acetonide in

incubation buffer (10 nM final concentration) and 25 [1 of incubation buffer to

determine the amount of total radioactivity. After a 16-24 hour incubation period at

4C, the unbound glucocorticoid was removed by addition of a 5% suspension of

activated charcoal in buffer (200 1\). The mixture was incubated for 5 min on ice

and then centrifuged at 10,000 rpm for 5 min in a micro-centrifuge (Fisher model

235A). The radioactivity (dpm) in 300 p1 of supernatant was determined using a

liquid scintillation counter (Beckman model LS 5000 TD, Palo Alto, CA).

All determinations were performed in triplicate and mean values for

the specific binding in sham rats collected after all intratracheal or intravenous

administration were used to determine 100 % free receptor level for individual data

sets. Specific binding estimates found in the individual tissues differed slightly

between IV(n=12, lung=1.25 nM, kidney=0.74 nM, liver=1.01 nM, and brain=0.35

nM) and IT (n=30, lung=1.01 nM, kidney=0.72 nM, liver=1.08 nM, and brain=0.40

nM) shams.

Non-Compartmental Analysis

In order to quantify the receptor binding in each tissue and degree of

pulmonary targeting the area-under-the-effect-curve (AUECorgan) up to six hours

was calculated for each investigation set by the trapezoidal rule from percent free

receptors vs. time profiles for both the local and systemic organs. The 12 hour time








point was not included in these calculations as the data density between 6 and 12

hours was not sufficient and flor ease of comparison with previous studies [125].

Pulmonary targeting was defined as:



PT = AUECLung AUECsys organ (eq. 3.1)



where AUECsys organ represents the AUEC of liver, spleen, kidney, or brain. All data

is reported as mean standard deviation of three or 6 independent experiments.

Differences in AUEC's between lung and systemic organs were tested for

significance using unpaired Student's T-test (p<0.05).

For comparison of the pulmonary targeting between different drugs

after IT administration, the difference in pulmonary targeting (using liver and

kidney as the systemic organ) was defined as



Diff. in PT = PTdrug 1 PTdrug 2 (eq. 3.2)



Where Drug 1 and 2 are TA, BUD, or FP. Differences in PT between drug

treatments were tested for significance using unpaired Student's T-test.

The mean pulmonary effect time was calculated from AUECoo and

AUMEC.o according to equation 3.3


MET = AUMECQ/AUEC,(3


(eq. 3.3)








where AUMECO-0 o is the area under the first moment curve, and AUECO_. is the

area under the curve. The area under the curve (AUECo-0) was calculated by

the trapezoidal rule from the E versus time curve (AUECo-012) and extrapolation

to infinity (AUEC12-,o) calculated by adding the ratio of the last E12 measured and

the first-order rate constant of the terminal phase (ke). The area under the first

moment curve (AUMECo_,) was calculated from the E t versus time curve

(AUMECo-12) and extrapolation to infinity (AUMEC12_,o) calculated by adding the

ratio of the last measured E12 t and the squared of the rate constant of

elimination (E12 t / ke + E12 / ke2).



Results


Receptor-Binding of TA after IT Administration in Rats

The resulting receptor occupancy versus time profiles after

intratracheal administration of triamcinolone acetonide dry powders are shown in

Figure 3.1 and AUEC's reported in Table 3.1. There was a significant difference

in receptor occupancies between lung versus kidney (p<0.01) and lung versus

brain (p<0.01), while lung versus liver (p>0.1) and lung versus spleen (p>0.4)

receptor occupancies resulted in close superimposition. The pulmonary MET

was 4.4 hours.

Receptor-Binding of TA after IV Administration in Rats

The resulting receptor occupancy versus time profile after

intravenously administered triamcinolone acetonide solutions is shown in Figure

3.2 and AUEC's reported in Table 3.1 as previously reported [160]. There was








no significant difference in receptor occupancies between lung versus liver

(p>0.20), resulting in close superimposition. The pulmonary MET was 2.8 hours.

Receptor-Binding of BUD after IT Administration in Rats

The resulting receptor occupancy versus time profiles after

intratracheally administered budesonide dry powder are shown in Figure 3.3 and

AUEC's reported in Table 3.1. There was a significant difference in receptor

occupancies between lung versus brain (p<0.01), while no targeting was

observed for lung versus liver (p>0.1), lung versus kidney (p>0.2), and lung

versus spleen (p>0.3). The pulmonary MET was 3.6 hours.

Receptor-Binding of BUD after IV Administration in Rats

The resulting receptor occupancy versus time profiles after

intravenously administered budesonide solutions are shown in Figure 3.3 and

AUEC's reported in Table 3.1. There was a significant difference in receptor

occupancies between lung versus brain (p<0.01), while no targeting was

observed for lung versus liver (p>0.4), lung versus kidney (p>0.3), and lung

versus spleen (p>0.3). The pulmonary MET was 3.1 hours.

Receptor-Binding of FP after IT Administration in Rats

The resulting receptor occupancy versus time profiles after

intratracheally administered fluticasone propionate dry powders are shown in

Figure 3.5 and AUEC's reported in Table 3.1. There was a significant difference

in receptor occupancies between lung versus liver (p<0.01), lung versus kidney

(p<0.01), lung versus spleen (p<0.01), and lung versus brain (p<0.01). The

pulmonary MET was 4.1 hours.








Receptor-Binding of FP after IV Administration in Rats

The resulting receptor occupancy versus time profiles after

intravenous administered fluticasone propionate solutions are shown in Figure

3.6 and AUEC's reported in Table 3.1. There was a significant difference in

receptor occupancies between lung versus liver (p<0.02) and lung versus brain

(p<0.01), while no targeting was observed for lung versus kidney (p>0.2) and

lung versus spleen (p>0.3). The pulmonary MET was 5.5 hours.

Comparison of Pulmonary Targeting between Drug Formulations

Differences in pulmonary targeting between TA vs. BUD, TA vs.

FP, BUD vs. FP (using liver and kidney for comparison) are reported in Table

3.2. Statistical significance was measured using unpaired Student's t-test

between individual AUEC's. There was no significant difference between TA vs.

BUD using kidney or liver as the systemic organ (p>0.4 and p>0.05,

respectively). For TA vs. FP there was a significant difference using liver as the

systemic organ (p<0.01), while there was no difference using the kidney as the

systemic organ (p>0.3). For BUD vs. FP both liver and kidney showed a

significant difference (p<0.01 for both).



Discussion


These experiments assessed the pulmonary targeting of

triamcinolone acetonide (TA), budesonide (BUD), and fluticasone propionate

(FP) after intratracheal (IT) and intravenous (IV) administration in vivo in rats. As

stated in Chapter 2, intravenous administration was compared with intratracheal











Lung vs. Kidney *


5 10
time (hrs)


5 10
time (hrs)


Lung vs. Spleen


Lung vs. Brain *


0 5 10
time (hrs)


Figure 3.1:


5 10 15
time (hrs)


Receptor occupancy time profiles after intratracheal administration
of triamcinolone acetonide-dry powder (100 p[g/kg): A) lung *
versus liver U (n=9**), B) lung versus spleen U (n=9**), C) lung
* versus kidney U (n=9**), and D) lung versus brain U, (n=3).


* denotes p < 0.05 between AUEC's (0-6 hrs).
** n=6 previously reported from [151].


Lung vs. Liver






















Lung vs. Liver


5 10
time (hrs)


Figure 3.2: Receptor occupancy time profile after intravenous administration of
triamcinolone acetonide solution (100 pjg/kg): A) lung versus liver
U (n=6**).


** previously reported from [151].











Lung vs. Kidney


0 5 10
time (hrs)


Lung vs. Spleen


5 10
time (hrs)


Figure 3.3:


0 5 10


time (hrs)


Lung vs. Brain *


5 10 15
time (hrs)


Receptor occupancy time profiles after intratracheal administration
of budesonide-dry powder (100 p!g/kg): A) lung versus liver a
(n=6), B) lung versus spleen U (n=6), C) lung versus kidney U
(n=6), and D) lung versus brain U, (n=6).


* denotes p < 0.05 between AUEC's (0-6 hrs).


Lung vs. Liver











Lung vs. Kidney


5 10
time (hrs)


Lung vs. Spleen


0 5 10
time (hrs)


Lung vs. Brain *


0 5 10
time (hrs)


Figure 3.4:


5 10
time (hrs)


Receptor occupancy time profiles after intravenous administration
of budesonide solution (100 jg/kg): A) lung versus liver U (n=3),
B) lung versus spleen N (n=3), C) lung versus kidney M (n=3),
and D) lung versus brain M, (n=3).


* denotes p < 0.05 between AUEC's (0-6 hrs).


Lung vs. Liver













Lung vs. Kidney *


5 10
time (hrs)


Lung vs. Spleen *


5 10
time (hrs)


Lung vs. Brain *


5 10
time (hrs)


Figure 3.5:


5 10
time (hrs)


Receptor occupancy time profiles after intratracheal administration
of fluticasone propionate-dry powder (100 tg/kg): A) lung versus
liver N (n=9**), B) lung versus spleen U (n=9**), C) lung *
versus kidney 0 (n=9**), and D) lung versus brain n, (n=6).


* denotes p < 0.05 between AUEC's (0-6 hrs).
** n=3 previously reported from [151].


Lung vs. Liver *


15













Lung vs. Kidney


0 5 10 15
time (hrs)


Lung vs. Spleen


0 5 10
time (hrs)


Figure 3.6:


5 10
time (hrs)


Lung vs. Brain *


0 5 10
time (hrs)


Receptor occupancy time profiles after intravenous administration
of fluticasone propionate solution (100 gg/kg): A) lung versus
liver U (n=6), B) lung versus spleen U (n=6), C) lung versus
kidney N (n=6), and D) lung versus brain U, (n=3).


* denotes p < 0.05 between AUEC's (0-6 hrs).


Lung vs. Liver *








Mean area-under-the-effect-curve (AUEC) and pulmonary targeting


(PT) values for each glucocorticoid.
TAIT TAIV BUD IT BUD IV FP IT FP IV
(n=9)* (n=6)** (n=6) (n=3) (n=9)*** (n=3)

AUEC (%*hr)

AUECiung 419 76 303+81 245 102 350 55 317 90 396 110

AUECiiver 379 48 322 77 197+62 337 104 109 40 211 127

AUECkidney 322 41 204 57 334 +65 194 79 349 108

AUECspieen 415 43 223 +64 327 65 229 110 381 62

AUECbrain 68 40 72 61 124 64 76 58 108 20

Pulmonary Targeting (%*hr)

AUECiung- 52 47 -19 6 49 64 13 52 208 77 185 74
AUECiiver

AUECiung- 110 90 41 63 17 18 123 79 48 74
AUECkidney

AUECiung- 17 58 22 64 24 18 87 + 55 16 64
AU ECspleen

AUECiung- 290+137 17346 226 26 216 110 231 120
AUECbrain

Pulmonary Mean Effect Time (hr)

METiung 4.4 2.8 3.6 3.1 4.1 5.5


* n=6 previously reported from [151], n=3 for brain.
** previously reported from [160].
** n=3 previously reported from [151], n=6 for brain.
Boldface denotes p < 0.05 between AUEC's (0-6 hrs).


Table 3.1:















Differences in PT and calculated P-values (unpaired T-test)
comparing pulmonary targeting of TA vs. BUD, TA vs. FP, BUD vs.
FP after IT administration (using the liver and kidney for systemic
organs only).

Difference in PT

TA vs. BUD PTiiver 3.9

TA vs. BUD PTkidney 69

TA vs. FP PTiiver 156

TA vs. FP PTkidney 13

BUD vs. FP PTiiver 160

BUD vs. FP PTkidney 82

Boldface denotes p < 0.05 between AUEC's (0-6 hrs).


Table 3.2:








administration to calculate differences in pulmonary targeting and MET due to

absorption in the lungs.

Several in vitro and in vivo studies have shown differences in the

topical and systemic potency [161], receptor affinity [162, 163], and

pharmacokinetic properties [11, 74, 78] of inhaled glucocorticoids. In addition,

pharmacokinetic / pharmacodynamic (PK/PD) models have been developed to

assess the extent of glucocorticoid systemic effects such as lymphocyte and

cortisol suppression [117]. These studies, however, give no direct indication of

the extent of pulmonary selectivity, as it is determined by both the extents of

local, as well as systemic effects. Furthermore, pulmonary targeting is difficult to

evaluate in humans because good surrogate markers for pulmonary effects are

not available for pharmacological studies [8]. In this study we used the ex vivo

receptor-binding assay previously described to assess pulmonary selectivity of

three commercially available inhaled glucocorticoids in different organs.

Our group previously assessed pulmonary targeting of TA after

intravenous administration by monitoring the receptor occupancy in lung and liver

only. We have shown that the IV administration of TA resulted in liver receptor

occupancies that were not significantly different from those in lung, indicating no

targeting. This is in agreement with the expected lack of targeting upon IV

administration.

In the present study we have considered not only the liver as a

marker for the extent of systemic exposure, but other tissues such as the kidney,

spleen, and brain. This was primarily done because of concerns that the high








intrinsic hepatic clearance of fluticasone propionate may affect the receptor

binding results. The overall level of pulmonary targeting was determined by

comparing the lung AUEC (% free receptors vs. time) to the systemic organ

AUEC (% free receptors vs. time) up to six hours as previously described [125].

Although differences in the AUEC's up to six hours are representative of the level

of pulmonary targeting, for the same drug differences in AUEC's between

administration routes (IV vs. IT) can be observed most likely due to differences in

absorption.

Upon intravenous administration of BUD we found that lung, liver,

kidney, and spleen had similar receptor binding profiles, but that the brain had

significantly less receptor binding, similar to IT administration of TA. Although

the brain has been shown to have a high concentration of glucocorticoid

receptors, cumulative brain receptor binding was less pronounced than those

observed in the other systemic organs, indicating that the distribution of drug to

this organ is affected by other factors [164]. These factors might include a lack of

efficient distribution across the blood-brain barrier or the effect of a p-glycoprotein

efflux pump [97, 98]. The similar binding profiles for lung, liver, kidney, and

spleen are in agreement with the expected distribution pattern of glucocorticoids

to highly perfused organs.

The intravenous administration of FP resulted in similar receptor

binding profiles for lung, kidney, and spleen. Again, the brain receptor

occupancies were significantly different from those in the lung. But contrary to

BUD and TA, the hepatic receptor occupancies for FP were significantly less








than the pulmonary receptor occupancies as well. This difference was not due to

metabolism of FP during ex vivo incubation, which was confirmed by the FP-

receptor complex demonstrating high stability under normal assay conditions

(unpublished results). This significant difference in lung and liver receptor

occupancies after IV administration was not expected, since the free drug

concentrations and receptor occupancies should be similar throughout the body

(assuming no tissue sequestration). But if the high intrinsic hepatic clearance of

FP is considered, the lower liver receptor occupancy observed may be

understandable. Because FP is highly metabolized in the liver (which is also

indicated by its oral bioavailability of lower than 1%), at any given time point both

free drug concentrations and liver receptor occupancies should be lower than

those in the other organs. These findings suggest that the monitoring the

receptor binding in the liver and lung alone may not be an appropriate method to

assess the extent of systemic effects of drugs with high intrinsic hepatic

clearance such as fluticasone propionate.

The intratracheal administration of TA, BUD, and, to a certain

degree, FP resulted in close superimposition of lung and spleen receptor

occupancies. It is well known that glucocorticoids induce the redistribution of

several blood cells to different lymphatic organs including spleen [165]. Recently

it has also been suggested that the glucocorticoid receptor density in immune

organs, such as the spleen, is higher than other organs (higher Bmax), which may

amplify the receptor occupancy after glucocorticoid administration compared to

other systemic organs [166]. This would indicate that the spleen might not be an








adequate systemic organ to assess the degree of glucocorticoid spill over into

the systemic circulation.

The intratracheal administration of BUD dry powder (100 gg/kg)

resulted in liver, kidney, and spleen receptor occupancies that were not

significantly lower than the lung receptor occupancy. Similar findings have been

reported using the Sephadex-induced lung edema model, where budesonide

displayed similar local vs. systemic effects [167]. The pulmonary MET after IV

administration was 3.1 hours compared to after IT administration of 3.6 hours,

showing a relatively similar difference in mean effect times (0.5 hour) compared

with the mean residence times (0.2 hour) as seen in Chapter 2. This data

suggests that fast dissolution and absorption of budesonide dry powders results

in limited pulmonary targeting.

Intratracheal administration of TA dry powder (100 jig/kg) resulted

in similar receptor occupancies for spleen and lung, while receptor occupancies

for kidney and brain were significantly lower than those for lung. Although these

results suggest that there is a higher pulmonary targeting for TA than BUD, the

kidney receptor occupancies for previously reported data sets showed high non-

specific binding [151]. If the last three data sets performed were considered

separately (with modifications made to decrease non-specific binding),

pulmonary targeting was not statistically significant between the lung and kidney

receptor occupancies. The difference in pulmonary mean effect times (1.6

hours) showed a greater difference than the mean residence times (0.2 hour)








found in Chapter 2, but may also be due to differences in experimental

techniques in previously assessing TA after IV administration [160].

The intratracheal administration of FP dry powder resulted in

receptor occupancies that were significantly lower for liver, kidney, spleen, and

brain than those for lung. The lower hepatic receptor occupancies is likely due to

the combined effects of high intrinsic clearance (similar to IV results) and slower

absorption of pulmonary-delivered FP, suggesting that the kidney may be a more

applicable organ to assess FP pulmonary selectivity. The pulmonary mean effect

time after IV administration was 5.5 hours compared to the MET after IT

administration of 4.1 hours, which is comparable to the calculated mean

residence time in plasma after IT administration of 5.6 hours (Chapter 2). The

high pulmonary mean effect time after IV administration is mainly due to the

sustained receptor occupancy out to 12 hours, which should be confirmed

through further experiments.

Overall, fluticasone propionate showed the highest level of

pulmonary targeting in multiple organs. As seen in Chapter 3, the dissolution

rates in vitro and the absorption rates in vivo were similar for TA and BUD, while

FP was significantly slower. Fluticasone propionate showed superior pulmonary

targeting versus triamcinolone acetonide when comparing lung receptor

occupancies to those in liver and budesonide when comparing lung receptor

occupancies to those in liver and kidney. Considering the differences in receptor

binding of fluticasone propionate to triamcinolone acetonide and budesonide, we

can observe that drug formulations with slower dissolution and absorption rates








result in an increase in pulmonary targeting. To further analyze this relationship

of increasing pulmonary targeting by controlling the dissolution rate, a sustained

release formulation of budesonide will be evaluated in Chapter 4.



Conclusions


Budesonide did not display statistically significant pulmonary

targeting when comparing lung receptor occupancies to those in

liver and kidney.

Triamcinolone acetonide did not display statistically significant

pulmonary targeting when comparing lung receptor occupancies to

those in liver.

Fluticasone propionate showed superior pulmonary targeting when

comparing lung receptor occupancies to those in liver and kidney.

Fluticasone propionate, which had the lowest dissolution and

absorption rate as seen in Chapter 3, showed a high level of

pulmonary targeting.

Considering the differences in receptor binding of fluticasone

propionate to triamcinolone acetonide and budesonide, we can

observe that glucocorticoid powders with slower dissolution rates

have higher pulmonary targeting.













CHAPTER 4
CHARACTERIZATION, IN VITRO DISSOLUTION, AND IN VIVO PLASMA
CONCENTRATIONS AND PULMONARY TARGETING IN RATS OF A NOVEL
SUSTAINED-RELEASE FORMULATION OF MICROENCAPSULATED
BUDESONIDE



Introduction


Currently, dry-powder inhalers (DPI) are used to deliver various

drugs to the lungs for localized or systemic delivery. Although current

formulations and delivery systems are adequate for pulmonary drug therapy, they

are limited by potential problems with pulmonary deposition characteristics as

well as the residence time of the drug after inhalation ([14], also seen in Chapter

2). Previously, liposomes were used by our group as a model sustained release

system with a substantial improvement in pulmonary targeting in rats [125].

Liposomes and microspheres have been investigated as sustained release

delivery systems for the lung [34, 69], but because of complicated manufacturing

and wet processing, a novel dry-coating technique previously developed for

engineered particulates using pulsed laser deposition (PLD) is proposed [146]. It

is proposed that modification of the release rate of the drug from dry powders by

applying a biodegradable polymer coating would greatly enhance the pulmonary

residence time, and thus improve pulmonary targeting.








Over the past few years, the pulsed laser deposition (PLD)

technique has emerged as one of the simplest and most versatile methods for

the deposition of thin films of a wide variety of materials [168]. The stoichiometric

removal of the constituent species from the target during ablation (i.e. monomer

and nanoclusters of polymer) from a polymer target, as well as the relatively

small number of control parameters, are the two major advantages of PLD over

some of the other physical vapor-deposition techniques. No studies have

currently been performed using biodegradable polymers for coating materials by

PLD, so comparison of the molecular structure of the deposited films to original

material was performed to ensure that the polymer structure remained intact after

deposition.

Overall the objective of this section was to use PLD to ablate a

target of a biodegradable polymer, poly(lactic-co-glycolic) acid (PLGA 50:50), to

coat budesonide (BUD) micronized drug particles for 10 (BUD10) and 25

(BUD25) minute runs. Characterization of films deposited on silicon wafers or

glass slides was performed using SEM, FTIR, and NMR to characterize polymer

structure and morphology. Then BUD10 and BUD25 coated powders were

tested in vitro to assess differences in the dissolution rates. Then BUD25 coated

drug formulation was administered intratracheally in vivo in rats to monitor

plasma concentration and improvement in pulmonary targeting. Comparison of

the plasma concentrations after IT administration of the coated powders with

uncoated BUD powders and IV administration of BUD solution in Chapter 2, as

well as with FP after IT administration, will be performed to compare absorption








rates. Finally, the pulmonary targeting of coated BUD25 powders after IT

administration was compared with the pulmonary targeting of uncoated BUD and

FP powders after IT administration and BUD solution after IV administration in

Chapter 3.



Hypothesis


Sustained release formulations of budesonide with a slower

dissolution rate in vitro will have a slower absorption rate and improved

pulmonary targeting in vivo.



Materials and Methods


Chemicals

Analytical grade chemicals were obtained from Sigma Chemical

Co. (St. Louis, MO.). Micronized budesonide was obtained from Sicor (Milan,

Italy). Poly(lactic-co-glycolic acid) 50:50 DL high intrinsic viscosity (Lot# 3097-

204) was purchased from Medisorb Medical Technologies, Inc., Cinncinnati,

Ohio. Lactose monohydrate NF extra fine (particle size 10-30 microns) was

obtained from EM industries Hawthorne, NY. Radiolabeled triamcinolone

acetonide ({6,7-3H} TA, 35.4 Ci/mmol) was purchased from DuPont NEN

Research Products (Boston, MA). All other reagents were of analytical grade.








Pulsed Laser Deposition (PLD) Setup

We have used the PLD procedure to deposit films of biodegradable

polymers, namely poly(lactic-co-glycolic acid) (PLGA), onto glucocorticoid drug

particles in the micrometer range. The PLGA target was heat pressed in a 0.15

cm x 3.5 cm x 3.5 cm mold using a carver press at 120 C. The coating

procedure consists of the polymer target (PLGA) and fluidized bed of micronized

drug powder (500 mg batch) contained within a vacuum chamber. The 248 nm

laser pulse at 150 mJ and 5 hertz enters the vacuum chamber through a quartz

window and ablates the polymer target. The surface of the target material is

rapidly heated and expands from the surface into the vacuum atmosphere to

form a plume. The plume of low-molecular weight polymer chains then adheres

to the drug particle as agglomerates. Preliminary coatings on silicon wafers were

performed to examine plume uniformity and coating thickness. Coating runs

were performed using 500 mg of free drug powders and coated for 10 minutes

(BUD10) and 25 minutes (BUD25).

SEM Analysis

Polymer coatings on silicon wafers and micronized drug powders

were analyzed using a Joel model 6400 EDS scanning electron microscope

(SEM) to obtain information on the size, shape, and surface morphology.

Micrographs of powder samples were prepared by applying a thin layer of carbon

paint onto a graphite sample mount and spreading approximately one milligram

on top. Sample mounts then underwent a carbon evaporation process prior to














polymer target pulsed laser beam




plume



fluid ization -
system core particles
system



vacuum chamber



Figure 5.1: Pulsed laser deposition (PLD) setup for coating drug particles with
polymer coating.








characterization. Characterization was performed at 1-2 kEV in vacuum.

Magnification of representative micrographs was x 5,500.

NMR Analysis

For characterization of polymer structure, proton nuclear magnetic

resonance (1H-NMR) analysis was performed as previously reported for PLGA

[169] using a Varian NMR (Model VXR, 300 MHz). Samples for analysis on

silicon wafers after 2-minute runs were dissolved in deuterated chloroform with

1% TMS (Sigma, Inc.) and approximately 1 ml transferred to NMR glass tubes for

analysis. Standard parameters were acquisition time 4.0 sec, relaxation delay

1.0 sec, 16 scan repetitions, and pulse width 3,600 hz were used (standard 1H-

NMR program).

FTIR Analysis

Fourier-transform infrared spectroscopy (FTIR) was also used to

characterize the polymer structure as previously reported [170] using a Nicolet

Magna 760 FTIR with OMNIC 3.0 software. Diffuse reflectance of films on glass

slides was monitored using the DRIFTS module and subtracting spectra for

uncoated glass slide. For PLGA analysis 10 mg of polymer was dissolved in 2 ml

of methylene chloride and cast on a glass slide and the solvent evaporated. For

deposited PLGA, a glass slide was placed under the PLGA target and deposited

for 5 minutes.

In Vitro Dissolution

In vitro dissolution of free drug (BUD), 10 minute coated drug

(BUD10), and 25 minute coated drug (BUD25) formulations (500 mg of 2% drug