Biopharmaceutical aspects relevant to pulmonary targeting of inhaled glucocorticoids

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Biopharmaceutical aspects relevant to pulmonary targeting of inhaled glucocorticoids application to liposomes and dry powders
Alternate title:
Application to liposomes and dry powders
Suarez, Sandra, 1965-
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ix, 156 leaves : ill. ; 29 cm.


Subjects / Keywords:
Dosage ( jstor )
Glucocorticoids ( jstor )
Inhalation administration ( jstor )
Intranasal administration ( jstor )
Intravenous injections ( jstor )
Liposomes ( jstor )
Liver ( jstor )
Lungs ( jstor )
Pharmacokinetics ( jstor )
Receptors ( jstor )
Department of Pharmaceutics thesis Ph.D ( mesh )
Dissertations, Academic -- College of Pharmacy -- Department of Pharmaceutics -- UF ( mesh )
Drug Carriers ( mesh )
Drug Delivery Systems ( mesh )
Glucocorticoids -- administration & dosage ( mesh )
Glucocorticoids -- pharmacokinetics ( mesh )
Glucocorticoids -- pharmacology ( mesh )
Liposomes ( mesh )
Lung ( mesh )
Powders ( mesh )
Rats ( mesh )
Vehicles ( mesh )
bibliography ( marcgt )
non-fiction ( marcgt )


Thesis (Ph.D.)--University of Florida, 1997.
Bibliography: leaves 145-155.
General Note:
General Note:
Statement of Responsibility:
by Sandra Suarez.

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Full Text








I extend my appreciation and grateful thanks to Dr. Guenther Hochhaus for his

guidance, patience and continuous encouragement during the course of the work

presented in this dissertation. I would like to thank the members of my supervisory

committee, Dr. Hartmut Derendorf, Dr. Jeffrey Hughes, and Dr. Charles E. Wood, for

their inestimable and kind advice throughout my doctoral research. I take this opportunity

to express may gratitude and esteem to Dr. Ricardo Gonzalez-Rothi for all his invaluable

help inside and outside of school.

I would like to thank all the graduate students and the secretaries, especially

Patricia Kahn, for their support. Technical assistance of Dave Succy and Jufey Tang was

invaluable to this work. I thank my sister Elena, my aunt Conchita and my friends Judith,

Jim and Suliman for all their help and moral support. Finally, I thank my parents and

sisters for supporting me in many ways.



ACKNOWLEDGMENTS ............. ............................................................ iii

ABSTRACT ...................... ............... viii


1. IN TRO D U CTION ................................ ........................................................... 1

2. B A CK G R O UN D ......................................................................... ....................3

A sthm a ..................... ........................................................................................... 3
Definition ............. ................................................. 3
Pathogenesis. ................................ ... .....................................................4
Therapy................................................. ................................................... 7

Glucocorticoids ....... ............................. ..................... 8
M olecular M echanism s........................................................ .....................9
Target G enes in A sthm a. ............................................. ....................... ... 10
Cellular Effects.............................................................................................. 11
O their Effects..................................... ..................................................... 12

The Pulmonary Route.............................................. ....................... ........................ 13
Structure and Function of the Respiratory Tract ............................................. 13
Mechanism of Drug Deposition................................................ .................... 15
Factors Controlling the Fate of Aerosol in the Respiratory Tract .................... 17
D position ................... ................ ......... ........................................... 17
Pulmonary Clearance Mechanisms ...................................................20
Absorption Kinetics. ............................................................................ 20
Drug Dissolution or Release ........................................... ..................21


Pulm onary T targeting ................................................................ .............................. 22
Pulmonary Selectivity of Inhaled Glucocorticoids.............................................22
Effect of Clearance, Volume of Distribution and Oral
Bioavailability.................... .............................. ................... 23
Effect of Pharmacodynamics.................................. ..................... 25
Effect of Release Rate and Dose. ............................................25
Improvement of Airway Selectivity...................... ....................... 26

L iposom es ........................................ .... ............................................... 28
Liposome Structure and Classification...................... .................. 29
Chem ical Com position ..................................................... ..................... 30
Methods of Preparation................... .......... ......................31
Chemical Stability...................................... ................................................... 33
Oxidation of Phospholipids ...............................................33
Hydrolysis of Phospholipids................................................ 34
Physical Stability. ........................................... ......................................... 37
Biological Stability. ........................................ .......................................41
Pharmacokinetics of Liposomes.................................................................. 41
Disposition of Liposomes Following Intravenous Administration..............41
Disposition of Liposomes Following Pulmonary Administration ...............46

Synopsis of Literature Review 50

3. RESEARCH PROPOSAL....................................................... ......................52

A im # ............................................................................. ........................... 5 2
Hypothesis......................... ...........................53
R ationale. ........................................ ......................................... 53
A im # 2 ......................................................... ...................................... ...... 55
Hypothesis............. ...............................................55
Rationale. ........................................... ......................................... 55
A im # 3 ............................................................... ......................................... 56
Hypothesis..................................................... 56
Rationale. .............................. ..................................................... 56
A im # 4 ......................................................................... ............................... 57
Hypothesis......................... ......... .......................... 58
R ationale. ....................................................... .................................. 58
A im # 5 ............................................................................ ................ .......... 59
H ypothesis......................... ............................. ..................... 59
R ationale. ......................... ....................................... ................. .. 59

4. ANALYTICAL M ETHODS............................ ................................................. 60

Characterization of Liposomes .............................. ...........................60
Size M easurement........................ ....................... .....................60
Lipid D eterm ination............................................................................... 62
Analysis of Triamcinolone Acetonide Phosphate by HPLC ....................64
In vitro Stablity at 37 C ..................................... ............. ............ 66
Receptor Binding Assay ................................................................... 67
HPLC/RIA Determination of Triamcinolone Acetonide .....................................70

ACETON IDE ..... ...... ........................................................72

Introduction .............................................. ................ ................... 72
Results ........................................................ 80
D discussion .................................. ........................................ .................... 85

AND INTRAVENOUS ADMINISTRATION ......................................88

Introduction ..................................................... ...................................... 88
M ethods....................... .... ........................................ 88
Results ........... .................................................. 94
D iscu ssion ....................................................................... ........................... 10 1

AS A MODEL DOSAGE FORM ......................................................... 107

Introduction .................................................................. ............................ 107
M ethods........................................................................... .................... 108
R esults................................................................... ......................................... 114
D iscussion................................................... ....................... ............. 121

EX VIVO RECEPTOR BINDING ASSAY. ........................................ 125

Introduction ................................................................ ....................... 125
M ethods.................................................................... ................ 126
R esults...................................... ............................................................ .. 13 1
Discussion.................................................................................... 138

9. CON CLU SION S...................................................................... ................... 143

LIST OF REFERENCES......................................................................... ....... 145

BIOGRAPHICAL SKETCH................................................... ...................... 157

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



Sandra Suarez

August 1997

Chairman: Guenther Hochhaus, Ph.D.
Major Department: Pharmaceutics

Little information is available regarding the biopharmaceutical factors that govern

pulmonary targeting. Pharmacokinetics/pharmacodynamic simulations have shown that

pulmonary targeting can be improved by optimization of dose and drug release rate.

Liposomes were used as a model dosage form to show the relevance of dose and release

rate on pulmonary targeting in in vivo situations. Liposomes were composed of two

phospholipids and triamcinolone acetonide phosphate (TAP-lip). TAP-lip and the drug in

solution were administered intratrachealy (IT) into anesthetized rats. Pulmonary targeting

was determined by simultaneously monitoring receptor occupancy in lung and liver. The

IT administration of the TAP-lip formulation resulted in higher pulmonary targeting and

longer mean pulmonary effect times (MET) than that after the IT administration of the

drug in solution. Thus, liposomes may represent a valuable approach to optimize the

delivery of glucocorticoids via topical administration. The pharmacokinetics and

pharmacodynamics of triamcinolone acetonide (TA) after IT and intravenous (IV)

administration of TAP-lip, and after IT instillation of the drug in solution, were also

investigated. Plasma and lung TA concentrations were determined by HPLC/RIA. No

changes in the pharmacodynamics (ECso and E_), were observed, indicating that

pharmacokinetics is the driving force to enhance pulmonary targeting. The intratracheal

administration of TAP in different release rate preparations resulted in mean effect times

(MET) and pulmonary targeting which were higher for the slowest release preparation.

The administration of escalating doses of TAP in 800 nm liposomes showed a bell shaped

curve, suggesting that there is an optimum dose for which maximum pulmonary targeting

can be achieved. Differences in pulmonary targeting between triamcinolone acetonide

(TA) and fluticasone propionate (FP) dry-powders were assessed after IT administration

by the ex vivo receptor assay. No significant differences in pulmonary targeting were

observed between these two drugs. Higher pulmonary targeting and longer MET values

were obtained after the IT administration of the liposomal formulation than those after the

IT administration of TA-dry powder. These studies clearly demonstrate the relevance of

dose and release rate for the optimization of pulmonary selectivity as well as the suitability

of the ex vivo animal model to assess pulmonary targeting.


Asthma is an inflammatory disease of the airways in which many cells play a role

including eosinophils and mast cells. Asthma is perhaps the only treatable condition whose

severity is on the rise. This problem requires the development of drugs which are more

effective and safer for the treatment of the disease.

Inhaled glucocorticoids have become first-line therapy for the treatment of

moderate to severe asthma as a results of glucocorticoid action at the cellular level to

attenuate the underlying disease process. These drugs are administered directly into the

respiratory airways using different devices such as metered dose inhaler (with and without

spacer devices), nebulizer and dry powder inhaler.

Inhalation therapy makes possible the administration of small doses of the drug

directly into the target site, a rapid onset of action as well as a low systemic exposure,

minimizing the risk of systemic side effects.

Several studies have shown that pulmonary selectivity is possible if a higher local

to systemic drug ratio is achieved. In attempts to increase this ratio drugs with high

systemic clearance and low oral bioavailability have been introduced. Thus, the latest

generations of inhaled glucocorticoids have oral bioavailability values lower than <1%

(fluticasone propionate) and clearance values closer to the hepatic blood flow (1.2 L/min).

Despite the advantages of inhaled glucocorticoids over oral therapy, there are still

concerns regarding side effects, especially suppression of growth in children and

osteoporosis, since inhaled glucocorticoids are likely to be administered over long periods

of time.

Recent computer simulation studies [1] have shown that pulmonary targeting can

be modulated if drug release rate and dose are optimized. These theoretical studies

demonstrated that there is an optimal dose and release rate for which maximum pulmonary

targeting can be achieved. It was important to demonstrate using in vivo experiments

whether these two factors are relevant in pulmonary selectivity. To show the importance

of these two factors, liposomes were used as a model dosage form due to their flexibility

in release rate characteristics, and their compatibility with lung surfactant components.

Furthermore, since inhaled glucocorticoids differ in their dissolution rate in vitro it was

also important to explore differences in release rate among these drugs (triamcinolone

acetonide and fluticasone propionate) based on the fact that pulmonary targeting is

affected by release rate.


2.1 Asthma

2.1.1 Definition

Asthma is considered an inflammatory disease, often described as chronic

desquamating eosinophilic bronchitis [2]. Asthma is a common disease of the airways

which affects 5% of the adult and 10-15% of the child population [3, 4]. Asthma has

become more prevalent during the last decades, and the severity and mortality have

increased [5]. Asthma is generally characterized by a combination of cough, dyspnea,

chest tightness, wheezing and sputum production [6].

2.1.2 Pathogenesis Inflammatory Cells

Asthma is a chronic inflammatory disease in which many cells are involved,

including eosinophils, macrophages, mast cells, epithelial cells and lymphocytes (see

Figure 2.1). 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 reflexes [3]. For example, macrophages products, such

as IL-1 may prime eosinophils to respond to other secretary stimuli, and perhaps it is the

sequence of inflammatory cell interactions that makes the inflammatory response in asthma

different from that of other conditions [7].

Several studies have reported that alveolar macrophages are key to the

pathophysiology of several respiratory diseases including asthma. The activation of these

cells induces the release of different inflammatory mediators: a) platelet-activating factor

(PAF), a mediator that selectively attracts human eosinophils and causes eosinophil

infiltration of airways and epithelial damage; b) gamma interferon, a powerful lymphokine

which modulates lymphocyte and fibroblast function; c) tumor necrosis factor; d)

interlukine 1 (IL-1), which activates lymphocytes and fibroblasts; e) antigen presentation

to lymphocytes; f) alveolar macrophage derived growth factors for fibroblast etc. [3, 8-


Eosinophils and epithelial cells play an important role in the inflammatory process

as well. Upon activation, eosinophils release a variety of mediators including leukotriene

C4, PAF, oxygen radicals and also basic proteins, which are toxic and lead to epithelial

damage [11]. Epithelial cells release inflammatory mediators like LTB4 and 15-

lipoxygenase products which are chemotactic to other inflammatory cells [3]. Inflammatory mediators

Different mediators have been implicated in asthma, and they may have a variety of

effects on the airways that account for most of the pathologic features of asthma (see

Table 2.1). Mediators such as histamine, prostaglandins, and leukotrienes contract airway

smooth muscle directly, increase airway mucous secretion, and attract and activate other

inflammatory cells, which in turn release inflammatory mediators.

Macrophage I Mast Cell

T-lymphocyte 1 Neutrophil

0 Eosinophil

Globet cell Epithelial shedding

Plasma leak. ;y nerve


Figure 2.1 Cellular interactions. Adapted from reference [3]. The role of cytokines in allergic cell recruitment

As stated previously, asthma is characterized by the recruitment and migration of

inflammatory cells, primarily eosinophils into the airways; cytokine generation in the

airways may trigger such infiltration. It is believed that four classes of cytokines are

important in allergic cell recruitment [12]: 1) non-specific endothelial activators (TNF-a

and IL-1); 2) specific endothelial activators (IL-4 and IL-3); 3) those that activate

eosinophil functions and prolong their survival (IL-3, IL-5, granulocyte-macrophage

colony-stimulating factor (GM-CSF), and interferon gamma), and 4) those that directly

stimulate cell migration (RANTES). Neural mechanisms

Neural mechanisms (cholinergic, adrenergic and non-adrenergic-non-cholinergic)

may be important in producing asthma symptoms (Figure 2.2). Factors that are under

autonomic control include submucosal gland secretion, bronchial vascular tone and

permeability, airway smooth muscle tone and probably secretion from inflammatory cells


Table 2.1 Inflammatory mediators implicated in asthma [13].

Mediator Broncho Airway Microvascular Chemotaxis Bronchial
constriction secretion leakage hyperresponsiveness

Histamine + + + +

Prostaglandins D2, F, ++ + ? +

Prostaglandin E2 + +

Leucotriene B4 ++

Leukotrienes C4, D4,
++ 4+ ++ ?

Platelet activating + +- ++ ++


Bradykinin + + ++

Adenosine + 7 ? ?

Substance P + ++ ++

Serotonin ? +

Oxygen radicals + ? +

Throboxane ++ +

2.1.3 Therapy of Asthma

The philosophy in the management of asthma has become the prevention and

control of the disease analogous to the management of other chronic diseases such as

hypertension [14]. In patients whose allergens are found at home or in the workplace the

first priority is to prevent exposure or reduce it greatly, since continuous exposure can

lead to chronic severe asthma that can no longer be controlled by avoiding the allergen

[15]. What then is the best way to attenuate asthma?

Five classes of medication are effective in the treatment of asthma:

anticholinergics, beta-adrenergic agonists, theophylline, sodium cromoglycate, and

glucocorticoids [14]. Many controlled trials have now established that inhaled

glucocorticoids are the most effective because of their effects at the cellular levels to

attenuate the underlying disease process [16-19].

Symphate- c ner

Figure 2.2. Neural mechanisms. Adapted from reference [3].

2.2 Glucocorticoids

Glucocorticoids consist of 21 carbon atoms with four rings, three six-carbon rings

(A, B, and C), and a five carbon ring (D) (Figure 2.3). Most of the anti-inflammatory

glucocorticoids are characterized by lipophilic susbtituents in positions 16 and 17; CH3, F

and Cl in positions 6 and 9; double bound in carbons 1,2. Other essential features consist of

the following: 1) a ketone oxygen at C-3, 2) an unsaturated bond between C-4 and C-5, 3)

a hydroxyl group at C-11, and 4) a ketone oxygen at C-20. By modifying the basic

structure of glucocorticoids, it appeared possible to modify the following functions [20-


Receptor binding: raising the affinity for the glucocorticoid receptor (GR),

reducing the affinity for the mineralocorticoid receptor, and reducing the

affinity for corticosteroid-binding globulin (CBG).

Biotransformation: modulating the metabolism via oxido-reductive or

hydrolytic pathways.

Other physicochemical aspects: uptake, tissue binding and systemic disposition.

C-22 -CH2OH
c-11 C=0
HO -o-C
C-1 I /CH3

A F C-16

C-4 B
Figure 2.3 The chemical structure of glucocorticoids as typified by triamcinolone
acetonide [23].

2.2.1 Molecular Mechanisms: the Glucocorticoid Receptor

The glucocorticoid receptor (GR; type II steroid receptors) belongs to the

superfamily of steroid receptors. They represent transactivating proteins for genomic

modulation. The glucocorticoid receptor is comprised of three domains: the ligand-

binding, the DNA-binding and the modulatory domain [24]. The GR consists of

approximately 780 amino-acids, with about 280 in the ligand-binding, 70 in the DNA-

binding, and 430 in the modulatory domains. In its inactivated state, GR is completed

together with two molecules of heat shock protein 90-kD (HSP-90) and one molecule of

the immunophilin p-59 (Figure 1.4). It is believed that these proteins cover the DNA-

binding domain of the receptor [25] and that this complex does not show any biological


When the glucocorticoid receptor is activated, it dissociates from the complex with

HSP-90 and p-59 and exposes the DNA-binding domain with its two zinc fingers [26].

Two activated receptors will form a homodimer which is able to interact with specific

DNA sequences called glucocorticoid response elements (GREs). Binding of the liganded

GR to DNA usually results in activation of transcription through positive GREs (pGREs),

but binding to less common so-called negative GREs (nGREs) can result in repression of

transcription. The degree of the pharmacological effect is proportional to the degree of

receptor occupancy. Such a correlation is one of the reasons for using receptor occupancy

as a method to evaluate pulmonary targeting in our study

In recent years, it was found that GR can also modulate transcription through non-

nuclear pathways. In this case, the activity of other transcription factors, such as AP-1

(activating protein-1) and NF-kB (nuclear factor kB), is modulated by direct interaction of

the activated glucocorticoid receptor with these two factors [27]. These newly discovered

pathways are extremely important because they provide a cross-talk 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. The repression of such genes is likely to

underlie the antiarthritic and immunosuppressive effects of glucocorticoids.

Figure 2.4. The Glucocorticoid receptor. Adapted from [18].

2.2.2 Target Genes in Asthma

The target genes involved in asthma are listed in Table 2.2. Glucocorticoids may

be effective in controlling asthma by decreasing or increasing gene transcription.

Although it is not yet possible to be certain of the most critical aspects of steroid

action in asthma, it is likely that their inhibitory effects on cytokine synthesis are of

particular relevance [16]. Steroids inhibit the transcription of several cytokines that are

relevant in asthma including IL-I, TNF a, granulocyte-macrophage colony-stimulating

factor, IL-3, IL-4, IL-5, IL-6, and IL-8. Glucocorticoids increase the synthesis of

lipocortin-1 a 37-KD protein that has an inhibitory effect on phospholipase A2, and

therefore may inhibit the production of lipid mediators such as leukotrienes, prostaglandins

and platelet activating factor [28]. Glucocorticoids may be more effective in inhibiting

cytokine release from alveolar macrophages than in inhibiting lipid mediators and reactive

oxygen species [29].

2.2.3 Cellular Effects of Glucocorticoids

One of the best described actions of steroids in asthma is a reduction in circulating

eosinophils which may reflect an action on eosinophil production in the bone. Inhalation of

budesonide 800 gg twice a day produced a marked reduction in the number of low density

eosinophils, presumably reflecting inhibition of cytokine production in the airways marrow

[16]. Glucocorticoids are very effective in inhibiting the activation of lymphocytes and in

blocking the release of cytokines, which are likely to play an important role in the

recruitment and survival of several inflammatory cells involved in asthmatic inflammation.

While steroids do not appear to have a direct inhibitory effect on mediator release

from lung mast cells, chronic steroid treatment is associated with a marked reduction in

mucosal mast cell number. This may be linked to a reduction in IL-3 and stem cell factor

(SCF) production, which are necessary for mast cell expression in tissues [19]. Epithelial

cells may be one of the most important targets for inhaled glucocorticoids in asthma.

Glucocorticoids inhibit the increased transcription of the IL-8 gene induced by TNF-a in

cultured human airway epithelial cells in vitro, the transcription of the RANTES gene and

GM-CSF in an epithelial cell line [30].

Table 2.2 Effect of glucocorticoids on transcription of genes relevant to asthma [19].

Increased transcription
P2-adrenergic receptor
Secretory leukocyte inhibitory protein
Macrophage inhibitory factor

Decreased transcription
Cytokines (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-11, IL-12, IL-13, TNF-
alpha, GM-CSF, RATESS)
Inducible nitric oxide synthase (iNOS)
Inducible cyclo-oxygenase (COX-2)
Inducible phospholipase A2 (cPLA2)
NKI receptors
Adhesion molecules

2.2.3 Other Effects

Glucocorticoids exert a number of other effects:

Carbohydrate and protein metabolism (e.g. gluconeogenesis)
Lipid metabolism (e.g. redistribution of body fat)
CNS effects (mood, behavior, neural excitability)
Inhibition of cholesterol synthesis
Cortisol suppression

2.3 The Pulmonary Route

Localized delivery of drugs to the respiratory tract has become an increasingly

important and effective therapeutic method for treating a variety of pulmonary disorders

including asthma, bronchitis, and cystic fibrosis. Although the traditional form of

inhalation therapy dates back to the earliest records of ancient cultures, the advantages of

inhalation therapy have essentially remained the same. Relatively small doses are required

for effective therapy, reducing exposure of drug to the systemic circulation, and

potentially minimizes adverse effects [31]. Lower dosage regimens may provide

considerable cost saving especially with expensive therapeutic agents. Delivering small

doses of active ingredients directly to the lung effectively localizes the drug, thereby

maximizing therapeutic effect while minimizing unwanted side effects. As mentioned

earlier, these advantages are the basis of inhalation therapy of glucocortioids.

2.3.1 Structure and Function of the Respiratory Tract

The respiratory tract can be divided into upper and lower airways with the line of

division being the junction of the larynx and trachea [32]. The upper airways or

nasopharyngeal region consists of the nose, mouth, larynx, and pharynx. Below the

contours of the nasopharyngeal region, the lower airways resemble a series of tubes

undergoing regular dichotomous branching [33]. Successive branching from the trachea to

the alveoli reduces the diameter of the tubes, but markedly increases the surface area of

the airways, which allows gas exchange. The lower airways can be divided into three

physiological zones: conducting, transitional, and respiratory zones [33] (see Figure 1.6).

The conducting 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 no gas exchange

occurs. The transitional zone plays a limited role in gas exchange. The characteristic D

shape of the trachea is maintained by cartilage supported by smooth muscle fibers. The

epithelial layer of the trachea and main bronchi are made up of several cell types including

ciliated, basal, and globet. A large number of mucous- and serum-producing glands are

located in the submucosa.

The human lung consists of five lobules and ten bronchopulmonary segments.

Arranged to each segment are lung lobules composed of three to five terminal bronchioles.

Each bronchiole supplies the smallest structural unit of the lung, the acinus, which consists

of the alveolar ducts, alveolar sacs, and alveoli. At the level of small bronchi and

bronchioles, the amount and organization of cartilage diminishes as the number of

bronchial bifurcation increases. The acinus represents a marked change in morphology.

The primary cells of the epithelium are the type I pneumocytes, which cover 90% of the

entire alveolar surface. Type II pneumocytes are more numerous, but have a smaller total

volume, and are responsible for the storage and secretion of lung surfactant. Less

prevalent cell types include type III pneumocytes and alveolar macrophages. The alveolar

blood barrier in its simplest form consists of a single epithelial cell, a basement membrane,

and a single endothelial cell. While this morphological arrangement readily facilitates the

exchange, it can still represent a major barrier to large molecules [34]. The lung tissue is

highly vascularized, which makes pulmonary targeting difficult due to a fast absorption of

most of the drugs (especially lipophilic and low molecular weight drugs).

Figure 2.5 Organization of the airways. Adapted from reference [34].

2.3.2 Mechanisms of Drug Deposition

Drugs for inhalation therapy are administered in aerosol form. An aerosol is

defined as a suspension of liquid or solid in the form of fine particles dispersed in a gas. A

prerequisite for therapeutic efficacy is the ability of the aerosolized drug to reach the

peripheral airways. Herein lies the fundamental problem of inhalation therapy as the

anatomy and physiology of the respiratory tract has evolved to prevent the entry of

particulate matter [34]. The regional pattern of deposition efficiency determines the

specific pathways and rate by which deposited particles are ultimately cleared and

redistributed [35].

The mechanism by which particles may deposit in the respiratory tract are

impaction (inertial deposition), sedimentation (gravitational deposition), Brownian

diffusion, interception, and electrostatic precipitation [35]. The relative contribution of

each depends on characteristics of the inhaled particles as well as on breathing patterns

and respiratory tract anatomy. Impaction

Impaction may occur when a particle's momentum prevents it from changing

course in an area where there is a rapid change in the direction of bulk airflow. It is the

main deposition mechanism in the upper airways, and at near bronchial branching points.

The probability of impaction increases with increasing air velocity, rate of breathing, and

particle size [35]. Sedimentation

Sedimentation results when the gravitational force on a particle is balanced by the

sum forces due to the air resistance; inspired particles will then fall out of the air stream at

a constant rate [36]. This is an important mechanism in small airways having low air

velocity. The probability of sedimentation is proportional to residence time in the airway

and to particle size, and decreases with increasing breathing rate. Diffusion

The constant random collision of gas molecules with small aerosol particles pushes

them about in an irregular fashion called Brownian movement. Thus, even in the absence

of gravity, a particle in still air moves around in a "random walk" [36]. The effectiveness

of Brownian motion is inversely proportional to particle diameters for those particles < 0.5

micron [37], and is important in bronchioles, alveoli, and at bronchial airway bifurcation.

Molecular-sized particles may deposit by diffusion in the upper respiratory tract, trachea,

and larger bronchi.

2.3.3 Factors Controlling the Fate of Aerosols in the Respiratory Tract

The fate of inhaled particulates in the respiratory tract and therefore, the local

therapeutic activity, depends upon the dynamic interaction of different factors: 1)

deposition, 2) mucociliary clearance, 3) drug dissolution rate or release, 4) absorption, 5)

tissue sequestration, and 6) metabolism kinetics. Extensive literature exists on the

theoretical and experimental aspects of each of these factors [35, 36, 38-41]. Factors controlling respiratory drug deposition

The factors that control drug deposition are 1) characteristics of the inhaled

particles such as size, distribution, shape, electrical charge, density, and hygroscopicity; 2)

anatomy of the respiratory tract, and, 3) breathing patterns such as frequency, depth, and

flow rate. Particle characteristics

The particle size is a critical factor affecting the site of their deposition, since it

determines operating mechanisms and extent of penetration into the lungs. Aerosol size is

often expressed in terms of aerodynamic diameter (D..). Aerodynamic diameter is defined

as the diameter of a spherical particle having unit density that has the same settling

velocity from an air stream as the particle in question [35]. Thus particles that have higher

than unit density will have actual diameters smaller than their D.. An aerosol has a size

distribution arbitrarily characterized as monodispersed (uniform size distribution and

geometric standard deviation of< 1.2) or polydispersed (less uniform size distribution and

geometric standard deviation equal to or > 1.2). Respiratory tract anatomy

In air-breathing animals, respiratory anatomy has evolved in such a way as to

actively thwart inhalation of putative airborne particulates [42]. The upper airways (nose,

mouth, larynx and pharynx) and the branching anatomy of the tracheobronchial tree act as

a series of filters for inhaled particles. Thus, aerosol particles, whose diameter is greater

than 100 micron, generally do not enter the respiratory tract and are trapped in the naso-

oropharynx. Particles larger than 10 micron will not penetrate the tracheobronchial tree.

Particles must generally be less than 5 micron in order to reach the alveolar space [36]. On

the other hand, particles of 0.5 micron in diameter will penetrate the lung deeply, but have

a high tendency to be exhaled without deposition.

Airway geometry affects particle deposition in various ways. For example, the

diameter sets the necessary displacement by the particle before it contacts an airway

surface, cross-section determines the air velocity for a given flow rate, and variations in

diameter and branching patterns affect mixing between tidal and reserve air [35]. Thus,

since the respiratory system of animals and humans differs anatomically, differences in

deposition patterns are also expected. Respiratory patterns

The pattern of respiration during aerosol exposure influences regional deposition,

since breathing volume and frequency determine the mean flow rates in each region of the

respiratory tract which, in turn, influence the effectiveness of each deposition mechanism.

Turbulence tends to enhance particle deposition, with the degree of potentiation

depending of the particle size [35]. Rapid breathing is often associated with increased

deposition of larger particles in the upper respiratory tract, while slow, steady inhalation

increases the number of particles able to penetrate to the peripheral parts of the lungs [43,

44]. Byron (1986) [39] developed a mathematical model which allowed him to identified

the effect of particle size and breathing pattern on lung drug deposition. Slow breathing,

with or without breath-holding, showed broad maximum deposition in the ciliated airways

(tracheobronchial portion TB). The pulmonary maximum occurs between 1.5-2.5 and 2.5-

4 micron, with and without breath-holding, respectively. Rapid inhalation showed similar

trends; the TB maximum falls and shifts to between 3 and 6 micron. Pulmonary deposition

sharpens and occurs between 1.5-2 and 2-3 microns, with and without breath-holding,


When the above considerations are taken into account, the ideal scenario for an

aerosol would be 1) aerosol D. < 5 micron to minimize oropharyngeal deposition, 2)

slow, steady inhalation, and 3) a period of breath-holding on completion of inhalation. Pulmonary clearance mechanisms

Insoluble particulates are cleared by several pathways which today are only

partially understood. These pathways are known to be impaired in certain diseases and are

thought to depend on the nature of the administered material [45]. Swallowing,

expectoration, and coughing constitute the first sequence of clearance mechanisms

operand in the naso-oropharynx and tracheobronchial tree [42]. A major clearance

mechanism for inhaled particulate matter is the mucociliary escalator. It consists of ciliated

epithelial cells reaching from the naso-oropharynx and the upper tracheobronchial region

down to the most peripheral terminal bronchioles. Beating of the cilia, together with

mucus secreted by the globet cells, contributes to an efficient clearance mechanism.

Mucociliary clearance kinetics are difficult to quantify. Studies of clearance kinetics

usually involve administration of 3.32 micron (D.) insoluble iron oxide aerosol to normal

humans. Byron (1986) [39], using a mathematical model, analyzed the clearance data

resulting from the administration of iron oxide. He found that 48.9 % of the deposited

material remained 24 h following administration. The major clearance mechanism in the

alveolar regions of the lung is uptake by alveolar macrophages [42]. Absorption kinetics

The rate at which drugs are cleared from the airways depends greatly on certain

physicochemical properties of the drug, such as 1) molecular weight, 2) partition

coefficient and, 3) binding characteristics.

Enna and Shancker [46] have examined the lung absorption of saccharides and

urea of various molecular weights. They found that the first-order rate constant, k,

decreased with increasing molecular weight. Because of the low lipid solubility of these

hydrophilic compounds, these investigators proposed that absorption occurred through

aqueous channels of intercellular spaces in the lipid membrane. In another study, it was

found that those compounds with molecular weights less than 1000 D were absorbed at

faster rates (tin = 90 min) than the larger molecules (tin = 3 to 27 h). First order

absorption rate constants from the upper respiratory tract are approximately half of those

from the alveolar regions [40]. Solutes with molecular weights in the range of 100 to 1000

D are absorbed with half-lives between 8 and 40 min [47].

Some studies have reported that solutes penetrate the alveolar wall at rates which

increase with the lipid solubility of the compound [48]. Those compounds having partition

coefficients greater than 10i are absorbed rapidly. However, there appears to be no

continuity for others with partition coefficients less than 106 [49]. If the partition

coefficients and molecular weights of the compounds are taken into consideration

simultaneously, it may be possible to estimate which compound will be absorbed most

quickly. Drug dissolution or release

It has been reported that the physicochemical properties of the drug such as

particle size (surface area) and crystal form [50], as well as its lipophilicity [51], play a

central role in the dissolution rate. Drugs with higher lipophilicity and larger particle size

will dissolve slower. A drug with a fast release rate will be dissolved immediately after

pulmonary deposition and will be absorbed into the systemic circulation at a rate which

will be determined by its partition coefficient and molecular weight as mentioned earlier in

section See also section below for the effect of release rate on pulmonary


2.4 Pulmonary Targeting

The purpose of pulmonary targeting is the achievement of relatively high drug lung

concentrations relative to drug systemic concentrations, thereby reducing systemic side


2.4.1 Pulmonary Selectivity of Inhaled Glucocorticoids

Inhaled glucocorticoids (IG) were first introduced into asthma therapy to replace

oral glucocorticoids in patients with severe asthma. The advantage of inhalation therapy

versus oral therapy is the direct delivery to the lungs, combined with a relatively lower

dose, which may reduce the incidence of systemic side effects [52].

Beclomethasone dipropionate, triamcinolone acetonide, flunisolide, budesonide

and fluticasone propionate are currently available as aerosols in the US for the treatment

of asthma [51-53]. They are delivered by metered dose inhalers (with and without

spacers), dry powder inhalers and nebulizers.

Due to the apparent uniformity of the glucocorticoid receptor in the body, there

was no basis for differentiation of desirable from undesirable glucocorticoid effects at the

receptor level. However, through chemical manipulation of the glucocorticoid structure it

has been possible to develop synthetic derivatives better suited for pulmonary targeting.

Thus, glucocorticoids with lipophilic substituents in the 17a or 16o, 17a positions were

found to be better candidates for inhalation therapy, especially because of the

improvement of pharmacokinetic properties.

Although the pharmacokinetic properties of inhaled glucocorticoids favor its

pulmonary selectivity, one problem associated with the respiratory drug delivery is the

relatively rapid absorption (once they are dissolved) into the systemic circulation requiring

frequent administration. Since frequent administration of these drugs is associated with

high risk of systemic side effects (especially suppression of growth in children and

osteoporosis), current research is aimed at identifying the factors that can improve

pulmonary targeting is obvious.

Hochhaus et al. [1] have developed a pharmacokinetic/pharmacodynamic model to

aid prediction of the pharmacokinetic and biopharmaceutical factors which affect

pulmonary targeting. Contrary to previous mathematical models [39, 54], this model

integrated lung physiology with pharmacokinetic, pharmacodynamic and

biopharmaceutical drug properties, and thereby allowed prediction of pulmonary and

systemic effects by calculating pulmonary and systemic receptor occupancies, as

pharmacodynamic surrogate. This model, which gave the basis for the in vivo studies

presented in this thesis, is described below using glucocorticoids as a model. Effect of clearance, volume of distribution and oral bioavailability

Commercially available glucocorticoids differ in their clearance and oral

bioavailability. Oral glucocorticoids are characterized by a low clearance and high oral

bioavailability, while inhaled glucocorticoids have a high clearance and low oral

bioavailability (see Table 2.3). Inhaled glucocorticoids also differ in their volume of

distribution (Table 2.3).

The computer simulations described above showed that an increase in clearance

(from 6 L/hr to 60 L/hr) resulted in a dramatic decrease in systemic side effects from

61.3% to 12.4%, with a small decrease in pulmonary effects. However, the difference

between pulmonary and systemic effects increased, and as a consequence, so did

pulmonary targeting (defined in here as the ratio of pulmonary effects and systemic

effects). In contrast, changes in volume of distribution did not seem to significantly affect

pulmonary targeting. Thus, drugs with high systemic clearance are better candidates for

attaining pulmonary selectivity.

Only 10% to 30% of the delivered drug is deposited along the airways. The rest is

swallowed and absorbed into the systemic circulation (see Figure 2.6). The percentage of

systemically absorbed drug that is related to the oral route depends on the glucocorticoid

oral bioavailability.

Table 2.3 Pharmacokinetic

available glucocorticoids [1].







and pharmacodynamic parameters of commercially

Volume of
(Vdss) (L)


life (hr)


103 2











71 233

69 318 7.8 <1 90 1800

Thus drugs with low oral bioavailability will be a better drugs for inhalation therapy. Since

inhaled glucocorticoids differ in their oral bioavailability (Table 2.4) it may be interpreted

as differences in pulmonary selectivity. Effect of pharmacodynamics

It has been stated that only glucocorticoids with high relative binding affinity

(RBA) for the glucocorticoid receptor are active by inhalation, and that the minimum level

of RBA seems to be 20 times above of hydrocortisone [55]. In addition Brattsand et al.

[23], have mentioned that a high affinity for and intrinsic activity at the glucocorticoid

receptor seems necessary for gaining topical anti-inflammatory activity on the mucous

membranes. However, the pk/pd simulations [1] described above, have demonstrated that

a low receptor affinity can be compensated by an increase in dose, assuming that the

required dose can be delivered via inhalation. Thus, this suggests that glucocorticoids with

high receptor affinity may not be better drugs to enhance pulmonary targeting. Effect of release rate and dose

Glucocorticoids are removed relatively fast from the airways (once they have been

dissolved) due to their relatively high lipophilicity and because of the high pulmonary

blood flow. Hochhaus et al. [1] have theoretically demonstrated the relevance of release

rate on pulmonary selectivity. He showed that a glucocorticoid with fast dissolution rate

will be removed immediately from the airways by entering the systemic circulation (see

Figure 2.6), resulting in small mean residence time values. After a short time, the free drug

concentrations will be the same in lung and systemic organs, and minimal pulmonary

targeting will be observed. If the release rate decreases, the free drug concentrations in

lung will be higher over an extended period of time than that in the systemic organs,

resulting in more pronounced pulmonary effects and unchanged systemic effects. Thus, it

was concluded that sustained release rate is very beneficial for improving lung selectivity.

However, the author mentioned that for drugs deposited into the upper portion of the

airways, a further reduction in drug release will result in reduction in pulmonary targeting

due to elimination of the undissolved drug by mucociliary transport. Likewise, he showed

that when the dose is too small it could mean that the minimum effect concentration is not

reached either in the local organ (lung) or in the systemic organ (liver). When the dose is

too high toxic side-effects may result and pulmonary targeting is lost. Thus, there is an

optimum dose and release rate for which maximum pulmonary targeting can be achieved.

Some studies [23, 56] have suggested the importance of drug release rate in

pulmonary targeting. In these studies the intratracheal administration of glucocorticoids in

slow release preparations yielded higher pulmonary selectivity than that after the

intratracheal administration of the drugs in solution.

2.4.2 Improvement of Glucocorticoid Airway Selectivity

Although the benefits of inhaled over oral glucocorticoids are clear, there are still

some concerns regarding side effects since inhaled glucocorticoids are likely to be

administered over long periods of time. This issue requires new emerging technologies

that can lead to the enhancement of airway selectivity. For example, the design of

glucocorticoids with extra-hepatic metabolism will lead to derivatives with even higher

clearance and reduced systemic side effects; the use of drug delivery systems such as

liposomes, or new drug entities will increase the pulmonary residence time and thus, more

prolonged pulmonary effects. This thesis proposes the use of liposomes as a slow drug

delivery system to improve pulmonary selectivity

Figure 2.6 Pharmacokinetic/Pharmacodynamic model for pulmonary selectivity [1].

2.5 Liposomes

In the last two decades, it has been demonstrated that encapsulation of drugs into

liposomes can lead to the enhancement of therapeutic efficacy of drugs, reduction of their

toxicity and prolongation of their therapeutic effect. In addition, studies have been

performed on the use of liposome-based vaccines. Recently, a liposome formulation

containing the antifungal agent amphotericin has been approved and is presently used in

the clinic [57] This suggests that the spirit and enthusiasm to explore new research

strategies with liposomes are as alive now as they were in the early days of liposome


Since selective drug delivery to specific tissues is the main application of

liposomes, efforts have been principally concentrated on "site-directed targeting," for

instance, selective uptake. As a result of these studies, a detailed picture of the tissue

distribution of systematically administered liposomes has emerged [58]. However, the

kinetics of the disposition processes are still far from being characterized.

Liposomes are quite similar to natural membranes, and are thus inert and

biodegradable. Through variation in size, lipid composition, number of bilayers, charge

and surface characteristics, the pharmacokinetics of liposomes and therefore the

pharmacokinetics of the encapsulated drug, can be manipulated.

2.5.1 Liposome Structure and Classification

Liposomes are vesicular structures composed usually of phospholipid bilayers

which can entrap hydrophilic or hydrophobic materials within their aqueous compartment

or within the membrane. These vesicles are formed spontaneously when lipids are

dispersed in aqueous media. Depending on the method of preparation, the population of

vesicles can range in size from the smallest vesicles obtainable in theoretical grounds (25

nm in diameter), determined by the maximum possible crowding that headgroups will

tolerate as the curvature in the inner leaflet increases with decreasing radius, to liposomes

which are visible under a light microscope with a diameter of 1 micron [59].

Liposomes can be classified either on the basis of their structural properties or on

the basis of the preparation method used. Table 2.4 lists the different vesicles with the

corresponding acronyms.

2.5.2 Chemical Composition.

Liposome-forming phospholipids are obtained from natural sources or through

synthetic routes. Natural phospholipids are not easy to characterize in terms of their exact

phospholipid composition. In contrast, synthetic phospholipids can be well characterized

and can be obtained in highly purified forms [59]. Two sorts of natural phospholipids

exist: phosphodiglycerides and sphingolipids. In phosphodiglycerides, a three-carbon

glycerol bridge links a long chain fatty acid with a phosphoryl headgroup moiety; by

convention, the fatty acids are said to occupy positions 1 and 2 of the glycerol bridge

while the polar headgroup is in position 3.

Choline-containing phospholipids, also known as lecithin, are the most abundant

phosphodiglycerides in nature. They form the major phospholipid component of many cell

membranes. Because of their neutrality and chemical inertness, they are the most often

used lipids for the preparation of liposomes. The chemical structure of a number of

phospholipids regularly used for liposome preparation is show in Figure 2.6.

Table 2.4 Liposome Classification [60]

A. Based on Structural parameters
MLV Multilamellar large vesicles, > 0.5 pmr
OLV Oligolamellar vesicles, 0.1-1 pm
UV Unilamellar vesicles (all sizes)
SUV Small unilamellar vesicles, 20-100 pm
MUV Medium-sized unilamellar vesicles
LUV Large unilamellar vesicles, > 100 un
GUV Giant unilamellar vesicles vesicless with diameters > 1 mm)
MVV Multivesicular vesicles (usually large > 1 mm)

B. Based on Method of Liposome Preparation
REV Single or oligolamellar vesicles made by reverse-phase evaporation method
MLV-REV Multilamellar vesicles made by reverse phase evaporation method
SPLV Stable plurilamellar vesicles
FATMLV Frozen and thawed MLV
VET Vesicles prepared by extrusion methods
FPV Vesicles prepared by French press
FUV Vesicles prepared by fusion
DRV Dehydration-rehydration vesicles
BSV Bubblesomes

2.5.3 Method of Preparation

Liposomes are prepared by mechanical dispersion methods (such as hand-shaken

multilamellar vesicles, non-shaken vesicles, freeze-drying, sonicated vesicles methods,

etc), by solvent dispersion methods (such as ethanol injection, ether injection or water in

organic phase method) or by a detergent solubilization method [60].

Dependent on the selection of lipids, the preparation technique, and preparation

conditions, liposomes can vary widely in size, number, position of lamellae, charge, and

bilayer rigidity. These parameters influence the behavior of liposomes both in vivo and in

vitro. The opsonization process, leakage profiles, disposition in the body, and shelf life al

depend on the type ofliposome involved [60]. Therefore, it is important to select liposome

constituents and the preparation technique carefully and to characterize the produced

liposomes properly.

Phosphatidyl moiety


-0o Me








Common name





Figure 2.6 Some common naturally-occurring phosphatidyl phospholipids. Adapted
from reference [57]

2.5.4 Chemical Stability

From a pharmaceutical point of view, it is important to demonstrate that drugs or

dosage forms are sufficiently stable, so that they can be stored for a reasonable period of

time (at least 1 year and preferably) without changing into inactive or toxic forms.

Liposomal drug formulations also must comply with this rule.

The chemical stability of liposomes is focused mainly on phosphatidylcholine,

because as previously mentioned, it is the most commonly used lipid in pharmaceutical

liposome preparations. Two degradation pathways which might limit the shelf life of

liposome dispersions have been described for phospholipids in aqueous liposome

dispersions. These are the oxidative and the hydrolytic degradation pathways [57]. Oxidation of Phospholipids

Oxidation of phospholipids in liposomes mainly takes place at the unsaturated acyl

chain-carrying phospholipids, but saturated fatty acids can also be oxidized at high

temperatures [57].

The fatty acyl chains of phospholipid molecules are oxidized via a free radical chain

mechanism in the absence of specific oxidants. Exposure to electromagnetic radiation

and/or the presence of trace amounts of transition metal ions, initiate radical formation

(hydrogen atom abstraction) in the lipid chain (initial step). Since unsaturation permits

delocalization of the remaining unpaired electron along the lipid chain, lipids containing

polyunsaturated fatty acids are the most sensitive to radical formation. If oxygen is

present, the process develops further and via the formation of hydroperoxides, fission of

the fatty acid chain can occur. Changes in UV absorbance of lipids is the first sign of the

occurrence of radical chain reactions which can lead to oxidation [60].

As mentioned above, the autoxidation of lipids is accelerated by metal ions, light,

some organic molecules and also by high pH. It is clear then, that this oxidation process

can be minimized by the use of high quality raw materials which are purified from

hydroperoxides and transition metal ions, storage at low temperatures and the use of


Gonzalez-Rothi et al. [61] evaluated the in vitro stability of various formulations

of liposomes is several fluids. They found that liposomes consisting of unsaturated

phospholipids, lost around 40% of their content over 24h compared to liposomes made of

saturated phospholipids which retained more than 90% of the encapsulated material. It is

possible that the increase in permeability in the unsaturated formulation was due to a

higher degree of autoxidation process in relation to the saturated formulations. Hydrolysis of phospholipids

The liposome is inherently a stable system. In the absence of oxygen and free

radicals the only chemical reactions which result in a change in liposome properties are

those of hydrolysis.

The four ester bonds present in a phospholipid molecule may be subject to hydrolysis. The

carboxy esters at sn-I and sn-2 positions are hydrolyzed faster than the phosphate esters.

Hydrolysis of esters is catalyzed via two pathways [57]:

Acid catalyzed hydrolysis. In this pathway the ester is first protonated and

subsequently, a nucleophilic attack of water takes place.

Base catalyzed hydrolysis. This step proceeds via an addition-elimination

mechanism. Here the rate determining step is the attack of hydroxide ions on

the carbonyl carbon atom.

Two products are formed as a result of hydrolysis of the carboxy esters at the sn-1

and sn-2 positions: 2-acyl and 1-acyl lysophospholipids, respectively [62]. Further

hydrolysis of both lysophospholipids produces a glycerophospho compound.

Glycerophosphoric acid, the end hydrolysis product, is produced by hydrolysis of the

phosphate-headgroup ester.

The rate of hydrolysis can be affected by different factors:

ionic strength
buffer species
chain length
incorporation of cholesterol
incorporation of charged phospholipids The effect of pH

Grit et al. [63], studied the effect of pH on the hydrolysis of saturated soybean

phosphatidylcholine (PC) in a pH range from 4-9 at 40 *C and 70 TC. They found that the

hydrolysis rate reached a minimum at pH 6.5, and V-shaped pH profiles were obtained as

expected for an acid/base catalyzed ester hydrolysis. In another study the pH profile at

zero buffer concentration and at 72 C showed a minimum hydrolysis rate at about pH 6.5

for natural soybean PC [64]. The effect of temperature

The effect of temperature on the hydrolysis of phospholipids has been studied with

synthetic and/or natural PC. For all PC's the results could be described by Arrhenius

kinetics as a semilogarithmic linear relationship was found between the observed

hydrolysis rate constant and the reciprocal of the absolute temperature. For natural PC's,

Arrhenius kinetics held in a temperature range from room temperature up to 82 C [64].

However, for phospholipids composed of saturated fatty acids and with a liquid to gel

crystalline phase transition, biphasic Arrhenius curves were obtained with a discontinuity

around the transition temperature [62]. The effect of charged liposomes

Charged phospholipids are generally included in pharmaceutical liposome

formulations as they tend to improve the physical stability of liposomes by reducing the

rate of aggregation and fusion. Recently, hydrolysis of partially hydrogenated egg PC and

egg phosphatidylglycerol (PG) have been investigated under different conditions [57].

Different electrostatic profiles in the aqueous phase around the bilayers were obtained by

varying the charged phospholipid content (PG) of liposomes. Hydrolysis kinetics for both

lipids followed pseudo first order reaction kinetics. It was found that egg PG hydrolyses

faster than partially hydrogenated egg PC. The effect of incorporation of cholesterol

The incorporation of cholesterol into liposomal bilayers tends to increase the

retention of hydrophilic drugs, counteracts lipid phase transition and increases the rigidity

of fluid state bilayers, and thereby, the resistance to liposome degradation. The effect of

cholesterol on the properties of distearoylphosphatidylcholine (DPPC) vesicles was

studied using a fluorescence polarization technique by Papahadjopoulos et al. [65]. They

found that cholesterol abolishes the phase transition of phospholipids, producing a rather

rigid membrane over a wide temperature range, which is translated into a protective effect

against hydrolysis.

Based on the structural properties of phospholipids and cholesterol molecules,

Huang C. [66], proposed a model for the effects of cholesterol on the structural and

motional properties of phospholipid acyl chains. In this model, the 303-hydroxy group of

cholesterol is assumed to engage in hydrogen bonding with the carbonyl oxygen of the

fatty acyl groups in phospholipids. Depending on the strength of this interaction, the

hydration properties of both ester bond (sn-1 and sn-2) may change, which in turn can

change the hydrolysis kinetics. This effect is, in general, expected to be a protective effect,

as mentioned above.

2.5.5 Physical Stability

Chemical decomposition of phospholipids causes physical instability of the

liposome dispersion, which means that all the variables considered in chemical stability will

affect the physical stability of liposomes and therefore might interfere with the

introduction of liposomes in therapy.

The physical stability ofliposomes is influenced by two parameters:

1. Changes in the average particle size and size distribution due to vesicle

aggregation and fusion.

2. Loss of entrapped drug due to leakage.

Changes in the average particle size and size distribution are strongly affected by:

Phospholipid composition

Medium composition The effect of phospholipid composition

Liposomes which lack a net electrical charge tend to be less stable towards

aggregation than charged liposomes. Crommelin [67] studied the effect of including

charge-inducing agents (SA and PS) on the zeta potential of PC containing liposomes in

aqueous media with varying ionic strength. It was found that for negatively charged

liposomes, both at low and high ionic strength, no increase in particle size occurred after

storage at 40C for 14 days. Thus, aggregation can be prevented or slowed down by

incorporation of a charge-carrying lipid into the liposomal formulation. Liposome composition

Several studies have shown that the incorporation of cholesterol into liposomes

increases the physical stability of the formulations by increasing rigidity of the bilayer

which is translated in decreased permeability [65, 66, 68-71].

As mentioned above, lysophospholipids and free fatty acids are produced as a

result of phospholipid hydrolysis. These products have different amphiphilic properties

than the parent drugs and can produce morphological changes in the membrane and thus

changes in permeability of the bilayer [68-70].

The effect of lysophospholipids and fatty acids formed in situ was investigated

[57]. Liposomal dispersions were stored over time intervals at elevated temperatures to

produce degradation of the phospholipids. It was found that when up to 10%

lysophosphatidylcholine (LPC) was produced (corresponding to 10 % of hydrolysis), a

drop in the leak-in rate of calcein occurred. Above that degree of hydrolysis, the leak-in

rate started to increase as in exogenous LPC containing liposomes. They concluded that in

the initial stage the free fatty acids were also formed, thus, neutralizing the LPC-

destabilizing effect. Liposome size

The effect of vesicle size on the physical stability of liposomes depends on the lipid

composition and the medium considered.

Large neutral liposomes are less stable than smaller liposomes, because the

increased planarity of the membranes allows greater areas of membrane to come into

contact (Van der Waals interactions) with each other, and an increase in aggregation

produces an increase in permeability.

Small negatively charged unilamellar vesicles (40 nm) are less stable

(thermodynamically) than large vesicles. They fuse easily as a means of relieving stress

arising from the high curvature of the membrane [60]. In general, large multilamellar

liposomes are more stable against leakage than small unilamellar vesicles due to the

difference in membrane thickness.

Size-dependent stability of liposomes in biological fluids has been reported by

different authors. Tari et al. [72] studied the relationship between liposome size and its

stability in plasma. They found that small liposomes composed of pure succinylglycerol

were more stable than larger ones. However, in another study [71], it was found that small

liposomes consisting of egg phosphatidylcholine and cholesterol were less stable than

larger liposomes. As can be seen from these results stability of charged and neutral

liposomes with respect to the size is completely different (inverse) to that in buffers. This

may be due to a different degree of protein association on the surface of the bilayer, or

that the final concentration of plasma used in either case was different. It has been shown

[73] that at low serum concentrations liposome formulations consisting ofDPPC, PI, SA

and cholesterol remain intact independently of size, but as the serum concentration is

decreased, the size of the liposomes decreases with concomitant release of encapsulated

material. Presence of metal ions

Divalent cations besides enhancing fusion and aggregation of liposomes as

mentioned above, can cause an increase in the permeability of the bilayer.

The interaction of liposome bilayers composed of succinylglycerol with divalent

ions was investigated by Tari, et al. [72]. In this study, it was found that Ca2+ and Mg2+

produce a destabilization of the bilayer due to an isothermic phase change of the lipid

bilayer from liquid to a crystalline state which resulted in increased permeability of the

encapsulated material.

2.5.6 Biological Stability

When administered in vivo, liposomes are rapidly cleared by the reticular

endothelial system (RES), in such a way that large, negative liposomes consisting of

unsaturated phospholipids are taken faster than small, neutral liposomes.

Advances in therapeutic applications of liposomes have been achieved through

surface modifications increasing their biological stability, thus, reducing constituent

exchange and leakage as well as reducing unwanted uptake by cells of the RES.

Perhaps one of the earliest descriptions of liposomes exhibiting a substantially

prolonged circulation was of small, neutral, cholesterol-rich liposomes composed of

phospholipids with a high temperature phase transition. However, severe limitations to a

small particle and neutral rigid lipids in combination with a lipid dose-dependence reduces

their therapeutic usefulness [74]. Subsequent incorporation of specific natural glycolipids

such as monosalo-ganglioside (Gwi) or hydrogenated soy phosphatidylinositol was shown

to result in prolonged circulation and reduced RES uptake.

2.5.7 Pharmacokinetics of Liposomes

In contrast to more common drugs which form true solutions in body fluids,

phospholipid liposomes are colloidal suspensions. Accordingly, they are cleared from the

blood by mechanisms different from those governing the clearance of substances dispersed

at the molecular level. This has prevented the use of the pharmacokinetic models currently

applied to the analysis of the disposition kinetics of drugs. Although, some kinetic models

have been proposed to describe the disposition of certain types of liposomes, a model of

general applicability is still lacking and a quantitative analysis of liposome disposition is

hardy feasible [75].

The fate of liposomes administered to the body is drastically altered by

administration route, dose, size, formulation, and surface charge, and specific liposomal

surface properties modified by certain ligands, sugars, antibodies, etc. [76]. Disposition of liposomes following intravenous (IV) administration

Liposomes administered IV can interact either with phagocytic cells (fusion or

endocytosis) or with plasma lipoproteins. Liposomes, similar to any foreign particulate

matter, are rapidly removed from the circulation mainly by the Kupffer cells, and to a

lesser extent, by the other macrophage populations. The RES represents, therefore, a

major obstacle to an effective targeting of liposomes to other cell types.

The rate and uptake of liposomes by the RES depend on liposome size, physical

state, composition, and dosage [75]. Therefore, since the RES is the principal mechanism

for liposome clearance, liposome clearance will be affected by changing dose, chemical

composition etc.,

Internalization of phospholipid liposomes is also operated by non-phagocytic cells

such as fibroblasts, cells of the kidney, lymphocytes, hepatocytes, etc. This non receptor-

mediated endocytosis appears to be strictly dependent on the size of liposomes, with the

optimal size being 50-100 nm. Vesicles larger than 400 nm are not apt to be endocytosed


Phospholipid vesicles can interact with, and become destabilized by plasma

lipoproteins, mainly high density lipoproteins (HDL). This destabilization is the result of

the insertion of apoproteins into the liposomal bilayer and the exchange of lipidic material

between lipoproteins and liposomes [75]. The degree of liposomal destabilization, and

therefore, the biological stability and half-life depend again, on the size and composition of

the vesicles.

The size of the endothelial cells (about 70 nm in diameter) does not permit

accommodation of particles larger than about 50 nm, therefore phospholipid liposomes are

unable to leave the vascular space by passing across the cells of the capillary endothelium.

Thus, egress of liposomes from the circulation is dependent on the presence of pores like

those in fenestrated or discontinuous capillaries present in the liver, and appears a realistic

possibility only for SUV [77]. 1 Effect of vesicle size on the kinetics of liposomes

Small unilamellar vesicles (< than 100 nm) are taken up less rapidly than large

multilamellar vesicles and when those small liposomes go to the liver, there is a higher

proportion entering the hepatocytes rather than the Kupffer cells, since they are small

enough to pass through the fenestrae of the sinusoids and into the liver parenchyma.

Accordingly, SUV encounter longer half-lives (days) and smaller clearance than LMV


As noted above, liposomes are destabilized upon IV administration due to

unidirectional transport of phospholipids from the liposomes to HDL. Resistance to such a

disruption depends on the diameter of the vesicles; large liposomes are less readily

penetrated by apoproteins than are SUV and, therefore, exhibit greater stability.

A biphasic plasma decay curve is quite often observed following IV injection of

liposomes, with a faster initial decay followed by a slower one. For substances that form

true solutions in body fluids, such a profile is analyzed on the basis of a

multicompartmental open model.

Huang et al. [78], observed a biphasic decay and extrapolated the volume of

distribution to be 1.28 times larger than the volume occupied by the erythrocytes after the

IV administration of SUV (19 nm mean diameter) prepared from sphingomyelin and

cholesterol. Since the ratio of the volume of distribution of the central compartment (Vc)

to the extrapolated volume of distribution (Vd) was 0.78, they concluded that a 1

compartment body model was completely satisfactory for the disposition kinetics of this

liposome preparation. The value of the Vc/Vd ratio, the limits of which are 0 to 1, is

indicative of the multicomparmental character of a disposition kinetics. The smaller the

numerical value of the ration, the greater the multicompartmental character.

As mentioned above, large liposomes do not distribute outside the plasma

compartment. If egress from plasma is unidirectional, then it is an elimination process

(irreversible loss of the substance from the site of measurement) that cannot be responsible

for a biphasic decay. Then, what factors are responsible for this biphasic decay? This

question has been addressed by several investigators [79, 80].

Juliano and Stamp [79], by showing that small vesicles are cleared at a slower rate

than large vesicles, demonstrated that a biphasic plasma decay may result from

heterogeneity in size of the injected liposomes. They found that a biexponential clearance

pattern is observed with heterogeneous vesicles populations, whereas a monoexponential

decay is seen with liposome samples homogeneous in size.

Gregoriadis et al. [80], observed that the biphasic decay pattern of IV injected

MLV is converted into a linear one upon increasing the liposomes dose. They suggested

that the biphasic decline may be due to the presence of two elimination pathways: a faster

one, due to a saturable uptake by the RES, and a slower one, due to uptake by the liver

parenchymal cells. When the retention capacity of the RES becomes saturated, then only

the slower elimination pathway operates and the decay rate decreases. Effect oflipid composition and surface charge on the kinetics of liposomes

Upon exposure to blood, liposomes become coated (opsonized) with plasma

proteins (alpha 2-macroglobulin) that can mediate their uptake. Recognition and ingestion

of opsonized liposomes by phagocytic cells are mediated via binding of liposomal opsonins

to specific receptors on these cells. A major mechanism for liposome uptake is through

opsonization with IgG and subsequent Fc-mediated phagocytosis or endocytosis [81].

Nabila et al. [82] observed that in liposomes containing certain lipids, including

phosphatidylinositol, ganglioside GM1, and sulfogalactosyl ceramide, complement-

dependent phagocytosis of the liposomes was greatly suppressed. They suggested that

suppression of this opsonization process could be a contributing factor in the promotion of

increased circulation time of "stealth" liposomes and that complement opsonization

probably plays a role in vivo in removing liposomes from the circulation.

Stabilization of the liposomal structure and therefore, decreased in the clearance

rate can be obtained by inclusion ofunesterified cholesterol that, by decreasing the fluidity

of the lipid bilayer, renders it less easily penetrated by apoproteins and decreases

opsonization by plasma proteins [83]. For instance, it was found [84] that when

cholesterol is incorporated in liposomes, the half-life was as long as 20 h.

The effect of surface charge on liposomal clearance has been investigated

extensively [76, 77, 82, 83]. Senior et al. [83] found that negatively charged liposomes

disappear from the circulation faster than neutral liposomes. Another study, however,

indicated that this is not a general phenomenon and that some negatively charged lipids

such as phosphatidylinositol and GM, ganglioside can actually cause the retardation of

liposomal clearance, due to suppression of the complement-dependent phagocytosis [82].

On the other hand, it was found that phosphatidylserine (PS) incorporated in liposomes

accelerates clearance by the RES trapping via scavenger receptors on monophagocytes


Abraham et al. [85] studied the pharmacokinetics of 3H- triamcinolone acetonide-

21-palmitate entrapped in liposomes with neutral, negative and positive surface in rabbits

after single IV bolus injection. They found that positive liposomes had encountered a

larger initial apparent volume of distribution than neutral or negatively charged liposomes.

These results can be explained if it is considered that the rate of liposomal uptake by the

RES is as follows: positive negative > neutral liposomes. Disposition of liposomes following pulmonary administration

As mentioned above, one of the benefits of liposomes, as drug carriers is based on

their ability to favorably alter the pharmacokinetic profile of the encapsulated species, and

thus, provide selective and prolonged pharmacological effects at the site of administration.

Administration of liposomes to the respiratory tract is attractive because of the

accessibility of the lung as a target organ, the compatibility of liposomes and lung

surfactant components, and the need for sustained local therapy following inhalation. Due

to this, numerous studies have explored the effect of liposomal encapsulation on the

distribution and fate of compounds administered directly to the lung by either intratracheal

instillation or inhalation.

One of the first studies was reported by Juliano et al. [86], comparing the

effectiveness and distribution of free and encapsulated beta-cytosine arabinoside in MLV

following intratracheal administration to rats. They found that free ARA-C (arabinoside C)

was rapidly cleared from the lung (tiz = 40 min) and entered the systemic circulation,

while liposomal ARA-C displayed little redistribution to other tissues and persisted

throughout the lung (t12= 8h).

Farr et al. [87] investigated the pulmonary deposition and clearance of liposomes

by labeling MLV and SUV composes of DPPC with "m-technetium. Deposition of

nebulized vesicles was dependent on the droplet size of the aerosol produced, and not on

the vesicle size. For both preparations, over 75% of the vesicles were retained in the lung

after 6 hr, indicating that the mucociliary elevator was a major mechanism of clearance.

Since the removal of particulate matter by the mucociliary elevator becomes progressively

faster from the peripheral to central airways, the initial regional deposition of aerosol

droplets must be considered an important factor in determining the rate of vesicle

clearance. From a therapeutic point of view, alveolar deposition would be preferred, not

only on the basis of providing local, but also prolonged activity due to the inability of the

lung to clear vesicles as quickly from that site.

Woolfrey et al. [88] investigated the pulmonary absorption of liposome

encapsulated 6-carboxyfluorescein (CF) following IT instillation to anaesthetized rats. The

extent of CF absorption was influenced by the dose of lipid and the surface charge of the

liposome. Increasing the lipid dose resulted in an increase in the rate of absorption, but

decreased the extent of absorption. The authors suggested that the higher lipid dose

accelerated removal of the liposomal CF from the lung, perhaps by specific pathways

capable of removing particulate material. The absorption of CF from negatively charged

vesicles was at least twice as fast as from neutral vesicles.

Investigations by Mihalko and co-workers [89], examined the effect of liposomal

encapsulation on the pharmacokinetic fate of hydrophilic and lipophilic compounds

following intratracheal instillation. They found that for hydrophilic compounds, the lung

epithelium represented the major rate-limiting barrier for systemic absorption, not the rate

of the drug release. In contrast, IT administration of lipophilic compounds as either free or

encapsulated drug produced almost identical plasma levels suggesting that diffusion of the

lipophilic compound through vesicles bilayers and lung was unrestricted.

A similar comparison of hydrophilic and lipophilic compounds was investigated by

Meisner and colleagues [34] following endotracheal administration to rabbits. In this

study, liposomal encapsulation of atropine base maintained drug concentrations in the lung

approximately three-fold over 48-hr period while free atropine disappeared rapidly. After

48 hr, 21% of liposomal atropine was still associated with lung tissues as compared to 4%

when instilled in solution form. They concluded that the amount of drug available to lung

tissue from liposomes was controlled by the rate of release from the MLV.

Pulmonary delivery of liposomal encapsulated-glucocorticoids is rather scarce.

One possible explanation for this is that glucocorticoids are highly lipophilic drugs which

tend to escape very easily from liposomes when diluted in a larger volume, as has been

demonstrated for hydrocortisone [90] and for triamcinolone acetonide [91]. In order to

overcome this problem more hydrophilic derivatives of glucocorticoids have been used

[23, 92, 93]. For example the encapsulation of triamcinolone acetonide-21-palmitate was

85%, compared to 5% for the parent drug [93]. Likewise, Brattsand et al. [23]

demonstrated that budesonide 21-palmitate incorporated into liposomes, showed

prolonged retention time (half-life = 6 hr) compared to budesonide, after intratracheal


The practicability of aerosolizing liposomes for the inhalation and deposition of

their content in the lung has been demonstrated [87, 91]. Traditionally, aqueous liposomes

aerosols have been generated with nebulizers like the Collison and Hudson. However, it

has been shown that the loss of an encapsulated marker is enhanced significantly by the air

flow pressure, thus restricting the usefulness of nebulizers [94]. As an alternative to

nebulization, emphasis has been put on the use of lyophilized liposomes delivered as dry

powders with dry powder inhalers like the Spinhaler [95].

Biodistribution studies have demonstrated that liposomal encapsulation of

compounds can localize and maintain drug levels in the lung for an extended period of

time while decreasing the extent of systemic absorption. Pharmacologically, there is also

evidence that liposomal drugs can produce selective and prolonged effects with decreased

systemic toxicity. The pulmonary absorption of liposome-encapsulated solutes can be

influenced by the physicochemical properties of the entrapped species, the site of

deposition in the lung as mentioned earlier, as well as, the lipid composition of the

vesicles. There are 3 mechanisms that participate in the clearance of liposomes:

mucociliary escalator, phospholipid exchange with the phospholipid pool and endocytosis

by lung macrophages. Which mechanism takes place, depends also on the deposition in the

lung. A better understanding of the factors that alter absorption and clearance is essential

in designing a liposomal formulation that not only optimizes pharmacokinetics and

pharmacological profiles, but also satisfies pharmaceutical requirements.

2.6 Synopsis of Literature Review

Asthma is perhaps the only treatable condition whose prevalence and severity are

increasing. With the recognition of asthma as an inflammatory disease, glucocorticoids

have become the drugs of choice for the treatment of this disease. Glucocorticoids exert

their action directly by interaction of the receptor-glucocorticoid complex with certain

portions of the DNA, or indirectly by inhibiting the activation of certain cytosolic factor

such as AP-I and NF-kB.

Inhalation therapy of glucocorticoids has the advantage of localizing the drug in

the target organ, thereby reducing the dose, as well as, systemic exposure. Triamcinolone

acetonide, beclomethasone dipropionate, budesonide, flunisolide and fluticasone

propionate are the commercially available inhaled glucocorticoids for the treatment of

asthma. They are delivered using metered dose inhalers (with and without spacer devices),

nebulizers or dry powder inhalers. This relatively new generation of inhaled

glucocorticoids are known to achieve higher local/systemic drug ratios than their oral

counterparts, mainly because of their pharmacokinetic properties. They have higher

systemic clearance and lower oral bioavailability, which results in lower systemic drug

concentration and therefore higher benefit/risk ratio. However, there are still concerns

regarding side effects, especially the suppression of growth in children and osteoporosis,

since these drugs are likely to be administered for long periods of time. Thus,

identification of the factors that can improve pulmonary targeting is obvious.

Recently, it has been shown that not only clearance and oral bioavailability, but

also drug release rate (slow release rate) and dose play an important role in pulmonary


Liposomes (microscopic phospholipid vesicles) have been widely used due to their

flexibility in controlling release rate. In the last two decades, it has been demonstrated that

encapsulation of a drug into liposomes enhances its therapeutic efficacy, reduces its

toxicity and prolongs its therapeutic effects. The concept ofliposome administration to the

respiratory tract is attractive because of the accessibility of the lung as a target organ, the

compatibility of liposomes with lung surfactant components, and the need for sustained

local therapy following inhalation. As a consequence, numerous studies have explored the

effect of liposomal encapsulation on the distribution and fate of compounds administered

directly to the lungs by either intratracheal (IT) installation or inhalation.

Thus, the options for a further improvement of airway selectivity seem to be: 1)

the design of glucocorticoids with extra-hepatic metabolism which lead to derivatives with

even higher clearance and reduced systemic side effects and 2) the use of drug delivery

systems such as liposomes or new drug entities which increase the pulmonary residence

time. This thesis proposes the use of liposomes as a slow drug delivery system to improve

pulmonary targeting.


3.1 Objectives

One of the goals of the present study was to test whether the intratracheal (T)

administration of liposomes can improve pulmonary targeting. We also attempted to

demonstrate that pulmonary targeting of glucocorticoids can be enhanced by optimization

of dose and release rate. This goal was achieved by comparing pulmonary targeting of a

liposomal formulation with different release rates and doses of encapsulated drug and

relating them to pulmonary targeting of currently available inhaled glucocorticoids

(powder formulations). Our ultimate goal was to identify some essential biopharmaceutical

factors that are relevant for the development of aerosol dosage forms.

3.2 Specific aims

3.2.1 Aim # 1

To design a glucocorticoid-liposome dosage form suitable for pulmonary

administration and assess pulmonary targeting of both, the developed liposomal

glucocorticoid formulation and a control glucocorticoid in solution using and ex vivo

animal model: assess size, encapsulation efficiency, lipid content, and in vitro stability in

different biological fluids; and monitor glucocorticoid receptor occupancy in local organ

(lung) and in systemic organ (liver) upon intratracheal (IT) and intravenous (IV)

administration. Hypothesis

If the liposomal membrane serves as a rate limiting barrier for the release of the

water-soluble TAP, then upon IT administration, a selective local (lung) receptor

occupancy versus systemic (liver) receptor occupancy should be possible. In this way

pulmonary targeting of encapsulated material will be more pronounced than pulmonary

targeting of unencapsulated glucocorticoid (drug in solution). Rationale.

We hypothesized that liposomes are a suitable dosage form due to their ability to

act as drug carriers for pulmonary delivery and their flexibility in release rate.

Phosphatidylcholine (PC), a neutral phospholipid and phosphatidylglycerol (PG), a

negatively charged phospholipid, were chosen for the liposome preparation. This selection

was based on the fact that these 2 compounds are biocompatible with lung surfactant that

continuously lines the alveolar ducts. The lung surfactant pool is composed mainly of 60-

70%/ of phosphatidylcholine and 5-10%/ of phosphatidylglycerol [96]. The use of

negatively charged phospholipid not only increases the physical stability of the liposomes,

but also favors the uptake by macrophages [67, 71, 88], ultimately, enhancing pulmonary


Among the great number of currently available glucocorticoids for the treatment of

asthma, our group initially chose triamcinolone acetonide (TA) as a model drug. This drug

has the advantage of having a relatively short half-life, high receptor affinity, and low oral

bioavailability, pharmacokinetic characteristics that favor pulmonary targeting. However,

preliminary studies showed that while liposomes have a high loading capacity for TA

under equilibrium conditions, it is rapidly released from the liposome matrix upon dilution.

As the liposomes seemingly provide no barrier function for the lipophilic glucocorticoid

TA, we believed that a solution to this problem was to encapsulate a water-soluble

prodrug of TA. Thus, we selected the water-soluble triamcinolone acetonide phosphate

(TAP), a prodrug of TA for the liposomal formulation. Since TAP is rapidly cleaved

within minutes of administration (5-9 min) with complete conversion to its active

compound TA [97], we expected almost immediate receptor interaction of TA once TAP

is released from the liposomes.

The pharmacological effects of glucocorticoids are mediated through the

interaction with receptors localized in the cytoplasm of the cells [25]. In addition, the

degree of the pharmacological effects depends on the degree of receptor occupancy.

Therefore, by simultaneously monitoring lung and liver receptor occupancy using an ex

vivo animal model, it is possible to assess pulmonary and systemic side effects indirectly.

The use of liposomes has been suggested to provide sustained pulmonary release

for various drugs including glucocorticoids such as beclomethasone dipropionate and

dexamethasone [42, 98, 99]. However, such studies can not directly track

pharmacodynamically relevant concentrations as generally both, encapsulated

(pharmacologically inactive) and unencapsulated drug are being monitored. Because of

these limitations, such an experimental design is not suitable for showing pulmonary

targeting. The ex vivo receptor binding model employed in this study is able to quantify

the degree of pulmonary targeting by evaluating the difference between pulmonary and

hepatic receptor occupancy. To determine the effect of the route of administration of

pulmonary targeting we assessed lung and liver receptor occupancy after IT administration

and compared it to lung and liver receptor occupancy after IV administration.

3.2.2 Aim # 2

Assess the pharmacokinetics and pharmacodynamics of the liposomal formulation

after IT and IV administration and compare them to those after the intratracheal

instillation of the drug in solution. Hypothesis

The liposomal formulation will release the drug in a sustained release fashion, thus

retarding the absorption of drug from the airways. The retention of drug in the lungs will

result in a more prolonged mean residence time (change in the pharmacokinetics of the

drug) as well as in sustained mean pulmonary effect (change in the pharmacodynamics)

compared to those following instillation of the drug in solution. Rationale

A number of relatively low lipophilic glucocorticoids (such as methyl prednisolone

and triamcinolone acetonide ) are rapidly absorbed from the airways because of their fast

dissolution rate. Therefore, the encapsulation of these drugs into liposomes will control

the release rate of these drugs, enhancing their retention in the respiratory airways. It has

been stated that the overall effects (effect-time profile) depend not only on the

pharmacokinetics of the drug (concentration-time profile), but also on its intrinsic

pharmacodynamics (effect-concentration profile) [100]. Thus, a change in the

pharmacokinetics of the drug by slowing down its release rate will result in a favorably

change in the drug pharmacodynamics (increase in pulmonary effects).

3.23 Aim#3

To establish the relationship between liposome size and the release kinetics using

6-carboxyfluorescein (CF) as a marker: assess the efflux ofCF in the presence of

surfactant. Hvpothesis

If the interaction ofliposomes with the surfactant pool present in the airways is the

main mechanism of pulmonary clearance ofliposomes, then the drug encapsulated in large

vesicles will be retained longer than that encapsulated in small vesicles. Rationale

It is known that the efflux of drug across the bilayers is the rate limiting step for

the release of water soluble glucocorticoids [101]. In addition, it has been stated that the

stability of liposomes (regarding leakage) depends primarily on liposome size and type

[102]. Generally large multilamellar vesicles are more stable since only a portion of the

phospholipid is exposed to attack, whereas small vesicles are the least stable because of

the stress imposed by their curvature and the reduced number of lamellae in the

membranes, thus increasing solute permeability. Although the in vivo mechanisms) of

liposomes clearance may be completely different from the ones that govern the in vitro

stability, assessing drug leakage from different sizes of liposomes under the same in vitro

conditions may predict what happens in vivo.

CF has been widely used as a marker to determined the release kinetics from

liposomes [88, 103, 104]. Like TAP, CF is a water soluble drug and thus, we expect that

they partition in the same way in the liposome environment. A surfactant was included to

mimic somehow the conditions that liposomes may encounter when delivered into the


These experiments, together with the ex vivo release studies performed in aim

number 4, were intended to show that by changing the size of the liposomes and therefore

the release rate, it is possible to optimize pulmonary targeting. This goal was achieved by

assessing the release of 6-carboxyfluorescein from liposomes of different sizes in the

presence of surfactant and by monitoring receptor occupancy after intratracheal instillation

of TAP-liposomes of different sizes (see aim number 4).

3.2.4 Aim # 4

To determine the relationship between release rate, dose and pulmonary targeting:

monitor lung and liver receptor occupancy upon intratracheal administration of

glucocorticoid in solution and in liposomes with different release rates and different doses

of encapsulated material.

3,2.4.1 Hypothesis

Pulmonary selectivity can be optimized by modulating drug release rate and dose. Rationale

In attempts to increase localization of drug in the lung, drugs with higher clearance

and lower oral bioavailability have been developed. However, little is known about the

biopharmaceutical factors that affect pulmonary targeting. Pharmacokinetic-

pharmacodynamic simulations done by our group [1] revealed that there are at least 2

biopharmaceutical factors that can affect pulmonary targeting: dose and release rate of the

drug from the formulation (such as dissolution rate). Given a fixed dose, a value of release

rate (KA) that is too small can mean that the minimum effect concentration is never

reached either in the local organ (lung) or in the systemic organ (liver). When Krel is too

high, toxic side-effects may result and pulmonary targeting is lost. The same rationale is

applied for the dose. In this specific aim it was attempted to show experimentally that

there is an optimal dose and release rate for which maximum pulmonary targeting can be

achieved. For this purpose liposomes were used as a model dosage form due to their

inherent capacity to control release rate as previously shown in the stability studies.

In order to compare the effect of release rate on pulmonary targeting, 3 different

formulations were delivered intratrachealy to anesthetized male rats: triamcinolone

acetonide phosphate (TAP) in solution (immediate release preparation), TAP in 200 nm

liposomes (intermediate release preparation) and TAP in 800 nm liposomes (slow release

preparation). Glucocorticoid receptor occupancy in the local organ (lung) was compared

to glucocorticoid receptor occupancy in the systemic organ (liver). Pulmonary targeting

was calculated as the difference between the degree of lung receptor occupancy and the

degree of liver receptor occupancy. To determine the effect of dose similar experiments

were performed using escalating doses of TAP in 800 nm liposomes (TAP-lip 800 nm).

3.2.5 Aim # 5

Determine the pulmonary targeting of two currently available inhaled

glucocorticoids: triamcinolone acetonide phosphate and fluticasone propionate using an

ex-vivo animal model: monitor glucocorticoid receptor occupancy in local organ (lung)

and in different systemic organs (liver, spleen and kidney) upon intratracheal (IT)

administration of dry powders. Hypothesis

If pulmonary selectivity depends on release rate then glucocorticoids of different

dissolution rates will yield different pulmonary targeting Rationale

Computer simulations [1] have shown that pulmonary selectivity can be

modulated by optimizing drug release rate. In addition, the limited data available for

currently available inhaled glucocorticoids suggest differences in their dissolution rates

[105-108]. Based on these findings it is predicted that the pulmonary selectivity achieved

by these drugs should be different. In this particular aim, kidney and spleen (organs with

no metabolic activity) were included because of concerns on that the high intrinsic hepatic

clearance offluticasone propionate may affect liver receptor occupancy.


4.1 Characterization of Liposomes

4.1.1 Size Measurement

The size (volume-weighted) and size uniformity of liposomes were determined

using a submicron particle sizer (Nicomp, model 270). A drop of the liposome suspension

was placed into a disposable culture tube (Fisher Scientific) and diluted with PBS

(phosphate buffer saline) to obtain a photopulse rate of approximately 300 KHZ. The

sample was monitored for 20 minutes or until the residual was zero and the fit error was

smaller than two (the residual is a parameter which indicates the extent of a shift in the

baseline of the autocorrelation function which is needed to produce the best fit for the

Nicomp distribution analysis). Two different types of particle size distribution analyses

were obtained: Gaussian and Nicomp. If the Chi Squared was less than 3.0, Gaussian

analysis was displayed (Figure 4.1). If Chi Squared exceeded 3.0, the Nicomp distribution

was displayed (Figure 4.2). Generally, 200 nm liposomes were best described by a

Gaussian distribution, while 800 nm liposomes were best described by a Nicomp


4.1.2 Separation of Unencapsulated Material

Since the aqueous volume enclosed within the lipid membrane is usually only a

small proportion of the total volume (about 5-10%) and triamcinolone acetonide

phosphate is a water soluble compound, a step to remove unencapsulated material was

needed. A size exclusion chromatography method [60] was used with some modifications.

Figure 4.1 Volume weighted
TAP-lip 200 nm

a is u aom Sm I

Gaussian distribution analysis for DSPC:DSPG (9:1)

W 20 r tw n s se a
5Iu Ir) -, rIIt-> _

Figure 4.2 Volume weighted Nicomp distribution analysis for DSPC:DSPG (9:1)
TAP-lip 800 nm. Column preparation/processing of samples

Ten grams of Sephadex G-75 were allowed to swell in 120 ml of PBS for at least

10 hr at room temperature and stored at 4 C. A PD-10 column (Pharmacia, Sweden) was

plugged with a Whatman GF/B filter pad and the column was filled entirely with the

hydrated gel using a Pasteur pipette with the tip removed. A second Whatman GF/B filter

pad was attached on top of the hydrated gel, and 0.2 ml of liposome suspension

(undiluted) was applied dropwise to the top of the gel bed. The first 3 ml of PBS (mobile

phase) were added and the eluate discarded. After an additional 3 ml of PBS, the 4-6 ml

eluate was collected. Liposomes were present in the 4-6 ml eluate. Preliminary studies

using dyed liposomes prepared with the phospholipid rhodamine showed that 200 nm and

800 nm liposomes elute in the 4-6 ml fraction. Liposomes were stored at 4 oC under


4.1.3 Lipid Determination

The lipid contents of the liposomal preparations were determined by a slight

modification of a colorimetric method [109]. Processing of samples

Duplicate samples of 0.010 ml of the liposome suspensions were evaporated using

a Jouan concentrator for 1 hr followed by the addition of 3 ml of a mixture of 0.1M ferric

chloride hexahydrate (FeCI3.6H20) and 0.4M ammonium thiocyanate (NH4SCN) in

deionized water. The tubes were sonicated for 2 min, vortexed 15 seconds four times and

incubated at room temperature for 1 hr. Subsequently, 4 ml of chloroform were added, the

tubes were closed and vortexed three times for 20 seconds. The samples were kept at

room temperature for 2 hr or until a clear separation of the layers occurred. The aqueous

(upper) layer was removed using a Pasteur pipette and the optical density absorbancee) of

the chloroform layer was read at 488 nm using a UV/VIS spectrophotometer (Perkin

Elmer, model W-3A). Calibration curve

For the calibration curve, 0.01-0.1 ml aliquots of a mixture of 90% PC and 10%

PG (1 mg/ml total lipid in chloroform) were assayed in duplicate as described in section

4.1.3. Inter- and intra-dav variability

The intra-day and inter-day variability were determined for three control samples

(20, 60 and 100 gg/ml) prepared as shown in 4.3.1. Replicate (n=5) analysis of control

samples was performed on the same day and over a period of 7 days. Precision was

expressed as the relative standard deviation obtained for the inter-day and intra-day

analysis for each concentration. Accuracy was determined by comparing the resulting

mean concentration with the theoretical one at each concentration. The correlation

coefficients obtained for all calibration curves were always higher than 0.995. The

coefficient of variation obtained for the slope was smaller than 12%. The results of the

inter- and intra-day variability are shown in Table 4.1. Based on the results of the

validation, the method was considered adequate for the purposes of this study.

Table 4.1 Intra-day and inter-day variability for DSPC:DSPG determination

Theoretical Measured concentration Inter-day Intra-day
concentration (pg/ml) mean (SD) variability (%) variability (%)
20 21.2(2.7) 13.64 12.9
60 52.9(4.7) 4.96 8.8
100 101.7(4.5) 1.55 4.9

4.1.4 Analysis of triamcinolone acetonide phosphate by HPLC

The applied reversed phase HPLC method for the quantification of encapsulated

TAP has been previously described [110] and was used here with some modifications. Instrumentation

The analysis of TAP was performed using a Perkin Elmer series 3B pump with a

flow rate of 1.0 ml/min and a Nucleosil Cis (150 x 4.6 mm) column connected to a Perkin

Elmer ISS 200 autoinjector. A water Cis guard pack column was used to protect the

analytical column. 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 35:65 v/v acetonitrile: sodium phosphate buffer 0.05 M (pH 2). Treatment of samples

Liposomal TAP concentration was determined after gel filtration. Aliquots of 10 pl

of the liposome dispersion were disrupted by adding 480 pl of a mixture of 80:20

methanol:PBS and 10 pl of methylprednisolone (100 pg/ml ethanol solution; internal

standard). The mixture was vortexed for 1 min and 70 pl of the solution injected onto the

column. Quantitative analysis was achieved by comparison of peak heights with an internal

standard using the fitted calibration curve.

Each determination was taken as the mean of two replicate injections. The

calibration curve was prepared over the range of 75-800 pg/ml with duplicate samples. A

typical triamcinolone acetonide phosphate and internal standard (methylprednisolone)

chromatogram is shown in Figure 4.3.

Figure 4.3 Representative chromatogram of TAP (RT 3.75 min) and internal standard
(RT 6.22 min) inMeOH:PBS. Intra-day and inter-day variability

The intra-day and inter-day variability were determined for three control samples

(75, 30 and 600 g.g/ml) prepared as shown in The limit of quantification was

determined as the lowest TAP concentration that produced an inter-day variability smaller

than 20 %. The lower limit of quantification for this HPLC method was 30 pg/ml. The

coefficient of variation obtained for the slope was 7.5% which reflects the precision of the

method. The correlation coefficient values were higher than 0.995. The results of the

inter- and intra-day variability are shown in Table 4.2.

Table 4.2. Intra-day and inter-day variability for TAP reversed phase HPLC

Theoretical Measured Inter-day Intra-day
concentration concentration (tg/ml) variability (%) variability (%)
(jg/ml) mean (SD)
75 77.9 (6.7) 8.6 6.6
300 307.2 (10.9) 3.3 6.7
600 599.6 (12.7) 2.1 5.1

4.1.5 In vitro Stability at 37C

The in vitro stability of freshly prepared TAP-lip was tested at 370C using PBS, rat

lung lavage fluid (see 4.1.6), and tissue culture medium RPMI-1640m or tissue culture

medium with 10% fetal bovine serum. After gel filtration, an aliquot of the liposome

preparation was diluted 5-fold (75-fold from the initial preparation) with the respective

fluids. This resulted in a TAP concentration of 100 Ig/ml, low enough to simulate sink

conditions and high enough to be quantified by the HPLC system employed. After

incubation at 370C, aliquots of 200 pl (in duplicate) were removed at 10, 30 min, 1, 3, 6,

18, and 24 h and the samples were then passed through Sephadex G-50 dry minicolumns

of 1 ml bed size [60] as is described in Column preparation/processing of samples

Ten grams of Sephadex G-50 were allowed to swell in 120 ml of PBS in a glass

screw capped bottle for at least 4 hr at room temperature and stored at 4 C. The plungers

from 1-ml disposable plastic syringes were removed and plugged with a Whatman GF/B

filter pad. The syringes were filled to the top with the hydrated gel using a Pasteur pipette

with the tip removed, and centrifuged in a Dynac 11 centrifuge (Becton, Dickinson and

Company) at 2000 rpm for 5 min to remove excess of solution. Duplicated aliquots of 0.2

ml of the liposome suspension were applied dropwise to the top of the gel bed and spun

down at 2000 rpm for 2 min. Aliquots of 10 il eluates were analyzed by HPLC (see


4.1.6 Method for Recovering Lung Lavage Fluids

Rats were anesthetized and lung lavage fluid was obtained after tracheal

cannulation with a 21 gauge polyethylene catheter. Approximately 5 ml of sterile isotonic

saline were slowly injected to fill the lungs. The fluid was withdrawn by gentle aspiration,

and spun at 2000 rpm for ten minutes at 40C. The cell free supernatant was aspirated and

used on the same day to assess the in vitro stability ofliposomes.

4.2 Receptor Binding Assay

Receptor binding was performed as previously described [56]. Immediately after

decapitation, the lung, without trachea, and a lobe of the liver were rejected and placed on ice.

The weighed tissue was added to 10 ml volumes of ice-cooled incubation buffer (10 mM

Tris/HCl, 10 mM sodium molybdate, 2 mM 1,4-dithiothreitol) and 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 40C 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 pl) were transferred into microcentrifuge tubes that contained 25 pl of 3H-

triamcinolone acetonide in incubation buffer (final concentration: 0.25, 0.5, 2, 4, 10 and, 30

nM) and 25 pl of incubation buffer or 25 pl ofunlabeled TA (3 mM) to determine total and

non-specific binding respectively. Aliquots of 150 pl of the resultant cytosol preparations were

transferred into microcentrifuge tubes which contained 25 pl of 'H-triamcinolone acetonide in

incubation buffer (final concentration: 0.25, 0.5, 2, 4, 10 and 30 nM) and 25 p1 of incubation

buffer to determine the amount of total radioactivity. In some cases only high concentration (30

nM) of labeled material was used (see chapters 7 and 8). After a 16-24 hour incubation period

at 4C, the unbound glucocorticoid was removed by addition of a 2% suspension of activated

charcoal in buffer (200 pl). 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 pl of supernatant was determined using a liquid scintillation counter (Beckman model LS

5000 TD, Palo Alto, CA). All determinations were performed in triplicate.

Estimates for total bound 31'-TA (TAT), nonspecifically bound 'H-TA (TAm) and free

'H-TA (TAp) were used to determine the number of available binding sites (B.) and the

equilibrium binding constant (K&). The data were fitted to equation 4.1 using the non-linear

curve-fitting program MINSQ (Micromath, Salt Lake City, UT).

TAT = B.* TAF / (TAF +Kd) TANs (eq. 4.1)

In some cases, B- could not be determined by the above method, as the high in

vivo receptor occupancy did not allow a reliable estimate of B,, and Kd. In this case, B,

was estimated by a second method and expressed as the difference of TAT values and

TANs observed for incubation with 30 nM 3H-TA (highest tracer concentration). This was

justifiable as the comparison of calculated B,, values from both methods resulted in

similar values for B., (R2=0.985, n=45; see Figure 4.3).



0 1 2
Bmax experimental (nM)

Figure 4.3 Correlation between B,. calculated values (using equation 4.1), and
B,. experimental (obtained from the difference of TAT and TANs
observed values). R2=0.985, n=45.

4.3 Reverse HPLC and Radioimmunoassay (HPLC/RIA) Determination of Triamcinolone

4.3.1 Extraction Procedure

Plasma and lung samples were thawed at room temperature for one hour and

extracted twice with 2 ml of ethyl acetate as described in detail somewhere else [111]. The

organic solvent was evaporated under vacuum in a speed vacuum concentrator (Jouane).

The residue was reconstituted in 150 Wl of 50:50 mobile phase (acetonitrile:water) and the

resulting suspension centrifuged for five minutes. The superatant was used directly for

the HPLC separation.

4.3.2 Reverse Phase HPLC Procedure

The analysis of TA was performed using a Costametric mG pump (LDC/Milton

Roy) to deliver solvent at 0.9 ml/min to a Sperisorb ODSII (150 x 4.6 mm) C18 column

connected to a Perkin Elmer ISS 200 autoinjector and to a programmable Gilson model

203 fraction collector. The spectromonitor D (LDC) variable UV detector was attached to

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

mobile phase consisted of a mixture of acetonitrile: water (50:50 v/v). Retention times of

hydrocortisone and triamcinolone acetonide were verified before every experiment. After

extensive washing, plasma or lung extracts (200 il) were injected and the TA fractions

collected. Organic solvent containing TA (HPLC fractions collected) was removed under

vacuum. The residual material was extracted twice with ethyl acetate (1 ml), centrifuged

and the solvent evaporated until dryness. The resulting residues were reconstituted in 300

il of incubation buffer containing 0.001% Triton X-100 and centrifuged. The portions of

the supernatant were used for RIA procedure.

4.3.3 RIA Procedure

The dried residue was dissolved in 300 pi of 0.01 M PBS containing Triton X-100.

Lung or plasma samples (50 ul), 200 pl of assay buffer, 50 pl of antiserum working

solution (1:1500 antibody), and 50 pl of tracer solution (3H-TA, 3000 CPM) were placed

in microcentrifuge tubes and incubated for 24 h at 40 C. After incubation, 100 il of 1 %

activated charcoal was added to the samples. The samples were incubated for 5 min and

centrifuged at 10,000 rpm. Aliquots of the supernatant (600 pI) were mixed with 4 ml of

scintillation fluid and counted in a scintillation counter.

Calibration curves of the bound radioactivity (B) versus competitor concentration

(C) were fitter to the equation shown below using a non-linear curve fitting procedure

(Scientist, Micromath, Salt Lake City, Utah).

B= T T*CN/(CN + ICsoN) + NS

Estimates ifNS (non specific binding), N (Hill slope factor), IC5o (concentration to

decrease specific tracer binding by 50%), and total specific binding (T) were used to

transform CPM of the unknowns into the corresponding concentrations.

The limit of quantification was 0.15 ng/ml (accuracy 85-115% and precision 15 %)

and the limit of detection was 0.1 ng/ml. The inter-batch variability for three batches of

quality controls samples was in average 13, 11, and 11 % for 0.35, 0.75, and 1.5 ng/ml.

Accuracy was between 99 and 118 %. Intra-day variability for quality control samples was

5.2, 4.7, and 4.4 % for 0.35, 0.75, and 1.5 ng/ml, respectively.


5.1 Introduction

Glucocorticoids are beneficial in treating various pulmonary diseases, including

asthma, sarcoidosis, and other conditions associated with alveolitis. Although systemic

glucocorticoid therapy is effective in such conditions, prolonged administration carries the

risk of toxicity and side effects [112]. In attempts at reducing systemic side effects, several

clinically efficacious glucocorticoids, including triamcinolone acetonide (TA) are

employed for delivery as aerosols.

We have been interested in optimizing pulmonary targeting of glucocorticoids for

inhalation therapy. In a recent study, we showed that lung specificity is achieved when

glucocorticoid suspensions are administered intratracheally. In contrast, lung targeting is

not observed when a glucocorticoid solution is administered intratracheally, presumably

because of the fast absorption of the lipophilic steroid [56]. This suggests that pulmonary

targeting depends on slow release from the delivery form which results in a prolonged

pulmonary residence time.

The use of liposomes has been suggested to provide sustained pulmonary release

for various drugs including glucocorticoids such as beclomethasone dipropionate, and

dexamethasone [42, 98, 99, 113]. However, we have found that while liposomes have a

high loading capacity for lipophilic glucocorticoids such as TA under equilibrium

conditions, TA is rapidly released under non-equilibrium conditions from the liposome

matrix upon dilution or administration [91]. In retrospect, this is predictable, given the

observations of Schanker & co-workers [49] that lipophilic glucocorticoids cross

membranes practically unhindered. Our findings question the benefits of achieving

sustained pulmonary release from such preparations.

As liposomes seemingly provide no barrier function for such glucocorticoids we

hypothesized that by encapsulating water-soluble derivatives of TA, rather than the

lipophilic parent compounds we could overcome this problem. If the liposomal membrane

serves as a rate-limiting barrier for the release of a water-soluble TA derivative, then slow

drug release and consequent improvement in pulmonary targeting might be possible.

We therefore selected the water-soluble salt triamcinolone acetonide phosphate (a

prodrug of TA) for developing a formulation which captured the negatively charged TAP

within liposomes. This formulation was delivered either by intratracheal or intravenous

injection to rats and compared with intratracheally administered TAP-sol. Since

pharmacodynamic effects of glucocorticoids are receptor mediated, tracking receptor

occupancy in lung and liver using the above mentioned animal model allowed for indirect

assessment of pulmonary and systemic effects.

5.2 Materials and Methods

5.2.1 Materials

Analytical grade chemicals were obtained from Sigma Chemical Co. (St. Louis,

MO.); (1,2,4-3H) triamcinolone acetonide (45 Ci/mmol) was purchased from New-

England Nuclear (Boston, MA). Lipids were obtained from Avanti Polar Lipids

(Alabaster, AL.)

5.2.2 Drug Solutions

Triamcinolone acetonide phosphate (TAP) (provided as a gift from Dr. K.

Reininger, Bristol Myers Squibb, Regensburg, Germany) was dissolved in buffered saline

to a concentration of 15 mg/ml. Before dosing, the respective stock solutions were diluted

with buffered isotonic saline to 0.2 mg/ml of TAP.

5.2.3 Animals

All animal procedures were approved by the Animal Care Committee of the

University of Florida, an AAALAC approved facility. Specific-pathogen-free, non-

adrenalectomized male F-344 rats, weighing approximately 250 g were housed in a 12 hr

light/dark cycle, in a constant temperature environment. Animals were allowed free access

to water and rat chow, but were food-fasted overnight prior to each experiment.

5.2.4 Liposome preparation

Liposomes composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (89.4 mg)

and 1,2-distearoyl-sn-glycero-3-{phospho-rac-(l-glycerol)} (10.6 mg) in a 9:1 molar ratio

respectively, were dissolved in chloroform. The chloroform was removed by rotary

evaporation at 63C to obtain a dry lipid film. After adding one ml of 100 mg/ml TAP

solution in isotonic buffer saline (PBS, CellgroTM, pH 7.5) the lipids were dispersed by

shaking at 63C for 2hr. This crude liposome formulation underwent 10 cycles of freezing

(in dry ice and methanol) and thawing at 630C and was then extruded 30 times using a

LiposofastR extruder (Avestin Co. Ottawa, Ontario, Canada) through 0.2 mm

polycarbonate filters. Liposomes were sized using a submicron particle sizer (Nicomp

model 270) to ensure size uniformity of the formulations, and stored under nitrogen at


On the day of the experiment, 200 pl of liposomal dispersion were passed through

10 ml Sephadex G-75 in a PD-10 column (Pharmacia, Sweden) with a PBS mobile phase.

Liposomes were present in the first 4 to 6 ml of eluate (void volume). TAP concentrations

were monitored by HPLC (section 5.2.5) For animal experiments, liposomes were diluted

to a final concentration of TAP of 0.2 mg/ml. Isotonicity of liposomes was confirmed

prior to use with a vapor pressure osmometer M 5500 (Wescor, Inc. Logan, UT). Lipid

content was determined as previously described [109].

5.2.5 HPLC Method

Liposomal TAP concentration after gel filtration was determined in a 10 gl aliquot

of the liposome dispersion by adding 480 pl of a mixture of 80:20 methanol:PBS and 10

pl of methylprednisolone (100 gg/ml ethanol solution) as internal standard. The mixture

was vortexed for 1 min and 70 pl of the solution was injected onto a Nucleosil CIs (150 x

4.6 mm) column, using a mixture of acetonitrile:0.05 M sodium phosphate buffer (pH 2;

35:65 v/v), as mobile phase at a flow rate of 1 ml/min. UV detection was performed at

254 nm. The lower limit of detection was 30 pg/ml.

5.2.6 Method for recovering lung lavage fluids.

Rats were anesthetized and lung lavage fluid was obtained after tracheal cannulation with

a 21 gauge polyethylene catheter. Approximately 5 ml of sterile isotonic saline were

slowly injected to fill the lungs. The fluid was withdrawn by gentle aspiration, and spun at

2000 rpm for ten minutes at 40C. The cell free supernatant was aspirated and used on the

same day to assess the in vitro stability of liposomes.

5.2.7 In vitro Stability

The in vitro stability of freshly prepared TAP-lip was tested at 370C using PBS, rat

lung lavage fluid, tissue culture medium RPMI-1640m or tissue culture medium with 10%

fetal bovine serum. After gel filtration, an aliquot of the liposome preparation was diluted

5-fold (75-fold from the initial preparation) with the respective fluids (TAP final

concentration of 100 ug/ml, a concentration that simulates sink conditions and is high

enough to be quantified by the HPLC system employed).

After incubation at 37C,aliquots of 200 il (in duplicate) were removed at 10, 30

min, 1, 3, 6, 18, and 24 h and the samples were then passed through Sephadex G-50 dry

minicolumns of I ml bed size [60]. Aliquots of 10 pl eluates were analyzed by HPLC.

5.2.8 Administration of Drugs

The animals were anesthetized via intraperitoneal injection and the corresponding

TAP-lip or TAP-sol (160 tg/kg of TAP) administered [56]. Animals (one animal per time

point) were decapitated at 1, 2, 5, 6, 12, or 18 hours after IT or IV administration of Tap-

lip. The lungs and livers were immediately processed for receptor binding studies. A total

of 3 (12 and 18 hr) to 6 (0-6 hr) independent experiments were performed for a given time

point after IT administration of TAP-lip. For the IT administration of TAP-sol and the IV

administration of TAP-lip 4 and 2 animals were used per time point, respectively.

Experiments were performed on different days for every form (IV or IT) of

administration and for each type of preparation, e.g. TAP-sol or TAP-lip. A sham animal

(receiving either IV or IT buffered saline) was always included on the day of experiment.

Each animal represented a single time point with paired data for both liver and lung.

5.2.9 Receptor Binding Assays

Receptor binding assays were performed as previously described [56] with

modifications. Immediately after decapitation, the lung, without trachea, and a lobe of the

liver were rejected and placed on ice.

The weighed tissue was added to 10 ml volumes of ice-cooled incubation buffer

(10mM Tris/HCL, 10 mM sodium molybdate, 2mM 1,4-dithiothreitol) and homogenized

in a Virtis 45 homogenizer at 40% of full speed, for three periods of 5 seconds each with a

30 sec 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 40C 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


Aliquots of the supernatant (150l) were transferred into microcentrifuge tubes

that contained 25 ml of 3H-triamcinolone acetonide in incubation buffer (final

concentration: 0.25, 0.5, 2, 4, 10 and, 30 nM) and 25 ml of incubation buffer or 25 ml of

unlabeled TA (24mM) to determine total and non-specific binding respectively. Aliquots

of 150 ml of the resultant cytosol preparations were transferred into microcentrifuge tubes

which contained 25 ml of 3H-triamcinolone acetonide in incubation buffer (final

concentration: 0.25, 0.5, 2, 4, 10 and 30 nM) and 25 ml of incubation buffer to determine

the amount of total radioactivity.

After a 16-24 hour incubation period at 40C, the unbound glucocorticoid was

removed by addition of a 2% suspension of activated charcoal in buffer (200 ml). 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 ml of supernatant

was determined using a liquid scintillation counter (Beckman model LS 5000 TD, Palo

Alto, CA). All determinations were performed in triplicate.

Estimates for total bound 3H-TA (TAT), nonspecificaly bound 3H-TA (TANs) and

free 3H-TA (TAF) were used to determine the number of available binding sites (Bn.) and

the equilibrium binding constant (Kd). The data were fitted to equation 1 using the non-

linear curve-fitting program MINSQ (Micromath, Salt Lake City, UT).

TAT = B,* TA / (TAF + Kd) + TANs (eq. 5.1)

In instances where the high in vivo receptor occupancy did not allow for reliable

B. determination by the above method, B, was estimated as the difference of TAr

values and TANs observed for incubation with 30 nM 3H-TA (highest tracer

concentration). This was justifiable as the comparison of calculated B-, values from both

methods resulted in similar values for B, (R2=0.985, n=45).

Pulmonary and hepatic B,- values were converted into % of control (average

values for sham rats). Kd values were necessary only to calculate Bs, and are not given in

the result section.

For a given form of administration, differences between the pulmonary and hepatic

receptor occupancies were tested by paired student T test. Data analyzed represented the

pool of paired (hepatic and pulmonary) receptor occupancies for individual single time

points (not AUC's) of all animals included in a given experimental sub-set. Statistical

significance was assumed for p<0.05.

For assessing differential receptor occupancy between lung and liver, the

cumulative change from baseline (AUC) was calculated for the 6 and 18 hour investigation

period by the trapezoidal rule from percent occupied receptor (Ex)-time profiles. The

pulmonary targeting factor (T) was defined as

T=AUCL-,/AUCLiu (eq. 5.2)

A targeting factor of greater than 1 would indicate preferential lung targeting. The area

under the first moment curve (AUMCoo) was calculated by the trapezoidal rule from

Ex*tx versus tx-pairs. Effects after the last measurement points were extrapolated for

TAP-sol and data of reference 2 assuming a linear decline of the effect over time at late

time points [100]. These estimates were also used to derive AUCoo The mean pulmonary

effect times (MET) were calculated consequently from AUCoo and AUMCoo (MET=


5.3 Results

5.3.1 Characterization of Lip TAP

The volume weighted mean particle diameter of the liposome obtained using a

Gaussian distribution analysis was 207 16 nm (see Figure 4.1, chapter 4). A 40%

average loss of lipid resulted after extrusion and gel filtration of liposomes. The

encapsulation efficiency varied from 7 to 8.5% of the total amount of TAP, corresponding

to a molar drug/lipid ratio of 1:7.

5.3.2 In vitro Stability in Biological Fluids

Results of the liposome stability studies are shown in Figure 5.1. The TAP-lip

preparations retained greater than 75-80% of the entrapped drug through 24 hours when

incubated in various fluids.




0 6 12 18 24
Time (hr)

Figure 5.1. In vitro stability of DSPC:DSP TAP liposomes at 370C under sink
conditions in PBS (*), culture medium (D), lung lavage fluid (o), and
90% culture medium plus 10% fetal bovine serum (A). The percentage of
TAP remaining encapsulated in liposomes over time of incubation is

5.3.3 Receptor Binding Studies

To exclude potential assay artifacts (e.g. ex-vivo release of glucocorticoids from

intact liposomes present in rejected tissues), experiments were performed in which cytosol

from untreated animals was spiked with TAP-lip (TAP 200 pg/ml) and consequently

processed as described in Materials and Methods. There was a negligible difference

between the % of free receptors of cytosol spiked with liposomes (85% 10%, n=4) and

control cytosol not spiked with TAP-lip (B.x = 100%),

There were statistically significant differences in glucocorticoid receptor

occupancy between lung and liver after intratracheal administration of TAP-lip for the 18

hr period. Receptor occupancy was more pronounced for lung than for liver (Fig. 5.2b),

reflected also in an AUC based lung targeting factor of 1.6 (Table 5.1). In contrast, there

was no difference in these parameters when equivalent doses of TAP were administered IT

as a solution, with the lung and liver curves being superimposable (Fig. 5.2a, Table 5.1).

120 120

80- so-

40- 40-

0 0
I -- I2 0 I-- -- I-- -- I
0 6 12 18 0 6 12 18

120 -

0 6 12 18
Time (hr)

Figure 5.2. Lung (0) and liver (*) glucocorticoid receptor occupancy profiles after
administration of 100 Ig/kg of TAP: A) intratracheal instillation of TAP-
sol; B) intratracheal instillation of TAP-lip; C) intravenously administered
TAP-lip. Error bars represent mean SD

Table 5.1 Cumulative receptor occupancy (AUC), lung targeting factor and mean
pulmonary effect times (MET) after administration of TAP liposomes
(TAP-lip) and TAP solution (TAP-sol).

TAP-lip TAP-sol TAP-lip
it adm it adm iv adm
AUC (%*hr)

0-6hr -18hr 0-6hr 0-6r 0-18hr

LUNG 450 770 350 490 370

LIVER 280 620 340 500 600

Targeting factor 1.6 1.2 1.0 1.0 0.6

Mean Pulmonary Effect 5.7 3.0 3.8
Time (hr)

After IV administration of TAP-lip, no significant differences were observed in

receptor occupancy profiles of lung and liver (Fig. 5.2c, Table 1) for at least the first 6


The lung receptor occupancy profiles between the various TAP drug preparations

and methods of administration are compared in Figure 5.3 together with previously

reported data on triamcinolone acetonide solutions (TA-sol) [100]. As is shown in this

Figure, lung receptor occupancy was significantly sustained after IT administration of

TAP-lip relative to IT and IV of TAP-sol and TAP-lip respectively, and to IT or IV

administered TA-sol. The distinct sustained receptor occupancy characteristics of TAP-

Lip can be seen also from the estimates of the mean effect times (Table 5.1). These MET

values were 1.5-1.9 times longer than that of TAP-lip administered IV and TAP-sol

administered IT (Table 5.1), respectively.

0 6 12 18
Time (hr)

Figure 5.3. Lung glucocorticoid receptor occupancy profiles after administration of
160 pg/kg of TAP: Intratracheal TAP-sol (U), intratracheal TAP-lip (A),
intravenous TAP-lip (*). Comparison is made to intratracheal TA-sol (A)
and intravenous TA-sol (o) profiles from ref. [56].

5.4 Discussion

TA is a clinically established, potent and inhalable synthetic glucocorticoid which

binds to the glucocorticoid receptors. In contrast to TA, TAP (a prodrug of TA) is highly

water-soluble (sol in water > than 50 mg/ml), and although the encapsulation efficiency

was substantially less than that of TA [91], we found TAP-lip (unlike TA-lip) to be more

stable, even when incubated in buffers or biological fluids at 370C (Figure 5.1).

In the systemic circulation, TAP is cleaved rapidly to the pharmacologically active

triamcinolone acetonide [97]. Our results in rats are in agreement with prior observations,

as the receptor occupancy profiles after administration of the TAP-sol are very similar to

what we have previously shown for TA when administered in the same fashion (Figure

5.3), suggesting a fast activation of TAP. In addition, the distinct pulmonary targeting of

liposomal TAP suggests that this activation has to occur in the lung.

Incorporation of drugs into liposomes changes their pharmacokinetic behavior

after pulmonary instillation [99], resulting in longer mean residence time in the lung and

the potential for sustained pulmonary effects. Based on our experiments, intratracheal

administration of TAP-lip resulted in a pronounced and sustained occupancy of pulmonary

receptors when compared to pulmonary delivery of TAP-sol or intravenously administered

TAP-lip (Figure 5.3, Table 5.1). This suggests that the sustained receptor occupancy is

linked to the pulmonary administration of the liposomal preparation, while the use of

TAP-sol or the intravenous administration of TAP-lip do not result in the sustained

occupation of pulmonary receptors (Figure 5.3). These results are consistent with the in

vitro stability of the TAP liposomes in biological fluids (Figure 5.1) and prolonged

pulmonary release of TAP from the liposomes.

In pharmacokinetic-pharmacodynamic (PK-PD) simulations we have shown that

the MET depends on the release rate of the drug delivery system in such a way that the

slower the release, the larger the MET (unpublished observations). On the basis of this

relationship, the longer MET value found for TAP-lip argues also for a prolonged release

of this formulation.

Besides providing a sustained receptor occupancy, the main goal of this study was

to test whether liposomes can improve pulmonary targeting after topical delivery. Previous

studies [56] using TA suspensions and solutions suggested that pulmonary targeting, as

indicated by a ratio of the cumulative pulmonary and hepatic receptor occupancy, is

achieved only if the drug is absorbed slowly from the lung; e.g. due to a slow dissolution

rate of the instilled particles. This study shows that TAP-lip, with presumably prolonged

release characteristics, but not TAP-sol confers pulmonary targeting. The resulting

targeting factor of 1.6 was significantly larger than estimates for TAP-sol (1.0) or IV


Although achieving a pronounced targeting for tissues with high organ blood flow,

such as the airways is theoretically difficult [114], our results show that liposomes increase

the therapeutic availability of drugs in target tissue (presumably due to prolonged release)

therefore increasing tissue targeting.

Our study shows that triamcinolone acetonide phosphate can be incorporated in a

stable liposomal formulation which appears suitable for pulmonary delivery. With

intratracheal delivery, TAP-lip provides sustained receptor occupancy properties and

improved pulmonary targeting superior to TAP-sol alone. Whether this formulation will


ultimately result in superior clinical activity in pathologic states in humans cannot be

ascertained, but deserves further study.


6.1 Introduction

Because asthma is considered an inflammatory disease [28], glucocorticoids are

the drugs of choice for the treatment of this disease. Generally, glucocorticoids are

administered locally as aerosols in an attempt to reduce systemic side effects. However,

due to its rapid absorption into the systemic circulation, frequent administration of these

drugs is needed leading to potential side effects [115, 116].

This prompted the development of slow release preparations (such as liposomes)

for pulmonary delivery (for recent review see [42] to control the absorption of drugs from

the respiratory airways. Liposomes have been suggested to provide a prolonged release

reservoir and facilitate the intracellular targeting of drugs [42]. To date, only few reports

have addressed the use of liposome-encapsulated glucocorticoids for pulmonary delivery

[23, 99]. Furthermore, little is known about the pharmacokinetics and pharmacodynamics

ofliposomal encapsulated glucocorticoids for pulmonary delivery.

We have previously shown (chapter 5) that the intratracheal (IT) administration of

TAP in liposomes resulted in a more pronounced lung receptor occupancy compared to

TAP in solution or TAP administered intravenously (IV). In the present study, we

correlate the pharmacokinetics of TA after IT administration of TAP liposomes with its

pulmonary and systemic effects (receptor occupancy) and compare these with those of

TAP in solution administered IT and after IV administration of TAP liposomes.

6.2 Methods

The methods for liposome preparation and characterization, drug administration

and receptor binding were reported previously in chapter 5.

Briefly, triamcinolone acetonide liposomes composed of phosphatidylcholine,

phosphatidylglycerol and triamcinolone acetonide phosphate (TAP) were prepared by

dispersion and extruded through 200 nm polycarbonate filters. Encapsulation efficiency

and in vitro stability at 37 C were assessed by size exclusion chromatography (refer to

section 3.1.2 and 3.1.5). TAP liposomes (TAP-lip) or TAP in solution (TAP-sol) were

delivered to male rats either by IT instillation or IV administration (chapter 5).

Glucocorticoid receptor occupancy was monitored over time in the lung and liver using an

ex vivo receptor binding assay as a pharmacodynamic measure of glucocorticoid action

(section 3.2).

6.2.1 Ouantification ofTriamcinolone Acetonide (TA) in Plasma and Lung Samples

Following drug administration (100 gg/kg of TAP in either liposomes or solution),

animals were decapitated at 1, 2, 5, 6, 12, or 18 hours (chapter 5). Blood samples were

collected in glass tubes containing heparin and centrifuged at 2,000 rpm for 20 minutes.

The plasma was separated and stored at -20 C until analyzed. Lungs were rejected,

homogenized as described previously (chapter 5) and samples were frozen (- 20 "C) until


The plasma and lung concentrations of triamcinolone acetonide were determined

by radioimmunoassay after reversed-phase HPLC separation from metabolites and

endogenous glucocorticoids as described in section 3.3. Briefly, plasma and lung samples

(1 ml) were thawed for 1 h at room temperature and extracted twice with 2 ml of ethyl

acetate. The organic phase was evaporated under vacuum, reconstituted in 0.15 ml of

mobile phase and centrifuged for 5 minutes at 10,000 rpm. Aliquots (200 pl) of the

supernatant were injected onto a Spherisorb ODS2 (150x4.6 mm, 5pm) column, using

acetonitrile/water (50:50) as a mobile phase at a flow rate of 0.9 ml/min. The HPLC

fractions containing TA were collected, evaporated, and extracted twice with ethyl

acetate. The organic solvent was evaporated under vacuum, reconstituted in incubation

buffer and analyzed by radioimmunoassay. The limit of quantification was 0.15 ng/ml with

an average coefficient of variation of 11% in a concentration range of 0.35 to 1.5 ng/ml.

6.3 Data Analysis

6.3.1 Pharmacokinetic Analysis

Plasma and lung TA average concentrations over time were subjected to standard

compartmental and non-compartmental pharmacokinetic analysis. Compartmental analysis Lung concentrations

Average TA lung concentrations (CLTA) versus-time profiles upon IV and IT

administration of TAP-lip were fitted to a two-compartment body model using the least-

squares nonlinear regression method (SCIENTIST, Micromath, Salt Lake City, UT).

Average concentration of TA were fitted to the following equation:

CTAL=Ae't+Be (eq.6.1)

where CTAL is the total TA concentration, a, and P are first order rate constants, while A

and B represent hybrid constants. In the case of TAP-sol administered IT the equation

used to fit the lung data was:

CTAL = Ae (eq. 6.2)

where k. is the first order elimination constant and A is the concentration at time zero.

The area under the lung concentration-time curve (AUCot) after intravenous and

intratracheal administration was calculated by the trapezoidal rule. Interpolation to infinity

was done by dividing the last CTAL measured by the first-order rate constant of the

terminal phase. The total area under the curve was estimated by the summation of these

two components.

AUCo_ = (AUCo-t) + (AUCt.) (eq. 6.3)

The elimination half-life was calculated from 0.693/P where p is the slope of the

terminal phase of the log of the concentration-time profile.

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