Professional Documents
Culture Documents
POWDERS
By
SANDRA SUAREZ
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1997
DEDICATED TO MY PARENTS ELENA URIBE TREJO AND JOAQUIN SUAREZ VILLAGRAN AND MY SISTERS CARMEN, ROSALBA RAQUEL, LOURDES, ELENA AND MY BROTHERS JOAQUIN, ARTURO, EDUARDO, ROBERTO AND ROGELIO.
ACKNOWLEDGMENTS
I
extend
my
Hochhaus
for his
guidance,
patience and
continuous
presented
in this dissertation.
would
my
supervisory
committee, Dr. Hartmut Derendorf, Dr. Jeffrey Hughes, and Dr. Charles E.
Wood,
for
their inestimable
my
doctoral research.
to express
may
all
his invaluable
would
like to
thank
all
Patricia
Kahn, for
thank
my
sister Elena,
my
my
friends Judith,
all
their help
Finally,
thank
my
parents and
sisters for
me
in
many ways.
in
TABLE OF CONTENTS
page
ACKNOWLEDGMENTS
iii
ABSTRACT
viii
CHAPTERS
INTRODUCTION
2.
BACKGROUND
Definition
Asthma
Pathogenesis
3 3
4 7
8
Therapy
Glucocorticoids
Molecular Mechanisms
Target Genes in Asthma
Cellular Effects
9
10
11
Other Effects
12
13 13
15 17 17
Deposition
20 20
21
Drug
Dissolution or Release
Pulmonary Targeting Pulmonary Selectivity of Inhaled Glucocorticoids Effect of Clearance, Volume of Distribution and Oral
Bioavailability
22 22
23
25 25
Dose
Improvement of Airway
Liposomes Liposome Structure and Chemical Composition
Selectivity
26 28 29 30
31 33
Classification
Methods of Preparation
Chemical
Stability
Oxidation of Phospholipids
Hydrolysis of Phospholipids
Physical Stability
Biological Stability
33
34 37
41 41 41
Pharmacokinetics of Liposomes
Disposition of Liposomes Following Intravenous Administration
46
50
3.
RESEARCH PROPOSAL
Aim#
1
52 52
53 53 55 55 55
Hypothesis
Rationale
Aim #2
Hypothesis
Rationale
Aim #3
Hypothesis
Rationale
56
56
56 57 58 58 59 59 59
Aim #4
Hypothesis
Rationale
Aim #5
Hypothesis
Rationale
4.
ANALYTICAL METHODS
Characterization of Liposomes
60 60 60 62 64 66 67 70
Size Measurement
Lipid Determination
HPLC
In vitro Stablity
at
37 C
Acetonide
5.
72 72 74 80
85
Methods
Results
Discussion
6.
PHARMACOKINETICS AND PHARMACODYNAMICS OF TRIAMCINOLONE ACETONIDE PHOSPHATE IN LIPOSOMES AND IN SOLUTION AFTER INTRATRACHEAL AND INTRAVENOUS ADMINISTRATION
Introduction
88 88 88
Methods
Results
94
101
Discussion
7.
EFFECT OF DOSE AND RELEASE RATE ON PULMONARY TARGETING OF GLUCOCORTICOIDS USING LIPOSOMES AS A MODEL DOSAGE FORM
Introduction
107 107
108
Methods
Results
114
121
Discussion
8.
ASSESSMENT OF PULMONARY SELECTIVITY OF TRIAMCINOLONE ACETONIDE AND FLUTICASONE PROPIONATE USING AN EX VIVO RECEPTOR BINDING ASSAY
Introduction
125
125
Methods
Results
126
131
Discussion
138
9.
CONCLUSIONS
143
LIST OF REFERENCES
145
BIOGRAPHICAL SKETCH
157
in Partial Fulfillment
of the
August 1997
Chairman: Guenther Hochhaus, Ph.D.
pulmonary
targeting.
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.
phospholipids and triamcinolone acetonide phosphate (TAP-lip). TAP-lip and the drug in
solution
Pulmonary targeting
lung and
liver.
in
The
longer
mean pulmonary
effect times
(MET)
drug
in solution.
Thus, liposomes
may
delivery
of glucocorticoids
via
topical
administration.
The
pharmacokinetics
and
after
were
also
investigated.
TA
HPLC/RIA. No
that
changes
in
the pharmacodynamics
(EC 5 o and
E,,,),
pharmacokinetics
is
The
intratracheal
administration of
TAP
mean
effect times
targeting which
were higher
nm
liposomes showed a
bell
shaped
is
maximum pulmonary
targeting
in
(TA) and
were assessed
after
IT administration
assay.
No
significant differences in
observed between these two drugs. Higher pulmonary targeting and longer
MET
values
were obtained
after the
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
targeting.
CHAPTER 1 INTRODUCTION
Asthma
is
in
which many
cells play
a role
cells.
Asthma
is
severity
is
on the
rise.
effective
and
of the
disease.
Inhaled glucocorticoids
have become
results
first-line
of glucocorticoid action
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
powder
inhaler.
Inhalation therapy
directly
shown
that
pulmonary
is
systemic clearance and low oral bioavailability have been introduced. Thus, the latest
<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 be modulated have shown that pulmonary targeting can
[1]
if
drug release rate and dose are optimized. These theoretical studies
an optimal dose and release rate for which
is
maximum pulmonary
It
was important
in
pulmonary
selectivity.
To show
the importance
of these two
factors,
flexibility
and
their compatibility
it
was
among
acetonide and fluticasone propionate) based on the fact that pulmonary targeting
is
CHAPTER 2
BACKGROUND
2.1
Asthma
2.1.1 Definition
Asthma
is
considered
an inflammatory disease,
often
described
as
chronic
[2].
Asthma
is
common
which
affects
5%
become more
increased
[5].
prevalent during the last decades, and the severity and mortality have
Asthma
is
sputum production
[6],
2. 1.2
Pathogenesis
Asthma
is
in
which many
cells
cells
are involved,
cells,
epithelial
of
different mediators
mucus
hypersecretion, epithelial
damage and
as IL-1
may prime
it is
the
sequence of inflammatory
in
asthma
different
[7].
Several
have
reported
that
alveolar
(PAF), a mediator that selectively attracts human eosinophils and causes eosinophil
infiltration
of airways and
epithelial
damage; b)
fibroblast
gamma
interferon, a powerful
lymphokine
function;
c)
tumor necrosis
factor;
d)
to lymphocytes;
f)
alveolar
10].
Eosinophils and epithelial cells play an important role in the inflammatory process
as well.
Upon
C 4 PAF,
,
oxygen
[11].
radicals
epithelial
damage
Epithelial
release
inflammatory
mediators
like
LTB 4
[3],
and
15-
may have
a variety of
effects
on the airways
Table 2.1). Mediators such as histamine, prostaglandins, and leukotrienes contract airway
smooth muscle
inflammatory
directly, increase
cells,
which
in
Mast
Cell
Neutrophil
7)
Eosinophil
Epithelial shedding
casoiy nerve
Bronchoconstriction
Figure
2.
Cellular interactions.
[3].
2,1.2,3
The
role
of cytokines
As
stated previously,
asthma
is
inflammatory
cells,
airways
may
It
is
important in allergic
cell
(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
that directly
(RANTES).
2,
,2.4
may be
important
in
2.2).
autonomic control include submucosal gland secretion, bronchial vascular tone and
permeability, airway
cells
[3].
Table
2.
in
asthma
[13].
Mediator
Airway
secretion
Microvascular
leakage
Chemotaxis
Bronchial hyperresponsiveness
Histamine
Prostaglandins
+ +
+
?
D 2 F
,
++
Prostaglandin
E2
Leucotriene
B4 C4
,
Leukotrienes
E,
D4
Platelet activating
factor
Bradykinin
Adenosine
Substance P
Serotonin
Oxygen
radicals
Throboxane
2.1.3
Therapy of Asthma
The philosophy
in
the
whose
home
first
priority is to prevent
exposure or reduce
that can
it
exposure can
allergen
asthma
[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].
Adrenergic
^Cholinergic
Non-adrenergic
Non-cholinergic
J 4-4P
Syniphathetic nerve
Vaeus
Cholinergic efferent
Adrenal medulla
^ncffljffB
Airway lumen
Figure 2.2.
[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
CH
3,
and CI
in positions
1,2.
oxygen
at
C-20.
By modifying
the basic
it
22]:
receptor (GR),
affinity
(CBG).
via
Biotransformation:
modulating
the
metabolism
oxido-reductive
or
hydrolytic pathways.
C-22
CHUOH
l
cn
c=o
Figure 2.3
The chemical
acetonide [23].
by triamcinolone
2.2.1
II
steroid
They represent
is
genomic
binding,
the
[24].
The
GR
consists
of
DNA-
430
in the
modulatory domains. In
its
inactivated state,
GR
is
complexed
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
DNA-
binding domain of the receptor [25] and that this complex does not
show any
biological
activity.
receptor
is
activated,
it
dissociates
the
its
two
Two
homodimer which
is
GREs
(pGREs),
common
so-called negative
GREs (nGREs)
is
transcription.
The degree of
is
as a
our study
was found
that
nuclear pathways. In this case, the activity of other transcription factors, such as
(activating protein- 1) and
NF-kB
is
modulated by
direct interaction
10
two
factors [27].
pathways
are
extremely
important
because
they
provide
between
glucocorticoids on one hand, and polypeptide hormones and cytokines on the other.
Some
this
many
The
is likely
to
Figure 2.4.
[18]
2.2.2 Target
Genes
in
Asthma
asthma are
listed in
The
be
may
effective in controlling
11
Although
action in asthma,
it is
critical
aspects of steroid
it
is
TNF
a, granulocyte-macrophage colony-stimulating
factor,
IL-3,
IL-4,
IL-5,
IL-6,
lipocortin-1
37-KD
on phospholipase
A2
and
therefore
may
inhibit the
and
may be more
effective in inhibiting
in inhibiting lipid
oxygen species
[29].
One of the
eosinophils which
is
a reduction in circulating
may
reflect
in the
bone. Inhalation of
in
the
number of low
in the
density
of cytokine production
airways
marrow
in
of lymphocytes and
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.
effect
on mediator release
cells,
is
associated with a
marked reduction
in
cell
number. This
may be
cell
12
cells
may be one of
TNF-a
in
cultured
human airway
of the
RANTES
gene and
GM-CSF in an epithelial
Table 2.2
Effect of glucocorticoids
on
transcription
[19].
Increased transcription
Lipocortin-1
(32-adrenergic receptor
Endonucleasas
Secretory leukocyte inhibitory protein
Macrophage
inhibitory factor
Decreased transcription
1,
IL-12, IL-13,
TNF-
GM-CSF, RANTES,)
(COX-2)
A2 (CPLA2)
Endothelin-1
NKi
receptors
Adhesion molecules
Immunosuppression
Carbohydrate and protein metabolism
(e.g.
gluconeogenesis)
Hypocalcemia
Hypertension
CNS
effects
excitability)
Inhibition
of cholesterol synthesis
Cortisol suppression
13
2,3
including
asthma,
bronchitis,
and cystic
earliest
fibrosis.
Although the
traditional
form of
back to the
inhalation therapy have essentially remained the same. Relatively small doses are required
for
effective
therapy,
systemic circulation,
and
potentially
minimizes
adverse
effects
[31].
considerable cost saving especially with expensive therapeutic agents. Delivering small
doses of active ingredients directly to the lung effectively localizes the drug, thereby
effects.
As mentioned
earlier,
2.3.
The
respiratory tract can be divided into upper and lower airways with the line
of
and trachea
[32].
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
[33]. Successive
the alveoli reduces the diameter of the tubes, but markedly increases the surface area of
physiological zones: conducting, transitional, and respiratory zones [33] (see Figure 1.6).
movement of air
14
is
The
transitional
in
is
fibers.
The
cell
types including
ciliated, basal,
large
are
consists
of
five lobules
Each bronchiole
which consists
sacs,
and
alveoli.
At the
level
cartilage
diminishes as the
number of
The acinus
represents a
marked change
in
morphology.
The primary
cells
90%
of the
total
Type
II
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
While
this
basement membrane,
facilitates the
cell.
it
can
still
The lung
fast
tissue
is
difficult
due to a
absorption of
(especially lipophilic
15
Trachea
Bronchi
Bronchioles
Terminal
bronchioles
Transitional
bronchioles
Respiratory
bronchioles
Alveolar ducts
Alveolar sacs
Figure 2.5
2.3.2
An
aerosol
is
defined as a suspension of liquid or solid in the form of fine particles dispersed in a gas.
is
peripheral airways. Herein lies the fundamental problem of inhalation therapy as the
tract has
The
16
specific
pathways and
rate
redistributed [35],
particles
may
deposition),
sedimentation
(gravitational
deposition),
Brownian
of
diffusion, interception,
[35].
The
relative contribution
and respiratory
tract
anatomy.
2.3.2.1 Impaction
Impaction
a particle's
momentum
prevents
it
from changing
the
course
in
It is
in the
at near bronchial
branching points.
The
of breathing, and
2.3.2.2 Sedimentation
Sedimentation results
when
is
balanced by the
sum
fall
at
is
an important mechanism
in small
velocity.
The
probability
of sedimentation
is
and to particle
size,
rate.
17
2,3,2.3 Diffusion
collision
particles pushes
in
the absence
in
still
air
moves around
in a
"random walk"
[36].
The
effectiveness
is
<
0.5
is
important
in bronchioles, alveoli,
and
at
Molecular-sized particles
may
of Aerosols
in the
Respiratory Tract
The
therapeutic
fate
of inhaled particulates
local
activity,
factors:
1)
tissue
sequestration,
and 6) metabolism
kinetics.
theoretical
2.3,3.
The
1)
characteristics
of the inhaled
and hygroscopicity; 2)
tract,
18
2.3.3.1.
Particle characteristics
The
of
it
determines operating mechanisms and extent of penetration into the lungs. Aerosol size
often expressed in terms of aerodynamic diameter (D).
is
Aerodynamic diameter
is
defined
as the diameter of a spherical particle having unit density that has the
same
settling
velocity
from an
air
in
than unit density will have actual diameters smaller than their D.
An
distribution arbitrarily
geometric standard deviation of < 1.2) or polydispersed geometric standard deviation equal to or
(less
>
1.2).
anatomy
way
as to
[42].
(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
Particles
tree.
must generally be
less
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
it
contacts an airway
19
surface, cross-section determines the air velocity for a given flow rate,
and variations
in
tidal
and reserve
air [35].
Thus,
The
since breathing
of the
with
degree of potentiation
is
deposition of larger particles in the upper respiratory tract, while slow, steady inhalation
increases the
number of particles
44],
Byron (1986)
him to
identified
maximum
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
sharpens and occurs between 1.5-2 and 2-3 microns, with and without breath-holding,
respectively.
20
When
aerosol
the above considerations are taken into account, the ideal scenario for an
would be
1) aerosol
a<1
<
2,3,3.2
partially
known
[45].
Swallowing,
first
operand
in
[42].
A
It
major clearance
mechanism
is
consists
of ciliated
down
mucus
cilia,
cells,
difficult
to quantify.
usually involve administration of 3.32 micron (D, e ) insoluble iron oxide aerosol to normal
humans. Byron (1986) [39], using a mathematical model, analyzed the clearance data
resulting
that 48.9
of the deposited
in the
alveolar regions
of the lung
is
21
The
rate at
which drugs are cleared from the airways depends greatly on certain
such as
1)
molecular weight,
2)
partition
urea of various molecular weights. They found that the first-order rate constant,
decreased with increasing molecular weight. Because of the low
k,
lipid solubility
of these
aqueous channels of
it
was
at
than 1000
were absorbed
h).
(ti/2
- 90 min)
3 to
27
First order
absorption rate constants from the upper respiratory tract are approximately half of those
[40]. Solutes
40 min
[47].
studies have reported that solutes penetrate the alveolar wall at rates
which
compound
[48].
partition
10
[49].
If the
partition
coefficients
simultaneously,
may be
compound
will
be absorbed most
quickly.
22
2,3,3,4
Drug
It
dissolution or release
has been reported that the physicochemical properties of the drug such as
its
lipophilicity
drug with a
will
at
be determined by
its
partition coefficient
earlier in
section 2.3.3.3.
targeting.
targeting
is
2.4.1
Pulmonary
Selectivity
of Inhaled Glucocorticoids
introduced into asthma therapy to replace
first
The advantage of
inhalation therapy
is
combined with a
relatively
lower
dose, which
may reduce
flunisolide,
budesonide
in the
US
23
of asthma [51-53], They are delivered by metered dose inhalers (with and without
spacers), dry
powder
Due
was no
in the
body, there
receptor
level.
it
has been possible to develop synthetic derivatives better suited for pulmonary targeting.
in
the
positions
were
found
to
be
better
candidates
for
inhalation
therapy,
because
of the
its
pulmonary
selectivity,
is
the
is
associated with
high risk of systemic side effects (especially suppression of growth in children and
osteoporosis),
current research
is
aimed
at
identifying the
improve
pulmonary targeting
is
obvious.
Hochhaus
aid
et al. [1]
prediction
which
affect
pulmonary
integrated
models
[39,
54], this
model
and
lung
physiology
with
pharmacokinetic,
pharmacodynamic
systemic
receptor
occupancies,
as
24
2.4.1.1 Effect
oral bioavailability
Commercially
glucocorticoids
differ
in
their
clearance
and
oral
bioavailability,
while
inhaled
glucocorticoids
have a high
bioavailability (see
Table
2.3).
The computer
showed
that
an increase
in clearance
in
a dramatic decrease
in
61.3%
effects
increased,
in
changes
in
significantly affect
pulmonary
attaining
targeting. Thus, drugs with high systemic clearance are better candidates for
pulmonary
selectivity.
Only
10%
to
30%
is
is
2.6).
The percentage of
oral bioavailability.
25
Table 2.3
Pharmacokinetic
and
pharmacodynamic
parameters
of commercially
Clearance
(L/hr)
Volume of
distribution
Halflife(hr)
Oral
bioavailability
Plasma
binding
RBA
(Vdss) (L)
(%)
4.5
(%)
77
100
Dexamethasone
Triamcinolone
acetonide
16.8
100
80
37
103
23
71
233
Flunisolide
58
96
183
1.6
20
11
80
88
190
Budesonide
Fluticasone
84
2.8
935
69
318
7.8
<1
90
1800
propionate
it
may be
interpreted
2.4.
.2 Effect
of pharmacodynamics
It
has been stated that only glucocorticoids with high relative binding affinity
(RBA)
of
for the glucocorticoid receptor are active by inhalation, and that the
minimum
level
RBA
al.
[23],
and
mucous
26
[1] described
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
may
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
et
al.
[1]
rate
on pulmonary
selectivity.
He showed
will
be removed immediately from the airways by entering the systemic circulation (see
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
effects
effects.
Thus,
it
was concluded
is
airways, a further reduction in drug release will result in reduction in pulmonary targeting
transport. Likewise,
he showed
not
when
the dose
is
too small
it
minimum
effect concentration
is
(liver).
When
the dose
is
27
may
result
is lost.
Thus, there
is
an
Some
pulmonary
in
selectivity
after
the
of the drugs
in solution.
2.4.2
Selectivity
still
side
effects
be
new emerging
technologies
enhancement of airway
selectivity.
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
thus,
more
prolonged pulmonary
effects.
28
Swallowed
Fraction
Oropharynx
1
Aerosol Dose
Mucocilliary transport
Inlialed fraction
Drug powder
\ Kdiss
Dissolved Drug
LUNG
Kpul
GI
BODY
Drug
at
Ka
|
Systemic
Elimination
(t
i
First
Pass
'/i:Cl,Vd)
Figure 2.6
selectivity [1].
2.5
Liposomes
In the last
two decades,
it
liposomes can lead to the enhancement of therapeutic efficacy of drugs, reduction of their
toxicity
and prolongation of
their therapeutic
effect.
In addition,
and
is
presently used in
spirit
new
research
29
now
as they
were
in the early
days of liposome
research.
specific
tissues
is
the
main application of
As a
result
of these
of the tissue
distribution
[58].
However, the
kinetics
still
to
natural
and
number of
and
bilayers,
charge
and
surface
characteristics,
the
pharmacokinetics
of liposomes
therefore
the
2.5.1
Classification
bilayers
aqueous compartment
lipids
are
grounds (25
nm
in diameter),
determined by the
maximum
tolerate as the curvature in the inner leaflet increases with decreasing radius, to liposomes
which are
visible
under a
light
micron [59].
30
Liposomes can be
classified either
on the
basis
of their
structural properties or
on
lists
corresponding acronyms.
in highly purified
forms
[59].
Two
sorts
of natural phospholipids
a three-carbon
In
phosphodiglycerides,
glycerol bridge links a long chain fatty acid with a phosphoryl headgroup moiety;
by
is
in position 3.
known
phosphodiglycerides
in nature.
cell
membranes. Because of
used
their neutrality
show
in
Figure 2.6.
31
MLV
OLV
>
0.5 |im
UV
SUV
Unilamellar vesicles
(all sizes)
MUV
LUV GUV
MW
B. Based on
>
mm)
>
mm)
REV MLV-REV
SPLV
made by
Multilamellar vesicles
made by
method
FATMLV
VET FPV FUV
MLV
Vesicles prepared by extrusion methods Vesicles prepared by French press Vesicles prepared by fusion
DRV
BSV
Dehydration-rehydration vesicles
Bubblesomes
2.5.3
Method of Preparation
Liposomes are prepared by mechanical dispersion methods (such as hand-shaken
water
in
Dependent on the
selection
of
lipids, the
vitro.
The opsonization
all
[60], Therefore,
it is
32
constituents and the preparation technique carefully and to characterize the produced
liposomes properly.
Phosphatidyl moiety
Headgroup
Common name
Phosphatidyl
choline
Me
xT^s Me
^\NH,+
ethanolamine
rAcoo
serme
"O^Y^
OH
"
glycerol
OH
OH
OH^j
I
inositol
OH
Figure 2.6
Some common
Adapted
33
From
it is
this rule.
The chemical
stability
of liposomes
is
is
it
the most
commonly used
lipid in
pharmaceutical
Two
life
of
dispersions
have been
described
for
phospholipids
in
aqueous liposome
dispersions.
[57].
2.5.4.1 Oxidation
of Phospholipids
in
Oxidation of phospholipids
at the
unsaturated acyl
chain-carrying phospholipids, but saturated fatty acids can also be oxidized at high
temperatures [57].
The
mechanism
in
initiate radical
formation
containing
polyunsaturated fatty acids are the most sensitive to radical formation. If oxygen
present, the process develops further
is
fission
of
34
UV
absorbance of lipids
is
the
first
sign of the
As mentioned
is
It is
this
oxidation process
can be minimized by the use of high quality raw materials which are purified from
hydroperoxides and transition metal ions, storage
antioxidants.
at
Gonzalez-Rothi
et
al.
of liposomes
is
several
fluids.
They found
that
40%
material. It is
was due
to a
The liposome
is
which
result in a
change
in
those of hydrolysis.
The four
ester
bonds present
at sn-1
in a
phospholipid molecule
may be
subject to hydrolysis.
The
carboxy esters
esters.
Hydrolysis of esters
catalyzed via
two pathways
[57]:
this
is first
protonated and
35
Base catalyzed
hydrolysis.
is
on
Two
products are formed as a result of hydrolysis of the carboxy esters at the sn-1
1-acyl
lysophospholipids,
respectively
[62].
Further
of
both
lysophospholipids
produces
glycerophospho
compound.
is
phosphate-headgroup
ester.
The
rate
factors:
pH
temperature
ionic strength
buffer species
chain length
incorporation of cholesterol
incorporation of charged phospholipids
2.5.4.2.1
The
effect
of pH
Grit et
al.
of
pH
phosphatidylcholine (PC) in a
pH
40 C and 70
C.
They found
that the
minimum
at
pH
6.5,
and V-shaped
pH
profiles
were obtained
as
pH
profile at
minimum
pH
6.5
PC
[64].
36
2.5.4.2.2
The
effect
of temperature
The
effect
all
PC's the
results could
be described by Arrhenius
the
kinetics
as
a semilogarithmic linear
relationship
observed
hydrolysis rate constant and the reciprocal of the absolute temperature. For natural PC's,
in a
However,
crystalline
for phospholipids
composed of
2.5.4.2.3
The
effect
of charged liposomes
are
Charged
phospholipids
generally
included
in
pharmaceutical
liposome
formulations as they tend to improve the physical stability of liposomes by reducing the
rate
of aggregation and
PC
and
were obtained by
varying the charged phospholipid content (PG) of liposomes. Hydrolysis kinetics for both
lipids
followed pseudo
first
It
was found
that
egg
PG
hydrolyses
2.5.4.2.4
The
effect
of incorporation of cholesterol
cholesterol into liposomal bilayers tends to increase the
The incorporation of
retention
lipid
rigidity
37
of fluid
state bilayers,
cholesterol
was
They
al.
[65],
found that cholesterol abolishes the phase transition of phospholipids, producing a rather
rigid
range, which
is
against hydrolysis.
Based on the
structural properties
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 33-hydroxy group of
cholesterol
is
assumed to engage
in
in
fatty acyl
groups
may
kinetics.
This effect
is,
in general,
expected to be a protective
effect,
Chemical
decomposition
instability
of the
the
physical
stability
interfere
with the
38
The
is
influenced by
two parameters:
1.
Changes
in
2.
Changes
average particle size and size distribution are strongly affected by:
Phospholipid composition
Medium composition
2.5.5.1
The
effect
of phospholipid composition
electrical
aggregation than charged liposomes. Crommelin [67] studied the effect of including
charge-inducing agents
PC
containing liposomes in
It
was found
liposomes, both at low and high ionic strength, no increase in particle size occurred after
storage at 4C for 14 days. Thus, aggregation can be prevented or slowed
down by
2.4.5.2
Liposome composition
Several studies have
shown
increases the physical stability of the formulations by increasing rigidity of the bilayer
which
is
produced as a
39
than the parent drugs and can produce morphological changes in the
changes
in permeability
The
[57].
effect
of lysophospholipids and
fatty acids
formed in
situ
was
investigated
It
was found
that
when up
to
10%
to 10
of hydrolysis), a
drop
in
Above
that degree
exogenous
LPC
containing liposomes.
They concluded
that in
the
initial
stage the free fatty acids were also formed, thus, neutralizing the
LPC-
destabilizing effect.
2.5.5.3
Liposome
size
The
effect
stability
lipid
less
stable
because the
to
come
into
contact
and an increase
in
aggregation
produces an increase
Small
in permeability.
negatively
charged
unilamellar
vesicles
(40
nm)
are
less
stable
(thermodynamically) than large vesicles. They fuse easily as a means of relieving stress
arising
[60].
liposomes are more stable against leakage than small unilamellar vesicles due to the
difference in
membrane
thickness.
40
Size-dependent
stability
of liposomes
in biological fluids
al.
between liposome
size
and
its
stability in
composed of pure
it
succinylglycerol
were more
However,
in
was found
that small
less stable
than
larger liposomes.
in buffers.
This
may be due
of plasma used
in either
case
was
different. It
[73] that at
PI,
SA
is
independently of
size,
decreased, the size of the liposomes decreases with concomitant release of encapsulated
material.
Divalent
cations
besides
in
The
ions
composed of
it
was
investigated by Tari, et
al.
was found
that
Ca
and
Mg2+
lipid
from
which resulted
in increased permeability
of the
encapsulated material.
41
When
administered
in
vivo,
way
of
Advances
in
stability,
thus,
reducing constituent
cells
of the RES.
substantially
of liposomes exhibiting a
RES
uptake.
In contrast to
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
This has prevented the use of the pharmacokinetic models currently
at the
molecular
level.
some
kinetic
models
have been proposed to describe the disposition of certain types of liposomes, a model of
42
general applicability
is still
is
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]
2.5.7.1
and to a
lesser extent,
RES
represents, therefore, a
cell types.
The
state,
rate
RES
depend on liposome
size, physical
RES
is
the principal
mechanism
by changing
dose, chemical
composition
etc.,
Internalization
of phospholipid liposomes
is
also operated
by non-phagocytic
cells
such as fibroblasts,
cells
etc.
nm
[75].
(HDL). This
is
the result of
43
the insertion of apoproteins into the liposomal bilayer and the exchange of lipidic material
[75].
destabilization,
and
and composition of
the vesicles.
The
size
of the endothelial
cells
(about 70
nm
in diameter)
accommodation of particles
unable to leave the vascular space by passing across the cells of the capillary endothelium.
is
like
in
liver,
and appears a
realistic
SUV [77].
2.4.7.1
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
is
a higher
cells,
SUV
LMV
[75].
are destabilized
disruption depends
SUV and,
44
is
IV
injection
of
initial
true
solutions
in
body
fluids,
is
analyzed
on
the
basis
of a
Huang
distribution to
et
al.
[78],
be
after the
IV
administration of
SUV
to the extrapolated
volume of
of
this
of which are
to
1,
is
kinetics.
The
smaller the
As mentioned
above,
large
unidirectional, then
it
is
an elimination process
site
of measurement)
that cannot
be responsible
decay? This
this biphasic
Stamp
[79],
by showing
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
decay
is
45
Gregoriadis
et al.
[80],
MLV is converted
into a linear
may be due
parenchymal
cells.
When
RES becomes
the slower elimination pathway operates and the decay rate decreases.
kinetics
of liposomes
Upon exposure
proteins (alpha 2-macroglobulin) that can mediate their uptake. Recognition and ingestion
cells are
on these
cells.
is
opsonization with
[81].
Nabila et
phosphatidylinositol,
ganglioside
GM
i,
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
in the clearance
that,
by decreasing the
fluidity
of the
lipid
bilayer,
less
easily penetrated
46
For
instance,
it
was found
20
h.
[84]
that
when
cholesterol
is
half-life
was
as long as
The
effect
disappear from the circulation faster than neutral liposomes. Another study, however,
indicated that this
is
not a general
phenomenon and
that
some
GM
On
it
was found
RES
[77].
Abraham
et
al.
of H- triamcinolone acetonide-
21-palmitate entrapped in liposomes with neutral, negative and positive surface in rabbits
after single
IV bolus
injection.
They found
had encountered a
These
results
can be explained
is
RES
is
as follows: positive
2.5.7.2 Disposition of
As mentioned
is
based on
of administration.
because of the
Administration of liposomes
the
respiratory
tract
is
attractive
47
accessibility
surfactant components, and the need for sustained local therapy following inhalation.
Due
to this,
numerous
and
distribution
fate
instillation
or inhalation.
One of
the
first
studies
was reported by
Juliano et
al.
[86],
comparing the
MLV
C)
rats.
They found
that free
ARA-C
(arabinoside
was
rapidly cleared
(t 1/2
= 40 min) and
while liposomal
ARA-C
little
and persisted
al.
by labeling
MLV
and
SUV
composes of
DPPC
nebulized vesicles
was dependent on
on
the vesicle
size.
75%
in
the lung
clearance.
Since the removal of particulate matter by the mucociliary elevator becomes progressively
faster
initial
From
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.
48
Woolfrey
et
al.
[88]
investigated
the
pulmonary
absorption
of liposome
rats.
The
extent of
CF
absorption
was
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
accelerated removal of the liposomal
lipid
dose
CF
specific
pathways
CF from
negatively charged
vesicles
was
at least
compounds
following intratracheal
instillation.
They found
epithelium represented the major rate-limiting barrier for systemic absorption, not the rate
of the drug
release. In contrast,
encapsulated drug produced almost identical plasma levels suggesting that diffusion of the
lipophilic
compound through
vesicles bilayers
In this
in the
lung
approximately three-fold over 48-hr period while free atropine disappeared rapidly After
48
hr,
21%
still
compared to
4%
when
tissue
instilled in solution
They concluded
rate
that the
amount of drug
available to lung
MLV.
49
is
rather scarce.
One
is
which
when
demonstrated for hydrocortisone [90] and for triamcinolone acetonide [91]. In order to
overcome
this
85%, compared
demonstrated
5%
for the
parent
drug
[93].
Likewise,
Brattsand et
al.
[23]
that
budesonide
21-palmitate
incorporated
into
liposomes,
showed
(half-life
= 6
hr)
compared to budesonide,
after intratracheal
The
practicability
aerosols have been generated with nebulizers like the Collison and Hudson. However,
it
has been
shown
of an encapsulated marker
is
enhanced
significantly
by the
air
As an
alternative to
studies
have
demonstrated
that
liposomal
encapsulation
of
compounds can
localize
in
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
50
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
is
essential
in
pharmacological
pharmaceutical requirements.
Review
Asthma
increasing.
is
perhaps the only treatable condition whose prevalence and severity are
disease, glucocorticoids
this disease.
Glucocorticoids exert
portions of the
DNA,
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
counterparts,
51
systemic clearance and lower oral bioavailability, which results in lower systemic drug
concentration and therefore higher benefit/risk
ratio.
still
concerns
regarding side effects, especially the suppression of growth in children and osteoporosis,
since
these
drugs are
likely
to
Thus,
identification
of the factors
that can
is
obvious.
Recently,
it
has been shown that not only clearance and oral bioavailability, but
in
pulmonary
selectivity.
vesicles)
the last
two decades,
its
it
therapeutic efficacy,
reduces
its
and prolongs
its
therapeutic effects.
administration to the
respiratory tract
is
attractive
compatibility of liposomes with lung surfactant components, and the need for sustained
local therapy following inhalation.
As
a consequence,
numerous
fate
effect
of compounds administered
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
pulmonary
targeting.
3.1 Objectives
One of
was
to test
We
also attempted to
rate.
liposomal formulation with different release rates and doses of encapsulated drug and
relating
available
inhaled glucocorticoids
identify
some
essential biopharmaceutical
Aim #1
To
design
glucocorticoid-liposome
dosage
form
suitable
for
pulmonary
liposomal
administration
and
assess
pulmonary targeting
of both,
the
developed
size,
encapsulation efficiency,
lipid content,
and in
vitro stability in
in local
organ
52
53
(lung)
and
in
(IV)
administration.
3.2.1.1 Hypothesis
If the liposomal
membrane
water-soluble
a selective local
(lung)
receptor
way
will
3.2.1.2 Rationale
We
act
hypothesized that liposomes are a suitable dosage form due to their ability to
carriers
as
drug
for
their
flexibility
in
release
rate.
Phosphatidylcholine (PC),
were chosen
was based on
compounds
The lung
surfactant pool
is
70%
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
targeting.
Among
the great
number of currently
of
initially
54
half-life,
high receptor
affinity,
and low
oral
preliminary studies
showed
TA
it is
rapidly released
dilution.
As
the liposomes seemingly provide no barrier function for the lipophilic glucocorticoid
TA
we
was
to encapsulate a water-soluble
we
(TAP), a prodrug of
TA
TAP
is
rapidly cleaved
its
active
compound
is
TA
[97],
we
TAP
The pharmacological
effects
of glucocorticoids
are
mediated
through
the
of the
cells [25].
In addition, the
dexamethasone
[42,
98,
99],
However,
such
studies
can
not
directly
track
pharmacodynamically
relevant
concentrations
as
generally
both,
encapsulated
The ex
is
able to quantify
55
the degree of pulmonary targeting by evaluating the difference between pulmonary and
hepatic receptor occupancy.
To
pulmonary targeting
and compared
we assessed
it
IV
administration.
3.2.2
Aim #2
Assess the pharmacokinetics and pharmacodynamics of the liposomal formulation
after
after
the intratracheal
instillation
3.2.2.1 Hypothesis
drug
in
The
retention
of drug
result in a
in the
pharmacokinetics of the
drug) as well as
sustained
mean pulmonary
effect
(change
in the
pharmacodynamics)
instillation
of the drug
in solution.
3.2.2.2 Rationale
A number of relatively
and triamcinolone acetonide
)
low
methyl prednisolone
will control
It
has
(effect-time
profile)
56
profile),
but also on
its
intrinsic
pharmacodynamics
(effect-concentration
profile)
[100].
Thus,
change
in
the
down
its
change
in the
in
pulmonary
effects).
3.2 3
Aim #3 To
establish the relationship
between liposome
size
the presence
of
3.2.3.1 Hypothesis
If the interaction
in the
airways
is
the
in large
in
small vesicles.
3.2.3.2 Rationale
It is
known
bilayers
is
it
size
and type
more
of the
phospholipid
is
least stable
because of
in
number of lamellae
the
57
liposomes clearance
may be
stability,
same
in vitro
conditions
may
predict
what happens in
vivo.
CF
has been widely used as a marker to determined the release kinetics from
CF
is
we
expect that
the
same way
in
A surfactant
was included
to
that liposomes
in
aim
number
4,
that
was achieved by
instillation
4).
3.2.4
Aim #4
To
determine the relationship between release
rate,
monitor
lung
and
liver
receptor
occupancy
upon
intratracheal
of
glucocorticoid in solution and in liposomes with different release rates and different doses
of encapsulated material.
58
3.2.4.1 Hypothesis
Pulmonary
selectivity
3.2.5.2 Rationale
In attempts to increase localization of drug in the lung, drugs with higher clearance
little is
known about
the
pulmonary
targeting.
Pharmacokinetic-
biopharmaceutical factors that can affect pulmonary targeting: dose and release rate of the
rate).
is
mean
that the
minimum
reached either
in the local
organ (lung) or
result
in the systemic
organ
(liver).
When
Krel
is
too
may
is lost.
The same
rationale
is
it
was attempted
to
show
experimentally that
there
is
maximum pulmonary
targeting can be
their
shown
in
In order to
compare the
delivered
effect
different
formulations were
intratrachealy
anesthetized
male
rats:
triamcinolone
TAP
in
200
nm
TAP
in
800
nm
occupancy
in the local
to glucocorticoid receptor
occupancy
in the systemic
organ
Pulmonary targeting
59
was
calculated as the difference between the degree of lung receptor occupancy and the
liver
degree of
receptor occupancy.
To determine
of TAP
in
escalating doses
800
nm
3.2.5
Aim #5
Determine
the
pulmonary
targeting
of
two
currently
available
inhaled
ex-vivo animal model: monitor glucocorticoid receptor occupancy in local organ (lung)
and
in
different
systemic organs
(liver,
upon
intratracheal
(IT)
3.2.5.1 Hypothesis
If
pulmonary
selectivity
3.3.6.2 Rationale
Computer simulations
[1]
selectivity
can be
rate.
it is
may
4.1 Characterization
of Liposomes
4,1.1 Size
Measurement
size
The
suspension
was placed
PBS
KHZ. The
error
the
fit
was
the
two
(the residual
is
shift in
which
is
fit
for the
Nicomp
distribution analysis).
Two
different types
of
If the Chi
Squared was
less
4.1). If
Generally,
200
nm
nm
Nicomp
60
61
lipid
membrane
is
usually only a
phosphate
needed.
is
modifications.
Figure 4.1
DSPCDSPG
(9 1)
nm
VOLUME-TIT NI XMPI BnaBunoN
1
KL
UQL
; =
too
BO
:
60
40
|;
3D
JL
200 500
fK
to
20
>
50
fit*
(m
_>
100 <Uaiclas>
Figure 4.2
DSPC DSPG (9
1)
62
4.
1 .2. 1
Column
preparation/processing of samples
to swell in 120
ml of PBS for
at least
stored at 4 C.
A PD-10
plugged with a
Whatman GF/B
filter
with the
filter
gel,
was
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.
in the
using dyed liposomes prepared with the phospholipid rhodamine showed that 200
nm
and
800
nm
liposomes elute
in the
nitrogen.
The
lipid
4.1.3.1 Processing
of samp les
M ferric
in
chloride
0.4M ammonium
thiocyanate
(NH4SCN)
deionized water.
incubated at
room temperature
hr.
63
at
room temperature
(upper) layer
The aqueous
was read
at
488
nm
using a
UV/VIS spectrophotometer
(Perkin
4.
.3.2 Calibration
curve
90% PC
and
10%
PG
(1
mg/ml
total lipid in
in
4.1.3.
The
(20,
60 and 100
|ig/ml) prepared as
shown
in 4.3.1.
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.
The
correlation
all
calibration
The
coefficient
of variation obtained for the slope was smaller than 12%. The results of the
variability are
inter-
and intra-day
shown
in
validation, the
of this study.
64
Table
4.
DSPC:DSPG determination
Inter-day
Theoretical
Measured concentration
(ug/ml)
Intra-day
variability
concentration
mean (SD)
variability
(%)
(%)
(ug/ml)
20 60
100
21.2(2.7)
52.9 (4.7)
13.64
12.9
8.8
4.96
1.55
101.7(4.5)
4.9
4.1.4
An alysis
The
HPLC
of encapsulated
HPLC
[1
method
10]
4.1.4.1 Instrumentation
The
analysis
of
TAP was
pump
with a
mm) column
connected to a Perkin
autoinjector.
analytical column.
SM
Packard
HP 3394A
The
eluent
was monitored
M (pH
2).
Liposomal
TAP
concentration
was determined
of 10
ul
methanokPBS and
standard).
min and 70
ul
65
column. Quantitative analysis was achieved by comparison of peak heights with an internal
standard using the fitted calibration curve.
as the
mean of two
replicate
injections.
The
chromatogram
is
shown
in
Figure 4.3.
Figure
4.
(RT
in
MeOH:PBS.
The
(75,
were determined
in 4.1.4.1.
The
limit
of quantification was
TAP
HPLC
of the
66
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.
TAP
reversed phase
HPLC
determination.
Theoretical
Measured
concentration (ug/ml)
Inter-day
variability
Intra-day
variability
concentration
(%)
(%)
(ug/ml)
mean (SD)
77.9 (6.7)
8.6
3.3 2.1
75
6.6 6.7
5.1
300 600
307.2 (10.9)
599.6 (12.7)
4.
.5
In vitro Stability
at
37C
of freshly prepared TAP-lip was tested
The
in vitro stability
at
rat
medium RPMI-1640m or
filtration,
tissue culture
fetal
was
from the
initial
fluids.
This resulted in a
TAP
HPLC
p.1
(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
1
of
is
described in 4.1.5.1.
67
4.1.5.1
Column
preparation/processing of samples
to swell in 120 ml of
PBS
in
a glass
4 hr
at
stored at 4 C.
The plungers
plastic syringes
pad.
The
syringes
were
filled
1 1
Company)
at
2000 rpm
for 5
ml of the liposome suspension were applied dropwise to the top of the gel bed and spun
down
4.1.4).
at
ul eluates
were analyzed by
HPLC
(see
4.1.6
Method
for Recovering
Lung Lavage
and
Fluids
lung
lavage
fluid
was obtained
after
tracheal
were slowly
at
injected to
fill
the lungs.
The
fluid
was withdrawn by
supernatant
gentle aspiration,
and spun
2000 rpm
The
cell free
was
aspirated and
stability
of liposomes.
[56],
Immediately after
liver
ice.
The weighed
Tris/HCl, 10
tissue
was added
to 10
mM
45
mM sodium molybdate, 2 mM
and homogenized
in a Virtis
68
homogenizer
at
40% of
fiill
30 second
One
tenth
volume of
5%
incubation buffer)
was added
4C
at
was
centrifuged at
in
Beckman
JA-21
rotor
(Beckman
CA)
3 supernatant (150 ul) were transferred into microcentrifuge tubes that contained 25 ul of H-
10 and, 30
nM)and25
ul
TA (3 mM) to
determine
total
and
ul
were
in
nM) and 25
ul
of incubation
amount of total
radioactivity. In
concentration (30
nM) of labeled
at
material
was used
(see chapters 7
and
8).
2%
ice
suspension of activated
ul).
for 5
min on
at
10,000
rpm
for 5
min
in
in
300
ul
LS
in triplicate.
H-TA (TAT),
nonspecifically
bound
H-TA (TAnS)
sites
and
free
H-TA
fitted to
curve-fitting
program
MINSQ (Micromath,
Lake
City,
UT).
69
(eq.4.1)
some
cases,
B,
in
vivo receptor occupancy did not allow a reliable estimate of B mx and Kd. In this case, B,
as the difference of
TAT
values and
TANS
concentration). This
was
in
justifiable as the
4.3).
Bmax
experimental (nM)
Figure
4. 3
B max calculated values (using equation 4. 1 ), and experimental (obtained from the difference of TA and TAn T S observed values). R 2=0.985, n=45.
Correlation between
B ms
70
4.3 Reverse
HPLC
an d Radioimmunoassay
Acetonide
4.3.1 Extraction
Procedure
at
room temperature
for
somewhere
The
organic solvent
in a
The
residue
was
directly for
the
HPLC
separation.
HPLC
of
Procedure
The
Roy) to
analysis
TA
ODSn
(150 x 4.6
connected to a Perkin Elmer ISS 200 autoinjector and to a programmable Gilson model
203 fraction
collector.
variable
a Hewlett Packard
HP 3394A
The
eluent
was monitored
v/v).
Retention times of
hydrocortisone and triamcinolone acetonide were verified before every experiment. After
extensive washing, plasma or lung extracts (200 ul) were injected and the
collected.
TA
fractions
TA (HPLC
fractions collected)
vacuum. The residual material was extracted twice with ethyl acetate
and the solvent evaporated
until dryness.
The
resulting residues
were reconstituted
in
300
71
ul
of incubation buffer containing 0.001% Triton X-100 and centrifuged. The portions of
R1A procedure.
4.3.3
RIA Procedure
The
dried residue
was
dissolved in
300
ul
of 0.01
ul),
200
ul
of assay
buffer,
of antiserum working
in microcentrifuge tubes
activated charcoal
was added
for 5
min and
centrifuged at 10,000 rpm. Aliquots of the supernatant (600 ul) were mixed with 4 ml of
scintillation fluid
and counted
in a scintillation counter.
bound
(C) were
fitter
to the equation
(Scientist,
Micromath,
Salt
Lake
B= T - T*C N/(CN +
Estimates
if
IC !0
N
)
+ NS
slope factor), IC 50 (concentration to
NS
N (Hill
decrease specific tracer binding by 50%), and total specific binding (T) were used to
transform
The
and the
of quantification was
0.
15% and
precision 15
%)
of
limit
of detection was
0.1 ng/ml.
The
was
in
and
1.5 ng/ml.
%. Intra-day
samples was
and 4.4
CHAPTER 5
5.1 Introduction
in treating
alveolitis.
Although systemic
is
risk
at
clinically
glucocorticoids,
including
acetonide
(TA)
are
as aerosols.
We
in
we showed
is
achieved
when
is
not observed
when a
fast
glucocorticoid solution
is
because of the
targeting depends
on slow
release
results in a
prolonged
to provide sustained
pulmonary release
dexamethasone
high
have a
lipophilic
such
as
TA
under equilibrium
72
73
conditions,
TA
is
is
observations
of Schanker
practically
&
co-workers
[49]
that
lipophilic
glucocorticoids
cross
membranes
unhindered.
Our
findings
question
the benefits
of achieving
we
this
membrane
then slow
TA derivative,
in
possible.
We therefore
prodrug of TA) for developing a formulation which captured the negatively charged
within liposomes. This formulation
TAP
was delivered
with
either
by intratracheal or intravenous
administered
injection
to
rats
and
compared
intratracheally
TAP-sol.
Since
pharmacodynamic
occupancy
in
effects
lung and liver using the above mentioned animal model allowed for indirect
74
5.2 Materials
and Methods
5.2.1 Materials
(St. Louis,
MO);
(1,2,4-
MA).
Lipids
5.2.2
Drug Solutions
Triamcinolone acetonide phosphate (TAP) (provided as a
gift
from Dr. K.
buffered saline
Reininger, Bristol
in
to a concentration of
5.2.3
Animals
All animal procedures
University
of
Florida,
an
AAALAC
rats,
Specific-pathogen-free,
non12 hr
in a
were allowed
free access
75
5,2,4
Liposome preparation
Liposomes composed of l,2-distearoyl-sn-glycero-3-phosphocholine (89.4 mg)
in
were dissolved
at
in
chloroform.
rotary
evaporation
lipid film.
mg/ml
TAP
Cellgro
pH
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 63 C and was then extruded 30 times using a
extruder (Avestin
Liposofast"
Co.
Ottawa,
Ontario,
Canada)
through
0.2
mm
polycarbonate
filters.
particle sizer
(Nicomp
at
On
200
u.1
10 ml Sephadex
G-75
in a
PBS
mobile phase.
in the first
TAP
concentrations
HPLC
(section 5.2.5)
to a final concentration of
TAP
osmometer
M 5500 (Wescor,
Inc.
content
was determined
76
5.2.5
HPLC Method
Liposomal
TAP
was determined
in
a 10 ul aliquot
u.1
ul
internal standard.
The mixture
was vortexed
4.6
min and 70
ul
mm)
2;
at
ml/min.
UV
detection
was performed
at
limit
5.2.6
Method
fluid
was obtained
sterile isotonic
saline
were
fill
The
fluid
was withdrawn by
supernatant
2000 rpm
The
cell free
was
on the
in vitro stability
of liposomes.
The
lung lavage
in vitro stability
rat
fluid, tissue
culture
tissue culture
fetal
filtration,
5-fold
from the
initial
preparation)
(TAP
is
final
high
enough
to
be quantified by the
HPLC
system employed).
77
After incubation
at
37C,aliquots of 200
u.1
(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 ml bed size
[60]. Aliquots
minicolumns of
of 10
ul eluates
[56],
were decapitated
livers
at
1,
2, 5, 6, 12,
or 18 hours after IT or
IV
administration of Tap-
lip.
A total
of 3 (12 and 18
hr) to
point after IT administration of TAP-lip. For the IT administration of TAP-sol and the
IV
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.
sham animal
(receiving either
IV or IT buffered
saline)
[56]
with
modifications. Immediately after decapitation, the lung, without trachea, and a lobe of the
liver
ice.
The weighed
tissue
was added
(lOmM
Tris/HCL, 10
mM
sodium molybdate,
2mM
,4-dithiothreitol)
and homogenized
78
in a Virtis
45 homogenizer
at
40%
One
tenth
volume of
5%
activated charcoal
incubation buffer)
was added 4C
at
to the
the suspension
was
centrifuged at
Beckman
centrifuge
CA)
to obtain a clear
contained
25
ml
of
H-triamcinolone
acetonide
in
incubation
buffer
(final
unlabeled
TA (24mM)
of 150 ml of the
were transferred
acetonide
in
which contained 25
ml of
H-triamcinolone
incubation
buffer
(final
buffer to determine
the
amount of total
radioactivity.
was
removed by addition of a
2%
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
in triplicate.
H-TA (TAT ),
nonspecifically
bound
H-TA (TA NS )
sites
and
and
free
H-TA
(B^)
79
fitted to
equation
linear curve-fitting
program
MINSQ
F /
TAT =
In instances
BW TA
(TAF +
in
TANS
(eq. 5
B m*
B^
was estimated
with
as the difference of
3
TAT
values
and
TANS
observed
for
incubation
30
nM
H-TA B m%
(highest
tracer
concentration). This
was justifiable
methods resulted
B max (R
=0.985, n=45).
B mx
%
,
of control (average
and are not given
in
sham
rats).
to calculate
B max
For a given form of administration, differences between the pulmonary and hepatic
receptor occupancies were tested by paired student
test.
pool of paired (hepatic and pulmonary) receptor occupancies for individual single time
points (not
AUC's) of
all
significance
was assumed
for p<0.05.
For assessing
differential
receptor
occupancy between
lung
and
liver,
the
(AUC) was
period by the trapezoidal rule from percent occupied receptor (E )-time profiles. x
The
T=AUC Lmg/AUCLiver
(eq. 5.2)
would
The area
under the
first
80
E x*'x
versus
last
of the
effect
over time
at late
time points [100]. These estimates were also used to derive AUCoo.
effect times
(MET) were
AUMCoo/AUCoo).
5.3 Results
5.3.1 Characterization
of Lip
TAP
particle diameter
16 nm
A 40%
lipid
resulted
after
extrusion
and gel
filtration
of liposomes. The
8.5% of the
total
1 :7.
shown
in
incubated
in
various fluids.
81
Time
(hr)
Figure 5.1.
In vitro
stability
conditions in
of DSPC:DSP TAP liposomes at 37C under sink PBS (), culture medium (D), lung lavage fluid (o), and medium plus 10% fetal bovine serum (a). The percentage of
encapsulated
in
is
5.3.3
To
intact
(e.g.
liposomes present
in resected tissues),
from untreated animals was spiked with TAP-lip (TAP 200 ug/ml) and consequently
processed as described in Materials and Methods. There was a negligible difference
between the
= 100%).
differences
in
There
were
statistically
significant
glucocorticoid
receptor
82
reflected also in an
AUC based
was no
when
were administered IT
as a solution, with the lung and liver curves being superimposable (Fig. 5.2a, Table 5.1).
Time
(hr)
Figure 5.2.
liver
()
B)
intratracheal instillation
mean SD
S3
Table 5.1
pulmonary
effect
times
(MET)
after administration
).
of
TAP
liposomes
(TAP-lip) and
TAP
solution (TAP-sol
TAP-lip
it
TAP-sol
it
TAP-lip
adm
adm
ivadm
AUC (%*hr)
0-6hr
0-1 8hr
0-6hr
0-6hr
0-1 8hr
LUNG
LIVER
Targeting factor
450
770
350 340
1.0
490
500
1.0
370 600
0.6
280
1.6
620
1.2
(AUCWAUC
Time
(hr)
liv
) 5.7
3.0
3.8
After
IV administration of TAP-lip, no
significant differences
were observed
first
in
receptor occupancy profiles of lung and liver (Fig. 5.2c, Table 1) for at least the
hours.
profiles
TAP
drug preparations
As
is
shown
in this
was
IT administration of
The
Lip can be seen also from the estimates of the mean effect times (Table
These
MET
S4
IV and TAP-sol
Time
(hr)
Figure 5.3.
Lung
glucocorticoid receptor occupancy profiles after administration of 160 ng/kg of TAP: Intratracheal TAP-sol (), intratracheal TAP-lip (a), intravenous TAP-lip (). Comparison is made to intratracheal TA-sol (A)
ref. [56].
85
5.4 Discussion
TA
is
TA,
TAP
(a
prodrug of TA)
is
highly
was
we
stable,
in buffers
or biological fluids
37C (Figure
5. 1).
TAP
is
Our
similar to
what
we
TA
when
53), suggesting a
liposomal
distinct
pulmonary targeting of
TAP
in the lung.
pulmonary
longer
mean
on our experiments,
intratracheal
receptors
when compared
5.3,
TAP-lip (Figure
Table
5.1).
is
linked to the pulmonary administration of the liposomal preparation, while the use of
These
of the
TAP
liposomes
in biological
fluids
86
In phantiacokinetic-pharmacodynamic
that
the
MET
such a
way
that the
MET
(unpublished observations).
On
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
achieved only
if
the drug
is
e.g.
rate
of the
instilled particles.
release characteristics,
but not
targeting.
The
resulting
was
TAP-sol (1.0) or IV
liposomes.
Although achieving a pronounced targeting for tissues with high organ blood flow,
such as the airways
is
the therapeutic availability of drugs in target tissue (presumably due to prolonged release)
be incorporated
in
liposomal
formulation which
appears
suitable
for
pulmonary
delivery.
With
intratracheal delivery,
formulation will
87
humans cannot be
CHAPTER 6 PHARMACOKINETICS AND PHARMACODYNAMICS OF TRIAMCINOLONE ACETONIDE PHOSPHATE IN LIPOSOMES AND IN SOLUTION AFTER INTRATRACHEAL AND INTRAVENOUS ADMTNISTRATON
6.1 Introduction
Because asthma
the drugs
is
effects.
However,
due
to
its
drugs
is
pulmonary delivery
of drugs from
To
date, only
few reports
known about
delivery.
of
liposomes resulted
in
TAP
in solution or
TAP
we
its
TA
after
IT administration of
TAP
liposomes with
88
89
effects (receptor
6.2
Methods
The methods
in
chapter
5.
triamcinolone acetonide
filters.
and in
vitro stability at
37 C were assessed by
size exclusion
chromatography
(refer to
TAP
either
liposomes (TAP-lip) or
TAP
IV
in solution
(TAP-sol) were
(chapter
delivered
to
male
rats
by IT
instillation
or
administration
5).
in the
6.2.
(TA)
in
in either
at 1, 2, 5, 6, 12,
or 18 hours (chapter
5).
rpm
for
20 minutes.
resected,
Lungs were
(-
5)
20 C)
until
90
reversed-phase
HPLC
separation
at
acetate.
reconstituted in 0.15 ml of
ODS2
(150x4.6
using
HPLC
fractions
containing
TA
were
collected,
acetate.
The organic
solvent
reconstituted in incubation
1%
in a concentration
6.3
Data Analysis
TA
91
6.3.1.1
Compartmental analysis
6.3.1.1.1
Lung
concentrations
Average
TA
lung concentrations
(CLT al)
versus-time profiles
upon IV and IT
least-
(SCIENTIST, Micromath,
following equation:
Lake
City,
UT).
fitted to the
= Ae-al + Be-'"
(eq.6.1)
where Ctal
and
is
the total
TA concentration,
a, and p are
first
used to
CiAi^Ae" ""
1
(eq. 6.2)
where
k,
is
the
first
and
intratracheal administration
was
was done by
terminal phase.
CTAl
measured by the
of the
The
total area
two components.
AUCo-,. = (AUCo-,,) + (AUC,^)
(eq. 6.3)
The
elimination half-life
was
where p
is
92
For the
intratracheal administration
TA
plasma
concentrations (Cpta) versus time profiles were fitted to a one compartment body model
with
first
(SCIENTIST, Micromath,
TAP-lip, plasma
Lake
City,
TA
the
after
IT
instillation
fitted
to
e ")
ki
(eq.6.4)
A=D k/Vd
(ka-k,,) is
body
at
time zero, k a
is
the
is
the
first
AUC
and terminal
half-life
were
6.3.1.2
The non-compartmental
C PTA
Cpta (0*) was determined from the average plasma concentrations (C PTA ) versus-time
profiles for both treatments,
Mean
residence time
(MRT)
IV and
TAP-sol IT) and for TAP-lip IV plasma data was calculated from the
relationship:
93
MRT
where
^ = AUMCo-, /AUC _
under the
first
(eq. 6.5)
AUMC
^, is the area
AUC
the
under
*
first
moment C Ta
(AUMC
-,t)
was
infinity
(AUMC,^) was
rate
ratio
of the
last
C TAL*t measured
last
and the
first-order
measured
+ CtaiA.2)
Mean
from the
were calculated
relationship:
(eq. 6.6)
MRTIV
MRT
TAp. ro
rr
that glucocorticoids in solution are rapidly absorbed into the systemic circulation
upon
Mean absorption
which
time
(MAT) was
calculated as l/k
where
in the case
of TAP-lip IT
(flip-flop case),
was
6.3.2
Pharmacodynamic Analysis
Glucocorticoid receptor occupancy was considered as a pharmacodynamic end
point.
in
lung
(AUC E
i g )
and
liver
(AUC E
jver )
instillation
of TAP-lip and IT
administration of TAP-sol
5).
TA
94
occupied receptors and the percentage of liver occupied receptors respectively) via a
model:
E,
E^E^,*
Em,,
is
C/(EC 50 + C)
(eq. 6.7)
(maximum
receptor binding),
EC S0
is
the pseudo-steady-state
50%
of Em X and
is
either
TA
A confidence interval
in
(95%) based
test
was used
6.4 Results
6.4.1 Pharmacokinetics
The average
are
shown
in
listed in
fall
in
plasma
concentrations
following
IT administration of TAP-lip,
first
fit
was done by
Akaike
comparing the
respective
model
(MSC).
This
modified
[1 18].
MSC
and
0.
respectively.
lip
IT and TAP-sol IT plasma data (see assumption below), suggested the presence of a
95
average plasma levels following TAP-lip IT. The AUCo-,, for the
half-life
was
4.
hr.
Maximum TA
absorption time
levels
at 5
h after administration.
The mean
profile, the
was
5.8
h.
Time
Figure 6.1.
(hr)
Average plasma concentrations (mean SD) of triamcinolone acetonide after intratracheal administration of TAP-lip (), intravenous
administration of TAP-lip (A), and intratracheal administration
(). (TAP-lip 100 ug/kg).
of TAP-sol
n=
3 to 6.
6.4.2 Pharmacokinetics of
of TAP-lip
IV
shown
in
resulting
96
profiles
(MSC
The
AUC
_, for the
drug
half-life for
was
17
h.
2.3
h.
profile, the
AUC
_ was
0.
18
ug
was 2
hr.
hr.
Time
Figure 6.2.
(hr)
Average lung concentrations (mean SD) of triamcinolone acetonide after intratracheal administration of TAP-lip (), intravenous administration of TAP-lip (A), and intratracheal administration of TAP-sol (). (TAP-lip 100
ug/kg).
n=
3 to 6.
6.4.3 Pharmacokinetics
analysis
97
plasma concentration-time profile was best described by a one compartment open model
with
first
AUCo^
was 1 79 ng
after
was
h.
Maximum TA
levels
h.
1.6 h.
AUC
_,
was
0.4
ng h/g
lung.
The terminal
half-life
was
h, identical
to that obtained
when
analyzing plasma data. [117] have reported that the absorption of glucocorticoids in
solution after IT administration occurs unhindered. Therefore,
we assumed
intratracheal administration
of TAP-sol
is
equivalent to
its
is
equal to
MRT
hr.
is 1.6
The AUCtagiAUCp,
after
the
drug continuously
98
Table
6.
TAP-lip
IT administration
TAP-lip
TAP-sol
IT administration
IV administration
390.0
32.0
0.41
Compartmental analysis
A (ng/ml)
B
(ng/ml)
69.0
166.0
POO
AUCo^.
(ng.h/ml)
t
226
102
1.7
179
P (h)
4.1
Mhr)
k.(hr-')
1.1
0.5
2
0.6
kc
(hr-')
0.17
Non-compartmental analysis
C,ax
(ng/ml)
28
5
62
1
tma* (hr)
MAT (hr)
MRT(hr)
Table
6.2.
TAP-lip IT administration
TAP-lip
TAP-sol
IT
administration
IV
administration
Compartmental analysis
A (ug/g lung)
B
(ug/g lung)
2.3
0.42
0.25
0.54
0.9
0.025
3.2
-1
(hr
P(hr')
0.12
6.3 5.8
0.35
AUG,-,,
(ug h/g lung)
t,,3(hr)
t,,(hr)
0.15
2.0
0.37
1.1
K (hr"
AUCmns
0.6
'AUCpi, m ,
28
1.5
2.1
Non-compartmental analysis
MRT(hr)
6.4
2.9
16
99
6.4.4
Pharmacodynamics
In order to test for potential assay artifacts, such as
ex vivo release of
TAP
from
intact
liposomes followed by
its
cleavage to
TA
in
(TA
100 ug). The resulted mixtures were frozen overnight and consequently the amount of TA
analyzed by
HPLC
as described in the
methods
section.
We
found a 30
12 (n=3)
release
after freezing
the
was not
sufficient to
Table 6.3
Pharmacodynamic data obtained from curve fits of TAP-lip IT, TAP-lip IV, and TAP-sol IT average data *Data reproduced from chapter 5.
TAP-lip IT administration TAP-lip
IV
administration
TAP-sol IT administration
80
E (%)
Plasma Data
92
17
87
6
EC
5o
(ng/ml)
3
Plasma Data
En x (%)
Lung Data
EC50 (ng/g lung)
89
93
90
10
200
5.7
3.8
3.0
MET (hr)
100
The
effect (receptor
local (lung)
and
(liver),
respectively.
The
EC 5o
TAP-lip IV were 17
significant difference
7.0,
1.2,
and 3
1.3
among
77
The
from
significant difference
among
this
is likely
intact
100
-i
1
LU
10- 1
10
Concentration (ng/ml)
Figure 6.3.
Systemic effects
triamcinolone
(%
liver receptor
occupancy; mean
SD)
as a function of
intratracheal
acetonide
(plasma
concentrations)
after
administration of TAP-lip
(),
101
100
10-2
10 -1
Local effects
(% lung receptor occupancy; mean SD) of triamcinolone acetonide (lung concentrations) after intratracheal administration of TAPlip
(),
intravenous
administration of TAP-lip
6.5 Discussion
we
Based
on
our
studies,
the
average
lung
concentration-time
profile
after
intratracheal instillation
6.2).
similar
biphasic decline
was observed
7,
pulmonary
in
instillation
shown
in
chapter
we
found
in
vitro
experiments
the
efflux
of 6-
carboxyfluorescein from liposomes (with the same lipid composition and size as TAP-lip)
102
in the
presence of surfactant
was
it
is likely
is
due to a
differential
interaction
of
in
of TAP-
has been well characterized. Gregoriadis and Neerunjun [80] observed that the biphasic
decay pattern of intravenously injected liposomes may be due to the presence of two
elimination pathways: a faster one,
parenchymal
cells.
of glucocorticoids
in
solution from the airways occurs rather fast (dexamethasone half-life of absorption
=1.7
minutes).
we assumed
of TAP-sol
administered IT
The plasma
half-life
following TAP-lip IT
was
one obtained
after
TAP-sol IT
occurs
when
indicating the
therefore,
a longer pulmonary
half-life (6 hr)
A number of less
methyl prednisolone, and flunisolide) are rapidly absorbed from the airways because of
their fast dissolution rate. This suggests that
liposomes
may be used
as a
means of
in
103
We
have shown
in
chapter
5,
of TAP-lip
after the
resulted in a longer
mean pulmonary
TAP-sol IT or
MET
Table 6.3
in this
phosphate encapsulated
in
in
longer
mean
residence
6.
and
6.2),
pharmacokinetics of the
a change in
its
pharmacodynamics
(overall effects).
These longer
MRT
which are linked to a slow absorption into the systemic circulation also suggest
design of once a day dosing
that the
may be
Several
studies
half-life
after
intratracheal
instillation
pulmonary
half-lives
of the drugs
in
compared to control
our results as they demonstrate the potential of liposomes to dramatically modulate the
pharmacokinetics.
that
by including
cholesterol in the liposomal formulation, the half-life increased from 1.4 hr to 18 hr.
likely that the
It is
wide range
in half-lives
is
due to the
differential size
vesicles.
These studies
illustrate the
possibility to further
in
AUC
after
TAP-sol
IT.
Assuming
that the
no
104
AUC
after
to
102 ng h/ml
TAP-sol
IT.
Assuming
that the
theoretically,
no
the
The reason
may be
TAP
after
However, as
it
will
TA
released
TA released
show
formulation
in
at
of TAP-lip resulted
accumulation of drug
in
in
intratracheal administration
of TAP-sol resulted
in a similar
AUC^gAUCpu.
TAP-lip
is
ratio
of 2.1,
AUQ^iAUCpi,
linked to the
An
analysis
of
single lung:plasma
drug
ratio
values after TAP-sol (data not shown) administration were constant over time and close to
two, indicating that the extent of protein binding in both lung cytosol and plasma tissue
was
nearly similar.
EC 50
values
(Table 6.3) were obtained for lung and plasma data after TAP-sol IT and TAP-lip IV. The
significantly greater
4 ng/g
lung)
105
EC
5o
value
is
there
is
may be due
studies
showed a 30
12 conversion of
TAP
(release
TAP
which resulted
shift
It is
was not
This
consistency
in
the
intrinsic
pharmacodynamic
is
properties
after
liposomal
administration
was expected,
since there
no
link
EC S0
(receptor affinity).
It
has been stated [100] that the overall effects of a drug (effect-time profile)
relationship),
pharmacodynamic properties
in
(e.g.
receptor binding
affinity), in
such a
way
that
changes
the
overall
effects
may be due
to
changes
in
the
alters
its
no strong
indication
for
changes
in
its
intrinsic
EC 50 E^).
,
(prolonged
receptor
occupancy)
in
the
a higher
mean
more
prolonged pulmonary
administered
the
effects,
in
106
in
pharmacokinetics for
TA
is
likely to
is
needed to
further
CHAPTER 7
EFFECT OF DOSE AND RELEASE RATE ON PULMONARY TARGETING OF GLUCOCORTICOIDS USING LIPOSOMES AS A MODEL DOSAGE FORM
7.1 Introduction
Inhaled glucocorticoids are the drugs of choice for the treatment of chronic asthma,
because they attenuate locally the underlying inflammatory process [16]. Aerosolized
glucocorticoid
delivery
to
the
lungs
has
potentially
great
clinical
and therapeutic
advantages. However,
it is
putative advantages due to the necessary frequent administration of inhaled drugs [39,
121].
desired.
oral
bioavailability
[51].
to prolong pulmonary drug action and reduce systemic side effects (anticancer drugs
[122];
[23, 99]).
theoretical
of drugs
in the
Gonda 1988
[54],
of the respiratory
tract
upon aerosol
107
108
administration.
(mode of
and particle
size) as well as
suitability for
improve dosage regimens, these simulations were not intended to quantify systemic
effects,
Recently,
we
selectivity
model suggest
that
drug
targeting.
of release of
rate
instillation
liposome preparations with different release rate characteristics and different doses of
encapsulated material. Thus, this study provides experimental in vivo information on
additional biopharmaceutical factors that should be considered in the development
of
Methods
7.2.1
Chemicals
Triamcinolone acetonide 21 -phosphate dipotassium
salt
was donated by
Bristol
(6,7-
NEN
109
>99%),
l,2-distearoyl-sn-glycero-3-phosphocholine
(DSPC)
and
1,2-distearoyl-sn-
glycero-3-{phospho-rac-(l-glycerol)}
Polar Lipids
7.2.2 Animals
rats
chapter
5.
was
slightly
modified to prepare
solutions of
DSPC
solvent
(89.4
mg) and
DSPG
(10.1
in
a round bottom
flask.
The organic
1
63 C
until
a dry film
ml of 60 mg/ml of
TAP
in
shown
lipids
that
efficiency.
hr at 63 C, above the
of the
nm
polycarbonate membrane
filters (Poretics,
Co., Livermore,
CA)
Inc.,
resulting
vesicles
were
separated
from
non-encapsulated
drug
by
size
exclusion
size
encapsulation
efficiency
in
5.
The
110
encapsulation efficiency
7. 1).
The
content
was determined
as described below.
%E=
A"fTAP(mol)
Amount of total
lipid
(mol)
Liposomes
6-carboxyfluorescein
Liposomes
containing
entrapped
(CF-liposomes)
were
CF was
marker to be encapsulated
TAP,
it is
a water soluble
substance and would be expected to partition similarly within the liposomal matrix.
One ml
of 200
200 nm or
and
800
nm
polycarbonate
filters
resulting
liposomes
were
processed
5.
The
lipid
method [109] with minor modifications. Duplicates of 0.010 ml of the liposome dispersion
were evaporated
Inc.,
at
40 C
for
RC10-10 (Jouan
Winchester
VA)
in
deionized water.
The tubes
were sonicated
temperature for
for 2 min, vortexed 4 times (15 seconds each time) and incubated at
room
hr.
Ill
and vortexed
3 times (20
at
room temperature
of the layers had occurred. The aqueous (upper) layer was removed
at
and the optical density (absorbance) of the chloroform layer was measured
the calibration curve, 0.01-0.1 ml aliquots of a mixture of
90% DSPC
and
10% DSPG
chloroform
(1
mg/ml
total lipid)
were assayed
in duplicate.
The
effect
under sink conditions using 6-carboxyfluorescein as a marker and Triton X-100 (0.03%)
as a leakage inducer.
uM total phospholipid)
final
were
diluted 3-fold
concentration).
After
vortexing,
200
at
ul aliquots
incubated
37
C. Carboxyfluorescein efflux
5 hr
on a luminescence
LS50B
nm
after
addition
The percentage of
carboxyfluorescein released
was
I(%)
= ,00
*Sy
* 7*
where Ix
is
the
100%
112
fitted to
Lake
City,
UT) and
the half-life of
release
was
initial
TAP
800
nm
liposomes
TAP) were
administered by intratracheal
instillation as
described in chapter
5.
at 1, 2.5, 6,
lungs and livers were immediately processed for receptor binding studies.
total
of 3
independent experiments were performed on different days under the same conditions.
To
similar experiments
were
A previously described
was employed
TA,
uM) was
as the
maximum
specific binding
justifiable
comparison of calculated
Bx
(R =0.985,
4).
113
7,2.9
Data Analysis
paired t-test
liver.
set
receptor occupancies of
AUC's) included
in
a given
experimental sub-set.
Statistical significance
was assumed
for p<0.05.
To
(AUC) was
quantify the degree of pulmonary targeting, the cumulative change from baseline
from percent
occupied receptor (EJ-time profiles for both the local and systemic organs. Pulmonary
targeting (PT) for TAP-lip
800
nm
200
nm
PT=AUCLung-AUCLiv=,
(eq. 7.3)
was ^ s0
Differences
among TAP-lip 800 nm, TAP-lip 200 nm and TAP-sol and among
significance using
doses,
test
assuming
alpha<0.05.
The mean
effect time
(MET) was
AUC
and
AUMC
according to equation 7.4 assuming that receptor levels returned to baseline during the
observation period. The area under the
first
for
MET= AUMC/AUC
(
eq 7.4)
In instances
where the
MET
was
calculated from
AUG
and
AUMCt, where t
114
7.3 Results
7.3.1 Characterization
of liposomes
particle diameter
for the
nm
nm
0.014 (n=21)
initial
for the
800
used
nm
70%
of the
amount of
16.5
lipid
nm was
1 :7.
2.1
plot
showed a
released
about
80%
of CF with a
half-life
20% was
released with a
half-life
of 3.7
hr.
CF-lip 800
nm showed
initial
10% of the
CF O, =4
min) with a more prolonged terminal phase (t =9.0 hr) for the remaining
90%
of CF released.
115
Time
Figure 7.1.
//;
(hr)
of DSPC:DSPG CF-liposomes
at
37 C under sink
nm
liposomes
, 800 nm
remaining
liposomes
The
percentage
of
is
6-carboxyfluorescein
7.3.3
Receptor Binding
Time courses of
intratracheal administration
free
glucocorticoid
receptors
in
the
lung
and
liver
after
nm
are
compared
in
Figure 7.2 to
in solution
(TAP-sol) and
TAP
in
200
nm
nm
lip)
(chapter
5).
Pulmonary targeting (PT), denned as the difference between cumulative lung and
receptor occupancies,
liver
(PTtamp
goo
m=379 %*h,
No
nm
preparation
(PTTAiMip2oonm=150 (PTTAp^i=28
%*h
p<0.02).
TAP
in solution
%*h
p>0.2; Figures
showed
significant difference
116
among these
preparations.
The mean
effect times
(MET;
with the in vitro release studies; these studies demonstrated that with increasing liposome
size,
both in vitro
mean
effect time
were
increased.
The
bell
TAP
dose and
PT
with a
maximum pulmonary
7.5,
targeting
Table
7.2).
There
were
all
doses except
450
significant difference
rest
significant difference
7.5,
117
12
Time
(hr)
Time
(hr)
Time
Figure 7.2.
(hr)
Lung
and
liver
intratracheal instillation
of 100 ug/kg
of:
5);
B)
TAP-lip 200
represent
nm from
(from chapter
for
5);
mean S.D.
n=
3 to 6
118
Table
7.
pulmonary
solution
effect times
(MET)
of TAP
in
(TAP-sol,
immediate release;
100
ug/kg),
TAP
in
200
liposomes (TAP-lip 200 nm, intermediate release; 100 ng/kg) and 800 nm liposomes (TAP-lip 800 nm, slow release; 100 ug/kg).
nm TAP in
TAP-sol*
TAP-lip 200
nm*
TAP-lip 800
nm
(AUC %*h)
1070
LUNG
LIVER
Pulmonary Targeting (%*h)
350
340
770
620
700
28
150
370
(AUCWAUCW)
Pulmonary Targeting
liv
1.0
1.2
1.5
Effect
5.7
>6.2
Table
7.2.
Cumulative receptor occupancy (AUC), pulmonary targeting and mean pulmonary effect times (MET) after intratracheal administration of
escalating doses of TAP in
800
nm
liposomes.
DOSE (ug/kg)
2.5
7.5
25
100
450
(AUC %*h)
LUNG
LIVER
Pulmonary Targeting (%*h) AUCtag-AUCiivc Pulmonary Targeting
92
78
338
111
345
130
1074
1261
696
1080
227
.
213
379
176
(%*h) AUCi^AUCuvs,
3.0
2.7
1.5
1.2
2.5
3.9
>6.2
>8.1
119
TAP-sol
200
400
600
Pulmonary targeting as a function of drug release rate: TAP-sol (immediate release), TAP-Iip 200 nm (intermediate release), TAP-lip 800 nm (slow
release). Statistical significance
was assumed
for alpha
<
0.05.
120
Time
(hr)
Figure 7.4.
Lung
A)
and
liver
intratracheal instillation
of escalating doses of
7.5 ug/kg, n=3;
TAP
in
800
nm
liposomes:
B)
mean
S.D.
121
200
400
TAP
in
800
nm
mean
S.D.
7.4 Discussion
[1]
used
here
incorporates
pharmacokinetic
and
after inhalation,
and the
effects
of pharmacokinetics on pulmonary
Based on
model suggested
in the
that,
by
altering the
lung pulmonary
we were
interested in
vivo.
For
this purpose,
[56], This
We have reported in
chapter
5 that pulmonary targeting can be calculated as the ratio of cumulative receptor occupancy
122
in
lung versus liver (AUCmng/AUCuver). However, since computer simulations [56] have
shown
that
in these
terms reaches
its
maximum
as dose
approaches zero,
we
is
as the
when pulmonary
selectivity
is
we
selected liposomes as a
rate
on pulmonary
lipid
on our
in vitro
nm)
with longer
half-life
who
is
determined by the
number of lamellae
in
it
initial
for
800
nm
may be may
beneficial since
in the
lung
be attained, followed by a prolonged release for the remainder of the dosage form.
Computer simulations
shown
the advantage of
mean of
upon
some
demonstrated, that pulmonary selectivity depends on a slow release from the delivery form
123
we
in
higher
pulmonary
7.2; see
effects (Figure
MET Table
is
been shown to increase the cumulative systemic effect for a given dose [100]. Our
observations
indicate
that
pulmonary
selectivity
was
enhanced
because
the
free
in
rate
was slowed
in
down. The
clinical
that
by administering glucocorticoids
slow release preparations, the frequency of administration as well as the dose can be
decreased. This will lead to reduction of systemic side effects while pulmonary effects are
maintained.
These
findings
also
suggest
that
since
currently
available
inhaled
The
nm showed
a typical bell
maximum
at
7.5),
until
maximum was
)
reached
(Figure 7.4 panels B, C, D). Both the low (2.5 ug/kg) and high (450 ug/kg
doses of
TAP
resulted
in
close
consequently, in low pulmonary targeting (Figure 7.4A, 7.4E). Thus, these studies suggest
124
there
is
maximum pulmonary
selectivity is
observed
(e.g.
it
is
of
clinical
response in asthma of
show
that overdosing
and underdosing
will
always go
parallel
concept
may
ultimately be
of
clinical
importance: clearly,
treated
with high doses of inhaled glucocorticoids might be prone to increasing systemic side
effects at the
selectivity
of glucocorticoids have
included the development of drugs with higher systemic clearance (such as budesonide
[105]), and lower oral bioavailability (such as fluticasone propionate [128]), as well as the
design of pulmonary delivery devices with increased pulmonary deposition (such as dry
powder
inhalers [129]).
To our knowledge
The animal
experiments shown here together with previously reported computer simulations [1]
illustrate the
two
local treatment
of
airway diseases, as well as in the design of dosage regimens which would minimize
systemic side effects
CHAPTER 8 ASSESSMENT OF PULMONARY SELECTIVITY OF TRIAMCINOLONE ACETONIDE AND FLUTICASONE PROPIONATE USING AN EX VIVO RECEPTOR BINDING ASSAY
8.1 Introduction
new
treatment of asthma. These drugs tend to accumulate in the lungs, have a lower oral
bioavailability,
and a higher systemic clearance [23, 51, 53], factors that improve the
show
steroid potency [130], thus allowing clinical effects to be achieved at lower doses.
Triamcinolone acetonide
is
is
use in asthma [131]. Fluticasone- 1 7-propionate, has recently been approved in eight
countries as a nasal spray for seasonal rhinitis and for the treatment of asthma. Fluticasone
propionate
receptor
affinity
(eight
times
more than
%) among
inhaled glucocorticoids,
is
powder
We
have shown
in
chapter
is
affected
by the
rate.
shown
significant differences
pulmonary absorption
profile
125
126
also different.
In vitro (induction of protease inhibitor [133]; receptor binding [56, 134]) and in
vivo studies (skin-blanching [16]; inhibition of ear
is
determined
not
only
by
the
but
also
by
its
pharmacokinetics.
Some
studies
Cortisol [137],
two
However, these
studies give
no indication of pulmonary
effects.
it
is
their
pulmonary
selectivity
because of quantification
effects.
The goal of
currently
available
is
two
inhaled
glucocorticoids;
triamcinolone
acetonide
and fluticasone
propionate using the ex vivo animal model previously described and to compare them with
data reported in chapter 5 for a glucocorticoid-encapsulated liposome formulation. In this study
we have
included not only the liver as a systemic organ to assess the extent of
systemic absorption, but also the kidney and spleen, because of concerns on high intrinsic
hepatic clearance
on
liver receptor
occupancy.
127
Methods
8.2.1 Materials
(St. Louis,
MO);
Research and Development Ltd. UK. Triamcinolone acetonide micronized was obtained
NF
extra fine
EM
industries
NEN*
Research Products (Boston, MA). All other reagents were of analytical grade.
8.2.2
Drug formulations
Fluticasone propionate
was dissolved
in a
mixture of
PEG
(3:1
intravenous administration.
mg
lactose.
8.2.3
Animals
All animal procedures
University of Florida, an
AAALAC
rats,
in
rat
chow, but
128
8.2.4
Drug Administration
1
8.2.4.
Intratracheal instillation
in
the operating
room
them accustomed to the new environment. The day of the experiment the
handled gently to produce
rats
were
minimum
stress.
rats
were placed
in a rat
10%,
1.5
ml of xylazine
anesthesia
2%
and 0.5 ml of
acepromazine 1%)
at
the dose of
ml/kg.
The depth of
was checked by
tail
aseptically cleaned
vertical
incision
was made
originating
above the
sternal notch.
carefully dissected
the
third
rings.
of dry powders of 5
A mixture
mg
of
extra
fine
either
fluticasone
propionate (FP) or
in
and
instilled in the
A sham rat
was included
lactose).
in
each
set
mg
total
FP and
TA
respectively,
were performed
129
injection, animals
and the femoral vein exposed by dissection. Either 100|il of fluticasone propionate
solution (200u.g/ml) or lOOul of the vehicle (for the
sham
rat)
were slowly
injected using a
tuberculin syringe with a 27 gauge needle. Animals (one animal per time point) including
sham
rat,
were decapitated
at 1, 3, 6, 12,
or 18 hours after
instillation.
Lung,
liver,
spleen
studies.
total
of four
independent experiments were performed for a given time point after IV administration.
The
skin
was used
to close the
incision.
its
abdomen
in a
lamps and allowed to recover while observed (monitoring of the breathing rate by visual
inspection of the chest wall).
8,2,5
in
chapter 4
(30
uM)
was used
maximum
was
justifiable as the
comparison of calculated
Scatchard method resulted
B,
in similar
values for
(chapter 4).
130
8.2.6
Data Analysis
A
between
paired t-test
was used
Iung:liver, Iung:spleen,
pool of paired
receptor occupancies of
all
AUC's)
included in a given
experimental sub-set.
Statistical significance
was assumed
for p<0.05.
In order to quantify the degree of pulmonary targeting, the cumulative change from
baseline
(AUC E) was
percent occupied receptor (EJ-time profiles for both the local and systemic organs. Pulmonary
targeting
was defined
as
PT=AUCE Lg-AUCE ,.
where
(eq 8.0)
AUC
Lug-AUC E Kidney or as
the treatments
AUC
tested
e Lung-
E Lung-
AUC E spira,)
within and
among
were
for significance using Tukey's multiple comparison test assuming a<0.05. Differences in
AUCE
TA
nm
nm
data
reported in chapter
7),
were
effect
AUC
and
AUMC
the
first
determined for the pulmonary receptor occupancy by the trapezoidal rule from
versus t x -pairs.
E x *t x
AUC
and
AUMC were
a linear decline of the effect over time at late time points [100].
131
MET= AUMC/AUC
( eq .
8.1)
8.3 Results
shown
in
Figure
There was a
significant
(p<0.01, Table 8.2) and for lung versus kidney (p<0.04), while no significant difference
was found
for
lung versus
spleen
(p>0.6).
liver
(PT=400 %*hr),
little
8.1).
targeting
among
pairs
and lung/liver
was obtained
Mean
effect time
was
5.5 hr.
shown
in
Figure
There was a
liver
(p<0.1)
was found
lung versus spleen (p>0.4). Pulmonary targeting was most pronounced for the pair lung
versus liver
little
or no targeting
was observed
132
lung/spleen
8.1).
Multiple
comparison
was obtained
hr.
Mean effect
Table
8.1.
Cumulative receptor occupancy (AUC E ), pulmonary targeting and mean pulmonary effect time after IT and IV administration of fluticasone
propionate (FP, 100 ug/kg), IT administration of triamcinolone acetonide dry powder (TA, 100 ug/kg), IV administration of TA solution (100
Ug/kg), and IT administration of triamcinolone acetonide phosphate in 800
ran liposomes (TAP-lip
FPpw
IT adm
FPsol
TApw
IT
*
TA sol**
IVadm
TAP-lip 800
IVadm
adm
nm
IT adm*
AUC E (%
h)
LUNG
LIVER
756
724
655
280
320
1070
467
693
700
SPLEEN
716
KIDNFY
7Q7
609
Pulmonary targeting
%*hrfAUC,._-AlJC
70
10
(AUCel^-AUCbu)
(AUCel^-AUCesp,^,
(AUCELung-AUCEKMM,)
400 65
115
289
63
60
50
-40
370 100
140
120
+ 180
50
160 60
5.5
-40
115 + 80
4.7
Mean pulmonary
5.5
>6.2
al.
1995
[56].
133
Table 8.2
between
tissues.
Treatment
(AUCbl^-AUCbl)
0.01
0.01
(AUC
Lug-AUC B spleen,
FPIT
FPIV
TAIT
0.02
0.02
Table 8.3
among pulmonary
(AUC
E Lue -
AUC g organ).
Treatment
FPIT
NS
NS
(p> 0.05)
(P>0.05)
FPIV
TAIT
NS
(p> 0.05
NS
(p> 0.05)
NS
(p> 0.05)
shown
in
Figure
There was a
liver
(p<0.02) and lung versus kidney (0.02), while lung versus spleen receptor occupancy
resulted in close superimposition (p>0.9).
lung versus kidney (PT=115), followed by lung versus liver (PT=69) while no targeting
was observed
for
Multiple comparison
in
pairs
were no
significant (see
Table
8.3).
Mean
effect time
was
134
Figure 8.1
A) lung D versus
liver
, n=3; B)
n=3, after
versus kidney
intratracheal
powder (100
Hg/kg).
135
Figure 8.2
A) lung
versus kidney
in
n=3, after
intravenous
ug/kg).
of fluticasone propionate
solution
(100
136
12
Time
(hr)
Figure 8.3
for:
A) lung D versus
liver
, n=6; B)
n=6, after
versus kidney
intratracheal administration
Hg/kg).
137
Time
(hr)
Figure 8.4
Lung D and
liver
for:
A)
Intratracheal
in
800
nm
nm;
TAP
nm
138
AUC
Lung-AUC
e Kidney)
among
the treatment groups using Tukey's test revealed significant differences between
FP IV
:FP IT and
FP IV
TA IT,
FP IT
There was a
significant difference in
triamcinolone acetonide-dry
800
nm
Mean pulmonary
preparation
effect
time
(MET >
as a dry
powder (MET =
4.7; Table 8.
1).
8.4 Discussion
The
in
male
rats.
shown
systemic potency [135], receptor affinity [138, 139], and pharmacokinetic properties [22,
105, 107, 140] of inhaled glucocorticoids. In addition, pharmacokinetic/pharmacodynamic
PK/PD and
the
[141].
These
studies,
selectivity,
as
it
is
local, as well as
difficult to
evaluate because
pulmonary
human pharmacological
139
we
in
chapter
3,
to assess pulmonary
selectivity
In chapters 5
we
occupancy
in
lung and
liver.
we have
a marker for the extent of drug systemic exposure, but also tissues with no metabolic
activity
such as the kidney and spleen, because of concerns that the high
intrinsic hepatic
may
(AUC E i^=756
%*hr), kidney
(AUC E
ki(toc y
spleen
(AUC E
,p
occupancy was
significantly smaller
(AUC E u,=467
free
body (assuming no
understandable,
The
lower
liver receptor
occupancy observed
is
if
of FP. Because FP
is
(which
is
also indicated
by
its
oral bioavailability
at
free
liver
this,
showed
that the
IV
administration of
a close superimposition of
lung and liver receptor occupancies (see Table 8.1). These findings suggest that the liver
may
not be an appropriate organ to assess the extent of systemic absorption of drugs with
high intrinsic hepatic clearance such as fluticasone propionate. These results also suggest
that, the
IV
may
140
be
powerful
tool
to
assess
differences
in
intrinsic
hepatic
clearance
among
glucocorticoids.
The
were
of FP resulted
in receptor
occupancies which
significantly
lower for
liver
(AUC E
ii
VC
(AUC ELung=637
%*hr).
No
significant
(p>0.5).
i\
va
was
targeting
data
is
likely
of high
intrinsic
significant difference
between lung
is
The
intratracheal administration
in liver
(AUC E
were
ih.er=655
AUC E
Kidney=609
significantly
(AUC E
Lung
=724 %*hr).
(69
ivr
51
%*hr)
Table
This results suggested that the higher pulmonary targeting obtained upon IT
administration
was not
affected
by the drug
pulmonary
good makers
141
The
intratracheal
administration of
TA
administration of
FP
may
spill
this
pattern
is
yet to be determined.
It
is
known
that
redistribution
of several blood
cells to different
may be due
to this organ.
In the present study, the intratracheal administration of equal doses (100 ng/kg) of
FP and
TA
resulted in
no
significant difference in
defined as
was
of FP to
act as
We have
is
affected
by clearance, release
rate,
TA
affinities, differ
100 ng/kg
example, expressed as
TA
equivalents
(TA 100
p.g/kg versus
FP 900
absorption profiles of
TA
and FP are
relatively
similar,
targeting between
TA
exist
between these two drugs. Future studies are needed to determine the optimal dose of
is
able to achieve
maximum pulmonary
targeting.
142
The
intratracheal administration
in
mean pulmonary
effect time
(MET=5.5, Table
IT
administration of TA-dry
is
this finding
is similar.
In
contrast, however,
mean
The
intratracheal administration
in
800
nm
a significantly
AUCe Liver)
effect time
(MET)
fashion (Figure 8.4, Table 8.1), indicating the superiority of the liposomal formulation to
attain
pulmonary
selectivity.
CHAPTER 9 CONCLUSIONS
2.
at
and
in the
3.
Intratracheal
administration
of triamcinolone
acetonide
in
liposomes
(TAP-lip)
provided sustained lung receptor occupancy, and increased pulmonary targeting which
in
solution
or the intravenous
is
not only linked to the slow release characteristics of the drug, but also to the route
of administration.
4.
into
liposomes
changed
favorably
its
pharmacokinetics (longer mean residence time and higher local versus systemic drug
ratio),
as well as
its
overall
changing
its
intrinsic
143
144
5.
is
size:
the larger
6.
There
exists
stability
rate.
rate. It
suggests
may
they
may
pulmonary
targeting.
8.
Mean
effect
occupied)
is
inversely
proportional to release rate: the slower the release rate the longer the
mean
effect time.
may
There
Liver
is
can be achieved.
10.
may
11.
Apparently, there
is
in
pulmonary
the
selectivity
between
when
is
administered.
12.
Liposomal
TAP
glucocorticoids
to
the
lungs
via
topical
administration,
TA
dry-powder
formulation.
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The
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in
of
vivo lung
BIOGRAPHICAL SKETCH
15, 1965, to
in
at
the National
School of
as a
Mexico
City, in
in
1988. She
worked
&
five years.
She
entered graduate school in the College of Pharmacy at the University of Florida in January
1994. She
the pharmaceutical
June,
1997.
Her major
interests
lie
in
pharmacokinetics.
156
opinion it conforms to I certify that I have read this study and that in my scope and quality, acceptable standards of scholarly presentation and is fully adequate, in as a dissertation for the degree of Doctor of Philosophy.
C. Ibc'll^y
Guenther Hochhaus, Chair Associate Professor of Pharmaceutics
have read this study and that in my opinion it conforms to scholarly presentation and is fully adequate, in scope and quality, of standards acceptable as a dissertation for the degree of Doctor of Philosophy.
I
certify that I
Hartmut Derendorf
Professor of Pharmaceutics
I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.
,j.
--tughes
certify that
have read
this study
and
that in
is
my
opinion
it
conforms to
fully
Charles E.
L '4kJU, Wood
/^
Professor of Physiology
UNIVERSITY OF FLORIDA
3 1262
06554 8898