Autoradiographic examination of the organ distribution of 14C-labelled pentamidine in rats after intravenous and inhaled administration


Wingen, F.; Brägas, B.

Arzneimittel-Forschung 41(9): 937-945

1991


Experiments with Sprague Dawley rats showed, from a pharmacological point of view, the advantages of aerosolized pentamidine (over iv pentamidine) in the treatment of Pneumocystis carinii pneumonia.

Antibiotika
Bakterizide
Chemotherapeutika
Zytostatika
Antibacterials
Antibiotics
Chemotherapeutics
Cytostatics
Autoradiographic
Examination
of
the
Organ
Distribution
of
14
C-Labelled
Pentamidine
in
Rats
after
Intravenous
and
Inhaled
Administration
F.
Wingen
and
B.
Bragas
Summary
The
organ
distribution
of
'
4
C
pentamidine
was
studied
in
Sprague
Dawley
rats
by
means
of
whole-body
autoradio-
graphy
following
intravenous
and
inhaled
application
of
pentamidine
(Pentacarinat®,
CAS
140-64-7).
The
distri-
bution
time
after
intravenous
administration
of
5
mg
pen-
tamidine
per
kg
rat
was
30
min,
6
h,
24
h,
and
7
days,
respectively,
The
corresponding
times
after
administration
of
aerosolized
pentamidine
were
30
min
and
24
h.
The
distribution
of
radioactivity
was
also
determined
by
meas-
uring
radioactivity
in
punch
specimens
of
100µm
sections.
Besides
renal
excretion,
the
excretion
of
pentamidine
in
the
bile
and
via
the
salivary
glands
was
assessed
by
au-
toradiography.
Further
target
organs
included
the
spleen
and
the
bone
marrow.
As
early
as
after
30
min
no
radio-
activity
was
detectable
in
the
blood
vessels.
A
lack
of
ra-
dioactivity
in
the
brain
tissue
with
accumulation
of
pen-
tamidine
in
the
meninges
suggests
that
pentamidine
does
not
cross
the
blood/brain
barrier.
Following
intravenous
administration
the
lung
uptake
of
pentamidine
was
rel-
atively
low.
However,
increased
drug
concentrations
were
recorded
in
the
bronchial
system.
The
elimination
time
of
pentamidine
from
the
target
organs
was
long.
Lung
pen-
tamidine
concentrations
remained
almost
unchanged
for
a
period
of
7
days.
After
inhaled
administration
high
levels
of
pentamidine
were
measured
in
the
lung.
'
4
C
pentam-
idine
was
also
located
in
the
oropharyngeal
and
gastro-
intestinal
tract,
the
drug
stemming
from
pentamidine
in-
gested
or
licked
off
the
skin
by
the
animals.
No
further
target
organs
were
verifiable.
From
the
pharmacological
point
of
view,
these
studies
prove
the
advantages
of
pen-
tamidine
aerosol
in
the
treatment
of
Pneumocystis
carinii
pneumonia
(PCP)
and
provide
information
on
extrarenal
excretion
mechanisms
and
deep
compartments.
Zusammenfassung
Autoradiographische
Untersuchung
der
Organverteilung
von
Pentamidin
bei
Ratten
nach
intravenoser
und
Aerosol-
Verabreichung
Die
Organverteilung
von
'
4
C-Pentamidin
wurde
an
Spra-
gue-Dawley-Ratten
nach
intravenoser
Verabreichttng
von
Pentamidin
(Pentacarinat®,
CAS
140-64-7)
und
nach
In-
halation
als
Aerosol
ganztierautoradiographisch
unter-
sucht.
30
min,
6
h,
24
h
oder
7
Tage
nach
intravenoser
Gabe
von
5
mg
Pentamidin
pro
kg
Ratte
wurde
die
Ver-
teilung
der
Substanz
bestimmt.
Nach
Aerosol-Applikation
betrug
die
Verteilungszeit
30
min
bzw.
24
h.
Die
Radio-
aktivitatsverteilung
wurde
zuslitzlich
durch
Radioaktivi-
tiitsbestimmung
in
Stanzproben
von
100-,um-Schnitten
be-
stimmt.
Neben
der
Ausscheidung
uber
die
Niere
wurde
autoradiographisch
eine
Elimination
via
Leber
und
Spei-
cheldriisen
festgestellt.
Weitere
Zielorgane
neben
diesen
Exkretionsorganen
waren
die
Milz
und
das
Knochen-
mark.
Bereits
nach
30
min
zeigten
die
Blutgefdfle
keine
Radloaktivitat
mehr.
Die
fehlende
Aktivitat
im
Hirnge-
webe
bei
deutlicher
Pentamidin-Anreicherung
in
den
Hirnhauten
deutet
auf
eine
Undurchliissigkeit
der
Blut-
Hirn-Schranke
fur
Pentamidin
hin.
Nach
intravenoser
Gabe
zeigte
die
Lunge
eine
relativ
schwache
Pentamidin-
Aufnahme,
jedoch
mit
erhohter
Konzentration
im
Bron-
chialsystem.
Pentamidin
zeigte
in
den
Zielorganen
eine
hohe
Persistenz.
Die
Lungenspiegel
blieben
uber
einen
Zeitraum
von
7
Tagen
nahezu
unverandert.
Nach
inha-
lativer
Gabe
zeigte
sich
eine
deutliche
Anreicherung
in
der
Lunge.
Im
Oropharynx
und
im
Gastrointestinaltrakt
konnte
ebenfalls
'
4
C-Pentamidin
nachgewiesen
werden,
das
von
verschlucktem
und
von
der
Haut
abgelecktem
Pen-
tamidin
herriihrte.
Andere
Zielorgane
waren
nicht
er-
kennbar.
Die
Untersuchungen
belegen
aus
pharmakolo-
gischer
Sicht
die
Vorteile
der
Anwendung
von
Pentamidin-
Aerosol
bei
der
Pneumocystis-carinii-Pneumonie
und
ge-
ben
dariiber
hinaus
weitere
Hinweise
auf
extrarenale
Aus-
scheidungsmechanismen
und
tiefe
Kompartimente.
Key
words:
CAS
140-64-7
Chemotherapeutics
Pentacarinat®
Pentamidine,
organ
distribution
Clinical
Research
Department,
Rhone-Poulenc
Rorer
GmbH,
Cologne
(Fed.
Rep.
of
Germany)
Arzneim.-Forsch./Drug
Res.
41
(II),
Nr.
9
(1991)
Wingen
et
al.
Pentamidine
937
1.
Introduction
With
the
occurrence
of
the
acquired
immunodeficiency
syndrome
AIDS
there
has
been
a
drastic
increase
in
Pneumocystis
carinii
pneumonia
(PCP),
currently
the
most
frequent
life-threatening
opportunistic
infection.
About
80
%
of
AIDS
patients
will
develop
PCP
in
their
lifetime.
In
about
40
%
of
patients
the
diagnosis
of
AIDS
is
based
on
the
pneumonia.
At
present
trimethoprim
sul-
famethoxazole
and
pentamidine
(CAS
140-64-7)
are
the
two
first-line
drugs
in
the
treatment
and
prophylaxis
of
PCP
(Montgomery
1989).
Systemic
side
effects
are
fre-
quent
after
oral
and
parenteral
administration
of
these
drugs,
often
forcing
to
a
switchover
from
one
drug
to
the
other.
Waldman
et
al.
first
described
the
use
of
aerosol-
ized
pentamidine
in
rats
in
1973;
later
the
drug
was
de-
veloped
further
for
clinical
use
by
Debs
et
al.
(1987)
and
Girard
et
al.
(1987).
The
incidence
of
side
effects
was
found
to
be
considerably
reduced
by
a
decrease
in
extra-
pulmonary
drug
deposition
(Fox
and
Wingen
1989,
Leoung
et
al.
1990,
Montgomery
et
al.
1987).
Since
pentamidine
is
poorly
absorbed
in
the
gastrointes-
tinal
tract,
it
is
not
suitable
for
oral
administration.
A
rapid
decline
was
reported
in
serum
pentamidine
levels
after
intramuscular
or
intravenous
administration
of
4
mg/kg
pentamidine
(Bernard
et
al.
1985,
Conte
et
al.
1986,
Vohringer
et
al.
1989).
Target
organs
included
the
kidney
and
liver,
where
pentamidine
levels
five-
to
ten-
fold
those
in
the
lung
were
recorded
(Lanuoy
et
al.
1960,
Waalkes
et
al.
1970,
Waldman
et
al.
1973).
Pentamidine
is
eliminated
mainly
via
the
kidney,
hepatic
transfor-
mation
has
not
been
reported
(Goa
and
Campoli-Ri-
chards
1987).
The
aim
of
this
study
was
to
illuminate
the
organ
distribution
of
pentamidine
following
intravenous
and
inhaled
administration
to
rats
using
'
4
C-labelled
pen-
tamidine
and
whole-body
autoradiography
for
demon-
strating
target
organs
and
elimination
pathways.
2.
Materials
and
methods
2.1.
Radio-labelled
compound
Pentamidine
isethionate'
)
was
used
as
a
'
4
C
ring-labelled
sub-
stance
(Fig.
1):
1,5-di(4-amidino[
14
C]phenoxy)pentane-isethion
-
ate.
Calculated
formula:
C
23
H
36
N
4
0
10
S
2
;
molecular
weight
=
592.7;
specific
radioactivity:
532
kBq/mg
=
14.4
pCi/mg,
314.5
MBq/
mmol
=
8.5
mCi/mmol;
radiochemical
purity:
98
%
following
thin-layer
chromatography
and
radioscan;
chemical
purity:
96
%
after
HPLC.
Pentacarinat
®
;
manufacturer:
Rhone-Poulenc
Rorer
GmbH,
Cologne
(Fed.
Rep.
of
Germany).
c
,
NH
CH-OH
NH2
CH
2
S0
3
H
2
Pentamidine
isethionate:
MW
592.68
C201364401052
Fig.
1:
Structural
formula
of
pentamidine
isethionate.
X
=
site
of
'
4
C-la-
belling.
2.2.
Administration
Six
male
Sprague-Dawley
rats
(antral
Institute
for
Laboratory
Animal
Breeding,
Hannover,
FRG)
with
a
body
weight
of
247-
314
g
(age:
4
to
6
months)
received
I
4
C
pentamidine
either
in-
travenously
or
by
inhalation.
The
animals
were
kept
in
single
makrolon
cages,
water
and
food
were
available
ad
libitum
(Al-
tromin
diet
1320,
Altrogge,
Lage/Lippe,
FRG).
A
dose
of
5
mg/kg
rat
was
given
intravenously.
The
exact
dose
was
determined
by
weighing
the
contents
of
the
syringe
after
injection
(Table
1).
Total
injected
radioactivity
per
animal
was
655
to
751
KBq
=
17.7
to
20.3
µCi.
One
animal
each
was
killed
after
30
min,
6
h,
24
h
and
7
days
after
injection,
respectively,
by
CO
2
fumigation
and
prepared
for
whole-body
autoradiogra-
phy.
A
dose
of
4.3
mg/kg
rat
was
given
by
inhalation.
With
a
postu-
lated
equal
pulmonary
deposition
rate
of
5
%
by
way
of
the
air
jet
nebulizer
Respirgard
II
this
dose
corresponds
to
a
comparable
human
dose
of
300
mg
pentamidine.
The
Respirgard
II
nebulizer
intended
for
human
use
was
modi-
fied
for
the
present
study.
Since
the
inspiratory
pressure
of
the
two
rats
connected
to
the
device
was
too
low
to
open
the
inha-
lation
valve,
it
was
removed
and
replaced
by
a
Y
piece.
Polyeth-
ylene
funnels
were
placed
at
both
ends
of
the
Y
piece.
The
ani-
mals
were
fixed
at
fine
steel
tables
and
positioned,
their
nasal
and
oral
apertures
were
to
reach
the
flow
of
aerosolized
pentam-
idine.
The
inhalation
was
terminated
after
20
min,
the
amount
of
nebulized
solution
being
2
ml.
The
maximum
calculated
up-
take
of
radioactivity
was
218.7
KBq/rat
=
5.911.1Ci
(see
Table
1).
Animal
no.
5
was
killed
30
min,
animal
no.
6,
24
h
after
the
end
of
inhalation
by
CO
2
fumigation
and
prepared
for
autoradiog-
raphy.
2.3.
.Whole-body
autoradiography
The
sacrificed
animals
were
shampooed
and
washed
under
hot
water
to
remove
skin
contamination.
Subsequently
they
were
embedded
in
5
%
aqueous
carboxymethyl
cellulose
on
single
mi-
crotome
blocks
and
cooled
in
an
acetone/dry
ice
mixture
to
-
70
°C.
After
warming
up
to
-
30
°C,
using
a
PMV
cryomicro-
tome
(PMV
AB,
Stockholm
Sweden)
10-pm
and
100-pm
sagittal
sections
were
made
through
the
planes
of
eye/spleen
and
brain/
kidney.
Furthermore,
median
sections
were
made
(Ullberg
1977).
The
sections
were
dehydrated
in
a
cryostat
at
-30
V
for
12
h.
10-pm
sections
were
covered
with
Ultrofilm-3H
(LKB,
Munich-
Grafelfing,
FRG),
fixed
under
vacuum
and
kept
in
light-
and
air-
tight
containers
at
-
20
°C
for
14
days
(in
the
case
of
animals
no.
5
and
6,
for
8
weeks).
The
100-gm
sections
were
used
for
the
semiquantitative
deter-
mination
of
radioactivity
in
the
various
organs.
With
the
aid
of
a
pneumatic
punch
2
specimens
(0
4
mm)
per
organ
were
col-
lected
in
each
case
and
the
14
C
content
determined
by
liquid
scintillation
spectrometry
after
having
burnt
the
samples
in
a
sample
oxidizer
(Packard,
Model
306).
HNo
C
0-ICH215
-0
Table
1:
Experimental
design.
Animal
Body
weight
on
application
(8)
Body
weight
on
sacrifice
(8)
no.
(mg/kg)
Route
of
application
Volume
of
application
(ml)
Dose
applied
Radioactivity
applied
(µCi/Mbq)
Distribution
time
after
application
i
2
3
4
5
6
247
273
288
314
261
277
283
280
298
314
261
277
i.v.
i.v.
i.v.
i.v.
aerosol
aerosol
0.5
0.55
0.55
0.6
1
ml
1
ml
5.1
5.1
4.6
5.1
1
.
4
1
.
4
17.73/0.48
18.61/0.50
18.61/0.50
20.30/0.55
5
.
910
/0.16
5
.
910
/0.16
7
d
24
h
6
h
30
min
30
min
24
h
")
I
ml
'
4
C
parent
solution
+
5
m11-1
2
0;
in
20
min
2
ml
solution
were
nebulized,
(1
ml/rat).
938
Arzneim.-Forsch./Drug
Res.
41
(11),
Nr.
9
(1991)
Wingen
et
al.
-
Pentamidine
2.
C
d
e
Fig.
2:
Whole-body
autoradiographs
of
rats
30
min,
6
h,
24
h
and
7
days
(from
top
to
bottom)
after
i.v.
application
'
4
C
pentamidine
isethionate
at
a
dose
of
5
mg/kg
b.w.
Duration
of
exposure:
14
days.
Note
the
marked
and
persistent
accumulation
of
14
C
in
the
salivary
gland
(a),
liver
(b),
spleen
(c),
kidney
(d)
and
gastric
mucosa
(e).
after
administration
of
aerosolized
pentamidine:
Even
though
the
amount
of
the
administered
total
radioactiv-
ity
was
smaller
for
technical
reasons
compared
with
that
following
intravenous
application,
the
whole-body
auto-
radiographs
exhibit
a
clear
accumulation
of
pentamidine
in
the
lung
tissue
30
min
after
completing
inhalation.
Pentamidine
also
accumulates
in
the
gastrointestinal
tract
and
in
the
oropharynx
and
nose (Fig.
5).
No
accu-
mulation
is
verifiable
in
the
target
organs
found
after
in-
travenous
administration.
24
h
after
the
end
of
inhalation
the
distribution
pattern
in
the
lung,
trachea
and
oropharynx
is
similar
to
that
after
30
min
(Fig.
6).
No
radioactivity
is
any
more
recovered
in
the
gastrointestinal
tract
except
for
the
lower
intestinal
segments,
which
points
to
faecal
excretion
(Fig.
7).
Table
2
contains
a
semiquantitative
analysis
of
the
whole-
body
autoradiographs.
Determination
of
'
4
C
was
per-
formed
in
punch
specimens
of
100
gm
sections
with
a
diameter
of
4
mm.
The
mean
0-value
was
65
dpm.
In
analogy
to
the
autoradiographic
findings
the
liver,
spleen
and
kidney
were
the
organs
with
the
highest
levels
of
ra-
dioactivity.
Hepatic
radioactivity
remained
almost
un-
939
2.4.
Evaluation
The
whole-body
autoradiographs
were
evaluated
qualitatively
by
microscopy.
For
semiquantitative
analysis
of
the
'
4
C
distribution
in
the
individual
organs
and
tissues
cpm-values
(counts/min)
were
converted
to
dpm-units
(disintegrations/min)
using
exter-
nal
(for
sample
oxidizer)
and
internal
(for
liquid
scintillation
spectrometry)
standards.
Accumulation
ratios
of
the
target
or-
gans,
liver,
kidney
and
spleen,
were
calculated
in
comparison
with
the
lung.
3.
Results
Fig.
2
provides
an
overview
of
the
whole-body
autora-
diograps
30
min,
6
h,
24
h
and
7
days
after
intravenous
administration
of
5
mg/kg
'
4
C
pentamidine.
In
the
fol-
lowing
it
is
proceeded
on
the
consideration
that
the
dis-
tribution
of
the
'
4
C
radio-label
refers
to
the
distribution
of
'
4
C
pentamidine,
as
no
pentamidine
metabolites
have
so
far
been
identified.
As
early
as
after
30
min
distribution
a
substantial
accu-
mulation
of
"C
pentamidine
is
detectable
in
the
kidneys,
liver
and
spleen.
Drug
accumulation
is
also
demonstrable
in
the
salivary
glands,
small
intestine
and
bone
marrow
as
well
as
in
the
meninges.
Pentamidine
accumulates
in
a
smaller
degree
in
the
lung
parenchyma
(Fig.
3)
and
gas-
tric
mucosa,
in
the
pylorus
and
skin
and
muscles.
No
'
4
C
pentamidine
is
found
in
the
brain
tissue
or
in
the
stomach
and
large
intestine
contents.
Bone
tissue
too
is
devoid
of
4
C
pentamidine
except
for
the
periosteal
and
endosteal
areas.
A
slight
deposition
of
pentamidine
is
appreciable
in
the
tendons
of
muscles,
especially
in
the
muscles
of
the
extremities
and
neck
as
well
as
in
the
tongue
(Fig.
4).
The
described
distribution
pattern
of
pentamidine
re-
mains
largely
unchanged
after
a
period
of
6
h
and
24
h
up
to
7
days.
The
radioactivity
in
the
small
intestine
de-
creased
in
favour
of
an
increase
in
radioactivity
in
the
large
intestine.
The
rapid
occurrence
of
pentamidine
in
the
small
intestine
after
30
min
with
a
lack
of
radioactiv-
ity
in
the
stomach
contents
suggests
that
pentamidine
is
eliminated
in
the
bile.
Pentamidine
excreted
in
the
bile
to
the
intestine
is
eliminated
mainly
in
the
faeces.
As
to
the
gastric
mucosa
the
fundus
only
contains
little
radio-
activity,
whereas
radioactivity
is
clearly
demonstrable
in
the
pylorus
with
its
acid-forming
cells
even
after
7
days.
A
magnification
of
the
salivary
gland
(Fig.
4)
shows
dif-
ferent
pentamidine
concentrations
in
the
glandular
pa-
renchyma
and
glandular
ducts,
indicative
of
excretion
in
the
saliva.
Renal
radioactivity is
not
reduced
within
the
first
24
h;
after
7
days,
however,
total
activity
is
clearly
decreased,
with
a
remaining
high
accumulation
in
the
glo-
meruli
and
in
the
medullo-cortical
transitional
zone.
Based
on
the
autoradiographs,
there
are
three
elimina-
tion
pathways
for
pentamidine:
1.
elimination
via
the
kidney,
2.
elimination
in
the
bile,
3.
elimination
in
the
saliva.
Besides
the
excretory
organs,
pentamidine
persists
for
a
prolonged
period
in
the
heart
muscle,
thymus,
glandular
stomach,
bone
marrow
and
spleen,
especially
in
the
red
pulp.
Furthermore,
pentamidine
accumulates
in
the
me-
ninges,
whereas
no
pentamidine
uptake
is
established
in
the
brain
tissue
(Fig.
5).
The
lung
tissue
contains
an
amount
of
pentamidine
com-
parable
with
that
located
in
the
muscles.
A
higher
accu-
mulation
of
pentamidine
is yet
noted
in
the
bronchial
system,
particularly
visible
in
the
tracheal
bifurcation.
Apparently
pentamidine
accumulates
in
the
bronchial
epithelium
(Fig.
3).
Compared
with
the
marked
accumulation
of
intrave-
nously
administered
pentamidine
in
the
liver,
kidney,
spleen
and
salivary
glands
and
the
relatively
negligible
concentration
in
the
lung
tissue,
the
situation
is
different
Ar
zneim.-Forsch./Drug
Res.
41
(II),
Nr.
9
(1991)
ifikk
Wingen
et
al.
Pentamidine
Fig.
3:
Details
of
thoracic
organs
6
h
(top)
and
7
d
(bottom)
after
i.v.
application
of
14
C
pentamidine
isethionate
at
a
dose
of
5
mg/kg
b.w.
Persistent
increased
accumulation
of
'
4
C
in
the
vascular
walls
and
bronchial
system
as
compared
with
the
lung
parenchyma.
Left:
native
sections;
right:
autoradiographs.
changed
for
24
h,
dropping
within
seven
days
to
about
half
the
initial
value.
The
level
of
radioactivity
in
the
spleen
showed
a
more
rapid
fall.
The
transitory
increase
in
radioactivity
in
the
kidney
points
to
renal
excretion
of
pentamidine
promptly
released
from
other
compart-
ments.
Radioactivity
gradually
decreased
in
the
small
in-
testine,
while
there
Was
a
transient
increase
in
radioac-
tivity
in
the
large
intestine
contents,
which
suggests
faecal
excretion,
as
indicated
by
the
autoradiographs.
The
level
of
radioactivity
in
the
muscles
(extremities,
tongue,
heart)
was
almost
steady
throughout
the
observation
pe-
riod,
this
also
applies
to
skin
and
bone
marrow.
Lung
radioactivity
also
remained
nearly
unchanged
during
the
investigation.
The
relative
accumulation
of
'
4
C
pentamidine
in
the
ma-
jor
target
organs
compared
with
lung
accumulation
is
shown
in
Table
3.
The
liver/lung
accumulation
ratio
is
even
above
9
following
intravenous
administration.
Sim-
ilar
values
were
measured
in
the
case
of
the
kidney/lung
and
spleen/lung
ratios.
The
autoradiographs
and
the
se-
miquantitative
analysis
of
the
tissue
specimens
showed
that
pentamidine
accumulated
predominantly
in
the
liver,
kidneys
and
spleen
after
intravenous
administra-
tion.
Hepatic
accumulation
was
9.1-fold,
renal
accumu-
940
Arzneim.-Forsch./Drug
Res.
41
(11),
Nr.
9
(1991)
Wingen
et
al.
Pentamidine
30'
Fig.
4:
Details
of
the
head/neck
area
30
min
(top)
and
6
h
(bottom)
after
i.v.
application
of
'
4
C
pentamidine
isethionate
at
a
dose
of
5
mg/kg
b.w.
Ap-
preciable
secretion
of
'
4
C
in
the
salivary
gland
(a),
accumulation
in
streak
form
in
the
neck-
(b)
and
tongue
muscles
(c).
lation
12.5-fold
and
splenic
accumulation
7.2-fold
higher
than
that
in
the
lung.
After
administration
of
aerosolized
pentamidine
the
highest
"C
pentamidine
concentrations
were
recorded
in
the
lung;
most
of
the
other
target
organs
reached
the
de-
tection
limit,
so
that
calculated
ratios
would
hardly
be
conclusive.
The
detection
limit
is
around
10
nCi/g
tissue.
4.
Discussion
Whole-body
autoradiography
is
a
primarily
qualitative
method
e.g.
for
the
demonstration
of
target
organs
and
for
the
illumination
of
elimination
pathways.
It
should
be
noted
that
accumulation
of
active
substances
in
organs
and
tissues
is
not
a
precondition
or
a
warranty
for
phar-
macological
and
toxicological
effects.
The
sensitivity
of
the
target
cell
to
the
drug
is
equally
important.
This
holds
true
for
the
substance
under
study,
pentamidine,
which
Possesses
a
proven,
marked
therapeutic
and
prophylactic
effect
in
Pneumocystis
carinii
pneumonia
following
in-
Table
2:
Determination
of
'
4
C
in
punch
specimens
from
100
gm
whole
body
sections,
diameter
4
mm.
Sample
i.v.
30
min
6h
24h
7d
Baseline
45
85
76
58
Brain
107
68
160
87
Eye
83
104
135
165
Tongue
383
382
412
Lung
268
210
349
214
Thymus
164
175
235
266
Heart
433
711
476
272
Liver
1914
1902
1953
891
Spleen
1919
1246
905
807
Kidney
1974
2622
3183
919
Adrenal
gland
231
209
461
Pancreas
872
163
195
218
Stomach
contents
88
68
75
117
Small
intestine
contents
824
437
353
106
I
A
rge
intestine
contents
62
246
482
92
Appendage
contents
119
438
384
78
Testicle
96
184
120
103
Muscle
263
315
211
273
Skin
175
192
120
153
Bone/bone
marrow
406
234
414
321
Double
measurement
dpm/2
samples;
=
not
measured.
Table
3:
'
4
C
accumulation
ratios
of
target
organs
compared
with
the
lungs.
Accumulation
ratio
i.v.
30
min
6h
24h
7d
Liver/lung
7.1
9.1
5.6
4.2
Kidney/lung
7.4
12.5
9.0
4.3
Spleen/lung
7.2
5.9
2.8
3.8
travenous
and
intramuscular
administration,
even
though
pentamidine
uptake
is
clearly
lower
in
the
lung
than
in
other
organs,
particularly
the
kidney,
liver
and
spleen.
Nephrotoxicity
following
intravenous
adminis-
tration
is
a
relatively
frequent
side
effect
of
pentamidine,
whereas
liver
damage
is
a
more
rare
occurrence.
More-
over,
dyshaematopoiesis
(cumulation
in
the
spleen
and
bone
marrow)
is
relatively
rare.
The
lung
distribution
of
pentamidine
is
not
homogene-
ous
following
intravenous
administration,
a
marked
pen-
tamidine
accumulation
is
noted
in
the
bronchial
system
compared
with
the
lung
parenchyma.
Cellular
localisa-
tion
of
pentamidine
is
not
demonstrable
for
methodical
reasons;
however,
the
drug
apparently
accumulates
in
the
bronchial
epithelium.
This
could
explain
the
good
effi-
cacy
of
relatively
small
amounts
of
pentamidine
against
intra-alveolar
and
intrabronchiolar
Pneumocystis
carinii
organisms.
In
contrast
to
the
negligible
accumulation
of
pentamidine
in
the
lung
tissue
after
intravenous
administration,
as
ex-
pected
after
inhaled
administration
the
lung
is
the
main
target
organ.
Furthermore,
a
deposition
of
'
4
C
pentami-
dine
is
appreciable
in
the
oropharynx
and
in
the
lumina
of
the
gastrointestinal
tract.
The
nasal
deposition
also
meets
the
expectations,
especially
particles
larger
than
5
gm
are
deposited
in
the
upper
respiratory
tract.
The
Respirgard
II
nebulizer
for
use
at
the
clinic
is
equipped
with
a
valve
between
the
nebulizer
chamber
and
the
mouthpiece
filtering
out
particles
of
sizes
>
5
gm.
This
valve
had
to
be
removed
in
our
investigations,
as
it
can
only
be
opened
through
a
certain
negative
inspiratory
pressure
not
attained
by
the
experimental
animals
with
the
present
experimental
design.
Therefore,
particles
ex-
ceeding
5
gm
also
reached
the
upper
respiratory
tract
in
rats,
where
they
were
deposited.
Thus,
the
deposition
in
the
nasal
and
buccal
cavities
is
comparatively
higher
and
Arzneim.-Forsch./Drug
Res.
41
(II),
Nr.
9
(1991)
%%gen
et
al.
Pentamidine
941
Fig.
5:
Details
of
the
head
30
min
after
intravenous
application
(top)
of
5
mg/kg
and
after
inhalation
(bottom)
of
about
1.4
mg/kg
pentamidine
isothionate.
No
'
4
C
demonstrable
in
the
brain
(a),
radioactivity
clearly
detectable
in
the
me-
ninges,
e.g.
basal
area
and
tentorium
cerebelli
(b),
following
i.v.
application.
Marked
accumulation
of
'
4
C
in
the
nasal
(c)
and
buccal
cavities
(d)
following
inhalation.
Left:
native
sections,
right:
autoradiographs.
the
lung
deposition
lower
than
expected
in
human
use
of
the
Respirgard
II
nebulizer.
The
accumulation
in
the
lu-
mina
of
the
gastrointestinal
tract
may
be
explained
by
the
fact
that
the
contaminated
nasal
area
was
licked
an
pentamidine
deposited
in
the
oropharyngel
area
swal-
lowed
by
the
animals.
No
major
pentamidine
absorption
is
appreciable
in
the
lung
with
the
administered
dose
and
the
given
duration
of
inhalation,
as
in
that
case
drug
ac-
cumulation
would
have
to
expected
in
the
liver,
kidneys
and
spleen.
Following
intravenous
and
inhaled
administration,
as
early
as
after
30
min
distribution
no
radioactivity
is
de-
tectable
in
the
blood
vessels.
This
is
in
line
with
distri-
bution
studies
in
HIV-infected
patients
after
intravenous
and
inhaled
administration
reporting
rapidly
decreasing
or
non-detectable
blood
pentamidine
levels
(V6hringer
et
al.
1990).
In
the
present
study
besides
the
known
renal
excretion,
the
excretion
of
pentamidine
in
the
bile
was
demon-
strated.
As
early
as
after
30
min
drug
accumulation
was
verifiable
in
the
area
of
the
duodenum.
The
radio-la-
belled
substance
was
excreted
within
24
h
in
the
faeces.
The
third
excretory
organ
was
the
salivary
gland,
where
pentamidine
also
persisted
for
a
long
time.
This
might
942
Arzneim.-Forsch./Drug
Res.
41
(11),
Nr.
9
(1991)
Wingen
et
al.
Pentamidine
Fig.
6:
Details
of
rat
neck/thorax/epigastrium
24
h
after
inhaled
application
of
about
1.4
mg/kg
pentamidine
isethionate.
Appreciable
accumulation
of
'
4
C
only
in
the
lungs
(a)
and
trachea
(b).
Left:
native
section,
right:
autoradiograph.
Fig.
7:
Whole-body
autoradiograph
24
h
after
inhaled
application
of
about
1.4
mg/kg
pentamidine
isethionate.
Appre-
ciable
accumulation
of
'
4
C
in
the
lungs
(a),
oropharynx
(b)
and
rectal
contents
(c).
Top:
native
section;
bottom:
auto-
radiograph.
explain
the
bitter
taste
noted
by
some
patients
after
i.v.
administration.
Determination
of
the
pentamidine
con-
centration
in
the
saliva
could
perhaps
replace
the
esti-
mation
of
serum
drug
concentrations
in
the
framework
of
drug
monitoring,
as
reported
for
zidovudine
(Rolinski
et
al.
1990).
The
distribution
of
radioactivity
in
the
stomach
is
clearly
limited
to
the
anatomic
boundaries
between
the
fore-
and
glandular
stomach.
While
the
forestomach
only
shows
slight
drug
accumulation,
there
is
a
marked
and
persist-
ent
accumulation
of
pentamidine
in
the
glandular
stom-
ach.
Obviously
pentamidine
base
accumulates
in
the
area
of
acid-secreting
cells.
Ar
zneim.-Forsch./Drug
Res.
41
(II),
Nr.
9
(1991)
Wingen
et
al.
Pentamidine
943
The
lack
of
radioactivity
in
the
brain
tissue
with
a
distinct
and
persistent
accumulation
of
'
4
C
pentamidine
in
the
meninges
suggests
that
the
drug
hardly
crosses
the
blood/
brain
barrier.
A
quantitative
evaluation
of
whole-body
autoradio-
graphs
can
be
undertaken
by
means of
micro-scales
if
the
distribution
of
radioactivity
is
in
the
range
of
calibration
standards.
With
great
differences
in
accumulation
among
the
individual
compartments,
like
in
the
present
case,
no
densitometric
estimations
are
possible
because
of
super-
imposition.
For
the
quantitation
of
the
accumulated
amounts
we
therefore
chose
the
determination
of
'
4
C
in
punch
specimens
with
a
diameter
of
4
mm,
obtained
from
100
gm
whole
body
sections.
Like
densitometry
of
autoradiographs,
this
method
is
semiquantitative,
since
punch
specimens
of
major
organs
do
not
always
provide
a
cross
section
representative
of
the
whole
organ,
e.g.
when
the
majority
of
blood
vessels
are
affected.
This
can
be
partly
compensated
with
multiple
sample
collections.
In
punch
specimens
of
smaller
organs
the
tissue
under
study
is
not
always
separated
from
the
surrounding
tis-
sue,
e.g.
determination
of
radioactivity
in
the
bone
mar-
row
is
complicated
by
the
fact
that
bone
trabeculae
too
are
included
in
the
assay.
Overall,
the
combined
use
of
"C
determination
in
punch
specimens
and
qualitative
evaluation
of
whole-body
autoradiographs
permits
an
orienting
assessment
of
the
distribution
of
drugs,
above
all
demonstration
of
target
organs
and
elimination
path-
ways.
As
early
as
in
1970
Waalkes
et
al.
investigated
the
organ
distribution
of
non-radio-labelled
pentamidine
after
in-
Table
4:
Pentamidine
distribution
in
organs
of
mice
24
and
72
h
after
intraperitoneal
application
of
pentamidine
at
a
dose
of
10
mg/kg
b.w.
(acc.
to
Waalkes
et
al.
1970).
Organ
pentamidin
e
base/g
tissue
24
h
72
h
Kidney
52
20
Liver
28
10
Spleen
II
3
Other
abdominal/
pelvic
organs
8
2
Lungs
5
4
Carcass
3
2
Brain
<
0.3
<
0.2
traperitoneal
administration
of
10
mg/kg
to
mice.
Also
in
this
study
the
demonstrated
target
organs
were
the
kid-
neys,
liver
and
spleen,
which
showed
two-
to
ten-fold
higher
pentamidine
concentrations
compared
with
the
lung
after
24
h
(Table
4).
No
pentamidine
was
detectable
in
the
brain.
In
three
days
pentamidine
concentrations
decreased
in
the
target
organs
kidneys,
liver
and
spleen
to
a
third
of
the
24-h
value,
whereas
the
level
of
pentam-
idine
in
the
lung
remained
almost unchanged
over
this
period
of
time.
The
reduction
in
the
pentamidine
con-
centrations
in
the
kidneys,
liver
and
carcasses
was
mon-
itored
over
a
period
of
25
days
after
application;
during
this
period
there
was
a
decline
in
the
pentamidine
levels
in
the
kidneys
and
liver
by
a
factor
of
10
and
100,
re-
spectively.
In
this
study
Waalkes
reported
a
somewhat
more
rapid
reduction
in
the
kidneys
and
liver
after
intra-
peritoneal
administration
of
10
mg
pentamidine/kg
com-
pared
with
our
investigations
after
intravenous
admin-
istration
of
5
mg/kg
(Table
5).
Using
autopsy
specimens
from
AIDS
patients,
Bernard
et
al.
(1985)
studied
the
concentrations
of
pentamidine
in
some
organs
one
day
after
the
end
of
treatment
with
pentamidine
isethionate
i.v.
Depending
on
the
number
of
doses,
high
pentamidine
concentrations
were
estab-
lished
in
the
liver,
spleen,
kidneys
and
adrenal
glands.
Following
a
single
administration
the
lung
concentration
of
pentamidine
was
by
a
factor
of
44
below
that
in
the
liver
and
by
a
factor
of
10
below
that
in
the
kidney.
Fol-
lowing
multiple
administration
of
pentamidine
i.v.
rela-
tively
high
lung
concentrations
were
recorded
(Table
6).
The
high
levels
of
pentamidine
in
the
adrenal
glands
re-
ported
by
Bernard
et
al.
(1985)
were
not
confirmed
in
our
studies
in
rats.
Accumulation
of
'
4
C
pentamidine
in
the
adrenal
cortex
was
proved
by
autoradiography,
the
me-
dulla
only
contained
little
I
4
C
pentamidine.
This
discrep-
ancy
might
be
explained
if
the
biopsy
specimens
were
taken
from
the
cortical
region
in
the
study
concerned.
Girard
et
al.
(1987)
investigated
concentrations
of
pen-
tamidine
after
application
of
aerosolized
pentamidine
to
control
animals
and
immunosuppressed
rats
(Table
7).
14-fold
higher
pentamidine
concentrations
were
meas-
ured
in
the
lung
tissue
than
in
the
kidneys
of
healthy
con-
trols.
In
immunosuppressed
animals
with
Pneumocystis
carinii
pneumonia
lung
pentamidine
levels
were
lower.
At
all
doses
of
pentamidine,
lung
levels
were
approxi-
mately
fivefold
those
recorded
in
the
kidneys.
This
ratio
was
also
reported
by
Farinotti
et
al.,
who
studied
the
or-
Table
5:
Pentamidine
distribution
in
organs
of
mice
at
various
time
points
after
intraperitoneal
administration
of
10
mg
pentamidine/kg
b.w.
(method:
extraction
+
spectrofluorometry
(acc.
to
Waalkes
et
al.
1970).
Days
after
application
Total
dose
(mg)
µg/g
tissue
Total
tissue
content
Kidney
Carcass
Liver
mg
%
of
dose
1
0.25
52
28
4
0.15
60
3
0.28
20
10
2
0.07
25
4
0.25
12
6
2
0.05
20
6
0.24
13
5
2
0.05
21
8
0.26
13
4
2
0.05
19
15
0.27
10
2
1
0.04
15
25
0.26
5
0.3
<
0.4
0.01
4
n
=
2
animals
per
time
point,
concentrations
in
lig
pentamidine
base.
Table
6:
Pentamidine
accumulation
in
autopsy
specimens
following
intravenous
treatment
in
patients
(acc.
to
Bernard
et
al.
1985).
Patient
Number
of
doses
Pentamidine
levels
(p.g/g)
1
d
after
end
of
therapy
(death)
Lungs Liver
Spleen
Kidney
Suprarenal
gland
1
1
<
0.8
35
40
8.5
19
2
2
<
0.8
23
40
13
n.d.
3
5
42
112
28
62
n.d.
4
15
56
368
300
123
92
944
Arzneim.-Forsch./Drug
Res.
41
(II),
Nr.
9
(1991)
Wingen
et
al.
Pentamidine
Table
7:
Organ
levels
of
pentamidine
in
immunosuppressed
rats
after
3
weeks
treatment
(3
times/week)
with
pentamidine
aerosol
using
a
Bird
neb-
ulizer
(25
%
<
5
gm)
(acc.
to
Girard
et
al.
1987).
Dose
of
penta-
No.
of
Pentamidine
level
(gg/g)
Lungs
Liver
Kidney
Group
midine
animals
(mg/kg)
Control
8.7
3
210±20
0.58±0.06
15±4
After
immuno-
4.7
3
18±
8
0.30±0.10
4±1
suppression
6
4
42±
9
0.26±0.03
7±1
14.6
8
128±17
1.40±0.20
26±5
gan
distribution
of
pentamidine
mesylate
24
h
after
ad-
ministration
by
inhalation
to
rats
(Farinotti
et
al.
1990).
The
presented
whole-body
autoradiographs
show,
in
ac-
cordance
with
the
literature,
the
advantages
of
aerosol-
ized
pentamidine
use
in
Pneumocystis
carinii
pneumo-
nia
from
the
pharmacological
point
of
view.
Further-
more,
information
was
obtained
of
extrarenal
elimina-
tion
pathways such
as
excretion
in
the
bile
and
saliva.
Long
persistence
of
pentamidine
in
the
known
and
new
target
organs
was
proved.
5.
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ki
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Pentamidine
945