Pharmacologic aspects of pentamidine


Waalkes, T.P.; Makulu, D.R.

National Cancer Institute Monograph 43: 171-177

1976


Pentamidine is an aromatic diamidino compound synthesized originally for the therapy of trypanosomiasis. The pharmacologic effects of pentamidine vary, depending on its route of administration. In animals, the dominant effects have been a precipitous, transitory drop in blood pressure after injection and renal toxicity following repeated administration. To avoid the possibility of immediate toxic reactions associated with iv administration, we now usually give the drug im to humans. Further interest in pentamidine has been stimulated by its usefulness in the treatment of interstitial pneumonia caused by Pneumocystis carinii. In some patients receiving antineoplastic or immunosuppressive therapy who have superimposed P. carinii pneumonia, pentamidine may cause serious renal toxicity. Distribution and excretion studies in animals indicate pentamidine is deposited in tissues, with the greatest concentration in the kidneys, and gradually eliminated over a prolonged period. The mechanism of action of pentamidine against P. carinii or the means whereby fixation in tissues and subsequent toxicity occur have not been elucidated. Recent investigations to help clarify these points indicate that pentamidine inhibits dihydrofolate reductase in all tissues studied both in vitro and in vivo. In addition, pentamidine interacts and forms water-insoluble products with specific nucleotides and nucleic acids.

Pharmacologic
Aspects
of
Pentamidine
1
T.
Philip
Waalkes,
M.D.,
Ph.D.,"
and
David
R.
Makulu,
Ph.D.
2
SUMMARY—Pentamidine
is
an
aromatic
diamidino
compound
synthesized
originally
for
the
therapy
of
trypanosomiasis.
The
pharmacologic
effects
of
pentamidine
vary,
depending
on
its
route
of
administration.
In
animals,
the
dominant
effects
have
been
a
precipitous,
transitory
drop
in
blood
pressure
after
in-
jection
and
renal
toxicity
following
repeated
administration.
To
avoid
the
possibility
of
immediate
toxic
reactions
associated
with
iv
administration,
we
now
usually
give
the
drug
im
to
hu-
mans.
Further
interest
in
pentamidine
has
been
stimulated
by
its
usefulness
in
the
treatment
of
interstitial
pneumonia
caused
by
Pneumocystis
carinii.
In
some
patients
receiving
antineoplastic
or
immunosuppressive
therapy
who
have
superimposed
P.
carinii
pneumonia,
pentamidine
may
cause
serious
renal
toxicity.
Distribution
and
excretion
studies
in
animals
indicate
penta-
midine
is
deposited
in
tissues,
with
the
greatest
concentration
in
the
kidneys,
and
gradually
eliminated
over
a
prolonged
period.
The
mechanism
of
action
of
pentamidine
against
P.
carinii
or
the
means
whereby
fixation
in
tissues
and
subsequent
toxicity
occur
have
not
been
elucidated.
Recent
investigations
to
help
clarify
these
points
indicate
that
pentamidine
inhibits
dihydrofolate
reductase
in
all
tissues
studied
both
in
vitro
and
in
vivo.
In
addi-
tion,
pentamidine
interacts
and
forms
water
-insoluble
products
with
specific
nucleotides
and
nucleic
acids.—Natl
Cancer
Inst
Monogr
43:
171-176,
1976.
Pentamidine
(4,4'-diamidinophenoxypentane;
Lomi-
dine)
is
a
member
of
a
series
of
aromatic
diamidino
com-
pounds
synthesized
in
the
1930's
and
extensively
evaluated
for
trypanocidal
activity.
The
diamidino
derivatives
among
many
other
classes
of
compounds
were
studied
initially
to
fi
nd
less
toxic
and
more
effective
antitrypanosomal
agents
than
the
diguanidines
(1-3).
Based
primarily
on
compara-
tive
therapeutic
properties,
attention
narrowed
to
four
aromatic
4,4'-diamidino
compounds:
pentamidine,
stil-
bamidine
(4,4'-stilbenedicarboxamidine),
propamidine
(4,
4'-diamidinophenoxypropane),
and
phenamidine
(4,4'-
diamidinodiphenyl
ether).
Of
these
compounds,
pentami-
dine
and
stilbamidine
have
received
the
most
extensive
clinical
evaluation.
Pharmacologic
studies
of
pentamidine
have
been
ham-
pered
by
the
lack
of
sufficiently
sensitive
methods
for
analysis
of
the
drug
in
tissues
and
body
fl
uids.
Stilbamidine
(4,
5)
has
been
investigated
more
completely
because
of
its
strong
fluorescence,
unique
among
the
diamidino
com-
pounds.
Since
1941,
pentamidine
has
had
extensive
clinical
use
outside
the
United
States,
primarily
for
the
treatment
of
I
Presented
at
the
Symposium
on
Pneumocystis
carinii
Infection
(sponsored
by
the
National
Institute
of
Child
Health
and
Human
Development
and
the
National
Cancer
Institute),
held
in
Bethesda,
Md.,
on
Dec.
13
and
14,
1973.
2
Laboratory
of
Chemical
Pharmacology,
Experimental
Thera-
peutics,
Division
of
Cancer
Treatment,
National
Cancer
Institute,
National
Institutes
of
Health,
Public
Health
Service,
U.S.
Depart-
ment
of
Health,
Education,
and
Welfare,
Bethesda,
Md.
20014.
3
Present
address:
Oncology
Center,
The
Johns
Hopkins
Medical
Institutions,
Baltimore,
Md.
21205.
specific
tropical
diseases.
The
drug
is
regarded
by
many
authorities
as
the
agent
of
choice
in
the
therapy
of
early
African
sleeping
sickness
(6-8),
as
prophylaxis
against
Trypanosoma
gambiense
(9,
10),
and
of
value
in
the
treatment
of
leishmaniasis
resistant
to
sodium
antimony
tartrate
and
ethyl
stilbene
(11).
The
diamidines
also
have
antibacterial
properties
(12)
as
well
as
activity
against
specific
types
of
malaria
(13)
in
animals.
The
aromatic
diamidines
were
tried
initially
by
Ivady
and
Paldy
in
1958
(14)
for
the
therapy
of
interstitial
pneumonitis
due
to
Pneumocystis
carinii,
and
their
results
with
pentamidine
were
reported
in
1963
(15).
Subsequent
evaluations
have
substantiated
the
effectiveness
of
pentami-
dine
against
this
organism
in
animals
(16)
and
man
(17-22).
The
clinical
toxicity
of
pentamidine
as
a
single
agent
has
been
well
documented
(12).
In
recent
years,
P.
carinii
pneumonia
has
been
seen
as
a
complication
in
seriously
ill
patients
receiving
antineoplastic
or
immunosuppressive
drugs.
In
some
of
these
patients,
severe
renal
toxicity
developed
(21,
23,
24)
after
pentamidine
administration.
This
report
briefly
reviews
the
background
and
current
pharmacologic
knowledge
of
pentamidine
and
presents
and
elaborates
on
data
showing
tissue
storage
and
elimi-
nation.
Recent
preliminary
experimental
evidence
demon-
strating
that
pentamidine
is
an
inhibitor
of
dihydrofolate
reductase
(DHFR)
is
given
and
initial
results
showing
the
interaction
of
pentamidine
with
nucleotides
and
nucleic
acids
are
discussed.
These
various
biologic
activities
and
properties
of
pentamidine
should
provide
a
greater
insight
into
its
possible
therapeutic
mode
of
action
and
the
possible
basis
for
the
toxicity
associated
with
the
drug.
MATERIALS
AND
METHODS
Tissue
deposition
and
excretion
of
pentamidine.—Mice
were
given
ip
injections
of
10
mg
pentamidine
isethionate/kg
body
weight
(calculated
as
base).
The
animals
were
kept
in
individual
glass
metabolism
cages,
and
urine
was
col-
lected
free
of
feces
by
use
of
an
attached
anal
cup
(25).
A
few
drops
of
0.1
N
HC1
were
added
to
the
urine
collection
tube
to
maintain
an
acid
pH.
Each
day
the
glass
cage
was
washed
with
water,
the
fi
nal
urine
volume
was
adjusted
to
15
ml/24-hour
period
and
frozen
for
subsequent
analysis.
The
feces
were
removed
from
the
anal
cup
daily
and
frozen.
The
animals
were
killed
at
specified
intervals.
Organs,
tissues,
and
feces
were
weighed
and
homogenized
in
fi
ve
to
seven
times
the
volume
of
0.1
N
HC1.
Pentamidine
analysis.
—The
procedure
of
Waalkes
and
DeVita
(26)
was
used
for
the
assay
of
pentamidine.
Pen-
tamidine
was
extracted
under
basic
conditions
from
plasma,
urine,
and
tissue
samples
into
organic
solvents.
After
this
extraction
and
the
subsequent
concentration
of
the
pent-
amidine
into
acid,
the
reaction
between
pentamidine,
glyoxal,
and
benzaldehyde
to
form
a
fl
uorescent
end
product
was
performed
in
basic
medium
following,
in
general,
a
modification
of
the
method
for
'a
romatic
dia-
171
172
WAALKES
AND
MAKULU
midines
developed
by
Jackson
et
al.
(27).
After
extraction
of
this
product
into
organic
solvents,
the
fi
nal
determina-
tion
was
made
with
an
Aminco-Bowman
spectrophoto-
fl
uorometer.
Duplicate
samples
were
analyzed
in
all
cases.
Samples
of
urine
or
tissues,
identical
samples
with
known
amounts
of
pentamidine
added
to
determine
recovery,
standard
pentamidine
solutions
alone,
and
a
reagent
blank
were
run
through
the
entire
procedure.
DHFR
studies.
—Aromatic
diamidines
included
pentami-
dine
isethionate
(a
generous
gift
of
May
and
Baker,
Ltd.,
Dagenham,
England,
through
Rhodia,
Inc.,
New
York,
N.Y.),
stilbamidine,
and
propamidine
(Chemistry
Branch,
Division
of
Cancer
Treatment,
National
Cancer
Institute).
Other
chemicals
and
their
sources
were:
folic
acid
and
methotrexate
(Lederle
Laboratories,
Pearl
River,
N.Y.),
NADPH
(Sigma
Chemical
Co.,
St.
Louis,
Mo.),
and
pyrimethamine
(2,4-diamino-5-(p-chloropheny1)-6-ethyl-py-
rimidine)
(Burroughs
Wellcome
and
Co.,
Research
Triangle,
N.C.).
Dihydrofolate
(DHF)
was
prepared
from
folate
by
the
dithionate
method
of
Futterman
(28)
as
modified
by
Blakley
(29)
and
stored
under
nitrogen
at
-68
°
C.
Liver
or
kidney
from
male
Sprague-Dawley
rats
was
was
used
as
the
source
of
DHFR.
The
enzyme
was
purified
to
homogeneity
by
affinity
chromatography
as
described.
3
Enzyme
assay.
—Activity
of
DHFR
was
measured
by
spectrophotometric
method,
with
the
decrease
in
absorbance
used
that
occurs
at
340
nm
when
NADPH
and
DHF
are
converted
to
NADP
and
tetrahydrofolate,
respectively
(30).
A
value
of
12,000
was
used
for
the
molar
extinction
change
at
340
nm
which
accompanies
the
reaction
(31).
Unless
otherwise
indicated,
the
standard
assay
mixture
contained
a
fi
nal
volume
of
1
ml:
100
nm
Tris-HCl
buffer,
pH
7.0;
1
nm
KC1;
0.10
nm
NADPH;
0.05
nm
DHF
containing
1
1
nm
2-mercaptoethanol,
and
enzyme.
Enzyme
activity
was
assayed
at
27
°
C
with
a
Gilford
Model
2400-S
multiple
absorbance
recorder;
the
decrease
in
absorbance
at
340
nm
was
recorded
automatically
at
5
-
second
intervals.
Enzyme
activity
was
expressed
as
i.noles
of
substrate
reduced
per
hour
per
volume
of
enzyme
solution.
In
vivo
study.
—Male
Sprague-Dawley
rats
(300
g;
3/
group)
were
given
ip
injections
of
saline
(control),
pentami-
dine
(20
mg/kg
calculated
as
free
base),
pyrimethamine
(10
mg/kg),
or
methotrexate
(10
mg/kg).
Twenty-four
hours
later,
the
animals
were
killed.
The
kidneys
were
dissected
rapidly
and
homogenized
in
saline
(1
g/10
ml).
The
homogenate
was
centrifuged
for
1
hour
(17,000
Xg)
at
C
and
the
supernatant
used
for
assay
of
DHFR
as
previously
described.
Protein
concentration
was
deter-
mined
by
the
method
of
Lowry
et
al.
(32).
RESULTS
As
shown
in
text
-figure
1,
after
ip
injection
into
mice,
pentamidine
was
deposited
in
tissues
and
gradually
excreted
over
a
prolonged
time.
From
the
amount
of
pentamidine
per
gram
tissue,
the
greatest
amount
was
found
in
the
kidneys
with
the
next
highest
amount
in
the
liver
as
com-
pared
with
the
remaining
tissues
of
the
animal.
The
relative
rate
of
release
of
pentamidine
from
each
site
with
time
appeared
similar.
After
administration
of
pentamidine
to
mice,
the
drug
was
excreted
predominantly
in
the
urine
(text
-fig.
2)
but
also
3
Makulu
DR,
Moroson
BA,
Skeel
R,
et
al:
Dihydrofolate
reductase
from
normal
and
neoplastic
tissues:
Purification
by
affinity
chroma-
tography,
comparative
inhibition
by
methotrexate,
NSC-139105
(Baker's
soluble
Antifol),
and
by
antibody.
In
preparation.
was
found
in
the
feces.
The
relative
total
amounts
in
urine
and
feces
were
similar
at
each
time
interval
studied
:
ap-
proximately
4
to
1,
respectively.
Again,
prolonged
storage
in
tissues
was
evident
with
secondary
delayed
excretion.
The
results
summarized
in
text
-figure
3
demonstrate
that
pentamidine
inhibited
the
activity
of
liver
DHFR,
50
40
10
0
Kidneys
All
Other
Tissinre___
5
TEXT
-FIGURE
1.
—Amount
and
all
other
tissues
at
Average
of
3
animals
for
idine
base.
100-
80
I-
60
w
w
a-
40
20
10
15
TIME
-DAYS
20
25
of
pentamidine
in
mouse
kidneys,
livers,
intervals
after
ip
injection
of
10
mg/kg.
each
time
point.
Values
expressed
as
pentam-
18
41
TIME-
HOURS
Amount
in
Feces,
El
Urine
90
and
Tissues
TEXT
-FIGURE
2.
—Relative
amounts
of
pentamidine
recovered,
ex-
pressed
as
percent
at
various
intervals
in
feces,
urine,
and
tissue
after
ip
injection
of
10
mg/kg.
Actual
recovery
of
the
total
dose
of
pentam-
idine
administered
was
100%
for
1,
18,
and
41
hours
and
87%
for
90
hours.
Average
of
2
animals
for
each
time
interval.
NATIONAL
CANCER
INSTITUTE
MONOGRAPH
NO.
43
PHARMACOLOGIC
ASPECTS
OF
PENTAMIDINE
173
w
0
X
p
Id
0
<
no
E
ow
n
'a
>-cc
E
5
1
7
-
2
Pentamidine
Propamidine
0.4
0.8
DIAMIDINE
(µrnoles)
Pyrimethamine
Methotrexate
0.04
0.08
0.004
0.008
FOLATE
ANALOGS
(
µmoles)
TEXT
-FIGURE
3.
—Inhibition
of
DHFR
by
aromatic
diamidines
and
folate
analogues.
Conditions
as
stated
under
"Materials
and
Methods."
Enzyme
in
buffer
incubated
with
each
compound
for
2
minutes
at
27°
C
before
NADPH
(0.08
nm)
and
DHF
(0.05
nm
of
2-mercaptoethanol)
were
added
to
initiate
the
reaction.
Each
point
represents
mean
±
SD
for
three
or
four
observations.
the
degree
of
inhibition
being
proportional
to
the
quantity
of
the
compound
incubated
with
the
enzyme.
On
a
molar
basis
and
under
identical
conditions,
propamidine
was
a
more
effective
inhibitor
than
pentamidine,
whereas
metho-
trexate,
particularly,
and
also
pyrimethamine
were
con-
siderably
more
potent
than
either
pentamidine
or
propamidine.
Because
of
its
strong
absorbance
at
340
nm,
stilbamidine
could
not
be
included
in
these
studies.
The
data
in
table
1
demonstrate
that,
24
hours
after
administration
of
pentamidine,
methotrexate,
or
pyri-
methamine,
DHFR
activity
was
significantly
reduced
in
the
kidneys
of
treated
animals
in
contrast
to
those
of
untreated
controls.
The
effects
of
methotrexate
and
pyrimethamine
were
compared.
DISCUSSION
Much
of
the
data
on
the
biologic
effects
of
pentamidine
were
published
during
the
1940's
shortly
after
its
trypanoci-
dal
activity
had
been
demonstrated.
Pentamidine
has
been
studied
and
administered
almost
exclusively
by
iv,
sc,
or
im
injection.
Probably
the
most
extensive
physiologic
and
pharmacologic
study
of
the
aromatic
diamidines
was
re-
ported
by
Wien
(33)
in
1943.
In
mice
the
LD50
by
the
iv
route
was
28
mg/kg
and
by
the
sc
route,
64
mg/kg.
For
both
mice
and
rabbits,
the
acute
toxic
effects
appeared
similar.
Death
was
attributed
to
respiratory
failure
associated
with
general
depression
of
the
central
nervous
system,
occasional
feeble
clonic
convulsions,
and
lowering
of
body
temperature.
In
larger
animals,
after
iv
injection,
the
most
profound
effect
appeared
to
be
on
the
blood
pressure.
All
four
aromatic
diamidines
studied
after
iv
injection
produced
a
marked
transient
fall
in
blood
pressure
in
anesthetized
cats
and
de
-
cerebrated
animals.
The
effect
was
reduced
or
abolished
by
calcium
but
only
partially
inhibited
by
atropine.
The
drop
TABLE
1.—In
vivo
inhibition
of
renal
DHFR
by
pentamidine,
pyrimethamine,
and
methotrexate
Drug
administered
Enzyme
activity
(Anoles/hr/
mg
protein)
None
0.
34
±
0.
03
Pentamidine
(20
mg/kg)
0.
15±
0.
02
Pyrimethamine
(15
mg/kg)
0.
27
±
0.
0
Methotrexate
(10
mg/kg)
0.
09
Details
of
the
procedure
as
presented
under
"Materials
and
Methods."
Each
value
represents
the
meansn
for
three
determinations.
in
blood
pressure
was
attributed
to
peripheral
vasodilatation
and
not
to
a
direct
cardiac
or
central
nervous
mechanism.
Injection
(iv)
of
pentamidine
(10
mg/kg)
into
cattle,
sheep,
and
goats
(84)
produced
shock,
sometimes
fatal.
Marked
venous
congestion,
fl
abby
heart,
and
evidence
of
either
or
both
kidney
or
liver
damage
were
seen
on
post-
mortem
examination.
With
lower,
better
tolerated
doses,
kidney
damage
accompanied
by
rising
blood
urea
nitrogen
(BUN)
was
noted
before
evidence
of
hepatoxicity.
Blood
sugar
levels
did
not
change
in
animals
eventually
dying,
though
BUN
rose
50-100%.
Pathologic
examination
of
the
kidneys
of
rabbits
treated
with
large
doses
(20
mg/kg)
of
propamidine
and
stilbamidine
revealed
cloudy
swelling,
desquamatioe,
and
fatty
degener-
ation
of
the
renal
convoluted
tubules
(12).
Repeated
doses,
equivalent
to
therapeutic
levels
in
man,
caused
no
histo-
logic
changes.
By
either
the
sc,
im,
or
iv
routes,
similar
though
transient
impairment
of
renaffunction
was
observed
after
maximally
tolerated
doses.
Some
animals
died
sud-
denly
from
uremia
after
a
single
injection
of
a
large
dose
of
diamidine.
Repeated
administration
of
pentamidine
to
young
rats
inhibited
growth.
Inflammatory
changes
were
noted
frequently
at
the
site
of
injection
(33).
Wien
et
al.
(35)
evaluated
various
metabolic
changes
produced
in
animals
by
the
diamidines.
Blood
sugar
levels
were
increased
by
pentamidine
but
only
at,
or
near,
toxic
doses.
Repeated
administration
was
associated
with
deple-
tion
of
liver
glycogen.
Elevations
of
blood
urea
and
non
-
protein
nitrogen
levels
occurred
in
some
instances
on
doses
which
did
not
influence
blood
sugar
levels.
With
toxic
doses,
cloudy
swelling
of
the
kidneys
and
fatty
degeneration
of
the
liver
were
produced.
Repeated
administration
of
therapeutic
doses
caused
no
change
in
blood
elements,
but,
with
toxic
doses
in
guinea
pigs,
leukocytosis
with
increase
in
polymorphonuclear
cells
preceded
death.
Amidines
also
have
other
biologic
properties
which
may
contribute
to
their
pharmacologic
and
toxicologic
effects.
In
1944,
Blaschko
et
al.
(36,
37)
found
diamidines,
particularly
pentamidine
and
propamidine,
inhibited
liver
histaminase,
though
distinct
species
differences
were
noted.
In
1949,
MacIntosh
and
Paton
(38)
reported
specific
organic
bases,
including
pentamidine,
released
tissue
histamine.
That
same
year
Gemmill
(39)
published
data
indicating
pentamidine
as
well
as
other
amidines
inhibited
anaerobic
glycolysis
of
glycogen
to
lactate
in
muscle
extracts.
Excretion,
distribution,
and
quantitative
disposition
studies
for
pentamidine
were
hindered
initially,
due
to
a
lack
of
sufficiently
sensitive
assay
procedures.
Unique
to
the
aromatic
diamidines,
stilbamidine
is
a
strongly
fl
u-
orescent
compound.
This
characteristic
is
secondary
to
its
central
double
-bond
structure
not
present
in
the
other
diamidines.
Consequently,
tissue
distribution
studies
of
stilbamidine
could
be
done
in
animals
by
use
of
this
fl
u-
orescent
property.
In
these
animals
(4,
5),
prolonged
intense
fi
xation
to
renal
and
hepatic
tissue
was
found.
Subsequent
studies
(40)
with
"C
-labeled
stilbamidine
confirmed
these
results
with
evidence
of
residual
compound
for
6
months
or
more.
Stilbamidine
was
eliminated
both
in
urine
and
feces,
with
no
radioactivity
found
in
exhaled
respiratory
carbon
dioxide.
Although
to
a
lesser
degree,
prolonged
storage
of
the
compound
was
also
found
m
the
lungs
and
heart.
All
observers
noted
an
apparent
ab-
sence
of
a
circulating
form
of
the
diamidine
in
the
blood.
The
urinary
excretion
of
stilbamidine
and
several
of
its
derivatives,
after
repeated
daily
administration,
was
also
SYMPOSIUM
ON
PNEUMOCTSTIS
CARINII
INFECTION
174
WAALKES
AND
MAKULU
studied
in
animals
with
fl
uorescence
used
for
assay
(41).
An
initial
delay
in
excretion
was
found
until
apparent
tissue
saturation
had
occurred.
This
fi
nding
was
thought
to
represent
and
confirm
the
tissue
deposition
and
fi
xation.
Launoy
et
al.
(42,
43)
in
1960
studied
the
elimination
of
'
4
C
-labeled
pentamidine
in
both
mice
and
rats.
A
sample
of
radioactive
compound
was
synthesized
with
the
H
e
label
on
the
terminal
amidine
carbon,
and
a
second
sample
with
the
14
C
label
in
the
central
carbon
chain.
With
either
compound
sample,
a
similar
excretion
pattern
was
found
for
mice,
which
suggested
that
pentamidine
was
eliminated
intact
without
metabolic
change.
It
was
cal-
culated
that
approximately
half
the
injected
dose
in
mice
had
been
eliminated
within
5
days.
No
attempt
was
made
to
analyze
the
drug
separately
in
urine
and
feces.
A
major
portion
of
the
labeled
compound
was
found
in
the
kidneys
and
liver,
and
a
much
smaller
amount
dispersed
in
all
other
tissues,
though
the
blood
appeared
devoid
of
radioactivity.
In
a
longer
term
study
in
rats,
the
investigators
found
prolonged
tissue
storage
and
delayed
elimination
of
pentamidine.
With
an
improved
fl
uorimetric
assay
procedure,
a
pharmacologic
study
of
pentamidine
was
done
in
1970
(44).
This
included
disposition,
distribution,
and
excretion
of
the
drug
in
mice
and
plasma
levels
and
urinary
excretion
in
patients
with
neoplastic
disease
undergoing
treatment
for
P.
carinii
pneumonia.
Only
a
single
ip
injection
(10
mg/kg)
was
given
to
the
mice.
The
results
and
conclusions
from
these
experiments
were
similar
to
those
published
by
Launoy
et
al.
(42,
43),
which
indicated
pentamidine
is
excreted
unchanged
with
prolonged
tissue
fi
xation.
It
is
apparent
that
pentamidine
is
stored
(text
-fig.
1)
in
tissues,
predominantly
renal,
and
gradually
disappears
with
time.
Analyses
of
other
mouse
organs
in
addition
to
kidneys
and
liver,
including
lungs,
spleen,
abdominal
and
pelvic
tissues,
brain,
skin,
and
skeleton,
indicated
the
kidneys,
and
second-
arily
the
liver,
contained
the
greatest
concentration.
Essentially
no
compound
was
found
in
the
brain.
Pentamidine
excretion
(text
-fig.
2)
occurs
primarily
by
way
of
the
kidneys,
with
lesser
total
amounts
present
in
the
feces.
In
a
more
recent
study
in
rats,
pentamidine,
36
hours
after
a
single
ip
injection
(10
mg/kg),
was
present
in
all
anatomic
areas
of
the
kidneys
when
grossly
dissected
into
cortex,
medulla,
and
collecting
ducts.
Quantitatively,
the
greatest
amount
was
in
the
cortical
area,
with
actual
levels
of
37,
29,
and
26
µg/g
of
tissue,
respectively.'
Plasma
and
urine
levels
of
pentamidine
were
also
deter-
mined
at
intervals
in
7
patients
with
suspected
or
proved
P.
carinii
pneumonia.
The
dosage
was
4
mg/kg/day
given
im
for
10-12
days.
On
this
regimen,
the
plasma
levels
were
low
(0.3-0.5
µg/ml),
did
not
rise
appreciably
immediately
after
injection,
remained
essentially
the
same
throughout
each
24
-hour
period,
and
did
not
increase
with
succeeding
days
of
treatment.
If
the
plasma
level
rose,
the
usual
time
was
1
hour
after
the
injection.
The
highest
levels
were
in
patients
with
an
elevated
BUN.
The
amount
of
penta-
midine
in
the
urine
for
each
6
-hour,
period
of
a
day
was
determined.
For
5
patients,
of
the
total
excreted
in
the
urine
during
the
24
hours
after
the
injection
of
pentamidine,
half
to
two-thirds
occurred
during
the
fi
rst
6
hours
after
administration
of
the
drug.
The
amount
excreted
in
the
urine
each
day
was
approximately
20%
or
less
of
the
daily
dose.
No
attempt
was
made
to
determine
the
possible
amount
of
drug
excreted
by
way
of
the
gastrointestinal
tract.
4
Waalkes
TP,
Slawsky
R,
Adamson
R:
Unpublished
observations.
After
cessation
of
therapy,
the
duration
of
the
urinary
excretion
of
pentamidine
was
determined
for
3
patients.
Decreasing
amounts
of
the
compound
were
detected
up
to
6-8
weeks
after
termination
of
the
drug
therapy.
The
evalua-
tion
of
the
effect
of
pentamidine
on
renal
function
was
com-
plicated
by
other
factors.
Several
patients
with
increases
in
BUN
during
pentamidine
therapy
also
were
receiving
nephrotoxic
antibiotics.
For
those
patients
with
an
increase
in
BUN,
the
peak
elevation
occurred
8-14
days
after
initia-
tion
of
pentamidine
therapy,
with
return
to
normal
levels
between
days
17
and
30.
No
other
significant
or
consistent
hepatic
effects
and
no
changes
in
blood
sugar
levels
were
noted.
Neither
the
mechanism
of
action
of
pentamidine
nor
its
toxic
effects
on
the
kidney
have
been
fully
explained.
It
has
been
suggested
that
pentamidine
acted
through
inter-
ference
with
aerobic
glycolysis
(45).
Hawking
and
Smiles
(4),
investigating
the
trypanocidal
action
of
stilbamidine,
noted
the
fi
xation
of
the
drug
to
nucleoproteins.
In
pa-
tients
with
multiple
myeloma,
both
stilbamidine
and
pentamidine
induce
selective
basophilic
granulations
in
myeloma
cells.
These
granulations
(46,
47)
have
been
re-
ported
to
be
made
up
of
a
combination
of
RNA
and
dia-
midine,
which
suggests
the
possibility
of
incorporation
of
these
compounds
into
nucleic
acid
components
within
the
cell.
In
more
recent
studies
(48),
pentamidine
has
been
reported
to
inhibit
in
vitro
synthesis
of
DNA,
RNA,
phos-
pholipid,
nucleotides,
and
protein
in
cells
of
a
murine
ascites
tumor.
In
clinical
studies,
Robbins
et
al.
(49)
reported
megaloblastosis
of
the
bone
marrow
in
a
patient
treated
with
pentamidine.
This
was
accompanied
by
lowered
serum
folate
levels.
From
these
clinical
observations,
Rob-
bins
(50)
suggested
that
pentamidine
might
act
as
a
direct
inhibitor
of
folate
metabolism
in
a
manner
similar
to
the
effect
of
methotrexate.
However,
Frenkel
et
al.
(16)
showed
that
pretreatment
with
folinic
acid
did
not
influence
the
therapeutic
efficacy
of
pentamidine
in
rats
infected
with
P.
carinii.
To
clarify
some
of
these
actions
of
pentamidine,
in
particular
the
bone
marrow
megaloblastosis
following
its
administration,
experiments
were
initiated
to
examine
the
possible
effect
of
pentamidine,
propamidine,
pyrimethamine
and
methotrexate
on
DHFR.
The
last
compound
was
used
for
comparative
purposes
as
a
classical
example
of
a
chemi-
cal
inhibitor
of
DHFR.
Both
pentamidine
and
propamidine
in
vitro
inhibit
DHFR
(text
-fig.
3),
but
on
a
molar
basis
the
activity
is
considerably
less
than
that
observed
with
methotrexate
or
pyrimethamine.
Twenty-four
hours
after
administration
of
pentamidine
to
rats,
DHFR
activity
in
kidney
extracts
was
reduced
(table
1)
and
was
similar
to
the
effects
of
methotrexate
and
pyrimethamine.
In
contrast
to
the
results
obtained
by
the
in
vitro
experiments,
the
apparent
relative
amount
of
pentamidine
required
to
inhibit
DHFR
to
an
extent
similar
to
methotrexate
seemed
considerably
less.
This,
however,
may
be
due
to
the
more
prolonged
storage
of
pentamidine
in
the
tissues
after
in
vivo
administration.
Also
in
process
are
In
vitro
studies
with
human
tissues,
including
normal
liver
and
kidneys,
leukemic
spleen,
and
erythrocytes
obtained
from
a
patient
with
polycythemia
vera
as
sources
of
DHFR.
The
results
to
date
are
similar
to
those
shown
in
text
-figure
2.
In
addition,
investigations
to
clarify
the
nature
of
the
individual
inhibition
are
being
done
and
will
be
reported
in
detail
elsewhere.
5
The
results
5
Makulu
DR,
Waalkes
TP:
Unpublished
observations.
NATIONAL
CANCER
INSTITUTE
MONOGRAPH
NO.
43
PHARMACOLOGIC
ASPECTS
OF
PENTAMIDINE
175
of
these
experiments
indicate
that
the
inhibition
is
competi-
tive
with
respect
to
the
enzyme
substrate
and
dihydrofolate
and
noncompetitive
with
respect
to
the
coenzyme
NADPH.
In
a
further
attempt
to
elucidate
the
biologic
interactions
of
pentamidine,
studies
of
the
effects
of
the
aromatic
diami-
dines
on
nucleic
acids
have
been
made
(51).
By
in
vitro
experiments,
pentamidine
was
found
to
react
with
isolated
nucleic
acids
to
form
water
-insoluble
precipitates
at
neutral
pH.
This
effect
was
observed
with
all
types
of
DNA
and
RNA
of
bacterial,
fungal,
or
mammalian
origin.
In
sub-
sequent
studies,
aromatic
diamidines
were
found
to
interact
and
precipitate
with
all
polynucleotides,
nucleotide
tri-
phosphates,
and
nucleotide
diphosphates,
but
not
with
nucleotide
monophosphates,
nucleosides,
free
nucleic
acid
bases,
and
non
-nucleic
acid
diphosphates,
such
as
fructose
diphosphate,
or
with
inorganic
phosphates.
This
fi
nding
suggests
that
the
nucleotide
diphosphate
molecule
is
the
minimum
nucleic
acid
structure
necessary
for
interaction
with
aromatic
diamidines.
It
is
clear
that
pentamidine
in
animals
is
deposited
and
stored
for
prolonged
periods
in
tissues
and
that
probably
in
humans
a
similar
process
occurs.
The
maximum
concentra-
tion
in
the
kidneys
associated
with
prolonged
tissue
fi
xation
would
appear
to
establish
ideal
conditions
for
renal
mal-
function,
particularly
in
seriously
ill
patients.
The
inter-
action
of
pentamidine
with
nucleic
acids
and
its
inhibition
of
DHFR
might
well
play
roles,
both
in
its
renal
toxicity
and
its
ability
to
eradicate
the
P.
carinii
organism.
The
precise
mechanism
of
the
deposition
of
pentamidine
in
tissues
has
not
been
completely
clarified,
but
its
ability
to
react
and
form
precipitates
with
nucleotides
and
nucleic
acids
and
to
bind
and
inhibit
DHFR
suggests
these
types
of
combinations
within
tissues
may
be
important
factors.
Such
molecular
interactions
could
be
the
mechanism
whereby
the
maximum
deposition
of
pentamidine
occurs
in
the
kidneys,
followed
by
gradual
release
and
excretion
predominantly
in
the
urine.
The
importance
of
DHFR
in
intrinsic
cellular
reactions
also
leads
to
speculation
that
pentamidine
may
be
effective
against
P.
carinii
by
virtue
of
its
ability
to
inhibit
this
important
enzyme
system.
Although
no
enzymatic
studies
have
been
carried
out
directly
on
the
P.
carinii
organism,
recent
experiments
5
indicate
the
DHFR
of
an
extract
of
T.
cruzi
is
exquisitely
sensitive
to
pentamidine.
Previous
work
(52)
indicates
that
various
DHFR
inhibitors,
including
methotrexate,
are
also
active
inhibitors
of
the
enzyme
from
extracts
of
trypanosomes.
Nevertheless,
in
vivo,
no
known
DHFR
inhibitor
had
been
effective
against
tryp-
anosomes.
This
therapeutic
failure
is
speculated
to
be
secondary
to
plasma
protein
binding
and
poor
transport
into
the
parasites,
but
pentamidine
apparently
has
no
such
restrictions.
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H,
LOURIE
EM,
YORKE
W:
Studies
in
chemotherapy.
XIX.
Further
report
on
new
trypanocidal
substances.
Ann
Trop
Med
Parasitol
32:177-192,
1938
(2)
ASHLEY
JN,
BARBER
HJ,
Ewn.ts
AJ,
et
al:
A
chemothera-
peutic
comparison
of
the
trypanocidal
action
of
some
aromatic
diamidines.
J
Chem
Soc
20:103-116,
1942
(3)
LOURIE
EM,
YORKE
W:
Studies
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chemotherapy.
XXI.
The
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of
certain
aromatic
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Ann
Trop
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Parasitol
33:289-304,
1939
(4)
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F,
SMILES
J:
The
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of
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stilbene
in
trypanosomes
and
mice
as
shown
by
fl
uorescence.
Ann
Trop
Med
Parasitol
35:45-52,
1941
(5)
HENRY
AJ,
MANSOUR
R,
WATSON
AG,
et
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Storage
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bamidine
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animal
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)
(8)
(
9
)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(20)
(21)
(22)
(23)
(24)
(25)
(26)
(27)
(28)
(29)
(30)
(31)
(32)
(33)
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TL:
Trypanosomiasis
treated
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1942
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Sierra
Leone
by
antrypol,
tryparsamide,
pentamidine
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and
in
various
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39:99-124,
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39:327-329,
1946
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PE,
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1948
FULTON
JD:
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34:53-66,
1940
IvADY
G,
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L:
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1958
IVADY
G,
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G:
A
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for
inter-
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plasma
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Monatsschr
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111:297-299,
1963
FRENKEL
JK,
GOOD
JT,
SHULTZ
JA:
Latent
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Lab
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1966
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WS,
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Am
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1962
RODGERS
TS,
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MH:
Pneumocystis
carinii
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associated
with
hypogammaglobulinemia
responding
to
pentamidine.
Lancet
1:1042,
1964
MARSHALL
WC,
WESTON
HJ,
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M:
Pneumocystis
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1964
RIFKIND
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RB:
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65:
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1966
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1969
WESTON
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MG:
Pentamidine
isethion-
ate
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the
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Ann
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73:695-702,
1970
EMMER
M,
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VT:
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69:637-638,
1968
WANG
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Unusual
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tions
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1970
OLIVERIO
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The
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36
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1962
WAALKES
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The
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urine,
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tissues.
J
Lab
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75:871-878,
1970
JACKSON
DP,
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WJ,
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The
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aro-
matic
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FUTTERMAN
S:
Enzymatic
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dihydrofolic
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J
Biol
Chem
228:1031-1038,
1957
BLAKLEY
RL:
Crystalline
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Nature
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1960
PERKINS
JP,
HILLCOAT
BL,
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JR:
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ductase
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a
resistant
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of
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Purification
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1967
MATHEWS
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1963
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Protein
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Folin
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1951
WIEN
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The
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Ann
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Med
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37:1-18,
1943
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MAKULU
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FRENCH
MH,
MILNE
AH:
Some
metabolic
disturbances
in
do-
mestic
stock
following
injection
of
4,4'-diamidino-1,5-di-
phenoxypentane.
Vet
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53:413-416,
1941
(35)
WIEN
R,
FREEMAN
W,
SCOTCHER
NM:
The
metabolic
effects
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certain
aromatic
diamidines.
Ann
Trop
Med
Parasitol
37:19-33,
1943
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BLASCHKO
H,
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The
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minase
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Biochem
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49:250-253,
1951
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BLAscinco
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JM:
The
inhibition
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amine
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Br
J
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1955
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MACINTOSH
FC,
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WD:
The
liberation
of
histamine
by
certain
organic
bases.
J
Physiol
109:190-219,
1949
(39)
GEMMILL
CL:
The
effects
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and
related
compounds
on
glycolysis
in
muscle
extracts.
J
Pharmacol
Exp
Ther
96:173-
178,
1949
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REID
JC,
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JC:
The
distribution
and
excretion
of
stil-
bamidine-CH
in
mice.
Cancer
Res
11:188-193,
1951
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HAMPTON
JW:
The
excretion
of
stilbamidine
and
some
related
compounds
in
experimental
animals.
Ann
Trop
Med
Parasitol
41:226-233,
1947
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LAUNOY
L,
GUILLOT
M,
JONCHERE
H:
Study
on
the
storage
and
elimination
of
pentamidine
in
the
mouse
and
white
rat.
Ann
Pharm
Fr
18:273-284,
1960
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Study
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the
storage
and
elimination
of
pentamidine
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the
mouse
and
white
rat.
Ann
Pharm
Fr
18:424-439,
1960
(44)
WAALKES
TP,
DENHAM
C,
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VT:
Pentamidine:
Clinical
pharmacologic
correlations
in
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Clin
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11:505-512,
1970
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I,
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B:
The
development
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basophilic
inclusion
bodies
in
myeloma
cells
after
stilbamidine
treatment.
Ann
Intern
Med
27:541-547,
1947
(47)
SNAPPER
I,
MIRSKY
AE,
Ris
H,
et
al:
Development
of
inclusion
bodies
containing
ribose
nucleic
acid
in
myeloma
cells
after
injection
of
stilbamidine.
Determination
of
stilbamidine
in
myeloma
tissue.
Blood
2:311-322,
1947
(48)
BORNSTEIN
RS,
YARBRO
JW:
An
evaluation
of
the
mechanism
of
action
of
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isethionate.
J
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Oncol
2:393-
398,
1970
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ROBBINS
JB,
MILLER
RH,
AREAN
VM,
et
al:
Successful
treat-
ment
of
Pneumocystis
carinii
pneumonitis
in
a
patient
with
congenital
hypogammaglobulinemia.
N
Engl
J
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272:
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1965
(50)
Romums
JB:
Pneumocystis
carinii
pneumonitis—a
review.
Pediatr
Res
1:131-158,
1967
(51)
MAKULU
DR,
WAALKES
TP:
Interaction
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diamidines
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nucleic
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Possible
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for
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J
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54:305-310,
1975
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JAFFE
JJ,
MCCORMACK
JJ,
GUTTERIDGE
WE:
Dihydrofolate
reductase
within
the
genus
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Exp
Parasitol
25:
311-318,
1969