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MECHANISM OF ACTION OF A NEW ANTI-INFLAMMATORY AGENT, NAPROXEN (II)
Japan. J. Pharmacol. 26, 91-103 (1976)
91
MECHANISM OF ACTION OF A NEW ANTI-INFLAMMATORY AGENT, NAPROXEN (II) EFFECTS
OF NAPROXEN
MUCOPOLYSACCHARASE, COLLAGENOLYTIC Yoshio
ON
ACTIVITIES
ACID
PROTEASE
ENZYMES
SUZUKI,
OF AND
IN INFLAMED
TISSUES
Mikio ITO and Ichika YAMAGAMI
Department of Pharmacology, Faculty of Pharmacy, Meijo University, Tenpaku-ku, Nagoya 468, Japan Accepted November 11, 1975
Abstract-In
order
proxen,
the
nidase
(ƒÀ-Gase)
(CL)
in
model
in
and
caused
only
a
the
on
a
of
activity
LZ only.
lysosomal
by In
decrease
in
the
exudate,
but
CL
activities,
From
these
effects
of
naproxen
of
the
naproxen
may
are
be
partly
these
the
attributable
curative
slight
test,
not pred-
formed there
but
a weak that to
were did
already
showing concluded
inand
enzymes
indomethacin
induced
prednisolone it
strongly
indomethacin
granuloma and
enzyme
Prednisolone In
na-
inflammatory
naproxen
activity.
granuloma.
weight
was
significant inhibition
of
anti-inflammatory its
inhibitory
actions
enzymes.
Naproxen is a new type of non-steroidal Syntex Research.
all
naproxen
indomethacin
while
collagenolytic
on
of
of [ƒÀ-glucuro-
proliferative
did
with
results,
a
as
APase
the
and
of
test,
in
and
(APase)
preventive
effects
activities
properties
mucopolysaccharase
accumulation
inhibit
APase
of
means
the
exudate
to
Naproxen
and
protease
and
decrease.
anti-rheumatic these
of
acid
inhibitory failed
marked
volume
slight
inhibition
the
LZ
activities
investigated
and
anti-inflammatory
on
rats.
Although
inhibit
biochemical
(LZ)],
were
indomethacin
significantly
and
lysozyme
formation
evident,
nisolone
the
compound
filter-paper-implanted
prednisolone.
in
elucidate this
tissues
granuloma
quite
CL
of
inflamed
hibited
and
to
effects
anti-inflammatory
Its effectiveness for rheumatoid
agent (1) developed by
arthritis has been confirmed in animal
experiments (2, 3) and clinical studies (4-8). In order to adequately ascertain the therapeutic effects of naproxen, the biochemical mechanism of the anti-inflammatory
actions of this agent was investigated.
In previous
papers (9, 10), it is reported that by using the proliferative granulation model in filter-paperimplanted rats, investigation was conducted as to how naproxen would act on quantitative changes of the main connective tissue constituents (total mucopolysaccharide,
acid muco-
polysaccharide, glycoprotein, non-collagenic protein and collagen) in inflamed granulation tissue. It was found that the drug increased the 0.15 M NaCl-insoluble fraction of constituents of granulation tissue. It is known that the degradation of the constituents of connective tissue in inflammatory regions due to lysosomal enzymes vary according to quantitative
changes of the consti-
tuents of the same tissue, and also that there is an elevation of activities of several different hydrolytic enzymes originating from lysosomes present in synovial fluid and synovium of patients with rheumatoid arthritis and in inflammatory tissues (11-16). We also found that in rats with adjuvant-induced arthritis, there were apparent correlations between the
92
Y. SUZUKI,
M.
ITO
& I.
YAMAGAMI
intensity of the inflammation and activities of enzymes and further between depression of inflammation
by anti-inflammatory
drugs and their inhibitory actions on enzymatic ac-
tivities (17). In order to clarify the biochemical mechanism of the anti-inflammatory actions of naproxen, we made an attempt to determine whether or not the drug can indeed inhibit activities of hydrolytic
enzymes (mucopolysaccharase,
enzyme) in granuloma
acid protease and collagenolytic
and skin covering the granuloma pouch by employing the same
inflammatory model used in a previous study (10). The effects of naproxen on these enzymatic activities were then compared with its effects on the weight of the granuloma and the volume of exudate. MATERIALS
AND
METHODS
Drugs The chemical structure of naproxen (NAP) is shown in Fig. 1. Indomethacin (IDM) [Merck-Banyu], benzydamine hydrochloride (BEN) [Daiichi Seiyaku Co., FIG.
Ltd.] and prednisolone (PD) [Toyo Jozo
1.
Co., Ltd.] were employed for comparison. Induction
of inflammatory
Female lightly A
Wistar
the
tails.
No.
26,
been
A
day
by
units)
was
was
Arabic effects
The
on
was day
as of
Within fluid
into
given
each
drug
was
the
first
MW:
placed
filter
230.25
mp:
of
into
naproxen acid
153•`457•Ž
and
with
dorsal mm
region,
the
was
board, clipper.
the
base
Filter
skin
Procaine
wound
a
electric near
(Toyo
the
incision.
on
an
skin
sq.
dorsal
the
position
shaved on
25•~35
the
through
a prone
was made
paper
s.c.
in
dorsum was
incision
groups
of
the
orally were
solutions.
by
on way
and
8 days,
granuloma
in
pouch.
the
gavage
once
a
day
12.
on
in
of
12 the
of
of
Paper
which
penicillin
carefully
to
weight
had G
sutured.
(3
This
day
for
12
test,
and
control
drugs
two
curative
day
suspended days,
24.
effects
saline were
procedures. or
consecutive
until
physiological
of
dissolved
Here were
solution
expressed
each
assessed
in the by
and
same
percentage
group.
of granuloma
pouch As
drug
continued
individual the
consisted
each
and first
given
the
groups
volume
Here
were
Effects
experiments
0.
day
as
control
drug-treated
The
day
evaluated
same as
6.
on
commenced
of exudate
accumulated
the
started
was
in
of
of
propionic
of drugs
serving
drug
inhibition
Determination
and
of test
Animals the
to
implantation.
the
width
spatula
0 of
test
administered
25.
amount ratio
second
of
in
inserted
applied
divided
Gum
preventive
drug
were
preventive
skin
mm
steel
evaluation
g were
sterile was
stainless
day
the
the
25
rectangular
then
and
Animals Firstly, 1%
a
C14H1414O8
180-200
and
thickness)
considered
Administration
in
of
in
ether approx.
peace
mm
loosened
million
weighing
with incision
0.7
structure
lesion
rats,
anesthetized
horizontal
Chemical
d-2-(6-methoxy-2-naphthyl)
it was
was
formed
impossible
around to
separate
the
implanted clearly
filter the
paper
granuloma
ANTI-INFLAMMATORY pouch
from
sacrifice,
the
the
Separating weight and
surrounding
the
pouch wall
the
pouch
Preparation
pouch
wall
Inflamed
tissues
were
tissues
saline
solution.
The
As were
of
used
β-Gase
into finely
was
used
the
protease
determined
by
of
and
day
covering
granuloma
covering
enzyme
as
skin
scissors
and
centrifuged
at
enzyme
8.
After
the
pouch
the
pouch. and
granuloma
wet
pouch
activities.
24
covering
the
granuloma
homogenized
12,000
solution.
within
rpm
This
in
for
20
solution
a
pouch.
physiological
min
while
was
cooling
kept
at
0-3•Ž
hr.
of ƒÀ-glucuronidase
with
Hasebe's
(ƒÀ-Gase)
method
(12).
Ltd.)
of
was
used
activity
method
of
LZ as
activity,
et
lyophilized
lysozyme
the
(LZ)
determination
of
Co., Ltd.) was used as
Micrococcus
lysodeikticus
substrate.
[substrate:
Bertelli
and
For
glucuronide (Chugai Pharmaceutical
determination
(APase) the
with
activities
Industries,
Acid
the
skin
after
skin
activities
accordance
for
(Biochemical
performed dorsal
in
The
granuloma
determined
ρ-nitrophenyl
while
exudate
determination
was
were
enzyme
in
activity,
substrate,
for
cut
mucopolysaccharases,
determined
of
the
93
NAPROXEN
were
with
respectively.
homogenate
activities
Determination
assays
together
volume
divided
were
supernatant
enzymatic
skin,
later
5, all
OF
solution
inflamed
The
the
day
excised
determined,
were
Both
and
until
was
from were
of enzyme
at 0•Ž.
tissue
granuloma
of pouch
PROPERTIES
denatured
al.
(18),
hemoglobin
a partial
(Sigma)]
modification
of
was
the
also
method
of
Anson. The
unit
of
these
enzyme
activities
was
expressed
as
the
specific
activity
per
1 g of
wet
of
the
tissue. Collagenolytic method for
determination
into
of
sections
of
1-2
Five
ml
of
flask. 4 vol, 0.5
(CL)
of Nakagawa
0.11
vol,
M
0.2
mM
were gas.
proline
the
were
the
and
hyrolysate
activity
was
mg
was calculated
each
were
to
kept
frozen
the
from
acid
in
of 6 N
quantitatively amount
HCl by
of
an
make
3 vol)
liquid
re-extracted
105•Ž
for of
16
hr.
M
sulfate
was
then
10 mM,
then
thawed, from
added
substances
Kivirikko
MgCl2 0.16
containing
of
the
method
KCl
of 95% O2-CO2
It
was
M
0.154
a concentration
soluble
finely
7 vol,
atmosphere
solution
cut
Erlenmeyer
vol,
fumarate
activity.
TCA
hydroxyproline.
0.5
substrate
0.154
CL
(TCA)
at the
ml
vol,
dihydrostreptomycin
to
2.5%.
a 20
and
under
and
Na
the
was
buffer
of
supernatant
mg
95
MgSO4 M
as
phosphate G
40 hr
medium
trichloroacetic
heating
the
for
500
NaCl
M 0.1
Na
determination
a concentration by
analyzed
M
penicillin
incubation until
0.154
modification used
into
M
4 vol,
0.1
at 34•Ž
The
A cold make
1 vol,
potassium out
transferred
(0.154
a
was
weighing
then
pyruvate and
by itself
granuloma
KH2PO4
5 vol
of
granuloma tissue
solution
M Na
carried
added
hydrolyzed
M
0.16
centrifuged.
to
which
glucose
was
combined.
of
Krebs-Ringer 0.154
in
Granulation
piece
thickness,
5.4%
0.5
was
solution
in
3 vol,
was
and
combined centrated
A
3 vol,
and
mixture
homogenized residue
activity.
mm
Incubation
L-cysteine
then
CL
4 vol,
added.
determined
(19).
NaHCO8
Na-L-glutamate
was
Tsurufuji
modified
CaC12
1.3%
activity
and
were
Hydroxyproline et
al.
(20).
the to
the conin CL
94
Y. SUZUKI,
M.
ITO
& I.
YAMAGAMI
RESULTS Changes
in exudate
Exudate 8
to
day
rapidly
100
of
after (8.8•}2.1
hardly
detected
peared
on
by
12
on
day
in
exudate
The
exudate
on
day
8 was
maintained
Changes
of
Fig.
at
but
ing
the
granuloma
LZ
and
APase
out
the
158.2
5.5
still
activities
On hardly
of than the
these those other
detectable β-Gase
and
from
three the
high
of
ml
from
exudate
on
day
decreased
Each
the
level
g on
day
and exudate plot
hand,
there
in the
normal
8 to
they
day
normal
25 in
skin was
enzyme day
day
increased
8 and
thereafter,
reached and
was
peak
until
(2.7•}0.1
which
ap-
day
35.
weight
de-
g) The
100.
volume
during
denotes
the from
a high
of
mean
day 8 to day 100 value
4.3,
those skin
in the
with S.E. ob-
than and
the
normal
covering
the
day
8 to
activity
of
day CL
The
those
31.8
in
granuloma
implantation.
higher
were as
activities
100
significantly
viz., on
enzymes
LZ activities
g and
a constant
weight
day
were
period, as
in
intervals
volume
3.5•}0.6
sharply
2.1•}0.2
2.0•}0.4
of hydrolytic
pouch
times
the
various
activities
granuloma
experimental
and
higher
enzyme
variations
in
at
2,
was
volume
nearly
was
in granuloma
hydrolytic
3 shows
Fig.
volume
after implantation of filter paper. tained from 6 rats.
Variation
recorded
100.
thereafter,
FIG. 2.
were
indicated
the
25.
granuloma was
gradually
weight weight
As i.e.,
day
of
day
granuloma
granuloma
implantation.
ml)
weight
creased
and
and
implantation,
a peak
The
volume
volume
in
4.0
the
on
tissue,
granuloma
skin
activities normal
times
skin
and
cover-
of ƒÀ-Gase, skin
day
8
throughand
11.2,
respectively. pouch
were
The slightly
16. in
the
granuloma,
although
such
was
skin.
in granuloma
reached
the peak
on days
12 and 25, respectively,
while both APase and CL exhibited the highest activities on day 35. The activities of all enzymes thereafter showed a gradual decrease. Anti-inflammatory effects of NAP given for 12 consecutive days after implantation of the filter paper (Preventive test) Effects on granuloma weight and exudate volume:
As seen in Table 1, at the dose levels
ANTI-INFLAMMATORY
FIG.
3.
Changes
activities to S.E.
in ƒÀ-glucuronidase, in
day
PROPERTIES
100
granuloma after
obtained
lysozyme,
and
skin
implantation from
OF
of
covering filter
acid the
paper.
protease
granuloma Each
95
NAPROXEN
and
collagenolytic
pouch
plot
denotes
during mean
day
value
8
with
6 rats.
of 2.5 mg/kg, 10 mg/kg and 25 mg/kg, NAP exerted dose-dependent actions which inhibited weight of granuloma 29%, 46% and 71%, respectively, and the exudate volume 17%, 60% and about 100%, respectively.
The inhibitory actions of 10 mg/kg of NAP on granulation
and exudation were nearly equivalent those of 2.5 mg/kg of IDM and 5 mg/kg of PD. BEN in the dose of 50 mg/kg had no significant inhibitory action on either granuloma weight or exudate volume. TABLE
1.
Effects
volume beginning
of of
naproxen
and
filter-paper-implanted day
0
after
other rats
drugs following
implantation
Statistical difference from the control group (a, p<0.001; b, p<0.01; c, p<0.05)
on
granuloma oral,
daily•~12
weight
and
exudate
administrations
96
Y. SUZUKI, TABLE
2.
Effects
and
skin
oral,
of naproxen covering
and
the
daily •~12
M.
ITO
other
drugs
granuloma
& I.
on ƒÀ-glucuronidase
pouch
administrations
YAMAGAMI
of
activity
in
filter-paper-implanted
beginning
day
0 after
granuloma
rats
following
implantation
Statistical difference from the control group (a, p<0.001; b, p<0.01; c, p<0.05)
Effects in
on ƒÀ-Gase
Table
tration
of
NAP
stronger 53 % of
drugs
(5
of
was
Effects lustrated
on in
TABLE
10 by
the
2.5
25
mg/kg
PD
covering
of
30%
This
IDM.
was
showed
pouch:
after
the
inhibitory
The
which
BEN
granuloma
about
mg/kg.
(5 mg/kg),
experiment.
enzyme at
37 %. LZ
activity
Effects
and
skin
present
the
dose
Neither
3,
3.
oral,
and
skin inhibited
action
enzyme
the no
was
potent
significant
given
adminis-
tended
activity
most
As oral
to
be
inhibited
inhibitory
action
inhibitory
action
at
mg/kg.
NAP
Table
this
was
mg/kg
of
in
50
of by
mg/kg)
of
and
granuloma
produced
tested
level
30%
in granuloma in
administration
Activity about
doses
that
the
dose
activity in
than after
the
the
activity
2, ƒÀ-Gase
LZ
of covering
daily •~12
in
levels IDM
in activity
naproxen the days
skin of
nor
BEN
granuloma in
skin
other
administrations
Statistical difference from the control group (a, p<0.001; b, p<0.01; c, p<0.05)
mg/kg,
any
was
drugs pouch
granuloma
25
on of
the
the
lysozyme
day
inhibitory
12%,
activity
0
was
by
in
implantation
PD
actions.
pouch: 17%
rats after
inhibited
inhibition
granuloma
inhibited
filter-paper-implanted
beginning
pouch and
significant
covering
granuloma
granuloma
the and
showed
and
the
and
covering
10 mg/kg
and
granuloma following
As 28%
ilby
ANTI-INFLAMMATORY NAP
at
dose
the
doses
(50
mg/kg)
elicited
each
and
of
are
by
The
dose
skin
of
mg/kg)
induced
in
drugs
tested.
APase
activity
in
Table
any
of
enzymatic
the
and
granuloma
oral,
Effects
of
skin
covering
daily •~12
mg/kg, by
significant
97
NAPROXEN respectively,
IDM
(2.5
inhibition
in
mg/kg).
of
LZ
proportion None
activity,
to
of
BEN
though
PD
the
and
activity 25
skin in
mg/kg,
granuloma
pouch
covering
granuloma
while
inhibited
markedly
pouch:
Findings
the granuloma was
the
was
inhibited
enzyme
activity
inhibited
17 to
20% was
or
not
more
by
significantly
used. from
was
a
25
20%
covering
APase
drug
activity
4.
skin
10 and
ranging
and
OF
on ƒÀ-Gase.
in granuloma
4. both
other
levels
the
mg/kg
inhibited
inhibition
LZ
levels
10 was
on
covering
TABLE
(5
mg/kg,
activity
the
at dose
At in
PD
illustrated
inhibited
2.5
remarkable
activity
Effects
NAP
of The
a most The
by
levels
given.
PROPERTIES
2.5
to
25
pouch,
also
thus
inhibited
naproxen the
mg/kg,
and
NAP showing
27 % by
other
granuloma
administrations
drugs pouch beginning
a dose-dependent
PD
on of
30%
of
APase
inhibitory
activity action.
(5 mg/kg).
acid
protease
activity
filter-paper-implanted day
0 after
in rats
granuloma following
implantation
Statistical difference from the control group (b, p<0.01 c, p<0.05)
Effects on CL activity in granuloma:
Actions of drugs against CL activity were ex-
amined only in granuloma tissue and results are shown in Table 5.
The activity of this
enzyme was inhibited 25%, 37% and 59% by NAP at the dose levels of 2.5 mg/kg, 10 mg/kg and 25 mg/kg, respectively, viz., the action was dose-dependent.
CL activity was signi-
ficantly inhibited 49% and 56% by IDM (2.5 mg/kg) and PD (5 mg/kg), respectively. the other hand, BEN (50 mg/kg) produced no significant inhibition
On
of the enzymatic
activity. Anti-inflammatory effects of NAP given for 13 successive days starting day 12 after implantation of filter paper (Curative test) Effects on granuloma weight and exudate volume:
As shown in Table 6, the weight
of wet granuloma was decreased 11-16% after the administration of NAP at the dose levels of 5 to 25 mg/kg, but the inhibitory action was not significant.
Among the other drugs used,
98
Y. SUZUKI, TABLE
5.
Effects
of
granuloma
naproxen
of
ministrations
M.
ITO
and
& I.
other
drugs
filter-paper-implanted beginning
YAMAGAMI on
rats
day
0 after
collagenolytic
following
activity
oral,
in
daily •~12
ad-
implantation
Statistical difference from the control group (a, p<0.001 ; b, p<0.01; c, p<0.05)
TABLE
6.
Effects
volume
of of
beginning
naproxen
and
other
filter-paper-implanted day
12
after
drugs
rats
on
granuloma
following
oral,
weight
and
daily •~13
exudate
administrations
implantation
Statistical difference from the control group (a, p<0.001; b, p<0.01; c, p<0.05)
PD
was
42%
the
loss
over
40%
test,
PD
only
by
at
in
the
drug
The
Table
mg
in
2.5
LZ
by
25
in NAP
activity had
the
comparative
by
already
in
and
granuloma
of
the
preventive
volume
IDM.
(25
mg/kg)
in
granuloma:
the
on
covering
skin was
though
inhibition
As
in the
test.
was
inhibited
case
of the
In
fact,
slightly preventive
this
the
covering
the
inhibited
inhibition
granuloma
slightly was
not
pouch:
(11-16%)
As
by
NAP
significant.
No
other
inhibited
slightly
enzyme.
granuloma
pouch
was
activity
in
but
only.
returned study.
36%
case exudate
(81%).
mg/kg,
skin
the
and
granuloma
activity
in
The
mg/kg)
in
as
(5 mg/kg).
inhibition
to
pouch
inhibition PD
25
activity
activity
(13%)
granuloma
and
a significant
enzyme
on
with
strongest
g-Gase of
showed
Effects
up
7,
level
significantly
taken
(10 the
strong
seen
on ƒÀ-Gase
dose used
having
was
NAP
induced
Effects given
one
in weight
Because to
As
the
indicated
LZ
normal
by
in Table
day
25
8, the
(refer
the to
inhibitory
skin Fig.
covering 3),
actions
it was of
the not NAP
ANTI-INFLAMMATORY TABLE
7.
Effects
and
skin
oral,
of naproxen covering
daily •~13
and
the
PROPERTIES other
drugs
granuloma
pouch
administrations
OF
on ƒÀ-glucuronidase of
activity
in
filter-paper-implanted
beginning
day
12
after
99
NAPROXEN
rats
granuloma following
implantation
Statistical difference from the control group (c, p<0.05)
TABLE
8.
Effects
granuloma administrations
of of
naproxen
and
other
drugs
filter-paper-implanted beginning
rats
day
12
after
on following
lysozyme oral,
activity
in
daily •~13
implantation
Statistical difference from the control group (b, p<0.01; c, p<0.05)
(10 mg/kg and 25 mg/kg) and IDM (2.5 mg/kg) against LZ activity in the granuloma were slight but significant, viz., the enzymatic activities were inhibited 10-15% by these drugs. Such were not inhibited significantly either by BEN (50 mg/kg) or by PD (5 mg/kg). Effects on APase activity in granuloma and skin covering the granuloma pouch:
As
shown in Table 9, APase activity in the granuloma was inhibited 10% by NAP (2.5 to 25 mg/kg), but the inhibitory effect was not significant.
Likewise, the other drugs used were
not so effective as to demonstrate significant inhibition against the activity of APase. APase activity in the skin covering the granuloma pouch was inhibited by none of the drugs used herein. Effects on CL activity in granuloma:
As shown in Table 10, NAP at the dose levels of
2.5 mg/kg, 10 mg/kg and 25 mg/kg inhibited CL activity 20%, 23% and 29%, respectively, in proportion to the dosage increase.
The inhibitory actions of IDM (2.5 mg/kg) and PD
100
Y. SUZUKI, TABLE
9. and
Effects
of
skin
covering
oral,
naproxen
TABLE
10.
and
the
daily •~13
M.
ITO
other
drugs
granuloma
Effects
of
of
acid
naproxen
and
beginning
day
other
12
12
drugs rats
day
protease
after
activity
in
filter-paper-implanted
beginning
of filter-paper-implanted
ministrations
YAMAGAMI
on
pouch
administrations
granuloma
& I.
on
after
following
implantation
collagenolytic
following
granuloma
rats
activity
oral,
in
daily •~13
ad-
implantation
Statistical difference from the control group (b, p<0.01; c, p<0.05)
(5
mg/kg)
by
BEN
were (50
21%
and
30%,
respectively,
however,
no
significant
inhibition
was
seen
mg/kg).
DISCUSSION In the of
order
preventive the
the
relation
or
test
and
pouch
(5
mg/kg).
curative
LZ,
inhibitory
two test
were
actions
is summarized
the the
that
in NAP
inhibitory
curative
test,
and
on of
of
carried
APase
model
revealed
remarkably, In
mechanism
test
(ƒÀ-Gase,
and
the
preventive
exudation
the
biochemical
inflammatory
between
PD
the
enzyme
proliferative
The and
elucidate
hydrolytic
granuloma in
to
anti-inflammatory
out. CL)
weight
Inhibitory
in of
Table
11. dose
action however,
being NAP
actions
granuloma
and
granuloma
and
filter-paper-implanted
at the
action
level nearly as
rats
of
10 mg/kg equal
well
as
that IDM,
on
skin volume
were
of
activities
covering of
studied.
inhibited of IDM which
NAP,
the
exudate Cor-
granulation (2.5 is an
mg/kg) acidic
ANTI-INFLAMMATORY
PROPERTIES
OF
NAPROXEN
101
102
Y. SUZUKI,
non-steroidal
anti-inflammatory
M.
ITO
& I.
YAMAGAMI
agent exerted only slight reducing actions on pre-existing
granuloma and exudate, while PD, a steroidal anti-inflammatory agent strongly acted on these targets. On the other hand, BEN (50 mg/kg), a basic non-steroidal anti-inflammatory agent was proved to be ineffective in both tests.
These facts are essentially in accord with
those reported in the previous paper (10) and suggest the following : If administration
commences immediately after onset of inflammation, an acidic non-
steroidal anti-inflammatory
agent exerts much the same action as does a steroidal one.
However, such is almost ineffective when administration
is started in the active stage of
granulation. Consequently, in the granulation period corresponding to stage 3 of inflammation the inhibitory actions of adrenocorticoids, e.g. against the activities of fibroblasts or other cells synthesizing intercellular substances, far surpass those of acidic non-steroidal anti-inflammatory The
effects
covering
the
pH
3.1
as
(21,
22)
of
during
tissues
(24),
as
It
also
thritis
and
LZ
et al.
closely
related
strated
that
was
liberated
was
also
to
In
that
drugs
APase
pouch were
in the
activities
of
of 5 mg/kg
and
and of
well
granuloma
and
enzymes
3)
former
BEN
(50
skin
not
mg/kg)
of
activities
of covering
the
the
not
more inhibit
significantly
of strongly
than
activities
of LZ
LZ
activity
because
10 mg/kg
and in
the
NAP and only
2) in the APase in
the
found
covering
tests the
its inhibitory NAP,
of
was
skin
the
it
These
tissues.
of
pouch,
enzyme
connective
significantly
mg/kg
demon-
occurrence
Findings
10
(14)
the
granuloma
granuloma those
most
synovitis.
level
inhibit
fluid. the
this
patients,
in
other
dose
granuloma. did
to
activities
inhibited
in
IDM
comparable CL
in
and
was
although
participate
at the
ar-
synovial
his colleagues
these
ar-
diseases.
rheumatoid
the
inflammatory
cartilage
other
cathepsin-D
from
a
and
rheumatoid
with in
are
mucopolysac-
from
55%
and
obtained
NAP
of
it
and
There
with
arthritis,
system
study,
significantly
and
cells
activities
activities
enzymes,
and
granulation to
enzymatic
patients
at
tissue, of
damage
patients
skin
lysosomes
inflammation.
that
Harris
in
(23)
suffering
rheumatoid
lysosome
activity
the
and did
CL mg/kg
of
non-rheumatic
APase
2.5
were
inhibited•@ƒÀ-Gase the
as
though
the
present and
the
tissues
with
destruction
the LZ
as
1)
other
however,
tissue,
lesion
serum
to
synovial
of
in
lysosomal arthritis.
patients
inhibit ƒÀ-Gase,
granuloma
of
(15)
35%
of
that
associates
among
non-specific
hydrolases
test
as
rheumatoid
some
preventive
his
source
induce
patients
and
determined
inflammatory
these
fluid of
was
their
also
reported
fluid
granuloma
resorption
progress
synovial
the
in
activity
the
between (12)
CL
have
into
but
and
the in
that
culture in
in
and
of
to
and
relationship
high
was
said
infiltrate
Hasebe
and APase
degradation
outbreak
high
APase
inflammation
activities
as
lesion
arthritic the
strongly
other
Pruzanski
in
the
were
by
the
the
example,
their
during
suggest
in
LZ)
collagenase
rheumatoid
of
with
are
which
process
demonstrated
detected
reports
cells part
activity
(16)
herein.
take
with
reported
LZ,
evaluated enzymes
various
dealing
compared
revealed
Granda
these
only
For
of ƒÀ-Gase,
were
participating
reports
(ƒÀ-Gase
thritis,
kg,
and not
arthritis.
charases
activities
pouch
repairing
thus
of
was
on
All
they the
rheumatoid
the
NAP
leucocytes that
tissue
to
of
granuloma cathepsin-D.
is known
number
agents.
with
the
activity
of
actions
PD
in
dose
the of
on dose
10 mg/
in granulation skin
covering
ANTI-INFLAMMATORY
PROPERTIES
OF
NAPROXEN
103
the granuloma pouch. The inhibitory actions of the respective drugs examined on all these enzymes in the curative test were considerably weaker than those in the preventive test, i.e., NAP (10 mg/kg) as well as IDM (2.2 mg/kg) slightly inhibited LZ and CL activities and PD (5 mg/kg) also slightly inhibited CL activity only, although the inhibitory actions were significant in both cases. Thus it would appear that the mechanism of anti-inflammatory
and anti-rheumatic
actions of NAP may be explained in part by the inhibitory action against activities of hydrolytic enzymes of the lysosomal system. REFERENCES 1)
HARRISON,I.T., LEWIS, B., NELSON, P., ROOKS, W., ROSZKOWSKI,A., TOMOLONIS,A. AND FRIED, LH.: J. med. Chem. 13, 203 (1970) 2) ROSZKOWSKI,A.P., ROOKSII, W.H., TOMOLONIS,A.J. AND MILLER,L.M.: J. Pharmacol. exp. Ther. 179, 114 (1971) 3) OZAWA,H., IKEDA,M. AND ITO, N.: Pharmacometrics, 6, 201 (1972) (in Japanese) 4) KATONA,G., ORTEGA,E. AND ROBLES-GILL,J.: Clin. Trials J. 8, 3 (1971) 5) BLAU,R.A., LIU, G., WILLENS,R., BOOST,G. AND SEGRE,E.: Abstracts of XIII International Congress of Rheumatology, Edited by DIXON, A. ST. J., HOLT, P.J.L., JAYSON,M.I.V. AND WOOD, P.H.N., p. 149, Excerpta Medica, Amsterdam, Princeton, London, Geneva and Tokyo (1973) 6) HILL, H.F., ANSELL,B.M., HILL, A.G.S., MATTHEWS,J.A., MOWAT,A.G. AND GUMPEL,M.: ibid. p. 149 (1973) 7) TOMPKINS,R.B., ODONE, D., KUZELL, W., ALEXANDER,S., LUSSIER,A. AND DIAMOND,H.: ibid. g, 149 (1973) 8) OKADA,T. AND SAKUMA,A.: ibid. p. 153 (1973) 9) SUZUKI, Y. AND ITO, M.: ibid. p. 147 (1973) 10) SUZUKI, Y., ITO, M., HAMAGUCHI,Y. AND YAMAGAMI,I.: Folia pharmacol. japon. 70, 465 (1974) (in Japanese) 11) LEHMAN,M.A., KREAM,J. AND BROGNA,D.: J. Bone and Joint Surg. 46, 1732 (1968) 12) HASEBE,K.: Fukushima J. Med. SeL 15, 35 (1968) 13) HosmNo, T.: Japan. Rheum. Assoc. 8, 78 (1968) 14) HARRIS, E.D., COHEN, G.L. ANDKRANE, S.M.: Arthritis Rheum. 12, 92 (1969) 15) PRUZANSKI,W., SAITO, S. AND OGRYZLO,A.: Arthritis Rheum. 13, 389 (1970) 16) GRANDA,J.L., RANAWAT,C.S. AND POSNER,A.S.: Arthritis Rheum. 14, 223 (1971) 17) SUZUKI, Y., ITO, I., ITO, M. AND YAMAGAMI, Folia pharmacol. japon. 69, 947 (1973) (in Japanese) 18) BERTELLI,A., DONATI, L. AND MAREK,J.: Inflammation Biochemistry Drug Interaction, Edited by BERTELLI,A. AND HOUCK, J.C., p. 66, Excerpta Medica Foundation, Amsterdam, New York, London, Milan, Tokyo and Buenos Aires (1969) 19) NAKAGAWA,H. AND TSURUFUJI,S.: Biochem. Pharmacol. 21, 839 (1972) 20) KIVIRIKKO,K.I., LAITINEN, O. AND PROCKOP,D.J.: Analyt. Biochem., 19, 249 (1967) 21) KATO, K.: Metabolism and Disease 9, 259 (1972) (in Japanese) 22) KONNO,K.: Metabolism and Disease 9, 259 (1972) (in Japanese) 23) BAZIN, S. AND DELAUNAY,A.: Inflammation Biochemistry and Drug Interaction, Edited by BERTELLI,A. AND HOUCK, J.C., p. 21, Excerpta Medica Foundation, Amsterdam, New York, London, Milan, Tokyo and Buenos Aires (1969) 24) WEISSMANN, G.: New Engl. J. Med. 286, 141 (1972)
91
MECHANISM OF ACTION OF A NEW ANTI-INFLAMMATORY AGENT, NAPROXEN (II) EFFECTS
OF NAPROXEN
MUCOPOLYSACCHARASE, COLLAGENOLYTIC Yoshio
ON
ACTIVITIES
ACID
PROTEASE
ENZYMES
SUZUKI,
OF AND
IN INFLAMED
TISSUES
Mikio ITO and Ichika YAMAGAMI
Department of Pharmacology, Faculty of Pharmacy, Meijo University, Tenpaku-ku, Nagoya 468, Japan Accepted November 11, 1975
Abstract-In
order
proxen,
the
nidase
(ƒÀ-Gase)
(CL)
in
model
in
and
caused
only
a
the
on
a
of
activity
LZ only.
lysosomal
by In
decrease
in
the
exudate,
but
CL
activities,
From
these
effects
of
naproxen
of
the
naproxen
may
are
be
partly
these
the
attributable
curative
slight
test,
not pred-
formed there
but
a weak that to
were did
already
showing concluded
inand
enzymes
indomethacin
induced
prednisolone it
strongly
indomethacin
granuloma and
enzyme
Prednisolone In
na-
inflammatory
naproxen
activity.
granuloma.
weight
was
significant inhibition
of
anti-inflammatory its
inhibitory
actions
enzymes.
Naproxen is a new type of non-steroidal Syntex Research.
all
naproxen
indomethacin
while
collagenolytic
on
of
of [ƒÀ-glucuro-
proliferative
did
with
results,
a
as
APase
the
and
of
test,
in
and
(APase)
preventive
effects
activities
properties
mucopolysaccharase
accumulation
inhibit
APase
of
means
the
exudate
to
Naproxen
and
protease
and
decrease.
anti-rheumatic these
of
acid
inhibitory failed
marked
volume
slight
inhibition
the
LZ
activities
investigated
and
anti-inflammatory
on
rats.
Although
inhibit
biochemical
(LZ)],
were
indomethacin
significantly
and
lysozyme
formation
evident,
nisolone
the
compound
filter-paper-implanted
prednisolone.
in
elucidate this
tissues
granuloma
quite
CL
of
inflamed
hibited
and
to
effects
anti-inflammatory
Its effectiveness for rheumatoid
agent (1) developed by
arthritis has been confirmed in animal
experiments (2, 3) and clinical studies (4-8). In order to adequately ascertain the therapeutic effects of naproxen, the biochemical mechanism of the anti-inflammatory
actions of this agent was investigated.
In previous
papers (9, 10), it is reported that by using the proliferative granulation model in filter-paperimplanted rats, investigation was conducted as to how naproxen would act on quantitative changes of the main connective tissue constituents (total mucopolysaccharide,
acid muco-
polysaccharide, glycoprotein, non-collagenic protein and collagen) in inflamed granulation tissue. It was found that the drug increased the 0.15 M NaCl-insoluble fraction of constituents of granulation tissue. It is known that the degradation of the constituents of connective tissue in inflammatory regions due to lysosomal enzymes vary according to quantitative
changes of the consti-
tuents of the same tissue, and also that there is an elevation of activities of several different hydrolytic enzymes originating from lysosomes present in synovial fluid and synovium of patients with rheumatoid arthritis and in inflammatory tissues (11-16). We also found that in rats with adjuvant-induced arthritis, there were apparent correlations between the
92
Y. SUZUKI,
M.
ITO
& I.
YAMAGAMI
intensity of the inflammation and activities of enzymes and further between depression of inflammation
by anti-inflammatory
drugs and their inhibitory actions on enzymatic ac-
tivities (17). In order to clarify the biochemical mechanism of the anti-inflammatory actions of naproxen, we made an attempt to determine whether or not the drug can indeed inhibit activities of hydrolytic
enzymes (mucopolysaccharase,
enzyme) in granuloma
acid protease and collagenolytic
and skin covering the granuloma pouch by employing the same
inflammatory model used in a previous study (10). The effects of naproxen on these enzymatic activities were then compared with its effects on the weight of the granuloma and the volume of exudate. MATERIALS
AND
METHODS
Drugs The chemical structure of naproxen (NAP) is shown in Fig. 1. Indomethacin (IDM) [Merck-Banyu], benzydamine hydrochloride (BEN) [Daiichi Seiyaku Co., FIG.
Ltd.] and prednisolone (PD) [Toyo Jozo
1.
Co., Ltd.] were employed for comparison. Induction
of inflammatory
Female lightly A
Wistar
the
tails.
No.
26,
been
A
day
by
units)
was
was
Arabic effects
The
on
was day
as of
Within fluid
into
given
each
drug
was
the
first
MW:
placed
filter
230.25
mp:
of
into
naproxen acid
153•`457•Ž
and
with
dorsal mm
region,
the
was
board, clipper.
the
base
Filter
skin
Procaine
wound
a
electric near
(Toyo
the
incision.
on
an
skin
sq.
dorsal
the
position
shaved on
25•~35
the
through
a prone
was made
paper
s.c.
in
dorsum was
incision
groups
of
the
orally were
solutions.
by
on way
and
8 days,
granuloma
in
pouch.
the
gavage
once
a
day
12.
on
in
of
12 the
of
of
Paper
which
penicillin
carefully
to
weight
had G
sutured.
(3
This
day
for
12
test,
and
control
drugs
two
curative
day
suspended days,
24.
effects
saline were
procedures. or
consecutive
until
physiological
of
dissolved
Here were
solution
expressed
each
assessed
in the by
and
same
percentage
group.
of granuloma
pouch As
drug
continued
individual the
consisted
each
and first
given
the
groups
volume
Here
were
Effects
experiments
0.
day
as
control
drug-treated
The
day
evaluated
same as
6.
on
commenced
of exudate
accumulated
the
started
was
in
of
of
propionic
of drugs
serving
drug
inhibition
Determination
and
of test
Animals the
to
implantation.
the
width
spatula
0 of
test
administered
25.
amount ratio
second
of
in
inserted
applied
divided
Gum
preventive
drug
were
preventive
skin
mm
steel
evaluation
g were
sterile was
stainless
day
the
the
25
rectangular
then
and
Animals Firstly, 1%
a
C14H1414O8
180-200
and
thickness)
considered
Administration
in
of
in
ether approx.
peace
mm
loosened
million
weighing
with incision
0.7
structure
lesion
rats,
anesthetized
horizontal
Chemical
d-2-(6-methoxy-2-naphthyl)
it was
was
formed
impossible
around to
separate
the
implanted clearly
filter the
paper
granuloma
ANTI-INFLAMMATORY pouch
from
sacrifice,
the
the
Separating weight and
surrounding
the
pouch wall
the
pouch
Preparation
pouch
wall
Inflamed
tissues
were
tissues
saline
solution.
The
As were
of
used
β-Gase
into finely
was
used
the
protease
determined
by
of
and
day
covering
granuloma
covering
enzyme
as
skin
scissors
and
centrifuged
at
enzyme
8.
After
the
pouch
the
pouch. and
granuloma
wet
pouch
activities.
24
covering
the
granuloma
homogenized
12,000
solution.
within
rpm
This
in
for
20
solution
a
pouch.
physiological
min
while
was
cooling
kept
at
0-3•Ž
hr.
of ƒÀ-glucuronidase
with
Hasebe's
(ƒÀ-Gase)
method
(12).
Ltd.)
of
was
used
activity
method
of
LZ as
activity,
et
lyophilized
lysozyme
the
(LZ)
determination
of
Co., Ltd.) was used as
Micrococcus
lysodeikticus
substrate.
[substrate:
Bertelli
and
For
glucuronide (Chugai Pharmaceutical
determination
(APase) the
with
activities
Industries,
Acid
the
skin
after
skin
activities
accordance
for
(Biochemical
performed dorsal
in
The
granuloma
determined
ρ-nitrophenyl
while
exudate
determination
was
were
enzyme
in
activity,
substrate,
for
cut
mucopolysaccharases,
determined
of
the
93
NAPROXEN
were
with
respectively.
homogenate
activities
Determination
assays
together
volume
divided
were
supernatant
enzymatic
skin,
later
5, all
OF
solution
inflamed
The
the
day
excised
determined,
were
Both
and
until
was
from were
of enzyme
at 0•Ž.
tissue
granuloma
of pouch
PROPERTIES
denatured
al.
(18),
hemoglobin
a partial
(Sigma)]
modification
of
was
the
also
method
of
Anson. The
unit
of
these
enzyme
activities
was
expressed
as
the
specific
activity
per
1 g of
wet
of
the
tissue. Collagenolytic method for
determination
into
of
sections
of
1-2
Five
ml
of
flask. 4 vol, 0.5
(CL)
of Nakagawa
0.11
vol,
M
0.2
mM
were gas.
proline
the
were
the
and
hyrolysate
activity
was
mg
was calculated
each
were
to
kept
frozen
the
from
acid
in
of 6 N
quantitatively amount
HCl by
of
an
make
3 vol)
liquid
re-extracted
105•Ž
for of
16
hr.
M
sulfate
was
then
10 mM,
then
thawed, from
added
substances
Kivirikko
MgCl2 0.16
containing
of
the
method
KCl
of 95% O2-CO2
It
was
M
0.154
a concentration
soluble
finely
7 vol,
atmosphere
solution
cut
Erlenmeyer
vol,
fumarate
activity.
TCA
hydroxyproline.
0.5
substrate
0.154
CL
(TCA)
at the
ml
vol,
dihydrostreptomycin
to
2.5%.
a 20
and
under
and
Na
the
was
buffer
of
supernatant
mg
95
MgSO4 M
as
phosphate G
40 hr
medium
trichloroacetic
heating
the
for
500
NaCl
M 0.1
Na
determination
a concentration by
analyzed
M
penicillin
incubation until
0.154
modification used
into
M
4 vol,
0.1
at 34•Ž
The
A cold make
1 vol,
potassium out
transferred
(0.154
a
was
weighing
then
pyruvate and
by itself
granuloma
KH2PO4
5 vol
of
granuloma tissue
solution
M Na
carried
added
hydrolyzed
M
0.16
centrifuged.
to
which
glucose
was
combined.
of
Krebs-Ringer 0.154
in
Granulation
piece
thickness,
5.4%
0.5
was
solution
in
3 vol,
was
and
combined centrated
A
3 vol,
and
mixture
homogenized residue
activity.
mm
Incubation
L-cysteine
then
CL
4 vol,
added.
determined
(19).
NaHCO8
Na-L-glutamate
was
Tsurufuji
modified
CaC12
1.3%
activity
and
were
Hydroxyproline et
al.
(20).
the to
the conin CL
94
Y. SUZUKI,
M.
ITO
& I.
YAMAGAMI
RESULTS Changes
in exudate
Exudate 8
to
day
rapidly
100
of
after (8.8•}2.1
hardly
detected
peared
on
by
12
on
day
in
exudate
The
exudate
on
day
8 was
maintained
Changes
of
Fig.
at
but
ing
the
granuloma
LZ
and
APase
out
the
158.2
5.5
still
activities
On hardly
of than the
these those other
detectable β-Gase
and
from
three the
high
of
ml
from
exudate
on
day
decreased
Each
the
level
g on
day
and exudate plot
hand,
there
in the
normal
8 to
they
day
normal
25 in
skin was
enzyme day
day
increased
8 and
thereafter,
reached and
was
peak
until
(2.7•}0.1
which
ap-
day
35.
weight
de-
g) The
100.
volume
during
denotes
the from
a high
of
mean
day 8 to day 100 value
4.3,
those skin
in the
with S.E. ob-
than and
the
normal
covering
the
day
8 to
activity
of
day CL
The
those
31.8
in
granuloma
implantation.
higher
were as
activities
100
significantly
viz., on
enzymes
LZ activities
g and
a constant
weight
day
were
period, as
in
intervals
volume
3.5•}0.6
sharply
2.1•}0.2
2.0•}0.4
of hydrolytic
pouch
times
the
various
activities
granuloma
experimental
and
higher
enzyme
variations
in
at
2,
was
volume
nearly
was
in granuloma
hydrolytic
3 shows
Fig.
volume
after implantation of filter paper. tained from 6 rats.
Variation
recorded
100.
thereafter,
FIG. 2.
were
indicated
the
25.
granuloma was
gradually
weight weight
As i.e.,
day
of
day
granuloma
granuloma
implantation.
ml)
weight
creased
and
and
implantation,
a peak
The
volume
volume
in
4.0
the
on
tissue,
granuloma
skin
activities normal
times
skin
and
cover-
of ƒÀ-Gase, skin
day
8
throughand
11.2,
respectively. pouch
were
The slightly
16. in
the
granuloma,
although
such
was
skin.
in granuloma
reached
the peak
on days
12 and 25, respectively,
while both APase and CL exhibited the highest activities on day 35. The activities of all enzymes thereafter showed a gradual decrease. Anti-inflammatory effects of NAP given for 12 consecutive days after implantation of the filter paper (Preventive test) Effects on granuloma weight and exudate volume:
As seen in Table 1, at the dose levels
ANTI-INFLAMMATORY
FIG.
3.
Changes
activities to S.E.
in ƒÀ-glucuronidase, in
day
PROPERTIES
100
granuloma after
obtained
lysozyme,
and
skin
implantation from
OF
of
covering filter
acid the
paper.
protease
granuloma Each
95
NAPROXEN
and
collagenolytic
pouch
plot
denotes
during mean
day
value
8
with
6 rats.
of 2.5 mg/kg, 10 mg/kg and 25 mg/kg, NAP exerted dose-dependent actions which inhibited weight of granuloma 29%, 46% and 71%, respectively, and the exudate volume 17%, 60% and about 100%, respectively.
The inhibitory actions of 10 mg/kg of NAP on granulation
and exudation were nearly equivalent those of 2.5 mg/kg of IDM and 5 mg/kg of PD. BEN in the dose of 50 mg/kg had no significant inhibitory action on either granuloma weight or exudate volume. TABLE
1.
Effects
volume beginning
of of
naproxen
and
filter-paper-implanted day
0
after
other rats
drugs following
implantation
Statistical difference from the control group (a, p<0.001; b, p<0.01; c, p<0.05)
on
granuloma oral,
daily•~12
weight
and
exudate
administrations
96
Y. SUZUKI, TABLE
2.
Effects
and
skin
oral,
of naproxen covering
and
the
daily •~12
M.
ITO
other
drugs
granuloma
& I.
on ƒÀ-glucuronidase
pouch
administrations
YAMAGAMI
of
activity
in
filter-paper-implanted
beginning
day
0 after
granuloma
rats
following
implantation
Statistical difference from the control group (a, p<0.001; b, p<0.01; c, p<0.05)
Effects in
on ƒÀ-Gase
Table
tration
of
NAP
stronger 53 % of
drugs
(5
of
was
Effects lustrated
on in
TABLE
10 by
the
2.5
25
mg/kg
PD
covering
of
30%
This
IDM.
was
showed
pouch:
after
the
inhibitory
The
which
BEN
granuloma
about
mg/kg.
(5 mg/kg),
experiment.
enzyme at
37 %. LZ
activity
Effects
and
skin
present
the
dose
Neither
3,
3.
oral,
and
skin inhibited
action
enzyme
the no
was
potent
significant
given
adminis-
tended
activity
most
As oral
to
be
inhibited
inhibitory
action
inhibitory
action
at
mg/kg.
NAP
Table
this
was
mg/kg
of
in
50
of by
mg/kg)
of
and
granuloma
produced
tested
level
30%
in granuloma in
administration
Activity about
doses
that
the
dose
activity in
than after
the
the
activity
2, ƒÀ-Gase
LZ
of covering
daily •~12
in
levels IDM
in activity
naproxen the days
skin of
nor
BEN
granuloma in
skin
other
administrations
Statistical difference from the control group (a, p<0.001; b, p<0.01; c, p<0.05)
mg/kg,
any
was
drugs pouch
granuloma
25
on of
the
the
lysozyme
day
inhibitory
12%,
activity
0
was
by
in
implantation
PD
actions.
pouch: 17%
rats after
inhibited
inhibition
granuloma
inhibited
filter-paper-implanted
beginning
pouch and
significant
covering
granuloma
granuloma
the and
showed
and
the
and
covering
10 mg/kg
and
granuloma following
As 28%
ilby
ANTI-INFLAMMATORY NAP
at
dose
the
doses
(50
mg/kg)
elicited
each
and
of
are
by
The
dose
skin
of
mg/kg)
induced
in
drugs
tested.
APase
activity
in
Table
any
of
enzymatic
the
and
granuloma
oral,
Effects
of
skin
covering
daily •~12
mg/kg, by
significant
97
NAPROXEN respectively,
IDM
(2.5
inhibition
in
mg/kg).
of
LZ
proportion None
activity,
to
of
BEN
though
PD
the
and
activity 25
skin in
mg/kg,
granuloma
pouch
covering
granuloma
while
inhibited
markedly
pouch:
Findings
the granuloma was
the
was
inhibited
enzyme
activity
inhibited
17 to
20% was
or
not
more
by
significantly
used. from
was
a
25
20%
covering
APase
drug
activity
4.
skin
10 and
ranging
and
OF
on ƒÀ-Gase.
in granuloma
4. both
other
levels
the
mg/kg
inhibited
inhibition
LZ
levels
10 was
on
covering
TABLE
(5
mg/kg,
activity
the
at dose
At in
PD
illustrated
inhibited
2.5
remarkable
activity
Effects
NAP
of The
a most The
by
levels
given.
PROPERTIES
2.5
to
25
pouch,
also
thus
inhibited
naproxen the
mg/kg,
and
NAP showing
27 % by
other
granuloma
administrations
drugs pouch beginning
a dose-dependent
PD
on of
30%
of
APase
inhibitory
activity action.
(5 mg/kg).
acid
protease
activity
filter-paper-implanted day
0 after
in rats
granuloma following
implantation
Statistical difference from the control group (b, p<0.01 c, p<0.05)
Effects on CL activity in granuloma:
Actions of drugs against CL activity were ex-
amined only in granuloma tissue and results are shown in Table 5.
The activity of this
enzyme was inhibited 25%, 37% and 59% by NAP at the dose levels of 2.5 mg/kg, 10 mg/kg and 25 mg/kg, respectively, viz., the action was dose-dependent.
CL activity was signi-
ficantly inhibited 49% and 56% by IDM (2.5 mg/kg) and PD (5 mg/kg), respectively. the other hand, BEN (50 mg/kg) produced no significant inhibition
On
of the enzymatic
activity. Anti-inflammatory effects of NAP given for 13 successive days starting day 12 after implantation of filter paper (Curative test) Effects on granuloma weight and exudate volume:
As shown in Table 6, the weight
of wet granuloma was decreased 11-16% after the administration of NAP at the dose levels of 5 to 25 mg/kg, but the inhibitory action was not significant.
Among the other drugs used,
98
Y. SUZUKI, TABLE
5.
Effects
of
granuloma
naproxen
of
ministrations
M.
ITO
and
& I.
other
drugs
filter-paper-implanted beginning
YAMAGAMI on
rats
day
0 after
collagenolytic
following
activity
oral,
in
daily •~12
ad-
implantation
Statistical difference from the control group (a, p<0.001 ; b, p<0.01; c, p<0.05)
TABLE
6.
Effects
volume
of of
beginning
naproxen
and
other
filter-paper-implanted day
12
after
drugs
rats
on
granuloma
following
oral,
weight
and
daily •~13
exudate
administrations
implantation
Statistical difference from the control group (a, p<0.001; b, p<0.01; c, p<0.05)
PD
was
42%
the
loss
over
40%
test,
PD
only
by
at
in
the
drug
The
Table
mg
in
2.5
LZ
by
25
in NAP
activity had
the
comparative
by
already
in
and
granuloma
of
the
preventive
volume
IDM.
(25
mg/kg)
in
granuloma:
the
on
covering
skin was
though
inhibition
As
in the
test.
was
inhibited
case
of the
In
fact,
slightly preventive
this
the
covering
the
inhibited
inhibition
granuloma
slightly was
not
pouch:
(11-16%)
As
by
NAP
significant.
No
other
inhibited
slightly
enzyme.
granuloma
pouch
was
activity
in
but
only.
returned study.
36%
case exudate
(81%).
mg/kg,
skin
the
and
granuloma
activity
in
The
mg/kg)
in
as
(5 mg/kg).
inhibition
to
pouch
inhibition PD
25
activity
activity
(13%)
granuloma
and
a significant
enzyme
on
with
strongest
g-Gase of
showed
Effects
up
7,
level
significantly
taken
(10 the
strong
seen
on ƒÀ-Gase
dose used
having
was
NAP
induced
Effects given
one
in weight
Because to
As
the
indicated
LZ
normal
by
in Table
day
25
8, the
(refer
the to
inhibitory
skin Fig.
covering 3),
actions
it was of
the not NAP
ANTI-INFLAMMATORY TABLE
7.
Effects
and
skin
oral,
of naproxen covering
daily •~13
and
the
PROPERTIES other
drugs
granuloma
pouch
administrations
OF
on ƒÀ-glucuronidase of
activity
in
filter-paper-implanted
beginning
day
12
after
99
NAPROXEN
rats
granuloma following
implantation
Statistical difference from the control group (c, p<0.05)
TABLE
8.
Effects
granuloma administrations
of of
naproxen
and
other
drugs
filter-paper-implanted beginning
rats
day
12
after
on following
lysozyme oral,
activity
in
daily •~13
implantation
Statistical difference from the control group (b, p<0.01; c, p<0.05)
(10 mg/kg and 25 mg/kg) and IDM (2.5 mg/kg) against LZ activity in the granuloma were slight but significant, viz., the enzymatic activities were inhibited 10-15% by these drugs. Such were not inhibited significantly either by BEN (50 mg/kg) or by PD (5 mg/kg). Effects on APase activity in granuloma and skin covering the granuloma pouch:
As
shown in Table 9, APase activity in the granuloma was inhibited 10% by NAP (2.5 to 25 mg/kg), but the inhibitory effect was not significant.
Likewise, the other drugs used were
not so effective as to demonstrate significant inhibition against the activity of APase. APase activity in the skin covering the granuloma pouch was inhibited by none of the drugs used herein. Effects on CL activity in granuloma:
As shown in Table 10, NAP at the dose levels of
2.5 mg/kg, 10 mg/kg and 25 mg/kg inhibited CL activity 20%, 23% and 29%, respectively, in proportion to the dosage increase.
The inhibitory actions of IDM (2.5 mg/kg) and PD
100
Y. SUZUKI, TABLE
9. and
Effects
of
skin
covering
oral,
naproxen
TABLE
10.
and
the
daily •~13
M.
ITO
other
drugs
granuloma
Effects
of
of
acid
naproxen
and
beginning
day
other
12
12
drugs rats
day
protease
after
activity
in
filter-paper-implanted
beginning
of filter-paper-implanted
ministrations
YAMAGAMI
on
pouch
administrations
granuloma
& I.
on
after
following
implantation
collagenolytic
following
granuloma
rats
activity
oral,
in
daily •~13
ad-
implantation
Statistical difference from the control group (b, p<0.01; c, p<0.05)
(5
mg/kg)
by
BEN
were (50
21%
and
30%,
respectively,
however,
no
significant
inhibition
was
seen
mg/kg).
DISCUSSION In the of
order
preventive the
the
relation
or
test
and
pouch
(5
mg/kg).
curative
LZ,
inhibitory
two test
were
actions
is summarized
the the
that
in NAP
inhibitory
curative
test,
and
on of
of
carried
APase
model
revealed
remarkably, In
mechanism
test
(ƒÀ-Gase,
and
the
preventive
exudation
the
biochemical
inflammatory
between
PD
the
enzyme
proliferative
The and
elucidate
hydrolytic
granuloma in
to
anti-inflammatory
out. CL)
weight
Inhibitory
in of
Table
11. dose
action however,
being NAP
actions
granuloma
and
granuloma
and
filter-paper-implanted
at the
action
level nearly as
rats
of
10 mg/kg equal
well
as
that IDM,
on
skin volume
were
of
activities
covering of
studied.
inhibited of IDM which
NAP,
the
exudate Cor-
granulation (2.5 is an
mg/kg) acidic
ANTI-INFLAMMATORY
PROPERTIES
OF
NAPROXEN
101
102
Y. SUZUKI,
non-steroidal
anti-inflammatory
M.
ITO
& I.
YAMAGAMI
agent exerted only slight reducing actions on pre-existing
granuloma and exudate, while PD, a steroidal anti-inflammatory agent strongly acted on these targets. On the other hand, BEN (50 mg/kg), a basic non-steroidal anti-inflammatory agent was proved to be ineffective in both tests.
These facts are essentially in accord with
those reported in the previous paper (10) and suggest the following : If administration
commences immediately after onset of inflammation, an acidic non-
steroidal anti-inflammatory
agent exerts much the same action as does a steroidal one.
However, such is almost ineffective when administration
is started in the active stage of
granulation. Consequently, in the granulation period corresponding to stage 3 of inflammation the inhibitory actions of adrenocorticoids, e.g. against the activities of fibroblasts or other cells synthesizing intercellular substances, far surpass those of acidic non-steroidal anti-inflammatory The
effects
covering
the
pH
3.1
as
(21,
22)
of
during
tissues
(24),
as
It
also
thritis
and
LZ
et al.
closely
related
strated
that
was
liberated
was
also
to
In
that
drugs
APase
pouch were
in the
activities
of
of 5 mg/kg
and
and of
well
granuloma
and
enzymes
3)
former
BEN
(50
skin
not
mg/kg)
of
activities
of covering
the
the
not
more inhibit
significantly
of strongly
than
activities
of LZ
LZ
activity
because
10 mg/kg
and in
the
NAP and only
2) in the APase in
the
found
covering
tests the
its inhibitory NAP,
of
was
skin
the
it
These
tissues.
of
pouch,
enzyme
connective
significantly
mg/kg
demon-
occurrence
Findings
10
(14)
the
granuloma
granuloma those
most
synovitis.
level
inhibit
fluid. the
this
patients,
in
other
dose
granuloma. did
to
activities
inhibited
in
IDM
comparable CL
in
and
was
although
participate
at the
ar-
synovial
his colleagues
these
ar-
diseases.
rheumatoid
the
inflammatory
cartilage
other
cathepsin-D
from
a
and
rheumatoid
with in
are
mucopolysac-
from
55%
and
obtained
NAP
of
it
and
There
with
arthritis,
system
study,
significantly
and
cells
activities
activities
enzymes,
and
granulation to
enzymatic
patients
at
tissue, of
damage
patients
skin
lysosomes
inflammation.
that
Harris
in
(23)
suffering
rheumatoid
lysosome
activity
the
and did
CL mg/kg
of
non-rheumatic
APase
2.5
were
inhibited•@ƒÀ-Gase the
as
though
the
present and
the
tissues
with
destruction
the LZ
as
1)
other
however,
tissue,
lesion
serum
to
synovial
of
in
lysosomal arthritis.
patients
inhibit ƒÀ-Gase,
granuloma
of
(15)
35%
of
that
associates
among
non-specific
hydrolases
test
as
rheumatoid
some
preventive
his
source
induce
patients
and
determined
inflammatory
these
fluid of
was
their
also
reported
fluid
granuloma
resorption
progress
synovial
the
in
activity
the
between (12)
CL
have
into
but
and
the in
that
culture in
in
and
of
to
and
relationship
high
was
said
infiltrate
Hasebe
and APase
degradation
outbreak
high
APase
inflammation
activities
as
lesion
arthritic the
strongly
other
Pruzanski
in
the
were
by
the
the
example,
their
during
suggest
in
LZ)
collagenase
rheumatoid
of
with
are
which
process
demonstrated
detected
reports
cells part
activity
(16)
herein.
take
with
reported
LZ,
evaluated enzymes
various
dealing
compared
revealed
Granda
these
only
For
of ƒÀ-Gase,
were
participating
reports
(ƒÀ-Gase
thritis,
kg,
and not
arthritis.
charases
activities
pouch
repairing
thus
of
was
on
All
they the
rheumatoid
the
NAP
leucocytes that
tissue
to
of
granuloma cathepsin-D.
is known
number
agents.
with
the
activity
of
actions
PD
in
dose
the of
on dose
10 mg/
in granulation skin
covering
ANTI-INFLAMMATORY
PROPERTIES
OF
NAPROXEN
103
the granuloma pouch. The inhibitory actions of the respective drugs examined on all these enzymes in the curative test were considerably weaker than those in the preventive test, i.e., NAP (10 mg/kg) as well as IDM (2.2 mg/kg) slightly inhibited LZ and CL activities and PD (5 mg/kg) also slightly inhibited CL activity only, although the inhibitory actions were significant in both cases. Thus it would appear that the mechanism of anti-inflammatory
and anti-rheumatic
actions of NAP may be explained in part by the inhibitory action against activities of hydrolytic enzymes of the lysosomal system. REFERENCES 1)
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