Using hard capsules (SR318B) developed as a sustained release diclofenac sodium (DF-Na) preparation for
once-daily administration, we investigated the persistence of the analgesic effect after oral administration in the
canine urate-induced gonarthritis model. In the control group, injection of 2% urate into the knee joint induced gait
disorder due to pain 2 hr after administration and thereafter. Gait disorder peaked 6 hr after urate injection, and
gradually recovered after 12 hr. In the treatment group, SR318B at 1.0 mg DF-Na/kg body weight was orally administered
6 hr before urate injection, and the walking score significantly decreased 2 hr after urate injection compared
with the control group (p < 0.05). Although the analgesic effect was not observed at the peak of urate-induced pain,
the walking score significantly decreased 14, 16, and 18 hr after urate injection compared with the control group
(p < 0.05). The plasma diclofenac (DF) concentration peaked 6 hr after SR318B administration, and decreased to
about 1/3–1/5 12–18 hr after administration (peak of urate-induced pain), and the plasma level was below the quantification
limit in three of five animals 24 hr after administration. DF was detected in the synovial fluid 24 hr after
administration in all animals and the concentration was 0.03 ± 0.01 μg/ml (mean ± standard error). The above
findings showed that the SR318B exhibits a persistent analgesic effect in a canine urate-induced gonarthritis model.
Not only DF in the plasma but also the DF that transferred to the synovial fluid may be involved in this persistent
analgesic effect.
Key words —–— diclofenac sodium, urate-induced gonarthritis, sustained release preparation, dog
INTRODUCTION
Diclofenac sodium (DF-Na) is a phenylacetate
non-steroidal anti-inflammatory agent synthesized
by Ciba-Geigy of Switzerland in 1965 (Fig. 1). In
Japan, sustained release capsules for twice-daily administration
have been developed by SS Pharmaceutical
Co., Ltd. (Japan) and used to treat rheumatoid
arthritis, osteoarthritis, and lumbago.
In order to reduce adverse effects, persistent drug
effect, and improve patient compliance, our aim was
to develop a preparation for once-daily administration
by inhibiting a rapid increase in the blood DFNa
concentration and maintaining the blood level
for a prolonged time. We designed four preparation
methods: 1) hard capsules containing rapid release
granules and granules coated with a combination
of enteric and water-insoluble polymers; 2) hard
capsules containing rapid release granules and enteric
film-coated plain granules combined with an
organic acid; 3) hard capsules containing granules
coated with a combination of water-insoluble and
water-soluble polymers; and 4) hard capsules containing
granules double-coated with water-insoluble
and enteric films. We prepared 3–4 prototypes using
each method and performed pharmacokinetic
studies using beagles, and selected one preparation
from those prepared from each method.1) Next, we
administered the four preparations to healthy adult
men and investigated the safety and pharmacokinetics.
The hard capsules containing rapid release granules
and enteric film-coated plain granules combined
with an organic acid at a ratio of DF-Na of 3 : 7 were
the most appropriate for the sustained release preparation
for once-daily administration based on the
pharmacokinetics of diclofenac (DF).
Thus, we orally administered this sustained re-
*To whom correspondence should be addressed: Department of
Hygienic Chemistry, College of Pharmacy, Nihon University,
7–7–1 Narashinodai, Funabashi, Chiba 274–8555, Japan. Tel.:
+81-47-465-5694; Fax: +81-47-465-5637; E-mail: tezukam@
pha.nihon-u.ac.jp
No. 5 465
lease preparation containing diclofenac (SR318B)
to a canine urate-induced gonarthritis model2) and
investigated the persistence of the analgesic effect.
MATERIALS AND METHODS
Materials —–— The test substance SR318B contains
89.98 mg of rapid release granules and
52.68 mg of sustained release granules per capsule,
and 7.5 mg and 17.5 mg of DF-Na were contained
in the rapid release and sustained release granules,
respectively. The capsules were stored at room temperature
until use.
Urate crystals were provided by SSP Co., Ltd
(Japan). Heparin sodium solution for injection
(Shimizu Pharmaceutical Co., Ltd., Japan), pentobarbital
sodium (Tokyo Kasei Kogyo Co., Ltd., Japan),
water for injection, and physiological saline
(Otsuka Pharmaceutical Factory, Inc., Japan) were
used.
Animals —–— Ten male beagles aged six months
were purchased from Nalc Co. (body weight: 8.57–
10.03 kg). The animals were individually housed
in metal bracket cages under conditions at 21–26°C
temperature, 44–66% humidity, and 12-hr lighting
(8:00–20:00). Animals were given about 300 g of
solid food (LABO D STOCK, Nihon Nosan Kogyo,
Co., Japan) per day and tap water ad libitum, and
quarantined and acclimatized for 10 days. Animals
that showed no abnormalities during the quarantine/
acclimation period were used.
Methods —–—
Establishment of dosage and timing of urate administration:
In the preliminary study in which 0.3
and 1.0 mg DF-Na/kg body weight were orally administered
to dogs, the analgesic effect was observed
at a dose of 1.0 mg/kg, and a small amount of DF
was detected in the plasma and synovial fluid 24 hr
after administration. Therefore, 1.0 mg/kg was selected
for the dose. Since 2% urate crystal suspension
in physiological saline caused severe persistent
gait disorder, we considered that to evaluate the
analgesic effect of SR318B, it is necessary to inject
urate into the joint when the plasma DF concentration
is high, and selected 6 hr after administration
of the test substance for the time of administering
urate.
Test substance administration and judgment of
analgesic effect: Using five dogs per group, SR318B
was orally administered at a dose of 1.0 mg/kg body
weight between 21:00 and 22:00, and about 30 ml
of water was immediately administered orally. The
amounts of the rapid release and sustained release
granules contained in the capsule were adjusted by
body weight for each dog. 6 hr after administration
of SR318B, 0.5 ml of 2% urate crystal suspension
in physiological saline was injected into the knee
joint to induce inflammation, and walking was observed
2, 6, 12, 14, 16, and 18 hr after injection of
urate. Walking was evaluated by five steps: normal
walking (0 point), mild claudication (1), moderate
claudication (2), walking on tiptoe (3), and walking
on three legs (4), and the mean score was obtained
in the control and SR318B treatment groups.
Measurement of plasma and synovial diclofenac
concentrations: About 2 ml of blood was collected
from the cephalic vein using a heparinized syringe
2, 6, 12, 18, and 24 hr after administration in five
animals in the SR318B treatment group. At the final
observation, synovial fluid was collected from
the knee joint in all animals.
The collected blood and synovial fluid were centrifuged
at 4°C, 3000 rpm for 10 min, and the obtained
plasma and supernatant of synovial fluid were
stored at –20°C until analysis.
The plasma and synovial DF concentrations were
measured by high performance liquid chromatography.
1)
Statistical analysis: The mean and standard error
of the measured values were calculated in each
group. For analysis of significance in comparison
of the walking score between the SR318B treatment
and control groups, Wilcoxon rank sum test was
used.3) A difference with a significance level less
than 5% was regarded as significant.
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