EFFECT OF VALPROIC ACID ON FOOD INTAKE AND NOCICEPTION IN
MORPHINE-DEPENDENT MICE
Omer T Ginawi*, Othman A Al-Shabanah, Abdulrahman M Al-Matroudi, Tarig M H ElHadiyah
Department of Pharmacology, College of Pharmacy, King Saud University, P. O. Box 2457, Riyadh 11451,
Saudi Arabia
* Corresponding author
E-mail: otginawi@hotmail.com
EFFECT OF VALPROIC ACID ON FOOD INTAKE AND NOCICEPTION IN
MORPHINE-DEPENDENT MICE
Omer T Ginawi*, Othman A Al-Shabanah, Abdulrahman M Al-Matroudi, Tarig M H ElHadiyah
Department of Pharmacology, College of Pharmacy, King Saud University, P. O. Box
2457, Riyadh 11451, Saudi Arabia
Abstract
The anorectic and antinociceptive effects of valproic acid (VPA) were studied in
morphine-dependent mice in comparison with normal ones. For this purpose, the food
intake of animals deprived of food for 24 hours and the hot plate reaction time, were
studied. Morphine-dependency was induced by i.p. injections of morphine HCl (40 mg
/kg; twice daily for 3 days).
Morphine-dependent animals showed a significant decrease in food intake (p < 0.05),
when compared with control mice (non-morphinized). Acute administration of VPA
(100, 200 and 300 mg /kg, i.p.) significantly potentiated the anorexia observed in
morphine-dependent mice. VPA (200 and 300 mg /kg, i.p.) alone produced a significant
decrease in food intake (p < 0.05) in non-morphinized animals.
In the study of antinociception, a significant increase (p < 0.001) in hot-plate latency
was observed in morphine-dependent animals, as compared to control mice. Treatment
with VPA alone produced a significant increase (p < 0.05) in hot-plate latency in salinepretreated animals in comparison with saline-pretreated control group. However, the
administration of VPA (100 and 200 mg /kg, i.p.) to morphine-dependent animals
significantly decreased (p < 0.05) their hot-plate latency as compared to the control
group.
In conclusion, VPA exhibited anorectic and antinociceptive activities in mice. VPA
potentiated the anorexia seen in mice, which were rendered dependent to morphine,
whereas the drug inhibited the antinociceptive activity observed in these mice. It
seems from the present study that VPA is probably toxic in morphine-dependent
subjects, since it might potentiate the anorectic and inhibit the analgesic effects.
Introduction
Morphine is a known drug of abuse. It is effective in most kinds of acute and
chronic pain. Chronic morphine administration causes physical and psychological
dependence (1). Opioid-dependent population is increasing and now it may be
considered to represent a sizable proportion of patients throughout the world.
Morphine-dependent subjects may use other drugs for treatment of certain
ailments, or as an attempt to avoid the withdrawal syndrome from morphine, or to gain
additional euphoria. Knowledge that morphine produces a large number of effects and
that it interacts with a number of neurotransmitters makes probably a lot of
interactions possible. The CNS is a very complex structure; therefore most of the
interactions that might originate from combinations with morphine remain unknown. It
seems necessary to evaluate the possible interaction that may result from the use of
most groups of drugs in morphine-dependent subjects.
It was found that stimulation of  and  opiate receptors decreases gamaaminobutyric acid (GABA) release from GABA-ergic interneurons (2, 3). Concomitant
use of morphine, the well known  agonist, with GABA-ergic drugs may therefore
reduce the various effects of the latter drugs. In the literature, few reports are written
on the effects of currently used GABA-ergic drugs in morphine-dependent subjects.
Valproic acid (VPA), a GABA-ergic drug, is an oral anticonvulsant that is chemically
unrelated to other anticonvulsants. It inhibits most kinds of experimentally-induced
convulsions, and is effective in many kinds of epilepsy. The mechanism of action of VPA
at present is not clear; but it has been postulated that the drug effects are mediated
through effects on the function of brain GABA, specifically by increasing brain
concentrations of this inhibitory transmitter (4, 5).
GABA has been reported to participate in the feeding behaviour in rats (6, 7). Also,
increased GABA level in the cerebrospinal fluid has been shown to have antinociceptive
effect through GABA-B receptor stimulation at the dorsal horn of the spinal cord (8).
Thus, both morphine and VPA can affect the central GABA-ergic systems. The
present study was performed to study the possible interaction between morphine
dependency and VPA on the food intake and antinociceptive effect of these drugs in
mice.
Materials and methods
1. Animals:
Swiss albino male mice weighing 25-30g were used in the current study. They were
obtained from the Experimental Animal Care Center, College of Pharmacy, King Saud
University. The animals were housed, 8 mice per cage, under conditions of normal
room temperature (21-23 C), humidity and light cycle (7 a.m. to 7 p.m.). They were
given free access to food (standard lab chow, Grain Silos and Flour Mills Organization,
Riyadh), and water ad libitum.
2. Drugs:
The following drugs were used: morphine hydrochloride (Sandoz Ltd., Basle,
Switzerland), Naloxone hydrochloride dihydrate (Fluka chemie. AG, Switzerland) and
valproic acid (Sigma chemical CO., USA). All drugs were dissolved in normal saline.
Doses were expressed as mg/kg body weight of the salt. Volume injected per animal
was 10 ml /kg.
3. Development of dependence in animals:
Animals were rendered morphine dependent by intraperitoneal injection of morphine
HCl (40 mg /kg) twice daily for 3 days, according to the method of Saelens et al. (9).
Morphine dependence was confirmed in a group of 15 mice by the method of naloxone
withdrawal jumping. Animals, which were pretreated with morphine, were injected i.p.
with 30 mg/kg of naloxone HCl, 2 hours after the last injection of morphine. The
animals were individually placed in glass cylinders (diameter and height of the cylinder
were 10 and 12 cm, respectively).
The number of jumps by each animal was
subsequently recorded for a period of 10 minutes. The number of jumps of each animal
was significantly increased in comparison with the jumps registered for the control
group. Animals subjected to this test were not used in subsequent experiments.
4. Treatment protocols:
Animals were randomly assigned to four groups (each group consisted of 8 mice). In
general, the design shown in the following table was adopted. The 2 nd treatment was
given 2 h after the last injection of the pretreatment. Appropriate drug dosages were
determined from pilot experiments.
Animals pretreated with morphine were referred to as morphinized and animals
pretreated with saline were referred to as non-morphinized.
1st treatment
2nd treatment
Group
(bid for 3 days, i.p.)
( acute, i.p.)
Group1 (non-morphinized control)
Saline
Saline
Group 2 (morphinized control)
Morphine
Saline
Group 3 (non-morphinized)
Saline
VPA
Group 4 (morphinized)
Morphine
VPA
5. Procedures:
5.1. Evaluation of anorectic activity:
Groups of animals were deprived of food for 24 h before the experiment. The
animals, however, were allowed free access to water. Each group was placed in a cage
and a weighed amount of the standard lab chow (≈ 20g) was placed at the top of the
animal’s cage. Reweighing of the animal’s food was done after 1, 2, 3, 4, and 5 h, in
order to determine the amount of food intake by the animals in each group. The
decrease in food intake was taken as an index for anorectic activity of the test drug in
food-deprived mice.
5.2. Evaluation of antinociception by the hot plate method:
An electrically heated hot plate was used. The temperature of the hot plate was
controlled at 55 ± 1º C. Groups of 8 mice were used for each dose. The animals were
individually placed on the hot plate. A stopwatch was used to record the time from
placing the animal on the hot plate until the animal either licked its forepaws or jumped
off the plate. The hot plate latency was recorded 30 min before and after 30, 60 and 90
min following drug administration.
6. Statistical analysis:
Data were expressed as mean ± SEM. One-way ANOVA was used for statistical
comparison between the means of different groups in each test. For significant results,
a post-hoc comparison of any two means was made with Tukey-Kramer multiple
comparisons test. All calculations were carried out using GraphPad InStat-2 software
program. The level of significance was adopted at p < 0.05.
Results
1. The effect of valproic acid on food intake in food-deprived non-morphinized mice:
As shown in Fig 1, VPA (100 mg /kg) did not alter the food intake of food-deprived
non-morphinized mice, at all time intervals. Only during the first hour after food
presentation, VPA (200 mg /kg) significantly reduced (p < 0.05) the food intake of
non-morphinized mice, as compared to control group (almost 50% reduction). VPA at
the dose of 300 mg/kg produced significant reduction of food intake in nonmorphinized mice at 1 h (p < 0.001) and 2 h (p < 0.05) time intervals (reductions
were about 31 and 38%, respectively), as compared to control animals.
2. The effect of valproic acid on food intake in food-deprived morphinized mice:
From Fig 2, no change in food intake by VPA (100 mg /kg) was seen after 1, 3 and,
5 hours as compared to morphinized control. However, at 2 h VPA (100 mg /kg) caused
a significant reduction (p < 0.05) in food intake and at 4 h it caused a
significant
increase (p < 0.05) in the food intake of morphinized groups as compared to control.
VPA (200 mg /kg) significantly reduced (p < 0.01) the food intake in morphinized fooddeprived mice only at 2 h as compared to control. Also, VPA (300 mg /kg) significantly
reduced food intake in morphinized groups at 1, 2 and 4 h as compared to control (p <
0.001, p < 0.01 and p < 0.01, respectively). Also, a significant increase in food intake
(p < 0.05) was observed at 5 h after VPA administration.
3. The antinociceptive effect of valproic acid in non-morphinized mice:
The effect of VPA (100, 200 and 300 mg /kg, i.p) administered to non-morphinized
mice in the hot plate test is shown in Fig 3. VPA (100 mg /kg) significantly increased
the hot plate latency at 30 and 60 min, as compared to their respective control (p <
0.01 and p < 0.05, respectively). VPA (200 mg /kg), on the other hand, did not result
in any changes in nociception as compared to saline control. VPA (300 mg /kg)
significantly increased the hot plate latency at 30 and 60 min, as compared to their
respective control (p < 0.001 and p < 0.01, respectively).
4. The antinociceptive effect of valproic acid in morphinized mice:
The effect of valproic acid in morphinized mice is shown in Fig 4. At 60 min post
VPA (100 mg /kg) injection there was a significant decrease (p < 0.05) in the hot-plate
latency, as compared to morphinized control. Also, VPA (200 mg /kg) significantly
decreased (p < 0.05) the hot-plate latency at 30 and 60 min as compared to control.
The higher dose of VPA of 300 mg /kg did not change the hot plate latency at any time
intervals post its injection in morphinized mice.
On the hot plate latency, the effect of VPA was greater in morphinized than nonmorphinized animals. This is indicated by the observation that the hot-plate latencies of
VPA 100 and 300 mg /kg doses at 90 min time interval were significantly increased (p
< 0.01) in morphinized as compared with non-morphinized mice. At 60 and 90 min,
VPA 200 mg /kg significantly increased (p < 0.01) the hot plate latency of morphinized
mice as compared to non-morphinized animals.
Discussion
In the current study, treatment with morphine, twice daily for 3 days, reduced food
intake of albino male mice. This is in agreement with Buck and Marrazzi (10) and
Shimomura et al. (11). Some authors, however, reported increases in food intake by
chronic morphine (12). Discrepancies between their results and our results may be
related to differences in species, doses, methods used to measure food intake, routes
of injection and observation intervals. Of the possible mechanisms by which morphine
reduces food intake is its ability to increase 5-hydroxytryptymine (5-HT) (13) and
dopamine (DA) (1, 3, 14) release, which are known to be anorectic (15).
It has been observed that acute VPA reduced food intake in mice in a dosedependent manner. Wolden-Hanson et al. reported that valproate induced obesity in
rats (16). VPA acts through GABA, which is thought to play a role in the central
regulation of feeding behaviour (17). Two reciprocal GABA-sensitive feeding systems
have been suggested to exist in the hypothalamus (18). It has been demonstrated that
GABA receptor agonists induce feeding in rats (19). Possibly, there are species
differences between rats and mice that might contribute to the central effects of
endogenous GABA on feeding behaviour.
In the present study, VPA at high doses produced synergism in decreasing food
intake in morphinized mice as compared to morphinized control. This could be
explained by the synergism between the GABAergic and opioid systems, since each of
them reduces food intake in mice. But, what is difficult to be explained is why low
doses of VPA antagonize the anorectic activity of morphine as compared to morphinized
control. It is known that morphine causes increased 5-HT and DA release, via inhibition
of GABA release. So, the administration of an exogenous GABA potentiating agent
should block morphine-induced increases of 5-HT and DA efflux. Thus, one of the
possible mechanisms that could be postulated to explain the interaction of VPA and
morphine is as follows: VPA increases GABA level, which may in turn block morphineinduced increases of 5-HT and DA efflux, hence leading to decreased morphine
anorexia.
In agreement with previous studies (20), morphine administration produced
antinociception in mice. VPA also produced antinociception in mice, in agreement with
previous studies (21, 22). VPA alone at the doses of 100 and 300 mg/ kg significantly
increased the hot plate latency, although it failed to do so at the dose of 200 mg/ kg.
These results indicate that VPA analgesic property could be dose specific, or
alternatively, larger groups of animals were needed to manifest the expected
antinociception by VPA 200 mg/kg. In the present study rapid onset of antinociceptive
action of VPA was observed.
VPA has been reported to be an inhibitor of GABA-transaminase and succinic
semialdehyde dehydrogenase. Thus, increased GABA by VPA is a possible explanation
for its antinociceptive action. Also, Vion-Dury et al. (22) reported that the analgesic
properties of VPA might be related to activation of pro-enkephalin system in rat brain.
In the present study, VPA failed to potentiate the analgesic effect of morphine in
morphinized mice. On the contrary, VPA (at low doses), antagonized the analgesic
effect of morphine.
The manipulation of 5-HT level, by increasing or decreasing its
release for example, in the CNS has been reported to affect morphine analgesia. It is
known that morphine administration evokes 5-HT release in the CNS through inhibition
of GABA release. Thus, the administration of an exogenous GABA-ergic drug is
expected to block morphine-induced increase in 5-HT and DA efflux. Tao and Auerbach
(13) have reported that muscimol (GABA-A agonist) infusion in the dorsal raphe
nucleus blocked the effect of morphine sulfate (20mg/kg s.c.) on 5-HT efflux. This
could be one of the possible mechanisms to explain the VPA-produced antagonism of
morphine analgesia in morphinized mice. In addition, it has been reported by Baf et al.
(23) that VPA significantly decreases 5-HT level in the hypothalamus and cerebellum;
this is another possible mechanism to explain the antagonism of VPA to morphine
analgesia.
In conclusion, morphine-dependence has resulted in increased VPA anorectic
activity. VPA failed to potentiate the analgesic activity observed in morphine-dependent
animals. Thus, VPA is possibly a toxic drug if used in morphine addicts. This is because
VPA potentiates the anorexia and inhibits the analgesia induced by morphine
administration.
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Figure legends
Fig 1. The effect of valproic acid (100, 200 and 300 mg /kg, i.p.) on food intake of
non-morphinized food-deprived male mice.
*p< 0.05, **p< 0.01, ***p<0.001 as compared to non-morphinized control.
In this and subsequent figures:
Vertical bars indicate SE of the means.
s = significant difference between the 4 groups (One-Way ANOVA).
Ns = no significant difference between the 4 groups (One-Way ANOVA).
Fig 2. The effect of valproic acid (100,200 and 300 mg /kg, i.p.) on food intake of
morphinized food-deprived male mice.
*p<0.05, **p<0.01, ***p<0.001 as compared to morphinized control.
Fig 3. The effectof valproic acid (100, 200 and 300 mg /kg, i.p) on hot-plat
latency of non-morphinized male mice.
*p<0.05, **p<0.01,***p<0.001, as compared to non-morphinized control.
Fig 4. The effect of valproic acid (100, 200 and 300 mg /kg, i.p.) on hot-plate latency
of morphinized male mice.
*p<0.05, as compared to morphinized control.
7
Control (non-morphinized)
contorl
VPA
100mg/kg
vpa
100
VPA
200mg/kg
vpa
200
VPA
300mg/kg
vpa
300
s
6
s
Food intake (g)
5
s
Ns
Ns
**
4
3
***
2
*
1
0
1
2
3
Time (h)
Fig 1.
4
5
Control (morphinized)
morphine
control
VPA 100 mg/kg
morphinized/vpa
100mg/kg
VPA 200 mg/kg
morphinized/vpa
200 mg/kg
VPA 300 mg/kg
morphinized/vpa
300 mg/kg
6
s
s
*
s
5
Food intake (g)
*
s
4
3
s
2
***
1
*
**
** **
0
1
2
3
Time (h)
Fig 2.
4
5
Control (non-morphinized)
VPA 100 mg/kg
VPA 200 mg/kg
VPA 300 mg/kg
s
s
##
###
***
**
s
20
**
*
18
Hot plate latency (sec)
16
14
12
10
8
6
4
2
0
1
30
2
Time (min)
Fig 3.
60
3
90
Control (morphinized)
VPA 100 mg/kg
VPA 200 mg/kg
VPA 300 mg/kg
25
s
Ns
s
20
*
hot plat latency (sec)
*
*
15
10
5
0
30
60
Time (min)
Fig 4.
90