Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
391
EVALUATION THE ANTINOCICEPTIVE EFFECTS OF
METOCLOPRAMIDE AND THEIR INTERACTION WITH
DIPHENHYDRAMINE IN ACUTE MODEL OF PAIN IN MALE
MICE
Taimaa A. Al-Najmawy , G. A-M. Faris***
Department of physiology , College of Veterinary Medicine, University of Mosul,
Mosul, Iraq
Keywords: Antinociceptive, Metoclopramide, Writhes .
Corresponding Author: [email protected]
ABSTRACT
The aim of this study was to evaluate the analgesic effect of metoclopramide
alone or as a combination with diphenhydramine. The type of interaction between two
drugs also determined at level of acute pain centrally and peripherally using hot-plate
and writhing test in male mice. The individual ED50 value of metoclopramide and
diphenhydramine for induction of antinociceptive effect was at 30.7 mg/kg,ip and
0.57mg/kg, sc ,respectively. While combined this ED50 value of diphenhydramine and
metoclopramide at several ratios (1:1, 0.5:1, 0.5:0.5 and 0.25:1) respectively,
produced synergism interaction between two drugs at all ratios (except the final ratio,
produced antagonism). This combination decreased the ED50 of each drug and
produced good antinociceptive effect in male mice at centrally level. Concomitant
administration of metoclopramide ip and diphenhydramine sc at double doses of ED50
for each drug, also produced good analgesic effect at visceral pain level, which
induced by acetic acid in writhing test. Avery effective and safe antinociceptive effect
and also achieved using combination of metoclopramide and diphenhydramine at
sedative doses (not analgesic).The results suggested that synergism (super-additive)
interaction of diphenhydramine and metoclopramide combination was a good and safe
antinociceptive effect produced by a combination of sedative dose for each drug . The
combination of these drugs very important in practice of the veterinary medicine.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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INTRODUCTION
Metoclopramide is a benzamide derivatives, gastroprokinitic agent, used as
antiemetic in animals (1,2 and 3) and human (4,5), its used also in a cases of reflux
esophagitis in dog (6) and man (7). This effect related to their antagonize of dopamine
D2-receptors centrally and peripherally (2,7 and 8), inhibition of serotonin receptors
5-HT3 (9,10) and agonist of 5-HT4 receptors (11,12). As well as to indirect
cholinergic activity (13) that lead to promotes release the neurotransmitter
acetylcholine (11). Metoclopramide has sedative and hypnotic effects alone (14) or
with Ketamine in chicken (15), the sedative effect also reported in man (5). The drug
has been successes as analgesic in human (16, 17), mice (18, 19) rats (20) and in dogs
(21). Many combinations with metoclopramide used to improve the analgesic effect
such as metoclopramide with diphenhydramine in human to treat headache (22),
decrease emesis and nausea after surgery (23, 24). Metoclopramide also used with
tramadol to produce analgesia, that equal to morphine (opioid agent) after surgery in
man (25). The mechanism related with the analgesic effect of metoclopramide not
understands yet.
Diphenhydramine is an ethanolamine derivative, which cross blood brain barrier
(26, 27). It’s the oldest first generation H1-receptors antagonist, in human and animals
(28, 29). The drug is commonly used as anti-allergy medication for treatment of
rhinitis, anaphylaxis, colds, motion sickness, agitation and insomnia (30, 31). It has
also anticholinergic properties (29, 27) ant muscarinic, and antiadrenergic in human
(32, 33) and animals (34). Used to treat cancer pain (35), and as local analgesic by
block sodium channel (32,36). It has anti-inflammatory properties in mice (37).
Combination of diphenhydramine with Ibuprofen, produced synergism effects in
human by increase analgesia and anti-inflammatory action of Ibuprofen (38).
Diphenhydramine also potentiate the analgesic effect of morphine in mice (39).
However; the antinociceptive effect of metoclopramide and diphenhydramine
combination in mice not examined in previous studies , and the aim and introduction
of the current study was evaluated the antinociciption effect of this combination in
male mice, as well as detected the type of interaction by using thermal method (hot
plate) (40,42 and 66) and chemical method (writhing reflex) (19,41 and 42) .
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
393
MATERIALS AND METHODS
In this study we used male albino Swiss mice weighing (20-35) g, housed in
animal house appurtenant to Veterinary Medicine of the university of Mosul/Iraq. At
standard circumstance of temperature (22± 2c°), ventilation and 10 hr light/14 hr dark.
Animals housed in rodent plastic cages (17x20x30) cm, food and water were given ad
libitum and complete care of mice until reaching 2 months. The doses of
metoclopramide and diphenhydramine were prepared by dissolve pure powder of each
drugs in physiological saline solution directly before the experiment, the volume of
administration of each drugs was at 5ml/kg, body weight in all experiments.
Experiment 1: Determine the individual median analgesic dose (ED50) of
metoclopramide (ip) and diphenhydramine (sc) in male mice by up and-down method
(43).
6 male mice were used for each drug, weight (19-30) g. The initial dose of
metoclopramide (Vaikunth,India) was at 20 mg/kg i.p., while for diphenhydramine
(Samanta Organics PVT,India) was at 1 mg/kg s.c., the doses were choices from
preliminary experiments and previous studies (19,44), The increase or decrease in
the later doses of metoclopramide and diphenhydramine was at 5,0.25 mg/kg,
respectively . Analgesic effect was measured using the hot plate test (thermal method)
(45), at a temperature of 56 C°. Animals were placed individually on the hot plate
(Heidolph Me Hei-standard, Germany) before administration of drug and recorded
response as latency time in seconds (first removal of fore or hind paw and licking
or/and jumping, shaking) , then recorded latency time 10 min after injection of
metoclopramide i.p. or diphenhydramine s.c. by placed the same mice on the hot
plate. The cut-off time for analgesia was 20 s (the maximum time was allowed the
animal to stay on the hot plate to avoid damage of the paw tissue (19,42,66). The
ED50 value was calculated according to the formula (43):
ED50= Xf + Kd
Xf: final dose
K: table value
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
394
d: increase or decrease in dose( constant )
Dixon table:
Stander
error
Second part K represent series tests that begin as at
from series O OO OOO OOOO
XOOO 0.157- 0.154- 0.154- 0.154- OXXX 0.61
XOOX 0.878- 0.861- 0.860- 0.860- OXXO
XOXO 0.701 0.737 0.741 0.741 OXOX
XOXX 0.084 0.169 0.181 0.182 OXOO
XXOO 0.305 0.372 0.380 0.381 OOXX
XXOX 0.305- 0.169- 0.144- 0.142- OOXO
XXXO 1.288 1.500 1.544 1.549 OOOX
XXXX 0.555 0.897 0.985 1.000 OOOO
Second part
from series
X XX XXX XXXX
- K represent series tests that begin as at
Experiment 2: Determination the type of drug interaction between metoclopramide
and diphenhydramine as a combination for induction antinociceptive effect in male
mice: At Ratio of 1:1
7 male mice were used at 2 months age , weighing (20-28) g selected randomly.
We tested the mice as same previous experiment on hot pate to determined latency
time. The initial doses administrated to the first mice as a combination were the ED50
of metoclopramide and that of diphenhydramine (30.7mg/kg, and 0.57mg/kg)
respectively, which detected previously in experiment 1. The increase and decrease in
the ED50 value of metoclopramide and diphenhydramine was (7.5 mg/kg and 0.14
mg/kg) respectively. Then the individual ED50 of each drug alone and that of a
combination of two drugs at ratio 1:1 subjected to isobolographic analysis, to explore
the type of interaction between two drugs (46,47,48,51) in producing antinociceptive
effect in mice.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
395
Dependent on the Isobolographic analysis, used diagram paper and pointed the
individual ED50 value of diphenhydramine on the X axis and that of metoclopramide
on the Y axis, then drew a straight diagonal line between the individual ED50. The
ED50 value for the combination then detected as if pointed above (right) or below
(left) the line indicate antagonism or synergism interaction respectively , while
located on the diagonal line represent additive (no interaction) (15,42,48,49).
For more explore the type of interaction , we used the interaction index from
equation : Y= da/Da + db/Db (48), as: Da and Db was individual value of ED50 of
metoclopramide and diphenhydramine respectively , while da and db was the value of
combination the ED50 of both drugs. If Y value equal 1 means no interaction
(additive), Y > 1 that means sub additive (antagonism) interaction, while Y < 1 means
synergism (super-additive) interaction. The percentage of reduction in the ED50 of
both drugs was calculate by following ]formula:
% in reducing of ED50= ED50 (individual) – ED50 (combination)/ED50 (individual)
As the same of previous experiment, we injected metoclopramide i.p. with
diphenhydramine s.c. as a combination at various ratio : 0.5:1 , 0.5:0.5 , 0.25:1
respectivley and detected the type of interaction for antinociceptive effect at those
ratios as previously.
Experiment 3: Effect of metoclopramide and diphenhydramine on the visceral pain in
male mice (writhing reflex) chemical method (41,42,45).
Randomly selective 20 male mice weighing (18-30)g which divided in to 4
groups, each group consist of 5 animals : Group 1 (control) injected with 1% Acetic
acid ip at (0.1 ml/10 g) which causes visceral pain (writhing reflex), the onset of
writhes and the number of writhes recorded during 20 min after injection (19,42).
Group 2, 3 individually injected with metoclopramide ip or diphenhydramine sc at
double doses of ED50 (61.4 mg/kg and 1.14 mg/kg) for each drug, respectively 15 min
before injection of acetic acid ip in the same animal. Then recorded the onset of
writhes and the number of writhes during 20 min, while the animals in group 4
injected with metoclopramide ip and diphenhydramine sc at (61.4 and 1.14) mg/kg,
respectively (as a combination )15 min before injection of acetic acid ip in the same
animal , the onset and number of writhes also recorded during 20 min . In order to
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
396
calculate the percentage of reduction in the number of writhes in each group used the
following formula (40,42):
( N control – N test/ N control ) X100
N: number of writhes for each group.
Experiment 4: Effect of metoclopramide and diphenhydramine alone or as a
combination at sedative doses (non-analgesic) on the acute pain in male mice.
Divided 20 mice weighing (20-33)g ,selective randomly; in to 4 groups, each
group consist of 5 animals treated as follows: Group 1 received normal saline(0.9%)
ip at (5 ml/kg) body weight, Group 2,3 were individually received sedative doses
(non analgesic doses) of metoclopramide ip at 10 mg/kg or diphenhydramine sc at
0.5 mg/kg (the doses choice according preliminary experiments). While Group 4
received a combination of metoclopramide ip and diphenhydramine sc at non
analgesic doses (10 and 0.5) mg/kg , respectively. Each mice was tested in all groups
before and 10 min after injection of drug on the hot plate as previously mentioned in
experiment 1.
Statistical analysis:
The data in experimental 3 and 4 were analyzed by One Way analysis of variance
(ANOVA), then to detect the significant between groups the data subjected to the
Least significant test LSD (50). While the score data such as writhing test were
statistically analyzed by Mann- whitney U test (50,51,52). The level of significance in
all experiments was at (p < 0.05).
RESULTS
Experiment 1: Determine the individual median analgesic dose (ED50) of
metoclopramide (ip) and diphenhydramine (sc) in male mice by up and-down method.
Several doses of metoclopramide and diphenhydramine injected individually in
numbers of mice to detect the individual ED50 value for metoclopramide and
diphenhydramine explore (30.7 mg/kg and 0.57 mg/kg) respectively (Tab.1). Animals
that treated with metoclopramide and diphenhydramine appeared symptom of
sedation, quiet and reduce movement.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
397
Table(1).Median analgesic dose of metoclopramide and diphenhydramine
individually in male mice by hot plate test:
Variable Metoclopramide ip Diphenhydramine SC
ED50 30.7 mg/kg 0.57 mg/kg
Range of the doses used 35-20 mg/kg 1-0.5 mg/kg
Initial dose 20 mg/kg 1 mg/kg
Last dose 35 mg/kg 0.75 mg/kg
Increase or decrease in the 5 mg/kg o.25 mg/kg
dose
Number of mice used OOXOOX 6 XXOXOX 6
X analgesia O no analgesia
Experiment 2: Determination the type of drug interaction between metoclopramide
and diphenhydramine as a combination for induction antinociceptive effect in male
mice:
The individual ED50 of metoclopramide and diphenhydramine which induced
analgesia in male mice were 30.7 mg/kg and 0.57mg/kg respectively , when combined
metoclopramide ip with diphenhydramine sc at different ratio of ED50 value for
each drug produce reduction in the ED50 value for each drug at several ratio :1:1,
0.5:1, 0.5:0.5 to (69.3 %, 66.7 %);( 67.9% 35 %) and (59.7 %, 68.4 %) respectively,
shown its in Tab 2,3,4 and 5 respectively.
Depended on Isobolographic analysis, determined the type of interaction between
both drugs at each ratio, that is shown in (figure 1, 2, 3 and 4) respectively. All the
value of ED50 for each combined was located under the diagonal line which
connected between the individual ED50 value for metoclopramide and that for
diphenhydramine , that indicated the synergism interaction between the two drugs at
each ratio as shown in Figure (1,2,3), except at ratio 0.25:1 the value of ED50 for
combined of the individual ED50 of two drugs located above the diagonal line,
therefor the combination of metoclopramide and diphenhydramine at this ratio was
antagonist (Figure 4).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Table (2). Determination the median analgesic doses (ED50) of metoclopramide ip
and diphenhydramine sc as a combination in male mice at ratio 1:1
Variable Metoclopramide and Diphenhydramine
Metoclopramide Diphenhydramine
ED50 9.44 mg/kg 0.19 mg/kg
Range of the doses used 30-7.5 mg/kg 0.57-0.15 mg/kg
Initial dose 30 mg/kg 0.57 mg/kg
Last dose 15 mg/kg 0.29 mg/kg
Increase or decrease in the 7.5 mg/kg o.14 mg/kg
dose
Number of mice used XXXOXOX 7 XXXOXOX 7
% Reduced in ED50 69.3% 66.7%
Y (interaction index) 0.63 0.63
X analgesia O no analgesia
Diphenhydramine ED50 mg/kg SC
Figure 1: Determination the type of interaction between metoclopramide ip and
diphenhydramine sc by Isobolographic analysis at a ratio 1:1. The interaction between
two drugs was synergism.
ED50 of metoclopramide at (30.7) mg/kg , IP
ED50 of diphenhydramine at (o.57) mg/kg , SC
Metoclopramide ED50 mg/kg IP
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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the ED50 of metoclopramide ip at (9.44) mg/kg and ED50 of diphenhydramine sc at
(0.19) mg/kg at 1:1
Table (3). Determination the median analgesic doses (ED50) of metoclopramide ip and
diphenhydramine sc as a combination in male mice at ratio 0.5:1
Metoclopramide and Diphenhydramine
Metoclopramide Diphenhydramine
Variable
ED50 9.85 mg/kg 0.37 mg/kg
Range of the doses used 15-7.5 mg/kg 0.57-0.29 mg/kg
Initial dose 15 mg/kg 0.57 mg/kg
Last dose 11.25 mg/kg 0.43 mg/kg
Increase or decrease in the 3.75 mg/kg o.14 mg/kg
dose
Number of mice used XXOOXX 6 XXOOXX 6
% Reduced in ED50 67.9% 35%
Y (interaction index) 0.96 0.96
X analgesia O no analgesia
Diphenhydramine ED50 mg/kg SC
Figure 2: Determination the type of interaction between metoclopramide ip and
diphenhydramine sc by Isobolographic analysis at ratio 0.5:1. The interaction
between two drugs was synergism.
ED50 of metoclopramide at (30.7) mg/kg , IP
ED50 of diphenhydramine at (o.57) mg/kg , SC
Metoclopramide ED50 mg/kg IP
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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the ED50 of metoclopramide ip at (9.85) mg/kg and ED50 of diphenhydramine
sc at (0.37) mg/kg
Table(4). Determination the median effected analgesic doses (ED50) of
combination for metoclopramide ip and diphenhydramine sc in male mice at
ratio 0.5:0.5
Variable Metoclopramide and Diphenhydramine
Metoclopramide Diphenhydramine
ED50 12.37 mg/kg 0.18 mg/kg
Range of the doses used 15-11.25 mg/kg 0.28-0.14 mg/kg
Initial dose 15 mg/kg 0.28 mg/kg
Last dose 15 mg/kg 0.28 mg/kg
Increase or decrease in the dose 3.75 mg/kg o.14 mg/kg
Number of mice used XOXOX 5 XOXOX 5
% Reduced in ED50 59.7% 68.4%
Y (interaction index) 0.72 0.72
X analgesia O no analgesic
Diphenhydramine ED50 mg/kg SC
Figure 3: Determination the type of interaction between metoclopramide ip and
diphenhydramine sc by Isobolographic analysis at a ratio 0.5:0.5. The interaction
between two drugs was synergism.
ED50 of metoclopramide at (30.7) mg/kg , IP
ED50 of diphenhydramine at (o.57) mg/kg , SC
Metoclopramide ED50 mg/kg IP
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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the ED50 of metoclopramide ip at (12.37) mg/kg and ED50 of diphenhydramine sc at
(0.18) mg/kg
Table (5). Determination the median effected analgesic dose (ED50) of combination
for metoclopramide ip and diphenhydramine sc in male mice at ratio 0.25:1
Variable Metoclopramide and Diphenhydramine
Metoclopramide Diphenhydramine
ED50 8.83 mg/kg 0.67 mg/kg
Range of the doses used 9.4-7.5 mg/kg 0.71-0.57 mg/kg
Initial dose 7.5 mg/kg 0.57 mg/kg
Last dose 7.5 mg/kg 0.57 mg/kg
Increase or decrease in the 1.9 mg/kg o.14 mg/kg
dose
Number of mice used OXOXO 5 OXOXO 5
Y (interaction index) 1.46 1.46
X analgesia O no analgesia
Diphenhydramine ED50 mg/kg SC
Figure 4: Determination the type of interaction between metoclopramide ip and
diphenhydramine sc by Isobolographic analysis at ratio 0.25:1. The interaction
between two drugs was antagonism.
ED50 of metoclopramide at (30.7) mg/kg , IP
ED50 of diphenhydramine at (o.57) mg/kg , SC
Metoclopramide ED50 mg/kg IP
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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The ED50 of metoclopramide ip at (8.83) mg/kg and ED50 of diphenhydramine sc at
(0.67) mg/kg
Experiment 3: Effect of metoclopramide and diphenhydramine on the visceral pain in
male mice (writhing reflex) chemical method.
Injection of metoclopramide alone at (double doses ED50) ip (61.4) mg/kg in
male mice significantly increased the time of onset and decreased the writhing
number in comparison with the control and diphenhydramine group Table (6). While
there was no significant effect of diphenhydramine at dose of (1.14) mg/kg on the
onset and number of writhing. While there was a significant increase in the time of
onset and decreased the number of writhing in group 4 (combination of two drugs at
double doses of ED50 (61.4, 1.14) mg/kg respectively by (80%), in comparison with
control (0%), diphenhydramine (3%) and metoclopramide (42%), at a level of
significant (p < 0.05) (Table 6, Figure 5).
Table (6). Analgesic effect of metoclopramide ip and diphenhydramine alone or as a
combination on writhing reflex (chemical methoed) in male mice.
% Reduce in
writhing number
Writhing number
Mean ±SE
Latency to Onset
of writhing
Mean ±SE
Treatments
Control (1% acec acid),ip 2.25± 0.47 35.5± 6.27 0%
Metoclopramide 61.4 mg/kg, 6.25± 0.25*a 20.5± 3.32*a 42%
ip
Diphenhydramine 2.75± 0.25 34.5± 0.95 3%
1.14mg/kg, sc
Metoclopramide 61.4mg/kg, 6.75±0.25* a 7± 1.47*ab 80%
ip + Diphenhydramine
1.14mg/kg, sc
Values are mean ± SE of five mice /group
Diphenhydramine was injected directly after metoclopramide
* : significantly different from the control group at p<0.05
a :significantly different from diphenhydramine at dose (1.14) mg/kg at p<0.05
b :significantly different from metoclopramide at dose (61.4)mg/kg at p<0.05
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Control Metoclopramide Diphenhydramine Metoclopramide with
diphenhydramine
Figure 5. Effect of metoclopramide and diphenhydramine alone or as a combination
on the visceral pain in male mice (writhing reflex) chemical method
Experiment 4: Effect of metoclopramide and diphenhydramine alone or as a
combination in sedative doses (non-analgesic) on the acute pain in male mice.
Administration of metoclopramide at (10) mg/kg, ip and diphenhydramine at
(0.57) mg/kg, sc ( sedative doses) each alone produced non-significant antinociceptive
effect 30 min after injection, in comparison with control group, , while a combination
of metoclopramide ip and diphenhydramine sc (at sedative doses 10, 0.5 mg/kg
respectively) produced significant antinociceptive effect at 100% in group 4 in
compared with the control group and groups of metoclopramide or diphenhydramine
each alone. (Tab. 7).
Table (7). Effect of metoclopramide and diphenhydramine in sedative doses (nonanalgesic)
on the acute pain, hot plate (thermal method) in male mice.
% of analgesic
pain
Latency me aer 30
min
Mean ±SE
Latency time Mean
±SE
(Base line)
Treatment
Control (normal saline 0.9%) ip 5.1 ± 1 4.06 ± 1.03 0%
Metoclopramide 10 mg/kg ip 4.74 ± 0.8 4.2 ± 0.75 0%
Diphenhydramine 0.5mg/kg sc 6 ± 1.7 3.9 ± 0.4 0%
Metoclopramide 10 mg/kg ip + 4.7 ± 1.06 8 ± 1.15*ab 100%
Diphenhydramine 0.5mg/kg sc
% the reduce in writhing number
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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Values are mean ± SE of five mice /group
Diphenhydramine was injected directly after metoclopramide
* : significantly different from the control group at p<0.05
a :significantly different from metoclopramide alone at dose (10) mg/kg, at p<0.05
b :significantly different from diphenhydramine at dose (0.5)mg/kg, at p<0.05
DISCUSSION
In this work we acted more focus on the analgesic effect of metoclopramide alone
or as a combination with diphenhydramine. Whereas metoclopramide is antiemetic
drug, but recently many reports have been proved the analgesic effect of
metoclopramide alone (17,18) or as a combined with other analgesic drugs such as
ketamine (19) or with tramadol (25). In the current study, the analgesic effects of
metoclopramide evaluated by using the hot-plate test to exam the centrally pain and
chemical test (writhing reflex) to evaluate viscerally pain in mice(19,42,64). As well
as determined the analgesic effects of metoclopramide and diphenhydramine as a
combination by using ED50 value, the type of interaction between them also
evaluated using isobolographic analysis and interaction index (Y value)(46,47,48).
Diphenhydramine is H1 receptor antagonist, it's the oldest first generation antihistamine
drug (26,27,28), several study confirmed the analgesic effect of
diphenhydramine alone (35,37) or as a combined with other drugs such as with
tramadol (44), ibuprophin (38) and morphine (39), However; the present study has
been determined the analgesic effect of metoclopramide and diphenhydramine each
alone or as a combination in mice, which was not reported in previous study. The
combination of drugs may produce antagonism, additive or synergism interaction. In
the present study we established the synergism interaction between two drugs at level
of antinociceptive effects. The individual ED50 value for each drug also detected by
using up and down method (43) which was (30.7) mg/kg and (0.57) mg/kg for
metoclopramide and diphenhydramine respectively , this data was in accordance with
previous study for metoclopramide (19) and diphenhydramine (44) in mice.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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By used different ratios of ED50 for each drug (1:1, 0.5:1: and 0.5:0.5) , the result
was synergism interaction for each ratio and this may be due to each drug act on
different receptors (pharmacodynamics) to produce their analgesic effect. As
metoclopramide act as analgesic drug by antagonism of D2-receptors which
correlated with opioid system without connected with opioid receptors (56). Also the
drug has been increased prolactin hormone, which connected with opioid system (57,
58) as well as effect as agonist of serotonin 5-HT4 (61), in addition to alter Ca+ cross
through cell membrane (59,60). While the analgesic effect of diphenhydramine is due
to block sodium channels, that similar to the mechanism of local anesthetic (36). A
good visceral antinociceptive effect of this combination also confirmed in the current
study by using writhing test (chemical method induced by acetic acid 1%). This effect
similar to the combination effect of metoclopramide with ketamine in mice (19), that
related to the central and peripheral analgesic effect of metoclopramide as well as
present of diphenhydramine with it as a combination , other suggested mechanism for
the analgesic effect of the metoclopramide and diphenhydramine combination that
metoclopramide metabolize by CYP450 2D6 iso-form (65), the first generation H1–
antihistamine diphenhydramine also metabolize by the same enzyme in the liver, as
well as it is CYP2D6 inhibitor(62,66,67), so it can inhibit the metabolism of
metoclopramide , this may be resulting the level of metoclopramide and stay for
prolong time in the body which produce effective analgesia for longer time,
Therefore; we demonstrate that the combination of metoclopramide and
diphenhydramine can be use as analgesic agent to control the acute pain centrally
through increase threshold of the pain (change the latency time) and peripherally as
decrease the numbers for writhing reflex in animals comparison with control group,
also we succeed in confirmed the good analgesic effect of metoclopramide and
diphenhydramine at sedative doses (non-analgesic doses) for each drug when injected
as a combination without any overt side effects on mice except docile, this effects not
reported previously.
CONCLUSION
The present study explored the antinociceptive effect of metoclopramide and
diphenhydramine each alone; in addition the combination of two drugs produced
synergism interaction. Also a combination of sedative doses of each drug produced
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
406
effective and safe analgesic effect in male mice which very important in Veterinary
practice.
REFERENCES
1. Jacoby, H.I. (2017). Reference Module in Biomedical Sciences: Gastric Emptying.
Dis. Res. Consultants, Brigntine, NJ, United States.
2. Kosecki, S.K. (2003). Metoclopramide: dopamine receptor antagonist, antiemetic ,
gastroprokinetic agent. Compend Contin Educ Pract Vet.; 25: 826-900.
3.Wilson, D.V., Evans, A.T., and Mauer, W.A. (2006) Influence of
metoclopramide On gastroesophageal reflux in anesthetized dogs.American
J.Vet.Res.; 67(1): 26-31.
4. Katzung BG. (2012). Basic and clinical pharmacology.12th ed.,New York
:McGraw-Hill. 2012,pp.1091-1099.
5.Pang WW, Wu HS, Lin CH, Chang DP and Huang MH. (2002) Metoclopramide
decreases emesis but increases sedation in tramadol patient-controlled analgesia.
2002; Can J Anaesth. 49(10): 1029- 1033.
6.Aiello, S.E E.d. (1998): Merck Veterinary Manual. Merck and Co. Inc.,
Whitehouse Station, NJ.
7.Rang, H.P, Dale, M.M, Ritter, J.M and Moore, P.K. (2003) Pharmacology.5th
ed., Edinburgh; New York: Churchill Livingstone.; pp. 374-375.
8.Peterson SE, Selvaggi KJ, Scullion BF and Blinderman CD. (2018).
Hematology. 7th ed.,Chapter 91-Pain Management and Antiemetic Therapy in
Hematologic Disorders. Elsevier Inc.,pp. 1473-1487.
9.Walkembach, J., Brüss, M., Urban, B.W., and Barann, M . (2005). Interactions
of metoclopramide and ergotamine with human 5- HT3A receptors and human 5-HT
reuptake carriers. Br J Pharmacol.; 146(4): 543-552.
10.Lee, A. and Kuo, B. (2010). Metoclopramide in the treatment of diabetic
gastroparesis. Expert Rev Endocrinol Metab.;5(5):653-662.
11. Tonini, M., Cipollina, L., Poluzzi, E., Crema, F., Corazza, G.R. And De Ponti,
F. (2004) Review article: clinical implication of enteric and central D2 receptor
blockade by antidopaminergic gastrointestinal prokinetics. Aliment Pharmacol.
Ther.;19(4): 379-390.
12. Harada, T.,Hirosawa, T.,Morinaga, K. and Shimizu, T. (2017).
Metoclopramide -induced Serotonin Syndrome.Intern. Med.;56: 737- 739.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
407
13. Petroianu, G., Kuhn, F., Thyes, C., Ewald, V. and Missler, A. (2003). In Vitro
Protection pf Plasma Cholinesterases by Metoclopramide from Inhibition by Paraxon.
J Appl. Toxicol.; 23(1):75- 79.
14. Al-Zubaidy, M.H.I and Mohammad, F.K. (2005). Metoclopramide –
induced central nervous system depression in the chicken. BMC Vet. Res.;1:1-6.
15. Mohammad, F.K., Al-Zubaidy, M.H.I and Alias, A.S. (2007). s Sedative and
hypnotic effects of combined administration of metoclopramide and ketamine in
chickens. Lab. Anim.2007; 36(4): 35-39.
16. Friedman, B.W., Esses, D., Solorzano, C. , Dua, N., Greebwald, P., Radulescu,
R., Chang, E., Hochberg, M., Campbell, C., Aghera, A., Valentin, T.,
Paternoster, J., Bijur, P., Lipton, R.B. and Gallagher, E.J. (2008). A randomized
controlled trial of prochlorperazine versus metoclopramide for treatment of acute
migraine. Ann Emerg Med.; 52 (04): 399-406.
17. Salazar, G., Fragoso, M., Vergez, L., Sergio, P. and Cuello, D. (2011).
Metoclopramide as an analgesic in severe migraine attacks: an open, single-blind,
parallel control study. Recent Pat CNS Drug Discov.;1;6(2): 141-145.
18. Ramswamy, S. and Bapna, J.S. (1986). Analgesic effect of metoclopramide and
its mechanism. Life Sci.; 38(14): 1289-1292.
19.Mohammad, F.K., Al-Baggou, B.K., Naser, A.S. (2012) . Antinociception by
metoclopramide, Ketamine and their combinations in mice. Pharmacol. Reports.; 64,
299-304.
20. Ceyhan, A., Ustun, H., Altunatmaz, K., Ide, T. and Unal, N.(2005). Is
metoclopramide an alternative to tramadol in the management of post-operative pain?
An experimental study. J. Vet. Med. Aphysiol. Pathol. Clin. Med.;52(5): 249-253.
21.Tavakoli, A., Mehrjerdi, H.K. and Haghighi , A. (2009). Analgesic Effects of
Metoclopramide Following Conventional Ovariohysterectomy in Bitches. Iranian J of
Vet. Sur.; 4(1-2): 77-84.
22. Friedman, B.W., Adewunmi, V., Campbell, C., Solorzano, C., Esses, D., Bijur,
P.E. and Gallagher, E.J. (2013). A Randomized Trial of Intravenous Ketorolac
Versus Intravenous Metoclopramide Plus Diphenhydramine for Tension- Type and
All Nonmigraine, Noncluster Recurrent Headaches. Ann Emerg Med.; 62(4): 311-
318.
23. Lacasse, A., Logoutte, A., Ferreira, E. and Berard, A. (2009). Metoclopramide
and diphenhydramine in the treatment of hyperemesis gravidarum: effectiveness and
predictors of rehospitalisation. Eur JObstet Gynecol Reprod Biol.; 143(1): 43-49.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
408
24. Lu, C.W., Jean, W.H., Wu, C.C., Shieh, J.S. and Lin, T.Y. (2010). Antiemetic
efficacy of metoclopramide and diphenhydramine added to patient –controlled
morphine analgesia : a randomized controlled trial. Eur J Anesthesiol. ; 27(12): 1052-
1057.
25. Pang, W., Liu, Y.C., Maboudou, E., Chen, T.X., Chois, J.M., Liao, C.C. and
Wu, R.S. (2013). Metoclopramide improves the quality of tramadol PCA
indistinguishable to morphine PCA: a prospective,randomized,double blind clinical
comparison. Pain Med.; 14(9): 1426-1434.
26. Skidgel, R. A. and Erdos, E. G. (2006). Histamine, Bradykinin, and thrir
antagonists. In: Brunton L L., Lazo J S, and Parker editors, Goodman and Gilman's
the Pharmacological basis Therapeutics. 11th ed., McGraw-Hill Co., Inc., New York.
:645 – 667.
27. Murphy, L . (2012). Antihistamines. Merck Veterinary Manual.9ed. White house
station, New Jersy : Merck Sharp and Dohme Corp.
28. Adams, H. R. (2001). Veterinary pharmacology and therapeutics. 8th ed . A
Black well Publishing CO. Lowa State Press.,PP. 1013-1014.
29. Hevesi, D. (2007). George, Rieveschl, 91e , Allergy Reliever, Dies. The New York
Times.
30. Donald, C.P. and Pharm, D. (2005). Veterinary drug handbook.5th ed.,
Blackwell Publishing professional, south state Avenue, Ames, Iowa., pp.267-271.
31.Hofmeister, E.H. and Egger, C.M. (2005). Evaluation of diphenhydramine as a
sedative for dogs. J AM Vet Med Assoc.; 226(7): 1092-1094.
32. Fujita, T., Ohue, M., Fujii, Y., Jotoku, T., Miyauchi, A. and Takagi, Y. (2013).
antihistamine diphenhydramine ointment on bone-joint-muscle pain as assessed by
skin impedance. Pharmacology.;92(3-4): 158-166.
33.Liu, H. and Farley, J.M. (2005). Effects of first and second generation
antihistamines on muscarinic induced mucus gland cell ion transport. BMC
Pharmacology. ;5:8
34. Katzung, B.G. (2006). Histamine, serotonin, and the ergot alkaloids: Introduction.
In: Katzung, B.G. (editor ), Basic and Clinical Pharmacology. 10th ed., McGraw-Hill
Co., Inc., U.S.A., pp. 377-410.
35. Santiago-Palma, J. Fischberg, D., Kornick, C., Khjainova, N. and Gonzales,
G. (2001). Diphenhydramine as an analgesic adjuvant in refractory cancer pain. J Pain
Symptom Manage.; 22(2): 699-703.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
409
36. Kim, Y.S., Shin, Y.K., Lee, C. and Song, J. (2000). Block of sodium currents in
rat dorsal root ganglion neurons by diphenhydramine. Brain Research .; 881(2): 190-
198.
37.Abdulqader, R.F. (2014). The analgesic and anti-inflammatory effects of
Diphenhydramine in mice. Iraqi J.Vet.Sci.; 28(2): 149-152.
38.Sunshine, A., Laska, E.M. and Siegel, C.E. (1991). Analgesic and Antiinflammatory
compositions comprising diphenhydramine and methods of using same.
United States Patent.; 19: 54-74.
39.Sansone, M., Castellano, C. and D'Amato, F.R. (1986). Enhancement of
morphine-induced hyperactivity by antihistaminic druge in mice. Arch Int
Pharmacodyn Ther.1986; 284(2): 239-245.
40.Adzu, B., Amos, S., Wambebe, C. and Gamaniel, K. (2001). Antinociceptive
activity of Zizyphus spina-christi root bark extract. Fitoterapia.; 72(4): 344-350.
41.Chou, S.C., Chiu, Y.J., Chen, C.J., Lin, Y.C., Wu, C.H., Chao, C.T., Chang,
C.W. and Peng, W.H. (2012). Analgesic and Anti-Inflammatory Activities of the
Ethanolic Extract of Artemisia morrisonensis Hayata in mice. Evid Based
Complement Alternat Med., 138954. doi: 10,1155/138954.
42.Hijazi, M.A., Mallah, A.E., Aboul-Ela, M. and Ellakany, A. (2017). Evaluation
of Analgesic Activity of Papaver libanoticum Extract in Mice: Involvement of opioids
Receptors. Evid Based Complement Alternat Med.;13.
43.Dixon, W.J. (1980). Efficient analysis of experimental observation. Ann Rev
Pharmacol Toxicol.; 20:441-462.
44.Al-Jader, G.H. and Taqa, G.A. (2014). Isobolographic analysis of the
antinociceptive interaction between tramadol and diphenhydramine in Mice. Inter J
Enhan. Res. Sci. Technology and Engineering, JSSN.; 3(2): 45-53.
45.Acharya, S.D., Ullal, S.D., Padiyar, S., Rao, Y.D., Upadhyaya, K., Pillai, D.
and Raj, V. (2011). Analgesic effect of extracts of Alpinia galangal rhizome in mice.
Zhong xi yi Jie He xue Bao .; 9(1):100-104.
46.Puig, M.M., Pol, O. and Warner, W. (1996). Interaction of morphine and
clonidine on gastrointestinal transiet in mice. Anesthesiol.; 85: 1404-1412.
47.Puig, M.M., Warner, W. and Pol, O. (2000). Intestinal inflammation and
morphine tolerance after the Interaction between morphine and clonidine on
gastrointestinal transiet in mice. Anesthesiol.; 93:219-230.
48.Tallarida, R.J. (2000). The interaction index: a measure og drug synergism. Pain.;
98:163-168.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
410
49.Gonzalez, C., Zegpi, C., Noriega, V., Prieto, J.C and Miranda, H.F. (2011).
Synergism between dexketoproten and meloxicam in an orofacial formalin test was
not modified by opioid antagonist. Pharmacol Rep. ; 63, 433-440.
50.Bluman, A.G. (2007). Elementary statistics, Astep by step approach. 6th ed.,
McCraw-Hill companies, NewYork, USA.
51.Daniel, W.W. (2010). Biostatics basic concepts and methodology for health
sciences. Wiely J Son. INC 9th ed.; p. 346.
52.Cleophas, T.J., Zwinderman, A.H., Cleophas, T.F. and Cleophas, E.P. (2009)
Statistics applied to clinical Trials. 4th ed.; pp. 375-477.
53.Derbent, A., Uyar, M., Demirag, K., Uyer, M., Kurtoglu, E. and Goktay, A.
(2005). Can antiemetics really relieve pain. Advances in Therapy.;22(4): 307-312.
54.Tavakoli, A. and Mehrjerdi, H.K. (2011). Enhancing analgesic effects of
lidocaine in rabbit epidural analgesia using metoclopramide or tramadol. Iranian J of
Vet. Sur.; 3(2): 41-48.
55.Childress, K.M.S., Dothager, C., Gavard, J.A., Lebovitz, S., Laska, C. and
Mostello, D.J. (2018). Metoclopramide and Diphenhydramine : A Randomized
controlled Trial of a treatment for Headache in pregnancy when Acetaminophen
Alone is ineffective (MAD Headache study ). Amer J Perinatol.; 10: 1055/s-0038-
1646952.(Epub ahead of print).
56.Kurtipek, O., Oral, M., Teltik, H., Asik, I., Ates, Y., Kuzu, I., Erdemli, E.,
Okten, F., and Tüzüner, F. (1999). Histopathologic changes after repetitive
peridural administration of metoclopramide in dogs. Anesth Analg.1999; 88: 100-102.
57.Lisander, B. (1993). Evaluation of the analgesic effect of metoclopramide after
opioid-free analgesia. Br J Anesth.; 70:631-633.
58.Lisander, B. and Kandler, B . (1993). Analgesic action of metoclopramide in
prosthetic hip surgery. Acta Anasthesiol Scand.; 37: 49-53.
59. Ganta, R. and Fee, J.P.H . (1992). Pain on injection of propofol comparison of
lidocaine with metoclopramide. Br J Anaesth.; 69: 316-317.
60.Ozucelik, D.N., Karaca, M.A. and Sivri, B. (2005). Effectiveness of pre-emptive
metoclopramide infusion in alleviating pain. Discomfort and nausea associated with
nasogastric tube insertion:A randomized, double-blind, placebocontrolled trial. Int J
Clin Pract.; 59(12): 1422-1427.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
411
61.Fisher, A.A. and Davis, M.W. (2002). Serotonin syndrome caused by selective
serotonine reuptake-inhibitors-metoclopramide interaction. Ann Pharmacother.;
36(1): 67-71.
62.Brown, N.J. and Roberts, LJ. (2001). Histamine, Bradykinin, and their
antagonists. In: Hardman, J.G., Limbird, L.E. and Gilman , A.G. ed, Goodman and
Gilman's the Pharmacological basis of therapeutics. 10th ed .,McGraw-Hill Co., Inc.,
New York., pp. 645-667.
63.Montoro, J., Barta, J., Sastre, J., Davila, I., Ferrer, M., Mullol, J., Cuvillo, A.,
Jauregui, I. and Valero, A. (2013). H1 Antihistamine and Benzodiazepam
Pharmacological Interactions and their impaction cerebral function. J Investing
Allergel Clin Immunol.; 23(1): 17-26.
64.Ghosh, M.N . (1984). Fundamental of experiment pharmacology, Scientific book
Agency Calcutta, 2en edition., p. 144-145.
65.Desta, Z., Wu, G.M., Morocho, A.M. and Flockhart, D.A . (2002). The
gastroprokinetic and antiemetic drug metoclopramide is a substrate and inhibitor of
cytochrome P450 2D6. Drug Metab Dispos.; 30(3): 336-343.
66.Gelotte, C.K., Zimmeman, B.A. and Thompson, G.A. (2018). Single-Dose
Pharmacokinetic study of Diphenhydramine Hcl in Children and Aldolescents. Clin
pharmacol Drug Dev.; 7(4): 400-407.
67. Lessard, E., Yessine, M.A., Hamelin, B.A., Gauvin, C., Labbe, L., O'Hara, G.,
LeBlanc, J. and Turgeon, J. (2001). Diphenhydramine alters the disposition of
venlafaxine through inhibition of CYP2D6 activity in humans. J Clin
Psychopharmacol.; 21(2): 175–184.
