The Interaction Between Gabapentin and Amitriptyline in the Rat Formalin Test After Systemic Administration
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The Interaction Between Gabapentin and Amitriptyline in the
Rat Formalin Test After Systemic Administration
Caroline E. Heughan, MSc, and Jana Sawynok, PhD
Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada
We examined the effects of systemically administered doses of gabapentin and amitriptyline produced an ad-
gabapentin on flinching and biting/licking behaviors ditive effect against biting/licking behaviors, as re-
produced by 2.5% formalin in the rat, compared these vealed by isobolographic analysis. These increments
with those of amitriptyline, and determined the effects had no effect on flinching behaviors except at the ED25
of combinations of gabapentin with amitriptyline. ⫹ ED25 doses, at which flinching was increased, again
Gabapentin produced a dose-related inhibition of revealing additivity between the two drugs. Flinching
Phase 2, but not Phase 1, flinching and biting/licking behaviors in rats do not reflect the analgesic properties
behaviors. In contrast, amitriptyline produced an in- of systemically administered amitriptyline observed in
crease in Phase 2 flinching behaviors while simulta- humans and may not be useful for predicting an effect
neously decreasing biting/licking behaviors. Fifty per- of combinations of drugs with amitriptyline. Biting/
cent effective dose (ED50) values against biting/licking licking behaviors do reflect analgesic properties for
behaviors were 22.9 ⫾ 1.3 mg/kg and 8.5 ⫾ 1.3 mg/kg both drugs and may be more useful in this regard.
for gabapentin and amitriptyline, respectively. Combi-
nations of increasing fractional increments of ED50 (Anesth Analg 2002;94:975–80)
A
variety of animal test models have been devel- produces synergistic effects, minimal doses could be
oped to examine various aspects of chronic used, and this would minimize adverse effects. How-
pain. Chronic pain is not simply continuing or ever, if two drugs simply exhibit additive effects, but
persistent acute pain; it involves adaptive changes in their side effect profiles are different and noninteract-
pain signaling in primary afferent neurons, in the ing, this could still serve to augment pain relief and
spinal cord, and at supraspinal sites, and it can exhibit minimize side effects.
distinct neurochemical features (1– 4). The pharmaco- Amitriptyline (an antidepressant) and gabapentin
logical treatment of chronic pain is not as effective as (an anticonvulsant) represent different classes of ad-
the treatment of acute pain, because these underlying juvants used for the treatment of chronic pain. Ami-
changes alter the sensitivity of the system to tradi- triptyline is widely used, and its efficacy has been
tional analgesics (nonsteroidal antiinflammatory established with metaanalysis and systematic ap-
drugs and opioids), and chronic pain therapy has proaches (9,10). Gabapentin is used to treat neuro-
come to rely on a group of drugs classified as adju- pathic pain in particular (11). Both drugs exhibit a
vants (e.g., antidepressants, anticonvulsants, local an- comparable efficacy in diabetic neuropathy when
esthetics, and ␣-adrenergics) (5,6). These drugs have compared directly in a single study (12) and when
other primary pharmacological indications, but they number-needed-to-treat scores are generated (6). Al-
can modify aspects of the changes that occur with though these drugs produce a comparable incidence
chronic pain and can lead to pain relief. Adjuvants, of side effects, the pattern of these effects differs some-
however, generally do not produce complete pain re- what (12).
lief, and their actions can be limited by adverse effects. The formalin test is a model of continuing pain
One potential strategy for minimizing side effects is to involving continued activation of sensory afferents,
use combinations of drugs (7,8). If a drug combination inflammation, and central sensitization (13,14). Acti-
vation of excitatory amino acid receptors in the spinal
This work was supported by the Canadian Institutes of Health cord contributes to central sensitization and chronic
Research. pain, and the formalin test has proved useful for re-
Accepted for publication December 7, 2001. vealing the pharmacology of this system (14,15). Both
Address correspondence and reprint requests to Jana Sawynok,
Department of Pharmacology, Dalhousie University, Halifax, Nova amitriptyline (16) and gabapentin (17,18) may modify
Scotia B3H 4H7, Canada. Address e-mail to sawydalu@is.dal.ca. aspects of excitatory amino acid function in the spinal
©2002 by the International Anesthesia Research Society
0003-2999/02 Anesth Analg 2002;94:975–80 975976 PAIN MEDICINE HEUGHAN AND SAWYNOK ANESTH ANALG
GABAPENTIN AND AMITRIPTYLINE 2002;94:975–80
cord to produce analgesia. We recently reported the The dose of drug required to produce 50% of the
analgesic properties of amitriptyline in the rat forma- maximum possible decrease in behavior (ED50) was
lin test after systemic administration (19). In this obtained for each drug by using linear regression.
study, we determined the analgesic properties of Subsequently, dose-response information for the con-
gabapentin after systemic administration in the rat current administration of both drugs was obtained by
formalin model and compared these with those pro- using constant dose ratios of the respective ED50 value
duced by amitriptyline. We also examined the effects for each drug (i.e., ED50 ⫹ ED50, ED37.5 ⫹ ED37.5, ED25
of combinations of gabapentin with amitriptyline to ⫹ ED25, and ED12.5 ⫹ ED12.5). The ED50 value for the
determine whether these two adjuvants could pro- drug combination was calculated by linear regression
duce additive or synergistic effects. Gabapentin has and isobolographic analysis, as previously described
been examined in combination with morphine (20), (27,28). The theoretical and actual ED50 values for the
ibuprofen (21), clonidine (22), and a non-N-methyl-d- drug combination were compared by using Student’s
aspartate excitatory amino acid receptor antagonist t-tests.
(23) in various pain models, whereas amitriptyline has Gabapentin was supplied by Parke Davis (Ann Ar-
been administered in combination with opioids, bor, MI). Amitriptyline hydrochloride and formalin
neostigmine, and clonidine (24,25). There have been were purchased from the Sigma Chemical Co. (St.
no reports on the effects of the combination of gaba- Louis, MO).
pentin with amitriptyline in any model, but the effects
of this combination in the formalin model are of par-
ticular interest because of its potential relevance to
chronic pain. Results of this study have been pre- Results
sented in abstract form (26). The IP injection of gabapentin 10 –50 mg/kg before
2.5% formalin produced a reduction in both flinching
and biting/licking behaviors, particularly in the later
Methods part of the second phase of activity (Figs. 1 and 2).
Experiments were conducted with male Sprague- Phase 1 behaviors were unaltered. A larger dose
Dawley rats (125–150 g; Charles River, Quebec, Can- (70 mg/kg) produced sedation (somnolence and flac-
ada). Rats were housed in pairs with a 12:12-h light/ cidity) (data not shown). Amitriptyline augments
dark cycle at 22°C, with free access to food and water. flinching but decreases biting/licking behaviors (Fig.
All experiments were approved by the University 2), and these actions were expressed uniformly
Committee on Laboratory Animals, and experiments throughout the second phase of activity. Regression
were conducted in accordance with International As- analysis revealed an ED50 of 22.9 ⫾ 1.3 mg/kg for
sociation for the Study of Pain and the Canadian gabapentin and 8.5 ⫾ 1.3 mg/kg for amitriptyline for
Council on Animal Care standards. Phase 2 biting/licking responses. Combinations of
Formalin 2.5% was administered in a volume of 50 fixed increments of ED50 values for the two drugs had
L by subcutaneous injection into the dorsal surface of no significant effect on flinching behaviors except at
the hindpaw at Time 0. Gabapentin and drug combi- the ED25 ⫹ ED25 doses, at which an augmentation in
nations were administered systemically as a 20-min flinching was observed (Fig. 3A). However, these
intraperitoneal (IP) pretreatment in a volume of 5 mL/ combinations exhibited an additive effect against
kg. Each behavioral testing session was preceded by a biting/licking behaviors (Fig. 3B). This was verified by
20-min acclimatization to the 28 ⫻ 28 ⫻ 28-cm Plexi- isobolographic analysis, with no significant difference
glas observation chamber. After injection of formalin, between theoretical and actual ED50 values for the
rats were returned to the chamber. Flinching behav- drug combination (Fig. 4).
iors (lifting, shaking, or rippling of the haunch, with
episodes scoring as a single event) were recorded as a
cumulative number of events, and biting/licking be-
haviors were recorded as time spent exhibiting that Discussion
behavior, for 60 min after formalin injection (Phase 1 This study demonstrates that systemic administration
⫽ 0 –12 min; Phase 2 ⫽ 16 – 60 min). Two rats in of gabapentin in rats inhibits both flinching and
adjacent chambers were observed at a time, with ob- biting/licking behaviors produced by formalin. Other
servations occurring in alternate 2-min bins. Values studies have previously noted the efficacy of gabap-
were not corrected for time, so scores represent ap- entin in the formalin test when given both systemi-
proximately half of the total behaviors. Statistics were cally (21) and spinally (29 –31). The suppression of
performed on the time-course and dose-response data behaviors was observed in Phase 2, but not in Phase 1,
by using analysis of variance followed by the Student- as noted in each of those reports, and was prominent
Newman-Keuls test for multiple groups and time in the later part of Phase 2, as also noted previously
courses. (21). The observation that spinal gabapentin is lessANESTH ANALG PAIN MEDICINE HEUGHAN AND SAWYNOK 977
2002;94:975–80 GABAPENTIN AND AMITRIPTYLINE
Figure 1. Time course of suppression of 2.5% formalin-evoked (A)
flinching and (B) biting/licking behaviors produced by systemic
(intraperitoneal) pretreatment with gabapentin (GP). Values depict
mean ⫾ sem for six per group; ns indicates P ⬎ 0.05 compared with
the Saline-Pretreated group.
Figure 2. Comparison of the effects of systemically-administered
gabapentin (GP) and amitriptyline (AMI) in the 2.5% formalin
potent when administered after the formalin and pref- (FOR) test against Phase 2 (A) flinching and (B) biting/licking
behaviors. Data for amitriptyline redrawn (19) with permission.
erentially suppresses Fos-like immunoreactivity in- Lines were fit by linear regression analysis of individual groups. *P
duced by larger concentrations of formalin suggests ⬍ 0.05 compared with the Saline-Pretreated group; n ⫽ 6 per group.
that gabapentin acts on elements of central sensitiza-
tion that are recruited during the second phase of
activity (31). Gabapentin also produces analgesic
could contribute to analgesia. Even though gabapentin
properties in other inflammatory pain tests and nerve
injury models that are considered to be relevant mod- interacts with specific heterodimers of ␥-aminobutyric
els of chronic pain in humans (11). Although the spe- acid type B receptors (35), a selective antagonist for
cific mechanisms by which gabapentin produces an- this receptor did not block antihyperalgesic effects of
algesia are not resolved, a spinal component of action gabapentin (36).
is clearly involved. Thus, gabapentin is not only active In contrast to gabapentin, systemic administration
in behavioral models after spinal application, but it of amitriptyline augments flinching behaviors while
also inhibits the formalin-evoked expression of Fos- simultaneously inhibiting biting/licking behaviors;
like immunoreactivity in the spinal cord (31) and af- the mechanisms underlying this duality of action are
fects excitatory transmission mediated by glutamate in considered elsewhere (19). Multiple mechanisms, in-
the superficial spinal cord (17,18). Gabapentin binds to cluding inhibition of biogenic amine reuptake, inter-
the ␣2␦ subunit of voltage-dependent Ca2⫹ channels actions with opioid and adenosine systems, and inter-
(32) and inhibits Ca2⫹ influx and neurotransmitter actions with N-methyl-d-aspartate receptors and ion
release at central sites (33,34), and these actions also channels, are thought to contribute to antinociception978 PAIN MEDICINE HEUGHAN AND SAWYNOK ANESTH ANALG
GABAPENTIN AND AMITRIPTYLINE 2002;94:975–80
Figure 4. Isobologram for the gabapentin (GP) and amitriptyline
(AMI) combination against biting/licking behaviors in Phase 2 of
the 2.5% formalin test in rats. Points represent 50% effective dose
(ED50) ⫾ sem. There was no significant difference between the
theoretical and actual ED50 values for the drug combination, indi-
cating an additive drug interaction.
licking behaviors were reduced in a dose-dependent
manner with fixed-dose increments of ED50 values,
and isobolographic analysis indicated a strictly addi-
tive effect. The drug combination also produced an
additive effect on flinching behaviors, even though
effects of the two individual drugs on this behavior
differed qualitatively. Thus, at the ED25 ⫹ ED25 dose,
there was an increase in flinching responses reflecting
an earlier expression of the facilitating effect of ami-
triptyline, whereas at larger doses, flinches were no
longer augmented, indicating that some degree of
suppression of enhanced behaviors was now occur-
Figure 3. Effects of gabapentin and amitriptyline at fixed-dose com- ring because of the activity of gabapentin.
binations of 50% effective dose (ED50) (1 ⫹ 1), and fractional doses
thereof, on Phase 2 (A) flinching and (B) biting/licking behaviors
Given that gabapentin and amitriptyline both pro-
produced by 2.5% formalin (FOR). Data are presented as mean ⫹ duce pain relief in humans (9 –11), biting/licking be-
sem for six per group. *P ⬍ 0.05 compared with the Saline- haviors seem to be a more appropriate end point to
Pretreated Formalin group. consider in the formalin model for amitriptyline com-
binations. Whether the additive analgesia observed
resulting from systemic administration of amitripty- with this behavior also manifests clinically as an ad-
line (37,38). ditive pain relief remains to be determined in a con-
Although the details of the mechanisms by which trolled and systematic manner. Before such a study, it
gabapentin and amitriptyline produce analgesia are would be prudent to determine (a) the effects of gaba-
not precisely defined, they represent two distinct pentin and amitriptyline combinations in other pre-
classes of drugs and seem to interact with different clinical models of chronic pain (there are more chronic
endogenous pain regulatory systems. Thus, it was models of inflammation and nerve injury in which
initially anticipated that these two drugs might recruit both gabapentin and amitriptyline exhibit analgesic
parallel or sequential mechanisms in pain transmis- activity), (b) whether there is an interaction with ad-
sion pathways and exhibit a synergistic analgesia. verse effects (although sedation was not quantitated
However, when gabapentin and amitriptyline were or examined systematically in this study, no sedation
combined in fixed fractional proportions, an additive was noted in the combination groups), and (c) the
effect was obtained with both behaviors. Biting/ effects of chronic administration of these two drugs (itANESTH ANALG PAIN MEDICINE HEUGHAN AND SAWYNOK 979
2002;94:975–80 GABAPENTIN AND AMITRIPTYLINE
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