Pain Physician 2021; 24:E511-E520 • ISSN 2150-1149

Animal Study

Investigation of the Combination of Pregabalin

with Duloxetine or Amitriptyline on the
Pharmacokinetics and Antiallodynic Effect During
Neuropathic Pain in Rats

Rafaela F. Rodrigues, MSc1, Taila Kawano, PhD1, Rodrigo V. Placido, MSc1,

Lellis H. Costa, MSc1, Márcia H.M.C. Podestá, PhD2, Rafaela S. Santos, MSc3,
Giovane Galdino, PhD3, Carlos M. Barros, MD4, and Vanessa B. Boralli, PhD1

From: 1Department of Clinical Background: Amitriptyline, duloxetine, and pregabalin are among the most
and Toxicological Analysis, Federal pharmacotherapeutic, effective treatments for neuropathic pain control. However, the evaluation
University of Alfenas, MG, Brazil;
2
Department of Pharmaceutical of synergism by combining these treatments is still poorly investigated.
Sciences, Federal University of
Alfenas, MG, Brazil; 3Sciences Objectives: To evaluate the pharmacokinetics of the combination of pregabalin plus duloxetine
of Motricity Institute, Federal and pregabalin plus amitriptyline, as well as the effect of these on neuropathic pain on rodent
University of Alfenas, MG, Brazil; model.
4
Federal University of Alfenas, MG,
Brazil Study Design: The experimental study.
Address Correspondence: Setting: The research took place in the research laboratories at the Federal University of Alfenas
Vanessa B. Boralli, PhD
after ethics committee approval.
Department of Clinical and
Toxicological Analysis, Federal
University of Alfenas
Methods: This study used male Wistar rats weighing between 220 and 250 g. The animals
Rua Gabriel Monteiro da Silva 700 were randomly divided into the following groups: monotherapy (pregabalin, amitriptyline,
Alfenas - MG, Brazil, CEP: 37130-001 duloxetine), combined therapy (pregabalin + amitriptyline, pregabalin + duloxetine), and
E-mail: vehicle (ultrapure water). Pharmacokinetic analysis of pregabalin or combination (pregabalin
[email protected]
+ amitriptyline or pregabalin + duloxetine) in the plasma were performed by ultraperformance
Disclaimer: This study was liquid chromatography tandem mass spectrometry. Neuropathic pain was induced by sciatic
supported by Coordenação de nerve constriction (chronic constriction injury [CCI]) model, and nociceptive threshold was
Aperfeiçoamento de Pessoal de measured by von Frey filaments test. In addition, to evaluate the influence of the treatments on
Nível Superior -- Brasil (CAPES) the motor coordination, the rotarod test was used.
[Finance Code 001].
Results: The pharmacokinetic disposition of pregabalin was changed in the association with
Conflict of interest: Each author
certifies that he or she, or a member amitriptyline, presenting a clearance reduction and consequently an increase in bioavailability.
of his or her immediate family, has Furthermore, after the 14th day of CCI, pregabalin was administered orally and induced
no commercial association (i.e., antiallodynic effect after 1, 2:15, 4, and 8 hours of its administration and showed the greatest
consultancies, stock ownership, antiallodynic effect after 4 hours of its administration. Moreover, this effect was prolonged
equity interest, patent/licensing
arrangements, etc.) that might pose (up to 8 hours) by combination with amitriptyline. Additionally, pregabalin and pregabalin +
a conflict of interest in connection duloxetine showed a hypoalgesic effect in sham rats. In addition, the rotarod test results showed
with the submitted manuscript. that drugs did not influence the motor coordination of the rats.
Manuscript received: 06-16-2020 Limitations: Potential competition mechanisms during the excretion of pregabalin, when
Revised manuscript received: pregabalin was combined with amitriptyline, were not investigated in this study.
11-14-2020
Accepted for publication:
Conclusions: The data demonstrated that combined therapy of pregabalin plus amitriptyline
11-20-2020
improved the bioavailability of pregabalin and potentiated the efficacy of the antiallodynic effect
Free full manuscript: of pregabalin alone, proving to be advantageous for the treatment of sciatic neuropathic pain.
www.painphysicianjournal.com
Key words: Neuropathic pain, pregabalin, duloxetine, amitriptyline, pharmacokinetic,
antiallodynic effect, combined therapy, rats

Pain Physician 2021: 24:E511-E520

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 Pain Physician: July 2021 24:E511-E520

N europathic pain is defined by the

International Association for the Study of
Pain as “pain caused by a lesion or disease of
the somatosensory nervous system” (1). In addition, it
is estimated that 8% to 10% of the global population
dry mouth, blurred vision, confusion, weight gain,
urinary retention, and tachycardia. Duloxetine mainly
presents nausea, somnolence, dizziness, and anorexia
as side effects (4-5).
For a better management of this type of pain, a
suffers from neuropathic pain (2-3). Thus it is important combination of 2 or more drugs with different mecha-
to recognize that neuropathy affects many aspects nisms of action can improve analgesic efficacy because
of patients’ daily lives and is associated with general different pain control pathways can be activated (13).
health problems, poor sleep, reduced quality of life, In addition, this type of therapeutic regimen is widely
and increased anxiety and depression (2). used to improve the effectiveness of drugs, reduce
In addition to being a chronic condition, neuro- toxicity, or treat coexisting diseases (14). The pain man-
pathic pain is difficult to treat, mainly because it re- agement is often clinically based on titration on poly-
sponds poorly to treatment with common analgesics. pharmacy focused on each patient (15). An extra effect
Therefore the pharmacotherapeutic treatment recom- of gabapentinoids combined with TCAs, duloxetine,
mended for neuropathic pain includes antidepressants, or opioids as compared with monotherapy has been
such as tricyclic antidepressants (TCAs) (e.g., amitripty- reported (16-20). However, this synergistic effect for
line), noradrenaline and serotonin reuptake inhibitors pain control is often done without concern for clinically
(e.g., duloxetine), and anticonvulsants (e.g., gabapen- significant pharmacokinetic interactions (14). Pharma-
tin and pregabalin) (4). These drugs are US Food and cokinetic drug interactions resulting from polyphar-
Drug Administration (FDA) approved for treatment of macy can interfere with drug absorption, distribution,
neuropathy, such as for diabetic peripheral neuropathy, metabolism, or excretion, changing the magnitude and
post-herpetic neuralgia, and chronic pain (5). duration of the effect, but the final response of the
Pregabalin has anticonvulsant, anxiolytic, and an- drug is preserved (21-22).
algesic properties (6). Its mechanism of action occurs It is known that rats are biologically and behavior-
by binding with great affinity and specificity to alpha- ally similar to humans, and many human symptoms can
2-delta-1 for the calcium channel voltage-dependent be applied to small rodents. However, it is important to
proteins in the central nervous system (CNS), prevent- recognize that humans differ from animals mainly on
ing the release of excitatory neurotransmitters (7). drug-metabolizing enzymes. Therefore animal models
Moreover, pregabalin can also increase the activity of are fundamental for understanding the mechanisms
the transporter of neuronal glutamate reuptake type 3 involved in neuropathic pain and for the development
and open the potassium channels ATP-sensitive (KATP) of effective and appropriate therapies for its manage-
(8), producing antinociception (9). ment in humans, always considering these genetic dif-
Additionally, amitriptyline and duloxetine are ferences in metabolism to make interspecies extrapola-
antidepressant drugs that act by inhibiting serotonin tion (23-26).
and norepinephrine reuptake in the CNS, leading to Thus the present study aimed to investigate and
higher levels of these neurotransmitters in the synaptic compare the use of pregabalin alone and combined
clefts, which may activate the descending pain inhibi- with duloxetine or amitriptyline in healthy rats after
tory pathway (10). Moreover, amitriptyline is also able oral single dose, and evaluate the correlation of phar-
to block channels of sodium, calcium, and potassium, macokinetic characteristics with antiallodynic effect in
reducing the conduction of nociceptive impulse (11), animals with neuropathic pain.
and duloxetine can block sodium channels voltage-
dependent in the CNS and peripheral nervous system
Methods
(PNS) (12).
However, these drugs have side effects that nega- Experimental Animals
tively impact the patient’s quality of life and continuity This study used conventional heterogeneous male
of treatment. Pregabalin may have cognitive effects Wistar rats (Rattus norvegicus albinus) weighing 220 to
including somnolence, difficulty focusing, and short- 250 g, aged 7 weeks, which were housed in groups of
term memory loss, as well as peripheral edema, dizzi- 5 per cage, containing shavings as bedding material,
ness, and weight gain as the main side effects. The side under 12-hour light/dark cycle at constant room tem-
effects of amitriptyline include somnolence, dizziness, perature (22°C ± 2°C) and humidity (60%). Water and

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 Combined Pregabalin Treatment and Neuropathic Pain in Rats

standard food were available ad libitum. All experi- rat was cannulated in the jugular vein for blood collec-
ments were carried out within the animals’ circadian tion (31). Twelve hours after cannulation, drugs were
cycle, respecting the light cycle, between 7 and 19 given (in combined treatment, the animals first received
hours. pregabalin and after 5 minutes they received one anti-
All procedures followed the committee for depressant) and after 0.083, 0.16, 0.25, 0.5, 0.75, 1, 1.5,
research and ethical issues of the International Asso- 2, 4, 6, 8, 10, 16, and 24 hours, 500 µL of blood samples
ciation for the Study of Pain (27), and the study was were collected; the volume was reposed with sterile sa-
approved by the ethics committee on the use of ani- line solution. Also, during the experiment, the animals
mals of Federal University of Alfenas, Brazil (protocol had water ad libitum and were fasting by the time of
number 670/2015). the procedure up to 2 hours after drug administration,
to avoid interference with absorption.
Drugs and Grouping Samples were collected in heparinized tubes and
Pregabalin was purchased from Pfizer (Karlsruhe, centrifuged; plasma was separated and stored at –70°C
Germany), and amitriptyline purchased from Merck until pharmacokinetics assays.
(Kenilworth, NJ). Both drugs were dissolved in distilled The pregabalin and diazepam (internal standard)
water. Duloxetine was purchased from Libbs (São were analyzed by ultraperformance liquid chromatog-
Paulo, Brazil) and prepared as suspension in water. All raphy mass spectrometry in positive electrospray ioniza-
drugs or vehicles were orally administrated by gavage tion, with monitoring of the following mass transitions:
(2.5 mL/kg) in single dose. The dose selection was done 158.00 > 140.80, 158.00 > 123.00, and 158.00 > 95.20 for
based on previous studies, briefly pregabalin 10 mg/ pregabalin; and 285.00 > 154.05, 285.00 > 192.90, and
kg, amitriptyline 1 mg/kg, and duloxetine 30 mg/kg 285.00 > 222.10 for diazepam. For analysis, a sample
(28-30). preparation with protein precipitation (with methanol)
All animals were randomized in a simple blind followed by high-speed centrifugation was done.
manner and allocated to an experimental group, ac- Mobile phase was performed with methanol: am-
cording to the treatment received orally: vehicle (ani- monium hydroxide solution pH 7.0 (90:10% v/v), the pH
mals treated with ultrapure water control), pregabalin was adjusted with acetic acid and stationary phase was
(animals treated with pregabalin), amitriptyline (ani- C18 column (pre-column and column). The method was
mals treated with amitriptyline), duloxetine (animals validated in accordance with the FDA validation guide-
treated with duloxetine), pregabalin + amitriptyline line (32), and the level of detection was 0.29 ng/mL of
(animals treated with pregabalin and amitriptyline), plasma. The method was accurate and linear from 10 to
pregabalin + duloxetine (animals treated with prega- 6,250 ng/mL of plasma.
balin and duloxetine). Pharmacokinetic parameters were calculated based
on plasma concentrations. To evaluate differences be-
Outcomes tween the groups, the apparent total clearance (ClT/f)
The primary outcome studied was pharmacoki- and area under the curve (AUC) parameters were used.
netics parameters of pregabalin in healthy animals, The pharmacokinetic analysis was performed using the
comparing the combined treatment (pregabalin + WinNonlin 4.0 software (Pharsight Corp, Mountain
antidepressants) with pregabalin alone. The secondary View, CA).
outcome studied was the measurement of neuropathic
pain invoked by chronic constriction injury (CCI) of Neuropathic Pain Model
sciatic nerve (by measuring mechanical allodynia with The CCI of sciatic nerve was used as a model for
von Frey hair) and how effective are the pharmacologic the induction of neuropathic pain (33). Briefly, animals
treatments, correlating with pharmacokinetics param- were anesthetized, and the sciatic nerve was exposed
eters. Also, analyses of the impact of pharmacologic and loosely ligated with 4-0 chronic gut thread at 4
treatment on motor coordination and balance. sites with an interval of 1 mm.
The 72 (72/129) rats were divided in the following
Pharmacokinetic Study 5 groups: vehicle, pregabalin, amitriptyline, duloxetine,
This study used 33 rats (33/129), divided into 3 pregabalin + amitriptyline, and pregabalin + dulox-
groups (n = 11): pregabalin, pregabalin + amitriptyline, etine. Each group had CCI animals (n = 6): animals that
and pregabalin + duloxetine. Before treatment, each underwent CCI and sham animals (n = 6): rats that had

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the sciatic nerve exposed but not ligated. In combined was assessed before drug administration and 1, 2:15, 4,
treatment, the animals first received pregabalin; after 8, and 24 hours after administration (Fig. 1B). Measure-
5 minutes they received the other drug. ments always occurred at the 12-hour light cycle.

Experimental Protocol of Mechanical Motor Coordination

Allodynia Assessment The 24 healthy rats (24/129) were randomly and in
First, rats were placed in the cages with a metal simple blind fashion divided in the following groups (n
mesh floor and allowed to acclimatize for at least 30 = 6/group): vehicle, pregabalin, pregabalin + amitripty-
minutes. Mechanical allodynia was determined by line, and pregabalin + duloxetine. In combined treat-
measuring paw withdraw in response to increasing ment, the animals first received pregabalin and after 5
stimulation with a series of calibrated von Frey fila- minutes they received another drug.
ments, ranging from 0.16 to 180 g. Each filament was Motor coordination and balance was assessed using
pressed over the plantar region of the hind paw for 6 a rotarod apparatus (Insight, Ribeirao Preto, Brazil). All
seconds and repeated 3 times, with an interval of at rats underwent a 2-day training program during which a
least 3 minutes (34). steady baseline level of performance was attained (35).
Based on a pilot experiment performed by our During that period, rats were trained to walk against
group (Fig. 1A and Supplementary Fig. S1), the evalu- the motion of a rotating drum at a constant speed of 16
ation of treatments on the neuropathic pain was rotations per minute (rpm) for a maximum of 2 minutes.
performed on the 14th day of CCI. The drugs were ad- Following the training days, a 1-day test was performed
ministered in a single dose. To avoid interference with using accelerating speed levels (5–16 rpm) mode of the
absorption, animals were fasting by 6 hours before the apparatus over 2 minutes. First, the baseline latency (BL)
administration and up to 2 hours after drug adminis- was measured, and new tests were performed after 30,
tration. To evaluate the effect in hours, each drug, or 60, and 120 minutes after drugs or vehicle administra-
combination, the nociceptive threshold measurement tion. Then, the mean latency to fall off the rotarod was
recorded.

Statistical Analysis
Data are presented as mean standard error of
mean (SEM) to behavior experiments and as median
for pharmacokinetic parameters. Two-way analysis of
variance (ANOVA) with repeated measures followed by
Bonferroni test were used for comparisons between-
groups of mechanical allodynia and motor test. For
pharmacokinetic data, the Mann-Whitney 2-tailed
test was applied. Statistical tests were performed with
GraphPad InStat (GraphPad, San Diego, CA); the level
of significance was set at 5%.

Results

Fig. 1. Schematic representation of experimental protocol of Pharmacokinetics of Pregabalin and

nociceptive threshold measurements. (A) Pilot experiment: Combinations
first, the BL of the nociceptive threshold in each rat was When then evaluated the median of each phar-
obtained. Then, neuropathic pain was performed by chronic macokinetic parameter (AUC and clearance); Table
constriction injury (CCI) or sham procedure was performed, 1 shows that in both parameters a significant differ-
and new measures of nociceptive threshold were done at
the 3rd, 6th, and from the 9th to 21st day after CCI. (B) ence of pregabalin + amitriptyline (P < 0.05) occurred
Evaluations of antiallodynic effect of each drug alone compared with pregabalin. There was no difference
or in combination was assessed at the BL, before drug (P > 0.05) of pregabalin + duloxetine compared with
administration, and at 1, 2:15, 4, 8, and 24 hours after drug pregabalin. The group that received pregabalin + ami-
administration on the 14th day following CCI. triptyline increased AUC by 4.8 times compared with

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 Combined Pregabalin Treatment and Neuropathic Pain in Rats

Table 1. Estimated pharmacokinetic parameters of pregabalin alone or combined.

Pregabalin Pregabalin (10 mg/kg) + Pregabalin (10 mg/kg) +
Parameter
(10 mg/kg) Amitriptyline (1 mg/kg) Duloxetine (30 mg/kg)
AUC 1717.0 8190.0* 3550.8
(h.ng/mL) (1049.5–2926.3) (3654.9–12348.0) (897.3–7800.1)
ClT/f 5854.0 1304.0* 3145.0
(L/h/kg) (3882.7–7395.5) (353.24–4664.4) (1067.9–6070.6)
ClT/f: total clearance of formation; AUC: area under the curve; h.ng/mL: nanogram per milliliter multiplied per hour (unit of AUC); L/h/Kg: liter
per hour per kilogram (unit of clearance).
*P < 0.05 indicates a statistical difference between pregabalin and other groups, Mann-Whitney U test.

the pregabalin group, and consequently reduced by

4.5 times the total clearance of formation of the group
that received pregabalin.
Pharmacokinetics profiles are represented in
Fig. 2; compartmental profiles show that pregabalin
combined with amitriptyline reached higher plasma
concentrations than pregabalin alone or pregabalin
combined with duloxetine.

Evaluation of Pregabalin Alone or Combined

in CCI-Induced Mechanical Allodynia
CCI-induced mechanical allodynia in all groups
at the 14th day before drug administration (P < 0.05,
F7,35= 7,507) when compared with sham animals (Fig. Fig. 2. Representative plasma concentration curves of
3). The mechanical allodynia induced by CCI continued pregabalin, pregabalin + duloxetine, and pregabalin
during the 24 hours tested in the vehicle, amitriptyline, + amitriptyline. Data are presented as the mean of 6
pharmacokinetic profiles obtained per group. Pharmacokinetic
and duloxetine groups (Fig. 3). tests were performed in healthy rats. Plasma samples were
We then evaluated the effect of drugs on CCI quantified by ultraperformance liquid chromatography with
animals after hours of their administrations; we found tandem mass spectrometry, and the data obtained were modeled
that only the groups of rats that received pregabalin, by the compartmental model.
or pregabalin combined (pregabalin + amitriptyline or
pregabalin + duloxetine), reduced the mechanical al-
lodynia, when compared with the CCI + vehicle group than pregabalin at the eighth hour, however, it was not
(Fig. 3). This effect was found after 1 (P < 0.05, F7,35 = significant (P > 0.05, F7,35 = 7,507) (Fig. 3).
7,507), 2:15 (P < 0.001, F7,35 = 7,507), 4 (P < 0.001, F7,35 The nociceptive threshold in the sham groups that
= 7,507), and 8 hours (P < 0.05, F7,35= 7,507) of prega- received drugs was not changed, except for animals
balin administration; 2:15 (P < 0.001, F7,35 = 7,507), 4 treated with pregabalin after 1 (P < 0.05, F6,30 = 3,502)
(P < 0.001, F7,35 = 7,507), and 8 hours (P < 0.001, F7,35 and 2:15 hours (P < 0.01, F6,30 = 3,502), and with pre-
= 7,507) of pregabalin + amitriptyline; and 4 (P < 0.001, gabalin + duloxetine, which was increased after 2:15
F7,35 = 7,507) and 8 hours (P < 0.01, F7,35 = 7,507) of (P< 0.01, F6,30 = 3,502), 4 (P < 0.001, F6,30 = 3,502), and
pregabalin + duloxetine (Fig. 3). When we compared 8 hours (P < 0.001, F6,30 = 3,502) compared with the ve-
pregabalin with pregabalin + amitriptyline and prega- hicle group (Fig. 4). These results suggest a hypoalgesic
balin + duloxetine, pregabalin showed higher antial- effect of pregabalin and pregabalin + duloxetine. Also,
lodynic effect (P < 0.05, F7,35 = 7,507, P < 0.001, F7,35 = the prolongation of the hypoalgesic effect of pregaba-
7,507, respectively) at the fourth hour. However, at the lin was observed after 4 and 8 hours (P < 0.001, F6,30 =
eighth hour, only the pregabalin + amitriptyline combi- 3,502 for both) after pregabalin + duloxetine adminis-
nation presented the antiallodynic effect higher than tration (Fig. 4). Nevertheless, a tendency of hypoalgesic
pregabalin (P < 0.001, F7,35 = 7,507). It was possible to effect was observed in pregabalin + amitriptyline after
observe that the pregabalin + duloxetine combination 1 and 2:15 hours; in duloxetine group at the first hour,
presented a tendency in an antiallodynic effect higher however, it was not significant (P > 0.05, F6,30 = 3,502).

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Fig. 4. Effect of pregabalin, duloxetine, amitriptyline,

pregabalin plus duloxetine, and pregabalin plus
Fig. 3. Effect in hours of pregabalin, duloxetine, amitriptyline, amitriptyline after 14 days sham rats. After 14 days of BL,
pregabalin plus duloxetine, and pregabalin plus amitriptyline sham rats were pretreated with oral administration of each
at the 14th day of neuropathic pain. After BL, rats were drug or combination. Data are expressed as the mean ±
undergoing the neuropathic pain model, and at the 14th day SEM of the nociceptive threshold of 6 animals per group.
they were pretreated with oral administration of each drug or *P < 0.05, **P < 0.01, ***P < 0.001 indicates a statistical difference
combination. Data are expressed as the mean ± SEM of the between sham rats that received vehicle (Sham + Veh) and other
nociceptive threshold of 6 animals per group. sham groups. !!!P < 0.001 indicates a statistical difference between
***P < 0.001, **P < 0.01, and *P < 0.05 indicates a statistical differ- sham rats that received pregabalin (Sham + Preg) and other sham
ence between animals with neuropathic pain and sham animals. !!!P groups. Sham + Veh, sham rats that received vehicle; Sham +
< 0.001, and !P < 0.05 indicates a statistical difference between CCI + Preg, sham rats that received pregabalin; Sham + Amit, sham rats
Preg and other CCI groups. ###P < 0.001, and #P < 0.05 indicates a that received amitriptyline; Sham + Preg + Amit, sham rats that
statistical difference between CCI + Veh and other CCI groups. Sham received pregabalin plus amitriptyline; Sham + Preg + Dul, sham
+ Veh, sham rats that received vehicle; CCI + Veh, animals underwent rats that received pregabalin plus duloxetine; 2-way ANOVA with
chronic constriction surgery and then received vehicle; CCI + Preg, repeated measures followed by Bonferroni test.
animals underwent chronic constriction surgery and then received
pregabalin; CCI + Amit, animals underwent chronic constriction
surgery and then received amitriptyline; CCI + Dul, animals under-
went chronic constriction surgery and then received duloxetine; CCI Pregabalin is not metabolized in vivo by cytochromes
+ Preg + Dul, animals underwent chronic constriction surgery and (CYP) enzymes (36), being excreted practically un-
then received pregabalin plus duloxetine; CCI + Preg + Amit, animals changed in the urine (37). Moreover, its absorption
underwent chronic constriction surgery and then received pregabalin
occurs by the L-transporter system and by the sodium
plus amitriptyline; 2-way ANOVA with repeated measures followed by
Bonferroni test. channels located in the apical membrane of the intes-
tine (38-39), not being a substrate the P-glycoprotein
(P-gp) (40). Conversely, amitriptyline is metabolized
Evaluation of Pregabalin Alone or Combined by several CYP isoforms, mainly CYP2C19 and CYP2D6
on the Motor Coordination and Balance of (41), which is a substrate of P-gp (42) and is excreted as
Rats inactive metabolites in the urine (43). Also, duloxetine
The rotarod results demonstrated that the drugs is metabolized by several CYP isoforms, mainly CYP1A2
in single dose did not alter (P > 0.05) the time of stay and CYP2D6 (15), which is a P-gp inhibitor (44), and
of animals in the test after 30, 60, and 120 minutes of most part is excreted in the urine mainly as conjugated
drug administration (Fig. 5). This result concludes that metabolites (45). Therefore neither amitriptyline or du-
the antiallodynic effect found by pregabalin alone and loxetine have absorption, distribution, and metabolic
combined was only sensory and not motor. pharmacokinetic interactions described with pregaba-
lin. Therefore it is important to note that for amitrip-
Discussion tyline and duloxetine, which have enzymes involved in
The present study demonstrated, for the first their metabolisms with polymorphisms that stratify the
time to our knowledge, a reduction of total apparent population from slow to ultrafast metabolizers, there
clearance and an increase of pregabalin bioavailabil- will be differences for these drugs between humans
ity when pregabalin was combined to amitriptyline. and animal models (23). Thus the increased bioavail-

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 Combined Pregabalin Treatment and Neuropathic Pain in Rats

ability of pregabalin combined with amitriptyline may

be associated with a competition during the pregabalin
excretion because the renal drug–drug interaction in-
volving the inhibition of tubular secretion mediated by
transporters most commonly manifests as an increase
in one of the substances in the plasma (46), but more
studies need to be performed to evaluate this process.
In addition, the present study also demonstrated
that pregabalin, pregabalin plus amitriptyline, or pre-
gabalin plus duloxetine were efficient in reduce me-
chanical allodynia induced by neuropathic pain. Further-
more, we found that combined with amitriptyline, the
pregabalin-induced antiallodynic effect was prolonged
and this response was associated with pharmacokinetics Fig. 5. Motor behavior was performed by rotarod test. After
findings, showing this correlation for the first time, to BL, healthy rats were pretreated with oral administration
our knowledge. Therefore pregabalin combined with of vehicle, pregabalin, or pregabalin and combinations.
amitriptyline has been demonstrated to be a great treat- New measurements of the motor behavior were performed
ment strategy for neuropathic pain control because our after drug administration (30, 60, and 120 min). Data are
expressed as the mean ± SEM. No statistical difference was
study has shown that only one administration of prega- observed; 2-way ANOVA with repeated measures followed by
balin + amitriptyline induced antiallodynic effects for 8 Bonferroni test.
hours, prolonging the antiallodynic effect of pregabalin
in monotherapy, and these findings corroborate with a
study that investigated the effect of combination of pre- amitriptyline and duloxetine did not show antiallo-
gabalin and amitriptyline in 1269 patients with severe dynic effect after single-dose administration, and only
neuropathic pain (47).The authors found a reduction pregabalin reduced mechanical allodynia. To achieve
of Visual Analog Scale score in just 7 days after treat- the analgesic effect, the antidepressants amitriptyline
ment (47). Moreover, another study investigated the and duloxetine need to recruit secondary downstream
safety and the efficacy of pregabalin and amitriptyline mechanisms and long-term neuroplasticity, exerting
as monotherapy or as low-dose combination in 92 pa- action only when administered repeatedly. However,
tients showing similar pain reduction profile for evalu- gabapentinoids are effective for neuropathic pain with
ated therapies, and the low-dose combination had less acute or chronic administration (13). This profile of pain
adverse effects than monotherapy (20). reduction is also demonstrated in a study with patients
Our study also showed that the antiallodynic ef- presenting with postherpetic neuropathic pain, which
fect of pregabalin combined with duloxetine had no found better results at pain relief with pregabalin com-
significant prolonged effect when compared with pared with amitriptyline (49).
pregabalin treatment alone, and pregabalin alone Moreover, we found a hypoalgesic effect of prega-
was more effective in reducing neuropathic pain than balin and pregabalin + duloxetine. This effect may be
the combination with duloxetine (Fig. 3). This agrees related to the inhibition of the development of central
with a multicentric study on diabetic peripheral neuro- sensitization (13), mainly by binding to the voltage-gat-
pathic pain, in which the patients demonstrated that ed calcium-channels, that leads to the reduction of the
combined pregabalin + duloxetine was less effective release of excitatory neurotransmitters that mediate
than monotherapy; however, the monotherapy dose pain, such as substance P, calcitonin gene-related pep-
used was higher (19). Nonetheless, a reduction of tide, and glutamate (7), and consequently triggering
the neuropathic pain induced by paclitaxel in women nociceptive impulses even beyond the baseline thresh-
treated for ovarian cancer was found after duloxetine old. Thus we hypothesize that the prolonged hypoal-
+ pregabalin therapy (48). These findings reinforcing gesic effect of pregabalin + duloxetine group occur as
that this combined therapy may be effective, safe, and a result of synergism between these 2 drugs with hy-
well tolerated, but the effects may vary according to poalgesic effect. In accordance with our findings, other
the type of neuropathic pain and/or the dose used. studies also demonstrated a tendency for hypoalgesia
In addition, when monotherapies were compared, in sham rats treated with pregabalin (50-51).

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 Pain Physician: July 2021 24:E511-E520

The hypoalgesic effect of pregabalin and pre- in pregabalin excretion when it was combined with
gabalin + duloxetine demonstrated in this study may antidepressants, and if this pathway is related to the
improve the drug choice for other painful conditions increase of pregabalin bioavailability when combined
involving both the CNS, such as other types of neuro- to amitriptyline.
pathic pain, and the PNS, such as postoperative pain.
Studies show that pregabalin was effective to reduce
Conclusions
postoperative pain after eyelid surgery (52), reduce Taken together, the present study concludes that
consumption of opioids and analgesics postoperatively combination of pregabalin + amitriptyline may be a
(53-54), and reduce preoperative anxiety (55). safe, effective, and advantageous strategy to neuro-
By the rotarod test, the present study demon- pathic pain compared with pregabalin alone because
strated that pregabalin administered alone or in com- this combination showed a better pharmacokinetic
bination, did not alter motor coordination or balance profile, with increased pregabalin bioavailability,
in rats, validating the obtained antiallodynic effect. A and proved to be the most effective treatment for
study performed with 65 patients with chronic diabetic neuropathic pain induced by sciatic nerve injury be-
peripheral neuropathic pain had already shown that cause of its greater efficacy and higher duration of
none of these treatments affected patients’ motor the effect.Furthermore, the combination pregabalin +
coordination (56). In addition, the authors have shown amitriptyline, despite the side effects of amitriptyline,
that both treatments increased the performance on is inexpensive in relation to the pregabalin + dulox-
sensory motor tasks (56). etine combination. In addition, we conclude that the
Thus combination of pregabalin with either TCAs hypoalgesic effect shown by pregabalin and pregaba-
or serotonin-norepinephrine reuptake inhibitors is de- lin + duloxetine could help the physicians in the drug
scribed as a treatment option if a patient cannot toler- choice as a safe, effective, and advantageous strategy
ate high-dose monotherapy (57), mainly when using 2 for other peripheral pain conditions, such as reduc-
drugs with different mechanisms of action. ing postoperative pain. Therefore the findings of this
study provide scientific information on animals, which
Limitations can serve as a basis for more effective treatments in
In this study, it was not possible to verify alteration humans.

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Supplementary Table S1. Baseline data of all animals (before
any procedure).
Baseline Data
Mean Microbiological Drug Prior
Group
Weight Status Treatment
PK Preg 237.8 Healthy Anesthes
PK Preg + Amit 238.5 Healthy Anesthes
PK Preg + Dul 237.3 Healthy Anesthes
Pilot CCI 235.6 Healthy Anesthes
Pilot SH 235.2 Healthy Anesthes
Veh CCI 237 Healthy Anesthes
Veh SH 237.6 Healthy Anesthes
Fig. S1. Pilot experiment: first, the BL of the
Preg CCI 232.2 Healthy Anesthes
nociceptive threshold in each rat was obtained. After
Preg SH 237.2 Healthy Anesthes that, neuropathic pain was performed by CCI, or
Preg + Amit CCI 234.5 Healthy Anesthes sham procedure was performed and new measures of
nociceptive threshold were done at the third, sixth, and
Preg + Amit SH 233.2 Healthy Anesthes
from ninth to 21st day after CCI. The animals did not
Preg + Dul CCI 234 Healthy Anesthes receive any drug. Data are expressed as the mean ± SEM
Preg + Dul SH 234 Healthy Anesthes of the nociceptive threshold of 5 animals per group. **P
< 0.01 and *P < 0.05 indicates a statistical difference
Amit CCI 232 Healthy Anesthes
between animals with neuropathic pain (CCI) and sham
Amit SH 234.2 Healthy Anesthes animals (SH), Mann-Whitney test.
Dul CCI 233.8 Healthy Anesthes
Dul SH 234.8 Healthy Anesthes
Rotarod Veh 237.8 Healthy None
Rotarod Preg 235.6 Healthy None
Rotarod Preg +
236 Healthy None
Amit
Rotarod Preg +
232.7 Healthy None
Dul