with nerve injury in rats (9–11) and humans (12–15).Thus, it is likely that low doses of anticonvulsants and Bvitamins might have synergistic effects in reducing neu-ropathic pain and side effects.

The present study was designed to assess the oral ef-ficacy of two anticonvulsants (gabapentin and carba-mazepine) and two B vitamins (benfotiamine and cyano-cobalamin) on the reduction of tactile allodynia in rats.Moreover, the possible synergistic interaction betweenthe anticonvulsants and either of the B vitamins wasassessed in the rat spinal nerve ligation model (16).

MATERIALS AND METHODS

AnimalsFemale Wistar rats aged 6–7 weeks (weight range

120–140 g) from our own breeding facilities were used inthis study. Animals had free access to food and drinking

INTRODUCTIONPeripheral nerve injury can result in neuropathic pain

characterized clinically by spontaneous burning pain,allodynia to touch and hyperalgesia to noxious stimuli (1,2). Currently there is no treatment to prevent the devel-opment of neuropathic pain, and the pharmacotherapy ofthis type of pain has been disappointing. Patients are usu-ally treated with tricyclic antidepressants, anticonvul-sants or, more recently, opioids (3). Although these drugshave limited efficacy and undesirable side effects (4–6),anticonvulsants have become the mainstay in the treat-ment of chronic neuropathic pain (7).

There is a challenge to develop effective therapies totreat neuropathic pain, and B vitamins could be an option.Preliminary studies have demonstrated that a mixture ofB vitamins (thiamine and cyanocobalamin) increases theanalgesic effect of gabapentin in patients with diabetesand neuropathic pain (8). Moreover, there is evidencethat B vitamins are useful in painful disorders associated

Methods Find Exp Clin Pharmacol 2008, 30(6): 431-441Copyright 2008 Prous Science, S.A.U. or its licensors. All rights reserved.CCC: 0379-0355/2008DOI: 10.1358/mf.2008.30.6.1232963

Synergistic Antiallodynic Interaction between Gabapentin or Carbamazepine and either Benfotiamine or Cyanocobalamin in Neuropathic RatsTeresa Mixcoatl-Zecuatl1, Geovanna N. Quiñónez-Bastidas2, Nadia L. Caram-Salas3, Mónica Ambriz-Tututi3, Claudia I. Araiza-Saldaña3, Héctor I. Rocha-González3, Roberto Medina-Santillán4, Gerardo Reyes-García4 and Vinicio Granados-Soto3

1Centro de Investigación y de Estudios Avanzados, Unidad Monterrey, Apodaca, Nuevo León; 2Facultad de CienciasQuímicas, Universidad Autónoma de Sinaloa, Culiacán, Sinaloa; 3Departamento de Farmacobiología, Centro de Investi-gación y de Estudios Avanzados, Sede Sur, D.F.; 4Escuela Superior de Medicina del Instituto Politécnico Nacional, D.F.,México

SUMMARYAnticonvulsants, including gabapentin and carbamazepine, have shown activity against several types of neuropathic pain; however, they have limit-

ing side effects that may minimize their use. In this study the possible synergistic interaction between anticonvulsants and benfotiamine or cyanocobala-min on spinal nerve ligation-induced tactile allodynia was assessed. Oral administration of gabapentin (15–300 mg/kg), carbamazepine (10–300 mg/kg),benfotiamine (30–600 mg/kg) or cyanocobalamin (0.3–6.0 mg/kg) significantly reduced tactile allodynia in rats. Maximal antiallodynic effects werereached with gabapentin 300 mg/kg (~70%), carbamazepine 300 mg/kg (~66%), benfotiamine 600 mg/kg (~51%) and cyanocobalamin 6 mg/kg(~59%). At the highest tested doses, gabapentin, but not carbamazepine, benfotiamine or cyanocobalamin, significantly reduced motor coordination.Coadministration of gabapentin or carbamazepine with benfotiamine or cyanocobalamin in a fixed ratio markedly reduced spinal nerve ligation-induced tactile allodynia, showing a synergistic interaction between anticonvulsants and B vitamins. Data indicate that combinations of anticonvul-sants with benfotiamine or cyanocobalamin are able to reduce tactile allodynia without affecting motor coordination in rats, and suggest the possibleclinical use of these combinations in the treatment of neuropathic pain in humans.

Key words: Gabapentin - Carbamazepine - Benfotiamine - Cyanocobalamin - Synergistic interaction - Tactile allodynia

erence 18) for the pattern of positive and/or negative re-sponses, and δ = the mean difference (in log units) be-tween stimulus strengths. Allodynia was considered to bepresent when paw withdrawal thresholds were < 4 g.

DrugsGabapentin and carbamazepine were purchased from

Sigma (St. Louis, MO, USA). Benfotiamine (an analogueof vitamin B1, or thiamine) and cyanocobalamin (vitaminB12) were kindly provided by Merck S.A. de C.V.(Mexico, D.F.). All drugs were dissolved in saline.

Study designRats received an oral administration of saline (4 ml/kg)

or increasing doses of gabapentin (15–300 mg/kg), car-bamazepine (10–300 mg/kg), benfotiamine (30-600mg/kg), cyanocobalamin (0.3–6.0 mg/kg) or their combi-nations in a fixed ratio of 0.5:0.5 (Tables 1–4) and theeffect was assessed for the following 4 h. Doses for oraladministration were selected based on a previous report(10) and on pilot experiments in our laboratory. The ob-server was unaware of the treatment for each animal.

Motor coordination testSince gabapentin and carbamazepine reduce motor

coordination (20, 21), the effects of the highest testeddoses of individual drugs, as well as those of combina-tions, on motor function were assessed. Ten independentgroups with 5 rats in each group were examined formotor coordination in a treadmill apparatus (rotarod test)before and after receiving an oral administration of gaba-pentin (100 and 300 mg/kg), carbamazepine (300 mg/kg),benfotiamine (600 mg/kg), cyanocobalamin (6 mg/kg),gabapentin + benfotiamine (26.3 + 218.4 mg/kg), ga-bapentin + cyanocobalamin (26.3 + 2.3 mg/kg), carba-mazepine + benfotiamine (51.5 + 218.4 mg/kg), carbama-zepine + cyanocobalamin (51.5 + 2.3 mg/kg) or vehicle(4 ml/kg). Animals were placed on a cylinder (7 cm indiameter) rotating at a speed of 10 rpm. Rats were trainedto walk on the cylinder for 3 consecutive sessions; on thefourth, they received the drug or vehicle treatment (time0), and the number of falls during a 5-min period wascounted after 2 h. Time was chosen based on the time toreach the maximal antiallodynic effects.

water before experiments. Efforts were made to mini-mize animal suffering and to reduce the number of animalsused. Rats were used once only. Experiments were car-ried out at the same hours of the day (10 a.m. to 4 p.m.).All experiments followed the Guidelines on Ethical Stand-ards for Investigation of Experimental Pain in Animals(17). Additionally, the study was approved by our localEthics Committee.

Measurement of antiallodynic activityRats were prepared according to the method of Kim

and Chung (16). Animals were anesthetized with a mix-ture of ketamine and xylazine (45/12 mg/kg i.p.). Aftersurgical preparation and exposure of the dorsal vertebralcolumn, the left L5 and L6 spinal nerves were exposedand tightly ligated with 6-0 silk suture distal to the dorsalroot ganglion. For sham-operated rats, the nerves wereexposed but not ligated. After closing the incisions, ani-mals were allowed to recover for 10 days. Rats exhibit-ing motor deficiency (such as paw dragging) were dis-carded from the study (< 5%).

Tactile allodynia was determined according to a pre-viously reported method (18). Briefly, rats were trans-ferred to a clear plastic, wire mesh-bottomed cage andallowed to acclimatize for 30 min. Von Frey filaments(Stoelting, Wood Dale, IL, USA) were used to determinethe 50% paw withdrawal threshold using the up–downmethod of Dixon (19). A series of filaments, starting withone that had a buckling weight of 2 g, were applied inconsecutive sequence to the plantar surface of the righthind paw with a pressure causing the filament to buckle.Lifting of the paw indicated a positive response andprompted the use of the next weaker filament, whereasabsence of paw withdrawal after 5 sec indicated a nega-tive response and prompted the use of the next filament ofincreasing weight. This paradigm continued until four moremeasurements were made after the initial change of the be-havioral response or until five consecutive negative (as-signed a score of 15 g) or four consecutive positive (assigneda score of 0.25 g) responses had occurred. The resultingscores were used to calculate the 50% response thresholdby using the formula: 50% g threshold = 10(Xf +κδ)/10,000, where Xf = the value (in log units) of the finalvon Frey filament used, κ = the value (from table in ref-

432 T. Mixcoatl-Zecuatl et al./Methods Find Exp Clin Pharmacol 2008, 30(6): 431-441

TABLE 1. Doses (mg/kg) used in the study of the synergistic interaction between gabapentin and benfotiamine in rats previously submittedto L5/L6 spinal nerve ligation.

Gabapentin Benfotiamine Gabapentin Benfotiamine Total dosealone alone in the combination in the combination in the combination

15 30 3.2 27.3 30.530 100 6.5 54.6 61.1

100 300 13.1 109.2 122.3300 600 26.3 218.4 244.7

Doses in the combination were calculated considering a fixed ratio (0.5:0.5).

T. Mixcoatl-Zecuatl et al./Methods Find Exp Clin Pharmacol 2008, 30(6): 431-441 433

effect (ED50 ± standard error) was calculated using least-squares linear regression according to Tallarida et al.(22). Subsequently, an experimentally derived dose–re-sponse curve was obtained by concurrent delivery of twodrugs in a constant fixed ratio (0.5:0.5) based on the ED50values of each individual agent according to the follow-ing equation (22):

ED50T= f (ED50 D1) + (1-f)(ED50 D2)

In the equation, ED50T is the theoretical ED50, f is thefixed ratio for each drug (0.5), ED50 D1 is the ED50 ofindividual drug 1, and ED50 D2 is the ED50 of individualdrug 2. The value ED50T represents the highest value ofthe total dose in the combination, i.e., 244.7 for thegabapentin + benfotiamine combination (Table 1). Oncethis value is calculated, the next value in the combinationis obtained dividing by 2, 4 and 8. The proportion of eachdrug to the total dose in the combination is calculatedaccording to the following equations:

Data analysisAll experimental results are expressed as the mean ±

SEM for 6 animals per group. Curves were constructedplotting the threshold for paw withdrawal as a function oftime. An increase of 50% withdrawal threshold was con-sidered an antiallodynic effect. Area under the 50% with-drawal threshold–time curve (AUC) was calculated bythe trapezoidal method. Antiallodynic effect was expres-sed as the maximum possible effect (%MPE) and calcu-lated as:

% MPE = ([AUCcompound – AUCsaline]/[AUCsham – AUCsaline]) × 100

In order to demonstrate a possible interaction betweenthe anticonvulsants and the B vitamins, isobolographicanalyses were used. Thus, from the dose–response curvesof each individual agent, the dose resulting in 50% of the

TABLE 2. Doses (mg/kg) used in the study of the synergistic interaction between gabapentin and cyanocobalamin in rats previously submit-ted to L5/L6 spinal nerve ligation.

Gabapentin Cyanocobalamin Gabapentin Cyanocobalamin Total dosealone alone in the combination in the combination in the combination

15 0.3 3.2 0.2 3.530 1 6.5 0.5 7.1

100 3 13.1 1.1 14.3300 6 26.3 2.3 28.6

Doses in the combination were calculated considering a fixed ratio (0.5:0.5).

TABLE 3. Doses (mg/kg) used in the study of the synergistic interaction between carbamazepine and benfotiamine in rats previously sub-mitted to L5/L6 spinal nerve ligation.

Carbamazepine Benfotiamine Carbamazepine Benfotiamine Total dosealone alone in the combination in the combination in the combination

10 30 6.4 27.3 33.730 100 12.8 54.6 67.4

100 300 25.7 109.2 134.9300 600 51.5 218.4 269.9

Doses in the combination were calculated considering a fixed ratio (0.5:0.5).

TABLE 4. Doses (mg/kg) used in the study of the synergistic interaction between carbamazepine and cyanocobalamin in rats previously sub-mitted to L5/L6 spinal nerve ligation.

Carbamazepine Cyanocobalamin Carbamazepine Cyanocobalamin Total dosealone alone in the combination in the combination in the combination

10 0.3 6.4 0.2 6.730 1 12.8 0.5 13.4

100 3 25.7 1.1 26.9300 6 51.5 2.3 53.9

Doses in the combination were calculated considering a fixed ratio (0.5:0.5).

by these drugs was significant (P < 0.05) and dose-de-pendent (Fig. 2). Maximal antiallodynic effect was reachedwith gabapentin 300 mg/kg (~70%; Fig. 2A), carba-mazepine 300 mg/kg (~66%; Fig. 2B), benfotiamine 600mg/kg (~51%; Fig. 2C) and cyanocobalamin 6 mg/kg(~59%; Fig. 2D); higher doses of any drug did not pro-duce greater antiallodynic effects. Gabapentin 300 mg/kgsignificantly reduced motor coordination (P < 0.05), whilegabapentin 100 mg/kg, carbamazepine, benfotiamine andcyanocobalamin produced a nonsignificant reduction(Table 5).

C1D1 = (pD1)(ED50T); pD1 = (0.5)(ED50 D1/ED50T)

In this equation, C1D1 is the contribution of drug 1 to thetotal dose in the combination (greatest value).

C1D2 = (pD2)(ED50T); pD2 = (0.5)(ED50 D2/ED50T)

In this equation, C1D2 is the contribution of drug 2 to thetotal dose in the combination (greatest value).

Detailed information on the resulting composition,obtained with this procedure, of the different combina-tions used in this study is shown in Tables 1–4.

The experimentally derived ED50 value of the combi-nation was then plotted on the isobologram and statisti-cally compared with the theoretical additive ED50 value(22). The theoretical and experimental ED50 values of thestudied combinations were also contrasted by calculatingthe interaction index (γ) as follows:

γ = ED50 of combination (experimental)/ED50 of combination (theoretical)

The interaction index indicates what portion of the ED50of the individual drugs accounts for the correspondingED50 in the combination, i.e., values near 1 correspond toan additive interaction, values > 1 imply an antagonisticinteraction and values < 1 indicate a synergistic interac-tion.

StatisticsIn order to demonstrate dose-dependency, dose–re-

sponse curves from individual drugs and combinationswere analyzed by one-way analysis of variance(ANOVA) followed by Tukey’s test for post hoc compar-ison. The Kruskal–Wallis analysis of variance was usedto test differences in rats submitted to the rotarod test.Statistical significance between the theoretical additiveED50 and the experimentally derived ED50 values wasevaluated using Student’s t-test (22). Statistical signifi-cance was considered to be achieved when P was < 0.05.

RESULTS

Antiallodynic activity of gabapentin, carbamazepine,benfotiamine and cyanocobalamin

Ligation of L5/L6 spinal nerves produced tactile allo-dynia in rats submitted to the surgery compared to sham-operated rats. Allodynia was observed in the rats 24 hafter spinal nerve ligation and remained that way for at least8 days after the experiments were carried out (Fig. 1A).

Oral administration of gabapentin, carbamazepine,benfotiamine and cyanocobalamin, but not vehicle, re-duced tactile allodynia induced by ligation of L5/L6spinal nerves (Fig. 1B). The antiallodynic effect produced

434 T. Mixcoatl-Zecuatl et al./Methods Find Exp Clin Pharmacol 2008, 30(6): 431-441

FIG. 1. A: Time course of paw withdrawal threshold in rats submittedto the ligation of L5/L6 spinal nerves compared to sham-operated rats.Withdrawal threshold was assessed at day 1, 3, 5, 7 and 8 after surgery.B: Time course of the antiallodynic effect observed after acute oraladministration of gabapentin (GBP), carbamazepine (CBZ), benfoti-amine (BEN) or cyanocobalamin (B12) in rats previously submitted tospinal nerve ligation. The sham group (black triangles) is placed as areference of the maximum possible effect. In all cases data are present-ed as mean ± SEM for 6 animals, although SEM were omitted in thetreatments with gabapentin, carbamazepine, benfotiamine andcyanocobalamin for the sake of clarity.

tions did not significantly modify motor coordination inthe rotarod test (Table 5). When data for combinationswere submitted to isobolographic analysis, the experi-mental values of ED50 were lower than those expectedfrom a purely additive interaction. The theoreticallyadditive dose line depicts all points of the combinationsyielding an effect of 50% according to an additive inter-action. For the four combinations, the experimental ED50values clearly were situated below the theoretically addi-tive dose line, indicating a synergistic interaction be-tween these drugs (Figs. 5A–5D). Comparison of exper-imental and theoretical ED50 values by Student’s t-testyielded statistically significant differences (P < 0.05) for

Antiallodynic activity of the combination of gabapentin or carbamazepine with eitherbenfotiamine or cyanocobalamin

Oral coadministration of gabapentin (Figs. 3A, 4A,4B) or carbamazepine (Figs. 3B, 4C, 4D) with eitherbenfotiamine or cyanocobalamin in a fixed ratio (0.5:0.5)significantly reduced tactile allodynia in neuropathicrats. At the maximal doses tested, the combination ofgabapentin or carbamazepine with either benfotiamine orcyanocobalamin completely recovered normal sensitivityin the neuropathic rats (Figs. 3A and 3B). In all cases,combination of drugs produced a significant (P < 0.05)and dose-dependent antiallodynic effect in the neuro-pathic rats (Figs. 4A–4D). Of note, these dose combina-

T. Mixcoatl-Zecuatl et al./Methods Find Exp Clin Pharmacol 2008, 30(6): 431-441 435

FIG. 2. Antiallodynic effect of the oral administration of gabapentin (GBP, A), carbamazepine (CBZ, B), benfotiamine (BEN, C) and cyanocobalamin(B12, D) in rats previously submitted to spinal nerve ligation. Data are presented % of maximum possible effect (%MPE) as a function of dose. Thesham group (gray bar) is placed as a reference of the maximum possible effect. Data are the mean ± SEM for 6 animals. *Significantly different fromthe vehicle (Veh) group (P < 0.05), as determined by analysis of variance followed by Tukey’s test.

this subunit is upregulated in dorsal root ganglion andspinal cord of neuropathic rats (30). Other mechanisms,such as reduction in the release of excitatory amino acidsthrough an indirect interaction with NMDA receptors,have been suggested (31–33). Our group recently sug-gested that the antiallodynic effect of gabapentin mightresult from activation of the nitric oxide (NO)–cyclicGMP–protein kinase G (NO–cGMP–PKG) and K+ chan-nel pathways, which would lead to hyperpolarization ofsensory neurons and a reduction of neuropathic pain inrats (34, 35).

the four combinations (Tables 6 and 7). However, analy-sis of the interaction index showed the best synergismwith the carbamazepine + cyanocobalamin combination(Table 7).

DISCUSSION

Antiallodynic effect of gabapentinOral administration of gabapentin significantly reduced

tactile allodynia in neuropathic rats. Our results agreewith the literature in showing that systemic gabapentin isable to reduce neuropathic pain in nerve-injured rats (10,23–26). This antiallodynic effect was accompanied by asignificant reduction of motor coordination in the rotarodsystem with the highest dose tested. Our data agree withprevious observations showing that gabapentin inducesrotarod deficits or pronounced sedation at antinocicep-tive doses (20, 27). Antiallodynic effects of gabapentinhave been correlated with electrophysiological studieswhere the drug is able to reduce neuronal responses inspinal nerve-ligated rats (28). To date, sites and mecha-nisms responsible for the antiallodynic effect of ga-bapentin remain unclear. However, it is possible that theantiallodynic effect of gabapentin could result from itsbinding to the α2δ subunit of L-type voltage-dependentCa2+ channels, reducing Ca2+ currents, as previously re-ported (29). Furthermore, there is evidence showing that

436 T. Mixcoatl-Zecuatl et al./Methods Find Exp Clin Pharmacol 2008, 30(6): 431-441

TABLE 5. Effect of the high tested doses of gabapentin, carba-mazepine, benfotiamine, cyanocobalamin and their combination onmotor coordination in rats.

Number of falls Number of fallsTreatment before treatment after treatment

Vehicle 1/5 1/5Gabapentin, 300 mg/kg 0/5 15/5*Gabapentin, 100 mg/kg 1/5 4/5Carbamazepine, 300 mg/kg 0/5 2/5Benfotiamine, 600 mg/kg 1/5 1/5Cyanocobalamin, 6 mg/kg 0/5 0/5Gabapentin + benfotiamine,

26.3 + 218.4 mg/kg 0/5 2/5Gabapentin + cyanocobalamin,

26.3 + 2.3 mg/kg 0/5 0/5Carbamazepine + benfotiamine,

51.5 + 218.4 mg/kg 1/5 1/5Carbamazepine + cyanocobalamin,

51.5 + 2.3 mg/kg 1/5 2/5

Rats were assessed on the rotarod test before and 2 h after drug admin-istration (n = 5). * Significantly different from the vehicle group (aftertreatment), by the Kruskal–Wallis one-way analysis of variance fol-lowed by Tukey’s test.

FIG. 3. Time course of the antiallodynic effect observed after acute oralcoadministration of gabapentin (GBP, A) or carbamazepine (CBZ, B)with either benfotiamine or cyanocobalamin in rats previously submit-ted to spinal nerve ligation. Data are presented as mean ± SEM for 6animals.

that the antiallodynic effect of carbamazepine could bedue to its sedative effects, as the highest tested dose (300mg/kg) produced only a slight rotarod deficit. Moreover,the fact that systemic carbamazepine is also able toreduce spinal neuronal responses in spinal nerve-ligatedrats is consistent with our data (28).

It has been reported that the antiallodynic action ofcarbamazepine could be due to the blockade of Na+ chan-nels (42, 43). More recently, Cardenas et al. (44) foundthat carbamazepine interacts with a slow inactivation stateof the NaV1.8-like sodium channels. However, it has alsobeen hypothesized that carbamazepine could enhance K+

currents or modulate the GABAergic system as mecha-nisms to explain its antiallodynic effect (45, 46).

Antiallodynic effect of carbamazepine Carbamazepine significantly reduced tactile allody-

nia in neuropathic rats. At the highest effective dose, car-bamazepine produced nonsignificant rotarod deficits.The data agree with previous observations showing thatsystemic administration of carbamazepine is able to re-duce neuropathic pain in several models of nerve injuryin the rat (36–40), including the Kim and Chung model(41). Our data disagree, however, with those of Hunter etal. (23), where carbamazepine was inactive against tac-tile allodynia in the same model. Differences could bedue to dose, since Hunter et al. administered carba-mazepine at 1–30 mg/kg, whereas doses of 10–300mg/kg were used in our study. However, it is unlikely

T. Mixcoatl-Zecuatl et al./Methods Find Exp Clin Pharmacol 2008, 30(6): 431-441 437

FIG. 4. Antiallodynic effect of the acute oral coadministration of gabapentin (GBP) + benfotiamine (BEN) (A), gabapentin + cyanocobalamin (B12)(B) or carbamazepine (CBZ) + benfotiamine (C) and carbamazepine + cyanocobalamin (D) in a fixed dose ratio in rats previously submitted to spinalnerve ligation. Data are mean ± SEM for 6 animals. *Significantly different from the vehicle (Veh) group (P < 0.05), as determined by analysis ofvariance followed by Tukey’s test.

438 T. Mixcoatl-Zecuatl et al./Methods Find Exp Clin Pharmacol 2008, 30(6): 431-441

TABLE 6. Effective doses calculated after oral administration of gabapentin alone or combined with either benfotiamine or cyanocobalaminin a fixed ratio (0.5:0.5) in rats previously submitted to L5/L6 spinal nerve ligation.

Individual drug or combination ED50 (mg/kg) Individual drug or combination ED50 (mg/kg)

Gabapentin 52.6 ± 12.0 Gabapentin 52.6 ± 12.0Benfotiamine 436.8 ± 64.3 Cyanocobalamin 4.6 ± 0.6Experimental combination 128.4 ± 7.9* Experimental combination 13.5 ± 1.7*Theoretical combination 244.7 ± 32.7 Theoretical combination 28.6 ± 6.0Interaction index 0.52 ± 0.07 Interaction index 0.47 ± 0.11

ED50, effective dose resulting in a 50% reduction of tactile allodynia. Data are mean ± SE of the estimate. *Significantly different from the theo-retical combination data (P < 0.05), using Student’s t-test.

FIG. 5. Isobologram showing the synergistic interaction between gabapentin (GBP) with either benfotiamine (BEN, A) or cyanocobalamin (B12, B)and carbamazepine (CBZ) with either benfotiamine (C) or cyanocobalamin (D) in rats previously submitted to spinal nerve ligation. Horizontal andvertical bars indicate SEM. The oblique line between the x axis and y axis is the theoretical additive line. The point in the middle of this line is thetheoretical additive point (T) calculated from the separate ED50 values. The experimental point (E) lies below the additive line, indicating a significantsynergism (P < 0.05), as determined by Student’s t-test.

ous data demonstrating that thiamine or cyanocobalaminsignificantly increased the antiallodynic effect of dexa-methasone (a steroidal antiinflammatory drug) in ratssubmitted to spinal nerve ligation (10). In addition, theoral administration of the mixture of thiamine, pyridoxineand cyanocobalamin enhances the antiallodynic activityof either systemic or spinal gabapentin (25, 54). Themechanism of action of these combinations remains to beelucidated, but could be due to a pharmacokinetic inter-action or to the different mechanisms and sites of actionof the individual drugs.

Final considerationsTreatment of neuropathic pain remains a challenge

for the physician. Current treatment includes antidepres-sants (tricyclic [imipramine, clomipramine and amitrip-tyline], selective serotonin reuptake inhibitors [paroxe-tine and fluoxetine] and serotonin–norepinephrinereuptake inhibitors [duloxetine and venlafaxine]), anti-convulsants (carbamazepine, gabapentin and phenytoin),local anesthetics (lidocaine), and more recently, opioids(tramadol and oxycodone). Analgesic efficacy dependson the neuropathic condition, but in general the efficacyorder is serotonin–norepinephrine reuptake inhibitors> tricyclic antidepressants = anticonvulsants = opioids >selective serotonin reuptake inhibitors (3). In this study,we have combined two anticonvulsants with benfoti-amine or cyanocobalamin and found synergistic effects.However, it is likely that drugs from the group ofdual serotonin–norepinephrine reuptake inhibitors couldalso produce synergistic effects with these vitamins.Currently, experiments are under way to examine thispossibility.

In summary, gabapentin and carbamazepine, as wellas benfotiamine and cyanocobalamin, reduced tactileallodynia induced by spinal nerve ligation in rats. Theantiallodynic effect of gabapentin, but not that of carba-mazepine, benfotiamine and cyanocobalamin, wasaccompanied by rotarod deficits. In addition, a synergis-tic interaction, without rotarod deficits, was observedwith the four tested combinations, with the best syner-gism achieved with the carbamazepine + cyanocobal-amin combination. These results suggest that these com-

Antiallodynic effect of vitamins (benfotiamine and cyanocobalamin)

In this study we have observed that systemic admin-istration of benfotiamine and cyanocobalamin, but notvehicle, significantly reduced tactile allodynia in rats sub-mitted to ligation of L5/L6 spinal nerves. At the highesttested doses, these drugs did not produce rotarod deficitsin the rats. These results agree with previous observa-tions from our group showing that cyanocobalamin andbenfotiamine are able to reduce tactile allodynia in thespinal nerve ligation model in the rat (10, 11). Therefore,results of this study confirm the efficacy of benfotiamineand cyanocobalamin in this type of pain. Moreover, ourdata agree with those of others showing that cyanocobal-amin is able to reduce thermal hyperalgesia, but notmechanical allodynia, in dorsal root ganglion compres-sion and chronic constriction injury models (9). Resultsalso agree with clinical observations showing thatcyanocobalamin and benfotiamine reduce signs ofpainful neuropathy (15, 47–49).

The mechanism of the antiallodynic action producedby benfotiamine and cyanocobalamin is unknown. How-ever, it has been suggested that B vitamins may con-tribute to the recovery of nerve function in conditions ofnerve injury (50, 51). Moreover, since benfotiamine isable to inhibit the diacylglycerol–protein kinase C (PKC)pathway, it has been speculated that the observed antial-lodynic effect of this drug may be due to inhibition of thispathway (11, 52, 53).

Antiallodynic effect of the combinations of gabapentin or carbamazepine with eitherbenfotiamine or cyanocobalamin

Besides producing antiallodynic effects by themselves,drugs in combination produced a dose-dependent andsignificant antiallodynic effect, in the absence of rotaroddeficits, in neuropathic rats. Isobolographic analysesshowed a functional synergistic interaction for the fourcombinations in spinal nerve-ligated rats. Low doses ofthe combinations produced a complete reduction of tac-tile allodynia in the rats. To our knowledge, this is thefirst report of a synergistic interaction between anticon-vulsants and B vitamins. These results agree with previ-

T. Mixcoatl-Zecuatl et al./Methods Find Exp Clin Pharmacol 2008, 30(6): 431-441 439

TABLE 7. Effective doses calculated after oral administration of carbamazepine alone or combined with either benfotiamine or cyanocobal-amin in a fixed ratio (0.5:0.5) in rats previously submitted to L5/L6 spinal nerve ligation.

Individual drug or combination ED50 (mg/kg) Individual drug or combination ED50 (mg/kg)

Carbamazepine 103.0 ± 8.4 Carbamazepine 103.0 ± 8.4Benfotiamine 436.8 ± 64.3 Cyanocobalamin 4.6 ± 0.6Experimental combination 127.6 ± 21.6* Experimental combination 15.3 ± 3.9*Theoretical combination 269.9 ± 32.4 Theoretical combination 53.8 ± 4.2Interaction index 0.47 ± 0.09 Interaction index 0.28 ± 0.07

ED50, effective dose resulting in a 50% reduction of tactile allodynia. Data are mean ± SE of the estimate. *Significantly different from the theo-retical combination data (P < 0.05), using Student's t-test.

13. Stracke, H., Lindemann, A., Federlin, K. A benfotiamine-vitamin Bcombination in treatment of diabetic polyneuropathy. Exp ClinEndocrinol Diabetes 1996, 104(4): 311-6.

14. Abbas, Z.G., Swai, A.B. Evaluation of the efficacy of thiamine andpyridoxine in the treatment of symptomatic diabetic peripheral neu-ropathy. East Afr Med J 1997, 74(12): 803-8.

15. Kuwabara, S., Nakazawa, R., Azuma, N. et al. Intravenous methyl-cobalamin treatment for uremic and diabetic neuropathy in chron-ic hemodialysis patients. Intern Med 1999, 38(6): 472-5.

16. Kim, S.H., Chung, J.M. An experimental model for peripheral neu-ropathy produced by segmental spinal nerve ligation in the rat. Pain1992, 50(3): 355-63.

17. Zimmermann, M. Ethical guidelines for investigations on experi-mental pain in conscious animals. Pain 1983, 16(2): 109-10.

18. Chaplan, S.R., Bach, F.W., Pogrel, J.W., Chung, J.M., Yaksh, T.L.Quantitative assessment of tactile allodynia in the rat paw. J NeurosciMethods 1994, 53(1): 55-63.

19. Dixon, W.J. Efficient analysis of experimental observations. AnnuRev Pharmacol Toxicol 1980, 20: 441-62.

20. Boyce, S., Wyatt, A., Webb, J.K. et al. Selective NMDA NR2B anta-gonists induce antinociception without motor dysfunction:Correlation with restricted localisation of NR2B subunit in dorsalhorn. Neuropharmacology 1999, 38(5): 611-23.

21. Idänpään-Heikkilä, J.J., Guilbaud, G. Pharmacological studies on arat model of trigeminal neuropathic pain: Baclofen, but not carba-mazepine, morphine or tricyclic antidepressants, attenuates theallodynia-like behaviour. Pain 1999, 79(2-3): 281-90.

22. Tallarida, R.J., Stone, D.J., Raffa, R.B. Efficient designs for study-ing synergistic drug combinations. Life Sci 1997, 61(26): PL 417-25.

23. Hunter, J.C., Gogas, K.R., Hedley, L.R. et al.The effect of novelanti-epileptic drugs in rat experimental models of acute and chron-ic pain. Eur J Pharmacol 1997, 324(2-3): 153-60.

24. Abdi, S., Lee, D.H., Chung, J.M. The anti-allodynic effects ofamitriptyline, gabapentin, and lidocaine in a rat model of neuro-pathic pain. Anesth Analg 1998, 87(6): 1360-6.

25. Reyes-García, G., Caram-Salas, N.L., Medina-Santillán, R., Gra-nados-Soto, V. Oral administration of B vitamins increases theantiallodynic effect of gabapentin in the rat. Proc West PharmacolSoc 2004, 47: 76-9.

26. Urban, M.O., Ren, K., Park, K.T. et al. Comparison of the antinoci-ceptive profiles of gabapentin and 3-methylgabapentin in rat mod-els of acute and persistent pain: Implications for mechanism ofaction. J Pharmacol Exp Ther 2005, 313(3): 1209-16.

27. Jones, D.L., Sorkin, L.S. Systemic gabapentin and S(+)-3-isobutyl-gamma-aminobutyric acid block secondary hyperalgesia. BrainRes 1998, 810: 93-9.

28. Chapman, V., Suzuki, R., Chamarette, H.L.C., Rygh, L.J., Dicken-son, A.H. Effects of systemic carbamazepine and gabapentin onspinal neuronal responses in spinal nerve ligated rats. Pain 1998,75(2-3): 261-72.

29. Gee, N.S., Brown, J.P., Dissanayake, V.U.K., Offord, J., Thurlow,R., Woodruff, G.N. The novel anticonvulsant drug, gabapentin(neurontin), binds to the α2δ subunit of a calcium channel. J BiolChem 1996, 271(10): 5768-76.

30. Luo, Z.D., Calcutt, N.A., Higuera, E.S. et al. Injury type-specificcalcium channel alpha 2 delta-1 subunit up-regulation in rat neu-ropathic pain models correlates with antiallodynic effects of gaba-pentin. J Pharmacol Exp Ther 2002, 303(3): 1199-205.

31. Singh, L., Field, M.J., Ferris, P. et al. The antiepileptic agentgabapentin (neurontin) possesses anxiolytic-like and antinocicep-tive actions that are reversed by D-serine. Psychopharmacology1996, 127(1): 1-9.

32. Partridge, B.J., Chaplan, S.R., Sakamoto, E., Yaksh, T.L. Charac-terization of the effects of gabapentin and 3-isobutyl-γ-aminobu-

binations could be useful for treating neuropathic pain inhumans.

ACKNOWLEDGEMENTSThis work was carried out at the Departamento de Far-

macobiología, Cinvestav, Sede Sur. The authors greatlyappreciate the technical and bibliographic assistance ofGuadalupe C. Vidal-Cantú, M.Sc., and Héctor Vázquez,B.Sc., respectively. Teresa Mixcoatl-Zecuatl is a post-doctoral fellow from Cinvestav, Unidad Monterrey.Nadia L. Caram-Salas, Mónica Ambriz-Tututi, Claudia I.Araiza-Saldaña and Héctor I. Rocha-González areCONACYT fellows. In addition, Nadia L. Caram-Salasand Claudia I. Araiza-Saldaña are the recipients of the“Apoyos Integrales para la Formación de Doctores enCiencias” fellowship. Geovanna N. Quiñónez-Bastidas isa visiting student of the “Verano de la InvestigaciónCientífica” program and recipient of the “AcademiaMexicana de Ciencias” fellowship. This work is part ofthe B.Sc. dissertation of Geovanna N. Quiñónez-Bastidas.

REFERENCES 1. White, S. Assessment of chronic neuropathic pain and the use of

pain tools. Br J Nurs 2004, 13(7): 372-8.2. Vera-Portocarrero, L.P., Zhang, E.T., Ossipov, M.H. et al. Descend-

ing facilitation from the rostral ventromedial medulla maintainsnerve injury-induced central sensitization. Neuroscience 2006,140(4): 1311-20.

3. Finnerup, N.B., Otto, M., McQuay, H.J., Jensen, T.S., Sindrup, S.H.Algorithm for neuropathic pain treatment: An evidence based pro-posal. Pain 2005, 118(3): 289-305.

4. McQuay, H., Carroll, D., Jadad, A.R., Wiffen, P., Moore, A. Anti-convulsant drugs for management of pain: A systematic review. BrMed J 1995, 311(7012): 1047-52.

5. Kingery, W.S. A critical review of controlled clinical trials for per-ipheral neuropathic pain and complex regional pain syndromes.Pain 1997, 73(2): 123-39.

6. Woolf, C.J., Mannion, R.J. Neuropathic pain: Aetiology, symptoms,mechanisms, and management. Lancet 1999, 353(9168): 1959-64.

7. Sindrup, S.H., Jensen, T.S. Efficacy of pharmacological treatmentsof neuropathic pain: An update and effect related to mechanism ofdrug action. Pain 1999, 83(3): 389-400.

8. Medina-Santillán, R., Morales-Franco, G., Espinoza-Raya, J., Gra-nados-Soto, V., Reyes-García, G. Treatment of diabetic neuropathicpain with gabapentin alone or combined with vitamin B complex.Preliminary results. Proc West Pharmacol Soc 2004, 47: 109-12.

9. Wang, Z.B., Gan, Q., Rupert, R.L., Zeng, Y.M., Song, X.J.Thiamine, pyridoxine, cyanocobalamin and their combination in-hibit thermal, but not mechanical, hyperalgesia in rats with prima-ry sensory neuron injury. Pain 2005, 114(1-2): 266-77.

10. Caram-Salas, N.L., Reyes-García, G., Medina-Santillán, R., Gra-nados-Soto, V. Thiamine and cyanocobalamin relieve neuropathicpain in rats: Synergy with dexamethasone. Pharmacology 2006,77(2): 53-62.

11. Sánchez-Ramírez, G.M., Caram-Salas, N.L., Rocha-González, H.I.et al. Benfotiamine relieves inflammatory and neuropathic pain inrats. Eur J Pharmacol 2006, 530(1-2): 48-53.

12. Bernstein, A.L., Loitz, C.S. Clinical and electrophysiological studyof the treatment of painful diabetic neuropathy with pyridoxine.Curr Top Nutr Dis 1988, 19: 411-23.

440 T. Mixcoatl-Zecuatl et al./Methods Find Exp Clin Pharmacol 2008, 30(6): 431-441

45. Olpe, H., Kolb, C.N., Hausdorf, A., Haas, H.L. 4-Aminopyridineand barium chloride attenuate the anti-epileptic effect of carba-mazepine in hippocampal slices. Experientia 1991, 47(3): 254-7.

46. Waldmeier, P.C., Baumann, P.A., Wicki, P., Feldtrauer, J.J., Stierlin,C., Schmutz, M. Similar potency of carbamazepine, oxcarbazepine,and lamotrigine in inhibiting the release of glutamate and otherneurotransmitters. Neurology 1995, 45(10): 1907-13.

47. Woelk, H., Lehrl, S., Bitsch, R., Kopcke, W. Benfotiamine in treat-ment of alcoholic polyneuropathy: An 8-week randomized con-trolled study (BAP I Study). Alcohol Alcohol 1998, 33(6): 631-8.

48. Winkler, G., Pal, B., Nagybeganyi, E., Ory, I., Porochnavec, M.,Kempler, P. Effectiveness of different benfotiamine dosage regimensin the treatment of painful diabetic neuropathy. Arzneimit-telforschung 1999, 49(3): 220-4.

49. Haupt, E., Ledermann, H., Kopcke, W. Benfotiamine in the treat-ment of diabetic polyneuropathy – A three-week randomized, con-trolled pilot study (BEDIP study). Int J Clin Pharmacol Ther 2005,43(2): 71-7.

50. Tong, H.I. Influence of neurotropic vitamins on the nerve conduc-tion velocity in diabetic neuropathy. Ann Acad Med Singapore1980, 9(1): 65-70.

51. Fujii, A., Matsumoto, H., Yamamoto, H. Effect of vitamin B com-plex on neurotransmission and neurite outgrowth. Gen Pharmacol1996, 27(6): 995-1000.

52. Hammes, H.P., Du, X., Edelstein, D. et al. Benfotiamine blocksthree major pathways of hyperglycemic damage and preventsexperimental diabetic retinopathy. Nat Med 2003, 9(3): 294-9.

53. Babaei-Jadidi, R., Karachalias, N., Ahmed, N., Battah, S.,Thornalley, P.J. Prevention of incipient diabetic nephropathy byhigh-dose thiamine and benfotiamine. Diabetes 2003, 52(8): 2110-20.

54. Reyes-García, G., Medina-Santillán, R., Rocha-González, H.I., Gra-nados-Soto, V. Synergistic interaction between spinal gabapentinand oral B vitamins in a neuropathic pain model. Proc West Phar-macol Soc 2003, 46: 91-4.

Address for correspondence: Vinicio Granados-Soto, Ph.D.,Departamento de Farmacobiología, Centro de Investigación y deEstudios Avanzados, Sede Sur, Calzada de los Tenorios 235, ColoniaGranjas Coapa, 14330 México, D.F., Mexico. E-mail: vgranados@prodigy.net.mx

tyric acid on substance P-induced thermal hyperalgesia. Anesthe-siology 1998, 88(1): 196-205.

33. Shimoyama, M., Shimoyama, N., Hori, Y. Gabapentin affects glu-tamatergic excitatory neurotransmission in the rat dorsal horn.Pain 2000, 85(3): 405-14.

34. Mixcoatl-Zecuatl, T., Medina-Santillán, R., Reyes-García, G., Vidal-Cantú, G.C., Granados-Soto, V. Effect of K+ channel modulators onthe antiallodynic effect of gabapentin. Eur J Pharmacol 2004,484(2-3): 201-8.

35. Mixcoatl-Zecuatl, T., Flores-Murrieta, F.J., Granados-Soto, V. Thenitric oxide-cyclic GMP-protein kinase G-K+ channel pathway par-ticipates in the antiallodynic effect of spinal gabapentin. Eur J Phar-macol 2006, 531(1-3): 87-95.

36. Hofmann, H.A., De Vry, J., Siegling, A., Spreyer, P., Denzer, D.Pharmacological sensitivity and gene expression analysis of thetibial nerve injury model of neuropathic pain. Eur J Pharmacol2003, 470(1-2): 17-25.

37. Ardid, D., Lamberty, Y., Alloui, A., Coudore-Civiale, M.A.,Klitgaard, H., Eschalier, A. Antihyperalgesic effect of levetiracetamin neuropathic pain models in rats. Eur J Pharmacol 2003, 473(1):27-33.

38. De Vry, J., Kuhl, E., Franken-Kunkel, P., Eckel, G. Pharma-cological characterization of the chronic constriction injury modelof neuropathic pain. Eur J Pharmacol 2004, 491(2-3): 137-48.

39. Decosterd, I., Allchorne, A., Woolf, C.J. Differential analgesic sen-sitivity of two distinct neuropathic pain models. Anesth Analg 2004,99(2): 457-63.

40. Lynch, J.J., Wade, C.L., Zhong, C.M., Mikusa, J.P., Honore, P.Attenuation of mechanical allodynia by clinically utilized drugs ina rat chemotherapy-induced neuropathic pain model. Pain 2004,110(1-2): 56-63.

41. Shao, B., Victory, S., Ilyin, V.I. et al. Phenoxyphenyl pyridines asnovel state-dependent, high-potency sodium channel inhibitors. JMed Chem 2004, 47(17): 4277-85.

42. Macdonald, R.L., Kelly, K.M. Antiepileptic drug mechanisms ofaction. Epilepsia 1995, 36(Suppl. 2): S2-S12.

43. Czapinski, P., Blaszczyk, B., Czuczwar, S.J. Mechanisms of actionof antiepileptic drugs. Curr Top Med Chem 2005, 5(1): 3-14.

44. Cardenas, C.A., Cardenas, C.G., de Armendi, A.J., Scroggs, R.S.Carbamazepine interacts with a slow inactivation state of NaV1.8-like sodium channels. Neurosci Lett 2006, 408(2): 129-34.

T. Mixcoatl-Zecuatl et al./Methods Find Exp Clin Pharmacol 2008, 30(6): 431-441 441