1 Introduction

Neuropathic pain may affect as much as 3% of the population, and these patients are a challenge to manage due to the severity of the pain and the lack of effective treatment [1, 2]. Many patients experience impaired mood and quality of life, with depression and sleeping disorders as common complications [3, 4].

Gabapentin is recommended as a first-line drug in the treatment of neuropathic pain [5, 6], based on evidence from numerous clinical trials [7, 8, 9]. Nevertheless, gabapentin as monotherapy may be limited by incomplete efficacy and dose-limiting adverse effects (sedation, dizziness, ataxia), resulting in unsatisfactory pain relief in a high proportion of patients. This is reflected in the estimated number needed to treat; approximately four patients need to be treated with gabapentin to reduce the pain by 50% in one patient [10]. Hence, one strategy to improve pain relief and/or reduce adverse effects is the use of a combination of analgesic drugs acting through different mechanisms [11].

Gabapentin is identified as a ligand to the α₂δ subunit of the voltage-gated calcium channel [12]. Binding to this site inhibits calcium influx at presynaptic nerve terminals, and subsequently reduces the release of several neurotransmitters. The antihyper-sensitivity actions of gabapentin are correlated with up-regulation of the α₂δ subunit in the spinal cord and/or dorsal root ganglia in experimental models of neuropathic pain [13]. Even though the spinal cord has been in focus as the primary site of the analgesic actions of gabapentin, it has in recent experimental studies been demonstrated that gabapentin also acts on supraspinal structures to stimulate the descending noradrenergic endogenous pain inhibitory system [14, 15, 16, 17, 18, 19]. This finding suggests a new treatment approach for gabapentin, i.e. to combine with a drug that prolongs the action of noradrenaline.

The present study evaluated the effect of co-administered gabapentin and venlafaxine in a rat model of neuropathic pain. Venlafaxine facilitates neurotransmission in the brain by blocking presynaptic reuptake of serotonin and noradrenaline [20], and may serve as a suitable adjuvant to augment the analgesic effect of gabapentin. It was further tested whether the treatments were associated with ataxia and other side effects that may interfere with the effect measurements.

Finally, the pharmacokinetics of gabapentin was investigated alone and in combination with venlafaxine to elucidate any alterations which may have consequences for the pharmacological effect and safety of the drug.

2 Materials and methods

All experiments were approved by the Animals Experiments Inspectorate, The Danish Ministry of Justice (no. 2007/561-1284 C7), and were in accordance with the ethic standards of IASP’s guidelines for pain research in animals [21].

3 Results

3.2 Pharmacological effect on mechanical hypersensitivity

3.2.1 Gabapentin

Gabapentin (60, 100 and 200 mg/kg) dose-dependently reversed hypersensitivity in response to von Frey stimulation (Fig. 1 and Table 1). The overall effect of gabapentin, measured as AUE_{0–3 h}, was significantly better than vehicle at the two highest doses tested (100 mg/kg; p = 0.0007 and 200 mg/kg; p = 0.0002). The time course of effect was typically a delay in the onset of action during the first hour, with maximum effect occurring 1.5–2.5 h following drug administration. During this interval, all doses significantly reduced the mechanical hypersensitivity in comparison to baseline (p < 0.05). Also, all doses produced peak withdrawal thresholds which were significantly different from vehicle (60 mg/kg; p = 0.02, 100 mg/kg; p = 0.0007 and 200 mg/kg; p = 0.0002).

Fig. 1

Withdrawal threshold in response to von Frey stimulation to the paw ipsilateral to SNI following subcutaneous administration of (A) gabapentin (60 mg/kg, n = 6; 100 mg/kg, n = 5; 200 mg/kg, n = 6), (B) venlafaxine (40 mg/kg, n = 6; 60 mg/kg, n = 6; 80 mg/kg, n = 8) and (C) co-administered gabapentin and venlafaxine (60 + 60 mg/kg, n = 8). Dotted line represents vehicle (n = 10). Data are presented as mean ± SEM. * p < 0.05 vs. time 0 (Friedman repeated measures one-way ANOVA with Dunn’s multiple comparison test).

Table 1

Summary of results from the von Frey and the Rotarod tests. Data are presented as mean (SEM).

Treatment	von Frey test				Rotarod test
	n	AUE0-3h(gh)	Maximum effect (g)	Onset–offset (hours post-dosing)	n	Minimum performance (percentage of baseline)	Onset–offset (hours post-dosing)
Vehicle	10	3.7 (0.6)	2.1(0.5)
Gabapentin
60 mg/kg	6	16.8 (5.4) ns	10 (3.5) *	1.5–2.5
100 mg/kg	5	25.9 (2.7) ***	16 (2.6) ***	1.5–2.5	6	59.8 (16.8) ns
200 mg/kg	6	33.6 (5.4) ***	23 (3.0) ***	1.5–2.5	5	2.6(0.6) *	<1.0–>3.0
Venlafaxine
40 mg/kg	6	5.4(1.6) ns	3.1(1.0) ns
60 mg/kg	6	16.5 (5.5) **	10 (3.4) **	1.0–2.0
80 mg/kg	8	12.8 (2.2) ***	8.1(1.1) ***	<0.5–2.0	5	95.9 (2.5) ns
Co-administration
60 + 60 mg/kg	8	32.7 (6.9) ***	16 (3.0) ***	1.0–>3.0	5	88.6 (7.0) ns

1. von Frey test: “AUE_{0–3 h}” is the area under effect curve during the time course of testing and “Maximum effect” is the peak withdrawal threshold measured at any time point during the testing period; ** p <0.01 and *** p <0.001 vs. vehicle (Mann–Whitney test). “Onset–offset” refers to the time interval where the withdrawal threshold is significantly higher than corresponding value at time 0 (Friedman repeated measures one-way ANOVA with Dunn’s multiple comparison test). Rotarod test: “Minimum performance” is the shortest time on the rod, expressed as percentage of baseline, at any of the time points during the testing period; * p <0.05 vs. baseline (Wilcoxon signed rank test). “Onset–offset” is the time interval where the performance time is significantly shorter than baseline (Wilcoxon signed rank test).

3.2.2 Venlafaxine

Venlafaxine (40, 60 and 80 mg/kg) reduced mechanical hypersensitivity in a dose-dependent manner (Fig. 1andTable. 1), with the two highest doses being significantly better in comparison to vehicle (60 mg/kg; p = 0.008 and 80 mg/kg; p <0.0001). Venlafaxine was characterized by a rapid onset of action, which reached its maximum approximately 1 h after dosing, and returned to baseline levels before the end of the 3 h time course of testing. The withdrawal thresholds were significantly higher than baseline between 1.0 and 2.0 h for 60 mg/kg, and between 0.5 and 2.0 h for 80 mg/kg (p <0.05). The peak withdrawal thresholds achieved from each dose were significantly higher than vehicle for the two highest doses tested (60 mg/kg; p = 0.003 and 80 mg/kg; p = 0.0002), but not for 40 mg/kg.

3.2.3 Co-administered gabapentin and venlafaxine

The combination of gabapentin (60 mg/kg) and venlafaxine (60 mg/kg) produced a significant reduction in mechanical hypersensitivity compared to vehicle (Fig. 1Cand table. 1; p <0.0001). The overall effect was also significantly improved compared to venlafaxine 60 mg/kg alone (p <0.02), but not in comparison to gabapentin 60 mg/kg (Fig. 2). The withdrawal thresholds were significantly higher than baseline threshold from 1 h post-dosing until the end of the 3 h testing period (p <0.05), thus, the duration of effect was prolonged as compared to each drug administered alone.

Fig. 2

Effect of co-administered gabapentin (60 mg/kg s.c.) and venlafaxine (60 mg/kg s.c.) in the von Frey test, in comparison to each drug administered alone. (A) The overall treatment effect is estimated from the area under effect curve during the time course of testing (AUE_{0–3 h}). (B) The maximum treatment effect is the peak withdrawal threshold measured at any time point during the testing period. Each bar represent mean + SEM. * p <0.05 (Mann–Whitney test).

3.3 Treatment-induced ataxia

Administration of gabapentin 100 mg/kg resulted in a trend towards impaired performance in the Rotarod test (Fig. 3Aand Table. 1). This effect increased over the 3 h time course of testing, but did not reach significance in comparison to baseline. Gabapentin 200 mg/kg caused pronounced ataxia with somnolence, which was present at all time points (Fig. 3A and Fig. 1; p <0.05). Neither venlafaxine 80 mg/kg nor the combination of gabapentin and venlafaxine (60 + 60 mg/kg) was associated with reduced performance times in the Rotarod test (Fig. 3B, C and Fig. 1).

Fig. 3

Performance time, calculated as percentage of baseline, in the Rotarod test following subcutaneous administration of (A) gabapentin (100 mg/kg, n = 6; 200 mg/kg, n = 5), (B) venlafaxine (80 mg/kg, n = 5) and (C) co-administered gabapentin and venlafaxine (60 + 60 mg/kg, n = 5). Non-SNI animals were used in the test. Data are presented as mean ± SEM. * p <0.05 vs. time 0 (Wilcoxon signed rank test).

3.4 Effect of venlafaxine on the pharmacokinetics of gabapentin

Following subcutaneous injection of gabapentin (60 mg/kg) in the rat, the drug rapidly reached the systemic circulation with maximum plasma concentrations within 15–30 min. Concomitant administration with venlafaxine (40 mg/kg s.c.) did not alter the plasma concentration–time curve of gabapentin (Fig. 4), and consequently, the pharmacokinetic estimates for gabapentin were almost identical in the two scenarios of presence and absence of venlafaxine (Table. 2).

Fig. 4

Plasma concentration–time profile for subcutaneously administered gabapentin (60 mg/kg) in presence (—) and absence (– – –) of co-administered venlafaxine (40 mg/kg s.c.). Data are presented as mean ± SD of four samples.

4 Discussion

Available clinical treatment algorithms for the management of neuropathic pain recommend combination therapy in cases of insufficient efficacy with first-line medications as monotherapy [5, 6]. The pharmacological treatment alternatives for neuropathic pain are limited, and do at most only offer partial pain relief in 50% of the neuropathic pain patients. Therefore, the use of combination pharmacotherapy in the clinic is substantial albeit in absence of evidence from clinical trials on the co-administered drugs [11].

The potential benefits of combining drugs include improved and prolonged duration of analgesic effect and fewer or milder adverse effects with lower doses of each drug. However, combination therapy might also be complicated by some concerns. Firstly, the risk for pharmacokinetic drug interactions, which may result in reduced effect or increase of unwanted effects, should always be considered before a second drug is added. Secondly, large differences in the duration of the pharmacological effect require different dosing schedules (e.g. dosing twice daily for drug A and three times daily for drug B), which are more difficult to follow and may have a negative impact on patient adherence.

The present study investigated the combination of gabapentin and venlafaxine in the SNI model of neuropathic pain. Encouraging results were obtained when the combination was evaluated for its effect on nerve injury-induced hypersensitivity in the von Frey test:

• The overall effect, measured as AUE, was similar to the highest dose of gabapentin tested alone.

• The maximum effect was in the same range as the intermediate dose of gabapentin tested alone.

• The duration of effect was prolonged in comparison to when gabapentin or venlafaxine were administered alone.

Sedation, dizziness and ataxia are reported as common and dose-limiting adverse effects of gabapentin in the clinic [10]. Furthermore, when performing preclinical studies involving measurements of stimuli-evoked hypersensitivity, these properties may result in an overestimated analgesic effect. Ataxic effects were in this study assessed in the Rotarod test. The highest dose of gabapentin was shown to massively reduce the performance time. Obvious somnolence may further have contributed to the impaired motor function. On the other hand, ataxia or somnolence was not detected for the combination, indicating more reliable results from the von Frey test as well as an improved side effect profile in comparison to equipotent doses of gabapentin.

Taken together, the results from the von Frey and the Rotarod tests indicate that co-administration of gabapentin and venlafaxine, in the doses combined here, provide an additive-like effect on mechanical hypersensitivity, but not on ataxic adverse effects. In order to accurately classify combination effects into additive, antagonistic or synergistic, an isobolographic study design should be applied [25]. Such design is time-consuming and requires a high number of animals to be sacrificed, but may for instance give valuable information on whether an observed combination effect is present over a broad range of dose pairs or only for a specific dose pair of the two drugs. The interpretations made in this study are, however, supported by an isobolographic study in which gabapentin reduced hypersensitivity in an additive manner when combined with the serotonin and noradrenaline reuptake inhibitor duloxetine [26].

Single drug administration gives no information about effects that may occur following long term exposure, e.g. tolerance development or effect potentiation. In fact, a recently published study reported that gabapentin inhibits excitatory synapse formation in vitro and in vivo [27]; a process which probably occurs within a longer time frame. Thus, a single administration design may not fully reflect the complex mechanism of gabapentin. The new findings are supported by a study, in which repeated intraperitoneal administration of 30 mg/kg gabapentin once a day produced a gradually increasing analgesic effect in spinal cord injured rats [28].

From a pharmacokinetic point of view, gabapentin may be an attractive agent for combination therapy due to its uncomplicated pharmacokinetic profile; the drug is not an inducer or inhibitor of the CYP system, is not metabolised itself and binding to plasma proteins is insignificant [29]. Thus, potential targets for pharmacokinetic interactions with other drugs are few. The pharmacokinetic investigation in this study, performed in the rat, confirmed that neither distribution nor elimination of gabapentin was affected by co-administered venlafaxine. The effect of gabapentin on the pharmacokinetics of venlafaxine was not studied, since any alterations are unlikely to occur on the basis of the pharmacokinetic properties of gabapentin.

By relating the pharmacodynamic and the pharmacokinetic time profiles of gabapentin from this study, an effect delay of approximately 1 h is observed. This delay may be a reflexion of the distribution from the subcutaneous site of administration to the target site, where receptor binding subsequently gives rise to analgesia. Intrathecal and intracerebroventricular administrations of gabapentin have previously resulted in immediate analgesic effects (within 15 min) in nerve injured rats and mice [14, 18].

A previously reported antagonistic interaction on coadministered gabapentin and venlafaxine could not be confirmed in the study [30]. Confoundingly, these studies were performed in the same experimental model (the SNI model), the same route of administration was used (subcutaneous injection) and similar doses were tested. As mentioned above, the results from the pharmacokinetic investigation clearly demonstrated that venlafaxine did not alter the pharmacokinetics of gabapentin, thus a pharmacokinetic explanation as previously suggested for the observed antagonistic interaction is not likely. However, inbred Brown Norwegian rats were used in the first study, while outbred Sprague–Dawley rats were used throughout the present study. A preliminary study where sparse blood samples were drawn from Brown Norwegian rats during the elimination phase of gabapentin only did, however, not show any differences in the pharmacokinetics of gabapentin due to co-administered venlafaxine.

Serotonin and noradrenaline are both substrates in the descending inhibitory pathways, producing anti-nociception. However, serotonin is also active in the descending excitatory pathways, which are pro-nociceptive. The role of serotonin in nociception is not yet fully understood, although recent studies suggest that the net effect of serotonin is pro-nociceptive rather than antinociceptive [31, 32, 33]. Differences in the balance between proand anti-nociceptive effects of serotonin in different rat strains might be an explanation for the diverse study outcomes, but needs further substantiation. Hence, research is required to increase the knowledge about serotonergic actions in nociception; whether the pro-nociceptive and anti-nociceptive actions vary between subjects and its implications for treatment outcome.

The clinical evidence of co-medication with gabapentin and venlafaxine is sparse. The combination has so far only been evaluated for its effect on painful diabetic neuropathy in patients whose pain did not improve with gabapentin monotherapy. This randomized, double-blind, placebo-controlled, 8-week clinical trial concluded that patients who received gabapentin plus venlafaxine (n = 6) showed significant improvement in pain reduction, mood disturbance, and quality of life when compared with patients treated with gabapentin plus placebo (n = 6). An uncontrolled follow-up study was later undertaken on 38 patients, resulting in the same conclusion [34]. Another study showed that the combination of gabapentin and the tricyclic antidepressant drug nortriptyline was superior to each drug alone for the treatment of diabetic polyneuropathy and postherpetic neuralgia [35].

In conclusion, the rationale for better effect and less adverse effects through combination therapy with gabapentin and venlafaxine in the management of neuropathic pain is supported by this preclinical study.