Gender differences in the antinociceptive effect of tramadol, alone or in combination with gabapentin, in mice

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1 J Biomed Sci (28) 15: DOI 1.7/s ORIGINAL PAPER Gender differences in the antinociceptive effect of tramadol, alone or in combination with gabapentin, in mice Xiaoli Dai Æ Claude D. Brunson Æ Robin W. Rockhold Æ Horace H. Loh Æ Ing K. Ho Æ Tangeng Ma Received: 11 December 27 / Accepted: 28 March 28 / Published online: 3 May 28 Ó National Science Council Taipei 28 Abstract Gender difference in the antinociceptive effect of tramadol and gabapentin (alone or in combination) were investigated in mice. For investigation of acute antinociceptive effect, tramadol and gabapentin were administered to mice by intraperitoneal injection and per os, respectively, and antinociceptive activity was measured by the tail-flick test 3 min after drug administration. For investigation of the development of antinociceptive tolerance to analgesics, mice were injected with tramadol (6 mg/kg), alone or in combination with gabapentin ( mg/kg), twice daily for seven consecutive days and the tail-flicks were tested on experimental days 1, 3, 5 and 7. Results showed there was a lower ED value of tramadol antinociception in males than in females, indicating that females were less sensitive to the drug. Gabapentin produces a limited antinociception in both males and females. The combination of gabapentin and tramadol produced synergistic effect X. Dai C. D. Brunson Department of Anesthesiology, University of Mississippi Medical Center, Jackson, MS 39216, USA R. W. Rockhold I. K. Ho T. Ma (&) Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, MS 39216, USA tma@pharmacology.umsmed.edu H. H. Loh Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN 55455, USA I. K. Ho Division of Mental Health and Substance Abuse Research, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan without gender difference. Repeated administration of tramadol produced antinociceptive tolerance in both genders. Gabapentin produced synergistic effect in tramadoltolerant mice and repeated administration of gabapentin did not alter the synergistic effect in tramadol-tolerant mice. Because females show a higher overall prevalence of pain and less sensitivity to opioids, our finding may suggest a clinical significance of combined use of the two drugs. Keywords Tramadol Gabapentin Pain Antinociception Tolerance Mice Tail-flick Opioid receptor Gender Introduction Opioids are the most powerful analgesics available. However, the use of strong opioid agents such as morphine is often limited by their intolerable adverse side effects. The opioid side effect problem has motivated the continuing search for new drugs that can provide adequate pain relief but with low side effects. Tramadol is a synthetic analgesic, which has been proven to be an effective therapeutic drug for treatment of pain [1]. Tramadol is classified as a weak or atypical opioid agent based on its agonist activity at the l- opioid receptor. Unlike morphine, a strong or typical opioid analgesic, tramadol has no clinically relevant respiratory depressant actions and has a low potential for abuse and dependence. It is therefore considered to be a safe analgesic drug. However, clinical trials and postmarketing surveillance studies have revealed side effects of this drug as well. The most common side effects that have been described are gastrointestinal and central nervous system symptoms including nausea, vomiting, dizziness, drowsiness and tiredness [2]. The maximum tolerated dose for tramadol is limited by these side effects.

2 646 J Biomed Sci (28) 15: Rational combination of opioids with non-opioid analgesics can enhance antinociceptive efficacy and reduce side effects. Therefore, combined pharmacotherapy for pain is frequently used by clinicians. Gabapentin is a novel anticonvulsant and an adjuvant analgesic. Gabapentin, alone or in combination with opioids, is often used for treatment of acute and chronic pain, including post surgical or neuropathic pain [3, 4]. Recent clinical and experimental animal study data demonstrate that gabapentin enhances morphine-induced antinociceptive activity [5 7]. It is unclear whether the combination of gabapentin with tramadol enhances their antinociceptive efficacy. There is a growing concern about gender differences in pain management. A considerable body of evidence indicates that there are gender differences in opioid antinociception in humans and experimental animals, with males generally displaying greater antinociceptive sensitivity than females [8 1]. Although it is proposed that the magnitude of this gender difference is inversely related to the efficacy of the agonist for opioid receptors, gender differences were found to be greater for intermediate to low efficacy agonists than for high efficacy agonists [11, 12]. As such, gender differences in tramadol antinociception remain unclear. The purposes of this study were to determine if: (1) there are gender differences of gabapentin and tramadol (alone and in combination)-produced antinociception in the tailflick test following acute administration, (2) repeated administration of tramadol produces tolerance, and (3) gabapentin potentiates antinociceptive response in tramadol-tolerant mice. Materials and methods Animals Adult male and female NIH Swiss mice (Harlan, USA), l- opioid receptor knockout mice and their wild-type controls were used in the study. The l-opioid receptor knockout mice were developed by Loh et al. [13]. Mice were maintained in the animal facilities on a 12-h light/dark cycle and at constant temperature (22 ± 2 C). Food and water were available ad libitum. All procedures for animal breeding, handling and experiments were approved by Institutional Animal Care Committee of the University of Mississippi Medical Center, and performed in compliance with the NIH Guide for the Care and Use of Laboratory Animals. Drugs and treatment protocols Tramadol hydrochloride (Sigma, USA) and gabapentin (Pfizer, USA) were dissolved in saline before each use and %MPE Tramadol (6 mg/kg, i.p.) Gabapentin ( mg/kg, p.o.) Time for tail-flick test after drug administration (min) Fig. 1 Time course of tramadol- and gabapentin-induced antinociception. Tramadol- and gabapentin-induced antinociceptive responses in the tail-flick are expressed as. Each symbol represents mean ± SEM from five animals administered to mice by intraperitoneal injection (i.p.) and per os (p.o.) by gavage, respectively. Tramadol showed a peak effect at around 3 min after administration and its effect was then gradually declined (Fig. 1). No peak antinociceptive effect was observed for gabapentin during the 3 h observation (Fig. 1). In the present study, therefore, tail-flick test was conducted at 3 min after drug administration. To determine the median effective dose (ED ) of tramadol-induced antinociception, mice were assigned to five or six groups with five animals per group. Each group of mice received one dose of tramadol, ranging from 32 9 mg/kg. The tail-flick latency of the mouse was measured 3 min after tramadol injection. The acute dose response data for tramadol-induced antinociception were fit to a sigmoidal function and the ED ± 95% was calculated using Graph Pad Prism Ò software (San Diego, USA). To investigate acute antinociceptive action of gabapentin and tramadol, alone or in combination, NIH Swiss male and female mice were assigned into experimental groups with eight mice per group. The tail-flick test was conducted 3 min after drug administration. To determine the antinociceptive effect of gabapentin in tramadol-tolerant mice, two groups of mice (eight or nine mice per group) from each gender were i.p. injected with tramadol (6 mg/kg, equal to the male antinociceptive ED ) twice daily (9 a.m. and 5 p.m.) for six consecutive days. On day 7, one group of mice was given gabapentin ( mg/kg, p.o.) plus tramadol (6 mg/kg, i.p.) and another group was given saline (p.o.) plus tramadol (6 mg/kg, i.p.). The tail-flick responses were measured on experimental days 1, 3, 5 and 7. Tramadol tolerance was determined by comparing the antinociceptive response during repeated administration of tramadol with the response to the first day of injection in the same animal.

3 J Biomed Sci (28) 15: The antinociceptive effect of gabapentin in tramadol-tolerant mice was evaluated by comparison of the tail-flick responses between the gabapentin/tramadol group and the saline/tramadol group as measured on day 7 (Fig. 4). To determine the effect of repeated administration of gabapentin plus tramadol on antinociceptive responsiveness, two groups of male mice with eight mice per group were used in the study. Gabapentin ( mg/kg, p.o.) or saline vehicle plus tramadol (6 mg/kg, i.p.) were administered to mice, twice daily for seven consecutive days. Tail-flick tests were conducted 3 min after drug administration in the morning on experimental days 1, 3, 5 and 7 (Fig. 5). The effect of repeated administration of gabapentin on antinociceptive activity was evaluated by comparison of antinociceptive responses between the gabapentin plus tramadol group and saline vehicle plus tramadol group on each test day. Tail-flick test confidence intervals were 59.2 ( ) mg/kg and 7.1 ( ) mg/kg in males and females, respectively, which indicate that females are less sensitive than males to tramadol-induced antinociception. A NIH Swiss male mice ED (mg/kg) = 59.2 ( ) 1 Tramadol dose (mg kg) Antinociceptive responses were determined with the tailflick test by measuring the latency of the tail-flick response [14]. The tail-flick latency was defined as the time between the onset of the heat stimulus and voluntary tail withdrawal. Mice were gently held while the tail was on apparatus (Columbus Instruments, USA). The tail-flick response was elicited by applying the radiant heat to the dorsal surface at cm from the tip of the tail. The intensity of the heat stimulus was adjusted to yield a baseline response of 3 4 s. A cutoff time of 1 s was used to avoid tissue injury. The response to a thermal nociceptive stimulus was expressed as percentage of maximum possible effect (), which was calculated as [(T 1 - T )/(T 2 - T )] 9. T and T 1 were the tail-flick latencies before and after drug administration, and T 2 was the cutoff time. Statistical analyses All statistical analyses were performed using SigmaStat 3.5 (Systat Software Inc, USA). Data were expressed as means ± standard errors of the mean (SEM). Two-way ANOVA followed by a post-hoc Student Newman Keuls test was used for analysis of all other data except ED s. A difference was considered significant at P \.5. B C NIH Swiss female mice ED (mg/kg) = 7.1 ( ) 1 Tramadol dose (mg kg) Wild-type (C57/BL6:129/Ola background) female mice ED (mg/kg) = 69.5 ( ) Results Female mice were less sensitive to tramadol-induced acute antinociceptive effect Acute i.p. injection of tramadol produced dose-dependent antinociceptive responses in the tail-flick assay in NIH Swiss mice (Fig. 2a and b). The ED s and their 95% 1 Tramadol dose (mg kg) Fig. 2 Acute tramadol-induced dose-antinociceptive response curves in NIH Swiss male (a) and female mice (b) as well as in female wildtype mice with C57/BL6 and 129/Ola genetic background (c). Tramadol-induced antinociceptive responses in the tail-flick are expressed as. Each symbol represents mean ± SEM from five animals

4 648 J Biomed Sci (28) 15: Table 1 Acute tramadol-induced antinociceptive and lethal effects in l-or knockout female mice Tramadol (mg kg -1 ) Animal number Mean ± SEM Death ±.7 (N = 4) None (N = 1) 5 Tramadol was i.p. injected to mice. The tail-flick was tested 3 min after tramadol injection. Tramadol-induced antinociceptive responses in the tail-flick are expressed as Male Female S S, G S, G S, G, T S, G, T Tramadol produced a limited antinociceptive effect in l-opioid receptor knockout mice Sal (S) Gab (G) Tra(T) Gab+Tra The antinociceptive effect of tramadol in female l-opioid receptor knockout mice and their wild-type controls was tested as well. Acute i.p. injection of tramadol produced dose-dependent antinociceptive effects in wild-type mice with a C57/BL6 and 129/Ola genetic background (Fig. 2c). The ED and 95% confidence intervals were 69.5 ( ) mg/kg, which were not different from NIH swiss female mice. However, tramadol did not produce dosedependent antinociceptive responses in l-opioid receptor knockout mice and thus no ED value could be estimated from the study. Tramadol at a dosage of 144 mg/kg (more than twice the ED value in wild-type mice) produced a MPE of 14.5% in l-opioid receptor knockout female mice. Higher doses of tramadol did not enhance an antinociceptive effect but led to significant animal fatality (Table 1). Gabapentin potentiated antinociceptive effects of tramadol in naïve mice without gender differences Two-way ANOVA revealed significant effects for tramadol, alone and in combination with gabapentin, in both genders (drug: F[3, 55] = 1, P \.1; gender: F[1, 55] = 1.4, P [.5); the drug 9 gender interactions: F[3, 55] = 7.1, P \.1). The post hoc analysis exhibited that in both genders the combination of gabapentin and tramadol produced a stronger antinociceptive effect than when used alone (Fig. 3). Although tramadol, when used alone, produced a stronger antinociceptive effect in males than that in females, the gender difference in antinociception was not observed when they were used in combination (Fig. 3). Gabapentin potentiated antinociceptive effects in tramadol-tolerant male and female mice The results in Fig. 4 show that repeated administration of tramadol produced antinociceptive tolerance and gabapentin can potentiate an antinociceptive effect in the tramadol-tolerant mice. Two-way ANOVA revealed significant antinociceptive effects of tested drugs in both males (day: Fig. 3 Acute gabapentin ( mg/kg, p.o.) and tramadol (6 mg/kg, i.p.), alone or in combination, -induced antinociceptive response in NIH Swiss male and female mice. Antinociceptive responses in the tail-flick are expressed as. Each column represents mean ± SEM from eight mice. Statistical analysis was performed by two-way ANOVA followed by Student Newman Keuls test. S P \.5 compared with the saline (Sal) vehicle group within the same gender; G P \.5 compared with gabapentin (Gab) group within the same gender; T P \.5 compared with tramadol (Tra) group within the same gender; P \.5 when compared in the same treatment between males and females A Male mice B Female mice Tramadol only Gabapentin plus tramadol (Day) Fig. 4 Antinociceptive effect of gabapentin in tramadol-tolerant male (a) and female (b) NIH swiss mice. Two groups of mice from each gender were i.p. injected with tramadol (6 mg/kg) twice per day from day 1 to day 6. On day 7, one group was administrated with gabapentin ( mg/kg, p.o.) plus tramadol (6 mg/kg, i.p.) and another group was administrated with saline (p.o.) plus tramadol (6 mg/kg, i.p.). The tailflicks of mice were measured 3 min after tramadol injection on experimental days 1, 3, 5, and 7. Antinociceptive activities in the tailflick are expressed as. Each column represents mean ± SEM from eight or nine mice. Statistical analysis was performed by two-way ANOVA followed by Student Newman Keuls test. P \.5 compared with the first injection on day 1 within corresponding group. P \.5 when compared between gabapentin plus tramadol group and saline plus tramadol group on day 7

5 J Biomed Sci (28) 15: F[3, 56] = 18.7, P \.1; drug: F[1, 56] = 5.8, P \.5) and females (day: F[3, 6] = 6.4, P \.1; drug: F[1, 6] = 4.1, P \.5). The drug 9 day interaction was not significantly different in males (F[3, 56] = 1.8, P [.5) and females (F[3, 6] = 2.4, P [.5). The post hoc analysis exhibited that repeated administration of tramadol caused a decrease in the MPE values as observed on days 3, 5, and 7 in males and on days 5 and 7 in females, which indicated the development of tolerance to tramadol. On day 7, however, administration of gabapentin to tramadol-tolerant mice significantly potentiated the antinociceptive response in both genders. Two-way ANOVA analysis also revealed the antinociceptive effect of repeated administration of both gabapentin and tramadol in male mice (day: F[3, 56] = 44.3, P \.1; drug: F[1, 56] = 18.1, P \.1). The drug 9 day interaction was not significantly different (F[3, 56] =.6, P [.5). The post hoc test exhibited that gabapentin retained the synergistic effect following repeated administration (Fig. 5). Discussion, (Day) The present study revealed that female mice were less sensitive to acute tramadol-induced antinociception, which Tramadol only Gabapentin plus tramadol,,, Fig. 5 Effect of repeated administration of gabapentin plus tramadol on antinociceptive response in NIH Swiss male mice. Two groups of mice received gabapentin ( mg/kg, p.o.) plus tramadol (6 mg/kg, i.p.) and saline vehicle (p.o.) plus tramadol (6 mg/kg, i.p.), respectively, twice per day for seven consecutive days. The tailflicks of mice were measured 3 min after first tramadol injection on experimental days 1, 3, 5, and 7. Antinociceptive activities in the tailflick are expressed as. Each column represents mean ± SEM from eight animals. Statistical analysis was performed by two-way ANOVA followed by Student Newman Keuls test. P \.5 compared with the first injection on day 1 within corresponding group. P \.5 compared with the first injection on day 3 within corresponding group. P \.5 when compared between gabapentin plus tramadol group and saline plus tramadol group on the same day is consistent with previous findings in the literature on antinociception of other opioids such as morphine [15, 16]. It has been proposed that ovarian hormones and periaqueductal gray l-opioid receptor density contribute to gender differences in antinociception produced by morphine [17]. Unlike the prototypical opioid morphine, tramadol is a weak (atypical) opioid agonist. Besides activation of l- opioid receptor, tramadol inhibits reuptake of serotonin and norepinephrine, which is proposed to enhance inhibitory effects on pain transmission in the spinal cord [18]. However, the present results demonstrate that the l-opioid receptor plays a critical role in tramadol-induced antinociception, as displayed by a limited antinociceptive response in l-opioid receptor knockout mice. Therefore, the mechanism underlying the gender differences in morphine antinociception may be responsible for tramadol as well. Repeated administration of opioids may lead to a decrease in its antinociceptive sensitivity, a phenomenon called antinociceptive tolerance. Reports in the literature dealing with antinociceptive tolerance to tramadol are not in agreement, including no, low and significant development of tolerance to this drug in experimental animals [19 22]. The results of the present study clearly revealed that repeated administration of tramadol produced significant antinociceptive tolerance in tail-flick responses in both male and female mice. It is known that development of antinociceptive tolerance to opioids depends on the dosage and duration of its administration. Different treatment protocols that have been used in different laboratories may partially explain these inconsistent results. The results suggest that long-term use of tramadol under certain conditions may lead to analgesic tolerance to the drug in patients. Tolerance is highly complex and involves a series of cellular events. Desensitization, internalization, and down-regulation of the l-opioid receptor are believed to be involved in the development of tolerance to opioids [23]. The mechanism responsible for the development of tramadol tolerance needs to be clarified. Gabapentin is an antiepileptic agent but exhibits antinociceptive effects in humans and some experimental animals. In this study, gabapentin showed a very limited antinociceptive effect in tail-flick tests (less than 1% MPE) in both genders of mice. However, gabapentin significantly enhanced the antinociceptive effect of tramadol in naïve and tramadol-tolerant mice. When gabapentin and tramadol were used in combination, they elicited similar antinociceptive responses in males and in females. There is a growing concern about gender differences in pain perception and analgesic responses. Women have a lower pain threshold and less tolerance to pain than men [8, 24, ], and thus females show a higher overall prevalence of pain than males [26]. As for the response to analgesics,

6 6 J Biomed Sci (28) 15: in the experimental animals, females exhibit less sensitivity to opioids such as morphine when compared to males [1, 11, 27, 28]. The same was true for tramadol as observed in the present study. In humans, it was reported that women required more morphine than men to achieve a similar degree of analgesia [8]. Because long-term use of opioid drugs at high doses may lead to severe side effects, including tolerance, current treatments for chronic pain remain insufficient for a large percentage of patients [29]. Therefore, there is a pressing need to develop effective analgesics without significant adverse side effects. Our finding, that there is no gender difference in gabapentin potentiation of the antinociceptive effect of tramadol, may be clinically relevant for the treatment of pain particularly in female patients. Gabapentin is a c-aminobutyric acid (GABA) derivative. GABA system seems not to be the target for gabapentin since it lacks an affinity for the GABA receptors and fails to inhibit GABA transaminase or GABA uptake [3 32]. Radioligand binding assays did not exhibit affinity of gabapentin for a number of common neurotransmitter receptors, including glutamatergic, adrenergic, cholinergic, dopaminergic, and opioidergic receptors [33]. Several lines of evidence have revealed gabapentin binding to the a2d subunit of voltage-sensitive Ca 2+ channels in animal brain tissues [34, 35]. It has been proposed that gabapentin binding to the a2d protein may selectively attenuate depolarization-induced Ca 2+ influx of presynaptic P/Q-type Ca 2+ channels and thus lead to a decrease in glutamate release in the central and peripheral nervous system [36 38]. Glutamate is an excitatory neurotransmitter in the nervous system of mammals. It is clear that noxious stimulus may result in glutamate release from the nociceptive afferent fibres synapse at the dorsal horn of the spinal cord, where glutamate is an important mediator for ascending pain transmission. Pharmacological blockade of the NMDA subtype of glutamate receptors can enhance antinociception of l-opioid, such as morphine, administered at the spinal cord level [39]. Therefore, we hypothesize that gabapentin, via activating the a2d subunit of Ca 2+ channels, reduces thermal stimulusinduced glutamate release in the tail-flick test and thereby enhances tramadol antinociception. Since estrogen can enhance glutamate binding to the NMDA receptor [4] and females were shown to be more sensitive to exogenous glutamate-induced pain [41, 42]. If gabapentin has a similar effect on both genders of mice in reducing thermal stimulus-induced glutamate release, it would show a higher efficacy in enhancing tramadol antinociception in the females. 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