Dose-response relationship analysis of pregabalin doses and their antinociceptive effects in hot-plate test in mice

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1 Pharmacological Reports 2010, 62, ISSN Copyright 2010 by Institute of Pharmacology Polish Academy of Sciences Short communication Dose-response relationship analysis of pregabalin doses and their antinociceptive effects in hot-plate test in mice Jarogniew J. uszczki 1,2 Department of Pathophysiology, Medical University of Lublin, Jaczewskiego 8, PL Lublin, Poland Isobolographic Analysis Laboratory, Institute of Agricultural Medicine, Jaczewskiego 2, PL Lublin, Poland Correspondence: Jarogniew J. uszczki, Abstract: The aim of this study was to determine the analgesic effects of pregabalin (a third-generation antiepileptic drug) using the acute thermal pain model (hot-plate test) in mice. Linear regression analysis was used to evaluate a dose-response relationship between logarithms of pregabalin doses and their resultant maximum possible antinociceptive effects (MPAE) using the hot-plate test in mice. From the linear equation of the dose-response relationship, doses of pregabalin that increased antinociceptive effects by 20%, 30%, 40%, and 50% were calculated and amounted to 9.33, 24.80, 65.93, and mg/kg, respectively. In conclusion, pregabalin produces analgesic effects in a dose-dependent manner, as demonstrated using the hot-plate test in mice. Key words: acute thermal pain, dose-response relationship, hot-plate test, linear regression analysis, maximum possible antinociceptive effect, pregabalin Introduction Both preclinical studies on animals [18 21, 26 28, 33] and clinical settings in humans [2, 4, 11, 22, 30, 40] provide overwhelming evidence that some antiepileptic drugs exert analgesic effects. At present, several antiepileptic drugs bring pain relief to patients with trigeminal neuralgia (carbamazepine, lamotrigine and oxcarbazepine), diabetic peripheral neuropathy (topiramate, lamotrigine, gabapentin, and pregabalin), post-herpetic neuralgia (topiramate, gabapentin, and pregabalin), phantom limb pain (gabapentin and pregabalin), and other types of chronic pain [2, 4, 11, 30, 40]. Pregabalin ((S)-(+)-3-(aminomethyl)-5-methylhexanoic acid) and gabapentin (2-[1-(aminomethyl)-cyclohexyl]acetic acid) are both alkylated analogues of -aminobutyric acid (GABA) (Fig. 1). In the case of pregabalin, the drug has been shown to suppress tactile allodynia in rats subjected to ischemic sciatic nerve injury [41], reduce thermal and mechanical hypersensitivity in a murine chronic pain model (Seltzer model) based on partial ligation of the sciatic nerve [35], and suppress tactile allodynia in rats induced by spinal nerve ligation [15]. In regard to gabapentin, numerous animal studies suggest that this drug may be useful for controlling acute nociceptive pain as well as many different types of neuropathic pain [1, 6, 18, 20, 31]. 942 Pharmacological Reports, 2010, 62,

2 Antinociceptive effects of pregabalin in the hot-plate test in mice Jarogniew uszczki Fig. 1. Chemical structures of GABA, gabapentin and pregabalin Previously, it has been documented that three second-generation antiepileptic drugs (gabapentin, tiagabine and vigabatrin) produced antinociceptive effects in a dose-dependent manner using the hot-plate test in mice. In the case of vigabatrin, the experimentally derived doses of the drug that increased its antinociceptive effects by 15%, 20% and 25% (i.e., AEID 15 antinociceptive effect increasing dose 15, AEID 20, and AEID 25 values) were 144, 388 and 1016 mg/kg, respectively [26]. Moreover, the doses of gabapentin and tiagabine that increased the antinociceptive effects by 50% (AEID 50 values) using the hot-plate test in mice were 504 and 5.7 mg/kg, respectively [28]. Considering the facts that gabapentin and pregabalin (1) exert antinociceptive effects in various experimental models of acute and chronic pain (2), reduce neuropathic pain in patients with diabetic neuropathy and post-herpetic neuralgia (3), and are both antiepileptic drugs structurally related to GABA, it is of pivotal importance to determine the dose-response relationship for pregabalin using the acute thermal pain model (hot-plate test) in mice. Least-squares linear regression analysis was used to establish the dose-response relationship between pregabalin doses and their resultant antinociceptive effects, expressed as maximum possible antinociceptive effects (MPAE), using the hot-plate test (a standard model used to determine the antinociceptive efficacy of compounds with respect to acute thermal nociception) in mice. Material and Methods Animals and experimental conditions Experiments were performed on adult male Swiss mice weighing g. The animals were kept in colony cages with free access to food and tap water, under standardized housing conditions (12 h of a light-dark cycle, stable temperature of 22 ± 1 C for 24 h). After seven days of adaptation to laboratory conditions, the animals were randomly assigned to experimental groups consisting of eight mice. All tests were performed between 9.00 a.m. and 3.00 p.m. Procedures involving animals and their care were conducted in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and Polish legislation on animal experimentation. Additionally, all efforts were made to minimize animal suffering and to use only the number of animals necessary to produce reliable scientific data. The experimental protocols and procedures described in this study were approved by the First Local Ethics Committee at the Medical University of Lublin (license no.: 21/2007 and 59/2009) and the Second Local Ethics Committee at the University of Life Sciences in Lublin (license no.: 60/2009). Drug Pregabalin (Lyrica, Pfizer Ltd., Sandwich, Kent, UK) was suspended in a 1% solution of Tween 80 (Sigma, St. Louis, MO, USA) in saline and administered intraperitoneally (ip) in a volume of 5 ml/kg body weight. Pregabalin was administered 60 min before the hot-plate test. This pretreatment time was chosen based upon information about the biological activity of pregabalin from the literature [14, 15]. Hot-plate test The hot-plate test was conducted according to the procedure described by Eddy and Leimbach [10] with minor modifications. The device used consisted of an electrically heated surface and an open Plexiglas tube (17 cm high 22 cm diameter) to confine the animals to the heated surface (Ugo Basile, Varese, Italy). The temperature was set to 55.0 ± 0.1 C. Mice were placed separately on a heated surface, and the time interval (in s) between placement and shaking, licking, or tucking of the fore- or hindpaws was recorded by a stopwatch as the predrug latency response. Animals Pharmacological Reports, 2010, 62,

3 were tested once before baselines were taken, and this trial served as the control reaction time for the animals. Mice showing a reaction time greater than 10 s were excluded from the subsequent test. The predrug latencies were between 5 and 8 s. Subsequently, the animals were administered pregabalin alone at increasing doses; and at the time of peak antinociceptive activity (i.e., 60 min), the same procedure was repeated, and the animals were placed again on the heated surface. In other words, each animal was subjected to the hot-plate test twice. To perform the first evaluation of time to the first pain reaction in animals using the hot-plate test, the naive mice were randomly assigned to experimental groups (consisting of 8 mice per group) and consecutively numbered on their tails with multi-colored markers. Then, the animals were challenged with the hot-plate test to determine the latency of the first pain reaction for each mouse separately. Next, the marked animals received only pregabalin and, at the time to the peak of maximum antinociceptive effects, were subjected to the second evaluation of time to the first pain reaction in the same animals. In other words, both pre- and posttreatment times were recorded for the same animals. In the present study, pregabalin was administered systemically (ip) at doses ranging between and 200 mg/kg. A maximum cut-off time of 30 s was chosen to prevent injury to animals. Mice not responding within 30 s were removed from the heated surface and assigned a score of 30 s. The MPAE was defined as the lack of a nociceptive response in mice during the exposure to the heat stimulus, and the percentage of MPAE was calculated according to the formula presented by Schmauss and Yaksh [32] as follows: [(T T )/(T T )] 100; T and T are the latencies obtained before and after drug administration, and T is the cut-off time of 30 s. Next, pregabalin doses were transformed to logarithms to the base 10 and plotted on the X-axis of the Cartesian system of coordinates. Simultaneously, the MPAE corresponding to the antiepileptic drug doses was plotted on the Y-axis, and both values were analyzed with least-squares linear regression analysis according to Motulsky and Christopoulos [29]. Subsequently, doses of pregabalin increasing antinociceptive effects by 20%, 30%, 40% and 50% (AEID 20, AEID 30, AEID 40, and AEID 50 values) were calculated from the linear equation of the dose-response relationship. Least-squares linear regression analysis was performed using commercially available GraphPad Prism 4 (GraphPad Software Inc., San Diego, CA, USA). This experimental procedure has been described in more detail in earlier studies [26]. Results Pregabalin was administered systemically (ip), 60 min before the hot-plate test, and the latency to the first pain reaction using the acute thermal pain model in mice was lengthened in a dose-dependent manner. The experimentally derived MPAE values for pregabalin administered at doses ranging between mg/kg were between % (Tab. 1). Subsequently, the MPAE values for animals injected with increasing doses of pregabalin (expressed as logarithms to the base 10) were graphically plotted in rectangular coordinates of the Cartesian system and examined with least-squares linear regression analysis. This method allowed for the determination of the equation for dose-response relationship for pregabalin as follows: y = x (r = ), where y is the MPAE in %, x is the logarithm of a drug dose, and r is the coefficient of determination (Fig. 2). From this equation, one could readily denote doses of Tab. 1. Antinociceptive effects of pregabalin in the hot-plate test in mice Dose (mg/kg) Log dose MPAE (%) ± ± ± ± ± ± ± ± ± 3.23 Values are presented as the means of the maximum possible antinociceptive effect (MPAE) ± SE of eight mice. Pregabalin was administered ip at 60 min before the antinociceptive effect evaluation. The MPAE was defined as the lack of a nociceptive response in mice during the exposure to the heat stimulus (55.0 ± 0.1 C), and the percentage of MPAE was calculated according to the formula by Schmauss and Yaksh [32]. For more details, see Materials and Methods 944 Pharmacological Reports, 2010, 62,

4 Antinociceptive effects of pregabalin in the hot-plate test in mice Jarogniew uszczki Fig. 2. Dose-response relationship between pregabalin doses and their resultant MPAE using the hot-plate test in mice. Doses of pregabalin were transformed to logarithms to the base 10 (log), whereas the antinociceptive effects produced by pregabalin were transformed to maximum possible antinociceptive effect (MPAE in % ± SE as the error bars, n = 8animals). Pregabalin was administered ip at 60 min before the antinociceptive effect evaluation. The MPAE was defined as the lack of a nociceptive response in mice during the exposure to the heat stimulus (55.0 ± 0.1 C), and the percentage of MPAE was calculated according to the formula by Schmauss and Yaksh [32] as follows: [(T T )/(T T )] 100, where T and T are the latencies obtained before and after drug administration, and T is the cut-off time of 30 s. Log doses of pregabalin and their resultant MPAE were plotted into the Cartesian system of coordinates and analyzed with least-squares linear regression to determine the dose-response relationship between doses of pregabalin and their resultant antinociceptive effects using the acute thermal pain model (hot-plate test) in mice. Linear regression analysis allowed for the determination of the equation of the dose-response relationship for pregabalin as follows: y = x (r = ); where y is the MPAE in %, x is the logarithm of the drug dose to the base 10, and r coefficient of determination. From this equation one denotes the AEID, AEID!, AEID " and AEID # values (pregabalin doses that increased antinociceptive effects by 20%, 30%, 40% and 50%) in the hot-plate test. In this study, the experimentally derived logarithms of AEID, AEID!, AEID " and AEID # values were 0.970, 1.394, 1.819, and 2.244, respectively, which corresponded to pregabalin doses of 9.33, 24.80, 65.93, and mg/kg. For more detailed information see the legend to Table 1 pregabalin that increased antinociceptive effects by 20%, 30%, 40%, and 50% (AEID 20, AEID 30, AEID 40 and AEID 50 values), which were 0.970, 1.394, 1.819, and 2.244, respectively, corresponding to pregabalin doses of 9.33 mg/kg for AEID 20, mg/kg for AEID 30, mg/kg for AEID 40 and mg/kg for AEID 50 using the hot-plate test in mice (Fig. 2). Discussion Results presented herein indicate that pregabalin produced antinociceptive effects in a dose-dependent manner using the acute thermal pain model (hot-plate test) in mice. Linear regression analysis of this doseresponse relationship for the determination of AEID 20, AEID 30, AEID 40 and AEID 50 values, i.e., doses of pregabalin that increased the antinociceptive effects by 20%, 30%, 40% and 50% using the hot-plate test in mice. The procedure for the determination of MPEA for pregabalin doses and the calculation of AEID 20, AEID 30, AEID 40 and AEID 50 values can be readily adapted to perform similar experiments in preclinical studies for other antiepileptic drugs so as to characterize the analgesic potential of classical, second- and third-generation antiepileptic drugs. Thus, one can classify antiepileptic drugs by considering their antinociceptive properties and the potency of antiepileptic drugs in terms of reduction of acute thermal pain. It should be stressed that the method described in this study allows to unequivocally determine doses of various drugs that fulfill identical criteria, i.e., producing an increase in antinociceptive effects by 20%, 30%, 40%, and 50%. There is no doubt that the AEID values are very helpful during the assessment of the analgesic potency of drugs because they can provide an indication of the efficacy of these drugs, offering an antinociceptive effect at low doses. Pharmacological Reports, 2010, 62,

5 It is important to note that the determination of the AEID 50 value for pregabalin in this study ( mg/kg) allowed the direct comparison of the potency of the drug with gabapentin. Previously, it had been reported that the AEID 50 value for gabapentin using the hot-plate test in mice was 504 mg/kg [28]. Thus, one can ascertain that pregabalin produced analgesic effects at dose ranges almost 3 times lower than gabapentin, when both antiepileptic drugs were administered systemically (ip). These differences in analgesic effects between gabapentin and pregabalin are consistent with other studies documenting that pregabalin was much more effective than gabapentin in the suppression of acute and chronic pain in rodents [14, 15], which may be at least partly attributed to their differential affinities for the 2 auxiliary subunits or system of L-amino acid transporters mediating their influx and efflux across the blood-brain barrier [12, 17, 23, 34, 37 39]. Noteworthy, pregabalin and gabapentin bind with high affinity to the 2 auxiliary subunits of voltage-gated calcium channels [13, 17] and thus reduce calcium influx at nerve terminals. In turn, the reduction of calcium influx reduces the release of excitatory neurotransmitters (such as glutamate, noradrenaline, dopamine and substance P) from presynaptic terminals [7 9, 16]. Experimental studies have revealed that the binding of pregabalin and gabapentin to the 2 auxiliary subunits of the calcium channels is necessary and sufficient for analgesic and anticonvulsant effects [36]. For instance, transgenic mice expressing the mutant gene for the 2 auxiliary subunit of the calcium channels (R217A mutant mice) have much smaller quantities of drug binding in the forebrain and spinal cord. They also completely lack analgesic-like actions of gabapentin and pregabalin with unaltered analgesia from morphine and amitriptyline [13]. Moreover, a mutation in the 2 auxiliary subunit of the calcium channel protein selectively reduces [ 3 H]gabapentin binding [5]. It should be stressed that doses of pregabalin used in this study to determine the acute antinociceptive effect using the hot-plate test in mice were in a similar range to those producing anticonvulsant effects in the mouse maximal electroshock-induced seizure (MES) model. For instance, the experimentally derived AEID 50 value for pregabalin in the present study was mg/kg, whereas the ED 50 value for pregabalin (i.e., the dose of pregabalin protecting 50% of the animals tested against MES-induced seizures in mice) was mg/kg [25]. In contrast, doses of pregabalin producing the analgesic activity in chronic neuropathic pain models considerably differ from those observed in acute pain models. It has been documented that pregabalin administered ip at doses up to 30 mg/kg alleviated thermal and mechanical hypersensitivity in the plantar test after peripheral nerve injury in mice with partially ligated sciatic nerves (the Seltzer model). In contrast, pregabalin at the dose of 30 mg/kg (ip), did not affect acute thermal and mechanical nociception of naive mice in the plantar test [35]. The apparent discrepancy between doses of pregabalin producing analgesic actions in acute and chronic (neuropathic) pain models is probably evoked by various molecular mechanisms of the action of pregabalin. Recently, it has been reported that pregabalin activates the descending noradrenergic system to facilitate spinal noradrenaline turnover resulting in analgesic effects mediated by spinal 2 -adrenoceptors after peripheral nerve injury in the mouse partial sciatic nerve ligation model [35]. Moreover, the antiallodynic effect of pregabalin is correlated with upregulation of 2 subunits of voltage-dependent calcium channels in the spinal cord and/or dorsal root ganglia [24]. Pregabalin impairs anterograde trafficking of the 2-1 subunit resulting in its decrease in presynaptic terminals, which reduces neurotransmitter release and spinal sensitization in rats with unilateral lumbar spinal nerve ligation [3]. The abovementioned facts could, in part, explain the difference in doses of pregabalin required to evoke analgesic effects in acute and chronic models of pain in experimental animal models. Also noteworthy, the acute antinociceptive effects produced by pregabalin may be of great importance for patients with epilepsy, who, similar to people without epilepsy, suffer from incidental pains (headaches, migraine, odontalgias, algomenorrhoeas, etc.) [11, 22, 30]. Thus, by offering antiseizure protection and simultaneously producing analgesic effects, pregabalin may eliminate the incidental intake of analgesic drugs that may interact with applied antiepileptic drugs, changing their pharmacological and therapeutic profiles. It seems that pregabalin, by inducing antiseizure and analgesic effects, could substantially reduce the incidental polytherapy associated with treatment of pain in patients with epilepsy; thus, pregabalin would be able to ameliorate the quality of living for epileptic patients. 946 Pharmacological Reports, 2010, 62,

6 Antinociceptive effects of pregabalin in the hot-plate test in mice Jarogniew uszczki Conclusions In a dose-dependent manner, pregabalin exerted antinociceptive effects during the hot-plate test in mice. The procedure describing the calculation of doses to increase the antinociceptive effects by 20%, 30%, 40%, and 50% (AEID 20, AEID 30, AEID 40 and AEID 50 values) using the hot-plate test can become a paradigm in preclinical studies allowing for the characterization of the antinociceptive properties of antiepileptic drugs and for comparing the potency of antiepileptic drugs with respect to their relief from pain. Acknowledgments: This study was supported by grants from the Medical University of Lublin and Institute of Agricultural Medicine (Lublin, Poland). Professor J.J. uszczki is a recipient of the Fellowship for Leading Young Researchers from the Ministry of Science and Higher Education (Warszawa, Poland). References: 1. 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