Subcutaneous, intrathecal and periaqueductal grey administration of ...

European Journal of Pharmacology 573 (2007) 75 – 83 www.elsevier.com/locate/ejphar

Subcutaneous, intrathecal and periaqueductal grey administration of asimadoline and ICI-204448 reduces tactile allodynia in the rat Nadia L. Caram-Salas a , Gerardo Reyes-García b , Gerd D. Bartoszyk c , Claudia I. Araiza-Saldaña a , Mónica Ambriz-Tututi a , Héctor I. Rocha-González a , Rosaura Arreola-Espino d , Silvia L. Cruz a , Vinicio Granados-Soto a,e,⁎ b

a Departamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados, Sede Sur, México, D.F., Mexico Sección de Estudios de Posgrado e Investigación, Escuela Superior de Medicina del Instituto Politécnico Nacional, México, D.F., Mexico c Global Preclinical R&D, Merck KaA, D-64271 Darmstadt, Germany d Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacan, Mexico e Centro de Investigación y de Estudios Avanzados, Unidad Monterrey, Monterrey, Nuevo León, Mexico

Received 1 March 2007; received in revised form 11 June 2007; accepted 12 June 2007 Available online 29 June 2007

Abstract The purpose of this study was to assess the possible antiallodynic effect of asimadoline ([N-methyl-N-[1S)-1-phenyl)-2-(13S))-3hydroxypyrrolidine-1-yl)-ethyl]-2,2-diphenylacetamide HCl]) and ICI-20448 ([2-[3-(1-(3,4-Dichlorophenyl-N-methylacetamido)-2-pyrrolidinoethyl)-phenoxy]acetic acid HCl]), two peripheral selective κ opioid receptor agonists, after subcutaneous, spinal and periaqueductal grey administration to neuropathic rats. Twelve days after spinal nerve ligation tactile allodynia was observed, along with an increase in κ opioid receptor mRNA expression in dorsal root ganglion and dorsal horn spinal cord. A non-significant increase in periaqueductal grey was also seen. Subcutaneous (s.c.) administration of asimadoline and ICI-204448 (1–30 mg/kg) dose-dependently reduced tactile allodynia. This effect was partially blocked by s.c., but not intrathecal, naloxone. Moreover, intrathecal administration of asimadoline or ICI-204448 (1–30 μg) reduced tactile allodynia in a dose-dependent manner and this effect was completely blocked by intrathecal naloxone. Microinjection of both κ opioid receptor agonists (3–30 μg) into periaqueductal grey also produced a naloxone-sensitive antiallodynic effect in rats. Our results indicate that systemic, intrathecal and periaqueductal grey administration of asimadoline and ICI-204448 reduces tactile allodynia. This effect may be a consequence of an increase in κ opioid receptor mRNA expression in dorsal root ganglion, dorsal horn spinal cord and, to some extent, in periaqueductal grey. Finally, our data suggest that these drugs could be useful to treat neuropathic pain in human beings. © 2007 Elsevier B.V. All rights reserved. Keywords: Asimadoline; ICI-204448; Neuropathic pain; Periaqueductal grey; Spinal cord; κ opioid receptor agonists

1. Introduction κ opioid receptor agonists are potent analgesics that have been tested in a variety of experimental pain models. Unlike μ opioid receptor agonists, κ opioid receptor agonists do not induce euphoria, respiratory depression or gastrointestinal transit inhibition (Horwell, 1988). Unfortunately, κ opioid receptor agonists ⁎ Corresponding author. Cinvestav, Unidad Monterrey, Avenida Cerro de las Mitras 2565, Colonia Obispado, 64060 Monterrey, Nuevo León, Mexico. Tel.: +52 81 8220 1740; fax: +52 81 8220 1741. E-mail address: [email protected] (V. Granados-Soto). 0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.06.034

can produce side effects such as sedation and dysphoria that limit their clinical usefulness (Pfeiffer et al., 1986). These central side effects can be avoided by administration of low, systemically inactive doses of κ opioid receptor agonists directly into the injured tissue, where they can produce potent antinociception by activation of peripheral opioid receptors. An alternative strategy is to administer κ receptor agonists with restricted access to the central nervous system (Barber and Gottschlich, 1997; Husbands, 2004). Asimadoline (EMD-61753, [(N-methyl-N-[1S)-1-phenyl)-2(3S))-3-hydroxypyrrolidine-1-yl)-ethyl]-2,2-diphenylacetamide HCl)] and ICI-204448 [(2-[3-(1-(3,4-Dichlorophenyl-N-

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methylacetamido)-2-pyrrolidinoethyl)-phenoxy]acetic acid HCl)] are κ selective agonists with restricted ability to cross the blood–brain barrier (Gottschlich et al., 1994; Barber et al., 1994). These compounds contain hydrophilic and hydrophobic groups that determine their peripheral κ opioid receptor selectivity and high potency. The antinociceptive effect of asimadoline has been extensively studied in inflammatory pain models (Barber et al., 1994; Binder and Walker, 1998; Machelska et al., 1999; Binder et al., 2000) and in humans (Machelska et al., 1999; Delvaux et al., 2004). However, its effect in neuropathic pain still needs characterization. It has been reported that local peripheral administration of asimadoline (Walker et al., 1999) and ICI-204448 (Keïta et al., 1995) reduces the pain generated after chronic constriction of the sciatic nerve, but there is no information about the antiallodynic efficacy in other models of neuropathic pain. Even though these drugs were designed for peripheral administration, they are valuable pharmacological tools that can be used by other routes of administration in order to establish the nervous regions and receptor subtypes involved in neuropathic pain. Therefore, the purpose of this study was to assess the putative antiallodynic activity of asimadoline and ICI-204448 after subcutaneous, spinal and periaqueductal grey matter administration in female rats. The results were correlated with the levels of expression of κ opioid receptor mRNA in dorsal root ganglion, spinal cord and periaqueductal grey of neuropathic rats. 2. Materials and methods 2.1. Animals Female Wistar rats aged 6–7 weeks (weight range, 120– 140 g) from our own breeding facilities were used in this study. Animals had free access to food and water before experiments. Efforts were made to minimize animal suffering and to reduce the number of animals used. All experiments followed the Guidelines on Ethical Standards for Investigation of Experimental Pain in Animals (Zimmerman, 1983) and were approved by our local Ethics Committee. 2.2. Measurement of tactile allodynia Rats were prepared according to the method described by Kim and Chung (1992). Animals were anesthetized with a mixture of ketamine/xylazine (45/12 mg/kg, i.p.). After surgical preparation and exposure of the dorsal vertebral column, the left L5 and L6 spinal nerves were exposed and tightly ligated with 6-0 silk suture distal to the dorsal root ganglion. In sham operated rats, the nerves were exposed but not ligated. After closing the incisions, animals were allowed to recover for 10 days. Rats exhibiting motor deficiency (such as paw-dragging) were discarded from the study (less than 5%). Tactile allodynia was determined by measuring paw withdrawal threshold in response to probing with a series of calibrated fine filaments (von Frey filaments). The strength of von Frey stimuli ranged from 0.4 to 15 g. Withdrawal threshold

was determined by increasing and decreasing stimulus strength eliciting paw withdrawal (Chaplan et al., 1994). The stimulus intensity required to produce a response in 50% of the applications for each animal was defined as “50% withdrawal threshold”. All nerve-ligated rats were verified to be allodynic (responding to a stimulus lower than 4 g). Non-allodynic rats were discarded (less than 5%). 2.3. Spinal surgery Ten days after the first surgery rats were anesthetized with a ketamine/xylazine mixture (45/12 mg/kg, i.p.) and placed in a stereotaxic head holder in order to expose the atlantooccipital membrane (Yaksh and Rudy, 1976). After piercing the membrane, a PE-10 catheter (7.5 cm) was passed intrathecally to the level of the thoracolumbar junction and the wound sutured. Rats were allowed to recover from surgery for at least 5 days in individualized cages before use. Animals showing any signs of motor impairment were euthanized with CO2. 2.4. Periaqueductal grey surgery Under ketamine/xylazine anaesthesia (45/12 mg/kg, i.p.), rats were stereotaxically implanted with a 23-gauge stainlesssteel guide cannula aimed at the periaqueductal grey (9 mm guide; AP 1.2 mm, ML 0.6 mm, DV-5.1 mm from lambda; Paxinos and Watson, 1986). Periaqueductal grey was chosen as a control site in the midbrain because it plays an important role in the descending regulation of pain (Pertovaara and Almeida, 2006). The guide cannula was held in place with dental cement affixed to a screw in the skull. A removable 30gauge stainless-steel stylet was inserted into the guide cannula following surgery. Rats were allowed to recover for a week prior to testing and they were housed individually for recovery with food and water available ad libitum. Compounds were administered through a 30-gauge injection cannula that extended 1 mm beyond the tip of the guide cannula. The injection cannula was connected to a 1 μl syringe (Hamilton Co., Reno, NV) with PE-20 tubing filled with sterile water. All microinjections were given in a volume of 0.5 μl over 30 s while the rat was gently restrained. The injection cannula remained in place for an additional 30 s in the guide cannula. At the end of experiments, the position of the cannula was verified histologically. 2.5. Histology Rats were anesthetized with a ketamine/xylazine mixture (45/12 mg/kg, i.p.) immediately afer testing. The microinjection site at periaqueductal grey was marked by injecting 0.5 μl of cresyl violet stain into the same site of drugs. The brain was removed and placed in formalin (4%). After at least 3 days, the brain was cut in coronal sections (50 μm thick), mounted on a gelatinized slide, stained with haematoxylin and eosin, and viewed under a microscope to assess cannula placement.


2.6. Drugs Asimadoline and ICI-204448 were obtained from Merck (Darmstadt, Germany). Naloxone was purchased from Sigma (St. Louis, MO, USA). All drugs were dissolved in 0.9% sterile saline. 2.7. RNA isolation Animals were sacrificed with CO2 12 days after nerve ligation. Ipsilateral lumbar (L5-L6) dorsal root ganglion, dorsal spinal cord and periaqueductal grey region were carefully excised from experimental and sham rats. Sections of these tissues were homogenized in 1 ml Tri-zol Reagent (Invitrogen, CA) and centrifuged at 14,000 rpm during 20 min. The aqueous phase was precipitated with isopropanol and the final total RNA pellet was re-suspended in 50 μl of RNAsecure™ re-suspension solution (Ambion, TX). An aliquot of RNA was used to measure its concentration spectrophotometrically at 260/ 280 nm and the rest was stored at −70 °C for 24 h. 2.8. Reverse transcriptase-polymerase chain reaction (RT-PCR) The cDNA was synthesized using a Superscript™ firststrand synthesis system (Invitrogen, CA). After synthesis, cDNA was amplified with specific primers for κ opioid receptor and glyceraldehyde 3-phosphate dehydrogenase (see Table 1; Rosin et al., 2000). Glyceraldehyde 3-phosphate dehydrogenase was used as internal control. Five μg of total RNA extract were incubated with 50 U of SuperScript™ in a buffer containing 200 mM Tris–HCl (pH 8.4), 500 mM KCl, 25 mM MgCl2, 0.1 M DTT, 10 mM dNTP mix (10 mM each dATP, dCTP, dGTP, dTTP) and oligo (dT)12–18 (0.5 μg). The mixture was incubated at 42 °C for 50 min, and the reaction was stopped by heating at 70 °C for 15 min. RNA was removed by adding 2 U of E. coli RNase H (Invitrogen, CA) to facilitate the synthesis of double stranded cDNA. After the reverse transcriptase reaction, 1 μl of reaction mixture was added to the polymerase chain reaction mixture, which had a final volume of 50 μl containing 1 μl of 50X Titanium™ Taq DNA polymerase (Clontech, CA) in a 10X Titanium™ PCR Buffer (Clontech, CA). The mixture was incubated in a thermocycler Mastercycler gradient (Eppendorf)

Table 1 Reverse transcriptase-polymerase chain reaction primers for κ opioid receptor and glyceraldehyde 3-phosphate dehydrogenase (G3PDH, taken from Rosin et al., 2000) Gene κ

Primer

Sequence

Forward 5′-AGT CCC CCA TCC AGA TTT TCC-3′ Reverse 5′-ACG GCA ATG TAA CGG TCC ACA-3′ G3PDH Forward 5′-GGC AAG TTC AAC GGC ACA GTC-3′ Reverse 5′-GAT GCA GGG ATG ATG TCC TGG-3′

Position GenBank Product size (bp) 261-282 U00442

475

716-737 225-246 X02231 674-696

470

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using 40 cycles (in the case of κ opioid receptor) or 30 cycles (in the case of glyceraldehyde 3-phosphate dehydrogenase) using the following profile: an initial denaturation step at 95 °C for 1 min, 60 °C for 1 min (annealing), 72 °C for 3 min (elongation) and 72 °C for 10 min (final extension) (Rosin et al., 2000). Polymerase chain reaction product (10 μl) and 100 bp markers (10 μl; Invitrogen, CA) were mixed with 2 μl of loading buffer (Invitrogen, CA). 2.9. Electrophoresis Samples were separated in 2% agarose gels (ICN, Ohio) at 80 V for 90 min. Gels were stained with ethidium bromide (Sigma, St. Louis MO) for 20 min. Finally, the optical density of bands was measured in a Transilluminator Epichem Darkroom (UVP Bioimaging Systems) and processed with Labworks 4.5 Software for image acquisition and analysis. 2.10. Study design Independent groups of animals (n = 6) were used for each experimental condition. As we intended to use female rats throughout the study, we performed an experiment to determine if there were differences in neuropathic pain manifestation or in the antiallodynic effect of asimadoline between males and females. For this purpose the effect of asimadoline (30 mg/kg, s.c.) in rats of both sexes was determined. Once the absence of sex differences was established, we used only female rats. For the systemic study, rats received a subcutaneous administration of asimadoline or ICI-204448 (1–30 mg/kg) 30 min before assessing withdrawal threshold in neuropathic rats. For the spinal study, rats were intrathecally injected with asimadoline or ICI-204448 (1–30 μg, − 30 min). For the periaqueductal grey study, animals were injected into this region with saline or 3–30 μg asimadoline or ICI-204448 (− 30 min). Animals receiving saline by the different routes of administration used in this study served as controls. Due to inherent variations of effect duration, paw withdrawal threshold was evaluated during 3.5 h for subcutaneous and intrathecal administration and for 2.5 h for periaqueductal grey administration. In an attempt to block the effects of asimadoline and ICI204448, naloxone was administered ten minutes before each κ opioid receptor agonist using several routes of administration. The doses of naloxone were 1 mg/kg s.c., for the systemic study and 30 μg for the spinal and periaqueductal grey experiments. All doses were selected based on previous reports from the available literature (Catheline et al., 1998; Eliav et al., 1999; Field et al., 1999; Lozano-Cuenca et al., 2005) and on pilot experiments in our laboratory. It is worth mentioning that there were no reports for spinal and central doses. Somnolence was observed after spinal and periaqueductal administration of both κ opioid receptor agonists at 100 μg. This adverse effect disappeared when doses were reduced to those reported in this study. The observer was unaware of the treatment each animal received. Rats in all groups were observed with respect to changes in behaviour or motor function induced by the treatments. This

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Reverse transcriptase-polymerase chain reaction data were expressed as ratio of optical density values (mean ± S.E.M. for 4 to 5 rats) of κ opioid receptor/glyceraldehyde 3-phosphate dehydrogenase. One- or two-way analysis of variance (ANOVA), followed by the Tukey's test, was used to compare differences between treatments in behavioral experiments. Unpaired Student's t-test was used to test differences in the expression of mRNA between neuropathic and sham-operated rats. Differences were considered to reach statistical significance when P b 0.05.

Fig. 1. A) Time course of paw withdrawal threshold in male and female Wistar rats submitted to ligation of L5/L6 spinal nerves compared to sham-operated rats. Withdrawal threshold was assessed at day 1, 3, 5, 7, 9 and 12 after surgery. B) Time course of tactile allodynia after administration of saline or asimadoline (30 mg/kg, s.c.) to male and female rats at day 12 after ligation of L5/L6 spinal nerves as compared to sham-operated rats. Data are the mean ± S.E.M. for 6 animals.

was assessed, but not quantified, by testing the animals' ability to stand and walk in a normal posture, as proposed elsewhere (Chen and Pan, 2003). 2.11. Data analysis and statistics Behavioral results are given as the mean ± S.E.M. for six to seven animals per group. Curves were constructed plotting the threshold for paw withdrawal as a function of time. An increase of 50% withdrawal threshold was considered an antiallodynic effect. Area under the 50% withdrawal threshold against time curve (AUC) for a period of 210 (systemic and intrathecal administration) or 150 min (periaqueductal grey administration) was calculated by the trapezoidal method. Percentage of maximum possible effect (%MPE) was calculated with the following equation: kMPE ¼ AUCcompound AUCvehicle =ðAUCsham AUCvehicle Þ 100:

Fig. 2. Time course of the antiallodynic effect of asimadoline (A) or ICI204448 (B) observed after systemic (s.c.), intrathecal (i.t.) and periaqueductal grey (PAG) administration in rats submitted to ligation of L5/L6 spinal nerves. In all cases, data are presented as mean ± S.E.M. for 6 animals. A) A significant difference (P b 0.05, by two-way ANOVA followed by the Tukey's test) was observed between vehicle and either subcutaneous or intrathecal asimadoline groups 30 min after starting thresholds evaluations and up to 210 min. In addition, a significant difference was observed between vehicle and PAG asimadoline group 15 min after injection and up to 150 min. B) A significant difference (P b 0.05, by two-way ANOVA followed by the Tukey's test) was observed between saline and intrathecal or subcutaneous ICI204448 groups 30 and 60 min respectively, after starting thresholds evaluations and up to 210 min. In addition, a significant difference was observed between vehicle and ICI-204448 group 15 min after injection and up to 120 min. In all cases, asterisks indicating significant differences were omitted for the sake of clarity.


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3. Results 3.1. Effect of spinal nerve ligation in male and female rats Ligation of L5/L6 spinal nerves produced tactile allodynia in male and female rats evidenced by a decrease in paw withdrawal threshold as compared to sham-operated rats. Tactile allodynia was observed from day 1 to day 12 (Fig. 1A). Once the neuropathic condition was established, asimadoline or saline was administered to male and female rats in order to determine the existence of sex differences. Asimadoline (30 mg/kg, s.c.) produced the same pattern of antiallodynic activity in both sexes (Fig. 1B). 3.2. Time course of the antiallodynic activity of asimadoline and ICI-204448 Fig. 2 shows the effects of asimadoline or ICI-204448 by different routes of administration in female rats. When given

Fig. 4. Antiallodynic effect of asimadoline (A) and ICI-204448 (B) in rats submitted to ligation of L5/L6 spinal nerves after intrathecal (i.t.) administration and effect of intrathecal naloxone (NLX) on asimadoline- and ICI-204448induced spinal antiallodynic activity. Rats were treated with vehicle or naloxone (− 40 min) and increasing doses of asimadoline or ICI-204448 (− 30 min) before starting threshold evaluations. Data are expressed as the % of maximum possible effect (%MPE). Bars are the mean ± S.E.M. for 6 animals. ⁎ significantly different from the saline (Sal) group, and # significantly different from asimadoline or ICI-204448 (30 μg) groups as determined by one-way ANOVA followed by the Tukey's test.

subcutaneously or intrathecally, these compounds produced a maximal antiallodynic effect in about 2 h, after which the effect gradually returned to control values in approximately 3.5 h. Interestingly, the maximal antiallodynic effect observed after microinjection of asimadoline or ICI-204448 into the periaqueductal grey was reached very rapidly, in close to 15 min, and then decayed gradually until it disappeared in about 2 h (Fig. 2). 3.3. Antiallodynic activity of subcutaneous asimadoline and ICI-204448

Fig. 3. Antiallodynic effect of asimadoline (A) and ICI-204448 (B) in rats submitted to ligation of L5/L6 spinal nerves after subcutaneous (s.c.) administration and effect of intrathecal (i.t.) or s.c. naloxone (NLX) on asimadoline- or ICI-204448-induced antiallodynic activity. Rats were pre-treated with vehicle or naloxone (−40 min) and increasing doses of asimadoline or ICI204448 (−30 min) before starting threshold evaluations. Data are expressed as the % of maximum possible effect (%MPE). Bars are the mean ± S.E.M. for 6 animals. ⁎ and & are significantly different from the saline (Sal) group, and # significantly different from asimadoline or ICI-204448 (30 mg/kg) groups as determined by oneway ANOVA followed by the Tukey's test.

Subcutaneous injection of asimadoline or ICI-204448 (1– 30 mg/kg), but not naloxone (1 mg/kg), significantly reduced (P b 0.05) tactile allodynia induced by ligation of L5/L6 spinal nerves in a dose-dependent manner (Fig. 3A and B). Moreover, the antiallodynic effect of asimadoline or ICI-204448 (30 mg/ kg, s.c.) was partially prevented by naloxone subcutaneous administration (1 mg/kg; Fig. 3A and B). Contrariwise, the spinal administration of naloxone (30 μg) did not reduce the antiallodynic effect of asimadoline or ICI-204448 (30 mg/kg, s.c.). No differences in maximal effects between asimadoline

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(∼ 50%) and ICI-204448 (∼ 45%) after subcutaneous administration were observed (Fig. 3A and B). Moreover, no side effects were detected at the greatest doses reported. 3.4. Antiallodynic activity of asimadoline and ICI-204448 in the spinal cord Intrathecal administration of asimadoline or ICI-204448 (1– 30 μg), but not naloxone (30 μg) or saline, dose-dependently reduced tactile allodynia (P b 0.05) induced by ligation of L5/L6 spinal nerves (Fig. 4A and B). The antiallodynic activity of both κ opioid receptor agonists (30 μg) was prevented by intrathecal naloxone (30 μg) (Fig. 4A and B). By this administration route, asimadoline reached a greater maximal effect (∼75%) than ICI204448 (∼45%) at the greatest dose tested (Fig. 4A and B). 3.5. Antiallodynic activity of asimadoline and ICI-204448 in periaqueductal grey Administration of asimadoline or ICI-204448 (3–30 μg), but not naloxone or saline, into periaqueductal grey significantly

Fig. 6. Expression of the κ opioid receptor mRNA in dorsal root ganglion (L5 and L6, n = 4), spinal cord (dorsal horn, n = 4) and periaqueductal grey (PAG, n = 5) of rats submitted to L5/L6 spinal nerve ligation. Tissues were harvested 12 days after surgery. G3PDH, glyceraldehyde 3-phosphate dehydrogenase. Data are the mean ± S.E.M. ⁎ significantly different from sham-operated rats, as determined by the Student's t-test.

reduced (P b 0.05) tactile allodynia in a dose-dependent manner (Fig. 5A and B). The maximal antiallodynic effect was reached with 30 μg of the κ opioid receptor agonists, but no differences in maximal effects between asimadoline (∼ 55%) and ICI204448 (∼ 45%) were observed after administration into periaqueductal grey (Fig. 5A and B). Pretreatment with naloxone (30 μg) significantly (P b 0.05) reduced the antiallodynic effect produced by asimadoline or ICI-204448 injected into periaqueductal grey (Fig. 5A and B). 3.6. mRNA determination of the κ opioid receptor in dorsal root ganglion, spinal cord and periaqueductal grey Based on the results obtained with κ opioid receptor agonists, we considered of interest to determine if the expression of κ opioid receptor mRNA was altered in rats with allodynia. Ligation of L5/L6 spinal nerves produced a significant increase of the κ opioid receptor mRNA expression in dorsal root ganglion and dorsal horn spinal cord of neuropathic rats as compared to sham-operated rats (P b 0.05, Fig. 6). This increase was observed 12 days after ligation, a time when tactile allodynia is usually established in rats (see below). In addition, a non-significant increase in κ opioid receptor mRNA expression in periaqueductal grey was also seen (Fig. 6). 4. Discussion 4.1. Antiallodynic effect of asimadoline in female and male rats

Fig. 5. Antiallodynic effect of asimadoline (A) and ICI-204448 (B) in rats submitted to ligation of L5/L6 spinal nerves after microinjection into the periaqueductal grey (PAG) and effect of microinjection of naloxone (NLX) into the same site on asimadoline- or ICI-204448-induced antiallodynic activity. Rats were treated with vehicle or naloxone (− 40 min) and increasing doses of asimadoline or ICI-204448 (− 30 min) before starting threshold evaluations. Data are expressed as the % of maximum possible effect (%MPE). Bars are the mean ± S.E.M. for 6 animals. ⁎ significantly different from the saline (Sal) group, and # significantly different from asimadoline or ICI-204448 (30 μg) groups as determined by one-way ANOVA followed by the Tukey's test.

It has been suggested that sex plays an important role in the antinociceptive effects of opioids (Barrett, 2006). In our conditions, spinal nerve ligation produced the same pattern of tactile allodynia development in both male and female rats. Moreover, subcutaneous administration of asimadoline produced similar antiallodynic effects in male and female neuropathic rats. To our knowledge, this is the first report about the efficacy of κ opioid receptor agonists in both sexes. These results disagree with data showing that systemic administration


of κ opioid receptor agonists was generally more potent and effective as antinociceptive agents in male than in female rats, when pain was induced by thermal or mechanical stimuli (Craft and Bernal, 2001; Barrett et al., 2002). Contrariwise, intraperitoneal administration of U50,488H induces a greater antinociception in female compared to male rats (Holtman and Wala, 2006). Several variables may influence the magnitude of sex differences in opioid antinociception in rodents such as rodent strain, opioid efficacy/selectivity and intensity of the noxious stimulus used in the pain test (Craft and Bernal, 2001; Stoffel et al., 2005). However, with the current conditions we were not able to observe any difference between male and female Wistar rats. 4.2. Antiallodynic activity of asimadoline and ICI-204448 In this study, we have observed that subcutaneous administration of asimadoline or ICI-204448 significantly reduces tactile allodynia in female rats submitted to spinal nerve ligation (Kim and Chung model). The antiallodynic effect of these drugs was partially blocked by subcutaneous, but not intrathecal, administration of naloxone. Since these drugs are selective peripheral κ opioid receptor agonists (Barber et al., 1994; Gottschlich et al., 1994), our data suggest that activation of κ opioid receptors located at the periphery reduces tactile allodynia induced by spinal nerve ligation. These results confirm earlier evidence about the efficacy of local peripheral administration of asimadoline (Walker et al., 1999) and ICI204448 (Keïta et al., 1995) to rats submitted to chronic constriction of the common sciatic nerve model. In addition to their peripheral effect, we have shown that administration of asimadoline or ICI-204448 into the spinal cord significantly reduces L5/L6 spinal nerve ligation-induced tactile allodynia. This spinal effect can be blocked by the microinjection of naloxone into the spinal cord. To our knowledge, this is the first report about the antiallodynic efficacy of these two selective κ opioid receptor agonists in the spinal cord and on a well-established model of neuropathic pain in the rat. Our results however, agree with previous observations showing that central κ opioid receptor agonists such as U69,593 (intravenous administration; Catheline et al., 1998), GR89,696 (intrathecal injection; Eliav et al., 1999) and U50,488H (intraplantar and intrathecal administration; Sounvoravong et al., 2004; Bileviciute-Ljungar and Spetea, 2004) reduce tactile allodynia or autotomy in rats with sciatic nerve ligation. Contrariwise, intrathecal injection of U-50,488H did not alleviate spinal cord ischemia-induced allodynia in rats (Hao et al., 1998). Differences could be due to the various models used to produce allodynia. 4.3. Effect of the κ opioid receptor agonists in periaqueductal grey Microinjection of asimadoline or ICI-204448 into periaqueductal grey produced a significant naloxone-sensitive antiallodynic effect in neuropathic rats. To our knowledge, there are no previous studies about the efficacy of κ opioid receptor agonists

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in periaqueductal grey of neuropathic rats. However, it has been previously reported that activation of κ opioid receptors in periaqueductal grey presynaptically inhibits GABAergic synaptic transmission in vitro and that this effect is produced through an increase of inwardly rectifying K+ conductance in a subpopulation of mouse periaqueductal grey neurons (Vaughan et al., 2003). Moreover, it has been suggested that these actions may lead to analgesia via presynaptic desinhibitory mechanisms (Porreca et al., 2002; Vaughan et al., 2003; Fields, 2004). κ opioid receptor agonists also have postsynaptic actions which might produce antinociception (Vaughan et al., 2003). Thus, it is tempting to suggest that asimadoline and ICI-204448, microinjected into periaqueductal grey, could reduce tactile allodynia in rats by activating a descending pathway which projects via the rostral ventromedial medulla to modulate nociceptive transmission at the spinal level (Fields et al., 1991) as μ opioid receptor agonists do (Moreau and Fields, 1986; Fields, 2004). This suggestion agrees with the fact that all components (offcells, GABAergic cells, κ opioid receptors) of this pathway are present in periaqueductal grey (Heinricher et al., 1987; Mansour et al., 1996). On the other hand, time to reach the maximal antiallodynic effect with both κ opioid receptor agonists was about 2 h for subcutaneous and intrathecal administration. Surprisingly, microinjection into periaqueductal grey produced a fast peak effect (approximately 15 min) which then decayed gradually in about 2 h. Our data agree with a previous report showing that the maximal antiallodynic effect of subcutaneous asimadoline in diabetic rats is reached in close to 2 h (Jolivalt et al., 2006). However, the rapid onset of κ opioid receptor agonists in periaqueductal grey has not been published before and their underlying mechanisms in neuropathic pain remain to be determined. 4.4. Determination of mRNA and its relationship with the effects of κ opioid receptor agonists Antiallodynic activity of asimadoline and ICI-204448 in the periphery, spinal cord and periaqueductal grey agrees with the location of κ opioid receptors in the nervous system including peripheral afferents (Jolivalt et al., 2006), dorsal root ganglion (Minami et al., 1995; Sung et al., 2000), spinal cord (Yaksh, 1997; Minami, 2004; Xu et al., 2004) and periaqueductal grey (Mansour et al., 1996; Gutstein et al., 1998). Accordingly, in our conditions spinal nerve ligation produced tactile allodynia along with an increase in κ opioid receptor mRNA expression in dorsal root ganglion and dorsal horn spinal cord. A nonsignificant increase in κ opioid receptor mRNA expression in periaqueductal grey was also observed. This last result could be explained by the fact that only a small sample of tissue can be obtained from periaqueductal grey. Our data agree with previous observations about the up-regulation of κ opioid receptor mRNA and protein in peripheral afferents (Jolivalt et al., 2006), dorsal root ganglion and spinal cord of mice with mechanical allodynia following nerve injury (Sung et al., 2000; Xu et al., 2004). However, to our knowledge this is the first report showing up-regulation of κ opioid receptor mRNA in

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periaqueductal grey. Although it is likely that a post-translational degradation may occur to κ opioid receptor mRNA, the increase observed may explain the efficacy of asimadoline and ICI-204448 after systemic and intrathecal administration to neuropathic rats. 4.5. Final considerations The antiallodynic effect of asimadoline and ICI-204448 can be explained by activation of κ opioid receptors which block voltage-dependent Ca2+ channels and open K+channels decreasing neuronal excitability and consequently tactile allodynia in the rats. Besides, asimadoline and other κ opioid receptor agonists are able to reduce tetrodotoxin-sensitive and tetrodotoxin-resistant sodium currents on primary sensory neurons (Joshi and Gebhart, 2003), which would further lead to a reduction of neuropathic pain (Pace et al., 2006). In summary, this study has shown that subcutaneous administration of peripheral κ opioid receptor agonists asimadoline and ICI-204448 reduces tactile allodynia induced by spinal nerve ligation. Moreover, microinjection of these drugs into spinal cord and periaqueductal grey also produces a significant antiallodynic effect. The antiallodynic effect of these drugs could be explained by an increase in mRNA expression for the κ opioid receptor in dorsal root ganglion, spinal cord and marginally in periaqueductal grey. The present results suggest that κ opioid receptor agonists may have potential for the treatment of neuropathic pain in humans. Acknowledgements Authors greatly appreciate the technical assistance of Guadalupe C. Vidal-Cantú and Isaí Méndez-Ocaña and bibliographic assistance of Héctor Vázquez. We kindly appreciate the technical support of Dr. Claudia González-Espinosa in the RT-PCR experiments. Nadia L. Caram-Salas, Héctor I. Rocha-González, Rosaura Arreola-Espino, Claudia I. AraizaSaldaña and Mónica Ambriz-Tututi are CONACYT fellows. In addition, Nadia L. Caram-Salas and Claudia I. Araiza-Saldaña are the recipients of the “Apoyos Integrales para la Formación de Doctores en Ciencias” fellowship. This work is part of the Ph.D. dissertation of Nadia L. Caram-Salas. Partially supported by Conacyt grant M-43604 (SLC). References Barber, A., Bartoszyk, G.D., Bender, H.M., Gottschlich, R., Greiner, H.E., Harting, J., Mauler, F., Minck, K.O., Murray, R.D., Simon, M., Seyfried, C.A., 1994. A pharmacological profile of the novel, peripherally-selective kappa-opioid receptor agonist, EMD 61753. Br. J. Pharmacol. 113, 1317–1327. Barber, A., Gottschlich, R., 1997. Novel developments with selective, nonpeptidic kappa-opioid receptor agonists. Expert Opin. Investig. Drugs 6, 1351–1368. Barrett, A.C., 2006. Low efficacy opioids: implications for sex differences in opioid antinociception. Exp. Clin. Psychopharmacol. 14, 1–11. Barrett, A.C., Smith, E.S., Picker, M.J., 2002. Sex-related differences in mechanical nociception and antinociception produced by mu-and kappaopioid receptor agonists in rats. Eur. J. Pharmacol. 452, 163–173.

Bileviciute-Ljungar, I., Spetea, M., 2004. Contralateral, ipsilateral and bilateral treatments with the kappa-opioid receptor agonist U-50,488H in mononeuropathic rats. Eur. J. Pharmacol. 494, 139–146. Binder, W., Walker, J.S., 1998. Effect of the peripherally selective kappa-opioid agonist, asimadoline, on adjuvant arthritis. Br. J. Pharmacol. 124, 647–654. Binder, W., Carmody, J., Walter, J., 2000. Effect of gender on anti-inflammatory and analgesic actions of two kappa-opioids. J. Pharmacol. Exp. Ther. 292, 303–309. Catheline, G., Guilbaud, G., Kayser, V., 1998. Peripheral component in the enhanced antinociceptive effect of systemic U-69,593, a kappa-opioid receptor agonist in mononeuropathic rats. Eur. J. Pharmacol. 357, 171–178. Chaplan, S.R., Bach, F.W., Pogrel, J.W., Chung, J.M., Yaksh, T.L., 1994. Quantitative assessment of tactile allodynia in the rat paw. J. Neurosci. Methods 53, 55–63. Chen, S.R., Pan, H.L., 2003. Antinociceptive effect of morphine, but not mu opioid receptor number, is attenuated in the spinal cord of diabetic rats. Anesthesiology 99, 1409–1414. Craft, R.M., Bernal, S.A., 2001. Sex differences in opioid antinociception: kappa and ‘mixed action’ agonists. Drug Alcohol Depend. 63, 215–228. Delvaux, M., Beck, A., Jacob, J., Bouzamondo, H., Weber, F.T., Frexinos, J., 2004. Effect of asimadoline, a kappa opioid agonist, on pain induced by colonic distension in patients with irritable bowel syndrome. Aliment. Pharmacol. Ther. 20, 237–246. Eliav, E., Herzberg, U., Caudle, R.M., 1999. The kappa opioid agonist GR89,696 blocks hyperalgesia and allodynia in rat models of peripheral neuritis and neuropathy. Pain 79, 255–264. Fields, H.L., 2004. State-dependent opioid control of pain. Nat. Rev. 5, 565–575. Fields, H.L., Heinricher, M.M., Manson, P., 1991. Neurotransmitters in nociceptive modulatory circuits. Ann. Rev. Neurosci. 14, 219–245. Field, M.J., Carnell, A.J., Gonzalez, M.I., McCleary, S., Oles, R.J., Smith, R., Hughes, J., Singh, L., 1999. Enadoline, a selective kappa-opioid receptor agonist shows potent antihyperalgesic and antiallodynic actions in a rat model of surgical pain. Pain 80, 383–389. Gottschlich, R., Krug, M., Barber, A., Devant, R.M., 1994. Kappa-opioid activity of the four stereoisomers of the peripherally selective kappaagonists, EMD 60,400 and EMD 61,753. Chirality 6, 685–689. Gutstein, H.B., Mansour, A., Watson, S.J., Akil, H., Fields, H.L., 1998. Mu and kappa opioid receptors in periaqueductal grey and rostral ventromedial medulla. Neuroreport 9, 1777–1781. Hao, J.X., Yu, W., Wiesenfeld-Hallin, Z., Xu, X.J., 1998. Treatment of chronic allodynia in spinally injured rats: effects of intrathecal selective opioid receptor agonists. Pain 75, 209–217. Heinricher, M.M., Cheng, Z.F., Fields, H.L., 1987. Evidence for two classes of nociceptive modulating neurons in the periaqueductal grey. J. Neurosci. 7, 271–278. Holtman, J.R., Wala, E.P., 2006. Characterization of the antinociceptive effect of oxycodone in male and female rats. Pharmacol. Biochem. Behav. 83, 100–108. Horwell, D.C., 1988. Kappa opioid analgesics. Drugs Future 13, 1061–1070. Husbands, S.M., 2004. Kappa-opioid receptor ligands. Expert Opinion Ther. Pat. 14, 1725–1741. Jolivalt, C.G., Jiang, Y., Freshwater, J.D., Bartoszyk, G.D., Calcutt, N.A., 2006. Dynorphin A, kappa opioid receptors and the antinociceptive efficacy of asimadoline in streptozotocin-induced diabetic rats. Diabetologia 49, 2775–2785. Joshi, S.K., Gebhart, G.F., 2003. Nonopioid actions of U50,488 enantiomers contribute to their peripheral cutaneous antinociceptive effects. J. Pharmacol. Exp. Ther. 305, 919–924. Keïta, H., Kayser, V., Guilbaud, G., 1995. Antinociceptive effect of a kappaopioid receptor agonist that minimally crosses the blood–brain barrier (ICI 204448) in a rat model of mononeuropathy. Eur. J. Pharmacol. 277, 275–280. Kim, H.K., Chung, K., 1992. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain 50, 355–363. Lozano-Cuenca, J., Castañeda-Hernández, G., Granados-Soto, V., 2005. Peripheral and spinal mechanisms of antinociceptive action of lumiracoxib. Eur. J. Pharmacol. 513, 81–91. Machelska, H., Pfluger, M., Weber, W., Piranvisseh-Volk, M., Daubert, J.D., Dehaven, R., Stein, C., 1999. Peripheral effects of the kappa-opioid agonist

N.L. Caram-Salas et al. / European Journal of Pharmacology 573 (2007) 75–83 EMD 61753 on pain and inflammation in rats and humans. J. Pharmacol. Exp. Ther. 290, 354–361. Mansour, A., Burke, S., Pavlic, R.J., Akil, H., Watson, S.J., 1996. Immunohistochemical localization of the cloned kappa 1 receptor in the rat CNS and pituitary. Neuroscience 71, 671–690. Minami, M., 2004. Molecular pharmacology of opioid receptors. Nippon Yakurigaku Zasshi. 123, 95–104. Minami, M., Maekawa, K., Yabuuchi, K., Satoh, M., 1995. Double in situ hybridization study on coexistence of mu-, delta-and kappa-opioid receptor mRNAs with preprotachykinin A mRNA in the rat dorsal root ganglia. Brain Res. Mol. Brain Res. 30, 203–210. Moreau, J.L., Fields, H.L., 1986. Evidence for GABA involvement in midbrain control of medullary neurons that modulate nociceptive transmission. Brain Res. 397, 37–46. Pace, M.C., Mazzariello, L., Passavanti, M.B., Sansone, P., Barbarisi, M., Aurilio, C., 2006. Neurobiology of pain. J. Cell Physiol. 209, 8–12. Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordinates, 2nd ed. Academic Press, San Diego, CA. Pertovaara, A., Almeida, A., 2006. In: Cervero, F., Jensen, T.S. (Eds.), Descending Inhibitory Systems. . Handbook of Clinical Neurology, vol. 81. Elsevier, Amsterdam, pp. 179–192. Pfeiffer, A., Knepel, W., Braun, S., Meyer, H.D., Lohmann, H., Brantl, V., 1986. Effects of a kappa-opioid agonist on adrenocorticotropic and diuretic function in man. Horm. Metab. Res. 18, 842–848. Porreca, F., Ossipov, M.H., Gebhart, G.F., 2002. Chronic pain and medullary descending facilitation. Trends Neurosci. 25, 319–325. Rosin, A., van der Ploeg, I., Georgieva, J., 2000. Basal and cocaine-induced opioid receptor gene expression in the rat CNS analyzed by competitive reverse transcription PCR. Brain Res. 28, 102–109.

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Sounvoravong, S., Takahashi, M., Nakashima, M.N., Nakashima, K., 2004. Disability of development of tolerance to morphine and U-50,488H, a selective kappa-opioid receptor agonist, in neuropathic pain model mice. J. Pharmacol. Sci. 94, 305–312. Stoffel, E.C., Ulibarri, C.M., Folk, J.E., Rice, K.C., Craft, R.M., 2005. Gonadal hormone modulation of mu, kappa, and delta opioid antinociception in male and female rats. J. Pain 6, 261–274. Sung, B., Loh, H.H., Wei, L., 2000. Association of kappa opioid receptor mRNA upregulation in dorsal root ganglia with mechanical allodynia in mice following nerve injury. Neurosci. Lett. 291, 163–166. Vaughan, C.W., Bagley, E.E., Drew, G.M., Schuller, A., Pintar, J.E., Hack, S.P., Christie, M.J., 2003. Cellular actions of opioids on periaqueductal grey neurons from C57B16/J mice and mutant mice lacking MOR-1.Br. J. Pharmacol. 139, 362–367. Walker, J., Catheline, G., Guilbaud, G., Kayser, V., 1999. Lack of crosstolerance between the antinociceptive effects of systemic morphine and asimadoline, a peripherally-selective kappa-opioid agonist, in CCI-neuropathic rats. Pain 83, 509–516. Xu, M., Petraschka, M., McLaughlin, J.P., Westenbroek, R.E., Caron, M.G., Lefkowitz, R.J., Czyzyk, T.A., Pintar, J.E., Terman, G.W., Chavkin, C., 2004. Neuropathic pain activates the endogenous kappa opioid system in mouse spinal cord and induces opioid receptor tolerance. J. Neurosci. 24, 4576–4584. Yaksh, T.L., 1997. Pharmacology and mechanisms of opioid analgesic activity. Acta Anaesthesiol. Scand. 41, 94–111. Yaksh, T.L., Rudy, T.A., 1976. Chronic catheterization of the spinal subarachnoid space. Physiol. Behav. 17, 1031–1036. Zimmerman, M., 1983. Ethical guides for investigations on experimental pain in conscious animals. Pain 16, 109–110.