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Ketoprofen and antinociception in hypo-oestrogenic Wistar rats fed on a high sucrose diet
European Journal of Pharmacology 788 (2016) 168–175
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Ketoprofen and antinociception in hypo-oestrogenic Wistar rats fed on a high sucrose diet Osmar Antonio Jaramillo-Morales, Josué Vidal Espinosa-Juárez, Betzabeth Anali García-Martínez, Francisco Javier López-Muñoz n Departamento de Farmacobiología, Cinvestav-Sede Sur, Delegación Tlálpan, México D.F. C.P. 14330, Mexico
art ic l e i nf o
a b s t r a c t
Article history: Received 4 May 2016 Received in revised form 8 June 2016 Accepted 17 June 2016 Available online 23 June 2016
Non-steroidal anti-inflammatory drugs such as ketoprofen are the most commonly used analgesics for the treatment of pain. However, no studies have evaluated the analgesic response to ketoprofen in conditions of obesity. The aim of this study was to analyse the time course of nociceptive pain in Wistar rats with and without hypo-oestrogenism on a high sucrose diet and to compare the antinociceptive response using ketoprofen. Hypo-oestrogenic and naïve rats received a hyper caloric diet (30% sucrose) or water ad libitum for 17 weeks, the thermal nociception (“plantar test” method) and body weight were tested during this period. A biphasic response was observed: thermal latency decreased in the 4th week (hyperalgesia), while from 12th to 17th week, thermal latency increased (hypoalgesia) in hypo-oestrogenic rats fed with high sucrose diet compared with the hypo-oestrogenic control group. At 4th and 17th weeks, different doses of ketoprofen (1.8–100 mg/kg p.o.), were evaluated in all groups. The administration of ketoprofen at 4th and 17th weeks showed dose-dependent effects in the all groups; however, a greater pharmacological efficacy was observed in the 4th week in the hypo-oestrogenic animals that received sucrose. Nevertheless, in all the groups significantly diminish the antinociceptive effects in the 17th week. Our data showed that nociception was altered in the hypo-oestrogenic animals that were fed sucrose (hyperalgesia and hypoalgesia). Ketoprofen showed a dose-dependent antinociceptive effect at both time points. However, hypo-oestrogenism plus high-sucrose diet modifies the antinociceptive effect of ketoprofen. & 2016 Elsevier B.V. All rights reserved.
Keywords: High-sucrose diet Nociception Ketoprofen Efficacy
1. Introduction Pain is a condition that affects thousands of people in the world. However, it has been shown that individuals with increased body weight are more likely to have problems with pain (Marcus, 2004; Wilson et al., 2010). Controversies exist regarding the perception of pain in obese subjects. Some studies report a hyperalgesic state associated with obesity (Roane and Martin, 1990; Sugimoto et al., 2008; Buchenauer et al., 2009), while others have proposed the existence of hypoalgesic processes (Ramzan et al., 1993; Zhang et al., 1994; Sugimoto et al., 2008). Moreover, the most frequent cases that have been documented clinically in obese people involve back pain (Hitt et al., 2007; D’Arcy, 2011) and n Correspondence to: Lab. No. 7 “Dolor y Analgesia” del Departamento de Farmacobiología, Cinvestav-Sede Sur, Calz. de los Tenorios No. 235 Col. Granjas Coapa, Delegación Tlálpan, México D.F. C.P. 14330, Mexico. E-mail addresses: [email protected] (O.A. Jaramillo-Morales), [email protected] (J.V. Espinosa-Juárez), [email protected] (B.A. García-Martínez), fl[email protected], fl[email protected] (F.J. López-Muñoz).
http://dx.doi.org/10.1016/j.ejphar.2016.06.030 0014-2999/& 2016 Elsevier B.V. All rights reserved.
arthritis pain (Marcus, 2004), triggered mainly due to a mechanical effect on the joints by weight gain. Obesity has also been associated with other chronic pain conditions, such as migraine and headache (Goadsby et al., 2002; Rossi et al., 2013). For this reason, it is common for these patients to be treated with analgesic drugs. The most widespread drug therapy currently used to relieve nociceptive and inflammatory pain involves the non-steroidal antiinflammatory analgesics (NSAIDs) and opioids (Ong et al., 2007). NSAIDs are the most widely prescribed drugs in clinical medicine; they are heterogeneous substances with varying nonsteroidal chemical structures (Laine, 2001). This group of drugs, which are indicated in the treatment of acute and chronic pain (Whiteside et al., 2004; Ong et al., 2007), is characterized by analgesic antiinflammatory and antipyretic properties (Dworkin y Gitlin, 1991). Ketoprofen is a member of this group. As other NSAIDs, ketoprofen exerts its analgesic effect through at least three mechanisms of action, clearly identified as: 1) inhibition of prostaglandin synthesis (Avouac and Teule, 1988; Kubota et al., 1997), 2) interaction with the serotonergic system (Díaz-Reval et al., 2001, 2004) and 3) inhibition of proinflammatory cytokines (Choi et al., 2013). Preclinical studies have indicated that ketoprofen exhibits
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dose-dependent effects in different models of acute and inflammatory pain (Díaz-Reval et al., 2001, 2004; Aguilar-Carrasco et al., 2014). However, no studies have evaluated the analgesic response of ketoprofen in conditions where there is an increase in body weight; therefore, the antinociceptive effect it may have under these conditions is unknown. The effects may differ because pathological conditions can change the activity of drugs, mainly through pharmacokinetic and/or pharmacodynamic alterations (Lloret et al., 2009). Therefore, the objectives of this study were to evaluate nociception in hypo-oestrogenic and naïve female Wistar rats under conditions of increasing weight up to a state of obesity achieved through the provision of a high sucrose diet (30% in drinking water) and to analyse the antinociceptive response to ketoprofen.
2. Materials and methods 2.1. Animals and housing conditions Female Wistar rats [Crl(WI)fBR] weighing 180–200 g at the time of surgery were used in this study. All animals were obtained from the animal breeding facility of the Centre of Investigation and Advanced Studies (Cinvestav, Sede Sur). The animals were housed under standardized conditions in a room on a 12 h light/dark cycle with food and water available ad libitum before treatment. All experimental procedures were approved by the Local Ethics Committee for the Management of Laboratory Animals of the Department of Pharmacobiology of Cinvestav, Sede Sur, following the Guidelines on Ethical Standards for Investigations of Experimental Pain in Animals (Zimmermann, 1983) and the Official Mexican Norm (NOM-062-ZOO-1999). All tests were performed during the light phase. The number of experimental animals was kept to a minimum, and the rats were killed immediately after the experiment by CO2 overdose. 2.2. Compounds Refined sucrose was obtained commercially from Supplies BenHill, S. A. de C. V. (Mexico City, Mexico). The sugar was dissolved in water to 30% (wt/vol). Ketoprofen was obtained from Sigma Chemical Company (St. Louis, MO, USA). Ketoprofen was suspended in 0.5% carboxymethylcellulose and was administered orally in an application volume of 4 ml/kg body weight. The doses mentioned in the text refer to the salts of the substances used. 2.3. Surgical technique (Ovariectomy) Briefly, the ovariectomy consisted of the following procedures. All animals were anaesthetized by an intraperitoneal (i.p.) injection of 50 mg/kg ketamine and 10 mg/kg xylazine mixture for bilateral removal of the ovaries. Briefly, the abdominal and pelvic areas of the animals were shaved and cleaned. A longitudinal incision of approximately 1.5 cm was made, the skin was separated from the muscle, and a second incision of approximately 0.5 cm was made in the muscle to exteriorize the ovaries. The fallopian tubes were ligated and cut below the ligature. After the excision, the incision was sutured. This surgery caused a state of hypooestrogenism in experimental animals. The ovariectomized female rat model has been commonly used as a direct cause of increased body weight (Stubbins et al., 2012; Da Silva et al., 2014) and as a model for menopause in humans (Diaz Brinton, 2012). All procedures were carried out under aseptic conditions.
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2.4. Measurement of antinociceptive activity To avoid additional stress, the rats were allowed to acclimate to the testing site until exploratory behaviour diminished for at least 10 min before stimulation was initiated. Responses to thermal nociception were evaluated using the Hargreaves method (UGOBASILE, Varese, Italy) (Hargreaves et al., 1988). Briefly, a radiant heat source with a locator light was positioned under the plantar surface and the latency to withdrawal the right hind paw was recorded for each animal. A resting period of 5 min followed to avoid any sensitization. The intensity of the lamp was set at 60 Hz, and a cut-off time of 30 s (s) was determined necessary to avoid tissue damage. The light beam was directed on the plantar surface of the hind paw until the animal responded, or when it came to cutting time (30 s), the lamp was turned off, whichever occurred first. The latency of the paw withdrawal from the incident light was recorded with a built-in timer, which displayed reaction time in 0.01 s increments. The latency of withdrawal was defined as the time between the initiation of the light and the moment at which the animal withdrew from the tip of the light quickly, either generating a limb-licking behaviour or jumping away from the heat source. Three readings were made per animal. Data are expressed as the withdrawal latency and are measured in seconds. 2.5. Experimental design On the 15th postoperative day (ovariectomy), the time course of the thermal latency was initiated. Female Wistar rats weighing 250–270 g each were randomized into four groups (n ¼6) as follows: hypo-oestrogenic-control (Ov-Ctrl), hypo-oestrogenic-sucrose (Ov-Suc) (both were ovariectomized), naïve-control (NaïveCtrl) and naïve-sucrose (Naïve-Suc) (both were non-operated). Before the start of the diet, baseline thermal threshold responses of all groups were measured at time 0 (immediately after receiving the respective diet). During the 17-weeks treatment, all groups were given free access to food (pellets of LabDiet 5008). Additionally, sucrose fed rats received 30% commercially refined sucrose (wt/vol) in drinking water given ad libitum. In contrast, the control group received filtered drinking water ad libitum. During this period, thermal nociception was assessed weekly using Hargreaves method (UGO-BASILE, Varese, Italy) (Hargreaves et al., 1988), and body weight measurements were taken. The effects of the acute administration of a drug (ketoprofen) on response thresholds to thermal stimuli were tested at week 4 and week 17 post-treatment with sucrose or water ad libitum in ovariectomized rats. Baseline thermal nociception was assessed before pharmacological testing. The experimental protocol consisted of two sets of experimental groups in which the antinociceptive effects produced by ketoprofen, given individually, were studied with the corresponding vehicle (0.5% carboxymethylcellulose). In the first set of experimental groups, each dose of ketoprofen (1.8, 5.6, 10, 17.8 or 31.6 mg/kg, p.o.) was given in a volume of 4 ml/kg to six hypo-oestrogenic and Naïve rats (that had received the respective diet for 4 weeks) to obtain the doseresponse curves (DRC). In the second set, hypo-oestrogenic and Naïve animals that were administered either sugar-free or sucrose diets for 17 weeks were treated at the end of this period with ketoprofen at different doses (10, 31.6 or 100 mg/kg p.o.) along with the corresponding vehicle to test for antinociceptive effects. The rats were tested every 30 min in the 240 min (4 h) post-administration period. 2.6. Statistical analyses All data are expressed as the mean 7S.E.M. and were checked for normality using the Shapiro-Wilk test. The thermal nociceptive
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(P o0.001) between the hypo-oestrogenic and Naïve sucrose-fed groups versus controls were observed; the Ov-Suc group was considered obese from this point.
responses with respect to body weight were analysed using an analysis of variance (ANOVA) test, followed by the post-hoc Tukey test. The cumulative antinociceptive effect during the entire 4 h observation period was determined as the area under the curve (AUC) of the time course. The AUCs for all the doses of the assayed drug were calculated using the trapezoidal method (Gibaldi, 1991). In all of the statistical analyses, P o0.05 was considered statistically significant. The analyses were performed using GraphPad Prism 6.0 software (GraphPad Software, Inc., La Jolla, CA, USA).
3.2. High-sucrose diet causes a biphasic response to thermal stimulation in hypo-oestrogenic rats To prove that the weight gain associated with increased body fat correlated with nociceptive processes, we analysed a time course of thermal stimulation responses throughout the 17-weeks period of the experimental diet, as shown in Fig. 2. The Ov-Suc group exhibited a biphasic response during administration of the high sucrose diet: 1) in the first four weeks of the high-sucrose diet, a decrease in thermal latency was observed in the Ov-Suc compared with that of the Ov-Ctrl group. In the fourth week, the Ov-Suc animals had a thermal latency of 7.670.2 s, whereas the animals in the control group had a thermal latency of 10.5 70.3 s, showing a statistically significant difference (P o0.001) and a hyperalgesic state. 2) From weeks 12th to 17th, the Ov-Suc animals showed an increase in thermal latency compared with the Ov-Ctrl animals. At the 17th week, the animals on a high-sucrose diet had a thermal latency of 14.4 70.4 s, which was significantly greater than that of the animals in the control group (11.470.1; Po 0.001), showing a hypoalgesic state. The Naïve-Suc group showed a decrease in latency, in the first four weeks of the highsucrose diet versus Naïve-Ctrl. At the 4th week the Naïve-Suc
3. Results 3.1. Weight gain with high sucrose diet in ovariectomized rats Fig. 1 shows the weight gain expressed in grams over the 17weeks time course for sucrose-fed and control animals. The OvSuc group showed a statistically significant increase at the 3rd week versus the Ov-Ctrl and Naïve-Ctrl group. During this time, the hypo-oestrogenic rats on the high-sucrose diet weighed 357.475.0 g, 9% more than the Ov-Ctrl and Naïve-Ctrl rats (P o0.001). This significant difference between groups persisted for the remainder of the study. The Naïve-Suc group showed a statistically significant increase at the 8th week weighed 378.0 712.1 g versus the Naïve-Ctrl group weighed 338.475.1 g (P o0.001). At 12th week, differences of approximately 20%
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Weeks Fig. 2. Responses to thermal stimulus of the Naïve-Ctrl, Ov-Ctrl and Naïve-Suc, Ov-Suc groups over 17 weeks. Hyperalgesia (first four weeks) and hypoalgesia state (weeks 12–17): a biphasic response is observed. Each point represents the mean 7 S.E.M. n ¼10;*Po 0.001, Ov-Suc vs. Ov-Ctrl; &Po 0.001, Naïve-Suc vs. Naïve Ctrl.
O.A. Jaramillo-Morales et al. / European Journal of Pharmacology 788 (2016) 168–175
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Fig. 3. Time course of anti-nociceptive effects produced by ketoprofen on the Ov-Suc (Panel A), Ov-Ctrl (Panel B), Naïve-Suc (Panel C) and Naïve-Ctrl (Panel D) groups at 4 week. Rats were treated with vehicle (0.5% carboxymethylcellulose) or increasing doses of ketoprofen (1.8, 5.6, 10, 17.8 or 31.6 mg/Kg, p.o.). Time course of antinociceptive effects of ketoprofen on the Ov-Suc (Panel E) and Ov-Ctrl (Panel F), Naïve-Suc (Panel G) and Naïve-Ctrl (Panel H) groups at 17 weeks. Rats were treated with vehicle (0.5% carboxymethylcellulose) or increasing doses of ketoprofen (10, 31.6 or 100 mg/Kg, p.o.). Each point represents the means 7 S.E.M., n ¼ 6/group. ***Po 0.001, **Po 0.01, *Po 0.05 rats treated with ketoprofen vs. vehicle.
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group had a thermal latency of 12.2 7 0.9 s; meanwhile the animals in the Naïve-Ctrl group had a thermal latency of 14.7 71.3 s exhibiting a statistically significant difference (P o0.001).
Antinociception (AUC)
Fig. 3 shows the 4-h time courses testing the effects of ketoprofen (1.8, 5.6, 10, 17.8 or 31.6 mg/kg) in animals with 4 weeks of treatment (Panels A, B, C and D), and the effects of ketoprofen administered at various doses (10, 31.6 or 100 mg/kg) at 17th week to both groups on thermal hyperalgesia after a single p.o. administration (Panels E, F, G and H). These time points were chosen due to the hyperalgesic and hypoalgesic response observed at 4th and 17th weeks, respectively. Prior to drug administration, the mean baseline response thermal latency of all of the hypo-oestrogenic female rats were 11.271.0 s and Naïve animals were 15.4 7 0.4 s as per the Plantar test method. In the tests performed at 4th or 17th weeks, the hypo-oestrogenic and Naïve rats fed with sucrose or water ad libitum were administered with vehicle (0.5% carboxymethylcellulose) showed the same percent baseline response thermal latency throughout the observation period. Ketoprofen shows antinociceptive effects, achieving its maximum effect at 30 min post-administration with the dose of 31.6 mg/kg p.o., showing an increase in thermal latency value by 4.3 70.4 s (Panel A). The effects were preserved during the entire time course. Moreover, in the Ov-Ctrl (Panel B) and Naïve-Suc (Panel C) group, 31.6 mg/kg ketoprofen showed its maximum effect at 90 min postadministration and 30 min post-administration respectively; this effect decreased 30 min later to its basal state. Lower doses did not achieve an effect different from vehicle treatment. An analysis of increase in maximum effect (Emax) from the corresponding time courses showed an increase in the values obtained from ketoprofen in animals that received a high-sucrose diet for 4 weeks compared with the control group and animals fed with the diet or water for 17 weeks (Panels E, F, G, H). The overall effects, expressed as the area under the curve (AUC) of the respective time courses during the first 4 h post-administration of ketoprofen (only 31.6 mg/kg, p.o.), were analysed to detect any anti-hyperalgesic effects (Fig. 4, Panel A). In this case, the area values were calculated by using the values of the thermal responses from the graphs of the time courses (Fig. 3, panels A, B, C and D) to clarify the anti-hyperalgesic effects shown by the corresponding treatments. Ketoprofen (31.6 mg/Kg) administered in Ov-Suc rats with 4 weeks of diet, showed 781.37 95.4 area units (au); thus, this dose had an anti-hyperalgesic effect and resulted in a significantly different response from the Ov-Ctrl, Naïve-Ctrl and Naïve-Suc groups (Student's t-test, Po 0.001), as shown in Fig. 4 (Panel A). Similarly, Naïve-Ctrl, Naïve-Suc, Ov-Ctrl and Ov-Suc groups under the same conditions, but administrated with 100 mg/Kg Ketoprofen, were evaluated at the 17th week (Fig. 4, Panel B). Ketoprofen showed a tendency to achieve an antinociceptive effect, but the effect was not statistically significant in comparison to the effect of vehicle in the control group. The maximum dose tested in animals fed with the diet for 17 weeks was 100 mg/kg, but adverse effects related to bloody diarrheal were observed with this dose. Therefore, in groups with 4 weeks of treatment, the maximum dose assessed was 31.6 mg/kg. Fig. 5 shows the DRC, expressed as the area under the curve (AUC), of the 4-h time courses post-administration of ketoprofen. The antinociceptive effects of ketoprofen administered at various doses, as measured in the Plantar test, increased in a dose-dependent manner at 4th and 17th week (Panels A and B) but displayed different efficacies at 4th week (Panel A). Ketoprofen (31.6 mg/Kg) showed a greater antinociceptive efficacy in the
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Ketoprofen (100 mg/Kg, p.o.) Fig. 4. Panel A: Anti-nociceptive effects (AUC¼ area under the curve) of ketoprofen (31.6 mg/kg, p.o.) on the Naïve-Ctrl, Naïve-Suc, Ov-Suc, and Ov-Ctrl groups at 4 weeks. Panel B: Anti-nociceptive effects (AUC) of ketoprofen (100 mg/kg, p.o.) on the Naïve-Ctrl, Naïve-Suc, Ov-Suc, and Ov-Ctrl groups at 17 weeks. Each point represents the AUC 7S.E.M., n ¼6/group. ***Po 0.001, Ov-Suc vs. Ov-Ctrl, Naïve-Ctrl and Naïve-Suc; N.S., no-significance.
Ov-Suc group (781.3795.4 au) than in the Ov-Ctrl (111.5734.8 au), Naïve-Ctrl (31.3 727.3 au) or Naïve-Suc group (70.5760.6 au) at 4 weeks. The maximum value of the AUC obtained in the Plantar test (for antinociceptive effects) under these experimental conditions was 800 au. The estimated doses required for producing 5% of the maximum effect, or ED5 values, were different for the OvSuc (2.3 mg/Kg) Ov-Ctrl (13.6 mg/Kg), Naïve-Ctrl (51.4 mg/Kg) and Naïve-Suc (19.65 mg/Kg) groups. The maximum value of the AUC obtained in the animals at 17 weeks under these experimental conditions was 250 au (Fig. 5, Panel B). The antinociceptive effects of ketoprofen were increased in a dose-dependent manner and displayed similar efficacies in both groups (Ov-Suc and Ov-Ctrl). Ketoprofen (100 mg/kg) showed an antinociceptive efficacy of 229.1 794.3 au in the Ov-Ctrl group, 135.6757.4 au in the Ov-Suc group, 73.8 764.9 au in the NaïveCtrl group and 111.2760.3 au in the Naïve-Suc. The estimated doses required to produce 25% of the maximum effect, or ED25 values, were different for the Ov-Suc (37.1 mg/kg), Ov-Ctrl (20.2 mg/kg), Naïve-Ctrl (81.66 mg/Kg) and Naïve-Suc (43.0 mg/ kg) groups.
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Ketoprofen (mg/Kgp.o.) Fig. 5. Panel A: Dose-response curves for the anti-nociceptive effects of ketoprofen on the Naïve-Ctrl, Naïve-Suc, Ov-Ctrl, and Ov-Suc groups at 4 weeks. Rats were treated with vehicle (0.5% carboxymethylcellulose) or increasing doses of ketoprofen (1.8, 5.6, 10, 17.8 or 31.6 mg/Kg, p.o.). ***Po 0.001, Ov-Suc vs. Ov-Ctrl, NaïveCtrl, and Naïve-Suc. Panel B: Dose-response curves for the anti-nociceptive effects of ketoprofen at 17 weeks. Rats were treated with vehicle (0.5% carboxymethylcellulose) or increasing doses of ketoprofen (10, 31.6 or 100 mg/Kg, p.o.). Each point represents the AUC 7S.E.M., n ¼ 6/group.
4. Discussion The aims of this study were 1) to characterize the nociceptive response using the plantar test method in ovariectomized Wistar rats fed a high sucrose diet (30%) over 17 weeks and 2) to evaluate the potential antinociceptive effects of ketoprofen under these conditions. The model of hypo-oestrogenism has previously been used as a mechanism for increasing body weight (Stubbins et al., 2012; Da Silva et al., 2014). Additionally, we used a high sucrose diet to increase the state of obesity to better reflect the current paradigm of obesity in humans wherein drinks with high sugar content are considered to be one of the major causes (Swinburn et al., 2004; Trumbo and Rivers, 2014). In our experiments, the animals showed a significant increase in body weight compared to the animals of the control group at the fourth week of treatment as result of the hyper caloric diet, and this weight gain continued to increase during the time course. These results are consistent with those from previous studies (Ramirez, 1987; Kawasaki et al., 2005).
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We showed herein for the first time that body weight gain caused by diet high in sucrose, is associated with a biphasic nociceptive response to a thermal stimulus in ovariectomized Wistar rats. These findings are similar to those obtained by Sugimoto et al. in 2008 using a genetic model of obesity, wherein a progression from hyperalgesia to hypoalgesia was observed in 8- and 32-weekold obese male Zucker rats. In the first 4 weeks of our experiments, animals fed the high-sucrose diet were in a hyperalgesic state, with a statistically significant decrease in their thermal latency compared with animals of the control group. The results are in agreement with similar results obtained by another study demonstrating that weight gain (over 25 days on a diet containing 20% sucrose) causes an increased sensitivity to noxious thermal stimulation (Roane and Martin, 1990). In contrast, in our experiments, the animals that received sucrose for a longer period showed an opposite response to that observed at the 4-week time point. The results revealed that in the sucrose-fed group, a significant increase in thermal latency occurred over weeks 12–17 compared with the control group, indicating a state of hypoalgesia. This biphasic effect observed may be caused by the pathophysiological mechanisms underlying obesity, including increases of the adipose tissue leading to the release of various substances, such as adipokines, cytokines, hormones, and opioids (Ramzan et al., 1993; Trayhurn, 2005; Deng and Scherer, 2010). But due to physiological changes in obesity, we cannot also rule out the involvement of pharmacokinetic changes or alterations. It is known that the perception of pain is a problem encountered in clinical practise. It has been proposed that this type of pain can be treated generally with NSAIDs (Laine, 2001). However, no studies have evaluated the analgesic response of these drugs with respect to the progression of obesity. We decided to evaluate ketoprofen because it has been extensively studied in rat models of persistent pain (Díaz-Reval et al., 2001, 2004; Girard et al., 2008; Aguilar-Carrasco et al., 2014). However, limited information is available on the individual administration of this drug in obese subjects. Some evidence suggests that obesity alters the effects of analgesic drugs (Ertugrul et al., 2010). Moreover, studies have shown that it may not even be necessary to increase doses for obese subjects to achieve effects similar to those in a normalweight subject (Lloret et al., 2009; Patanwala et al., 2014). In this study, we demonstrated an antihyperalgesic dose-dependent response to ketoprofen. Our findings are in agreement with those from other studies demonstrating that ketoprofen is useful for the treatment of chronic inflammatory pain (Díaz-Reval et al., 2001, 2004; Girard et al., 2008; Aguilar-Carrasco et al., 2014). The antinociceptive effects of ketoprofen may involve the inhibition of cyclooxygenase (COX), leading to blockage of prostaglandin (PG) biosynthesis (Avouac and Teule, 1988; Kubota et al., 1997); this inhibition is related to the anti-inflammatory, analgesic, and antipyretic properties of ketoprofen. Furthermore, ketoprofen administration significantly decreases inflammatory cytokine levels (TNF-α, and IL-1β) (Choi et al., 2013). In agreement with previous studies, obesity is regarded as a low-grade inflammatory disorder that involves the expansion of adipose tissue, which results in an increase in the circulating levels of pro-inflammatory adipokines (Leptin, tumour necrosis factor-α and interleukin-1 β) and the expression of COX-2 (Kershaw and Flier, 2004; Deng and Scherer, 2010; Hsieh, et al., 2009). Indeed, it has been established that these adipokines contribute to the development of pain and hyperalgesia (Ferreira et al., 1988; Cunha et al., 1992; Wieseler-Frank et al., 2005). Therefore the antinociceptive effect of ketoprofen may be due to its inhibition of these pathophysiological mechanisms involved in obesity. The antinociceptive effects of ketoprofen at two different states of weight gain were evaluated, and two different physiological effects on nociception were obtained. In the evaluation of
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ketoprofen at 4 weeks, diverse responses were obtained between the sucrose-fed and control groups. The DRC of ketoprofen showed a greater antinociceptive efficacy in the Ov-Suc group, but it also showed a great ED5 values, a less latency period (time to achieve the maximum effect), and a better effect on the time course. In contrast, at 17th week, ketoprofen showed antinociceptive effects different to those obtained in the fourth week with the high sucrose diet. Generally, it is known that the effect of some drugs is modified under conditions of obesity, and that these changes may be due to pharmacokinetic and/or pharmacodynamic alterations (Lloret et al., 2009). With respect to modifications of pharmacokinetic processes, it has been suggested that fundamental alterations may occur in the volume of distribution (Lloret et al., 2009). One of the most common changes in obesity is an increase in body fat, in which the adipose tissue constitutes a warehouse for liposoluble drugs. Deposition of the drugs in the adipose tissue can thus decrease the desired pharmacological effect. Obesity may also increase the levels of plasma proteins which may be capable of binding the drug and decrease the amount of free drug, thus affecting drug distribution (Lloret et al., 2009). Another important process that can be modified is metabolism. It is common for obese patients to have fatty livers or other diseases such as metabolic syndrome that modify metabolism (Mazoit et al., 1990; Richardson et al., 2006). Such patients can also be found to have alterations in their pharmacodynamics that can affect drug efficacies (Lloret et al., 2009). One of the characteristics of ketoprofen is its liposolubility and affinity for plasma proteins (Meunier and Verbeeck, 1999). By other hand, at the fourth week, when the greater antinociceptive effect of ketoprofen is obtained, is precisely when the lower threshold thermal response is exhibited. This could be due to changes in the expression of inflammatory mediators by expanding adipose tissue, and these changes may make the effect of ketoprofen more pronounced. In contrast, at 17th week, alterations in other inflammatory mediators may compensate for the decrease in the threshold. This may be explained by the release of substances such as 17-β-estradiol and β-endorphins that have anti-hyperalgesic effects (Ramzan et al., 1993; Barnes et al., 2006; Haghighi et al., 2014), or the release of adiponectin, which has anti-inflammatory effect (Trujillo and Scherer, 2005). It is probable that these phenomena mask the effects of ketoprofen to some extent. In conclusion, this study provides the first evidence showing that the hypo-oestrogenism plus high-sucrose diet causes changes in both nociception and antinociceptive effects of ketoprofen in hypooestrogenic Wistar rats. These results may be due to pharmacodynamic changes that might involve inflammatory cytokine levels (TNF-α, and IL-1β) and/or β-endorphins. However, we cannot rule out that other pharmacokinetic alterations may be influencing this process, for which further studies would be needed. Therefore, to establish the proper use of this drug in the clinic, it will be important to consider these results and formulate appropriate clinical studies to establish the adequate dose in patients.
5. Conflicts of interest The authors declare no conflicts of interest.
Acknowledgements We wish to thank L. Oliva and J. S. Ledesma for technical assistance. Jaramillo-Morales thanks fellowship by National Council for Sciences and Technology (CONACYT) 243335.
References Aguilar-Carrasco, J.C., Rodríguez-Silverio, J., Jiménez-Andrade, J.M., Carrasco-Portugal, M., del, C., Flores-Murrieta, F.J., 2014. Relationship between blood levels and the anti-hyperalgesic effect of ketoprofen in the rat. Drug Dev. Res. 75, 189–194. Avouac, B., Teule, M., 1988. Ketoprofen: the European experience. J. Clin. Pharmacol. 28, S2–S7. Barnes, M.J., Holmes, G., Primeaux, S.D., York, D.A., Bray, G.A., 2006. Increased expression of mu opioid receptors in animals susceptible to diet-induced obesity. Peptides 27, 3292–3298. Buchenauer, T., Behrendt, P., Bode, F.J., Horn, R., Brabant, G., Sthephan, M., Nave, H., 2009. Diet-induced obesity alters behavior as well as serum levels of corticosterone in F344 rats. Physiol. Behav. 98, 563–569. Choi, E.K., Kim, S.H., Kang, I.C., Jeong, J.Y., Koh, J.T., Lee, B.N., Oh, W.M., Min, K.S., Nör, J.E., Hwang, Y.C., 2013. Ketoprofen inhibits expression of inflammatory mediators in human dental pulp cells. J. Endod. 39, 764–767. Cunha, F., Poole, S., Lorenzetti, B., Ferreira, S., 1992. The pivotal role of tumor necrosis factor alpha in the development of inflammatory hyperalgesia. Br. J. Pharmacol. 107, 660–664. Da Silva, R.P., Zampieri, T.T., Pedroso, J.A., Nagaishi, V.S., Ramos-Lobo, A.M., Furigo, I. C., Câmara, N.O., Frazão, R., Donato, J., 2014. Leptin resistance is not the primary cause of weight gain associated with reduced sex hormone levels in female mice. Endocrinology 155, 4226–4236. D’Arcy, Y., 2011. Managing pain in obese patients. Nurse Pract. 36, 28–32. Deng, Y., Scherer, P.E., 2010. Adipokines as novel biomarkers and regulators of the metabolic syndrome. Ann. N. Y. Acad. Sci. 1212, E1–E19. Diaz Brinton, R., 2012. Minireview: translational animal models of human menopause: challenges and emerging opportunities. Endocrinology 8, 3571–3578. Díaz-Reval, M.I., Ventura-Martínez, R., Déciga-Campos, M., Terrón, J.A., Cabré, F., López-Muñoz, F.J., 2004. Evidence for a central mechanism of action of S( þ )-ketoprofen. Eur. J. Pharmacol. 483, 241–248. Díaz-Reval, M.I., Ventura-Martínez, R., Hernández-Delgadillo, G.P., DomínguezRamírez, A.M., López-Muñoz, F.J., 2001. Effect of caffeine on antinociceptive action of ketoprofen in rats. Arch. Med. Res. 32, 13–20. Dworkin, H., Gitlin, J., 1991. Clinical aspects of depression in chronic pain patients. Clin. J. Pain. 7, 79–94. Ertugrul, D.T., Tutal, E., Yildiz, M., Akin, O., Yalçin, A.A., Ure, O.S., Yilmaz, H., Yavuz, B., Deveci, O.S., Ata, N., Küçükazman, M., 2010. Aspirin resistance is associated with glycemic control, the dose of aspirin, and obesity in type 2 diabetes mellitus. J. Clin. Endocrinol. Metab. 95, 2897–2901. Ferreira, S., Lorenzetti, B., Bristow, A., Poole, S., 1988. Interleukin-1β as a potent hyperalgesic agent antagonized by a tripeptide analogue. Nature 334, 698–700. Gibaldi, M., 1991. Estimation of area under the curve. In: Gibaldi, M. (Ed.), Biopharmaceutics and Clinical Pharmacokinetics., 4th ed. Lea & Febiger, Philadelphia, pp. 377–378. Girard, P., Verniers, D., Coppé, M.C., Pansart, Y., Gillardin, J.M., 2008. Nefopam and ketoprofen synergy in rodent models of antinociception. Eur. J. Pharmacol. 584, 263–671. Goadsby, P.J., Lipton, R.B., Ferrari, M.D., 2002. Migraine-current understanding and treatment. N. Engl. J. Med. 346, 257–270. Haghighi, A., Melka, M.G., Bernard, M., Abrahamowicz, M., Leonard, G.T., Richer, L., Perron, M., Veillette, S., Xu, C.J., Greenwood, C.M., Dias, A., El-Sohemy, A., Gaudet, D., Paus, T., Pausova, Z., 2014. Opioid receptor mu 1 gene, fat intake and obesity in adolescence. Mol. Psychiatry 19, 63–68. Hargreaves, K., Dubner, R., Brown, F., Flores, C., Joris, J., 1988. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32, 77–88. Hitt, H.C., McMillen, R.C., Thornton-Neaves, T., Koch, K., Cosby, A.G., 2007. Comorbidity of obesity and pain in a general population: Results from the southern pain prevalence study. J. Pain 8, 430–436. Hsieh, P.S., Jin, J.S., Chiang, C.F., Chan, P.C., Chen, C.H., Shih, K.C., 2009. COX-2 mediated inflammation in fat is crucial for obesity-linked insulin resistance and fatty liver. Obesity 17, 1150–1157. Kawasaki, T., Kashiwabara, A., Sakai, T., Igarashi, K., Ogata, N., Watanabe, H., Ichiyanagi, K., Yamanouchi, T., 2005. Long-term sucrose-drinking causes increased body weight and glucose intolerance in normal male rats. Br. J. Nutr. 93, 613–618. Kershaw, E.E., Flier, J.S., 2004. Adipose tissue as an endocrine organ. J. Clin. Endocrinol. Metab. 89, 2548–2556. Kubota, T., Komatsu, H., Kawamoto, H., Yamada, T., 1997. Studies on the effects of anti-inflammatory action of benzoyl-hydratropic acid (ketoprofen) and other drugs with special reference to prostaglandin synthesis. Arch. Int. Pharmacodyn. Ther. 237, 169–176. Laine, L., 2001. Approaches to nonsteroidal anti-inflammatory drug use in the highrisk patient. Gastroenterology 120, 594–606. Lloret, L.C., Decleves, X., Oppert, J.M., Basdevant, A., Clement, K., Bardin, C., Scherrmann, J.M., Lepine, J.P., Bergmann, J.F., Mouly, S., 2009. Pharmacology of morphine in obese patients: clinical implications. Clin. Pharm. 48, 635–651. Marcus, D.A., 2004. Obesity and the impact of chronic pain. Clin. J. Pain. 20, 186–191. Mazoit, J.X., Sandouk, P., Scherrmann, J.M., Roche, A., 1990. Extrahepatic metabolism of morphine occurs in humans. Clin. Pharmacol. Ther. 48, 613–618. Meunier, C.J., Verbeeck, R.K., 1999. Glucuronidation kinetics of R,S-ketoprofen in adjuvant-induced arthritic rats. Pharm. Res. 16, 1081–1086.
O.A. Jaramillo-Morales et al. / European Journal of Pharmacology 788 (2016) 168–175
Ong, C.K., Lirk, P., Tan, C.H., Seymour, R.A., 2007. An evidence-based update on nonsteroidal anti-inflammatory drugs. Clin. Med. Res. 5, 19–34. Patanwala, A.E., Holmes, K.L., Erstad, B.L., 2014. Analgesic response to morphine in obese and morbidly obese patients in the emergency department. Emerg. Med. J. 31, 139–142. Ramirez, I., 1987. Feeding a liquid diet increases energy intake, weight gain and body fat in rats. J. Nutr. 117, 2127–2134. Ramzan, I., Wong, B.K., Corcoran, G.B., 1993. Pain sensitivity in dietary-induced obese rats. Physiol. Behav. 54, 433–435. Roane, D.S., Martin, J.R., 1990. Continuos sucrose feeding decreases pain threshold and increases morphine potency. Pharmacol. Biochem. Behav. 35, 225–229. Richardson, T.A., Sherman, M., Kalman, D., Morgan, E.T., 2006. Expression of UDP glucuronosyltransferase isoform mRNAs during inflammation and infection in mouse liver and kidney. Drug Metab. Dispos. 34, 351–353. Rossi, H.L., Luu, A.K., DeVilbiss, J.L., Recober, A., 2013. Obesity increases nociceptive activation of the trigeminal system. Eur. J. Pain 17, 649–653. Stubbins, R.E., Holcomb, V.B., Hong, J., Núñez, N.P., 2012. Estrogen modulates abdominal adiposity and protects female mice from obesity and impaired glucose tolerance. Eur. J. Nutr. 51, 861–870. Sugimoto, K., Rashid, I.B., Kojima, K., Shoji, M., Tanabe, J., Tamasawa, N., Suda, T., Yasujima, M., 2008. Time course of pain sensation in rat models of insulin resistance, type 2 diabetes, and exogenous hyperinsulinaemia. Diabetes Metab. Res. Rev. 24, 642–650.
175
Swinburn, B.A., Caterson, I., Seidell, J.C., James, W.P., 2004. Diet, nutrition and the prevention of excess weight gain and obesity. Public. Health Nutr. 7, 123–146. Trayhurn, P., 2005. Endocrine and signaling role of adipose tissue: new perspectives on fat. Acta Physiol. Scand. 184, 285–293. Trujillo, M.E., Scherer, P.E., 2005. Adiponectin-journey from an adipocyte secretory protein to biomarker of the metabolic syndrome. J. Intern. Med. 257, 167–175. Trumbo, P.R., Rivers, C.R., 2014. Systematic review of the evidence for an association between sugar-sweetened beverage consumption and risk of obesity. Nutr. Rev. 72, 566–574. Whiteside, G., Harrison, J., Boulet, J., Mark, L., Pearson, M., Gottshall, S., Walker, K., 2004. Pharmacological consideration of a rat model of incisional pain. Br. J. Pharmacol. 141, 85–91. Wieseler-Frank, J., Maier, S., Watkins, L.R., 2005. Immune-to-brain communication dynamically modulates pain: physiological and pathological consequences. Brain Behav. Immun. 19, 104–111. Wilson, A.C., Samuelson, B., Palermo, T.M., 2010. Obesity in children and adolescents with chronic pain: associations with pain and activity limitations. Clin. J. Pain. 26, 705–711. Zhang, T., Reid, K., Acuff, C.G., Jin, C.B., Rockhold, R.W., 1994. Cardiovascular and analgesic effects of a highly palatable diet in spontaneously hypertensive and Wistar-Kyoto rats. Pharmacol. Biochem. Behav. 48, 57–61. Zimmermann, M., 1983. Ethical guidelines for investigation of experimental pain in conscious animals. Pain 16, 109–110.
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Ketoprofen and antinociception in hypo-oestrogenic Wistar rats fed on a high sucrose diet Osmar Antonio Jaramillo-Morales, Josué Vidal Espinosa-Juárez, Betzabeth Anali García-Martínez, Francisco Javier López-Muñoz n Departamento de Farmacobiología, Cinvestav-Sede Sur, Delegación Tlálpan, México D.F. C.P. 14330, Mexico
art ic l e i nf o
a b s t r a c t
Article history: Received 4 May 2016 Received in revised form 8 June 2016 Accepted 17 June 2016 Available online 23 June 2016
Non-steroidal anti-inflammatory drugs such as ketoprofen are the most commonly used analgesics for the treatment of pain. However, no studies have evaluated the analgesic response to ketoprofen in conditions of obesity. The aim of this study was to analyse the time course of nociceptive pain in Wistar rats with and without hypo-oestrogenism on a high sucrose diet and to compare the antinociceptive response using ketoprofen. Hypo-oestrogenic and naïve rats received a hyper caloric diet (30% sucrose) or water ad libitum for 17 weeks, the thermal nociception (“plantar test” method) and body weight were tested during this period. A biphasic response was observed: thermal latency decreased in the 4th week (hyperalgesia), while from 12th to 17th week, thermal latency increased (hypoalgesia) in hypo-oestrogenic rats fed with high sucrose diet compared with the hypo-oestrogenic control group. At 4th and 17th weeks, different doses of ketoprofen (1.8–100 mg/kg p.o.), were evaluated in all groups. The administration of ketoprofen at 4th and 17th weeks showed dose-dependent effects in the all groups; however, a greater pharmacological efficacy was observed in the 4th week in the hypo-oestrogenic animals that received sucrose. Nevertheless, in all the groups significantly diminish the antinociceptive effects in the 17th week. Our data showed that nociception was altered in the hypo-oestrogenic animals that were fed sucrose (hyperalgesia and hypoalgesia). Ketoprofen showed a dose-dependent antinociceptive effect at both time points. However, hypo-oestrogenism plus high-sucrose diet modifies the antinociceptive effect of ketoprofen. & 2016 Elsevier B.V. All rights reserved.
Keywords: High-sucrose diet Nociception Ketoprofen Efficacy
1. Introduction Pain is a condition that affects thousands of people in the world. However, it has been shown that individuals with increased body weight are more likely to have problems with pain (Marcus, 2004; Wilson et al., 2010). Controversies exist regarding the perception of pain in obese subjects. Some studies report a hyperalgesic state associated with obesity (Roane and Martin, 1990; Sugimoto et al., 2008; Buchenauer et al., 2009), while others have proposed the existence of hypoalgesic processes (Ramzan et al., 1993; Zhang et al., 1994; Sugimoto et al., 2008). Moreover, the most frequent cases that have been documented clinically in obese people involve back pain (Hitt et al., 2007; D’Arcy, 2011) and n Correspondence to: Lab. No. 7 “Dolor y Analgesia” del Departamento de Farmacobiología, Cinvestav-Sede Sur, Calz. de los Tenorios No. 235 Col. Granjas Coapa, Delegación Tlálpan, México D.F. C.P. 14330, Mexico. E-mail addresses: [email protected] (O.A. Jaramillo-Morales), [email protected] (J.V. Espinosa-Juárez), [email protected] (B.A. García-Martínez), fl[email protected], fl[email protected] (F.J. López-Muñoz).
http://dx.doi.org/10.1016/j.ejphar.2016.06.030 0014-2999/& 2016 Elsevier B.V. All rights reserved.
arthritis pain (Marcus, 2004), triggered mainly due to a mechanical effect on the joints by weight gain. Obesity has also been associated with other chronic pain conditions, such as migraine and headache (Goadsby et al., 2002; Rossi et al., 2013). For this reason, it is common for these patients to be treated with analgesic drugs. The most widespread drug therapy currently used to relieve nociceptive and inflammatory pain involves the non-steroidal antiinflammatory analgesics (NSAIDs) and opioids (Ong et al., 2007). NSAIDs are the most widely prescribed drugs in clinical medicine; they are heterogeneous substances with varying nonsteroidal chemical structures (Laine, 2001). This group of drugs, which are indicated in the treatment of acute and chronic pain (Whiteside et al., 2004; Ong et al., 2007), is characterized by analgesic antiinflammatory and antipyretic properties (Dworkin y Gitlin, 1991). Ketoprofen is a member of this group. As other NSAIDs, ketoprofen exerts its analgesic effect through at least three mechanisms of action, clearly identified as: 1) inhibition of prostaglandin synthesis (Avouac and Teule, 1988; Kubota et al., 1997), 2) interaction with the serotonergic system (Díaz-Reval et al., 2001, 2004) and 3) inhibition of proinflammatory cytokines (Choi et al., 2013). Preclinical studies have indicated that ketoprofen exhibits
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dose-dependent effects in different models of acute and inflammatory pain (Díaz-Reval et al., 2001, 2004; Aguilar-Carrasco et al., 2014). However, no studies have evaluated the analgesic response of ketoprofen in conditions where there is an increase in body weight; therefore, the antinociceptive effect it may have under these conditions is unknown. The effects may differ because pathological conditions can change the activity of drugs, mainly through pharmacokinetic and/or pharmacodynamic alterations (Lloret et al., 2009). Therefore, the objectives of this study were to evaluate nociception in hypo-oestrogenic and naïve female Wistar rats under conditions of increasing weight up to a state of obesity achieved through the provision of a high sucrose diet (30% in drinking water) and to analyse the antinociceptive response to ketoprofen.
2. Materials and methods 2.1. Animals and housing conditions Female Wistar rats [Crl(WI)fBR] weighing 180–200 g at the time of surgery were used in this study. All animals were obtained from the animal breeding facility of the Centre of Investigation and Advanced Studies (Cinvestav, Sede Sur). The animals were housed under standardized conditions in a room on a 12 h light/dark cycle with food and water available ad libitum before treatment. All experimental procedures were approved by the Local Ethics Committee for the Management of Laboratory Animals of the Department of Pharmacobiology of Cinvestav, Sede Sur, following the Guidelines on Ethical Standards for Investigations of Experimental Pain in Animals (Zimmermann, 1983) and the Official Mexican Norm (NOM-062-ZOO-1999). All tests were performed during the light phase. The number of experimental animals was kept to a minimum, and the rats were killed immediately after the experiment by CO2 overdose. 2.2. Compounds Refined sucrose was obtained commercially from Supplies BenHill, S. A. de C. V. (Mexico City, Mexico). The sugar was dissolved in water to 30% (wt/vol). Ketoprofen was obtained from Sigma Chemical Company (St. Louis, MO, USA). Ketoprofen was suspended in 0.5% carboxymethylcellulose and was administered orally in an application volume of 4 ml/kg body weight. The doses mentioned in the text refer to the salts of the substances used. 2.3. Surgical technique (Ovariectomy) Briefly, the ovariectomy consisted of the following procedures. All animals were anaesthetized by an intraperitoneal (i.p.) injection of 50 mg/kg ketamine and 10 mg/kg xylazine mixture for bilateral removal of the ovaries. Briefly, the abdominal and pelvic areas of the animals were shaved and cleaned. A longitudinal incision of approximately 1.5 cm was made, the skin was separated from the muscle, and a second incision of approximately 0.5 cm was made in the muscle to exteriorize the ovaries. The fallopian tubes were ligated and cut below the ligature. After the excision, the incision was sutured. This surgery caused a state of hypooestrogenism in experimental animals. The ovariectomized female rat model has been commonly used as a direct cause of increased body weight (Stubbins et al., 2012; Da Silva et al., 2014) and as a model for menopause in humans (Diaz Brinton, 2012). All procedures were carried out under aseptic conditions.
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2.4. Measurement of antinociceptive activity To avoid additional stress, the rats were allowed to acclimate to the testing site until exploratory behaviour diminished for at least 10 min before stimulation was initiated. Responses to thermal nociception were evaluated using the Hargreaves method (UGOBASILE, Varese, Italy) (Hargreaves et al., 1988). Briefly, a radiant heat source with a locator light was positioned under the plantar surface and the latency to withdrawal the right hind paw was recorded for each animal. A resting period of 5 min followed to avoid any sensitization. The intensity of the lamp was set at 60 Hz, and a cut-off time of 30 s (s) was determined necessary to avoid tissue damage. The light beam was directed on the plantar surface of the hind paw until the animal responded, or when it came to cutting time (30 s), the lamp was turned off, whichever occurred first. The latency of the paw withdrawal from the incident light was recorded with a built-in timer, which displayed reaction time in 0.01 s increments. The latency of withdrawal was defined as the time between the initiation of the light and the moment at which the animal withdrew from the tip of the light quickly, either generating a limb-licking behaviour or jumping away from the heat source. Three readings were made per animal. Data are expressed as the withdrawal latency and are measured in seconds. 2.5. Experimental design On the 15th postoperative day (ovariectomy), the time course of the thermal latency was initiated. Female Wistar rats weighing 250–270 g each were randomized into four groups (n ¼6) as follows: hypo-oestrogenic-control (Ov-Ctrl), hypo-oestrogenic-sucrose (Ov-Suc) (both were ovariectomized), naïve-control (NaïveCtrl) and naïve-sucrose (Naïve-Suc) (both were non-operated). Before the start of the diet, baseline thermal threshold responses of all groups were measured at time 0 (immediately after receiving the respective diet). During the 17-weeks treatment, all groups were given free access to food (pellets of LabDiet 5008). Additionally, sucrose fed rats received 30% commercially refined sucrose (wt/vol) in drinking water given ad libitum. In contrast, the control group received filtered drinking water ad libitum. During this period, thermal nociception was assessed weekly using Hargreaves method (UGO-BASILE, Varese, Italy) (Hargreaves et al., 1988), and body weight measurements were taken. The effects of the acute administration of a drug (ketoprofen) on response thresholds to thermal stimuli were tested at week 4 and week 17 post-treatment with sucrose or water ad libitum in ovariectomized rats. Baseline thermal nociception was assessed before pharmacological testing. The experimental protocol consisted of two sets of experimental groups in which the antinociceptive effects produced by ketoprofen, given individually, were studied with the corresponding vehicle (0.5% carboxymethylcellulose). In the first set of experimental groups, each dose of ketoprofen (1.8, 5.6, 10, 17.8 or 31.6 mg/kg, p.o.) was given in a volume of 4 ml/kg to six hypo-oestrogenic and Naïve rats (that had received the respective diet for 4 weeks) to obtain the doseresponse curves (DRC). In the second set, hypo-oestrogenic and Naïve animals that were administered either sugar-free or sucrose diets for 17 weeks were treated at the end of this period with ketoprofen at different doses (10, 31.6 or 100 mg/kg p.o.) along with the corresponding vehicle to test for antinociceptive effects. The rats were tested every 30 min in the 240 min (4 h) post-administration period. 2.6. Statistical analyses All data are expressed as the mean 7S.E.M. and were checked for normality using the Shapiro-Wilk test. The thermal nociceptive
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(P o0.001) between the hypo-oestrogenic and Naïve sucrose-fed groups versus controls were observed; the Ov-Suc group was considered obese from this point.
responses with respect to body weight were analysed using an analysis of variance (ANOVA) test, followed by the post-hoc Tukey test. The cumulative antinociceptive effect during the entire 4 h observation period was determined as the area under the curve (AUC) of the time course. The AUCs for all the doses of the assayed drug were calculated using the trapezoidal method (Gibaldi, 1991). In all of the statistical analyses, P o0.05 was considered statistically significant. The analyses were performed using GraphPad Prism 6.0 software (GraphPad Software, Inc., La Jolla, CA, USA).
3.2. High-sucrose diet causes a biphasic response to thermal stimulation in hypo-oestrogenic rats To prove that the weight gain associated with increased body fat correlated with nociceptive processes, we analysed a time course of thermal stimulation responses throughout the 17-weeks period of the experimental diet, as shown in Fig. 2. The Ov-Suc group exhibited a biphasic response during administration of the high sucrose diet: 1) in the first four weeks of the high-sucrose diet, a decrease in thermal latency was observed in the Ov-Suc compared with that of the Ov-Ctrl group. In the fourth week, the Ov-Suc animals had a thermal latency of 7.670.2 s, whereas the animals in the control group had a thermal latency of 10.5 70.3 s, showing a statistically significant difference (P o0.001) and a hyperalgesic state. 2) From weeks 12th to 17th, the Ov-Suc animals showed an increase in thermal latency compared with the Ov-Ctrl animals. At the 17th week, the animals on a high-sucrose diet had a thermal latency of 14.4 70.4 s, which was significantly greater than that of the animals in the control group (11.470.1; Po 0.001), showing a hypoalgesic state. The Naïve-Suc group showed a decrease in latency, in the first four weeks of the highsucrose diet versus Naïve-Ctrl. At the 4th week the Naïve-Suc
3. Results 3.1. Weight gain with high sucrose diet in ovariectomized rats Fig. 1 shows the weight gain expressed in grams over the 17weeks time course for sucrose-fed and control animals. The OvSuc group showed a statistically significant increase at the 3rd week versus the Ov-Ctrl and Naïve-Ctrl group. During this time, the hypo-oestrogenic rats on the high-sucrose diet weighed 357.475.0 g, 9% more than the Ov-Ctrl and Naïve-Ctrl rats (P o0.001). This significant difference between groups persisted for the remainder of the study. The Naïve-Suc group showed a statistically significant increase at the 8th week weighed 378.0 712.1 g versus the Naïve-Ctrl group weighed 338.475.1 g (P o0.001). At 12th week, differences of approximately 20%
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Weeks Fig. 2. Responses to thermal stimulus of the Naïve-Ctrl, Ov-Ctrl and Naïve-Suc, Ov-Suc groups over 17 weeks. Hyperalgesia (first four weeks) and hypoalgesia state (weeks 12–17): a biphasic response is observed. Each point represents the mean 7 S.E.M. n ¼10;*Po 0.001, Ov-Suc vs. Ov-Ctrl; &Po 0.001, Naïve-Suc vs. Naïve Ctrl.
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Fig. 3. Time course of anti-nociceptive effects produced by ketoprofen on the Ov-Suc (Panel A), Ov-Ctrl (Panel B), Naïve-Suc (Panel C) and Naïve-Ctrl (Panel D) groups at 4 week. Rats were treated with vehicle (0.5% carboxymethylcellulose) or increasing doses of ketoprofen (1.8, 5.6, 10, 17.8 or 31.6 mg/Kg, p.o.). Time course of antinociceptive effects of ketoprofen on the Ov-Suc (Panel E) and Ov-Ctrl (Panel F), Naïve-Suc (Panel G) and Naïve-Ctrl (Panel H) groups at 17 weeks. Rats were treated with vehicle (0.5% carboxymethylcellulose) or increasing doses of ketoprofen (10, 31.6 or 100 mg/Kg, p.o.). Each point represents the means 7 S.E.M., n ¼ 6/group. ***Po 0.001, **Po 0.01, *Po 0.05 rats treated with ketoprofen vs. vehicle.
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group had a thermal latency of 12.2 7 0.9 s; meanwhile the animals in the Naïve-Ctrl group had a thermal latency of 14.7 71.3 s exhibiting a statistically significant difference (P o0.001).
Antinociception (AUC)
Fig. 3 shows the 4-h time courses testing the effects of ketoprofen (1.8, 5.6, 10, 17.8 or 31.6 mg/kg) in animals with 4 weeks of treatment (Panels A, B, C and D), and the effects of ketoprofen administered at various doses (10, 31.6 or 100 mg/kg) at 17th week to both groups on thermal hyperalgesia after a single p.o. administration (Panels E, F, G and H). These time points were chosen due to the hyperalgesic and hypoalgesic response observed at 4th and 17th weeks, respectively. Prior to drug administration, the mean baseline response thermal latency of all of the hypo-oestrogenic female rats were 11.271.0 s and Naïve animals were 15.4 7 0.4 s as per the Plantar test method. In the tests performed at 4th or 17th weeks, the hypo-oestrogenic and Naïve rats fed with sucrose or water ad libitum were administered with vehicle (0.5% carboxymethylcellulose) showed the same percent baseline response thermal latency throughout the observation period. Ketoprofen shows antinociceptive effects, achieving its maximum effect at 30 min post-administration with the dose of 31.6 mg/kg p.o., showing an increase in thermal latency value by 4.3 70.4 s (Panel A). The effects were preserved during the entire time course. Moreover, in the Ov-Ctrl (Panel B) and Naïve-Suc (Panel C) group, 31.6 mg/kg ketoprofen showed its maximum effect at 90 min postadministration and 30 min post-administration respectively; this effect decreased 30 min later to its basal state. Lower doses did not achieve an effect different from vehicle treatment. An analysis of increase in maximum effect (Emax) from the corresponding time courses showed an increase in the values obtained from ketoprofen in animals that received a high-sucrose diet for 4 weeks compared with the control group and animals fed with the diet or water for 17 weeks (Panels E, F, G, H). The overall effects, expressed as the area under the curve (AUC) of the respective time courses during the first 4 h post-administration of ketoprofen (only 31.6 mg/kg, p.o.), were analysed to detect any anti-hyperalgesic effects (Fig. 4, Panel A). In this case, the area values were calculated by using the values of the thermal responses from the graphs of the time courses (Fig. 3, panels A, B, C and D) to clarify the anti-hyperalgesic effects shown by the corresponding treatments. Ketoprofen (31.6 mg/Kg) administered in Ov-Suc rats with 4 weeks of diet, showed 781.37 95.4 area units (au); thus, this dose had an anti-hyperalgesic effect and resulted in a significantly different response from the Ov-Ctrl, Naïve-Ctrl and Naïve-Suc groups (Student's t-test, Po 0.001), as shown in Fig. 4 (Panel A). Similarly, Naïve-Ctrl, Naïve-Suc, Ov-Ctrl and Ov-Suc groups under the same conditions, but administrated with 100 mg/Kg Ketoprofen, were evaluated at the 17th week (Fig. 4, Panel B). Ketoprofen showed a tendency to achieve an antinociceptive effect, but the effect was not statistically significant in comparison to the effect of vehicle in the control group. The maximum dose tested in animals fed with the diet for 17 weeks was 100 mg/kg, but adverse effects related to bloody diarrheal were observed with this dose. Therefore, in groups with 4 weeks of treatment, the maximum dose assessed was 31.6 mg/kg. Fig. 5 shows the DRC, expressed as the area under the curve (AUC), of the 4-h time courses post-administration of ketoprofen. The antinociceptive effects of ketoprofen administered at various doses, as measured in the Plantar test, increased in a dose-dependent manner at 4th and 17th week (Panels A and B) but displayed different efficacies at 4th week (Panel A). Ketoprofen (31.6 mg/Kg) showed a greater antinociceptive efficacy in the
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Ketoprofen (100 mg/Kg, p.o.) Fig. 4. Panel A: Anti-nociceptive effects (AUC¼ area under the curve) of ketoprofen (31.6 mg/kg, p.o.) on the Naïve-Ctrl, Naïve-Suc, Ov-Suc, and Ov-Ctrl groups at 4 weeks. Panel B: Anti-nociceptive effects (AUC) of ketoprofen (100 mg/kg, p.o.) on the Naïve-Ctrl, Naïve-Suc, Ov-Suc, and Ov-Ctrl groups at 17 weeks. Each point represents the AUC 7S.E.M., n ¼6/group. ***Po 0.001, Ov-Suc vs. Ov-Ctrl, Naïve-Ctrl and Naïve-Suc; N.S., no-significance.
Ov-Suc group (781.3795.4 au) than in the Ov-Ctrl (111.5734.8 au), Naïve-Ctrl (31.3 727.3 au) or Naïve-Suc group (70.5760.6 au) at 4 weeks. The maximum value of the AUC obtained in the Plantar test (for antinociceptive effects) under these experimental conditions was 800 au. The estimated doses required for producing 5% of the maximum effect, or ED5 values, were different for the OvSuc (2.3 mg/Kg) Ov-Ctrl (13.6 mg/Kg), Naïve-Ctrl (51.4 mg/Kg) and Naïve-Suc (19.65 mg/Kg) groups. The maximum value of the AUC obtained in the animals at 17 weeks under these experimental conditions was 250 au (Fig. 5, Panel B). The antinociceptive effects of ketoprofen were increased in a dose-dependent manner and displayed similar efficacies in both groups (Ov-Suc and Ov-Ctrl). Ketoprofen (100 mg/kg) showed an antinociceptive efficacy of 229.1 794.3 au in the Ov-Ctrl group, 135.6757.4 au in the Ov-Suc group, 73.8 764.9 au in the NaïveCtrl group and 111.2760.3 au in the Naïve-Suc. The estimated doses required to produce 25% of the maximum effect, or ED25 values, were different for the Ov-Suc (37.1 mg/kg), Ov-Ctrl (20.2 mg/kg), Naïve-Ctrl (81.66 mg/Kg) and Naïve-Suc (43.0 mg/ kg) groups.
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4. Discussion The aims of this study were 1) to characterize the nociceptive response using the plantar test method in ovariectomized Wistar rats fed a high sucrose diet (30%) over 17 weeks and 2) to evaluate the potential antinociceptive effects of ketoprofen under these conditions. The model of hypo-oestrogenism has previously been used as a mechanism for increasing body weight (Stubbins et al., 2012; Da Silva et al., 2014). Additionally, we used a high sucrose diet to increase the state of obesity to better reflect the current paradigm of obesity in humans wherein drinks with high sugar content are considered to be one of the major causes (Swinburn et al., 2004; Trumbo and Rivers, 2014). In our experiments, the animals showed a significant increase in body weight compared to the animals of the control group at the fourth week of treatment as result of the hyper caloric diet, and this weight gain continued to increase during the time course. These results are consistent with those from previous studies (Ramirez, 1987; Kawasaki et al., 2005).
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We showed herein for the first time that body weight gain caused by diet high in sucrose, is associated with a biphasic nociceptive response to a thermal stimulus in ovariectomized Wistar rats. These findings are similar to those obtained by Sugimoto et al. in 2008 using a genetic model of obesity, wherein a progression from hyperalgesia to hypoalgesia was observed in 8- and 32-weekold obese male Zucker rats. In the first 4 weeks of our experiments, animals fed the high-sucrose diet were in a hyperalgesic state, with a statistically significant decrease in their thermal latency compared with animals of the control group. The results are in agreement with similar results obtained by another study demonstrating that weight gain (over 25 days on a diet containing 20% sucrose) causes an increased sensitivity to noxious thermal stimulation (Roane and Martin, 1990). In contrast, in our experiments, the animals that received sucrose for a longer period showed an opposite response to that observed at the 4-week time point. The results revealed that in the sucrose-fed group, a significant increase in thermal latency occurred over weeks 12–17 compared with the control group, indicating a state of hypoalgesia. This biphasic effect observed may be caused by the pathophysiological mechanisms underlying obesity, including increases of the adipose tissue leading to the release of various substances, such as adipokines, cytokines, hormones, and opioids (Ramzan et al., 1993; Trayhurn, 2005; Deng and Scherer, 2010). But due to physiological changes in obesity, we cannot also rule out the involvement of pharmacokinetic changes or alterations. It is known that the perception of pain is a problem encountered in clinical practise. It has been proposed that this type of pain can be treated generally with NSAIDs (Laine, 2001). However, no studies have evaluated the analgesic response of these drugs with respect to the progression of obesity. We decided to evaluate ketoprofen because it has been extensively studied in rat models of persistent pain (Díaz-Reval et al., 2001, 2004; Girard et al., 2008; Aguilar-Carrasco et al., 2014). However, limited information is available on the individual administration of this drug in obese subjects. Some evidence suggests that obesity alters the effects of analgesic drugs (Ertugrul et al., 2010). Moreover, studies have shown that it may not even be necessary to increase doses for obese subjects to achieve effects similar to those in a normalweight subject (Lloret et al., 2009; Patanwala et al., 2014). In this study, we demonstrated an antihyperalgesic dose-dependent response to ketoprofen. Our findings are in agreement with those from other studies demonstrating that ketoprofen is useful for the treatment of chronic inflammatory pain (Díaz-Reval et al., 2001, 2004; Girard et al., 2008; Aguilar-Carrasco et al., 2014). The antinociceptive effects of ketoprofen may involve the inhibition of cyclooxygenase (COX), leading to blockage of prostaglandin (PG) biosynthesis (Avouac and Teule, 1988; Kubota et al., 1997); this inhibition is related to the anti-inflammatory, analgesic, and antipyretic properties of ketoprofen. Furthermore, ketoprofen administration significantly decreases inflammatory cytokine levels (TNF-α, and IL-1β) (Choi et al., 2013). In agreement with previous studies, obesity is regarded as a low-grade inflammatory disorder that involves the expansion of adipose tissue, which results in an increase in the circulating levels of pro-inflammatory adipokines (Leptin, tumour necrosis factor-α and interleukin-1 β) and the expression of COX-2 (Kershaw and Flier, 2004; Deng and Scherer, 2010; Hsieh, et al., 2009). Indeed, it has been established that these adipokines contribute to the development of pain and hyperalgesia (Ferreira et al., 1988; Cunha et al., 1992; Wieseler-Frank et al., 2005). Therefore the antinociceptive effect of ketoprofen may be due to its inhibition of these pathophysiological mechanisms involved in obesity. The antinociceptive effects of ketoprofen at two different states of weight gain were evaluated, and two different physiological effects on nociception were obtained. In the evaluation of
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ketoprofen at 4 weeks, diverse responses were obtained between the sucrose-fed and control groups. The DRC of ketoprofen showed a greater antinociceptive efficacy in the Ov-Suc group, but it also showed a great ED5 values, a less latency period (time to achieve the maximum effect), and a better effect on the time course. In contrast, at 17th week, ketoprofen showed antinociceptive effects different to those obtained in the fourth week with the high sucrose diet. Generally, it is known that the effect of some drugs is modified under conditions of obesity, and that these changes may be due to pharmacokinetic and/or pharmacodynamic alterations (Lloret et al., 2009). With respect to modifications of pharmacokinetic processes, it has been suggested that fundamental alterations may occur in the volume of distribution (Lloret et al., 2009). One of the most common changes in obesity is an increase in body fat, in which the adipose tissue constitutes a warehouse for liposoluble drugs. Deposition of the drugs in the adipose tissue can thus decrease the desired pharmacological effect. Obesity may also increase the levels of plasma proteins which may be capable of binding the drug and decrease the amount of free drug, thus affecting drug distribution (Lloret et al., 2009). Another important process that can be modified is metabolism. It is common for obese patients to have fatty livers or other diseases such as metabolic syndrome that modify metabolism (Mazoit et al., 1990; Richardson et al., 2006). Such patients can also be found to have alterations in their pharmacodynamics that can affect drug efficacies (Lloret et al., 2009). One of the characteristics of ketoprofen is its liposolubility and affinity for plasma proteins (Meunier and Verbeeck, 1999). By other hand, at the fourth week, when the greater antinociceptive effect of ketoprofen is obtained, is precisely when the lower threshold thermal response is exhibited. This could be due to changes in the expression of inflammatory mediators by expanding adipose tissue, and these changes may make the effect of ketoprofen more pronounced. In contrast, at 17th week, alterations in other inflammatory mediators may compensate for the decrease in the threshold. This may be explained by the release of substances such as 17-β-estradiol and β-endorphins that have anti-hyperalgesic effects (Ramzan et al., 1993; Barnes et al., 2006; Haghighi et al., 2014), or the release of adiponectin, which has anti-inflammatory effect (Trujillo and Scherer, 2005). It is probable that these phenomena mask the effects of ketoprofen to some extent. In conclusion, this study provides the first evidence showing that the hypo-oestrogenism plus high-sucrose diet causes changes in both nociception and antinociceptive effects of ketoprofen in hypooestrogenic Wistar rats. These results may be due to pharmacodynamic changes that might involve inflammatory cytokine levels (TNF-α, and IL-1β) and/or β-endorphins. However, we cannot rule out that other pharmacokinetic alterations may be influencing this process, for which further studies would be needed. Therefore, to establish the proper use of this drug in the clinic, it will be important to consider these results and formulate appropriate clinical studies to establish the adequate dose in patients.
5. Conflicts of interest The authors declare no conflicts of interest.
Acknowledgements We wish to thank L. Oliva and J. S. Ledesma for technical assistance. Jaramillo-Morales thanks fellowship by National Council for Sciences and Technology (CONACYT) 243335.
References Aguilar-Carrasco, J.C., Rodríguez-Silverio, J., Jiménez-Andrade, J.M., Carrasco-Portugal, M., del, C., Flores-Murrieta, F.J., 2014. Relationship between blood levels and the anti-hyperalgesic effect of ketoprofen in the rat. Drug Dev. Res. 75, 189–194. Avouac, B., Teule, M., 1988. Ketoprofen: the European experience. J. Clin. Pharmacol. 28, S2–S7. Barnes, M.J., Holmes, G., Primeaux, S.D., York, D.A., Bray, G.A., 2006. Increased expression of mu opioid receptors in animals susceptible to diet-induced obesity. Peptides 27, 3292–3298. Buchenauer, T., Behrendt, P., Bode, F.J., Horn, R., Brabant, G., Sthephan, M., Nave, H., 2009. Diet-induced obesity alters behavior as well as serum levels of corticosterone in F344 rats. Physiol. Behav. 98, 563–569. Choi, E.K., Kim, S.H., Kang, I.C., Jeong, J.Y., Koh, J.T., Lee, B.N., Oh, W.M., Min, K.S., Nör, J.E., Hwang, Y.C., 2013. Ketoprofen inhibits expression of inflammatory mediators in human dental pulp cells. J. Endod. 39, 764–767. Cunha, F., Poole, S., Lorenzetti, B., Ferreira, S., 1992. The pivotal role of tumor necrosis factor alpha in the development of inflammatory hyperalgesia. Br. J. Pharmacol. 107, 660–664. Da Silva, R.P., Zampieri, T.T., Pedroso, J.A., Nagaishi, V.S., Ramos-Lobo, A.M., Furigo, I. C., Câmara, N.O., Frazão, R., Donato, J., 2014. Leptin resistance is not the primary cause of weight gain associated with reduced sex hormone levels in female mice. Endocrinology 155, 4226–4236. D’Arcy, Y., 2011. Managing pain in obese patients. Nurse Pract. 36, 28–32. Deng, Y., Scherer, P.E., 2010. Adipokines as novel biomarkers and regulators of the metabolic syndrome. Ann. N. Y. Acad. Sci. 1212, E1–E19. Diaz Brinton, R., 2012. Minireview: translational animal models of human menopause: challenges and emerging opportunities. Endocrinology 8, 3571–3578. Díaz-Reval, M.I., Ventura-Martínez, R., Déciga-Campos, M., Terrón, J.A., Cabré, F., López-Muñoz, F.J., 2004. Evidence for a central mechanism of action of S( þ )-ketoprofen. Eur. J. Pharmacol. 483, 241–248. Díaz-Reval, M.I., Ventura-Martínez, R., Hernández-Delgadillo, G.P., DomínguezRamírez, A.M., López-Muñoz, F.J., 2001. Effect of caffeine on antinociceptive action of ketoprofen in rats. Arch. Med. Res. 32, 13–20. Dworkin, H., Gitlin, J., 1991. Clinical aspects of depression in chronic pain patients. Clin. J. Pain. 7, 79–94. Ertugrul, D.T., Tutal, E., Yildiz, M., Akin, O., Yalçin, A.A., Ure, O.S., Yilmaz, H., Yavuz, B., Deveci, O.S., Ata, N., Küçükazman, M., 2010. Aspirin resistance is associated with glycemic control, the dose of aspirin, and obesity in type 2 diabetes mellitus. J. Clin. Endocrinol. Metab. 95, 2897–2901. Ferreira, S., Lorenzetti, B., Bristow, A., Poole, S., 1988. Interleukin-1β as a potent hyperalgesic agent antagonized by a tripeptide analogue. Nature 334, 698–700. Gibaldi, M., 1991. Estimation of area under the curve. In: Gibaldi, M. (Ed.), Biopharmaceutics and Clinical Pharmacokinetics., 4th ed. Lea & Febiger, Philadelphia, pp. 377–378. Girard, P., Verniers, D., Coppé, M.C., Pansart, Y., Gillardin, J.M., 2008. Nefopam and ketoprofen synergy in rodent models of antinociception. Eur. J. Pharmacol. 584, 263–671. Goadsby, P.J., Lipton, R.B., Ferrari, M.D., 2002. Migraine-current understanding and treatment. N. Engl. J. Med. 346, 257–270. Haghighi, A., Melka, M.G., Bernard, M., Abrahamowicz, M., Leonard, G.T., Richer, L., Perron, M., Veillette, S., Xu, C.J., Greenwood, C.M., Dias, A., El-Sohemy, A., Gaudet, D., Paus, T., Pausova, Z., 2014. Opioid receptor mu 1 gene, fat intake and obesity in adolescence. Mol. Psychiatry 19, 63–68. Hargreaves, K., Dubner, R., Brown, F., Flores, C., Joris, J., 1988. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32, 77–88. Hitt, H.C., McMillen, R.C., Thornton-Neaves, T., Koch, K., Cosby, A.G., 2007. Comorbidity of obesity and pain in a general population: Results from the southern pain prevalence study. J. Pain 8, 430–436. Hsieh, P.S., Jin, J.S., Chiang, C.F., Chan, P.C., Chen, C.H., Shih, K.C., 2009. COX-2 mediated inflammation in fat is crucial for obesity-linked insulin resistance and fatty liver. Obesity 17, 1150–1157. Kawasaki, T., Kashiwabara, A., Sakai, T., Igarashi, K., Ogata, N., Watanabe, H., Ichiyanagi, K., Yamanouchi, T., 2005. Long-term sucrose-drinking causes increased body weight and glucose intolerance in normal male rats. Br. J. Nutr. 93, 613–618. Kershaw, E.E., Flier, J.S., 2004. Adipose tissue as an endocrine organ. J. Clin. Endocrinol. Metab. 89, 2548–2556. Kubota, T., Komatsu, H., Kawamoto, H., Yamada, T., 1997. Studies on the effects of anti-inflammatory action of benzoyl-hydratropic acid (ketoprofen) and other drugs with special reference to prostaglandin synthesis. Arch. Int. Pharmacodyn. Ther. 237, 169–176. Laine, L., 2001. Approaches to nonsteroidal anti-inflammatory drug use in the highrisk patient. Gastroenterology 120, 594–606. Lloret, L.C., Decleves, X., Oppert, J.M., Basdevant, A., Clement, K., Bardin, C., Scherrmann, J.M., Lepine, J.P., Bergmann, J.F., Mouly, S., 2009. Pharmacology of morphine in obese patients: clinical implications. Clin. Pharm. 48, 635–651. Marcus, D.A., 2004. Obesity and the impact of chronic pain. Clin. J. Pain. 20, 186–191. Mazoit, J.X., Sandouk, P., Scherrmann, J.M., Roche, A., 1990. Extrahepatic metabolism of morphine occurs in humans. Clin. Pharmacol. Ther. 48, 613–618. Meunier, C.J., Verbeeck, R.K., 1999. Glucuronidation kinetics of R,S-ketoprofen in adjuvant-induced arthritic rats. Pharm. Res. 16, 1081–1086.
O.A. Jaramillo-Morales et al. / European Journal of Pharmacology 788 (2016) 168–175
Ong, C.K., Lirk, P., Tan, C.H., Seymour, R.A., 2007. An evidence-based update on nonsteroidal anti-inflammatory drugs. Clin. Med. Res. 5, 19–34. Patanwala, A.E., Holmes, K.L., Erstad, B.L., 2014. Analgesic response to morphine in obese and morbidly obese patients in the emergency department. Emerg. Med. J. 31, 139–142. Ramirez, I., 1987. Feeding a liquid diet increases energy intake, weight gain and body fat in rats. J. Nutr. 117, 2127–2134. Ramzan, I., Wong, B.K., Corcoran, G.B., 1993. Pain sensitivity in dietary-induced obese rats. Physiol. Behav. 54, 433–435. Roane, D.S., Martin, J.R., 1990. Continuos sucrose feeding decreases pain threshold and increases morphine potency. Pharmacol. Biochem. Behav. 35, 225–229. Richardson, T.A., Sherman, M., Kalman, D., Morgan, E.T., 2006. Expression of UDP glucuronosyltransferase isoform mRNAs during inflammation and infection in mouse liver and kidney. Drug Metab. Dispos. 34, 351–353. Rossi, H.L., Luu, A.K., DeVilbiss, J.L., Recober, A., 2013. Obesity increases nociceptive activation of the trigeminal system. Eur. J. Pain 17, 649–653. Stubbins, R.E., Holcomb, V.B., Hong, J., Núñez, N.P., 2012. Estrogen modulates abdominal adiposity and protects female mice from obesity and impaired glucose tolerance. Eur. J. Nutr. 51, 861–870. Sugimoto, K., Rashid, I.B., Kojima, K., Shoji, M., Tanabe, J., Tamasawa, N., Suda, T., Yasujima, M., 2008. Time course of pain sensation in rat models of insulin resistance, type 2 diabetes, and exogenous hyperinsulinaemia. Diabetes Metab. Res. Rev. 24, 642–650.
175
Swinburn, B.A., Caterson, I., Seidell, J.C., James, W.P., 2004. Diet, nutrition and the prevention of excess weight gain and obesity. Public. Health Nutr. 7, 123–146. Trayhurn, P., 2005. Endocrine and signaling role of adipose tissue: new perspectives on fat. Acta Physiol. Scand. 184, 285–293. Trujillo, M.E., Scherer, P.E., 2005. Adiponectin-journey from an adipocyte secretory protein to biomarker of the metabolic syndrome. J. Intern. Med. 257, 167–175. Trumbo, P.R., Rivers, C.R., 2014. Systematic review of the evidence for an association between sugar-sweetened beverage consumption and risk of obesity. Nutr. Rev. 72, 566–574. Whiteside, G., Harrison, J., Boulet, J., Mark, L., Pearson, M., Gottshall, S., Walker, K., 2004. Pharmacological consideration of a rat model of incisional pain. Br. J. Pharmacol. 141, 85–91. Wieseler-Frank, J., Maier, S., Watkins, L.R., 2005. Immune-to-brain communication dynamically modulates pain: physiological and pathological consequences. Brain Behav. Immun. 19, 104–111. Wilson, A.C., Samuelson, B., Palermo, T.M., 2010. Obesity in children and adolescents with chronic pain: associations with pain and activity limitations. Clin. J. Pain. 26, 705–711. Zhang, T., Reid, K., Acuff, C.G., Jin, C.B., Rockhold, R.W., 1994. Cardiovascular and analgesic effects of a highly palatable diet in spontaneously hypertensive and Wistar-Kyoto rats. Pharmacol. Biochem. Behav. 48, 57–61. Zimmermann, M., 1983. Ethical guidelines for investigation of experimental pain in conscious animals. Pain 16, 109–110.