Involvement of imidazoline and opioid receptors in the enhancement ...

541

Involvement of imidazoline and opioid receptors in the enhancement of clonidine-induced analgesia by sulfisoxazole Mustufa Boxwalla, George Matwyshyn, Bhagya L. Puppala, Shridhar V. Andurkar, and Anil Gulati

Abstract: Clonidine, an a2-adrenergic agonist, has been demonstrated to produce significant analgesia and potentiate morphine analgesia. Endothelin (ETA) receptor antagonists have also been found to potentiate the antinociceptive response to morphine. Clonidine and ET have been reported to have cardiovascular interactions involving the sympathetic nervous system, but it is not known whether ETA receptor antagonist affects clonidine analgesia. This study examined the influence of sulfisoxazole (ETA receptor antagonist) on clonidine analgesia. Male Swiss Webster mice were used to determine antinociceptive response of drugs by measuring tail-flick latency. The effect of clonidine (0.3, 1.0, and 3.0 mg/kg, i.p.) alone or in combination with sulfisoxazole (25, 75, and 225 mg/kg, p.o.) on analgesia and body temperature was determined. Clonidine produced a dose-dependent analgesia and hypothermia. Sulfisoxazole (25, 75, and 225 mg/kg), when administered with clonidine (0.3 mg/kg), significantly potentiated (31% increase in area under the curve (AUC)) the analgesic effect of clonidine. Yohimbine (a2-adrenergic receptor antagonist) did not affect analgesic effect of clonidine plus sulfisoxazole. Idazoxan (I1-imidazoline and a2-adrenergic receptor antagonist) reduced (47% decrease in AUC) the analgesic effect of clonidine plus sulfisoxazole. Treatment with naloxone reduced (46% decrease in AUC) the analgesic effect of clonidine plus sulfisoxazole. The effect of another ETA receptor antagonist, BMS-182874 (2, 10, and 50 mg, i.c.v.) was studied, and it was found that the dose of 10 mg significantly potentiated (26% increase in AUC) the analgesic effect of clonidine. These results indicate that sulfisoxazole, an ETA receptor antagonist, potentiates the analgesic effect of clonidine, which could be mediated through I1-imidazoline receptors and opioid receptors. Key words: adrenergic receptor, analgesia, endothelin, sulfisoxazole, clonidine, nociception, yohimbine, idazoxan, BMS182874, imidazoline. Re´sume´ : On a de´montre´ que l’agoniste adreńergique-a2 clonidine a un effet analge´sique important, et qu’il potentialise l’analge´sie induite par la morphine. On a aussi montre´ que les antagonistes du rećepteur ETA potentialisent la re´ponse antinociceptive a` la morphine. On a rapporte´ qu’il existe une interaction entre la clonidine et l’ET dans les effets cardiovasculaires impliquant le syste`me nerveux sympathique, mais on ignore si un antagoniste du rećepteur ETA aura un impact sur l’analge´sie produite par la clonidine. La pre´sente e´tude a examine´ l’effet du sulfisoxazole (un antagoniste du rećepteur ETA) sur l’analge´sie induite par la clonidine. On a utilise´ le test de latence de re´traction de la queue chez des souris Webster suisses maˆles pour examiner la re´ponse antinociceptive des me´dicaments. On a de´termine´ l’effet de la clonidine (0,3, 1,0 et 3,0 mg/kg, i.p.), seule ou combineé avec le sulfisoxazole (25, 75 et 225 mg/kg, p.o.), sur l’analge´sie et la tempe´rature corporelle. La clonidine a produit une analge´sie et une hypothermie. Combine´ avec la clonidine (0,3 mg/kg), le sulfisoxazole (25, 75 et 225 mg/kg, p.o.), a potentialise´ de manie`re significative (augmentation de 31 % de l’ASC) l’effet analge´sique de la clonidine. La yohimbine (un antagoniste des rećepteurs a2-adreńergiques) n’a pas modifie´ l’effet analge´sique de la combinaison clonidine–sulfisoxazole. L’idazoxane (un antagoniste des rećepteurs a2-adreńergiques et de l’imidazole I1) a diminue´ (diminution de 47 % de l’ASC) l’effet analge´sique de la combinaison clonidine–sulfisoxazole. Le naloxone a atteńue´ (diminution de 46 % de l’ASC) l’effet analge´sique de la combinaison clonidine–sulfisoxazole. Un dose de 10 mg d’un autre antagoniste du rećepteur ETA, BMS-182874 (2, 10 et 50 mg, i.c.v.), a significativement potentialise´ (augmentation de 26 % de l’ASC) l’effet analge´sique de la clonidine. Ces re´sultats indiquent que le sulfisoxazole, un antagoniste du rećepteur ETA, potentialise l’effet analge´sique de la clonidine, et que cet effet pourrait eˆtre ve´hicule´ par les rećepteurs de l’imidazoline I1 et les rećepteurs opioı¨des. Mots-cle´s : rećepteur adreńergique, analge´sie, endothe´line, sulfisoxazole, clonidine, nociception, yohimbine, idazoxane, BMS-182874, imidazoline. Received 26 May 2009. Accepted 19 January 2010. Published on the NRC Research Press Web site at cjpp.nrc.ca on 13 May 2010. M. Boxwalla and B.L. Puppala. Department of Pediatrics and Neonatology, Advocate Lutheran General Children’s Hospital, Park Ridge, IL 60068, USA. G. Matwyshyn, S.V. Andurkar, and A. Gulati.1 Department of Pharmaceutical Sciences, Midwestern University Chicago College of Pharmacy, Downers Grove, IL 60515, USA. 1Corresponding

author (e-mail: [email protected]).

Can. J. Physiol. Pharmacol. 88: 541–552 (2010)

doi:10.1139/Y10-007

Published by NRC Research Press

542

Can. J. Physiol. Pharmacol. Vol. 88, 2010

[Traduit par la Re´daction]

_______________________________________________________________________________________ Introduction Clonidine, an a2-adrenergic agonist, produces analgesia in rodents (Paalzow 1974), but animals treated repeatedly (twice daily for 7 days) with the drug develop tolerance to this effect. It has been proposed that clonidine activates adrenergic and opioid antinociceptive mechanisms in the diencephalic periventricular gray, dorsal raphe nuclei, and periaqueductal gray (Wang et al. 1985). On the other hand, acute clonidine administration enhances the analgesic effect of morphine (Paalzow 1978), pentazocine (Gordon et al. 1992), fentanyl (Constant et al. 1998), and bupivacaine (Hutschala et al. 2004). Although clonidine can suppress opioid withdrawal (Cristea et al. 1993; Ozdogan et al. 2004), and part of its analgesic effect is naloxone sensitive, possibly mediated by release of a b-endorphin-like opioid (Kunos et al. 1987), there is no cross-tolerance between analgesia produced by clonidine and morphine (Spaulding et al. 1979). Theoretically, these properties render clonidine a suitable analgesic substitute in patients tolerant to opioids (Milne et al. 1985). Endothelin (ET) ETA receptor antagonists potentiate the antinociceptive response to morphine in rodents (Bhalla et al. 2002, 2005; Gulati et al. 2004). Repeated or even single injections of ETA receptor antagonists were found to prevent or reverse the tolerance to morphine analgesia in rodents (Bhalla et al. 2003, 2005, 2006). However, ETA antagonists did not aggravate any side effects of morphine such as cataleptic action (Bhalla et al. 2002) or gastrointestinal transit (Matwyshyn et al. 2006). Therefore, ETA receptor antagonists selectively potentiate morphine analgesia without enhancing the side effects of morphine (Gulati et al. 2004). Sulfisoxazole is commonly used to treat bacterial infections of otitis media and has exhibited excellent antibacterial activity. When administered orally, sulfisoxazole is rapidly absorbed and excreted, and it is highly water soluble, thus reducing the renal toxicity inherent in the use of sulfonamides (Mandell and Sande 1991). The inhibitory effect of the sulfanilamides on the binding of ET-1 to ETA and ETB receptors was determined in membrane preparations. Sulfisoxazole was the most active sulfanilamide, with IC50 of 0.60 and 22 mmol/L for ETA and ETB receptors, respectively (Chan et al. 1994). The following reasons prompted us to conduct the present study: (1) clonidine has an analgesic effect and potentiates morphine analgesia (Paalzow 1978; Goudas and Carr 1996); (2) ETA receptor antagonists potentiate morphine analgesia (Bhalla et al. 2002; Gulati et al. 2004); (3) sulfisoxazole is an ETA receptor antagonist (Chan et al. 1994); and (4) clonidine and ETA receptor antagonists have been shown to have cardiovascular interactions (Gulati 1992; MutafovaYambolieva et al. 1992; Gulati and Srimal 1993; Gulati et al. 1997). However, to our knowledge, no study has been performed to determine the effects of the interaction between clonidine and ETA receptor antagonists on analgesia. The purpose of this study was to determine (1) the effect of sulfisoxazole (ETA receptor antagonist) on clonidine analge-

sia and (2) the involvement of a2-adrenergic, I1-imidazoline, and opioid receptors in sulfisoxazole enhancement of clonidine analgesia.

Materials and methods Animals Male Swiss Webster mice weighing 25–30 g (Harlan, Indianapolis, Ind.) were used. The animals were housed 5 per cage in a room with controlled ambient temperature (23 ± 1 8C), humidity (50% ± 10%), and 12 h light : 12 h dark cycle (0600–1800). Food and water were made available ad libitum. Experiments were carried out after the animals had been acclimated to this environment for at least 4 days. Animal care and use for experimental procedures were approved by the Institutional Review Board of Animal Care and Use. All anesthetic and surgical procedures were in compliance with the guidelines established by the Animal Care Committee. Drugs Sulfisoxazole (Sigma Chemical Company, St. Louis, Mo.) was dissolved in carboxymethyl cellulose. Clonidine, idazoxan, and naloxone (Sigma Chemical Company) were dissolved in sterile saline; yohimbine hydrochloride (Sigma Chemical Company) was dissolved in alcohol (1 part) and sterile saline (9 parts). BMS-182874, 5-(dimethylamino)-N(3,4-dimethyl-5-isoxazolyl)-1-naphthalene sulfonamide (Tocris Bioscience, Ellisville, Mo.) was dissolved in 20% DMSO. Determination of tail-flick latency Antinociceptive response to clonidine was determined by the tail-flick latency method of D’Amour and Smith (D’Amour and Smith 1941). Application of thermal stimulation (focused light) to the tail of an animal provokes withdrawal of the tail by a brief, vigorous movement. The reaction time of this movement was recorded as tail-flick latency using an analgesiometer. Tail-flick latencies to thermal stimulation were determined before and at 0, 30, 60, 90, 120, 180, 240, 300, and 360 min after injection of the drug or vehicle. A photoelectric cell stops the timer and switches off the lamp at the moment the tail is withdrawn (Bass and Vander Brook 1952). To prevent any burn injury to the mice, the maximum time of 10 s was used during the experiments. Antinociceptive response in each mouse was converted to a graphical presentation. Tail-flick latency values are expressed as means ± SE. Base-line tail-flick latency without any drug treatment ranged from 1.5 to 2.5 s. Determination of body temperature The change in body temperature in response to drugs was determined (Gulati and Bhargava 1988, 1990). The colonic temperature of each mouse was recorded before and at various times after the injection of clonidine for a period of 360 min using a Cole Palmer Animal Monitoring Thermometer with colonic probe (Vernon Hills, Ill.). The temperature Published by NRC Research Press

Boxwalla et al.

543

Fig. 1. Dose-response effect of clonidine on analgesia and body temperature in mice. Clonidine produced analgesia and a decrease in body temperature. The effect of clonidine on tail-flick latency was dose-dependent. Values are means ± SE; n = 6 per group. *, P < 0.05 for clonidine-treated group versus control group.

(8C) was plotted against time, and values were expressed as means ± SE. The dose of clonidine was selected based on past studies (Gulati 1992; Gulati et al. 1993; Gulati et al. 2009). The data were collected for 6 h based on the duration of action of drugs and the results of previous studies (Gulati and Bhargava 1988, 1990). Study design The dose-response effect of clonidine on analgesia and body temperature was determined in vehicle (1 mL/kg) and clonidine (0.3, 1.0 and 3.0 mg/kg) treated mice. The doseresponse effect of sulfisoxazole on analgesia and body temperature was determined in vehicle (carboxymethyl cellulose (CMC), 1 mL/kg) and sulfisoxazole (25, 75, and 225 mg/kg) treated mice. The dose-response effect of sulfisoxazole plus

clonidine on analgesia and body temperature was determined in mice treated with CMC (10 mL/kg) plus vehicle (1 mL/kg), CMC (10 mL/kg) plus clonidine (0.3 mg/kg), sulfisoxazole (25 mg/kg) plus clonidine (0.3 mg/kg), sulfisoxazole (75 mg/kg) plus clonidine (0.3 mg/kg), and sulfisoxazole (225 mg/kg) plus clonidine (0.3 mg/kg). Clonidine (i.p.) was administered immediately (5 min) after sulfisoxazole (p.o.). The effect of sulfisoxazole plus clonidine on analgesia and body temperature and its blockade by yohimbine was determined in mice treated with vehicle (1 mL/kg) plus CMC (10 mL/kg) plus vehicle (1 mL/kg); yohimbine (2 mg/kg) plus CMC (10 mL/kg) plus vehicle (1 mL/kg); yohimbine (2 mg/kg) plus CMC (10 mL/kg) plus clonidine (0.3 mg/kg); yohimbine (2 mg/kg) plus sulfisoxazole (225 mg/ kg) plus vehicle (1 mL/kg); and yohimbine (2 mg/kg) plus Published by NRC Research Press

544

Can. J. Physiol. Pharmacol. Vol. 88, 2010

Fig. 2. Dose-response effect of sulfisoxazole on analgesia and body temperature in mice. Sulfisoxazole did not produce any effect on analgesia or body temperature. Values are means ± SE; n = 6 per group.

sulfisoxazole (225 mg/kg) plus clonidine (0.3 mg/kg). The effect of sulfisoxazole plus clonidine on analgesia and body temperature and its blockade by idazoxan was determined in mice treated with vehicle (1 mL/kg) plus CMC (10 mL/ kg) plus vehicle (1 mL/kg); idazoxan (2 mg/kg) plus CMC (10 mL/kg) plus vehicle (1 mL/kg); idazoxan (2 mg/ kg) plus CMC (10 mL/kg) plus clonidine (0.3 mg/kg); idazoxan (2 mg/kg) plus sulfisoxazole (225 mg/kg) plus vehicle (1 mL/kg); and idazoxan (2 mg/kg) plus sulfisoxazole (225 mg/kg) plus clonidine (0.3 mg/kg). Yohimbine (i.p.) or idazoxan (i.p.) were administered 15 min before sulfisoxazole. The effect of sulfisoxazole plus clonidine on analgesia and body temperature and its blockade by naloxone was determined in mice treated with naloxone (1 mg/kg) plus CMC (10 mL/kg) plus vehicle (1 mL/kg); naloxone (1 mg/kg) plus CMC (10 mL/kg) plus clonidine (0.3 mg/kg); and naloxone (1 mg/kg) plus sulfisoxazole (225 mg/kg) plus clonidine (0.3 mg/kg). Naloxone (i.p.) was administered 10 min before the administration of sulfisoxazole. The effect of BMS-182874 plus clonidine on analgesia

and body temperature was determined in mice treated with BMS-182874 (2 mg) plus clonidine (0.3 mg/kg), BMS182874 (10 mg) plus clonidine (0.3 mg/kg), and BMS182874 (50 mg) plus clonidine (0.3 mg/kg). BMS-182874 (i.c.v.) was administered 15 min before clonidine. Statistics All data values are presented as means ± SE and represent the result of 6 animals per group. Area under the curve (AUC) was determined using GraphPad Prism version 4.00 for Windows (San Diego, Calif.). Multivariate analysis of variance followed by a post-hoc test (Bonferroni’s test) was performed using GraphPad Prism version 4.00 for Windows. A value of P