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BEHAVIORAL PHARMACOLOGY
-Epoxy-3-hydroxy-14
-methoxy-17-methylmorphinan-6-yl)amino]acetic Acid (HS-731) after Subcutaneous and Oral Administration in Rats with Carrageenan-Induced Hindpaw Inflammation
Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (I.B.-L., Y.G.); Department of Surgical Sciences, Section of Orthopaedics, Karolinska Institutet, Orthopaedic Research Laboratory M3:02, Karolinska Hospital, Stockholm, Sweden (I.B.-L.); and Department of Pharmaceutical Chemistry, Institute of Pharmacy and Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innsbruck, Austria (M.S., J.S., P.W., H.S.)
Received October 6, 2005; accepted December 6, 2005.
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Abstract |
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-epoxy-3-hydroxy-14
-methoxy-17-methylmorphinan-6-yl)amino]acetic acid (HS-731) after s.c. and oral administration in rats with carrageenan-induced hindpaw inflammation. Antinociceptive effects after s.c. administration were assessed 3 h after intraplantar carrageenan injection and compared with those of centrally acting µ-opioid agonists 14-methoxymetopon and morphine. Opioid agonists caused dose-dependent increases in inflamed paw withdrawal latencies to mechanical and thermal stimulation. The time course of action was different, in that HS-731 (20 µg/kg s.c.) produced significant long-lasting effects up to 4 h after administration, whereas 14-methoxymetopon (20 µg/kg) and morphine (2 mg/kg) reached their peak of action at 10 to 30 min, and their effect declined rapidly thereafter. Subcutaneous administration of the peripherally selective opioid antagonist naloxone methiodide inhibited antinociception elicited by HS-731 (20 µg/kg s.c.), whereas it was ineffective against 14-methoxymetopon (20 µg/kg s.c.). Moreover, the antinociception produced by 100 µg/kg s.c. HS-731 was dose-dependently reversed by s.c. naloxone methiodide. This indicates that HS-731 preferentially activates peripheral opioid receptors, whereas 14-methoxymetopon mediates analgesia via central mechanisms. Orally administered HS-731 significantly reduced hyperalgesia in the inflamed paw induced by carrageenan, which was reversible by s.c. administered naloxone methiodide. These results show that systemic (s.c. and oral) treatment with the µ-opioid agonist HS-731 produces potent and long-lasting antinociception through peripheral mechanisms in rats with carrageenan-induced hindpaw inflammation.
). Several studies, however, have challenged the notion that opioids act to produce antinociception exclusively through central mechanisms. Peripheral antinociception in response to different noxious stimuli (e.g., heat, pressure, and chemicals) has been demonstrated in a variety of models and species in which opioids are applied locally at doses that are systemically inactive. These peripheral analgesic effects are enhanced under hyperalgesic and inflammatory conditions (Stein et al., 2001, 2003). The local actions of opioids are mediated via µ-, 
-, and 
-opioid receptors and are reversible by antagonists (Stein et al., 1989; Perrot et al., 1999). Pharmacological, neuroanatomical, molecular, and electrophysiological studies have shown that such opioid receptors are expressed on peripheral terminals of sensory neurons where they can modulate both afferent and efferent neuronal functions, resulting in analgesic action (Ji et al., 1995; Schäfer et al., 1995; Coggeshall et al., 1997).
Opioids are the cornerstone of treatment of severe acute and chronic pain. Current pain therapy relies mostly on the use of opioid analgesics, such as morphine, fentanyl, and oxycodone. However, their clinical use is limited by a number of centrally mediated adverse actions (e.g., respiratory depression, nausea, sedation, tolerance, addiction) (MacPherson, 2002). This fact has stimulated the development of a new generation of opioid drugs acting selectively in the periphery and as a result lacking unwanted CNS side effects (Stein et al., 2003). Strategies to reduce their access to the CNS include chemical modifications at opioid compounds that increase their hydrophilicity, such as incorporation of highly polar hydrophilic substituents (DeHaven-Hudkins and Dolle, 2004). There is considerable evidence that peripherally restricted opioids are effective in different animal models, including inflammatory, visceral, and neuropathic pain, which are frequently resistant to conventional drug treatment in humans (Stein et al., 2003). Loperamide, used as an antidiarrheal drug, was reported as peripherally active antinociceptive µ-opioid agonist when applied locally in an injured paw of the rat (DeHaven-Hudkins et al., 1999). Asimadoline, a -opioid agonist with peripheral activity, induces potent analgesia in animals (Machelska et al., 1999; Walker et al., 1999). Peptidic -opioid ligands have also been identified as peripherally acting antinociceptive agents (Binder et al., 2001). Morphine-6-glucuronide, the active metabolite of morphine, which does not readily penetrate the blood-brain barrier, has been reported to have antinociceptive effects on inflammatory pain, while inducing less nausea and vomiting than morphine in humans (Lötsch and Geisslinger, 2001; Tegeder et al., 2003).
We have recently reported on a family of six-amino acid-substituted derivatives (e.g., glycine, alanine, and phenylalanine) of 14-O-methyloxymorphone, which has been developed in an effort to obtain µ-opioid receptor agonists with restricted access to the CNS (Schütz et al., 2003; Spetea et al., 2004). Potent antinociceptive actions of this class of opioids were described in the tail-flick and the formalin tests in the rat (Fürst et al., 2005). In the present study, we aimed to investigate the antinociceptive effect and the site of action of the 6-glycine conjugate of 14-O-methyloxymorphone, HS-731 (Fig. 1), after s.c. and oral routes of administration, on pain-related behavior in rats with unilateral carrageenan-induced hindpaw inflammation. In addition, antinociceptive effects on inflammatory pain after s.c. administration of 14-methoxymetopon (Fig. 1) (Schmidhammer et al., 1990; Fürst et al., 1993; Zernig et al., 2000; King et al., 2003), a centrally acting µ-opioid receptor agonist, are described.
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) and 14-methoxymetopon (Schmidhammer et al., 1990) were prepared according to the previously described procedures. Morphine (10 mg/ml) was purchased from Pharmacia and Upjohn (Stockholm, Sweden). Carrageenan (
-carrageenan) and naloxone methiodide were obtained from Sigma-Aldrich (St. Louis, MO). Carrageenan (1% solution) was prepared in sterile water. All drugs were dissolved in sterile 0.9% saline solution for s.c. injections or in sterile water for p.o. administration. Animals. Experiments were performed on freely moving male albino Sprague-Dawley rats (250 g; B&K Universal Lab, Sollentuna, Sweden) and were approved by the Ethics Committee for Animal Research (North Stockholm, Sweden). Every effort was made to minimize both the animal suffering and the number of animals used. The rats were housed four per cage for 5 days before use and thereafter acclimatized in the experimental facilities for 1 h before commencement of the experiments. Animals were housed at 21°C in a 12-h light/dark cycle and had free access to food and water. Each animal was used in only one experiment.
Carrageenan-Induced Inflammation. Unilateral inflammation was induced in nonanesthetized rats by injection of 100 µl of 1% carrageenan with a 27-gauge needle into the plantar surface of the right hindpaw. Carrageenan solution was prepared 24 h before each experiment. All treatments were performed 3 h after carrageenan injection. By use of coded syringes, the experimenter was blind to the drugs tested whenever possible. A maximal edema formation and significant nociceptive behavior was developed 3 h postcarrageenan injection (Perrot et al., 1999; Bileviciute-Ljungar and Spetea, 2001). Right and left hindpaw volumes were measured using a plethysmometer (type 7150; Ugo Basile, Comerio, Italy) and expressed in milliliters. The volume of displacement, which is equal to the paw volume, was read on a digital display. For each rat, two measurements were taken for each hindpaw, and the average was calculated and used to quantify percentage of changes. As a sign of inflammation, at 3 h postcarrageenan injection, the right hindpaw volume was increased by 60.9 ± 2.1% from 1.6 ± 0.01 to 2.6 ± 0.04 ml (p < 0.001; n = 70). No increase in the contralateral noninflamed paw volume was detected (1.6 ± 0.01 ml before carrageenan injection and 1.6 ± 0.02 ml at 3 h after induction of inflammation).
Behavioral Tests. Rats were accustomed to each testing condition three times daily for 3 days before the experiments were performed, using the Randall-Selitto and hot-plate test, and varying the testing 50/50 between the two tests. The hindpaw withdrawal latencies (HWLs) during noxious mechanical and thermal stimulation were tested as described previously (Bileviciute-Ljungar and Spetea, 2001). The order of measurements of HWL for the right (inflamed) and left (noninflamed) hindpaws was continually alternated to avoid any possible order effects, and the interval between different stimulations was approximately 3 to 5 min. Two measurements were carried out for each hindpaw and test, and the average value was used to quantify the percentage of changes. All assessments were made at the same time of the day.
The Randall-Selitto test (type 7200; Ugo Basile) was used to assess the HWL to mechanical stimulation. The animal was gently restrained in the experimenter's right hand, and the left hand was used to position the hindpaw of the rat on the plate by gently holding the leg of the animal at the lower part of the shinbone. A wedge-shaped, blunt piston with an area of 1 x 10 mm2 and a loading rate of 48 g/s was applied to the dorsal surface of the manually handled hindpaw, and the time required to initiate the withdrawal of the stimulated hindpaw was assessed and expressed in seconds. A cut-off time of 20 s was applied.
The withdrawal response to noxious heat was determined using the hot-plate test (Karolinska Institutet, Stockholm, Sweden). Rats were handled in the same manner as described above for the Randall-Selitto test. The entire ventral surface of each individual hindpaw was placed on a hot-plate, which was maintained at a temperature of 50°C. The time necessary to elicit paw withdrawal was measured in seconds and subsequently referred to as the HWL to thermal stimulation. The cut-off time was 50 s.
Experimental Protocols. Drugs (HS-731, 14-methoxymetopon, and morphine) or saline (control) were s.c. administered to rats in a volume of 100 µl under the skin of the neck 3 h after intraplantar injection of carrageenan. For dose-response studies, HS-731 or 14-methoxymetopon was injected at doses of 1, 5, and 20 µg/kg. Morphine was applied s.c. in a dose of 2 mg/kg. HWL to mechanical and thermal stimuli was evaluated before injection of carrageenan (basal values), before administration of the drug, 3 h postcarrageenan injection (pretreatment values), and at 10, 30, 60, 120, 180, and 240 min after drug administration.
To evaluate the site of action of HS-731 and 14-methoxymetopon, a dose of 20 µg/kg of the respective opioid agonist was s.c. coinjected with the peripherally selective opioid antagonist naloxone methiodide (20 µg/kg), 3 h after carrageenan injection, in a total volume of 100 µl. To further assess the site of antinociceptive action of 100 µg/kg HS-731, the opioid drug was s.c. coadministered with naloxone methiodide in doses of 100, 200, and 400 µg/kg. Based on the observation that significant antinociceptive effects of s.c. HS-731 were evident not earlier than 2 h after drug administration and more pronounced differences were observed in HWLs to noxious pressure compared with heat stimulus, only HWL to mechanical stimulation was determined at 30, 60, 120, 180, and 240 min after s.c. injection of 100 µg/kg HS-731 alone or together with different doses of naloxone methiodide.
To investigate the effects of oral administration of HS-731, doses of 5 and 10 mg/kg were administered to rats in a volume of 500 µlby using a silicon tip catheter (1.5 mm in diameter and 52 mm in length; AgnTho's AB, Stockholm, Sweden) at 3 h after carrageenan injection. HWL to mechanical stimulation was determined at 30, 60, 120, 180, and 240 min after p.o. drug administration. Control rats received the same volume of sterile water. Pilot studies were performed to identify the dose for oral administration. To assess the site of action of orally administrated HS-731, 400 µg/kg naloxone methiodide was s.c. injected in a volume of 100 µl, before 10 mg/kg of the oral opioid agonist.
Data Analysis. Statistical analysis was carried out using SPSS software (Statistical Product and Service Solutions, release 10.1; SPSS Inc., Chicago, IL). All data are expressed as the mean ± S.E.M. Each experimental group included six to nine rats. A p value <0.05 was considered statistically significant.
The t test for paired samples was used to compare basal versus pretreatment HWL values (3 h postcarrageenan injection). All changes in HWL are presented as percentage of changes from the pretreatment values obtained at 3 h after carrageenan injection that were calculated according to the following equation: % changes = 100 [(PX – P0)/P0], where P is the parameter of nociception, 0 (zero) is the time before drug administration at 3 h postcarrageenan injection, and X is the time after drug administration. Comparisons between the treatment groups throughout the study were performed using analysis of variance (ANOVA) followed by Bonferroni test.
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; Bileviciute-Ljungar and Spetea, 2001), intraplantar injection of carrageenan (1%; 100 µl) causes a significant hyperalgesia as evidenced by a reduction at 3 h postcarrageenan injection in the inflamed hindpaw thresholds to noxious pressure (2.8 ± 0.2 s; –52.0 ± 2.2%; p < 0.001; n = 149) and heat stimuli (8.7 ± 1.1 s; –26.2 ± 4.3%; p < 0.001; n = 78). In the noninflamed paw, a decrease in the HWL to mechanical stimulation was observed (4.9 ± 0.2 s; –15.4 ± 2.3%; p < 0.001; n = 149) at 3 h after carrageenan injection, whereas no significant changes in the HWL to thermal stimulation (9.3 ± 1.2; –26.2 ± 10.8%; n = 78) were noted. Antinociceptive Effects after Subcutaneous Administration of Either HS-731 or 14-Methoxymetopon. Subcutaneous administration of HS-731 and 14-methoxymetopon, 3 h after intraplantar injection of carrageenan, elicited time- and dose-dependent increases in inflamed paw withdrawal latencies to mechanical and thermal stimulation. A time-related antinociceptive effect was produced by 2 mg/kg morphine after s.c. administration in rats with carrageenan-induced hindpaw inflammation.
As shown in Fig. 2, treatment with HS-731, 14-methoxymetopon and morphine produced different time-courses of action. A dose of 20 µg/kg s.c. HS-731 caused a significant reduction in carrageenan-induced hyperalgesia in the inflamed paw not earlier than 2 h after drug administration. The effect produced by HS-731 had the longest duration with significant elevation of HWL to noxious pressure up to 4 h in the inflamed hindpaw after a single s.c. administration. The mean curve of HS-731 (20 µg/kg s.c.) reached a plateau between 2 and 4 h to mechanical stimulation, whereas a maximum effect occurred at 3 h to thermal stimulation (Fig. 2, A and B). In contrast, the onset of the antinociceptive response elicited in the inflamed paw by s.c. administered 20 µg/kg 14-methoxymetopon and 2 mg/kg morphine was rapid, with a peak of action at 10 to 30 min, to both mechanical and thermal stimulation (Fig. 2, C–F). This effect declined rapidly in the case of 14-methoxymetopon, whereas s.c. morphine still produced a significant increase in withdrawal thresholds to noxious heat at 2 h.
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When comparing antinociceptive effects of HS-731 versus 14-methoxymetopon, s.c. administration of 20 µg/kg HS-731 resulted in increased withdrawal latencies of inflamed hindpaws to mechanical stimulation at 3 h (p < 0.05), whereas 20 µg/kg 14-methoxymetopon prolonged latencies to thermal stimulation at 10 min after drug injection (p = 0.056). Administration of morphine (2 mg/kg s.c.) produced elevation in HWLs to mechanical pressure in the inflamed paw at 10 min (p < 0.01) and to thermal stimulation at 10 min (p < 0.05) and 30 min (p < 0.001), compared with animals that received 20 µg/kg s.c. HS-731. No significant difference in changes in HWL of inflamed paws to noxious pressure and heat was detected between the groups treated with 2 mg/kg morphine and 20 µg/kg 14-methoxymetopon (data not shown).
Subcutaneous administration of HS-731 (1, 5, and 20 µg/kg) did not produce any significant increases in withdrawal latencies of noninflamed hindpaws compared with the saline-treated group (data not shown). Nociceptive thresholds to mechanical stimulation were significantly elevated in noninflamed hindpaws at 10 and 30 min (p < 0.05) and at 60 min (p < 0.01) after 2 mg/kg s.c. morphine. A dose of 20 µg/kg s.c. 14-methoxymetopon prolonged latencies to mechanical stimulation in the left paw at 30 min after drug injection (p = 0.057) (data not shown).
Site of Action after Subcutaneous Administration of HS-731 and 14-Methoxymetopon. To establish the site of action (peripheral or central) of HS-731 and 14-methoxymetopon, the peripherally selective opioid antagonist naloxone methiodide was s.c. coadministered with each opioid agonist, 3 h after intraplantar injection of carrageenan in the rat. As shown in Fig. 3, s.c. naloxone methiodide (20 µg/kg) completely inhibited the effects of a 20-µg/kg dose of HS-731 on hyperalgesia. The HWLs to both mechanical and thermal stimulation of inflamed paws were significantly reduced by the antagonist at 2, 3, and 4 h, time points at which a potent antinociceptive action of HS-731 has been demonstrated (Fig. 3, A and C). In contrast, naloxone methiodide (20 µg/kg) was ineffective against the antinociception induced by s.c. administered 20 µg/kg 14-methoxymetopon at 10 and 30 min (Fig. 3, B and D).
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To examine whether the antinociceptive effect of 100 µg/kg s.c. HS-731 in rats with carrageenan-induced hyperalgesia is peripherally mediated, the antagonist effect of naloxone methiodide was assessed (Fig. 4). Concomitant s.c. administration of HS-731 with the antagonist abolished the prolonged HWL to mechanical stimulation in the inflamed paw in a dose-dependent manner compared with administration of the agonist alone. The action of 100 µg/kg HS-731 was fully reversed by 200 and 400 µg/kg opioid antagonist. A dose of 100 µg/kg naloxone methiodide induced a significant decrease in inflamed hindpaw withdrawal latencies at 30 min and 3 h, and a strong tendency toward an inhibitory effect was observed at 1 h (p = 0.072), 2 h (p = 0.074), and 4 h (p = 0.051) (Fig. 4). Moreover, naloxone methiodide s.c. coadministered in doses of 100, 200, and 400 µg/kg with the opioid agonist did not result in any statistically significant inhibition of mechanical hyperalgesia compared with vehicle-treated controls.
Antinociceptive Effects and Site of Action after Oral Administration of HS-731. Oral administration of HS-731, 3 h after intraplantar carrageenan injection, elicited dose-dependent antinociception in inflamed hindpaws to mechanical stimulation (Fig. 5). Compared with the vehicle-treated controls, the antinociceptive action was significant only for the higher tested dose of 10 mg/kg. The effect of p.o. HS-731 started at 30 min and lasted up to 4 h after drug administration. No significant increase in the HWL in the contralateral noninflamed hindpaws was observed after oral HS-731 (data not shown).
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When evaluating the antinociceptive effect in the inflamed paw of 10 mg/kg p.o. HS-731 (Fig. 5) with the action of 100 µg/kg agonist injected s.c. (Fig. 4), a very similar inhibition of mechanical hyperalgesia was detected (n = 8; ANOVA). On the basis of these observations, a dose of 400 µg/kg s.c. naloxone methiodide was used to antagonize the effect of 10 mg/kg p.o. HS-731. As shown in Fig. 6, s.c. pretreatment with the antagonist was able to reverse antinociception produced in the inflamed hindpaw by the orally administered HS-731. No statistical significance was observed compared with vehicle-treated controls.
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The investigated compounds HS-731 and 14-methoxymetopon are two µ-opioid agonists based upon in vitro binding assays and bioassay (Spetea et al., 2003, 2004). They interact with high affinity with µ-opioid receptors in rat brain membranes, 0.83 and 0.15 nM, respectively and showed several-fold greater affinity to µ-opioid receptors than morphine (Ki = 6.55 nM). In the mouse vas deferens bioassay, HS-731 was approximately 2-fold more potent than 14-methoxymetopon, indicating a strong agonist action (Spetea et al., 2004).
In agreement with our previous findings (Bileviciute-Ljungar and Lundeberg, 2000; Bileviciute-Ljungar and Spetea, 2001) and also those of others (Perrot et al., 1999, 2001), intraplantar carrageenan injection induces a significant reduction of thresholds to noxious stimulation (e.g., pressure and heat) and maximal paw edema at 3 h postcarrageenan. Once inflammation has developed, s.c. HS-731, 14-methoxymetopon, and morphine produced a significant attenuation in pain-related behavior by increasing HWLs to pressure and thermal stimuli applied to the inflamed paw. Our results on the inhibition of hyperalgesia by systemic morphine 3 h postcarrageenan support previous observations on the antinociceptive effect of intraperitoneal morphine, 60 min after intraplantar carrageenan injection in rats, with the same short onset (Joris et al., 1990).
Typically, hyperalgesia ceased rapidly upon s.c. administration of 14-methoxymetopon and morphine. The effect reached its peak at 10 to 30 min and declined rapidly thereafter. The fast onset with an early maximum effect that disappears shortly after drug application is a typical feature of the time course of action of opioid drugs (i.e., morphine, fentanyl, and etorphine) that mediate analgesia via central mechanisms and has been depicted under normal and inflammatory conditions (Millan et al., 1987; Aceto et al., 1997; Fürst et al., 2005). In contrast, s.c. HS-731 acted considerably longer (up to 4 h) and dose-dependent reduction of carrageenan-induced hyperalgesia. A long-lasting, dose-dependent antinociceptive effect was also evident after oral administration of HS-731. Similarly, prolonged antinociceptive effects have been reported after systemic administration of morphine-6-glucuronide, a hydrophilic molecule, in the hotplate and tail-flick test in mice (Frances et al., 1992; Lötsch et al., 2000). The long-lasting effects were attributed to a peripheral action that was also demonstrated in inflammatory pain in humans (Tegeder et al., 2003). Another peripheral µ-opioid agonist, loperamide, induces antinociception after local application at the site of inflammation in animals (DeHaven-Hudkins et al., 1999). Our present observations are consistent with recent findings on the potent antinociception elicited by s.c. HS-731 in the tail-flick and the formalin test in rats after s.c. administration (Fürst et al., 2005). Here, we report on the significant and long-lasting reduction in pain-related behavior after s.c. and oral administration of HS-731 in carrageenan-induced hyperalgesia in the rat.
In this study, the antinociceptive effect of 14-methoxymetopon was also investigated in rats with carrageenan-induced inflammation. Administration of a low dose (20 µg/kg s.c.) of 14-methoxymetopon produced a significant attenuation of nociceptive behavior in inflamed paws, demonstrated by increased HWL on application of pressure or heat, showing similar potency to morphine (2 mg/kg s.c.). The profile of the antinociceptive activity of 14-methoxymetopon identified in this study confirms previous reports on acute nociception using different pain stimuli and animal species (rat and mouse) (Schmidhammer et al., 1990; Fürst et al., 1993; Zernig et al., 2000; King et al., 2003). The analgesic effects of 14-methoxymetopon were reversed by naloxone and naltrexone, two opioid antagonists that readily cross the blood-brain barrier (Fürst et al., 1993; Zernig et al., 2000; King et al., 2003), indicating that 14-methoxymetopon has central actions. In the present study, a peripherally selective opioid antagonist, naloxone methiodide (Brown and Goldberg, 1985), was used to assess the site of action of 14-methoxymetopon after s.c. administration in rats with unilateral carrageenan-induced hyperalgesia. As expected, systemic naloxone methiodide failed to antagonize the antinociceptive effect of s.c. 14-methoxymetopon, confirming its central mode of action. In contrast to 14-methoxymetopon, the antinociceptive action on the inflamed paw of a low (20 µg/kg) and a higher (100 µg/kg) dose of s.c. HS-731 was abolished by systemic naloxone methiodide, demonstrating a peripheral opioid receptor-specific mechanism of action. The potent reduction of hyperalgesia after oral administration of HS-731 was also inhibited by s.c. naloxone methiodide, indicating that oral HS-731 preferentially activates peripheral opioid receptors. The present results on the antagonism caused by naloxone methiodide corroborate and extend recent findings in the tail-flick and the formalin test in rats (Fürst et al., 2005) on the peripheral site of action of systemic s.c. HS-731.
Both µ-opioid agonists HS-731 and 14-methoxymetopon are potent antinociceptive agents after s.c. administration under conditions of inflammatory pain. Their mechanism of action is consistent with agonist activity at the µ-opioid receptor, which they activate with low nanomolar potency (Spetea et al., 2003, 2004). However, the difference in the site of action is related to specific structural features (Fig. 1). The lipophilic opioid 14-methoxymetopon exerts its antinociceptive effect via central mechanisms, while HS-731 interacts primarily with opioid receptors located in the periphery. HS-731 contains a hydrophilic group (glycine residue) attached to the C6 position of the morphinan skeleton that is highly ionized at physiological pH. The inclusion of an ionizable group lowers lipophilicity and consequently reduces the penetration into the CNS, causing greater selectivity toward peripheral tissues upon systemic administration.
In the present study, extending our recent findings on antinociceptive effects of HS-731 and derivatives (Fürst et al., 2005), we report on peripherally mediated antinociception after systemic administration in the carrageenan-induced hyperalgesia model. In most of the reported works, peripheral analgesic effects were obtained after local application of µ-([D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, fentanyl, and morphine) (Stein et al., 1988, 1989; Perrot et al., 1999), -(U-50,488H and U-69,593) (Stein et al., 1989; Binder et al., 2001), and -opioid agonists ([D-Pen2,D-Pen5]-enkephalin) (Stein et al., 1989) in the inflamed tissue. Peripheral opioid agonists acting at µ-(loperamide) (DeHaven-Hudkins et al., 1999) and -receptors (asimadoline and FE 200665) (Machelska et al., 1999; Binder et al., 2001) were described as decreasing pain-related behavior after local application, but extremely low or no effect was observed after systemic administration. Systemic rather than local application of opioids with selective peripheral action would be more convenient in many clinical cases.
What are the mechanisms leading to antinociception of peripherally acting µ-opioid agonists such as HS-731? Recent research has shown that opioid-mediated analgesia is not restricted to the CNS. Intrinsic pain control can also occur in the periphery. There is increasing evidence that peripheral opioid antinociception is mediated by opioid receptors located on peripheral sensory neurons (Schäfer et al., 1995; Stein et al., 2003). Pharmacological characteristics of these peripherally occurring receptors are very similar to those of central opioid receptors (Hassan et al., 1993; Zöllner et al., 2003).
One underlying mechanism of peripheral opioid analgesia is an increased number of opioid receptors in the periphery under conditions of tissue injury such as inflammation (Stein et al., 2001, 2003). In dorsal root ganglia, the synthesis and expression of opioid receptors is increased by peripheral tissue inflammation (Ji et al., 1995; Schäfer et al., 1995; Zöllner et al., 2003). Subsequently, the axonal transport of opioid receptors is greatly enhanced (Hassan et al., 1993; Mousa et al., 2001), leading to their up-regulation and enhanced agonist efficacy on peripheral nerve terminals (Zöllner et al., 2003; Shaqura et al., 2004). The specific milieu (low pH and prostanoid release) of inflamed tissue increases opioid agonist efficacy by enhanced G protein coupling and by increased neuronal cyclic adenosine monophosphate levels (Selley et al., 1993; Zöllner et al., 2003; Shaqura et al., 2004). Inflammation also increases the number of sensory nerve terminals in inflamed tissue ("sprouting") and disrupts the perineural barrier (Antonijevic et al., 1995), thus facilitating the access of hydrophilic substances to their receptors. In peripheral tissues, opioids attenuate the excitability of the peripheral nociceptor terminals and the propagation of the action potential. Similar to their effects at the central terminals, opioids inhibit the release of excitatory proinflammatory neuropeptides from peripheral sensory nerve endings, resulting in analgesic actions (Stein et al., 2001, 2003). These mechanisms have been postulated to account for peripheral opioid analgesia (Stein et al., 2001, 2003) and thus may be responsible for the action of peripheral opioid agonists such as HS-731.
In conclusion, the present study shows that the µ-opioid receptor agonist HS-731 dose-dependently produces potent and long-lasting peripherally mediated antinociception after s.c. and oral administration in rats with carrageenan-induced hindpaw inflammation. In addition, the µ-opioid agonist 14-methoxymetopon induces antinociceptive effects in inflammatory hyperalgesia after s.c. administration, through central mechanisms. Our findings suggest that peripheral µ-opioid agonists such as HS-731 may find clinical applications as analgesics in inflammatory pain conditions. Opioid-induced CNS side effects, e.g., respiratory depression, sedation, tolerance, and addiction, can be minimized with drugs that do not readily cross the blood-brain barrier after systemic administration.
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I.B.-L. and M.S. contributed equally to this work.
ABBREVIATIONS: CNS, central nervous system; HS-731, 2-[(4,5-epoxy-3-hydroxy-14-methoxy-17-methylmorphinan-6-yl)amino]acetic acid bis(tetrafluoroborate); HWL, hindpaw withdrawal latency; U-50,488H, trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide; U-69,593, (+)-(5,7,8)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]benzeneacetamide; ANOVA, analysis of variance; FE 200655, H-D-Phe-D-Phe-D-Orn-morpholine amide; NalMeth, naloxone methiodide.
Address correspondence to: Dr. Indre Bileviciute-Ljungar, Department of Rehabilitation Medicine Stockholm, Bldg. 32, Danderyd University Hospital, S-182 88 Stockholm, Sweden. E-mail: indre.ljungar{at}ds.se
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