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Pain Medicine  |   June 2001
Analgesic and Antiinflammatory Effects of Two Novel κ-Opioid Peptides
Author Affiliations & Notes
  • Waltraud Binder, Ph.D.
    *
  • Halina Machelska, Ph.D.
    *
  • Shaaban Mousa, Ph.D.
    *
  • Thomas Schmitt, M.D.
    *
  • Pierre J. M. Rivière, Ph.D.
  • Jean-Louis Junien, Ph.D.
  • Christoph Stein, M.D.
    §
  • Michael Schäfer, M.D.
  • * Postdoctoral Fellow, § Professor and Chairman, ∥ Associate Professor, Department of Anesthesiology and Critical Care Medicine, Klinikum Benjamin Franklin, Freie Universität Berlin. † Head of Biology, Ferring Research Institute Inc. ‡ Chief Scientific Officer, Ferring Pharmaceuticals.
  • Received from the Department of Anesthesiology and Critical Care Medicine, Klinikum Benjamin Franklin, Freie Universität Berlin, Berlin, Germany; Ferring Research Institute Inc., San Diego, California; and Ferring Pharmaceuticals, Paris, France.
Article Information
Pain Medicine
Pain Medicine   |   June 2001
Analgesic and Antiinflammatory Effects of Two Novel κ-Opioid Peptides
Anesthesiology 6 2001, Vol.94, 1034-1044. doi:
Anesthesiology 6 2001, Vol.94, 1034-1044. doi:
OPIOID analgesics, despite their side effects, are unsurpassed in the pharmacologic treatment of severe pain. They exert their diverse physiologic effects through three distinct membrane-bound receptor subtypes—μ, δ, and κ—in the central nervous system 1 and in the periphery. 2 
In addition to analgesia, each opioid receptor subtype mediates specific additional responses. For example ,  euphoria, physical dependence, and respiratory depression are mainly associated with μ and δ receptors. 1 Spinal, supraspinal, and peripheral μ-receptors also mediate opioid-induced gastrointestinal transit inhibition and constipation. 3 In contrast, opioids acting at κ-receptors produce dysphoric rather than euphoric effects, which limits their dependence liability and produces little or no respiratory depression. Furthermore, κ-agonists do not inhibit intestinal transit or induce constipation. Thus, κ-opioids offer advantages in comparison to μ-opioids and are currently being pursued as analgesic agents.
However, centrally acting κ-opioids can produce psychotomimesis and may be aversive in humans. 4 A potentially successful strategy to eliminate the adverse side effects of κ-opioids is to restrict the access of these compounds to the central nervous system, as opioid analgesia can also be brought about by the activation of opioid receptors on peripheral nerve endings, particularly during inflammatory conditions. 5–7 Clinical studies have shown that intraarticular morphine relieves acute postoperative as well as chronic arthritic pain. 8 The analgesic effect is long-lasting, e.g.  , up to 2 and 7 days, respectively. This is not only explained by an inhibition in pain transmission but also by a local antiinflammatory effect. 8 
Previous attempts to design peripherally selective κ-agonists have not been successful. These attempts were based on small organic molecules. Recently, novel and peptidic κ-ligands were identified by positional scanning of a tetrapeptide combinatorial library screened in opioid receptor radioligand binding assays. 9 These new κ-ligands have an all D-amino acids sequence and show high selectivity for the κ-receptor. Furthermore, this new class of κ-ligands are potent κ-agonists in vitro  , potent antinociceptive agents in vivo  , and peripherally selective, as shown by the lack of sedative activity in the mouse rotarod after peripheral administration. 10 However, these series of new κ-agonists are short-acting. Chemical optimization has led to the characterization of a second generation of tetrapeptide κ-agonists with high peripheral selectivity and long duration of action. 10 In the current study we examined two of these novel compounds, FE 200665 and FE 200666, which show high affinity subnanomolar and high selectivity for human κ-opioid receptor versus  μ-opiod receptor (up to 90,000-fold) or δ-opiod receptor (85,000-fold). 10 The antinociceptive and antiinflammatory properties of these potentially peripherally selective κ-opioid peptides were assessed and compared with the standard κ-opioid U-69,593.
Materials and Methods
Induction of Arthritis
Experiments were conducted in male Wistar rats weighing 250–300 g. Rats were housed individually and kept in a temperature-controlled room (22°C ± 1) with a 12-h alternating light–dark cycle. To induce inflammation, rats were sedated by brief halothane anesthesia and received an intraplantar injection of 150 μl Freund complete adjuvant (FCA; Calbiochem, San Diego, CA) into the right hind paw. Experiments and animal care were conducted in accordance with standard ethical guidelines (Tierschutz komission, Berlin, Germany).
Nociceptive Thresholds
Between 4 and 6 days after induction of inflammation, mechanical nociceptive thresholds were assessed using a paw pressure analgesymeter (Ugo Basile, Comerio, Italy). The paw pressure threshold (PPT) required to elicit hind-paw withdrawal was determined by averaging three consecutive trials separated by 10 s. The sequence of left and right paws was alternated between animals to avoid bias. The experimenter was blind to the conditions used. Data are represented as percent maximum possible effect (MPE) with an arbitrary cutoff point of 250 g and are calculated by the formula: (PPT treated − PPT pretreated/250 − PPT pretreated) × 100.
Evaluation of Inflammation
Antiinflammatory effects were assessed by monitoring paw volume with a plethysmometer (Ugo Basile) and by histologic examination. Histologic sections were prepared as follows. Rats were deeply anesthetized with halothane and perfused transcardially with 100 ml of 0.1 m phosphate-buffered saline (pH 7.4) and 300 ml cold phosphate-buffered saline containing 4% paraformaldehyde and 14% saturated picric acid (pH 7.4) (fixative solution). The hind limbs were removed, postfixed for 42 h at 4°C, and washed for 30 min in distilled water. Decalcification was performed with commercially available hydrochloric acid–based decalcifying agent (Decal; Decal Chemical Corporation, Congers, NY). After decalcification, the tissues were washed in water for 30 min and cryoprotected overnight at 4°C in phosphate-buffered saline containing 10% sucrose. The tissues were then embedded in tissue-Tek compound (O.C.T.; Miles Inc., Elkhart, IN) and frozen at −20°C. Sections, 6-μm thick, were cut on a cryostat, mounted onto gelatin-coated slides, and stained with hematoxylin and eosin. The tissues were oriented to allow longitudinal sections to be cut so as to show the dorsoventral faces of the tarsal, metatarsal, and phalangeal joints, bones, and soft tissue on each slide. Each section was quantitatively evaluated by two observers, who were blind to the treatment regimen, using the following criteria: (1) periarticular inflammation (density of inflammatory cells); (2) pannus formation (degree of intrusion of granulation tissue into the joint space); and (3) periosteal reaction (extent of new bone formation). A rating scale from 0 to 8 was used for each variable (i.e.  , maximum obtainable score = 24). This procedure was based on the methodology originally described by Ackerman et al.  11 
Drugs
We used the κ-opioid agonists U-69,593 (Sigma, Deisenhofen, Bavaria, Germany), FE 200665 and FE 200666 (Ferring Research, San Diego, CA), and the opioid antagonists nor-binaltorphimine (nor-BNI) and naloxone methiodide (NLXM; Sigma). All opioid drugs were dissolved in distilled water and administered intraplantarly (100 μl), subcutaneously into a skin fold in the neck (200 μl), or intrathecally (10 μl). Antagonists were given either concomitantly or 5 min before agonist administration. Control animals received distilled water.
Implantation of Intrathecal Catheters
Rats were handled and trained in the test situation for 3 days before intrathecal catherization. Anesthesia was induced and maintained with 2% halothane via  a loose-fitting plastic mask. The intrathecal catheters were prepared according to a method described previously. 12 Briefly, a polyethylene tubing (PE-10; Portex, United Kingdom) was cut in 200-mm lengths and inserted 15 mm in a cervical direction, through a previously made incision at the L3–L4 level. The animals were allowed 4 days to recover from anesthesia and surgery. During this time, animals showing any sign of neurologic damage were discarded from the study.
Drugs were injected in a volume of 10 μl followed by 5 μl of vehicle to flush the catheter. The effectiveness of the catheter was tested by an injection of 10 μl lidocaine 2% (Sigma) 24 h before the experiment. Only animals experiencing an immediate yet reversible paralysis of their hind limbs were included in the study. After the experiments, in which the animals were used only once, the rats were killed, and the correct position of the catheter tip was confirmed at autopsy by an experienced investigator who was blinded to the experimental results.
Experimental Protocols
Nociceptive Thresholds.
Timecourse of Drug Action.
The antinociceptive effects of intraplantar or subcutaneous FE 200665, FE 200666, and U-69,593 were examined as a function of time, in both ipsilateral and contralateral paws (n = 6 per group). Baseline (pre-agonist) PPTs were obtained and then reevaluated at 5, 10, and 30 min, and 1, 6, 24, and 48 h after opioid administration. Control rats received distilled water.
Dose–Response Relations.
The antinociceptive dose–response relation of the opioid agonists FE 200665 and FE 200666 were determined (n = 6 per group) and compared with U-69,593. After baseline measurements, animals received intraplantar injections of water or FE 200665 at 3, 10, 30, or 100 μg/paw; FE 200666 at 1, 3, 10, 30, or 100 μg/paw; and U-69,593 at 25, 50, 100, or 200 μg/paw in both hind paws. Equivalent doses were also administered subcutaneously into a skin fold of the neck to exclude a central site of action.
Receptor Specificity and Site of Action.
The κ-opioid antagonist nor-BNI, as well as the peripherally selective opioid antagonist NLXM, were administered with FE 200665, FE 200666, or U-69,593 (intraplantarly) to determine the κ-specificity as well as the site of action (peripheral or central) of these opioid agonists (n = 6 per group). nor-BNI was administered either concomitantly (50, 100, or 200 μg/paw, intraplantarly) or immediately before the opioid agonists (400 μg/rat, subcutaneously). NLXM was administered subcutaneously 5 min before intraplantar opioid agonists (2.5, 5, and 10 mg/kg). Finally, to confirm peripheral selectivity, the most potent κ-opioid peptide, FE 200665, was administered intrathecally in a dose range of 1–10 μg. The most effective dose was then coadministered with NLXM (10 mg/kg subcutaneously). In addition, NLXM was administered intrathecally (10 μg) together with intraplantar FE 200665 (100 μg).
Antiinflammatory Effects.
Time Course and Dose Response.
The antiinflammatory effects of FE 200665, FE 200666, and U-69,593 were examined as a function of dose and time in both ipsilateral and contralateral paws (n = 6 per group). Baseline paw volumes were obtained and then reevaluated according to the time and dose schedule for antinociceptive effects. In addition, paw volume was also monitored daily from the induction of inflammation (day 0) until day 6, when the rats were killed. Drugs were administered either daily or by single injection simultaneously with FCA on day 0. After the animals were killed, the hind limbs were removed for histologic assessment.
Receptor Specificity and Site of Action.
The specificity and site of action of the antiinflammatory effects of the three κ-opioids were assessed by concomitant administration of nor-BNI (200 μg/day intraplantarly) or NLXM (10 mg/kg subcutaneously). The antagonists were injected immediately before κ-agonist administration either daily or by single injection on day 0.
Statistical Analysis
Data are represented as mean ± SEM. Dose–response curves and antagonist data were assessed by analysis of variance followed by a linear regression analysis or a post hoc  Dunnett test for non–linearly distributed antagonist data. Time course data were analyzed by two-way repeated-measure analysis of variance (treatment × time) followed by a post hoc  Dunnett test. All data were specifically tested for normality (Skewness and Kurtosis normality tests). Differences were considered significant at P  < 0.05. All tests were performed using Sigma Stat 2.03 statistical software. ED50values were calculated using Pharm/PCS pharmacological calculations with computer programs (Philadelphia, PA).
Results
Nociceptive Thresholds
Time Course of Drug Action.
Treatment with FE 200665 and FE 200666 produced a similar time course of drug action (figs. 1A–1D). A peak was observed 5 min after opioid administration for both drugs; thereafter, PPT decreased. Intraplantar FE 200665 produced a significant antinociceptive effect in inflamed paws until 30 min after drug administration. By 1 h, PPT had returned to baseline. In noninflamed paws, FE 200665 (100 μg) was markedly less potent but still produced a significant effect (33 ± 4.8%). FE 200666 produced a significant antinociceptive effect until 10 min after intraplantar administration in inflamed paws. Similar to FE 200665, administration into the noninflamed paw was much less effective (data not shown). The time course produced by U-60,593 was of longer duration. Significant elevation of PPT was observed until 1 h after intraplantar administration to inflamed paws, returning to baseline by 2 h. Maximum antinociception occurred at 10 min (fig. 1E). In all cases systemic administration resulted in a loss of potency (figs. 1B, 1D, and 1F). Control animals treated with distilled water showed no effect.
Fig. 1. Time course and dose–response of the antinociceptive effects (% maximum possible effect [MPE]) of FE 200665, FE 200666, and U-69,593 on paw pressure threshold in inflamed paws. FE 200665 administered (A  ) intraplantarly and (B  ) subcutaneously; FE 200666 administered (C  ) intraplantarly and (D  ) subcutaneously; and U-69,593 administered (E  ) intraplantarly and (F  ) subcutaneously. *Significant difference from controls, P  < 0.05, analysis of variance (Dunnett test).
Fig. 1. Time course and dose–response of the antinociceptive effects (% maximum possible effect [MPE]) of FE 200665, FE 200666, and U-69,593 on paw pressure threshold in inflamed paws. FE 200665 administered (A 
	) intraplantarly and (B 
	) subcutaneously; FE 200666 administered (C 
	) intraplantarly and (D 
	) subcutaneously; and U-69,593 administered (E 
	) intraplantarly and (F 
	) subcutaneously. *Significant difference from controls, P 
	< 0.05, analysis of variance (Dunnett test).
Fig. 1. Time course and dose–response of the antinociceptive effects (% maximum possible effect [MPE]) of FE 200665, FE 200666, and U-69,593 on paw pressure threshold in inflamed paws. FE 200665 administered (A  ) intraplantarly and (B  ) subcutaneously; FE 200666 administered (C  ) intraplantarly and (D  ) subcutaneously; and U-69,593 administered (E  ) intraplantarly and (F  ) subcutaneously. *Significant difference from controls, P  < 0.05, analysis of variance (Dunnett test).
×
Dose–Response Relations.
FE 200665, FE 200666, and U-69,593 dose-dependently increased PPT in inflamed paws (figs. 1A–1F) when administered intraplantarly. A comparable MPE of approximately 85% was observed for FE 200665 and U-69,593 in inflamed paws (figs. 1A and 1E). However, FE 200666 was markedly less effective, producing an MPE of only 35% (fig. 1C). Higher doses of FE 20066 (up to 100 μg) produced no additional effect (data not shown); therefore, its low activity is likely to be a result of decreased efficacy. Systemically administered, much higher doses were necessary to produce an equivalent antinociceptive response for all opioids tested (figs. 1B, 1D, and 1F). Indeed, subcutaneous administration of FE 200665 (0.2, 0.6, 2, 6, and 20 mg/rat) and FE 200666 (0.06, 0.2 0.6, 2, and 6 mg/rat) was only effective in inflamed paws at the higher doses tested, greater than 2 and 0.6 mg/rat, respectively (figs. 1B and 1D), and ineffective in noninflamed paws (data not shown). Subcutaneous administration of U-69,593 (200, 400, and 800 μg/rat) dose-dependently increased PPT in both paws—maximum 85% ipsilateral (fig. 1F), 42% contralateral (data not shown)—although the dose–response curve was shifted to the right, i.e.  , an eight times higher intraplantar dose was needed to produce the same MPE (figs. 1E and 1F).
Receptor Specificity
The κ-specificity of intraplantar FE 200665 and FE 200666 was determined using the κ-opioid antagonist nor-BNI and compared with that of U-69,593. nor-BNI dose-dependently attenuated opioid-induced antinociception for all three drugs in both inflamed and noninflamed paws (dose range, 50–200 μg/paw;fig. 2). The highest dose (200 μg/paw) completely abolished the antinociceptive response of all opioids tested (figs. 2A–2C). This indicates that the analgesic actions of these opioids are mediated via  κ-receptors.
Fig. 2. Dose–response effects of intraplantar nor-BNI on the antinociceptive action (% maximum possible effect [MPE]) of (A  ) FE 200665 (100 μg), (B  ) FE 200666 (30 μg), and (C  ) U-69,593 (100 μg). P  < 0.05, analysis of variance (linear regression).
Fig. 2. Dose–response effects of intraplantar nor-BNI on the antinociceptive action (% maximum possible effect [MPE]) of (A 
	) FE 200665 (100 μg), (B 
	) FE 200666 (30 μg), and (C 
	) U-69,593 (100 μg). P 
	< 0.05, analysis of variance (linear regression).
Fig. 2. Dose–response effects of intraplantar nor-BNI on the antinociceptive action (% maximum possible effect [MPE]) of (A  ) FE 200665 (100 μg), (B  ) FE 200666 (30 μg), and (C  ) U-69,593 (100 μg). P  < 0.05, analysis of variance (linear regression).
×
Peripheral Selectivity
Subcutaneous versus  Intraplantar nor-BNI.
The antinociceptive response produced by intraplantar FE 200665 and FE 200666 was completely abolished, and that of intraplantar U-69,593 was almost completely abolished, by intraplantar nor-BNI (200 μg/paw;figs. 3A–3C). Subcutaneous nor-BNI (400 μg/rat) had no effect on the intraplantar agonist response. The antinociceptive effects of subcutaneous FE 200665 (2 mg/rat) and FE 200666 (600 μg/rat) in both inflamed and noninflamed paws were also abolished by intraplantar nor-BNI (200 μg/paw), indicative of a peripheral mode of action (figs. 3A and 3B). Although intraplantar nor-BNI significantly attenuated subcutaneous U-69,593 (800 μg/rat) in inflamed paws, the antagonism was not complete (fig. 3C). However, higher doses of nor-BNI were completely able to abolish the antinociceptive effect of U-69,593 (data not shown).
Fig. 3. Effect of nor-BNI (200 μg intraplantarly or 400 μg subcutaneously) on the antinociceptive action (% maximum possible effect [MPE]) of (A  ) FE 200665 (100 μg/paw intraplantarly or 2 mg/rat subcutaneously), (B  ) FE 200666 (30 μg/paw intraplantarly or 600 μg/rat subcutaneously), and (C  ) U-69,593 (100 μg/paw intraplantarly or 800 μg/rat subcutaneously). *Significant difference from agonist-treated animals, P  < 0.05, analysis of variance (Dunnett test). i.pl. = intraplantar; s.c. = subcutaneous.
Fig. 3. Effect of nor-BNI (200 μg intraplantarly or 400 μg subcutaneously) on the antinociceptive action (% maximum possible effect [MPE]) of (A 
	) FE 200665 (100 μg/paw intraplantarly or 2 mg/rat subcutaneously), (B 
	) FE 200666 (30 μg/paw intraplantarly or 600 μg/rat subcutaneously), and (C 
	) U-69,593 (100 μg/paw intraplantarly or 800 μg/rat subcutaneously). *Significant difference from agonist-treated animals, P 
	< 0.05, analysis of variance (Dunnett test). i.pl. = intraplantar; s.c. = subcutaneous.
Fig. 3. Effect of nor-BNI (200 μg intraplantarly or 400 μg subcutaneously) on the antinociceptive action (% maximum possible effect [MPE]) of (A  ) FE 200665 (100 μg/paw intraplantarly or 2 mg/rat subcutaneously), (B  ) FE 200666 (30 μg/paw intraplantarly or 600 μg/rat subcutaneously), and (C  ) U-69,593 (100 μg/paw intraplantarly or 800 μg/rat subcutaneously). *Significant difference from agonist-treated animals, P  < 0.05, analysis of variance (Dunnett test). i.pl. = intraplantar; s.c. = subcutaneous.
×
Naloxone Methiodide.
The peripherally selective systemic opioid antagonist NLXM (2.5–10 mg/kg) dose-dependently attenuated intraplantar FE 200665–, FE 200666–, and U-69,593–induced antinociception in inflamed paws (figs. 4A–4C). The antinociceptive effects in noninflamed paws, as well as those of subcutaneous administration of these opioid agonists, were also abolished by 10 mg/kg NLXM (figs. 4A–4C).
Fig. 4. Dose–response effects of naloxone methiodide (NLXM, administered subcutaneously) on the antinociceptive action (% maximum possible effect [MPE]) of (A  ) FE 200665 (100 μg/paw intraplantarly or 2 mg/rat subcutaneously), (B  ) FE 200666 (30 μg/paw intraplantarly or 600 μg/rat intraplantarly), and (C  ) U-69,593 (100 μg/paw intraplantarly). P  < 0.05, analysis of variance (linear regression). i.pl. = intraplantar; s.c. = subcutaneous.
Fig. 4. Dose–response effects of naloxone methiodide (NLXM, administered subcutaneously) on the antinociceptive action (% maximum possible effect [MPE]) of (A 
	) FE 200665 (100 μg/paw intraplantarly or 2 mg/rat subcutaneously), (B 
	) FE 200666 (30 μg/paw intraplantarly or 600 μg/rat intraplantarly), and (C 
	) U-69,593 (100 μg/paw intraplantarly). P 
	< 0.05, analysis of variance (linear regression). i.pl. = intraplantar; s.c. = subcutaneous.
Fig. 4. Dose–response effects of naloxone methiodide (NLXM, administered subcutaneously) on the antinociceptive action (% maximum possible effect [MPE]) of (A  ) FE 200665 (100 μg/paw intraplantarly or 2 mg/rat subcutaneously), (B  ) FE 200666 (30 μg/paw intraplantarly or 600 μg/rat intraplantarly), and (C  ) U-69,593 (100 μg/paw intraplantarly). P  < 0.05, analysis of variance (linear regression). i.pl. = intraplantar; s.c. = subcutaneous.
×
Intrathecal Administration.
FE 200665 produced a dose-dependent antinociceptive response when administered intrathecally (fig. 5A). The peripherally selective opioid antagonist NLXM (10 μg/kg) was completely without effect when administered systemically with intrathecal FE 200665 (10 μg). Similarly, when NLXM was administered intrathecally (10 μg), it was completely ineffective against peripherally administered FE200665 (100 μg;fig. 5B).
Fig. 5. Antinociceptive effects (% maximum possible effect [MPE]) of intrathecal [i.t.] FE 200665. (A  ) Dose–response relation and (B  ) antagonism by naloxone methiodide (NLXM; administered subcutaneously and intrathecally). P  < 0.05, analysis of variance (linear regression). s.c. = subcutaneous; i.pl. = intraplantar.
Fig. 5. Antinociceptive effects (% maximum possible effect [MPE]) of intrathecal [i.t.] FE 200665. (A 
	) Dose–response relation and (B 
	) antagonism by naloxone methiodide (NLXM; administered subcutaneously and intrathecally). P 
	< 0.05, analysis of variance (linear regression). s.c. = subcutaneous; i.pl. = intraplantar.
Fig. 5. Antinociceptive effects (% maximum possible effect [MPE]) of intrathecal [i.t.] FE 200665. (A  ) Dose–response relation and (B  ) antagonism by naloxone methiodide (NLXM; administered subcutaneously and intrathecally). P  < 0.05, analysis of variance (linear regression). s.c. = subcutaneous; i.pl. = intraplantar.
×
Antiinflammatory Effects
Effect of Dose and Time Schedule.
Paw volume measurements were used to assess the antiinflammatory effects of all three κ-opioids. A significant decrease in ipsilateral (inflamed) paw volume was observed at 24 and 48 h after intraplantar and subcutaneous treatment with a single injection of FE 200665 (30 and 100 μg/paw intraplantarly and 2 mg/rat subcutaneously;fig. 6A). FE 200666 and U-69,593 also produced a reduction in paw volume at 24 and 48 h at 30 μg/paw intraplantarly and 100 μg/paw intraplantarly, respectively (figs. 6B and 6C). No effect was observed in noninflamed paws. The antiinflammatory effects of these κ-agonists became evident at a much later time points than their antinociceptive effects. Early and continuous treatment resulted in an increased effectiveness of all three opioids (fig. 7). There was little difference between a 6-day treatment course and treatment given only once on initiation of inflammation.
Fig. 6. Effect of intraplantar κ-opioids on ipsilateral paw volume 4 days after administration of Freund complete adjuvant, as a function of time. (A  ) FE 200665, (B  ) FE 200666, and (C  ) U-69,593. *Significant difference from controls, P  < 0.05, analysis of variance (Dunnett test).
Fig. 6. Effect of intraplantar κ-opioids on ipsilateral paw volume 4 days after administration of Freund complete adjuvant, as a function of time. (A 
	) FE 200665, (B 
	) FE 200666, and (C 
	) U-69,593. *Significant difference from controls, P 
	< 0.05, analysis of variance (Dunnett test).
Fig. 6. Effect of intraplantar κ-opioids on ipsilateral paw volume 4 days after administration of Freund complete adjuvant, as a function of time. (A  ) FE 200665, (B  ) FE 200666, and (C  ) U-69,593. *Significant difference from controls, P  < 0.05, analysis of variance (Dunnett test).
×
Fig. 7. Effect of daily treatment and a single injection of (A  ) FE 200665, (B  ) FE 200666, and (C  ) U-69,593 on paw volume as a function of time. *Significant difference from controls, P  < 0.05, analysis of variance (Dunnett test). FCA = Freund complete adjuvant.
Fig. 7. Effect of daily treatment and a single injection of (A 
	) FE 200665, (B 
	) FE 200666, and (C 
	) U-69,593 on paw volume as a function of time. *Significant difference from controls, P 
	< 0.05, analysis of variance (Dunnett test). FCA = Freund complete adjuvant.
Fig. 7. Effect of daily treatment and a single injection of (A  ) FE 200665, (B  ) FE 200666, and (C  ) U-69,593 on paw volume as a function of time. *Significant difference from controls, P  < 0.05, analysis of variance (Dunnett test). FCA = Freund complete adjuvant.
×
Receptor Specificity and Site of Action.
Concomitant administration of κ-antagonist nor-BNI (200 μg) and peripherally selective antagonist NLXM (10 mg/kg) significantly antagonized the antiinflammatory effects of FE 200665, FE 200666, and U-69,593 judged by paw volume (figs. 8A–8C) or histologic score (fig. 9). Histologic assessment concurred with paw volume measurements and showed an approximate 50% reduction in arthritic parameters for all three κ-opioids (fig. 9). Thus, the antiinflammatory as well as the antinociceptive effects of these κ-opioids are mediated via  κ-receptors in the periphery.
Fig. 8. Effect of nor-BNI (administered intraplantarly) or naloxone methiodide (NLXM; administered subcutaneously) on the antiinflammatory action of (A  ) FE 20065, (B  ) FE 200666, and (C  ) U-69,593. *Significant difference from antagonist-treated groups, P  < 0.05, analysis of variance (Dunnett test). FCA = Freund complete adjuvant.
Fig. 8. Effect of nor-BNI (administered intraplantarly) or naloxone methiodide (NLXM; administered subcutaneously) on the antiinflammatory action of (A 
	) FE 20065, (B 
	) FE 200666, and (C 
	) U-69,593. *Significant difference from antagonist-treated groups, P 
	< 0.05, analysis of variance (Dunnett test). FCA = Freund complete adjuvant.
Fig. 8. Effect of nor-BNI (administered intraplantarly) or naloxone methiodide (NLXM; administered subcutaneously) on the antiinflammatory action of (A  ) FE 20065, (B  ) FE 200666, and (C  ) U-69,593. *Significant difference from antagonist-treated groups, P  < 0.05, analysis of variance (Dunnett test). FCA = Freund complete adjuvant.
×
Fig. 9. Histologic assessment of the antiinflammatory effects of κ-opioid treatment and antagonism by nor-BNI (200 μg intraplantarly) or naloxone methiodide (NLXM; 10 mg/kg). Histologic scores judged by periarticular inflammtion, pannus, and new bone formation (maximum obtainable score = 24). *Significant difference from controls, P  < 0.05, analysis of variance (Dunnett test).
Fig. 9. Histologic assessment of the antiinflammatory effects of κ-opioid treatment and antagonism by nor-BNI (200 μg intraplantarly) or naloxone methiodide (NLXM; 10 mg/kg). Histologic scores judged by periarticular inflammtion, pannus, and new bone formation (maximum obtainable score = 24). *Significant difference from controls, P 
	< 0.05, analysis of variance (Dunnett test).
Fig. 9. Histologic assessment of the antiinflammatory effects of κ-opioid treatment and antagonism by nor-BNI (200 μg intraplantarly) or naloxone methiodide (NLXM; 10 mg/kg). Histologic scores judged by periarticular inflammtion, pannus, and new bone formation (maximum obtainable score = 24). *Significant difference from controls, P  < 0.05, analysis of variance (Dunnett test).
×
Discussion
The current study found the novel κ-opioid peptide FE 200665 to be a potent analgesic agent, with an antinociceptive effect comparable to that of U-69,593. Both FE compounds showed no overt central effects such as sedation or respiratory depression. Rivière et al.  10 previously showed via  the rotarod test in mice, that central nervous system–mediated sedation occurred at 643- and 84-fold higher doses in comparison to analgesic doses, for FE 200665 and FE 200666, respectively. All three opioids dose-dependently increased PPT (figs. 1A–1D), although FE 200666 was markedly less effective (33% MPE) in comparison to FE 200665 (85% MPE) and U-69,593 (86% MPE). The duration of the antinociceptive effects produced by these agents was noticeably less for the two opioid peptides than for U-69,593. Although the FE compounds maintained an analgesic effect for only 30 min, this is considerably longer than other κ-peptides, which has been attributed to the rapid degradation rate of peptides in vivo  . 13 The FE compounds, shown here to be peripherally selective, as well as U-69,593, also show greater antinociception in inflamed than in normal paws (fig. 2), consistent with current literature. The efficacy and potency of peripheral opioid analgesics in general is greatly enhanced during inflammatory conditions. 7,14 This phenomenon has been extensively examined during conditions such as neuropathic pain, 15 visceral pain, 16 bone damage, 17 and inflammation of subcutaneous tissue, viscera, or joints, induced by FCA, formalin, carrageenan, prostaglandin, or neurogenic inflammation. 5–7 
The receptor specificity of FE 200665, FE 200666, and U-69,593 were investigated with the κ-antagonist nor-BNI. All three opioids administered intraplantarly were dose-dependently antagonized by intraplantar nor-BNI (figs. 2A–2C), indicative of κ-opioid selectivity. The peripheral selectivity of these opioids was then assessed by the peripherally selective antagonist NLXM, a general opioid antagonist whose access to the brain is restriced via  the incorporation of a quarternary ammonium ion, as well as subcutaneous (distant) versus  intraplantar (local) treatment with nor-BNI. NLXM completely abolished the effect of FE 200665 and FE 200666 and markedly attenuated that of U-69,593. When these opioid agonists were administered subcutaneously, the antinociceptive effect of U-69,593 was not completely abolished by intraplantar nor-BNI, in contrast to the FE compounds (fig. 3). Thus, U-69,593 appears to have both a peripheral and a central site of action, depending on the dose and mode of administration. This is in agreement with other studies that have also observed a peripheral and central component in the site of action of this opioid in a model of neuropathic pain. 18 The peripheral selectivity of the more potent κ-opioid peptide FE 200665 was additionally examined via  the coadministration of subcutaneous NLXM with intrathecal FE 200665. The peripherally selective NLXM was no longer able to antagonize intrathecal FE 200665 despite the fact that this antagonist completely abolished the effect of intraplantar FE 200665. This provides further evidence that FE 200665 is unable to cross the blood–brain barrier.
The antiinflammatory effects of opioids have been extensively described. Gyires et al.  19 showed that the μ-opioid agonist morphine can inhibit carrageenan-induced paw swelling in the rat. Moreover, the effects of morphine were dose-dependent (ED30, 1.6 mg/kg) and partially antagonized by naloxone. In addition, opioid peptides such as dynorphin A and hemorphin-7 have also been shown to modulate inflammation. 20,21 Most studies indicate that κ-opioid agonists are able to attenuate experimental arthritis; they have been shown to attenuate the severity of adjuvant arthritis in a dose-dependent, stereoselective, and antagonist-reversible manner. 22–25 Recently, U-69,593 was shown to reduce paw and ankle edema in a model of carrageenin-induced inflammation. 26 High doses of morphine are also able to attenuate adjuvant arthritis, 27 but the dosages are much higher than used systemically in humans.
In our study, all three opioid agonists displayed potent antiinflammatory effects assessed by paw volume and histologic parameters. Administration of FE 200665, FE 200666, and U-69,593 at various time schedules showed that single treatment on initiation of inflammation was almost as effective as continuous treatment over 6 days (fig. 7) and was sustained well beyond the administration of the opioids. This implies that the later stages of hind-paw inflammation are dependent on very early factors, e.g.  , modulation of substance P release from primary afferent neurons or on the migration or activation of immune cells, induced rapidly on the initiation of inflammation and inhibited by a single κ-opioid treatment. When treatment was delayed until inflammation was established, i.e.  , 4 days after FCA injection, a significant reduction in paw volume was also observed with all κ-opioids tested (figs. 5A–5C). Intriguingly, the antiinflammatory response appeared to be delayed by 24–48 h after injection of the κ-opioids. This may be a result of differential effects of these opioids on plasma extravasation, edema, neurogenic inflammation, and immune cells. For example, a possible reason for this delayed effect may be that these drugs do not affect the initial plasma extravasation. The literature in this regard is controversial. Some studies, using the Evans blue dye leakage technique, have shown that κ-opioids are unable to affect plasma extravasation, 28 whereas others have found the opposite. 29,30 Opioids have been shown to modulate the release of substance P, a potent mediator of neurogenic inflammation. 31–33 This may be a mechanism, at least in part, by which opioids produce antiinflammatory effects. Second, opioids could act via  the immune system through the activation of opioid receptors that have been identified on various immune cells. 34 Opioids can modulate immune cell proliferation, chemotaxis, superoxide and cytokine production, and mast cell degranulation. 35 Opioids may diminish the synthesis or release of cytokines from macrophages and mast cells 35 or suppress T-lymphocyte function, 36 downregulate the expression of adhesion molecules, and reduce the migration of immune cells into the injured tissue. 37 Knockout mice provide unequivocal evidence for the involvement of opioids in immunosuppression. This has been shown by Gaveriaux-Ruff et al.  , 38 who found that the effects of morphine on immune responses were completely absent in μ-opioid receptor–deficient mice.
Little difference was observed in the antiinflammatory effects of FE 200665, FE 200666, and U-69,593, assessed either by paw volume or histology (infiltration of inflammatory cells, new bone and pannus formation). The antiinflammatory effects of all the κ-opioids tested were completely reversed by nor-BNI and NLXM, indicating that these effects are mediated via  κ-receptors in the periphery. This is in agreement with results of Wilson et al.  , 23 who showed that the centrally acting κ-opioid U-50,488H had greater antiinflammatory effects when administered locally rather than centrally, and that this effect was reversed by peripherally selective antagonists. The κ-opioid peptides investigated in this report represent a clear advancement compared with other currently available peripherally selective κ-agonists, such as asimadoline. Asimadoline (EMD 61753) was previously investigated in our laboratory, and although this κ-agonist showed promise in early animal studies, our investigations found that it was ineffective in patients who underwent knee surgery. Indeed, patients tended to report an increase in pain. 39 In addition, asimadoline was shown to produce non–opioid-mediated hyperalgesic and proinflammatory effects in FCA-treated rats. 39 Clearly, more effective peripheral κ-opioid agonists are required. In contrast, the current study found that FE 200665 was not only a potent analgesic agent but also showed marked antiinflammatory effects and no sign of hyperalgesia in FCA-treated animals.
In summary, FE 200665 shows potent antinociceptive and antiinflammatory effects that are mediated via  peripheral κ-opioid receptors. The magnitude of the response produced by the peripherally selective FE 200665 was comparable to that of the standard κ-opioid U-69,593, whereas FE 200666 was only weakly effective. Our findings suggest that κ-opioid peptides such as FE 200665 may lead to improved analgesic–antiinflammatory therapy compared with centrally acting opioids or standard nonsteroidal antiinflammatory drugs.
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Fig. 1. Time course and dose–response of the antinociceptive effects (% maximum possible effect [MPE]) of FE 200665, FE 200666, and U-69,593 on paw pressure threshold in inflamed paws. FE 200665 administered (A  ) intraplantarly and (B  ) subcutaneously; FE 200666 administered (C  ) intraplantarly and (D  ) subcutaneously; and U-69,593 administered (E  ) intraplantarly and (F  ) subcutaneously. *Significant difference from controls, P  < 0.05, analysis of variance (Dunnett test).
Fig. 1. Time course and dose–response of the antinociceptive effects (% maximum possible effect [MPE]) of FE 200665, FE 200666, and U-69,593 on paw pressure threshold in inflamed paws. FE 200665 administered (A 
	) intraplantarly and (B 
	) subcutaneously; FE 200666 administered (C 
	) intraplantarly and (D 
	) subcutaneously; and U-69,593 administered (E 
	) intraplantarly and (F 
	) subcutaneously. *Significant difference from controls, P 
	< 0.05, analysis of variance (Dunnett test).
Fig. 1. Time course and dose–response of the antinociceptive effects (% maximum possible effect [MPE]) of FE 200665, FE 200666, and U-69,593 on paw pressure threshold in inflamed paws. FE 200665 administered (A  ) intraplantarly and (B  ) subcutaneously; FE 200666 administered (C  ) intraplantarly and (D  ) subcutaneously; and U-69,593 administered (E  ) intraplantarly and (F  ) subcutaneously. *Significant difference from controls, P  < 0.05, analysis of variance (Dunnett test).
×
Fig. 2. Dose–response effects of intraplantar nor-BNI on the antinociceptive action (% maximum possible effect [MPE]) of (A  ) FE 200665 (100 μg), (B  ) FE 200666 (30 μg), and (C  ) U-69,593 (100 μg). P  < 0.05, analysis of variance (linear regression).
Fig. 2. Dose–response effects of intraplantar nor-BNI on the antinociceptive action (% maximum possible effect [MPE]) of (A 
	) FE 200665 (100 μg), (B 
	) FE 200666 (30 μg), and (C 
	) U-69,593 (100 μg). P 
	< 0.05, analysis of variance (linear regression).
Fig. 2. Dose–response effects of intraplantar nor-BNI on the antinociceptive action (% maximum possible effect [MPE]) of (A  ) FE 200665 (100 μg), (B  ) FE 200666 (30 μg), and (C  ) U-69,593 (100 μg). P  < 0.05, analysis of variance (linear regression).
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Fig. 3. Effect of nor-BNI (200 μg intraplantarly or 400 μg subcutaneously) on the antinociceptive action (% maximum possible effect [MPE]) of (A  ) FE 200665 (100 μg/paw intraplantarly or 2 mg/rat subcutaneously), (B  ) FE 200666 (30 μg/paw intraplantarly or 600 μg/rat subcutaneously), and (C  ) U-69,593 (100 μg/paw intraplantarly or 800 μg/rat subcutaneously). *Significant difference from agonist-treated animals, P  < 0.05, analysis of variance (Dunnett test). i.pl. = intraplantar; s.c. = subcutaneous.
Fig. 3. Effect of nor-BNI (200 μg intraplantarly or 400 μg subcutaneously) on the antinociceptive action (% maximum possible effect [MPE]) of (A 
	) FE 200665 (100 μg/paw intraplantarly or 2 mg/rat subcutaneously), (B 
	) FE 200666 (30 μg/paw intraplantarly or 600 μg/rat subcutaneously), and (C 
	) U-69,593 (100 μg/paw intraplantarly or 800 μg/rat subcutaneously). *Significant difference from agonist-treated animals, P 
	< 0.05, analysis of variance (Dunnett test). i.pl. = intraplantar; s.c. = subcutaneous.
Fig. 3. Effect of nor-BNI (200 μg intraplantarly or 400 μg subcutaneously) on the antinociceptive action (% maximum possible effect [MPE]) of (A  ) FE 200665 (100 μg/paw intraplantarly or 2 mg/rat subcutaneously), (B  ) FE 200666 (30 μg/paw intraplantarly or 600 μg/rat subcutaneously), and (C  ) U-69,593 (100 μg/paw intraplantarly or 800 μg/rat subcutaneously). *Significant difference from agonist-treated animals, P  < 0.05, analysis of variance (Dunnett test). i.pl. = intraplantar; s.c. = subcutaneous.
×
Fig. 4. Dose–response effects of naloxone methiodide (NLXM, administered subcutaneously) on the antinociceptive action (% maximum possible effect [MPE]) of (A  ) FE 200665 (100 μg/paw intraplantarly or 2 mg/rat subcutaneously), (B  ) FE 200666 (30 μg/paw intraplantarly or 600 μg/rat intraplantarly), and (C  ) U-69,593 (100 μg/paw intraplantarly). P  < 0.05, analysis of variance (linear regression). i.pl. = intraplantar; s.c. = subcutaneous.
Fig. 4. Dose–response effects of naloxone methiodide (NLXM, administered subcutaneously) on the antinociceptive action (% maximum possible effect [MPE]) of (A 
	) FE 200665 (100 μg/paw intraplantarly or 2 mg/rat subcutaneously), (B 
	) FE 200666 (30 μg/paw intraplantarly or 600 μg/rat intraplantarly), and (C 
	) U-69,593 (100 μg/paw intraplantarly). P 
	< 0.05, analysis of variance (linear regression). i.pl. = intraplantar; s.c. = subcutaneous.
Fig. 4. Dose–response effects of naloxone methiodide (NLXM, administered subcutaneously) on the antinociceptive action (% maximum possible effect [MPE]) of (A  ) FE 200665 (100 μg/paw intraplantarly or 2 mg/rat subcutaneously), (B  ) FE 200666 (30 μg/paw intraplantarly or 600 μg/rat intraplantarly), and (C  ) U-69,593 (100 μg/paw intraplantarly). P  < 0.05, analysis of variance (linear regression). i.pl. = intraplantar; s.c. = subcutaneous.
×
Fig. 5. Antinociceptive effects (% maximum possible effect [MPE]) of intrathecal [i.t.] FE 200665. (A  ) Dose–response relation and (B  ) antagonism by naloxone methiodide (NLXM; administered subcutaneously and intrathecally). P  < 0.05, analysis of variance (linear regression). s.c. = subcutaneous; i.pl. = intraplantar.
Fig. 5. Antinociceptive effects (% maximum possible effect [MPE]) of intrathecal [i.t.] FE 200665. (A 
	) Dose–response relation and (B 
	) antagonism by naloxone methiodide (NLXM; administered subcutaneously and intrathecally). P 
	< 0.05, analysis of variance (linear regression). s.c. = subcutaneous; i.pl. = intraplantar.
Fig. 5. Antinociceptive effects (% maximum possible effect [MPE]) of intrathecal [i.t.] FE 200665. (A  ) Dose–response relation and (B  ) antagonism by naloxone methiodide (NLXM; administered subcutaneously and intrathecally). P  < 0.05, analysis of variance (linear regression). s.c. = subcutaneous; i.pl. = intraplantar.
×
Fig. 6. Effect of intraplantar κ-opioids on ipsilateral paw volume 4 days after administration of Freund complete adjuvant, as a function of time. (A  ) FE 200665, (B  ) FE 200666, and (C  ) U-69,593. *Significant difference from controls, P  < 0.05, analysis of variance (Dunnett test).
Fig. 6. Effect of intraplantar κ-opioids on ipsilateral paw volume 4 days after administration of Freund complete adjuvant, as a function of time. (A 
	) FE 200665, (B 
	) FE 200666, and (C 
	) U-69,593. *Significant difference from controls, P 
	< 0.05, analysis of variance (Dunnett test).
Fig. 6. Effect of intraplantar κ-opioids on ipsilateral paw volume 4 days after administration of Freund complete adjuvant, as a function of time. (A  ) FE 200665, (B  ) FE 200666, and (C  ) U-69,593. *Significant difference from controls, P  < 0.05, analysis of variance (Dunnett test).
×
Fig. 7. Effect of daily treatment and a single injection of (A  ) FE 200665, (B  ) FE 200666, and (C  ) U-69,593 on paw volume as a function of time. *Significant difference from controls, P  < 0.05, analysis of variance (Dunnett test). FCA = Freund complete adjuvant.
Fig. 7. Effect of daily treatment and a single injection of (A 
	) FE 200665, (B 
	) FE 200666, and (C 
	) U-69,593 on paw volume as a function of time. *Significant difference from controls, P 
	< 0.05, analysis of variance (Dunnett test). FCA = Freund complete adjuvant.
Fig. 7. Effect of daily treatment and a single injection of (A  ) FE 200665, (B  ) FE 200666, and (C  ) U-69,593 on paw volume as a function of time. *Significant difference from controls, P  < 0.05, analysis of variance (Dunnett test). FCA = Freund complete adjuvant.
×
Fig. 8. Effect of nor-BNI (administered intraplantarly) or naloxone methiodide (NLXM; administered subcutaneously) on the antiinflammatory action of (A  ) FE 20065, (B  ) FE 200666, and (C  ) U-69,593. *Significant difference from antagonist-treated groups, P  < 0.05, analysis of variance (Dunnett test). FCA = Freund complete adjuvant.
Fig. 8. Effect of nor-BNI (administered intraplantarly) or naloxone methiodide (NLXM; administered subcutaneously) on the antiinflammatory action of (A 
	) FE 20065, (B 
	) FE 200666, and (C 
	) U-69,593. *Significant difference from antagonist-treated groups, P 
	< 0.05, analysis of variance (Dunnett test). FCA = Freund complete adjuvant.
Fig. 8. Effect of nor-BNI (administered intraplantarly) or naloxone methiodide (NLXM; administered subcutaneously) on the antiinflammatory action of (A  ) FE 20065, (B  ) FE 200666, and (C  ) U-69,593. *Significant difference from antagonist-treated groups, P  < 0.05, analysis of variance (Dunnett test). FCA = Freund complete adjuvant.
×
Fig. 9. Histologic assessment of the antiinflammatory effects of κ-opioid treatment and antagonism by nor-BNI (200 μg intraplantarly) or naloxone methiodide (NLXM; 10 mg/kg). Histologic scores judged by periarticular inflammtion, pannus, and new bone formation (maximum obtainable score = 24). *Significant difference from controls, P  < 0.05, analysis of variance (Dunnett test).
Fig. 9. Histologic assessment of the antiinflammatory effects of κ-opioid treatment and antagonism by nor-BNI (200 μg intraplantarly) or naloxone methiodide (NLXM; 10 mg/kg). Histologic scores judged by periarticular inflammtion, pannus, and new bone formation (maximum obtainable score = 24). *Significant difference from controls, P 
	< 0.05, analysis of variance (Dunnett test).
Fig. 9. Histologic assessment of the antiinflammatory effects of κ-opioid treatment and antagonism by nor-BNI (200 μg intraplantarly) or naloxone methiodide (NLXM; 10 mg/kg). Histologic scores judged by periarticular inflammtion, pannus, and new bone formation (maximum obtainable score = 24). *Significant difference from controls, P  < 0.05, analysis of variance (Dunnett test).
×