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BEHAVIORAL PHARMACOLOGY

FE200041 (D-Phe-D-Phe-D-Nle-D-Arg-NH₂): A Peripheral Efficacious Opioid Agonist with Unprecedented Selectivity

Department of Pharmacology, University of Arizona, Tucson, Arizona (T.W.V., J.L.); Ferring Research Institute Inc., San Diego, California (C.D.S., J.T., P.J.-M.R.); and Laboratories Fournier, Dijon, France (J.-L.J.)

Received January 12, 2004; accepted February 24, 2004.

	Abstract

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The side effects typically associated with the clinical profiles of opioid µ-receptor agonists have driven continuing efforts to identify novel efficacious analgesics, including agonists acting at opioid receptors. Unfortunately, the therapeutic potential of agonists seems limited by significant central nervous system side effects. Opioid agonists, however, exhibit potent peripherally mediated antihyperalgesic and antinociceptive effects, suggesting that a peripherally acting agonist may be efficacious in pain control with a more desirable safety profile than that associated with currently available opioids. Here, we report an all D-amino acid tetrapeptide characterized as a novel, highly selective opioid receptor agonist. FE200041 (D-Phe-D-Phe-D-Nle-D-Arg-NH₂) showed selectivity for the human opioid receptor of greater than 30,000- and 68,000-fold versus human µ opioid receptor and human -opioid receptor receptors, respectively, and efficacious agonist activity using in vitro tissue assays. FE200041 produced local, peripheral antinociception in the hindpaw ipsilateral, but not contralateral, to injection. Antinociceptive effects of FE200041 in the mouse acetic acid writhing assay lasted over 60 min and were antagonized by naloxone and by selective , but not µ, opioid receptor antagonists. FE200041 significantly inhibited acetic acid writhing and inhibited formalin-induced flinching in rats. FE200041 did not elicit sedation or motor impairment after systemic administration at a dose 10-fold higher than that needed to achieve antinociception. FE200041 is thus a potent peripherally restricted opioid agonist with no demonstrable side effects typical of agonists with central nervous system activity and with unprecedented selectivity for the opioid receptor. The pharmacology of this compound suggests the possibility of therapeutic application.

Many studies have shown that opiates have peripheral analgesic effects, especially under inflammatory or hyperalgesic conditions (Barber and Gottschlich, 1992; Junien and Wettstein, 1992; Stein, 1993). Agonists at opioid receptors have been shown to produce analgesia and decrease inflammation in models of rheumatoid arthritis after local administration (Wilson et al., 1996). Restricted CNS penetration is a common strategy to reduce central side effects of drugs with beneficial peripheral actions. For example, the antihistamines terfenadine, astemizole, and mequitazine do not cross the blood-brain barrier at therapeutic doses (Nicholson, 1987). Similar techniques have been attempted in the development of peripherally restricted opioid agonists, including ICI 204448 (Shaw et al., 1989), GR 94839 (Rogers et al., 1992), and EMD61753(asimadoline) (Barber et al., 1994). When asimadoline was tested in humans after postoperative knee surgery the patients tended to report an increase in pain (Machelska et al., 1999). Unfortunately, these compounds were discontinued in clinical trials due to either poor bioavailability, lack of efficacy, or CNS side effects at analgesic doses (Barber and Gottschlich, 1997). Peptidic opioid agonists, including E-2078 (Tachibana et al., 1988) and SK-9709 (Ambo et al., 1995), have been developed as analgesics and used years after their synthesis with the idea that a peptide would be less likely to cross the blood-brain barrier; however, studies demonstrated that both of these dynorphin peptidic fragments crossed the blood-brain barrier (Yu et al., 1997; Hiramatsu et al., 2001).

Recently, Dooley et al. (1998) reported the discovery of a high-affinity (K_i < 1 nM) and selective (µ/ and / ratios of >3000) opioid agonist using mixture-based positional scanning of a combinatorial tetrapeptide library. The tetrapeptide is composed entirely of D-amino acids and was identified as an agonist by its inhibition of forskolin-stimulated cAMP formation using R1G1 thymoma cell line membranes. We have further characterized this tetrapeptide, now termed FE200041, by radioligand binding and in vitro functional assays on transfected cells that express the human , µ, and opioid receptors. Additionally, FE200041 was studied in models of somatic as well as inflammatory pain in both mice and rats, and CNS effects were studied after peripheral administration. The data demonstrate that FE200041 is a highly selective opioid antinociceptive agent without central nervous system side effects at doses higher than those needed to elicit antinociception. The peripheral antinociceptive actions of FE200041 thus suggest that it is possible to develop peripherally restricted opioid peptides for use in controlling pain.

	Materials and Methods

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Radioligand Binding. All radioligands were from PerkinElmer Life and Analytical Sciences (Boston, MA). Radioligand binding analysis was carried out as described previously (Lai et al., 1994) using crude membrane preparations from HN9.10 cells that express the human , µ, or opioid receptor. The membranes were resuspended in ice-cold Tris-buffer (50 mM, pH 7.4) containing 0.5% bovine serum albumin, 30 µM bestatin, 10 µM captopril, 50 µg/ml bacitracin, and 100 µM phenylmethylsulfonyl fluoride. To characterize the binding affinity of [³H]FE200041 (23.6 Ci/mmol) for the human opioid receptor, the rate of association of the radioligand was determined by incubating membranes (50 µg) from transfected cells that express the human opioid receptor with 0.8 nM [³H]FE200041 for 0.5, 1, 5, 10, 15, 30, 60, 90, and 120 min at 25°C, done in triplicates. The rate of dissociation was determined by incubating membranes with 0.8 nM [³H]FE200041 for 2 h at 25°C, followed by incubation with 10 µM naloxone for 0.5, 1, 5, 10, 15, 30, 50, and 90 min, done in triplicates. The dissociation constant (k_-1) was calculated by plotting ln[B_t/B₀] against t, where B₀ and B_t are the amounts of radioligand bound at time 0 and specific time t, after the addition of naloxone to obtain the slope of -k_-1. The association rate constant (k₊₁) was based on the equation k_on = k_-1 + k₊₁[L], where [L] is the concentration of the radioligand, and k_on was determined by plotting ln[(B - B_t)/B] against t, where B is the amount of radioligand bound at equilibrium and B_t is that at time t, to obtain the slope of -k_on. The dissociation constant (K_D) for [³H]FE200041 was then calculated by the equation K_D = k_-1[L]/(k_on - k_-1) and expressed as the mean ± S.E.M. of three independent experiments.

For equilibrium saturation analysis, membranes were incubated with 12 concentrations of [³H]FE200041 (12.5 pM–4000 pM) for 90 min at 25°C in the presence or absence of 10 µM naloxone. The total reaction volume was 1 ml/assay tube, and all reactions were carried out in duplicates. At the end of the incubation, reactions were terminated by rapid filtration through Whatman GF/B filters (presoaked in polyethyleneimine) and washed with 2 x 4 ml of ice-cold 50 mM Tris. For competition analysis, 10 concentrations of FE200041 and either 1.6 nM [³H]U69,593 for opioid binding, 1 nM [³H][D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin for µ opioid binding, or 1 nM [³H]pCl-[D-Pen²,D-Pen⁵]-enkephalin for opioid binding, were carried out using membranes from transfected cells that express the , µ, or receptor, respectively. Nonspecific binding was defined as the amount of radioligand bound in the presence of 10 µM naloxone. Reactions were carried out in duplicates for 3 h at 25°C and terminated by rapid filtration as described above. Radioactivity was determined by liquid scintillation counting. Data were fitted by nonlinear least-squares analysis using GraphPad Prism (version 3.0). All analyses were based on three independent experiments.

GTPS Binding. GTPS binding was done using membranes from cells that express the human opioid receptor. The procedure for this analysis was based on that of Lorenzen et al. (1993). Reactions were initiated by the addition of an aliquot of membrane preparation (15 µg) to a final volume of 300 µl of incubation mix [50 mM HEPES, pH.7.4, 1 mM EDTA, 5 mM MgCl₂, 30 µM GDP, 1 mM dithiothreitol, 100 mM NaCl, 100 µM phenylmethylsulfonyl fluoride, 0.1% bovine serum albumin, 0.1 nM [³⁵S]GTPS (1250 Ci/mmol)] and indicated concentration range of agonist and incubated for 60 min at 30°C. Basal level of [³⁵S]GTPS binding was defined as the amount bound in the absence of agonist. Nonspecific binding was determined in the presence of 10 µM unlabeled GTPS. Reactions were performed in triplicate and terminated by rapid filtration through Whatman GF/B filters presoaked in water followed by four washes with ice-cold wash buffer (50 mM Tris, 5 mM MgCl₂, and 100 mM NaCl, pH 7.4). The radioactivity was determined by liquid scintillation counting. Data were fitted by nonlinear least-squares analysis using GraphPad Prism.

Animals. Male ICR mice (20–25g) and male Sprague-Dawley rats (200–250 g) were used in all experiments. Animals were purchased from Harlan (Indianapolis, IN), housed on a regular 12-h light/dark cycle (lights on at 6:00 AM), in a climate-controlled room with food and water ad libitum. All procedures were in accordance with the policies and recommendations of the International Association for the Study of Pain and the National Institutes of Health guidelines and use of laboratory animals as well as approved by the Animal Care and Use Committee of the University of Arizona. Groups of 6 to 15 were used in all experiments.

Mouse Isolated Vas Deferens. Male ICR mice (20–25 g) were used in all experiments. Mice were lightly anesthetized using ether and sacrificed. Tissue preparation was performed as described previously (Hughes et al., 1975). Briefly, the vas deferens was isolated and mounted in organ baths containing 20 ml of oxygenated Krebs' buffer solution omitting the MgSO₄ at 37°C. Using a Grass S48 stimulator, the tissues were stimulated transmurally between platinum wire electrodes at 0.1 Hz, with 2.0-ms pulses at supramaximal voltage. Contractions were recorded on a Grass 7D polygraph. Test compounds were added to the baths in 14- to 60-µl volumes. Each dose remained in bath for 4 min or until maximal inhibition was reached. Subsequent doses were added cumulatively to the bath at 4-min intervals to produce a concentration-response curve. Tissues were then washed extensively with fresh buffer until the original contraction height was reestablished.

Injections. For i.v. administration, animals were placed in restrainers and injections were made using a disposable 1-ml syringe equipped with a 30-gauge disposable needle. The needle was inserted into the tail vein at a 25° angle, and a small amount of blood was drawn back into the syringe before injection of either compounds or vehicle to ensure injection into the vein. Injection of compounds or vehicle was performed over a 5-s period for consistency purposes. After injection, the needle was removed from the tail vein and gentle pressure was applied at the site of injection to prevent loss of fluid from the site of injection. Subcutaneous (s.c.) injections were performed by manually holding the animal and inserting a 30-gauge disposable needle on a disposable 1-ml syringe into the abdominal region of the animal, ensuring that the needle remained between the muscle and the skin of the animal. Injections of compounds were performed over a 5-s period and were noted as positive by the development of an outpocketing of the skin at the site of injection. Intraperitoneal (i.p.) injections were made in similar manner to the s.c. injections except that the needle was placed completely under the muscle of the abdominal region and an outpocketing of the skin was not seen. Injections into the hindpaw (i.paw) of the animal were made using a Hamilton 100-ml syringe and a 30-gauge disposable needle. Injections into the paw were made on the plantar surface just below the skin in a volume of 50 µl/animal.

Acetic Acid Writhing Assay. As a measure of peripheral antinociception, FE200041 as well as the reference opioid agonists U50,488H and asimadoline were tested using the acetic acid writhing test. Animals were fasted 12 to 16 h before testing. The nociceptive behavior (writhes) was induced by diluted acetic acid (0.6%, 10 ml/kg, for mice and 2.5%, 0.5 ml/rat) administered i.p. at time 0 min. In all experiments with mice, compounds were given i.v. in the tail 5 min before acetic acid administration to determine the activity and potency, as well as at different intervals before acetic acid to determine their duration of action using a submaximally effective dose (A₈₀). In all experiments with rats, compounds were given s.c. 15 min before acetic acid administration to determine the activity and potency. The number of writhes was counted over a 15-min period starting from the time of acetic acid injection. Activity is expressed as a percentage and is calculated as follows: % antinociception = 100 x [(writhes in control group - writhes in treated group)/writhes in control group]. Potency (A₅₀) is calculated from the full dose-response curve. Antagonist studies were performed in mice as described above with antagonist administered before FE200041. The opioid antagonist naloxone (1 mg/kg) was given by the s.c. route 15 min before FE200041. The µ selective, nonequilibrium opioid receptor antagonist -funaltrexamine (-FNA) (10 mg/kg) was administered by the s.c. route 24 h before FE200041 administration (Jiang et al., 1989). The opioid receptor antagonist nor-binaltorphimine (nor-BNI) (10 mg/kg) (Marchand et al., 2003) was administered by the s.c. route 15 min before FE200041 administration (Portoghese et al., 1987). In addition, to determine a peripheral and non-CNS effect, the opioid receptor antagonist nor-BNI (10 µg/5 µl) (Horan et al., 1992) was administered by the i.c.v. route 10 min before an A₉₀ FE200041 administration. To achieve a time course, separate groups of mice were pretreated with an A₈₀ dose of FE200041 at either 5, 10, 15, 30, 60, or 120 min before receiving an i.p. injection of acetic acid. At all time points, the number of writhes was counted for 15 min immediately after the administration of acetic acid.

Formalin Flinch Test. As a measure of antinociception in both acute and inflammatory (tonic) pain, FE200041 was tested using the 2% formalin flinch assay in rats. Animals were habituated to the environment for approximately 30 min before testing. The formalin test was carried out in a 30 x 30 x 30-cm chamber with the front and bottom of the chambers made of clear Plexiglas. Mirrors were placed at a 45° angle under the chambers to give an unobstructed view of the paws. Animals were restrained manually, and 2% formalin (made fresh the day of the experiment) was injected subcutaneously (50 µl) into the plantar surface of the left hindpaw with a 30-gauge needle. The number of paw flinches was then recorded at 5-min intervals from the time of injection for a 60-min period. Elevations of the paw, licking and biting of the injected paw were counted as "flinches". The acute or "first phase" of the nociceptive response peaked at 5 min after formalin injection, and the inflammatory or "second phase" peaked at 30 min after formalin injection. The number of flinches in the first 10 min was representative of the acute phase, and the number of flinches from 10 min until 60 min was representative of the second phase. FE200041 or vehicle was administered by the i.v. route 5 min before formalin injection. Antinociception for either phase I or phase II was calculated as follows: % antinociception = 100 [(no. of flinches in control animal - no. of flinches in drug treated animal)/(no. of flinches in control animal)]. Potency (A₅₀) is calculated from the full dose-response curve for both phase I and phase II.

Radiant Heat Paw Withdrawal Assay. To evaluate the peripheral activity of FE200041, we tested the local administration of FE200041 and hindpaw withdrawal latencies using a thermal stimulus in mice. The method of Hargreaves et al. (1988) was used to assess paw withdrawal latency to a thermal nociceptive stimulus. Animals were placed in 3 cm x 3-cm Plexiglas boxes on top of a glass plate that was maintained at room temperature. Animals were allowed to habituate for a period of 45 min. For baseline paw withdrawal latencies, a heat source that increased in intensity from a non-noxious temperature to a noxious temperature (occurs within 1 to 16 s in naive animals) from under the glass plate was reflected onto the plantar surface of the right hindpaw with the focus of the light beam being no larger than a 3 to 5 mm in diameter, and the time to withdrawal the paw from the heat source was recorded. A maximum cut-off of 40 s was used to prevent tissue damage. Animals were administered an A₈₀ dose (10 µg/5 µl i.paw) of FE200041 or vehicle into the dorsal side of the right hindpaw, and paw withdrawal latencies were tested at 15-min intervals over a 60-min period. To determine a local, peripheral effect of FE200041, the agonist or vehicle was injected into the left hindpaw, and paw withdrawal latencies were tested at 15-min intervals over a 60-min period. Antinociception was calculated as follows: % antinociception = 100 [(test paw withdrawal latency - baseline paw withdrawal latency)/(cut-off for paw withdrawal latency of 40 s - baseline paw withdrawal latency)].

Rotarod Test. To evaluate CNS effects, including sedation or nonspecific motor effects, animals were tested for their ability to balance on a slowly rotating rod (rate of rotation was 10 revolutions per minute; diameter was 1.5 cm for mice and 6.5 cm for rats) after peripheral administration of test compounds or vehicle. Animals were conditioned before the experiment, and selected animals (those remaining on the rotarod for 180 sec) were injected by either the i.v. (mice) or s.c. (rats) route with test compounds. Animals were tested either 5, 10, or 15 min after i.v. or 15, 20, or 25 min after s.c. administration. The latency to fall off the rod was recorded with 180 s as the cut-off. Animals falling off the rod before the 180-s cut-off were considered to have motor impairment. Activity is expressed as a percentage and is calculated as follows: % activity = 100 x [(latency to remain on the rotarod in control group - latency in treated group)/(latency in control group)].

Statistics. Data were analyzed by analysis of variance, and where significance was indicated, followed by Student's t test for grouped data. The significance criterion was p < 0.05 throughout. Results are reported as the mean activity score and S.E.M.

Chemicals. FE200041 and asimadoline were dissolved in distilled water and provided by Ferring Research Institute Inc. (San Diego, CA). Naloxone was dissolved in distilled water and purchased from Sigma-Aldrich (St. Louis, MO). -FNA, (-)-U50,488, and nor-BNI were dissolved in distilled water and purchased from Tocris Cookson Inc. (Ellisville, MO). ICI 174,864 and D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH₂ were a gift from the National Institute on Drug Abuse via Multiple Peptide Systems (San Diego, CA). Radiolabeled [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (80 Ci/mmol), U69,593 (69 Ci/mmol), pCl-[D-Pen²,D-Pen⁵]-enkephalin (65 Ci/mmol), FE200041 (23.6 Ci/mmol), and [³⁵S]GTPS were purchased from PerkinElmer Life and Analytical Sciences.

	Results

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[³H]FE200041 Exhibits High Affinity and Selectivity for Human KOR (hKOR). Association and dissociation analysis of [³H]FE200041 using transfected cells that expressed the hKOR showed that the peptide had a dissociation constant of 0.43 ± 0.13 nM based on three independent determinations (Fig. 1, A and B). This value is in good agreement with the K_D value of 0.8 nM based on equilibrium saturation analysis (Fig. 1C). The receptor density based on the B_max value obtained from the saturation analysis was 3.5 pmol/mg of membrane protein. The equilibrium dissociation constants (K_i) for FE200041 for hKOR, hMOR, and hDOR based on competition analysis were 0.15 nM, 4.6 µM, and >10 µM, respectively. Thus, FE200041 was over 30,000-fold selective for hKOR over hMOR and over 68,000-fold selective for hKOR over hDOR (Table 1).

View larger version (15K): [in this window] [in a new window]   Fig. 1. Time course of association (A) and dissociation (B) of [3H]FE200041 in membrane preparations from transfected cells that express the hKOR. Data shown are representative of three independent determinations. The KD value determined from this experiment was 0.71 nM. Each data point is the mean of triplicate assay. Saturation radioligand binding using [3H]FE200041 on mouse cancer cells HN.9.10 stably transfected with the human opioid receptor (C). The binding capacity (Bmax) and dissociation constant (KD) for [3H]FE200041 on hKOR is 3.5 pmol/mg and 0.77 nM, respectively (n = 3).

FE200041 Is a Potent and Efficacious Agonist at the Opioid Receptor. In vitro analysis based on FE200041-induced specific binding of [³⁵S]GTPS to hKOR-containing cell membranes showed that the peptide activated GTP binding with an EC₅₀ value of 1.1 nM and an E_max value of 148% over basal level of GTP binding in the absence of FE200041 (Table 1). These values were similar to the fully efficacious agonist U69,593 (EC₅₀ of 1.6 nM and E_max of 86%) (T. W. Vanderah and P.J.-M. Riviere, unpublished observations), suggesting that FE200041 acts as a fully efficacious agonist in vitro. The EC₅₀ value thus approximated the affinity based on the dissociation constant of FE200041 for hKOR. In vitro bio-assay using mouse vas deferens (MVD) preparations showed that FE200041 inhibited the electrically evoked smooth muscle contraction in a dose-dependent manner. A complete washout after FE200041 administration restored full electrical-induced contraction. The IC₅₀ for FE200041 was 5.8 nM with an E_max value of 96% in the presence of the -antagonist ICI 174,864 (1 µM) and in the presence of the µ-opioid antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH₂ (1 µM). FE200041 inhibition of electrically induced contraction of the MVD was blocked by 1 µM naloxone.

FE200041 Produces Potent Antinociception through the Receptor. FE200041 exhibited antinociceptive activity in assays of acute and persistent nociception such as the acetic acid-induced writhing and formalin-induced paw flinch tests. FE200041, when administered i.v. 5 min before 0.6% acetic acid administration into the abdominal region of mice, resulted in a dose-related inhibition of writhes over a 15-min period compared with animals receiving only vehicle before acetic acid (Fig. 2). The A₅₀ for FE200041 in the acetic acid writhing test was 0.06 mg/kg i.v. (95% CI, 0.04–0.11). For comparison, the opioid agonist (-)-U50,488 and asimadoline were also tested in the mouse acetic acid writhing test after i.v. administration using the same dosing schedule as FE200041. (-)-U50,488 resulted in an A₅₀ of 0.10 mg/kg i.v. (95% CI, 0.06–0.18) and asimadoline resulted in an A₅₀ of 0.19 mg/kg i.v. (95% CI, 0.14–0.25) (Fig. 2). FE200041 was also tested after i.p. injection 5 min before 0.6% acetic acid administration in mice, resulting in an A₅₀ of 2.3 mg/kg (95% CI, 1.15–3.57) (data not shown). A time course for FE200041 using the acetic acid writhing test was performed using an A₈₀ dose (0.3 mg/kg i.v.) and testing groups of animals until the antinociceptive response was reduced to 20%. FE200041 showed significant antinociceptive activity given as much as 60 min before acetic acid administration (Fig. 3). The opioid antagonist naloxone significantly blocked the antinociceptive effects of i.v. administered FE200041 in the mouse acetic acid writhing test; the A₅₀ value of FE200041 in the presence of naloxone was 1.78 mg/kg (95% CI, 0.78–2.18), indicating a 29.6-fold rightward shift in the presence of naloxone. Likewise, an A₉₀ (1 mg/kg i.v.) antinociceptive dose of FE200041 (95.3 ± 1.8% activity) in the mouse acetic acid writhing test was significantly attenuated by pretreatment with the opioid receptor-selective antagonist, nor-BNI (10 mg/kg s.c.) (Portoghese et al., 1987), administered 15 min before FE200041 (29.8 ± 8.7% activity) (Fig. 4). However, the opioid µ receptor-selective antagonist -FNA (10 mg/kg s.c.) (Jiang et al., 1989), given as a pretreatment 24 h before testing did not attenuate the antinociceptive activity of FE200041 (80.5 ± 5.3% activity) (Fig. 4). To elucidate whether the antinociceptive effects of systemic FE200041 was due to penetration into the central nervous system, the selective opioid receptor antagonist nor-BNI (10 µg) was administered directly into the central nervous system by the i.c.v. route just before the systemic administration of FE200041. The A₉₀ (1 mg/kg i.v.) antinociceptive dose of FE200041 in the mouse acetic acid writhing test was not significantly attenuated by the pretreatment with nor-BNI (10 µg i.c.v.) (88.4 ± 8.4% activity). Antagonists alone had no effect on the number of acetic acid-induced writhes. The s.c. administration of FE200041 also resulted in a dose-related inhibition of writhes in rats over a 15-min period compared with animals receiving only vehicle. Animals were pretreated (15 min) with FE200041 before 2.5% acetic acid administration into the abdominal region. The A₅₀ for FE200041 in the rat acetic acid writhing test was 0.09 mg/kg s.c. (95% CI, 0.02–0.48).

View larger version (12K): [in this window] [in a new window]   Fig. 2. Dose-related inhibition of acetic acid (0.6%, 10 ml/kg i.p.)-induced writhing in mice by FE200041 (filled circle), (-)-U50,488 (filled triangle), or asimadoline (filled square) by the i.v. route of administration. Intravenous administration of FE200041 resulted in an A50 of 0.06 mg/kg (95% CI, 0.04–0.11). (-)-U50,488 resulted in an A50 of 0.10 mg/kg (95% CI, 0.06–0.18) and asimadoline resulted in an A50 of 0.19 mg/kg (95% CI, 0.14–0.25) (n = 6 to 8 for each point).

View larger version (10K): [in this window] [in a new window]   Fig. 3. Time-related inhibition of acetic acid-induced writhing in mice by an A80 dose of FE200041 was performed. The dose of 0.3 mg/kg i.v. of FE200041 was administered either 5, 10, 15, 30, 60, or 120 min before acetic acid administration in separate animals, and the number of writhes was recorded. FE200041 resulted in a significant antinociceptive effect (>20%) over a period of 60 min (n = 6 for each point).

View larger version (16K): [in this window] [in a new window]   Fig. 4. Inhibition of acetic acid-induced writhing in mice by FE200041 (1 mg/kg i.v.) was performed in the absence and presence of either the selective µ opioid receptor antagonist -FNA (10 mg/kg s.c.) or the selective opioid receptor antagonist nor-BNI (10 mg/kg s.c.; 10 µg/5 µl i.c.v.). -FNA was administered 24 h before FE200041 and did not block the antinociceptive effects of FE200041. In contrast, nor-BNI given s.c. 15 min before FE200041 significantly attenuated the antinociceptive effects of FE200041 from 96% activity in control animals to 28% activity (n = 8 for each column). However, nor-BNI given i.c.v. 5 min before FE200041 had no effect on the antinociceptive effects of FE200041 from 96% activity in control animals to 88% activity (n = 8 for each column).

In the formalin test, FE200041 resulted in a dose-related inhibition of 2% formalin-induced flinching in both phase I and phase II of the assay. Hindpaw injection of 2% formalin in vehicle-treated animals resulted in a typical response with two phases. The first phase was seen during the first 10 min, and the second phase was seen during the period from 10 to 60 min. A dose of 1 mg/kg i.v. of FE200041 resulted in approximately 95% inhibition of phase I flinching and approximately 80% inhibition in phase II flinching (Fig. 5, A and B). The A₅₀ for FE200041 in the rat 2% formalin flinching assay was 0.39 mg/kg i.v. (95% CI, 0.29–0.55) for phase I and 0.55 mg/kg i.v. (95% CI, 0.42–0.73) for phase II.

View larger version (25K): [in this window] [in a new window]   Fig. 5. A, dose-related inhibition of formalin-induced flinching in rats by FE200041 by the i.v. route of administration, vehicle (filled circle), 0.3 mg/kg (filled square), 0.6 mg/kg (filled triangle), 1 mg/kg (filled diamond). B, FE200041 significantly attenuated both phase I and Phase II of the formalin-induced paw flick assay with and A50 of 0.39 mg/kg i.v. (95% CI, 0.29–0.55) for phase I (closed circle) and 0.55 mg/kg i.v. (95% CI, 0.42–0.73) for phase II (closed square) (n = 6).

To further characterize the peripheral actions of FE200041, the radiant heat paw withdrawal assay was performed and FE200041 (10 µg/rat) was administered to the paw either ipsilateral or contralateral to that paw being tested. The i.paw administration of FE200041 resulted in a fully antinociceptive effect (79.9 ± 7.7% activity) when administered into the ipsilateral paw (the paw of thermal testing). The paw withdrawal latency of the ipsilateral paw after FE200041 administration was 36.2 ± 2.7 s, significantly higher than the pre-FE200041 administration baseline latency of 14.0 ± 0.5 s. When the paw contralateral to the side of FE200041 injection was tested, no change in paw withdrawal latency was observed (i.e., 13.8 ± 0.3 versus 13.5 ± 0.4 s) (Fig. 6). Administration of vehicle into the paw either ipsilateral or contralateral to the side of thermal testing did not result in significant changes from baseline paw withdrawal latencies.

View larger version (23K): [in this window] [in a new window]   Fig. 6. Local analgesic effects of FE200041 using the radiant heat test. FE200041 (10 µg/rat) was administered in either the ipsilateral hindpaw or contralateral hindpaw to testing with a thermal stimulus. FE200041 resulted in a significant increase in the ipsilateral paw withdrawal latency from a radiant heat source, whereas when administered in the contralateral paw to testing there was no difference in paw withdrawal latencies from baseline (n = 6).

FE200041 Did Not Elicit Significant CNS Effects. To evaluate possible CNS activity of FE200041, mice or rats given the peptide were monitored for signs of sedation or motor impairment based on the rotarod test. For these experiments, FE200041 was administered to mice i.v. at antinociceptive doses and at doses several times higher than those needed to produce antinociception. Antinociceptive doses of FE200041 did not interfere with rotarod performance for the duration of the experiment (180 s). Similarly, doses of FE200041 10-fold higher than those necessary to achieve antinociception did not interfere with rotarod performance in mice. The A₅₀ for FE200041 in the mouse rotarod test was 5.04 mg/kg i.v. (95% CI, 3.68–6.89) measured at 5 min after administration, the time of peak antinociceptive action. Thus, the sedation/motor impairment dose-response curve for FE200041 in the mouse rotarod test showed greater than a 84-fold shift to the right compared with the antinociceptive dose-response curve in the mouse acetic acid writhing test. However, when the agonists (-)-U50,488 and asimadoline were tested in the mouse rotarod test over the dose range in which these compounds were found to be active in the writhing test, only a 2-fold shift to the right for (-)-U50,488 (rotarod A₅₀, 0.19 mg/kg i.v.) and a 10-fold shift to the right for asimadoline (rotarod A₅₀, 1.94 mg/kg i.v.) was observed.

Similar results were found in rat rotarod performance. Rats remained on the rotarod for the duration of the experiment (180 s) at antinociceptive doses of FE200041. Every animal tested remained on the rotarod until cut-off (180 s) at all doses of FE200041 tested, including 1, 3, and 10 mg/kg s.c. (n = 6 for each dose). These doses were 10-fold higher than those necessary to produce antinociception in both the rat acetic acid writhing and 2% formalin flinch assay. Thus, no sedation or motor impairment was seen in any of the rats over the duration of the experiment.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Pharmacological characterization of FE200041 presented here shows that the all D-amino acid tetrapeptide FE200041 exhibits high affinity, selectivity, and agonist activity at the human opioid receptor. The selectivity of this peptide for the hKOR over the hMOR or hDOR of 31,000- to 68,000-fold is unprecedented and represents the most selective opioid agonist identified to date. In addition, in vitro and in vivo analyses reveal that FE200041 is a potent and efficacious agonist both at the cloned hKOR as well as at the rodent receptor in vivo. These findings support the possibility that FE200041 would likely be active as an analgesic agent in humans.

Previous studies have suggested a potential for therapeutic application of opioid agonists. For example, studies with enadoline (CI-977) (Hunter et al., 1990), a highly selective and potent opioid agonist, showed that this compound was as active as morphine (10 mg) as an analgesic at a dose of 25 µg in female patients (Pande et al., 1996). However, clinical use of enadoline was dose-limited due to neuropsychiatric side effects (Pande et al., 1996). Asimadoline resulted in a trend toward hyperalgesia on postoperative pain in patients who underwent knee surgery (Machelska et al., 1999). This may very likely be due to the limited doses of asimadoline that could be administered to prevent unwanted CNS-induced dysphoric effects. These findings, together with previous reports of dysphoric actions of CNS-penetrating agonists (Pfeiffer et al., 1986) as well as the many reports from preclinical investigations showing peripheral antinociceptive actions of agonists (Barber et al., 1992; 1994), led to investigations of compounds with restricted access to the CNS.

The present study shows that FE200041 exhibits potent antinociceptive actions after systemic administration. In the acetic acid-induced writhing test, which models persistent pain of moderate intensity, a 1 mg/kg i.v. dose of FE200041 produced a full antinociceptive effect in both mice and rats. This effect of FE200041 was mediated by opioid receptors as the antinociception was fully blocked by naloxone. The peripheral effects of FE200041 were demonstrated by administering the selective antagonist either systemically or centrally. The selective antagonist nor-BNI given peripherally reversed the antinociceptive effect of FE200041 but failed to antagonize the antinociceptive actions of FE200041 when the antagonist was administered by the i.c.v. route. These data, together with the failure of the peripherally administered µ opioid receptor antagonist -FNA, given at doses and times that have previously been shown to block the effects of selective µ opioid agonists (Portoghese et al., 1987; Jiang et al., 1989; Horan et al., 1992; Marchand et al., 2003) to antagonize the antinocicpetive effects of FE200041, provide further confirmation that the actions of FE200041 in vivo are the result of peripheral receptor activation. This conclusion is consistent with the selectivity of FE200041 for receptors in the in vitro assays as well as the lack of typical opioid-induced CNS side effects such as sedation as seen in the rotarod test.

The formalin flinch test (Dubuisson and Dennis, 1977; Dickenson and Sullivan, 1987; Wheeler-Aceto and Cowan, 1991) was used to determine the possible effect of FE200041 on acute C/A sensory afferent fiber activity (phase I) and subsequent tonic inflammatory pain (phase II). Both µ and opioid agonists such as morphine and enadoline, respectively, are effective in the inhibition of formalin-induced nociception in both the acute and tonic phase of this assay (Dickenson and Sullivan, 1987; Wheeler-Aceto and Cowan, 1991; Barber et al., 1994), suggesting that opioids may directly inhibit the activity of afferent fibers in normal and in inflammatory states. FE200041 at a dose of 1 mg/kg i.v. resulted in complete inhibition of 2% formalin-induced flinching in phase I and over 80% inhibition in phase II at 1 mg/kg after systemic administration in the rat. Thus, these data further substantiate the activity of FE200041 as an efficacious, systemically active selective agonist. The antinociceptive effect of i.v. FE200041 at a subeffective (A₈₀) dose persisted for over 60 min, suggesting sufficient duration of action which might be suitable for potential clinical application.

Importantly, the antinociceptive activity of FE200041 is likely to be mediated peripherally because the antinociceptive dose elicited no motor impairment in either mice or rats. Likewise, the antinociceptive dose of FE200041 did not result in signs of apparent sedation as measured in the rotarod assay. In contrast, the opioid agonists (-)-U50,488H and asimadoline, at doses that are antinociceptive in the writhing test, resulted in motor impairment as demonstrated by reduced latencies on the rotarod. This conclusion is supported by the observation that the antinociceptive effects of systemically administered FE200041 were not antagonized by the i.c.v. administration of the antagonist nor-BNI, suggesting that the antinociceptive effects of FE200041 are via its peripheral actions. Furthermore, FE200041 was tested by local administration into either the hindpaw ipsilateral or contralateral to testing with a thermal stimulus. These data demonstrated that FE200041 was active in the ipsilateral but not the contralateral paw, further demonstrating a local, peripheral antinociceptive effect. Thus, the probable peripheral site of antinociceptive activity of FE200041 coupled with a lack of CNS actions strongly suggest that this peptide has the potential to be developed as an analgesic without central side effects associated with µ opioid agonists such as morphine, or other agonists that have access to the CNS at analgesic doses, The present data also indicate that modified peptides with all D-amino acids may have increased bioavailability based on the long-lasting activity of FE200041. FE200041 showed antinociceptive actions for approximately 60 min after i.v. administration. Thus, FE200041 represents a prototypic opioid peptide that is highly efficacious as a peripherally acting, selective compound that is likely to have few side effects associated with conventional opioid agonists.

In summary, our data show that FE200041 is 1) highly selective for human opioid receptors, with in vitro and in vivo activity in the nanomolar and micromolar range, respectively; 2) a fully efficacious and peripherally active antinociceptive agent; 3) does not elicit typical CNS side effects associated with agonists; and 4) demonstrates a long-lasting time course of action compared with other peptidic agonists, suggesting suitability for systemic administration. These features suggest the possibility of future efforts to develop such peripherally restricted agonists as therapeutic agents for humans.

	Acknowledgements

 
We thank Peg Davis, Rachel Johnson, Shou-Wu Ma, and Dinesh Srinivasan for technical assistance.

	Footnotes

 
These studies were supported by Ferring Research Institute Inc.

DOI: 10.1124/jpet.104.065391.

ABBREVIATIONS: CNS, central nervous system; GTPS, guanosine 5'-O-(3-thio)triphosphate; i.paw, injection into the hindpaw; -FNA, -funaltrexamine; nor-BNI, nor-binaltorphimine; hKOR, human opioid receptor; hMOR, human µ opioid receptor; hDOR, human opioid receptor; MVD, mouse vas deferens; CI, confidence interval; FE200041, D-Phe-D-Phe-D-Nle-O-Arg-NH₂; ICI 204448, ((±)-[3-[1-[[3,4-dichlorophenyl)acetyl]methylamino]-2-(1-pyrrolindinyl)ethyl]phenoxy]acetic acid hydrochloride; GR 94839, 4-acetyl-1-(3,4-dichlorophenyl)acetyl]-2-[(3-hydroxy-1-pyrrolidinyl)methyl]piperazine; E-2078, N-methyl-Tyr¹,N-methyl-Arg⁷,D-Leu⁸dynorphin A[1-8]ethylamide; SK-9709, Tyr-D-Ala-Phe-Leu-Arg (CH₂NH) Arg-NH₂; U50,488, (-)3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]-cyclohexyl)benzeneacetamide; ICI 174,864, N,N-diallyl-Tyr-Aib-Aib-Phe-Leu; U69,593, (5,7,8)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro[4.5]dec-8-yl)-benzeneacetamide.

Address correspondence to: Dr. Todd W. Vanderah, Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, AZ 85724. E-mail: vanderah{at}u.arizona.edu

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