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ANALGESIA

The Hypnotic, Electroencephalographic, and Antinociceptive Properties of Nonpeptide ORL1 Receptor Agonists After Intravenous Injection in Rodents

Alan J. Byford, MSc^*, Alison Anderson^*, Philip S. Jones, PhD, Ronald Palin, PhD, and Andrea K. Houghton, PhD^*

From the Departments of *Pharmacology and Chemistry, Organon Laboratories Ltd, Newhouse, Lanarkshire, ML1 5SH, United Kingdom.

Address correspondence and reprint requests to Andrea K. Houghton, PhD, Department of Pharmacology, Organon Laboratories Ltd, Newhouse, Lanarkshire, ML1 5SH, United Kingdom. Address e-mail to a.houghton{at}organon.co.uk.

Abstract

BACKGROUND: Agonists at the opioid receptor-like receptor 1 (ORL1) induce motor impairment, sedation, and loss of righting reflex (LRR) in rodents. This receptor may provide a novel target in the field of anesthesia.

METHODS: We examined the hypnotic, electroencephalographic (EEG), and antinociceptive effects of two IV administered nonpeptide ORL1 agonists, (Ro 65-6570 and Org 26383), using LRR in mice and rats, percent EEG burst suppression in rats, and formalin paw test in mice.

RESULTS: In mice, Ro 65-6570 and Org 26383 produced LRR (hypnotic dose 0.6 and 3.7 µmol/kg for Ro 65-6570 and Org 26383, respectively). Naloxone had no significant effect on sleep times produced by both compounds. In rats, Ro 65-6570 (0.6–2.4 µmol/kg) and Org 26383 (4–8 µmol/kg) produced LRR and burst suppression activity in the EEG. Both sleep times and burst suppression activity were significantly reduced with a selective ORL1 antagonist. In mice, dose-dependent inhibition of formalin-induced nociceptive behaviors occurred (Phase 1 ED₅₀ 0.4 and 1.8 µmol/kg and Phase 2 ED₅₀ 0.4 and 4.2 µmol/kg for Ro 65-6570 and Org 26383, respectively).

CONCLUSIONS: These results show that Ro 65-6570 and Org 26383 (probably via the ORL1 receptor) behave as IV hypnotics and analgesics in mice and rats, and that the hypnotic and antinociceptive doses are similar.

The recently identified G-protein coupled ORL1 (opioid receptor-like receptor 1) receptor has been the subject of much investigation subsequent to the characterization of human (1) and rodent (2) isoforms. This receptor shares a high degree of sequence homology with other opioid (µ, , and ) receptors, although traditional opioid ligands do not bind with high affinity to the ORL1 receptor (2). The endogenous peptide ligand for the ORL1 receptor has been termed nociceptin (3) and orphanin FQ (4), the former being the term used herein.

Purported roles of the nociceptin ORL1 system include cognition, pain, locomotion, anxiety, neuroendocrine control, and modulation of cardiovascular and respiratory function (5). Localization and functional data, primarily in rodent species, support these roles. The function of the nociceptin ORL1 system in modulating sensory processing has been studied extensively (5–8). Both antinociceptive and pronociceptive responses have been observed after administration of nociceptin to rodents; the effect appears to be dependent on the dose of nociceptin administered and the route of administration, as well as the noxious stimulus modality used and the stress level of the animals. The complex nature of the results obtained with nociceptin has led pharmaceutical companies to develop both nonpeptide agonists and antagonists as putative analgesics (9). An effect of nociceptin much less studied is its ability to produce sedation and loss of responsiveness (anesthesia). After supraspinal injection of nociceptin (10 nmol) muscular flaccidity, ataxia, and loss of righting reflex (LRR) were observed (4). Furthermore, systemic administration of a nonpeptide agonist, Ro 64-6198, has been reported to elicit sedation and LRR (10).

Much of the work published on the nociceptin ORL1 system is based on data obtained using the endogenous peptide nociceptin. Because of the rapid metabolism of peptides and their limited blood-brain permeability, it is difficult to examine the effects after systemic administration. The purpose of the present study was to characterize the hypnotic, electroencephalographic (EEG), and antinociceptive properties of two nonpeptide ORL1 agonists after IV administration. The two compounds used, Ro 65-6570 and Org 26383, have been characterized using in vitro assays at Organon. Ro 65-6570 and Org 26383 bind to the ORL1 receptor with a k_i of 1 and 4 nM, respectively and have selectivity over the µ opioid receptor of 17- and 15-fold, respectively (data not shown). In a mouse vas deferens assay, Org 26383 did not demonstrate activity as a µ opioid receptor agonist (11). The compounds also behave as full agonists in a cAMP functional assay with a k_i of 6 and 38 nM for Ro 65-6570 and Org 26383, respectively. We have also used an ORL1 reversible competitive antagonist in some experiments to show that the biological effects of the agonists can be reversed and/or blocked. The antagonist used is a proprietary compound with k_i of 18 nM for the ORL1 receptor and selectivity of >500-old over other opioid receptors and at least 50-fold selectivity over -aminobutyric acid and glutamate receptors (NovaScreen).

METHODS

This study was performed under a project license issued by the UK Home Office under the Animals (Scientific Procedures) Act 1986. Animals (male ICR mice, 20–30 g and male Wistar rats, 269–446 g; Harlan, Bicester, UK) were housed at a constant temperature of 21 ± 1°C under a 12/12 h light–dark cycle with access to food and water ad libitum. Experiments were performed during the light period of their diurnal cycle.

Hypnosis: LRR
Groups of eight mice were injected (10 mL/kg over 10 s), via a tail vein, with Ro 65-6570 (0.35, 0.5, 0.6, 0.7, or 1 µmol/kg), Org 26383 (2.8, 3.3, 4, or 5.6 µmol/kg), or vehicle. Immediately after injection, the mouse was placed on its back in a clear Perspex box. If the animal remained on its back for at least 30 s it was deemed to have lost its righting reflex (LRR). If immediate LRR did not occur, the animal was observed to determine whether LRR occurred over time. Onset of LRR, and time the animals regained the righting reflex (GRR) were recorded. Sleep time was calculated as LRR-GRR. A heated mat was used to maintain the animal’s body temperature.

Hypnosis: Pharmacologic Reversal
The second experiment examined whether naloxone or a proprietary ORL1 receptor antagonist could reverse sleep after Ro 65-6570 or Org 26383 administration. Ro 65-6570 and Org 26383 were given at 2 x HD₅₀ (hypnotic dose), based on the first experiment. Ro 65-6570 (1.2 µmol/kg) and Org 26383 (7.8 µmol/kg) were administered IV to 10 animals each. Animals were placed in a Perspex box and the time to LRR was recorded. Two minutes after LRR, the animal received an IV injection of naloxone (3 mg/kg), the ORL1 antagonist (10 µmol/kg), or saline. Immediately after the second injection, the animal was returned to its box and subsequent sleep time (time from second injection to GRR) was recorded.

EEG: Burst Suppression Test
This model has been described in detail elsewhere (12). Briefly, rats were instrumented with extradural electrodes under either isoflurane or intraperitoneal (i.p.) pentobarbitone (60 mg/kg) anesthesia. The rats were given a minimum of 7 days to recover from surgery, and also between experiments, permitting animals to be tested with both drugs.

On the day of experimentation, the animals were removed from their holding room, weighed, and their tails warmed for approximately 2 min in a beaker of warm water (approximately 42°C) in order to facilitate insertion of a 25-gauge needle into a tail vein for drug administration. The rats were lightly restrained by hand, connected to the EEG recording equipment, and injected with test compound or vehicle (maximum 1 mL/kg over 10 s) using a computer-controlled infusion pump (Model 44, Harvard Apparatus, Edenbridge, UK). After injection, the animals were lightly restrained until LRR. If this did not occur, they were placed in their home cage for observation. If LRR occurred, the animals were placed on top of a sodium acetate heat mat (Prism Healthcare, San Antonio, TX) to avoid hypothermia. The EEG was recorded until after the GRR or for sufficient time to be sure that LRR was unlikely to occur. For reversal studies, either vehicle or the ORL1 receptor antagonist (1 mL/kg) was injected IV 5 min after dosing with the ORL1 receptor agonist.

Nociception: Formalin Paw Test
To record paw movement after formalin injection an Automated Nociception Analyzer (ANA Instrument, University of CA, San Diego) was used (13). Each mouse had a metal band attached to its left paw and was housed in a Perspex cylinder (23 cm high x 9 cm wide) to acclimatize for 1 h. After acclimatization, groups of six mice were injected IV via the tail vein with Ro 65-6570 (0.03, 0.1, 0.3, or 1 µmol/kg), Org 26383 (0.1, 0.3, 1, or 3 µmol/kg) or vehicle. Five minutes after drug treatment, animals received a 20 µL IV injection of 5% formalin into the dorsum of the left hindpaw. Animals were placed in the chambers of the Automated Nociception Analyzer and paw movements recorded for 30 min. Collection parameters used were acquisition rate = 1 kHz; DAQ card buffer size = 5000 samples; range window length = 129 samples; convolution length = 129 samples; peak threshold = 0.35 V; peak width = 350 samples; binning interval = 60 s.

In this study, responses (licking, flinching, and lifting of the paw) recorded 0–5 and 20–30 min after formalin injection were designated Phase 1 and Phase 2, respectively (14).

Drugs
The ORL1 receptor agonists, Ro 65-6570 [(R,S)-8-acenaphten-1-yl-1-phenyl-1,3,8-triaza-spiro [4,5]decan-4-one hydrochloride] and Org 26383 [(R,S)-1-{1-(3-(5-methoxy-2-methylphenoxy-4-methylpentyl)-piperidinyl}-1,3-dihydro-2H-benzimidazole-2-one] were both synthesized by Organon, and were dissolved in 10% Tween 80 (Sigma-Aldrich, Dorset, UK) diluted using saline (Aqupharm®1; NaCl Ph Eur 0.9% w/v, Animalcare, York, UK). Org 26383 is a proprietary Organon compound, whereas Ro 65-6570 was identified from a Roche patent (15). The proprietary ORL1 receptor antagonist was dissolved in either 10% Tween 80 or saline. Naloxone hydrochloride (Sigma-Aldrich) was dissolved using saline. Propofol was injected as the commercially available product (Diprovan®, Astra Zeneca, Macclesfield, UK).

Data Analysis
The dose required to cause a 50% inhibition of both phases of the formalin response (ED₅₀) and LRR in 50% of mice (HD₅₀), plus the 95% confidence limits (shown in parentheses) were calculated in XL Fit version 2 (IDBS Software, Guilford, UK) using curve 205 and constants of 0 and 100 for bottom and top, respectively. All other data are presented as the mean ± se of the mean (sem), and analyzed using nonparametric analysis of variance (Kruskal–Wallis test) followed by the Dunn’s post hoc test. For all tests, P < 0.05 was considered as statistically significant from control.

The burst suppression ratio (BSR) was calculated as the percentage of time per 15 s EEG epoch spent in suppression, where suppression was defined as an interval in which the amplitude of the time-differentiated EEG signal stayed within a –15 to 15 µV/s window for at least 100 ms.

The highest BSR value (BSR_max) between the times of injection and the GRR, plus the mean BSR value from 5–10 min (BSR_5–10) after injection were calculated. The 5–10 min after injection period was chosen, as during this time, the BSR response was relatively stable.

RESULTS

Hypnosis: LRR
Both of the ORL1 receptor agonists, Ro 65-6570 and Org 26383, caused LRR in mice. The calculated HD₅₀ values were 0.59 (0.54–0.64) µmol/kg and 3.67 (3.12–4.32) µmol/kg for Ro 65-6570 and Org 26383, respectively. The individual onset times and sleep times are tabulated in Table 1.

Hypnosis: Pharmacologic Reversal
In the sleep reversal experiment, sleep times for animals receiving the opioid receptor antagonist, naloxone, were not reduced. However, treatment with the ORL1 receptor antagonist reduced sleep times for Ro 65-6570 from 38.3 to 11.8 min and reduced sleep times for Org 26383 from 31.0 to 4.3 min (Table 2).

Burst Suppression Test
Injection of the vehicle (10% Tween 80/saline) had no effect on the EEG of the rat. We selected doses of Ro 65-6570 (0.6, 1.2, and 2.4 µmol/kg) and Org 26383 (4 and 8 µmol/kg) based on multiples of the mouse HD₅₀ values described earlier. LRR generally occurred within 1 min of injection. The onset and sleep times were dose-dependent (Table 3). LRR coincided with EEG burst suppression. Figure 1 shows the relationship between burst suppression and dose. At the end of the sleep period, the animals awoke rapidly and immediately started to explore despite mild ataxia. This coincided with the EEG disappearance of burst suppression. Both doses of propofol (34 and 48 µmol/kg) produced burst suppression and LRR with rapid onset and offset (Fig. 1 and Table 3).

View larger version (22K): [in this window] [in a new window]   Figure 1. Burst suppression ratio (BSR) responses after IV administration of (a) 0.6, 1.2, and 2.4 µmol/kg Ro 65-6570; and (b) 4 and 8 µmol/kg Org 28383, and 34 µmol/kg propofol. The lines represent the mean values from 4 to 8 rats.

IV injection of the ORL1 antagonist (10 µmol/kg) resulted in a rapid reversal of Ro 65-6570 (1.2 µmol/kg)-induced burst suppression (Fig. 2), with GRR occurring 2.1 ± 0.1 min after injection of the antagonist when compared with 39.8 ± 0.6 min in the vehicle treated group.

View larger version (13K): [in this window] [in a new window]   Figure 2. The effects of an ORL1 receptor antagonist on burst suppression ratio (BSR) responses after IV administration of 1.2 µmol/kg RO 65-6570. The ORL1 antagonist (10 µmol/kg) or vehicle (1 mL/kg) was injected 5 min after injection of RO 65-6570. The times for loss of righting reflex (LRR) and gain of righting reflex (GRR) (antagonist group only) are shown. The lines represent the mean values from four rats.

Formalin Paw Test
IV administration of both Ro 65-6570 and Org 26383, caused dose-dependent inhibition of formalin-induced nociceptive behaviors in both Phase 1 and 2 (Fig. 3a and b). The highest dose of Ro 65-6570 (1 µmol/kg) decreased Phase 1 and Phase 2 nociceptive responses to 11.9% and 9.9% of vehicle-treated mice, respectively. This inhibition was significant (P < 0.01 and P < 0.02 for Phase 1 and 2, respectively). Although this dose initially caused LRR, animals regained their righting reflex after formalin injection. The calculated ED₅₀ values for Phase 1 and 2 were 0.35 (0.0–0.66) µmol/kg and 0.43 (0.16–0.71) µmol/kg, respectively.

View larger version (19K): [in this window] [in a new window]   Figure 3. The effect of (a) Ro 65-6570; and (b) Org 26383 on the nociceptive behaviors produced by injection of formalin into the paw of the mouse. Data are mean ± sem, n = 6.

The highest dose of Org 26383 (5 µmol/kg) decreased Phase 1 and Phase 2 nociceptive responses to 4.5% and 10% of vehicle-treated mice, respectively. This inhibition was significant when compared with vehicle-treated mice (P < 0.001 and P < 0.02 for Phase 1 and 2, respectively). This dose initially caused LRR, animals regained their righting reflex after formalin injection. No LRR was observed after administration of lower doses. The calculated ED₅₀ values for Phase 1 and 2 were 1.79 (–0.64 to 4.23) µmol/kg and 4.18 (–3.40 to 11.75) µmol/kg, respectively.

DISCUSSION

The results from this study demonstrate that IV administration of two chemically unrelated nonpeptide ORL1 receptor agonists produced dose-dependent LRR in both rats and mice, confirming previous findings that ORL1 receptor agonists have hypnotic properties (4,10). Furthermore, the nonpeptide ORL1 receptor agonists are antinociceptive in the mouse formalin paw test. The decreased nociceptive behavior could have been due to a change in the behavioral state of the mice, but this is unlikely as formalin injection reversed the LRR, and doses which did not affect LRR (e.g., 3 µmol/kg Org 26383) also produced an inhibition of Phase 1 nociceptive counts.

EEG burst suppression has been observed in many species, including humans, after administration of different classes of anesthetic drugs (16–20). The burst suppression model used in this study has been shown to be useful for monitoring the effects of hypnotics in rats (12). The data from this study indicate that both Ro 65-6570 and Org 26383 have hypnotic and EEG effects similar to the IV hypnotics propofol and thiopental. However, unlike propofol and thiopental, whose effects are mediated by enhancement of the depressant effects of -aminobutyric acid, the mechanism of action of Ro 65-6570 and ORG 26383 appears to be mediated completely via the ORL1 receptor. This is supported by complete reversal of the effects by IV administration of a selective ORL1 receptor antagonist. The lack of involvement of the µ opioid receptor is suggested by the inability of naloxone to block the hypnotic effects of Org 26383 or Ro 65-6570 in the mouse LRR assay. We did not test the effects of naloxone on the antinociceptive effects of ORG 26383 and Ro 65-6570, a shortcoming of our study design.

This study supports the hypothesis that ORL1 receptor agonists are antinociceptive. There is evidence in the literature that the endogenous peptide for the ORL1 receptor can be antinociceptive or pronociceptive, depending upon the dose and route of administration (5). However, there are few published data reporting the effects of nonpeptide ORL1 agonists on nociceptive processing after systemic administration. We did not observe a pronociceptive effect for either compound.

In summary, these data suggest that ORL1 receptor agonists have hypnotic, EEG, and antinociceptive properties in rodents, mediated via a novel mechanism of action. These properties suggest that the ORL1 receptor may be a novel drug target for anesthetic and analgesic drug development.

Footnotes

Accepted for publication October 3, 2006.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Web of Science (4) Citing Articles via Google Scholar Google Scholar Articles by Byford, A. J. Articles by Houghton, A. K. Search for Related Content PubMed PubMed Citation Articles by Byford, A. J. Articles by Houghton, A. K. Related Collections Pain Mechanisms Pain Pharmacology