Behavioral and neurobiological effects of the enkephalinase inhibitor RB101 relative to its antidepressant effects

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Abstract

Nonpeptidic delta-opioid receptor agonists produce antidepressant-like effects in rodents, and compounds that inhibit the breakdown of endogenous opioid peptides have antidepressant-like effects in animal models. In this study, the behavioral effects of the enkephalinase inhibitor, RB101 (N-[(R, S)-2-benzyl-3-[(S)(2-amino-4-methyl-thio)-butyldithio]-1-oxopropyl]-l-phenylalanine benzyl ester), were examined. Specifically, the effects of RB101 on convulsive activity, locomotor activity, and antidepressant-like effects in the forced swim test were studied in Sprague–Dawley rats, and the opioid receptor types mediating these effects were examined by antagonist studies. In addition, the effects of RB101 on brain-derived neurotrophic factor (BDNF) mRNA expression were evaluated in relation to its antidepressant effects. RB101 produced delta-opioid receptor-mediated antidepressant effects (32 mg/kg i.v. and 100 mg/kg i.p.) and increased locomotor activity (32 mg/kg i.v.) in rats. RB101 did not produce convulsions or seizures and did not alter BDNF mRNA expression. In conclusion, RB101 has the potential to produce antidepressant effects without convulsions.

Introduction

Nonpeptidic delta-opioid receptor agonists demonstrate antidepressant-like activity in animal models of depression (Broom et al., 2002, Jutkiewicz et al., 2004, Saitoh et al., 2004). In addition, delta-opioid receptor agonists stimulate neurobiological correlates of antidepressant activity in rat brains. For example, a single administration of the delta receptor agonist (+)BW373U86 has been shown to increase brain-derived neurotrophic factor (BDNF) mRNA expression in rat frontal cortex, hippocampus, and basolateral amygdala (Torregrossa et al., 2004). Increases in BDNF have been suggested to be responsible for the clinical efficacy of antidepressant treatment (Duman, 2002, Vaidya and Duman, 2001). The rapid increase in BDNF mRNA expression produced by delta receptor agonists suggests that compounds acting on the delta-opioid receptor system may be faster acting than traditional antidepressants, and a promising system for the development of a new class of antidepressants. However, delta-opioid receptor agonists also induce convulsions in a number of species potentially limiting their therapeutic utility (Comer et al., 1993, Dykstra et al., 1993, Negus et al., 1994, Pakarinen et al., 1995, Hong et al., 1998, Broom et al., 2002, Jutkiewicz et al., 2004). In order to develop delta-opioid receptor agonists as potential treatments for depression, compounds without seizure activity would be ideal. This study examines an alternative mechanism of activating the delta-opioid receptor in order to achieve antidepressant-like effects without convulsions. These results are discussed as compared to the effects observed with nonpeptidic delta-opioid receptor agonists.

Opioid peptides have been demonstrated to have antidepressant actions in animal models of depression without producing overt convulsions. For example, enkephalins and endorphins decreased immobility in the forced swim test and in the learned helplessness paradigm in rats, demonstrating antidepressant-like effects (Kastin et al., 1978, Tejedor-Real et al., 1995, respectively); however, analogs of endogenous enkephalins and other opioid peptides have been shown to produce epileptiform activity and seizures (Haffmans and Dzoljic, 1983, Stutzmann et al., 1986, Walker and Yaksh, 1986, Snead, 1986, De Sarro et al., 1992). It has also been demonstrated that preventing the breakdown of endogenous opioid peptides with enkephalinase inhibitors produces antidepressant-like effects in animal models, but these compounds have not been evaluated in terms of convulsions or electroencephalographic changes. RB38A, a mixed inhibitor of enkephalinase, and RB38B, a selective inhibitor of endopeptidase EC 3.4.24.11, produced antidepressant-like effects in the learned helplessness paradigm following intracerebroventricular administration (Tejedor-Real et al., 1993, Tejedor-Real et al., 1995). These effects were blocked by the nonselective opioid receptor antagonist naloxone, demonstrating an opioid-mediated effect.

Oral administration of the enkephalinase inhibitor BL-2401 demonstrated antidepressant-like effects in the forced swim test in mice, and these effects were blocked by the nonselective opioid receptor antagonist naloxone (Kita et al., 1997). Enkephalinase inhibitors elevate levels of endogenous opioids that act at multiple opioid receptors; therefore, the receptor mediating this antidepressant activity was unknown. However, Baamonde et al. (1992) and Tejedor-Real et al. (1998) demonstrated that the selective delta-opioid receptor antagonist, naltrindole, blocked the antidepressant-like effects produced by the enkephalinase inhibitor RB101 in the learned helplessness model of depression in mice. To further support this finding, intravenous administration of the selective delta-opioid peptide BUBU (Tyr-D.Ser-(O-tert-butyl)-Gly-Phe-Leu-Thr(O-Tert-butyl-OH) demonstrated antidepressant-like effects in the learned helplessness paradigm (Tejedor-Real et al., 1998). Although the enkephalinase inhibitor RB101 demonstrated antidepressant-like effects through the delta-opioid receptor, its antinociceptive effects were mediated through the mu-opioid receptor (Noble et al., 1992).

The enkephalinase inhibitor RB101 appears to produce antidepressant-like effects similar to those observed with nonpeptidic delta-opioid receptor agonists. However, enkephalins are promiscuous ligands in that they bind to multiple opioid receptor subtypes, and the contribution of elevated enkephalin levels to the different behavioral effects produced by RB101 has not been fully elucidated. In addition, the effects of RB101 on convulsions and seizure-like electroencephalographic (EEG) changes have not been evaluated. The present study characterized the behavioral and physiological effects of RB101 in Sprague–Dawley rats and the neurobiological correlates of the antidepressant-like effects of RB101. In particular, RB101 was evaluated based on behavioral effects observed with delta-opioid agonists: convulsions, EEG, locomotor stimulation, and antidepressant-like effects; and the involvement of opioid receptor subtypes was examined. In addition, the antidepressant-like effects produced by RB101 may be induced by increases in BDNF. However, the effect of endogenous opioids on BDNF mRNA expression is unknown. Thus, changes in BDNF mRNA were measured following administration of RB101.

Section snippets

Subjects

Male Sprague–Dawley rats (250–300 g), obtained from Harlan Sprague Dawley (Indianapolis, IN), were housed in groups of three or four animals per cage. All animals were fed on a standard laboratory diet and maintained on a 12 h light/dark cycle with lights on at 6:30 am at an average temperature of 21 °C. Studies were performed in accordance with the Declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of

Results

In the forced swim test, RB101 administered i.v. produced a dose-dependent decrease in immobility (F(4, 29) = 11.89; P < 0.0001), a significant increase in swimming behaviors (F(4, 29) = 22.52; P < 0.0001), but did not alter climbing (F(4, 29) = 0.75; P = 0.57) (Fig. 1) when administered 30 min prior to swimming. A dose of 32 mg/kg RB101 (i.v.) significantly decreased immobility (P < 0.01) and significantly increased swimming (P < 0.01). This decrease in immobility was no longer observed 60 min after RB101

Discussion

The enkephalinase inhibitor RB101 (i.v.) demonstrated antidepressant-like effects in the forced swim test in rats, presumably by increasing endogenous levels of delta-opioid receptor-preferring neurotransmitter met-enkephalin (Daugé et al., 1996). This antidepressant activity was blocked with the selective delta-opioid receptor antagonist naltrindole, but not with mu-selective doses of naltrexone nor the kappa-selective receptor antagonist, nor-BNI. These findings confirm previous studies

Acknowledgements

This work was supported by grants from the Unites States Public Health Service Grants DA00254, T32 GM07767, and T32 DA07267.

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