Elsevier

Neuropharmacology

Volume 50, Issue 6, May 2006, Pages 677-689
Neuropharmacology

The in vivo properties of pagoclone in rat are most likely mediated by 5′-hydroxy pagoclone

https://doi.org/10.1016/j.neuropharm.2005.11.014Get rights and content

Abstract

The cyclopyrrolone pagoclone binds with roughly equivalent high affinity (0.7–9.1 nM) to the benzodiazepine binding site of human recombinant GABAA receptors containing either an α1, α2, α3 or α5 subunit. However, whereas it was a partial agonist at α1-, α2- and α5-containing GABAA receptors, pagoclone was a full agonist at receptors containing an α3 subunit. In the rat elevated plus maze assay pagoclone (3 mg/kg) had significant anxiolytic-like activity but at all three doses tested (0.3, 1 and 3 mg/kg p.o.) it produced a significant reduction in the total distance travelled. This sedative-like effect was confirmed in rat chain-pulling and spontaneous locomotor assays. Surprisingly, in the plasma and brain samples derived from the elevated plus maze assay, the major metabolite of pagoclone, 5′-hydroxy pagoclone, was present at 10–20-fold higher concentrations relative to the parent compound. In order to establish whether this metabolite might have pharmacological activity, we measured its affinity and efficacy profile and found that both were comparable to those of pagoclone with the exception that efficacy at the α1 subtype was considerably greater for 5′-hydroxy pagoclone compared with the parent. This metabolite had significant anxiolytic-like activity in the elevated plus maze but at these same doses (0.3–3 mg/kg p.o.) also produced sedation. It is therefore likely that in rats 5′-hydroxy pagoclone mediates the majority of the pharmacological actions following pagoclone administration.

Introduction

Since their introduction in the early 1960s, the 1,4-benzodiazepines, typified by diazepam, have been extensively used in the clinic due to their anxiolytic, sedative/hypnotic, myorelaxant, cognitive impairing and anticonvulsant activities (Sternbach, 1979). The diverse pharmacology of these compounds is now understood to be a consequence of modulation of GABAA receptors containing β and γ2 subunits and either an α1, α2, α3 or α5 subunit at a binding site that is physically distinct from, but allosterically coupled to, the agonist (GABA) binding site (Sieghart, 1995, Sieghart and Sperk, 2002). The classical benzodiazepines interact with these different GABAA receptor subtypes with equivalent affinity and efficacy and are therefore non-selective GABAA receptor modulators.

Depending upon the clinical setting, the various pharmacological attributes of the benzodiazepines may be either beneficial or a liability. For example, the myorelaxant and cognitive impairing properties may be beneficial when benzodiazepines are employed as premedication (Williams and Bowie, 1999, Buffett-Jerrott and Stewart, 2002) but are clearly liabilities for everyday living when given for other indications. Similarly, the sedative/hypnotic properties are useful for treating sleep disorders but are undesirable properties of an anxiolytic (Argyropoulos and Nutt, 1999, Lader, 1999). Accordingly, considerable efforts have been made in order to develop GABAA receptor modulators that are anxiolytic but which are devoid of sedative effects (Atack, 2005).

A key aspect of developing such non-sedating anxiolytics has been establishing which of the various GABAA receptor subtypes are associated with particular pharmacological activities of the benzodiazepines. In this regard, transgenic mouse studies in which particular GABAA receptor subunits have been either deleted or point-mutated have proved especially informative (Rudolph and Möhler, 2004). For example, the α5 subtype plays a role in cognitive processes (Collinson et al., 2002, Crestani et al., 2002) and compounds which selectively attenuate the effects of GABA at this subtype (i.e., α5-selective inverse agonists) enhance cognition in preclinical models (Maubach, 2003). In addition, the α1 subtype appears to be responsible for the majority of the sedation seen with non-selective benzodiazepines (Rudolph et al., 1999, McKernan et al., 2000). Moreover, the α1-selective compound zolpidem is used clinically as a hypnotic (Rush, 1998) and this hypnotic activity is associated with α1-containing GABAA receptors (Crestani et al., 2000). As a corollary, compounds devoid of efficacy at the α1 subtype are non-sedating in animal models (McKernan et al., 2000, Johnstone et al., 2004). In contrast, however, it remains uncertain whether the anxiolytic effects of diazepam are mediated via the α2 and/or α3 subtype. Thus, transgenic mice would suggest that the α2 subtype is responsible for the anxiolytic properties of diazepam (Löw et al., 2000) whereas pharmacological evidence implicates the α3 subtype (Atack et al., 2005, Dias et al., 2005). Nevertheless, it would seem that a compound with efficacy at the α2 and/or α3 subtypes but devoid of any α1 efficacy should be anxiolytic without the sedation associated with agonist activity at the α1 subtype (McKernan et al., 2000).

Pagoclone, which is the active (+)-enantiomer of the racemate RP 59037, was initially in clinical development for the treatment of panic attacks and generalized anxiety disorder (Sorbera et al., 2001, Bateson, 2003) but these indications are no longer being pursued due to lack of robust efficacy (Atack, 2005). However, pagoclone is being considered for the treatment of stuttering (Atack, 2005). Despite being evaluated clinically, the in vitro efficacy profile of pagoclone remains unclear. Hence, whilst it was initially described as a partial agonist (Piot et al., 1990), in a [35S]TBPS binding assay, a predictor of intrinsic efficacy (Supavilai and Karobath, 1983), pagoclone had an efficacy similar to the full agonist diazepam. On the other hand, in a [3H]Ro 15-1788 GABA shift assay, an alternative in vitro binding assay predictive of intrinsic efficacy (Braestrup et al., 1984), pagoclone behaved like a partial agonist (Doble et al., 1993). More recently, pagoclone has been reported to behave like an inverse agonist in that it has a GABA shift value of less than 1 (Cordon et al., 2001). Given this uncertainty, the initial purpose of the present study was to examine the intrinsic efficacy of pagoclone at different subtypes of the GABAA receptor in relation to the in vivo properties of this compound. During the course of these studies, it became apparent that the major metabolite of pagoclone, 5′-hydroxy pagoclone, occurs at much higher concentrations than the parent. Consequently, we undertook a series of studies to characterize the in vitro and in vivo properties of this compound to see whether it might contribute to the pharmacological effects observed after pagoclone administration. The in vitro affinity and efficacy of 5′-hydroxy pagoclone along with its in vivo anxiolytic- and sedative-like effects suggest that in rats it may, indeed, contribute an appreciable component of the in vivo properties of pagoclone, with sedation presumably being due to its full agonist activity at the α1 subtype.

Section snippets

Materials and methods

All animal procedures were performed in accordance with the U.K. Animals (Scientific Procedures) Act, 1986.

In vitro properties of pagoclone

Table 1 shows the affinity of the racemate RP 59037 and its separate enantiomers, of which the (+)-enantiomer is pagoclone, for the benzodiazepine binding site of human recombinant GABAA receptors containing different α subunits. RP 59037 has high and approximately equivalent affinity (Ki values = 1.4–10.5 nM) for recombinant human GABAA receptors containing either an α1, α2, α3 or α5 subunit (Table 1). These affinities are consistent with those seen against native rat brain receptors (Ki = 0.4–1.6 

Discussion

Pagoclone has attracted attention since it is reported to be anxioselective, in that, unlike existing clinically used benzodiazepines, there is a considerable separation between the doses required to cause sedation and those required to produce anxiolysis in preclinical species (Piot et al., 1990, Piot et al., 1992, Doble et al., 1993, Kinsora et al., 2000). Such a separation between anxiolysis and sedation was also described as occurring in man in Phase I studies in healthy normal volunteers (

Acknowledgements

We would like to thank Rosa Fradley for expert technical assistance with the locomotor activity experiment and Jennifer Chilenski for assistance with the preparative HPLC.

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