Pharmacological Properties of DOV 315,090, an ocinaplon metabolite

Background Compounds targeting the benzodiazepine binding site of the GABAA-R are widely prescribed for the treatment of anxiety disorders, epilepsy, and insomnia as well as for pre-anesthetic sedation and muscle relaxation. It has been hypothesized that these various pharmacological effects are mediated by different GABAA-R subtypes. If this hypothesis is correct, then it may be possible to develop compounds targeting particular GABAA-R subtypes as, for example, selective anxiolytics with a diminished side effect profile. The pyrazolo[1,5-a]-pyrimidine ocinaplon is anxioselective in both preclinical studies and in patients with generalized anxiety disorder, but does not exhibit the selectivity between α1/α2-containing receptors for an anxioselective that is predicted by studies using transgenic mice. Results We hypothesized that the pharmacological properties of ocinaplon in vivo might be influenced by an active biotransformation product with greater selectivity for the α2 subunit relative to α1. One hour after administration of ocinaplon, the plasma concentration of its primary biotransformation product, DOV 315,090, is 38% of the parent compound. The pharmacological properties of DOV 315,090 were assessed using radioligand binding studies and two-electrode voltage clamp electrophysiology. We report that DOV 315,090 possesses modulatory activity at GABAA-Rs, but that its selectivity profile is similar to that of ocinaplon. Conclusion These findings imply that DOV 315,090 could contribute to the action of ocinaplon in vivo, but that the anxioselective properties of ocinaplon cannot be readily explained by a subtype selective effect/action of DOV 315,090. Further inquiry is required to identify the extent to which different subtypes are involved in the anxiolytic and other pharmacological effects of GABAA-R modulators.


Background
GABA A receptors (GABA A -R) are pentameric membrane proteins that belong to the superfamily of cys-loop ligandgated ion channels (LGIC), which operate as GABA-gated Cl --selective channels. GABA A -R mediate most of the fast inhibitory neurotransmission in the CNS [1][2][3]. Initially, two subunits of the GABA A -R named α and β were purified [4,5] and subsequently their cDNAs were isolated [6]. Twenty related GABA A -R subunits have been so far identified in mammals (α 1-6 , β 1-4 , γ 1-3 , δ, ε, π, θ, and ρ 1-3 [7,8]), yielding a high degree of potential diversity. If all of these subunits could randomly co-assemble, more than one hundred thousand GABA A -R subtypes with distinct subunit composition and arrangement would be formed [9]. The composition of the most abundant GABA A -R type in the CNS is αβγ, and immunohistochemistry studies suggest that receptors containing α 1 , β 2/3 and γ 2 subunits are the most widespread GABA A -R subtype in adult mammalian brain and represent about 50% of the total receptor pool [2,10].
Typical αβγ GABA A -Rs harbor two agonist (GABA) binding sites located at the two α/β subunit interfaces [2,11]. The function of GABA A -Rs can be modulated by various compounds acting at different allosteric sites located on GABA A -Rs. The benzodiazepine (BZD) site, which is located at an α/γ interface [12,13], is the most frequently targeted site for therapeutic agents, and ligands that enhance GABA A -R function through positive modulation at this site possess anxiolytic, sedative, myorelaxant, anesthetic and amnestic properties [2,3,10,14]. Based on pharmacological studies in transgenic mice, it has been proposed that GABA A -Rs can be classified into the following pharmacological classes according to the effects of BZ site ligands: α 1 -containing receptors (GABA A1 ) that mediate sedative effects; α 2 -containing receptors (GABA A2 ) that mediate anxiolytic effects; α 3 -containing receptors (GABA A3 ) that mediate myorelaxation; and α 5 -containing receptors (GABA A5 ) that are involved in learning and memory processes [7,15,16]. This classification is consistent with the sedative/hypnotic profile of GABA A1 -preferring compounds such as zolpidem and zaleplon [17], but pharmacological studies in wild-type animals and in man have raised questions regarding the attribution of anxiolytic effects to GABA A2 receptors. In particular, a number of compounds have been identified that exhibit an anxioselective profile in vivo despite lacking the expected GABA A2 selectivity. A series of compounds with mixed preference for α 2 /α 3 -containing receptors has been reported to produce robust anxiolysis in animals without noticeable sedation, including one compound that exhibits selectivity for α 3 -containing receptors [18][19][20][21]. Other compounds, such as ocinaplon [22] and DOV 51,892 [23], are anxiolytic in humans and animals without undesired side effects such as sedation and myorelaxation, but do not exhibit strong selectivity among GABA A -Rs sensitive to benzodiazepines (that is, those receptors containing α 1-3 and/or α 5 -subunits) One hypothesis that could explain the anxioselective profile of ocinaplon is the presence of one or more biotransformation products that exhibit selectivity at GABA A2 receptors. To test this hypothesis, we characterized the pharmacological properties of the major biotransformation product of ocinaplon in dogs, rats and man, DOV 315,090 ( Fig. 1), using in vitro radioligand binding and two-electrode voltage-clamp electrophysiology. We now report that like its parent compound, DOV 315,090 acts as a positive modulator at GABA receptors, and like its parent, does not exhibit marked selectivity among α 1-3 and α 5 containing receptors. Thus, while DOV 315,090 may contribute to the pharmacological actions of ocinaplon, the anxioselective profile of ocinaplon cannot be Structures of diazepam, ocinaplon and DOV 315,090
For competition binding, 100 μg of membrane protein was incubated in 500 μl of PBS buffer with 0.5 nM [ 3 H]Ro15-1788 (78.6 Ci/mmol, PerkinElmer Life Sciences) in the presence of diazepam (1 nM -10 μM, Sigma-Aldrich), ocinaplon (0.1 -250 μM, DOV Pharmaceuticals) or DOV 315,090 (0.1 -50 μM, DOV Pharmaceuticals) for 1 h at 0°C. The samples were then diluted with 5 ml of ice-cold buffer and filtered under vacuum through glass-fiber filters (GF/B Whatman). Filters were washed 3 times with 5 ml of buffer and the radioactivity was quantitated by liquid scintillation counting in 5 ml of Ecolite scintillation fluid (ICN). Non-specific binding determined in the presence of 100 μM Ro 15-1788 (Sigma-Aldrich) was subtracted from total binding to calculate specific binding. Data were analyzed by non-linear regression (Prism, Graph-Pad software).

Recording of GABA-Gated Currents from GABA A Receptors Expressed in Xenopus Oocytes
cRNAs encoding GABA A -R α 1 , α 2 , α 3 , α 5 , β 2 and γ 2S subunits were injected into oocytes from Xenopus laevis. Fortyeight hours later, measurements of the effects of diazepam, ocinaplon and DOV 315,090 on GABA-gated Clcurrents from oocytes expressing GABA A -Rs were performed using a Warner TEVC amplifier (Warner Instruments, Inc., Foster City, CA) (Park-Chung et al., 1999). GABA (Sigma) was prepared as a 1 M stock solution in ND96. Microelectrodes of 1-3 MΩ when filled with 3 M KCl were used to record from oocytes in a recording cham-ber continuously perfused with ND-96 buffer solution. During data acquisition, oocytes were clamped at a holding potential of -70 mV. Drugs were applied by perfusion at a rate of approximately 50 μl sec -1 for 20 s followed by a 120 s wash. At the end of each experiment 3 μM of diazepam was applied as a potentiation control. All experiments were performed at room temperature (22-24°C).
GABA concentration-response data was obtained for each subunit combination, and the GABA EC 10 was determined by nonlinear regression using the logistic equation. This concentration of GABA was used for modulation studies. Peak current measurements were normalized and expressed as a fraction of the peak control current measurements. Control responses to an EC 10 concentration of GABA were re-determined after every 2 -4 applications of modulator + GABA. Percent potentiation is defined as [I (GABA + Drug) /I GABA )-1] × 100, where I (GABA + Drug) is the current response in the presence of diazepam, and I GABA is the control GABA current. Potentiation data from each oocyte was fitted to the equation Potentiation = E max × [Drug]/ ([Drug + EC 50 ) by non-linear regression (Prism, Graph-Pad software). Due to a decline in the response at high diazepam concentrations, concentrations of diazepam above 3 μM were excluded from the fit. Some oocytes expressing α1β1γ2 receptors appeared to exhibit a biphasic modulatory response to diazepam, suggesting the possible presence of an additional component of modulation with a sub-nM EC 50 . For 6 of 8 oocytes, the fit was significantly improved by adding a second, higher-potency component of modulation, but the affinity of this second component was not well resolved in fitting due to its small amplitude. Given the lack of consistency of this possible high affinity effect, we have omitted it in fitting our concentration-effect curves. The choice of fitting to a monophasic or biphasic equation had only a small effect on the EC 50 for the major component of modulation. For diazepam, the mean EC 50 of the major component was increased from 35 nM to 42 nM when a two-component fit was used for those oocytes in which it produced a significant improvement in the sum of squares.

Biotransformation of ocinaplon into DOV 315,090 in vivo
As shown in Figure 2, DOV 315,090 appears rapidly in plasma following i.v. or oral administration of a behaviorally active dose of ocinaplon (5 mg/kg) to rats. At 1 h, corresponding to the time at which the anticonflict effect of ocinaplon was evaluated [22], the plasma concentration of DOV 315,090 is ~38% of the concentration of parent compound. Figure 3 and Table 1 document the binding properties of diazepam, ocinaplon and DOV 315,090 in HEK293 cells expressing different GABA A -R subunit combinations. Examination of binding constants shows that ocinaplon and DOV 315,090 have lower affinity than diazepam at all of the receptor subunit combinations tested. The binding profile of DOV 315,090 is similar to that of ocinaplon, with little selectivity among the subunit combinations tested. In contrast to diazepam, which exhibits markedly lower affinity for α 1 β 2 γ 3 and α 2 β 2 γ 3 receptors than for α 1 β 2 γ 2 s and α 2 β 2 γ 2 s receptors, replacement of γ 2S with γ 3 had little effect on the affinity of either ocinaplon or DOV 315,090 for any subunit combination (Table 1). Also, whereas diazepam has similar affinity for α 1 -containing and α 2 -containing receptors, both ocinaplon and DOV 315,090 have 3-4 fold lower affinity for α 2 -containing receptors. Specific [ 3 H]Ro15-1788 or [ 3 H]flunitrazepam binding to membrane preparations from cells transfected with α 3 , β 2 and γ 3 subunits was not detected, suggesting that these subunits failed to assemble in the HEK293 cells.

Discussion
In the CNS, classical 1,4-BZDs such as diazepam, as well as other ligands of the BZD binding site, act on GABA A -Rs that are composed of α, β, and γ subunits. The majority of GABA A receptors contain α 1-6 , β 2/3 and γ 2 subunits, whereas the β 1 and γ 1/3 subunits have very restricted pat-terns of expression [2]. It has been shown that BZD pharmacology is primarily dependent upon the α subunit subtype present (α 1-3 or α 5 ), whereas receptors containing α 4 or α 6 subunits are insensitive to "classical" 1,4-BZDs [7,24,25]. Studies of animals in which genes coding for specific α subunits have been deleted or mutated to eliminate BZD sensitivity (e.g. the α 1 H101R mutation, which disrupts the BZD binding site) led to the hypothesis that the sedative effects of the BZDs are mediated by α 1 -subunit containing receptors (designated GABA A1 -R), whereas anxiolytic effects are mediated by α 2 -subunit containing receptors (GABA A2 -R) [7,17,26,27]. GABA A -Rs containing   α 5 subunits are thought to be responsible for the impairment of learning and memory that is induced by BZDs [28]. These finding raised the attractive prospect that BZDlike drugs that specifically target GABA A -Rs that contain a specific α-subunit will be able to produce the intended pharmacological effect (e.g sedation or anxiolysis) with reduced incidence of side effects. Because BZD-like drugs function as allosteric modulators and do not occupy the GABA binding site, specificity may be potentially achieved on the basis of either differences in potency or on differences in modulatory efficacy at specific receptor subtypes.

Receptor Type
Compounds such as zolpidem and zaleplon, which exhibit higher affinity for α1-containing receptors relative to other subtypes, have been promoted as sedative agents, driven in part by the hypothesis that selectivity for GABA A1 -Rs would translate into an improved side-effect profile, particularly with respect to tolerance, withdrawal, and abuse liability. Although these compounds are effective sedative agents, consistent with the identification of GABA A1 -Rs as mediating sedation, the selectivity of these compounds for GABA A1 -Rs vs. GABA A -Rs containing other α-subunits is generally an order of magnitude or less, and Potentiation of GABA-gated currents by diazepam, ocinaplon and DOV 315,090 Figure 4 Potentiation of GABA-gated currents by diazepam, ocinaplon and DOV 315,090. Rat GABA A -Rs consisting of α 1 β 2 γ 2S , α 2 β 2 γ 2S , α 3 β 2 γ 2S and α 5 β 2 γ 2S subunits were expressed in Xenopus oocytes. Potentiation was determined using an EC 10 concentration of GABA (~10 μM for α 1 β 2 γ 2S , α 2 β 2 γ 2S and α 3 β 2 γ 2S ; ~5 μM for the α 5 β 2 γ 2S ). Curves were calculated by normalizing values of relative currents obtained following administration of diazepam (❍), ocinaplon (•) or DOV 315,090 (ᮀ) in the presence of GABA (from at least four oocytes harvested from at least two batches) to the value obtained following application of GABA. The dose-response curves of diazepam were fitted up to 3 μM. Higher concentrations (in parentheses) were excluded from the fit due to a decline in potentiation at higher concentrations. Smooth curves are calculated based on mean parameter values given in Table 2. Asterisks indicate fits for which the extrapolated E max is more than 25% greater than the maximum potentiation observed at highest drug concentration.
it is unclear to what extent the hypothesized benefits are achieved in clinical practice [17].
However, the situation is less clear for compounds possessing anxiolytic properties. Recently published articles describe the pharmacological properties of two novel anxioselective compounds -ocinaplon [22] and DOV 51892 [23]. These compounds do not exhibit a marked selectivity among GABA A -Rs containing different diazepam-sensitive subunits (e.g. α 1-3 and α 5 ), yet are reported to be anxioselective, lacking sedative and myorelaxant side effects at anxiolytic doses. In particular, DOV 51892 exhibits higher efficacy than diazepam at GABA A1 -Rs.
The classic BZD diazepam has been shown to act with high efficacy and similar potency across a broad spectrum of GABA A -Rs [1,10,22] (Table 2). This lack of selectivity with respect to either potency or efficacy among the major GABA A -R types have been hypothesized to account for the side effects associated with the use of diazepam when used as an anxiolytic, which include sedation, myorelaxation, narcosis, and amnesia. However, as has been confirmed by in vivo behavioral studies, such side effects are not observed with ocinaplon (e.g. in motor activity test, inclined screen and rod walking) or for DOV 51892 (e.g. rotarod and grip strength tests), even at doses well in excess of those that enhanced punished responding in the thirsty rat test [22,23]. Further, ocinaplon is an effective anxiolytic in humans at doses that do not produce BZDlike side effects [22]. The present study was designed to test whether the anxioselective profile of ocinaplon is due to metabolism into subtype-selective metabolites. Our pharmacokinetic data demonstrate that in rats, the major metabolite of ocinaplon is a 4'-N' oxide, DOV 315,090. Whereas DOV 315,090 is active as a GABA A -R modulator, and its in vitro binding affinities for recombinant α 1 β 2 γ 2S , α 2 β 2 γ 2S , and α 3 β 2 γ 2S receptors differ only marginally from ocinaplon, its affinity for α 5 β 2 γ 2S receptors is only slightly lower than that of ocinaplon (~2-fold).
Comparison of the pharmacological profile of ocinaplon and DOV 315,090 using two electrode voltage clamp electrophysiology (Table 2) shows that the greatest difference in efficacy occurred at α 3 β 2 γ 2S receptors. Although a clear maximum was not attained due to solubility limits, the extrapolated maximum potentiation by DOV 315,090 was 1.87-fold greater, followed by a 1.45-fold difference at α 1 β 2 γ 2S receptors compared to ocinaplon. In contrast, maximum potentiation by DOV 315,090 was lower than that of ocinaplon at the α 5 β 2 γ 2S receptor subtype. The efficacies of DOV 315,090 and ocinaplon at α 2 β 2 γ 2S receptors were similar.
These results do not support the hypothesis that the anxioselective profile of ocinaplon is attributable to enhanced selectivity of its metabolite DOV 315,090 for α 2 -containing receptors. Thus, compared to ocinaplon, DOV 315,090 does not exhibit enhanced affinity or potency for α 2 -containing receptors over α 1 -containing receptors, whereas the difference in efficacy favors α 3 -, α 5 -, or α 1containing receptors over α 2 -containing receptors. The present experiments examined GABA A -Rs in two different heterologous expression systems (Xenopus oocytes and HEK 293 cells), which may be lacking modulatory proteins or regulatory mechanisms that are only present in neurons. While we cannot exclude the possibility that such interactions somehow confer differences in modulator binding or efficacy, such a hypothesis would require that such interactions modify the structure of the benzodiazepine binding site, which is located in the extracellular domain of the GABA A -R, in such a way as to selectively alter its interactions with different ligands.
In summary, transgenic mice in which the BZD recognition site of the α 2 subunit is disabled exhibit reduced diazepam sensitivity in behavioral tests considered to be predictive of anxiolytic activity, and a similar modification to the α 1 subunit reduces sensitivity in tests held to be predictive of sedation [15,26]. These observations have led to optimism that it will be possible to achieve the long-desired goal of developing a nonsedating anxiolytic [36]. And indeed, there has been substantial progress in identifying such compounds [19][20][21][22]31,[37][38][39][40], yet ironically, they do not in general conform to the expected paradigm of favoring α 2 -containing over α 1 -containing receptors. This suggests that anxiolysis in humans may prove to be more complex than is suggested by a simple reading of the results from transgenic mice in behavioral models thought to be indicative of anxiety. It remains to be determined whether differences in the design of the behavioral assays [41,42], interspecies differences [43,44], or a combination of these factors account for these discrepancies. Translating such promising results into clinically useful compounds is likely to require an improved understanding of the ways in which BZD-like ligands act at different GABA A -R subtypes and the consequences of these effects upon neural system-mediated behavioral outputs.

Conclusion
1. DOV315090 is a major metabolite of the anxioselective GABA A -R modulator ocinaplon.
3. The anxioselective properties of ocinaplon, demonstrated in both preclinical and clinical studies, are not a consequence of enhanced subtype selectivity by DOV315090.

Authors' contributions
DB carried out electrophysiological recordings. MCG carried out radioligand binding experiments. EK performed initial electrophysiological experiments. SD developed the data-acquisition hardware and software used in this study. TTG participated in the design of the study, performed the statistical analysis and participated in manuscript preparation. DHF participated in the design of the study and participated in manuscript preparation. PS directed development of ocinaplon at DOV Pharmaceuticals and participated in manuscript preparation. ASB identified major ocinaplon metabolite and participated in manuscript preparation. All authors read and approved the final manuscript.