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NEUROPHARMACOLOGY

Indiplon Is a High-Affinity Positive Allosteric Modulator with Selectivity for 1 Subunit-Containing GABA_A Receptors

Departments of Neuroscience (R.E.P., J.E.P., R.D., H.B., A.P.C., A.C.F.) and Molecular Biology (W.Y.), Neurocrine Biosciences Inc., San Diego, California

Received for publication October 6, 2005
Accepted January 5, 2006.

	Abstract

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Indiplon (NBI 34060) is a novel pyrazolopyrimidine currently in development for the treatment of insomnia. We have previously shown that indiplon exhibits high-affinity binding to native GABA_A receptors from rat brain and acts as a positive allosteric modulator of GABA_A receptor currents in cultured rat neurons (Sullivan et al., 2004). In this study, we examined the GABA_A receptor subunit selectivity of indiplon using electrophysiological techniques to record GABA-activated chloride currents from recombinant rodent GABA_A receptors expressed in human embryonic kidney 293 cells. Indiplon potentiated the GABA-activated chloride current in recombinant GABA_A receptors in a dose-dependent and reversible manner and was approximately 10-fold selective for 1 subunit-containing receptors over GABA_A receptors containing 2, 3, or 5 subunits. The EC₅₀ values were 2.6, 24, 60, and 77 nM for 122, 222, 332, and 522 receptors, respectively. Indiplon was approximately 10 times more potent than zolpidem and zopiclone and >100 times more potent than zaleplon. Moreover, indiplon, up to 1 µM, did not potentiate GABA_A receptors composed of 422 and 622 subunits. This mechanism of action is proposed to underlie the sedative-hypnotic effects of indiplon in animals and humans.

The GABA_A receptor is a hetero-oligomeric complex composed of five transmembrane spanning subunits from sixteen different genes, (1–6), (1–3), (1–3), , , , and (Barnard et al., 1998; Korpi et al., 2002; Whiting, 2003). In most neurons, two subunits, two subunits, and one subunit form the typical GABA_A receptor (Chang et al., 1996; Tretter et al., 1997). The , , , and subunits have some reported selective functions but are not yet fully understood. Theoretically, there are thousands of possible subunit combinations, but a limited number of subtype combinations have been found in native systems with 122, 232, and 332 being the most abundant (Whiting, 2003). The assembly of , , and subunits is required to produce functional GABA_A receptors that exhibit all of the pharmacological properties of native GABA_A receptors. Benzodiazepine binding occurs at the interface between and 2 subunits (Wieland et al., 1992). GABA elicits chloride currents in recombinant GABA_A receptors composed of only and subunits, but these currents are not potentiated by benzodiazepines (Schofield et al., 1987).

The diversity of subunits and their heterogeneous distribution between brain regions provokes the question of whether GABA_A receptors composed of different subunit combinations play different functional roles in the brain. GABA_A receptors containing 1, 2, 3, or 5 subunits in combination with subunits bind to and are potentiated by benzodiazepines. In contrast, 4 or 6 subunit-containing receptors are insensitive to the classic benzodiazepines like diazepam (with the exceptions of the antagonist Ro15-1788 (flumazenil) and the inverse agonist Ro15-4513 [ethyl 8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a]-[1,4]benzodiazepine-3-carboxylate], which show weaker agonist activity at 4 and 6). Genetically engineered mice have been generated in which a single histidine (His) to arginine (Arg) mutation has been introduced for individual subunits that render them insensitive to diazepam, whereas GABA_A receptor expression and GABA-evoked responses are normal (Mohler et al., 2002). These mouse lines have been used to explore the physiological roles of GABA_A receptor subunits in vivo. GABA_A receptors containing 1 subunits play a role in sedation, as the 1 (H101R) mutant mice are resistant to the sedative effects of diazepam (Rudolph et al., 1999; Low et al., 2000; McKernan et al., 2000) and zolpidem (Crestani et al., 2000). GABA_A receptors containing 2 subunits play a role in anxiety, as the 2 (H101R) mutant mice are resistant to the anxiolytic effects of diazepam (Low et al., 2000). The role of 3 subunit-containing GABA_A receptors is unclear, because the 3 (H126R) mutant mice do not show any missing diazepam responses (Low et al., 2000). The restricted expression of GABA_A receptors containing 5 subunits to the hippocampus suggests that they may play a role in learning and memory. This is supported by the 5 (H105R) mutant mice, which exhibit enhanced learning and memory (Collinson et al., 2002).

For the treatment of insomnia, zolpidem (Ambien) is currently the market leader, representing the class of so-called "nonbenzodiazepines" that also include the marketed drugs zopiclone (Imovane), eszopiclone (Lunesta), and zaleplon (Sonata). The advantages of zolpidem over the earlier benzodiazepine sedative-hypnotics, such as triazolam (Halcion), are a short duration of action with reduced residual sedation upon waking and fewer cognitive and psychomotor side effects (Mitler, 2000). In terms of GABA_A receptor pharmacology, zolpidem binds to the benzodiazepine site and acts as a positive allosteric modulator. Interestingly, zolpidem has highest affinity for GABA_A receptors containing 1 subunits, a feature that has been proposed to underlie its improved properties as a sedative-hypnotic drug (Sanger, 2004).

Indiplon (NBI 34060) is a novel pyrazolopyrimidine exhibiting high affinity for the benzodiazepine site on GABA_A receptors currently being developed for the treatment of insomnia (Sullivan et al., 2004). The present study was carried out to determine the mechanism of action of indiplon on both native and recombinant GABA_A receptors using an electrophysiological assay to measure functional chloride currents. In addition, we evaluated the subunit selectivity by testing indiplon on each of the six subunits (1-6) in combination with 22 (or 32) subunits. Finally, the activity of indiplon on native and recombinant GABA_A receptors was compared with the sedative-hypnotics zolpidem, zopiclone, and zaleplon.

	Materials and Methods

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Neuronal Cell Culture. Neuronal cultures were prepared from neonatal rats. Cerebral cortices from one rat were removed under a dissecting microscope and cut into small pieces (0.5 mm²). The tissue was digested in papain/DNAase (Papain Dissociation System; Worthington Biochemical Corp., Lakewood, NJ) for 45 min at 37°. The pieces were then triturated through a 5-ml serological pipette five to 10 times until the solution became cloudy. The dispersed cells were counted on a hemacytometer and plated at very low density (2000 cells per well) onto pre-existing monolayers of astrocytes growing on 12-mm diameter glass coverslips in serum-free basal medium Eagle (BME) containing B27 supplements (Gibco, Grand Island, NY). On the 4th day, 5'-fluoro-2'-deoxyuridine (10 µM) was added to prevent proliferation of non-neuronal cells. Cells were typically used for electrophysiological experiments at 7 to 14 days in vitro.

Astrocyte cultures were prepared from the same cell dissociate described above. The remaining cells were plated into a T-25 tissue culture flask in astrocyte medium (Dulbecco's modified Eagle's medium/F12 supplemented with 10% fetal bovine serum). After approximately 1 week, a monolayer of type 1 astrocytes was established. At this time, cytosine arabinoside (10 µM) was added to kill rapidly dividing microglial cells and fibroblasts. After 3 days, the flask was shaken overnight at 200 rpm to dislodge the contaminating top cells. The next day, the medium was replaced and the flask contained a nearly pure population of type 1 astrocytes. These cells were removed from the flask by shaking for 2 to 5 min in trypsin/EDTA. The cells were pelleted and resuspended in astrocyte medium and counted using a hemacytometer. The astrocytes (10,000 cells in 0.5-ml media) were plated onto poly-D-lysine/collagen-coated cover-slips (12 mm diameter) in 24-well tissue culture trays. After approximately 1 week, the astrocytes formed confluent monolayers. On the day neurons were plated onto the glial monolayers, the astrocytes medium was replaced with serum-free BME/B27.

Expression of Recombinant GABA_A Receptors. Recombinant GABA_A receptors were transiently expressed in HEK293 cells as heterotrimeric combinations of , , and subunits. In this study, subunit selectivity was examined by combining rat 1, 2, 4, 5, or 6 subunit with rat 2 and mouse 2S. All receptor subunits were subcloned into the mammalian expression vector pcDNA3. HEK293 cells were transiently transfected using the Effectene transfection kit (QIAGEN, Valencia, CA) using a ratio of 1:1:1 for , , and subunit DNA. Green fluorescent protein was coexpressed to identify positively transfected cells. Electrophysiological recordings were conducted 48 to 72 h after transfection on cells exhibiting green fluorescence. We did not see chloride currents from cells expressing the 3/2/2S combination. However, we did record chloride currents when 3 was expressed in combination with rat 3 and mouse 2S. Therefore, this combination was used to characterize the response of 3 subunit-containing GABA_A receptors. Although subunits affect the binding of GABA and other ligands, they have been shown to not influence the affinity of benzodiazepine site ligands (Hadingham et al., 1993; Graham et al., 1996).

Electrophysiological Recording of GABA Currents. Coverslips, upon which cells had been plated, were transferred to the recording chamber on an inverted microscope (Olympus IX70) and continuously perfused (1.5–2 ml/min) with external recording solution at room temperature. The composition of the external solution was 140 mM NaCl, 2.5 mM KCl, 2.5 CaCl₂, 1.3 mM MgCl₂, 10 mM glucose, and 10 mM HEPES, and the pH was 7.3. For neuronal recordings, the external was supplemented with 0.3 µM tetrodotoxin to block sodium currents and 10 µM NBQX to block AMPA receptor currents. The composition of the internal solution in the recording pipette was 125 mM CsCl, 10 mM NaCl, 1 mM MgCl₂, 5 mM EGTA, 0.5 mM CaCl₂, 10 mM HEPES, and the pH was 7.3.

A Multiclamp 700A patch-clamp amplifier and pClamp 9 software (Axon Instruments, Union City, CA) were used for electrophysiological recording. After gigaohm seals were formed between the patch electrodes (approximate resistance range: 1–3 M) and the cell, the whole-cell patch-clamp configuration was established by rupturing the membrane across the electrode tip. If the quality of the seal was judged to be poor, the electrode was replaced and the process was repeated with a different cell. Once a stable configuration had been achieved, recording was started in voltage-clamp mode, with the cell initially clamped at –70 mV.

GABA (Sigma-Aldrich, St. Louis, MO) was prepared as a 100 mM stock in water, and from this, a 300 µM working stock was prepared. Small aliquots were dispensed and stored at –20°, so that any given working stock was not subject to repeated freeze-thaw cycles. On each recording day, a fresh GABA test solution was prepared in external solution.

For dose-response curves, GABA was applied for 2 s to elicit an inward chloride current. GABA solutions (10 nM to 1 mM) were fed by gravity into an 8-to-1 micromanifold (Warner) with the outlet (PE-10 tubing) positioned near the cell. Solution exchange occurred in <50 ms. Cells were washed with external solution for 60 s between GABA applications to allow receptors to recover from desensitization.

To study the effects of positive allosteric modulators, GABA currents were repeatedly elicited by puffer application of 3 µM GABA every 12 s, and test substances were applied by bath perfusion. A pressurized (10 p.s.i.) puffer pipette (2 µm tip diameter) was positioned near the recorded cell, and GABA (3 µM) was applied by opening a computer-controlled solenoid valve (50–100 ms). This activated a peak inward current (200–2000 pA) that rapidly decayed. Because the small volume of GABA released from the puffer pipette was rapidly diluted in the external bath, the neurons were exposed to a maximum concentration of 3 µM GABA. This is less than the EC₅₀ of GABA for native and cloned GABA_A receptors (Fig. 2) and provided a reliable starting point to measure potentiation of the current by positive allosteric modulators. Drugs were applied for 3 min (15 evoked GABA currents), which was sufficient for an equilibrium response to be established. Drugs were washed out for at least 3 min. If the GABA current recovered to predrug control amplitude, a higher concentration of drug was applied. We found that when cells were treated with drugs that potentiated the GABA current by 2-fold, the potentiation by subsequent drug treatments was diminished relative to treatment with the same concentration of drugs to naive cells (never exposed to drugs). This was not due to rundown of the current, because the amplitude of the GABA-activated current was unchanged. We ended the experiment as soon as a concentration of drug was used that potentiated the GABA current by 2-fold. Therefore, full dose-response curves were not run on each cell, since this would result in an underestimation of the potentiation produced by higher concentrations. Each drug concentration was tested on four to 20 different cells. We recorded from 203 cultured neurons and 399 HEK293 cells from 31 transfections.

View larger version (22K): [in this window] [in a new window]   Fig. 2. Dose responses of GABA at native and cloned GABAA receptors. GABA was locally applied for 2 s to cultured neurons (native receptors) or HEK293 cells transiently transfected with cloned receptor subunits to elicit an inward chloride current. GABA was washed out for 60 s before applying the next higher concentration. The peak current was normalized to the maximum GABA-activated current, and symbols represent the average response ± S.E.M. for six to 15 cells.

View larger version (11K): [in this window] [in a new window]   Fig. 1. Structures of indiplon, zolpidem, zopiclone, and zaleplon.

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	Results

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GABA_A Receptor Dose Responses. For GABA dose-response curves, GABA (10 nM to 1 mM) was locally applied for 2 s to elicit an inward chloride current. Peak GABA currents were normalized to the maximum response elicited by 1 mM GABA, and dose-response curves were fitted to a Logistic function (Fig. 2). The EC₅₀ of GABA on native and cloned GABA_A receptors was around 10 µM: neurons (6.4 µM), 122s (13.4 µM), 222s (12.2 µM), 332s (45.7 µM), and 522s (10.2 µM). The lower affinity for 3 subunit-containing receptors has been previously reported (Bohme et al., 2004).

Drug Effects on Native GABA_A Receptors. Puffer application of 3 µM GABA elicited an inward current in cultured neurons (Fig. 3). We chose this concentration, because it was below the EC₅₀ (6.4 µM; Fig. 1) yet elicited a robust current that could be potentiated. The brief application of GABA (50–100 ms) used in our protocol minimized the desensitization of the GABA current that occurs during longer applications (5–10 s). Because the GABA current was repeatedly measured, the effect of test compounds could be normalized to the control current immediately before application of the test compound and the recovery of the GABA current could be readily determined after washout. Bath application of indiplon rapidly potentiated the inward current activated by 3 µM GABA, and this effect was readily reversed upon washout (Fig. 3). Indiplon did not activate a current in the absence of puffer applied GABA, indicating that it is a positive allosteric modulator rather than a direct agonist at GABA_A receptors.

View larger version (10K): [in this window] [in a new window]   Fig. 3. Indiplon potentiates chloride currents from native GABAA receptors. A neuron was voltage clamped at –70 mV, and puffer applications of 3 µM GABA (every 12 s) were used to activate inward currents (seen as downward deflections). A, concatenated display of GABA-activated currents (one of every five traces shown) before, during, and after a 3-min bath application of indiplon (10 nM and 1 µM; indicated by gray bars). Each current trace is 5-s long, but the time between current traces is 1 min. Indiplon dose-dependently and reversibly potentiated the GABA-activated currents. B, close-up of three superimposed GABAA receptor current traces: 1) control, 2) 10 nM indiplon, and 3) 1 µM indiplon.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Concentration-response curves for indiplon, zolpidem, zopiclone, and zaleplon on GABA-activated chloride currents from cultured neocortical neurons. The data points represent the average ± S.E.M. potentiation from four to nine different cells for each concentration for each drug. The population data were fitted to a single sigmoid function (Logistic 4 parameter).

Drug Effects on Recombinant GABA_A Receptors. Puffer application of 3 µM GABA also elicited an inward current in HEK293 cells expressing recombinant GABA_A receptors composed of , , and subunits. Representative GABA-activated chloride currents from 1/2/2S receptors are shown in Fig. 5. As in the neuronal recordings, bath application of indiplon rapidly and reversibly potentiated the inward current activated by 3 µM GABA but did not directly activate the GABA_A receptor current, indicating that it is a positive allosteric modulator rather than an agonist.

View larger version (9K): [in this window] [in a new window]   Fig. 5. Indiplon potentiates chloride currents from recombinant GABAA receptors. An HEK293 cell expressing 122 GABAA receptor subunits was voltage-clamped at –70 mV, and puffer applications of 3 µM GABA (every 12 s) were used to activate inward currents (seen as downward deflections). A, concatenated display of GABA-activated currents (one of every five traces shown) before, during, and after a 3-min bath application of indiplon (1 nM and 100 nM; indicated by gray bars). Each current trace is 5-s long, but the time between current traces is 1 min. Indiplon dose-dependently and reversibly potentiated the GABA-activated currents. B, close-up of three superimposed GABAA receptor current traces: 1) control, 2) 1 nM indiplon, and 3) 100 nM indiplon.

We tested the subunit selectivity of indiplon by expressing each subunit (one to six) in combination with 22. We were not able to record chloride currents from the 322 combination but did obtain functional currents from 332; consequently, this combination was used to test for activity at 3 subunit-containing GABA_A receptors. Indiplon potentiated 1 subunit-, 2 subunit-, 3 subunit-, and 5 subunit-containing GABA_A receptors but had no effect on 4 subunit and 6 subunit-containing GABA_A receptors at concentrations up to 1 µM (data not shown).

Concentration-response curves showed that indiplon more potently potentiated 1 subunit-containing GABA_A receptors (EC₅₀ of 2.6 nM) than 2 subunit- (24 nM), 3 subunit- (60 nM), or 5 subunit-containing (77 nM) GABA_A receptors (Fig. 6 and Table 1). Thus, indiplon showed at least 10-fold selectivity for 1 subunit-containing GABA_A receptors compared with the other subunits. The maximal potentiation of GABA currents from recombinant receptors was approximately 250%, with the exception of 222 in which E_max was 398% (Table 1).

View larger version (30K): [in this window] [in a new window]   Fig. 6. Concentration-response curves for indiplon, zolpidem, zopiclone, and zaleplon on 1 subunit-, 2 subunit-, 3 subunit-, and 5 subunit-containing recombinant GABAA receptors. The data points represent the average ± S.E.M. potentiation from four to 20 different cells for each concentration for each drug. The population data were fitted to a single sigmoid function (Logistic 4 parameter).

Zopiclone was also weaker than indiplon against all recombinant GABA_A receptors. It exhibited preference for 122 (158 nM) and 522 (146 nM) receptors compared with 222 (598 nM) and 332 (1187 nM) receptors.

As in recordings from neuronal GABA_A receptors, zaleplon was the weakest compound tested and showed only marginal selectivity for the various subunit-containing receptors: 122 (499 nM), 222 (1098 nM), and 332 (1514 nM). It exhibited much less activity at 532. Maximal efficacy was not obtained by 10 µM, the highest concentration tested, and we estimated the EC₅₀ to be >3 µM.

Interestingly, the EC₅₀ values for indiplon, zolpidem, and zopiclone on native GABA_A receptors fell in between the EC₅₀ values for 122 and 222 (Table 1). These results are compatible, because the native GABA_A receptors in the neocortex are thought to be primarily composed of 1- and 2 subunit-containing subtypes (Fritschy and Mohler, 1995). Zaleplon was found to be slightly less active at native GABA_A receptors than either 1 subunit- or 2 subunit-containing recombinant receptors.

	Discussion

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The present studies represent a comprehensive comparison of "nonbenzodiazepine" drugs at GABA_A receptor subtypes. Indiplon was shown to be a positive allosteric modulator of GABA_A receptors; it potentiated the chloride current evoked by subsaturating concentrations of GABA (3 µM) but did not directly gate the channel. Indiplon dose-dependently potentiated GABA currents from native (neuronal) as well as recombinant GABA_A receptors.

Indiplon was approximately 10 times more potent than zolpidem and zopiclone and over 100 times more potent than zaleplon on native GABA_A receptors from neocortical neurons and exhibited a similar advantage in potency against recombinant GABA_A receptor combinations (Table 2). This rank order agrees with our previously reported binding data (Sullivan et al., 2004). All compounds potentiated native GABA_A receptor currents by 200 to 300%. The E_max of indiplon was not statistically different from zolpidem, zopiclone, and zaleplon (one-way ANOVA). However, the E_max values varied more for cloned GABA_A receptors ranging from 242 to 553%. The greater variability in E_max values for recombinant GABA_A receptors is likely due to differences in subunit expression between batches of transiently transfected cells.

Binding studies have shown that zolpidem is approximately 5- to 10-fold more selective for 1 subunit-containing receptors than 2 and 3 subunit-containing receptors and has no detectable affinity for 5 subunit-containing receptors (Langer et al., 1992; Hadingham et al., 1993; Damgen and Luddens, 1999). This was confirmed by our electrophysiology results in which we found zolpidem to be 5.0- and 11.9-fold selective for 1 over 2 and 3 subunits, respectively. In contrast, indiplon exhibited even greater selectivity for 1 over 2 and 3 subunits with 9.3- and 23.5-fold selectivity, respectively.

There are only limited reports addressing the subunit selectivity of zopiclone and zaleplon. Zopiclone was slightly selective (5-fold) for binding to 1 and 5 over 3 subunit-containing receptors (combined with 22 subunits) with K_i values of 66.7, 52.7, and 262 nM, respectively (Graham et al., 1996). However, another report showed little subunit selectivity for zopiclone and reported mixed affinities for zaleplon on subunits depending upon which subunits they were combined with (Damgen and Luddens, 1999). Electrophysiological recordings were used to determine the K_d of (R,S)-zopiclone from kinetics (K_d = k_off/k_on) using a single concentration of zopiclone (20 µM) (Fleck, 2002). The K_d values are near-perfect matches for our EC₅₀ values: 122 (0.2 versus 0.157 µM), 222 (0.6 versus 0.598 µM), and 322 (1.4 versus 1.2 µM). Our electrophysiological data on zolpidem and zaleplon or rat receptors are also in very close agreement with results reported for human GABA_A receptors expressed in Xenopus oocytes (Sanna et al., 2002).

GABA_A receptors containing 1 subunits have been shown to mediate the sedative effects of diazepam (Rudolph et al., 1999; Low et al., 2000; McKernan et al., 2000) and zolpidem (Crestani et al., 2000). Indiplon was selective for 1 subunit-containing receptors (Table 3), and the degree of selectivity for 1 subunit-containing receptors over 2 and 3 subunit-containing receptors was even greater than zolpidem, an effective sedative-hypnotic whose 1 selectivity is thought to contribute to its therapeutic efficacy (Sanger, 2004). Unlike zolpidem, which showed no detectable activity at 5 subunit-containing receptors, indiplon did potentiate 522 receptors, albeit at 30 times the concentration required to potentiate 122 receptors. Indiplon has been demonstrated to exhibit sedative effects in rats that were blocked by the benzodiazepine site antagonist flumazenil (Foster et al., 2004). It is likely that the sedative actions of indiplon are mediated by its activity at 1 subunit-containing GABA receptors. Moreover, the sedative effects of indiplon in mice were achieved at a lower dose (ED₅₀ 2.7 mg/kg p.o.) than zolpidem (ED₅₀ 6.1 mg/kg p.o.) or zaleplon (ED₅₀ 24.6 mg/kg p.o.) (Foster et al., 2004). This rank order in vivo nicely matches the rank order for these compounds on native and recombinant GABA_A receptors in vitro reported in this study.

View this table: [in this window] [in a new window]   TABLE 3 Subunit selectivity of indiplon, zolpidem, zopiclone, and zaleplon EC50 values were normalized to the EC50 at recombinant 122 receptors. Zolpidem (up to 10 µM) did not potentiate 522 receptors, so relative selectivity was not calculated. The EC50 for zaleplon at 522 receptors could not be determined since the maximum potentiation was not achieved at 10 µM, the highest concentration tested. We estimated the EC50 to be >3 µM and therefore >6-fold less active than at 122 receptors.

The selectivity of a positive allosteric modulator for GABA_A receptor subtypes containing different subunits may influence its profile as a sedative-hypnotic. Behavioral sedation in mice induced by benzodiazepine and nonbenzodiazepine drugs is clearly due to enhanced GABA neurotransmission mediated by 1 subunit-containing GABA_A receptors (Rudolph et al., 1999; Crestani et al., 2000; McKernan et al., 2000; Kralic et al., 2002; Blednov et al., 2003). GABA_A receptors containing 5 subunits have received much attention in terms of their involvement in memory processes because of their localization and proposed physiological role in the hippocampus (Crestani et al., 2002). At the same time, evidence for an involvement of 1 subunit-containing GABA_A receptors in memory comes from observations that the effects of diazepam on passive avoidance in mice is absent in the 1 (H101R) knock-in mice (Rudolph et al., 1999). If 5 subunit-containing GABA_A receptors played a dominant role in the amnesic effects of benzodiazepine site ligands, then one would expect that zolpidem, which has no measurable affinity for this subtype, would be free of amnesic effects. This is not the case, since clinical studies have reported retrograde amnesia with zolpidem (Roehrs et al., 1994) and animal studies clearly show an effect of zolpidem on passive avoidance in the mouse (Foster et al., 2004). It seems reasonable to propose that the amnesic effects of hypnotics are inseparable from the hypnosis they produce, which in the case of zolpidem and possibly all benzodiazepine site ligands is most likely a result of positive allosteric modulation at 1 subunit-containing GABA_A receptors and not due to a specific interaction with 5 subunit-containing GABA_A receptors. Recent data using the knock-in approach have also indicated that tolerance to the sedative effects of diazepam is absent in 5 (H105R) mice, suggesting that activation of 5 subunit-containing GABA_A receptors is important for the development of tolerance (van Rijnsoever et al., 2004). In rodent studies, zolpidem has been reported to have a lower tolerance propensity than benzodiazepines (Perrault et al., 1992), although in primates, tolerance comparable with that of benzodiazepines was observed (Griffiths et al., 1992). The fact that tolerance can be observed with zolpidem would suggest that activity at 5 subunit containing GABA_A receptors is not an absolute requirement. Ethanol potentiates the sedative-hypnotic effects of benzodiazepine site ligands, an effect that has recently been linked with 2 subunit-containing GABA_A receptors (Tauber et al., 2003). This is supported by clinical studies that have found no major interaction between ethanol and zolpidem (Wilkinson, 1995), in contrast to the well known potentiation of the effects of ethanol by benzodiazepines (Hollister, 1990).

What are the implications of the GABA_A receptor subtype selectivity profile of indiplon? The high affinity of indiplon for 1 subunit-containing GABA_A receptors is consistent with the effects observed in rodents (Foster et al., 2004) and humans (Roth et al., 2003) after indiplon administration. Human electroencephalogram studies with indiplon have indicated that a plasma level of 5 ng/ml (13 nM) is associated with hypnotic effects (Jochelson et al., 2003). Because the effective concentration of indiplon at the relevant GABA_A receptor sites in the brain is likely to be at least one order of magnitude lower due to plasma and tissue binding, this corresponds well with the high affinity of indiplon observed in the present study for 1 subunit-containing GABA_A receptors. Compared with 1 subunit-containing GABA_A receptors, indiplon showed approximately 10-fold lower affinity at 2 subunit-containing GABA_A receptors, which might predict that anxiolytic effects and an interaction with ethanol are not prominent features of its pharmacology. Accordingly, no prominent pharmacodynamic interaction between indiplon and ethanol was observed in clinical studies (Berkowitz et al., 2003). Indiplon does show anxiolytic effects in the Vogel conflict test of anxiety in the rat, but over a dose range where sedative effects are also observed (Foster et al., 2004). This is in contrast to diazepam, which despite having no separation of affinity between 1 and 2 subunit-containing GABA_A receptors has anticonflict effects at doses lower than its sedative effects. Consequently, the greater selectivity of indiplon for 1 versus 2 subunit-containing GABA_A receptors is consistent with this profile. Indiplon has approximately 30-fold selectivity for 1 versus 5 subunit-containing GABA_A receptors, which might predict that indiplon has less propensity to produce amnesic effects and tolerance. However, this should be interpreted with caution because, as noted above, zolpidem has shown amnesic effects and evidence of tolerance, despite no measurable affinity for 5 subunit-containing GABA_A receptors. However, the combination of 30-fold selectivity for 1 versus 5 subunit-containing GABA_A receptors and a 1- to 2-h half-life (Foster et al., 2004) bodes well for a low incidence of residual amnesia and tolerance with indiplon. Indeed, a lack of tolerance to repeated indiplon administration has been observed in man (Jochelson et al., 2003; Scharf et al., 2005).

In conclusion, we demonstrate that indiplon has the highest affinity for GABA_A receptors of the currently known nonbenzodiazepine positive allosteric modulators and exhibits selectivity for 1 subunit-containing GABA_A receptors. The pharmacological profile of indiplon in animals is consistent with its 1 selectivity, and ongoing clinical trials will characterize in detail its hypnotic profile in humans.

	Acknowledgements

 
We thank Ray Gross for synthesizing indiplon and zaleplon, Richard Olsen for providing GABA_A receptor subunits clones, and Harvey Clark for assistance in preparing the figures.

	Footnotes

 
ABBREVIATIONS: Ro15-1788, flumazenil; Ro15-4513, ethyl 8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a]-[1,4]benzodiazepine-3-carboxylate; HEK, human embryonic kidney; NBQX, 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline; AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; DMSO, dimethyl sulfoxide; AUC, area under the curve; ANOVA, analysis of variance.

doi:10.1124/jpet.105.096701.

Address correspondence to: Dr. Robert E. Petroski, Department of Neuroscience, Neurocrine Biosciences Inc., 12790 El Camino Real, San Diego, CA 92130. E-mail: rpetroski{at}neurocrine.com

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