The neurotransmitter histamine is synthesized by neurons originating in the tuberomammillary nucleus of the posterior hypothalamus. These neurons project widely throughout the cortex, hippocampus, amygdala, and striatum (Brown et al., 2001). Histamine exerts its action by interacting with a group of G protein-coupled histamine receptors and plays important roles in mediating a variety of functions in the central nervous system (Brown et al., 2001). The H₃Ris presynaptically localized and functions both as an autoreceptor and a heteroreceptor by modulating the release of neurotransmitters, including histamine (Arrang et al., 1985), dopamine, acetylcholine, serotonin, and norepinephrine (Schlicker et al., 1994; Blandina et al., 1996; Brown et al., 2001). Activation of the H₃R results in the inhibition of neurotransmitter release. In contrast, blockade of the H₃R by selective antagonists or inverse agonists can reverse the histamine-mediated inhibition of neurotransmitter release. By virtue of the unique central nervous system localization of the H₃R (Drutel et al., 2001; Leurs et al., 2005) and its ability to regulate a variety of neurotransmitters that are thought to be involved in vigilance, cognition, and wakefulness, H₃R antagonists and inverse agonists are suggested to hold promise for a number of therapeutic applications (Hancock and Fox, 2004).

Selective H₃R antagonists have been extensively evaluated in several preclinical rodent models to establish their role in cognitive function and sleep-wake regulation. These models include social recognition (Fox et al., 2003a,b, 2005), novel object recognition (Fox et al., 2005; Ligneau et al., 2007; Medhurst et al., 2007a), spatial learning and memory in an adult rat water maze test (Fox et al., 2005), scopolamine-induced deficits in memory consolidation in a passive avoidance test (Medhurst et al., 2007b), and cortical electroencephalographic (EEG) recording (Barbier et al., 2004; Ligneau et al., 2007).

It has also been suggested that histamine plays an important role in the modulation of the sleep-wake cycle. Histaminergic neurons exhibit an increase in firing rate during waking (Brown et al., 2001). Depletion of histamine has been shown to lead to the reduction of awake time (Monti et al., 1988), whereas activation of the H₃R by selective agonists results in an increase in slow-wave sleep (Lin et al., 1990; Monti, 1993; Monti et al., 1996). In contrast, H₃R antagonists have been shown to increase wakefulness in cats, rats, and mice (Lin et al., 1990; Monti et al., 1991, 1996; Barbier et al., 2004; Ligneau et al., 2007; Parmentier et al., 2007). Recent studies with the H₃R antagonist thioperamide and JNJ-5207852 in H₃R knockout mice have confirmed the involvement of the H₃R in the wake-promoting activity of these compounds (Toyota et al., 2002; Barbier et al., 2004).

It is apparent that H₃R antagonists produce in vivo effects in preclinical models across a wide dose range. Several selective H₃R antagonists exhibit potent efficacy in social recognition models (Fox et al., 2003b, 2005). However, studies suggest that much higher doses of H₃R antagonists are required to produce robust wake activity. For example, the minimal effective i.p. dose for ciproxifan to reduce slow-wave activity was 10-fold higher than that required for the improvement of social memory, whereas the dose difference was 30-fold for A-304121 (Fox et al., 2003b). In the case of ABT-239, there was no detectable change in slow-wave EEG at 30 mg/kg, whereas it was effective in social recognition at 0.01 mg/kg (Fox et al., 2005). The present study sought to examine this phenomenon by correlating the wake-promoting activity of a number of H₃R antagonists with an ex vivo estimate of receptor occupancy. Our results with ciproxifan, thioperamide, and GSK189254 suggest that the relatively low levels of cumulative wake activity are linearly correlated with up to 80% of H₃R occupancy, after which an abrupt increase of waking activity is observed. These observations suggest that the relatively small increase in waking EEG activity observed at low levels of H₃R occupancy may be consistent with literature reports of enhanced attention and cognitive function. Robust waking activity is associated with higher levels of H₃R occupancy and may be mechanistically different from activities observed at low occupancy.

Materials and Methods

Animals. Adult Long Evans rats (350 g; Harlan, Indianapolis, IN) were used in these studies. All animals were maintained on a 24-h light/dark cycle (on at 7:00 AM/off at 7:00 PM), with food and water available ad libitum. All animals except those in the sleep study (see below) were group housed. All experimental animal procedures were conducted in accordance with the Institute of Laboratory Animal Resources (1996) and were approved by Cephalon's Institutional Animal Care and Use Committee.

Compounds. Ciproxifan and GSK189254 were purchased from ValuTek (Princeton, NJ). Thioperamide and d-amphetamine were purchased from Sigma-Aldrich (St. Louis, MO). [³H]N-α-Methylhistamine ([³H]NAMH, catalog no. NET 1027, 83 Ci/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA).

Ex Vivo [³H]NAMH Binding Assay in Rat Cortical Homogenate. Animals were dosed with vehicle (0.5% methylcellulose/0.2% Tween 80) or various concentrations of compound by i.p. injection. The animals were killed by decapitation 1 h after injection, and the cerebral cortex was dissected. The 1-h time point was chosen based on pharmacokinetic studies demonstrating that, after i.p. dosing, all of the compounds evaluated in the present study reached a plateau plasma level by 1 h that was maintained for at least 4 h. Therefore, 1 h represents the earliest time at which steady-state occupancy could be assessed. The cortices were homogenized in 50 mM HEPES, pH 7.4 (7.5 ml/2 g) with a polytron for 10 s. The cortical homogenate was added to a deep-well plate containing [³H]NAMH (0.15 nM), in a volume of 0.4 ml (final protein concentration 0.8 mg/assay) as described previously (Taylor et al., 1992). Reactions were incubated at 25°C for 1 h and stopped by rapid filtration onto glass filter paper (GF/B Whatman, catalog no. FPLR-24) using a 48-well cell harvester (Brandel Inc., Gaithersburg, MD). The filters were washed three times with ice-cold assay buffer and mixed with 5 ml of liquid scintillation cocktail (Ultima Gold; PerkinElmer Life and Analytical Sciences). Radioactivity trapped on the filters was measured in a liquid scintillation counter (PerkinElmer Life and Analytical Sciences). A limitation of ex vivo binding is the in vitro manipulation of the tissue, an issue that has been described in great detail for corticotrophin-releasing factor 1 (CRF1) binding (Li et al., 2003). Care was taken in our ex vivo assays to choose a minimal incubation time that allowed binding to reach equilibrium by assessing the time course of receptor binding for H₃ antagonists from 15 min to 3 h. These studies showed that receptor binding reached equilibrium at 1-h incubation and was stable for up to 3 h for compounds used.

Nonspecific binding was determined in the presence of 10 μM thioperamide. The nonspecific binding was 10% of total binding in vehicle-treated samples, indicating that 90% of the [³H]NAMH binding was specific. Each compound was tested in three independent experiments with a total of at least four animals per dose in the dose-response curve. The inhibition of specific [³H]NAMH binding, calculated as relative to vehicle-treated samples, was determined to provide an indication of receptor occupancy by the compound. The dose-response curve was fit to a four parameter logistic equation to yield the IC₅₀ value using Prism 4.0 (GraphPad Software Inc., San Diego, CA).

Compound Quantification. Plasma and brain samples were collected from the study animals in the ex vivo binding experiments and were submitted for quantitative analysis to determine the respective compound concentrations. Blood samples were collected into heparinized tubes and placed on wet ice until centrifuged (16,000g, 5 min) to separate plasma. The supernatant was collected and stored at -20°C pending analysis. At the time of analysis, 10 volumes of cold acetonitrile containing an internal standard (Alprenolol) was added to each sample, which was then vortexed and centrifuged. The supernatant was removed and placed into an auto sampler vial, and the amount of compound present in the samples was analyzed by liquid chromatography/mass spectrometry. The concentration of compound in the samples was quantified against either a rat plasma or brain standard curve made via serial dilution in a concentration range from 10 to 5000 ng/ml. Samples containing concentrations greater than 5% above the top of the standard curve were diluted 1:10 with acetonitrile. The brain/plasma ratio was calculated accordingly.

Determination of Sleep/Wake Activity. Cortical EEG and electromyographic (EMG) recordings from neck muscles were used to determine whether the reference H₃R antagonists altered sleep-wake activity in rats. Ciproxifan (0.3, 1, 3, 10, and 100 mg/kg), thioperamide (1, 3, 10, 30, and 100 mg/kg), GSK189254 (1, 3, 10, 20, and 30 mg/kg), or ABT-239 (1, 2, 3, and 10 mg/kg) was dosed by i.p. injection. EEG and EMG electrodes were chronically implanted under Nembutal anesthesia. EEG activity was recorded from screw electrodes over the frontal cortex (+3.0 mm AP from bregma, ±2.0 mm mediolateral) and hippocampus (-4.0 mm AP from bregma, ±2.0 mm mediolateral). The animals were allowed to recover from surgery for 2 weeks before recording, during which time they were housed in pairs in standard rat cages. The day before recording, rats were placed in individual containers (31 × 31 × 31 cm) in sound-attenuating cabinets and connected by cables to the recording equipment. Each cabinet contained a fan, a light (12-h light/dark cycle, ∼30 lux), and a speaker to provide background noise. The animals were dosed the following day 5 h after lights on (circadian time-5) and removed 22 h later and were otherwise not disturbed. EEG and EMG signals were amplified (10,000 and 1000, respectively), band pass filtered between 0.3 and 500 Hz for EEG and between 10 and 500 Hz for EMG, and digitized at 128 samples/s. Sleep/wake activity was evaluated using ICELUS sleep scoring software (M. Opp, University of Michigan, Ann Arbor, MI) by scoring EEG and EMG activity into wake, slow-wave sleep, and rapid-eye movement sleep in 6-s epochs according to standard criteria (Opp and Krueger, 1994; Edgar and Seidel, 1997). The EEG/EMG data were scored for 8 h postdosing and converted to percentage time awake. Given the 1-h occupancy determination time, wake would ideally be measured at 1 h. However, the manipulation and injection procedure produces a transient increase in wake activity that lasts for approximately 30 min (evident in vehicle curve in Fig. 3). The 2-h cumulative wake time was therefore chosen as the most representative measure of wake activity. Values of cumulative time awake were evaluated for 2 h postdosing for comparison with receptor occupancy.

Statistical Analysis. Values are listed as mean ± S.E.M. where appropriate. Treatment effects were evaluated using ANOVA. Treatment group comparisons were made using unpaired, two-tailed Student's t tests or Dunnett's test for multiple comparisons as appropriate.

Results

Ex Vivo Binding. An ex vivo binding assay was used to estimate in vivo occupancy by H₃R antagonists. Ciproxifan (0.1–10 mg/kg), thioperamide (0.3–100 mg/kg), GSK189254 (0.1–30 mg/kg), or ABT-239 (0.3–30 mg/kg) were administered by the i.p. route as described under Materials and Methods. All four compounds tested produced dose-dependent inhibition of specific ex vivo binding of [³H]NAMH to cortical homogenate with the following rank order of potency: ciproxifan > GSK189254 > ABT-239 > thioperamide (Fig. 1; Table 1). Maximal inhibition was obtained with all compounds and IC₅₀ values were between 0.44 and 5.7 mg/kg i.p. (Table 1). The plasma and brain concentrations were determined by liquid chromatography/mass spectrometry. All compounds showed a linear correlation between the inhibition of specific [³H]N-α-methylhistamine binding and plasma or brain compound concentration (Fig. 2). These data were used to calculate the OC₅₀ (brain concentration at 50% occupancy) for each compound (Table 1). Although these numbers are significantly higher than the K_i values typically reported for these compounds in rat cortex (for example, see Medhurst et al., 2007a), multiple factors other than binding affinity such as the distribution of the compound in brain tissue (Fox et al., 2005) and free fraction of the compound play a role in determining the OC₅₀. d-Amphetamine was also tested in the ex vivo H₃R binding assay. Although 0.3 and 1 mg/kg i.p. doses of d-amphetamine produced robust wake promotion activity, no detectable inhibition of the specific [³H]N-α-methylhistamine binding was observed from 0.003 up to 1 mg/kg (data not shown).

Wake Promotion of H₃R Antagonists. EEG/EMG recording was used to determine the ability of H₃R antagonists to affect arousal and sleep-wake states. Ciproxifan, thioperamide, GSK189254, or ABT-239 was administered i.p. The time course of wake activity of ABT-239 is shown in Fig. 3. A dose-dependent increase in cumulative wake activity for 2 h postdosing [2-h area under the curve (AUC)] was observed from 1 to 10 mg/kg i.p. for this compound (Fig. 4D). In contrast, slow-wave sleep and rapid-eye movement sleep showed cumulative decreases from 2 to 10 mg/kg (data not shown). Cumulative wake activity for the H₃R antagonists ciproxifan, thioperamide, and GSK189254 are also shown in Fig. 4. All four compounds enhanced wake in this assay: ciproxifan (0.3–30 mg/kg i.p.), thioperamide (30 and 100 mg/kg i.p.), GSK189254 (20–30 mg/kg i.p.), and ABT-239 (1–10 mg/kg i.p.) (p < 0.001, ANOVA).

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Fig. 1.

Inhibition of ex vivo [³H]NAMH binding in the rat cortex after i.p. administration of ciproxifan (A), thioperamide (B), GSK189254 (C), and ABT-239 (D) for 1 h. Data points are compared with vehicle-treated animals and expressed as percentage inhibition of specific binding. Data are expressed as mean ± S.E.M. of at least four animals per dose from a minimum of three independent experiments.

Correlation of Receptor Occupancy and Waking Activity of H₃R Antagonists. The fact that H₃R antagonists are reported to promote wakefulness at doses that are 10- to 100-fold higher than the minimal effective dose in other preclinical models prompted us to investigate the relationship between receptor occupancy and waking activity. For this purpose, we correlated the 2-h cumulative wake time of each H₃ compound with the respective ex vivo binding values. As shown in the Fig. 5, a modest increase in cumulative wake activity (2-h AUC) with increasing receptor occupancy was observed for ciproxifan, thioperamide, and GSK189254 up to relatively high occupancy values (Fig. 5). All four compounds showed highly linear correlations (R² > 0.8) over this portion of the occupancy versus wake activity curve. The slopes for ciproxifan, thioperamide, and GSK189254 were comparable and yielded an average increase of approximately 2 min/h of wake for every 10% increase in occupancy. In contrast to the modest increase in wake at low levels of H₃R occupancy for these compounds, a robust increase in wakefulness was observed at a particular level of receptor occupancy for each compound. For ciproxifan, thioperamide, or GSK189254, this upward increase occurred when the H₃R was at least 80% occupied. In the case of ABT-239, a slope of 8.8 min/h for every 10% increase in occupancy was observed, which was approximately 4-fold greater than the others. For ABT-239, a similar level of wake activity (∼100 min) was reached at around 80% of the H₃R occupancy without an abrupt increase up to 2 h postdosing.

Discussion

The results obtained in the present study demonstrated a consistent relationship between the in vivo effects and the level of H₃R occupancy produced by several selective H₃R antagonists. The ex vivo receptor binding assay has been useful in examining the ability of compounds to penetrate the blood-brain barrier and to bind receptors (Taylor et al., 1992). This approach has been used in previous reports for assessing the in vivo receptor binding of a variety of compounds, including H₃R antagonists thioperamide (Barnes et al., 1993; Bertoni et al., 2007), VUF 9153 (Barnes et al., 1993), JNJ-5207852 (Barbier et al., 2004), GSK189254 (Medhurst et al., 2007a), GSK207040, and GSK334429 (Medhurst et al., 2007b). In the present study, the data demonstrate a dose-dependent inhibition of the ex vivo binding by H₃R antagonists in rat cortical homogenate. As a negative control, d-amphetamine, a wake-promoting agent and a known inhibitor of dopamine transporter activity (Nishino et al., 1998; Wayment et al., 1998) and a dopamine release agent (Sulzer et al., 1993), does not inhibit specific binding of [³H]NAMH. We also demonstrate that receptor occupancy is correlated with the plasma and brain level of the compound. Ex vivo binding studies with the same H₃R antagonists employed in the present studies have been described previously (Medhurst et al., 2007a). Although there are significant methodological differences between these studies and the studies presented here (dosing route, dosing time, tissue preparation, and binding conditions), it is of interest to compare the results. Despite the differences in protocol, our results agree reasonably well. The OC₅₀ for GSK189254 in the two studies was within 3-fold (60 nM in Medhurst et al. and 167 nM in the present study). In addition, although Medhurst et al. did not report an OC₅₀ for the other H₃R antagonists, the percentage inhibition at the highest dose tested for ciproxifan (97% at 10 mg/kg compared with 99 ± 0.2% in the present study), thioperamide (1.3-fold difference with 80% at 10 mg/kg reported compared with 60 ± 5% in the present study), and ABT-239 (3.7-fold difference with 68% at 1 mg/kg reported compared with 18 ± 6% in the present study) all compare favorably. Although it could be argued that the present studies underestimate or that the previous studies overestimate in vivo occupancy, a true measure of in vivo occupancy must await the use of a sensitive PET ligand.

The relationship between receptor occupancy and behavioral effects of several targets has been studied, including CRF1 (Li et al., 2003), cannabinoid (Gifford et al., 1999), and dopamine D₂ receptor (Wadenberg et al., 2000). In the case of the CRF1 receptor, it was shown that some compounds produced a minimal anxiolytic effort at 50% occupancy (Heinrichs et al., 2002) but were ineffective in a defensive withdrawal test until higher level of receptor occupancy was reached (Arborelius et al., 2000). Likewise, studies with D₂ receptor showed that suppression of conditioned avoidance response by raclopride occurred at a D₂ occupancy of around 70 to 75%, but catalepsy occurred at D₂ occupancy > 80% in rats (Wadenberg et al., 2000). Here, we find that relatively high H₃R occupancy is required to produce robust waking activity. GSK189254 required 92% occupancy (20 mg/kg i.p.) to significantly increase waking, whereas thioperamide required 85% occupancy (30 mg/kg i.p.). Notably, we demonstrate here a significant wake-enhancing effect for ABT-239 that was not observed at similar doses previously (Fox et al., 2005). A possible reason for this discrepancy is that only delta power was used to evaluate changes in sleep/wake activity previously. Our data suggest that the increase in waking activity and receptor occupancy is linear up to relatively high occupancy. The slopes for the linear correlations shown in Fig. 5 are modest, indicating a small effect on wake at levels of H₃R occupancy below 80% for ciproxifan, thioperamide, and GSK189254 (2 min/h for every 10% increase in occupancy). Only when H₃R occupancy exceeded a certain threshold (∼80%) was an abrupt increase in waking activity detected. It is interesting to note that ABT-239 seems to have a waking versus occupancy profile different from the other antagonists. First, although the increase in wake was proportional to occupancy up to 80%, the rate of increase was greater (8.8 min/h per 10% increase in occupancy) (Fig. 5). Second, there was no abrupt increase in waking activity produced by ABT-239. However, there is evidently a distinct increase in excitability at higher levels. Tremor and seizures have been reported at above 28 mg/kg i.p. in rats (Fox et al., 2005). Although these observations cannot be explained based on the current data, the possibility exists that ABT-239 may possess additional activities that distinguish it from the other H₃R antagonists. Alternatively, other properties may contribute to the differential activity of the compound. For example, H₃Rs are known to exhibit constitutive activity (Rouleau et al., 2002). The degree of inverse agonism exhibited by an H₃R ligand, as well as variations in the degree of constitutive activity in various brain regions could produce significantly different in vivo behavioral effects at identical occupancy levels. Other factors including the pharmacokinetic and pharmacodynamic properties of the compounds may also play a role in the observed differential in vivo activity.

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Fig. 2.

Relationship between ex vivo H₃R binding and plasma (A) and brain (B) concentration of ciproxifan (a), thioperamide (b), GSK189254 (c), and ABT-239 (d) in individual animals. The curves were fit by linear regression with R² = 0.87, 0.90, 0.88, and 0.94 for ciproxifan, thioperamide, GSK189254, and ABT-239 in plasma and R² = 0.87, 0.91, 0.79, and 0.95 for ciproxifan, thioperamide, GSK189254, and ABT-239 in brain, respectively.

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Fig. 3.

Time course of wake enhancement produced by ABT-239 dosed at 1 to 10 mg/kg i.p., 5 h after lights on. Dosing occurs at time = 0, which wakes animals up for approximately 30 min. Time of lights off shown by black bar on time axis. N for each group shown in parentheses in legend. Values = mean + S.E.M. *, p < 0.05 versus vehicle, unpaired Student's t test for individual 0.5-h time points.

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Fig. 4.

Cumulative wake activity (2 h AUC) for ciproxifan (A), thioperamide (B), GSK189254 (C), and ABT-239 (D). Values = mean + S.E.M.; the number of animals in each treatment group is shown at bottom of the bar. Each compound showed a significant treatment effect (p < 0.001, ANOVA). *, p < 0.05 versus vehicle, Dunnett's test.

Our results suggest that the H₃R system is finely tuned to discriminate changes in receptor occupancy. A relatively small increase of EEG activity at low levels of receptor occupancy observed with ciproxifan, thioperamide, and GSK189254. However, these effects may be consistent with previously reported enhancement of attention and cognitive function by H₃R antagonists. Robust waking activity at higher occupancy may be mechanistically different from activities at low occupancy.

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Fig. 5.

Relationship between receptor occupancy and cumulative waking (2-h AUC): A, ciproxifan; B, thioperamide; C, GSK189254; and D, ABT-239. Solid line, linear correlation over selected points. Corresponding slope, R², and p value indicating difference of slope from zero are indicated for each compound. *, p < 0.05 for 2-h wake AUC versus vehicle for each point, Dunnett's test.

The blockade of H₃R is known to promote the release of a variety of neurotransmitters, most of which play important roles in arousal, cognition and sleep-wake regulation (Parmentier et al., 2007). Synaptic levels of dopamine, norepinephrine, and serotonin are regulated by autoreceptors, their respective transporters (Schmitz et al., 2001; Torres et al., 2003) and by recurrent inhibitory circuits (Le Roux et al., 2006), which would tend to oppose increased excitability. The sudden enhancement of waking at higher occupancy levels suggests that the homeostatic regulation of transmitter release may change or even be overwhelmed under these conditions. Alternatively, the enhanced excitability could result from the release of particular neurotransmitters over different occupancy ranges. It will therefore be informative to correlate receptor occupancy with the amounts of particular neurotransmitters released during in vivo microdialysis studies.

It is important to note that, although the compounds used in these studies are reported in the literature to be selective for the H₃R, we cannot completely exclude the possibility that the highest concentrations of H₃R antagonists could exert effects by binding to other receptors. The fact that these compounds represent multiple chemical scaffolds makes this an unlikely possibility; however, future studies with additional structurally diverse H₃R antagonists are needed.

Our observations suggest that the difference in efficacy produced by H₃R antagonists observed in the in vivo models can be explained by the degree of H₃R occupancy. This information can be valuable in understanding in vivo pharmacodynamics and is potentially useful in providing the guideline for the selection of dose regimens in clinical studies.