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CELLULAR AND MOLECULAR

In Vitro Pharmacology of Clinically Used Central Nervous System-Active Drugs as Inverse H₁ Receptor Agonists

Department of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands (R.A.B., H.T., R.L.); ACADIA Pharmaceuticals Inc., San Diego, California (R.A.B., M.W.N., T.T.S., E.S.B., U.H., M.R.B., D.M.W.); and Departments of Pharmacology (M.R.B.), Neurosciences (D.M.W.), and Psychiatry (D.M.W.), University of California, San Diego, California

Received January 2, 2007; accepted March 30, 2007.

	Abstract

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The human histamine H₁ receptor (H₁R) is a prototypical G protein-coupled receptor and an important, well characterized target for the development of antagonists to treat allergic conditions. Many neuropsychiatric drugs are also known to potently antagonize this receptor, underlying aspects of their side effect profiles. We have used the cell-based receptor selection and amplification technology assay to further define the clinical pharmacology of the human H₁R by evaluating >130 therapeutic and reference drugs for functional receptor activity. Based on this screen, we have reported on the identification of 8R-lisuride as a potent stereospecific partial H₁R agonist (Mol Pharmacol 65:538–549, 2004). In contrast, herein we report on a large number of varied clinical and chemical classes of drugs that are active in the central nervous system that display potent H₁R inverse agonist activity. Absolute and rank order of functional potency of these clinically relevant brain-penetrating drugs may possibly be used to predict aspects of their clinical profiles, including propensity for sedation.

Drugs with antihistaminergic activity have been traditionally classified as pharmacological antagonists of histamine at the H₁R, acting by competitively binding to the receptor, thereby blocking H₁R-mediated responses (Hill et al., 1997; Zhang et al., 1997). However, the techniques previously used to assess H₁R activity of commonly used therapeutics lack the ability to discriminate the functional nature of these interactions. More recent studies, using functional assays, have shown that some antihistamines possess negative intrinsic activity at the H₁R, which has led to the reclassification of these agents as H₁R inverse agonists (Weiner et al., 1999; Bakker et al., 2000, 2001). These observations raise important questions as to the critical physiological role of basal H₁R signaling and potential pharmacological importance of negative intrinsic versus neutral antagonistic activity of the multitude of clinically useful compounds that interact with H₁Rs.

We have used the cell-based functional assay receptor selection and amplification technology (R-SAT) to further explore the clinical pharmacology of a variety of CNS drugs as inverse agonists at the human H₁R. We demonstrate a strong correlation between the affinity of known histaminergic drugs at the H₁R, as determined by radioligand binding, and the inverse agonist potency, as determined by functional R-SAT and NF-B assays (Bakker et al., 2001). Subsequently, extensive R-SAT-based analysis of >130 clinically relevant neuropsychiatric drugs revealed that many of these drugs are potent H₁R inverse agonists, whereas none were found to be true neutral antagonists.

	Materials and Methods

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Materials. Cell culture media, penicillin, and streptomycin were obtained from Invitrogen (Merelbeke, Belgium). Calf serum (Invitrogen), Cyto-SF₃ (Kemp Laboratories, Frederick, MD), [³H]mepyramine (20 Ci/mmol), and myo-[2-³H]inositol (21 Ci/mmol) were purchased from NEN (Zaventem, Belgium). pNF-B-Luc was obtained from Stratagene (La Jolla, CA), pSI was obtained Promega (Madison, WI), and Lipofectamine was from QIAGEN GmbH (Dusseldorf, Germany).

The sources of many of the drugs used in this study have been reported previously (Wellendorph et al., 2002; Bakker et al., 2004). These chemical compounds are as follows: (±)-2-carboxypiperazine-4-yl)propyl-1-phosphonic acid, (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride, DS-121, JL-18, LY 53,857, m-chlorophenylpiperazine, MDL 10097, MK 212, SB 206553, SCH 12679, SCH 23390, SKF 38393, and SKF 83566.

Gifts of acrivastine (The Wellcome Foundation Ltd., London, UK), astemizole (Janssen Pharmaceutica NV, Beerse, Belgium), cyproheptadine hydrochloride (MSD, Haarlem, The Netherlands), d-chlorpheniramine maleate (A. Beld, Nijmegen, The Netherlands), diphenhydramine hydrochloride (Gist-Brocades, Delft, The Netherlands), levocabastine (Janssen Pharmaceutica NV), loratadine (Schering Plough, Bloomfield, NJ), mainserin hydrochloride and mirtazepine (Organon NV, Oss, The Netherlands), pcDEF₃ (Dr. J. Langer, Robert Wood Johnson Medical School, Piscataway, NJ), and ranitidine dihydrochloride (GlaxoSmithKline, Uxbridge, Middlesex, UK), and of the cDNA encoding the human histamine H₁R (Fukui et al., 1994) are greatly acknowledged.

Molecular Cloning. The genes coding for the human H₁R and the G_q subunit were cloned as described previously (Burstein et al., 1995; Bakker et al., 2004). All receptor and G protein constructs were fully sequence-verified by dideoxy chain termination methods. The sequence of the human H₁R used in this study corresponds to Gen-Bank accession no. D14436. All plasmid DNA used for transfections was prepared using resin-based mega-prep purifications following the manufacturer's protocol (QIAGEN GmbH).

Cell Culture and Transfection. COS-7 African green monkey kidney cells were maintained at 37°C in a humidified 5% CO₂, 95% air atmosphere in Dulbecco's modified Eagle's medium (DMEM) containing 2 mM L-glutamine, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 5% (v/v) fetal calf serum. COS-7 cells were transiently transfected using the DEAE-dextran method as described previously (Bakker et al., 2001). The total amount of DNA transfected was maintained constant by addition of pcDEF₃. NIH-3T3 cells were cultured in DMEM supplemented with 2 mM L-glutamine, 1% penicillin and streptomycin, and 10% bovine calf serum and maintained at 37°C in a humidified 5% CO₂, 95% air atmosphere. NIH-3T3 cells were transiently transfected using the SuperFect transfection reagent (QIAGEN GmbH) following the manufacturer's protocol.

R-SAT Assays. R-SAT assays were performed as described previously (Weiner et al., 2001; Bakker et al., 2004). On forming a monolayer, NIH-3T3 cells normally stop growing due to contact inhibition. In R-SAT assays, the activation of pathways, i.e., through the activation of GPCRs, that promote cell growth result in NIH-3T3 cells being able to overcome their contact inhibition and proliferate. These stimulatory effects can be readily quantified using a marker gene, which allows graded responses to be measured, permitting precise determinations of ligand potency and efficacy. In brief, on day 1, NIH-3T3 cells were plated into 96-well cell culture plates at a density of 7500 cells/well. On day 2, cells were transfected with 10 to 25 ng/well H₁R DNA, with or without 5 ng/well plasmid DNA encoding the various G subunits, and 20 ng/well plasmid DNA encoding -galactosidase. On day 3, the media were replaced with DMEM supplemented with 1% penicillin and streptomycin, 2% Cyto-SF₃, and varying drug concentrations. After 5 days of cell culture, media were removed, and the cells were incubated in phosphate-buffered saline containing 3.5 mM O-nitrophenyl--D-galactopyranoside and 0.5% Nonidet P-40 detergent. The 96-well plates were incubated at room temperature for up to 8 h, and the resulting colorimetric reaction was measured by spectrophotometric analysis at 420 nm on an automated plate reader (BioTek Instruments Inc., Burlington, VT). Data were analyzed by a nonlinear, least-squares curve-fitting procedure using GraphPad Prism (GraphPad Software Inc., San Diego, CA). All data shown are expressed as mean ± S.E.M.

Reporter-Gene Assay. COS-7 cells transiently cotransfected with pNFB-Luc (125 µg/1 x 10⁷ cells) and either pcDEF₃ or pcDEF₃hH₁ (25 µg/1 x 10⁷ cells) were seeded in 96-well blackplates (Corning Life Sciences, Acton, MA) in serum-free culture medium and incubated with drugs. After 48 h, cells were assayed for luminescence by aspiration of the medium and the addition of 25 µl/well luciferase assay reagent [0.83 mM ATP, 0.83 mM D-luciferin, 18.7 mM MgCl₂, 0.78 µMNa₂H₂P₂O₇, 38.9 mM Tris, pH 7.8, 0.39% (v/v) glycerol, 0.03% (v/v) Triton X-100, and 2.6 µM dithiothreitol]. After 30 min, luminescence was measured for 3 s/well in a Victor² multi-label counter (PerkinElmer Life and Analytical Sciences, Boston, MA). All data shown are expressed as mean ± S.E.M.

H₁R Binding Studies. Cells used for radioligand binding studies were harvested 48 h after transfection and homogenized in ice-cold H₁R binding buffer (50 mM Na₂/potassium-phosphate buffer, pH 7.4). The cell homogenates were incubated for 30 min at 25°C in a total volume of 400 µlofH₁R binding buffer with 1 nM [³H]mepyramine. The nonspecific binding was determined in the presence of 1 µM mianserin. The incubations were stopped by rapid dilution with 3 ml of ice-cold H₁R binding buffer. The bound radioactivity was separated by filtration through Whatman GF/C filters (Whatman, Maidstone, UK) that had been treated with 0.3% polyethyleneimine. Filters were washed twice with 3 ml of buffer, and radioactivity retained on the filters was measured by liquid scintillation counting. Binding data were evaluated by a nonlinear, least-squares curvefitting procedure using GraphPad Prism (GraphPad Software Inc.). Protein concentrations were determined according to Bradford (1976), using bovine serum albumin as a standard. All data shown are expressed as mean ± S.E.M.

	Results

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Signaling Characteristics of the Human H₁Ras Determined by R-SAT
Plasmid DNA encoding the human H₁R was transiently transfected into NIH-3T3 cells as part of the R-SAT assay. Titration of the amount of H₁R DNA used for transfection revealed robust functional responses to histamine over a 100-fold dose range of receptor DNA, from 0.5 to 50 ng of DNA per well of a 96-well cell culture plate (Fig. 1; Table 1). Histamine yielded an average biological response of 11.4 ± 0.8-fold in H₁R-expressing cells, and it was without effect in cells transfected with the marker gene alone. Transfection of the cells with increasing amounts of cDNA encoding the H₁R results in an increase in observed potencies for histamine, which reached a plateau pEC₅₀ of 7.3 ± 0.2 at 10 ng of DNA per well. As depicted in Fig. 1 and Table 1, mepyramine started to display negative intrinsic activity at the H₁R in cells transfected with 10 ng of cDNA per well encoding the H₁R. When expressed alone, a maximum of 6% of the total H₁R response corresponds to basal, agonist independent, signaling. As reported previously, constitutive receptor activity can be modulated by the expression of appropriate subunits of guanine nucleotide binding proteins (G proteins) (Burstein et al., 1995, 1997; Leurs et al., 2000; Bakker et al., 2001; Weiner et al., 2001), and this approach was therefore used in the present study to augment H₁R basal signaling properties. As depicted in Fig. 1 and Table 1, cotransfection of a cDNA encoding G_q (20 ng/well) enhanced the biological responses observed for the H₁R under all conditions studied, and it yielded an average biological response of 9.6 ± 0.9-fold for histamine. Agonist potencies were increased upon cotransfection of G_q compared with receptor alone, ranging from 11- to 26-fold more potent than that observed without G_q coexpression. The observed potencies for histamine reached a plateau pEC₅₀ of 8.5 ± 0.2 at 5 ng of receptor DNA per well in the G_q coexpression experiments. Coexpression of H₁Rs and G_q resulted in an increased basal response and a concomitant reduction in the -fold response upon histamine stimulation of the cells (Fig. 1B). Mepyramine behaved as an inverse agonist under all conditions studied, but it does not display a significant change in inverse agonist potency when increasing amounts of cDNA encoding the H₁R are used. Constitutive H₁R signaling was detectable in all coexpression experiments, ranging from 10 to 30% of the total biological response. Figure 1C depicts the relationship between the amount of transfected DNA and constitutive receptor signaling observed under these experimental conditions.

View larger version (12K): [in this window] [in a new window]   Fig. 1. Signaling properties of human H1Rs. Functional responses for histamine () and mepyramine () observed in R-SAT assays in 3T3 cells transfected with H1R cDNA amounts of 0.5 ng/well (A) and 10 ng/well (B). The effects of cotransfection of 20 ng of Gq per well are indicated for histamine () and mepyramine () for both receptor cDNA amounts (dashed lines). No drug values for each experimental condition are denoted at the left-hand side of the figure. Data are reported as a percentage of the total response determined by histamine response/mepyramine response. C, graphic depiction of the relationship between constitutive activity of the human H1R and concentration of receptor DNA used for transfections as part of the R-SAT assay. Percentage constitutive activity is calculated as (basal, no drug, response–mepyramine response)/(histamine response–mepyramine response). Closed squares represent receptor expressed alone (), whereas open squares depict coexpression with Gq at 20 ng/well (). The values are expressed as means ± S.E.M. of three to eight separate experiments from representative nine-point concentration response curves each performed in duplicate.

Constitutive H₁R Activity Is Not Due to Endogenous Histamine
We have reported previously the use of S-(+)--fluoromehtylhistidine, an irreversible inhibitor of histidine decarboxylase (Watanabe et al., 1990), together with serum-free assay conditions, to confirm that constitutive H₁R activity is not due to contamination with endogenous histamine (Bakker et al., 2000, 2001). To avoid a similar confounding factor in the R-SAT assays, synthetic serum, devoid of trace monoamines, replaced calf serum during cell culture. Moreover, in agreement with our previous findings in COS-7 cells, the addition of as much as 100 µM S-(+)--fluoromehtylhistidine to the cell culture media did not attenuate the basal H₁R-mediated signaling or the observed negative intrinsic activity displayed by mepyramine observed in this assay (data not shown).

Evaluation of R-SAT for Determining Functional H₁R Responses
Agonist Responses. Based on the potencies observed for histamine during the cDNA titration studies, 10 ng of H₁R DNA per well was chosen as the most appropriate assay condition to evaluate potential agonist activity of ligands at the human H₁R. The histamine-induced R-SAT responses were competitively antagonized by the classic H₁R inverse agonist mepyramine. Schild plot analysis of the competitive antagonism by mepyramine of the histamine-induced proliferation resulted in a pA₂ value for mepyramine of 8.3 (slope = 1.05 ± 0.03; r² = 0.997). A series of known histaminergic agonists were tested for functional activity at the human H₁R, where the most potent agonist was histamine itself with an EC₅₀ of 35 nM. Histamine yielded the largest -fold responses, consistent with its designation as a full agonist (intrinsic activity, ,of1). N-Methylhistamine and 2-(2-aminoethyl)-pyridine behaved as full agonists, with EC₅₀ values of 120 nM and 1.32 µM, respectively, whereas 6-[2-(4-imidazolyl)ethylamine]-N-(4-trifuormethylphenyl)-heptanecardoxamide dimaleate displayed only weak partial agonist activity (pEC₅₀ = 6.2 ± 0.2; = 0.27 ± 0.04). In contrast, both enantiomers of the H₃R-preferring agonist -methylhistamine displayed only weak partial agonist activity with EC₅₀ values greater than 10 µM, whereas the H_3/4 receptor-selective agonists imetit and immepip and the H₃R antagonist/H₄R agonist clobenpropit displayed no intrinsic activity at the H₁R (data not shown).

Inverse Agonist Responses. Based on the degree of basal signaling, and the potencies observed for mepyramine during the titration studies, 10 ng of H₁R cDNA per well cotransfected with 20 ng of G_q cDNA per well was chosen as the most appropriate assay condition to evaluate potential inverse agonist activity of ligands at the human H₁R. Mepyramine and astemizole consistently yielded the largest degree of inhibition of basal signaling, consistent with their designation as full inverse agonists ( =–1). As reported in Table 2, all 11 of the known H₁R antagonists that were tested in this manner behaved as inverse agonists. Ketotifen was the most potent, with an IC₅₀ of 0.21 nM. The rank order of potencies for these compounds was ketotifen > levocabastine > mepyramine > astemizole > triprolidine > chlorpheniramine > tripelennamine > acrivastine > diphenhydramine > loratidine. All of these compounds displayed high potency for the human H₁R, ranging from 0.21 to 126 nM; and all, with one notable exception, behaved as full inverse agonists. Interestingly, loratidine displayed partial efficacy ( =–0.77 ± 0.03; Table 2). The H₂R-selective inverse agonists cimetidine and ranitidine, the H₃R inverse agonist clobenpropit, and the H_3/4 receptor-preferring antagonists thioperamide and iodophenpropit all lacked activity as inverse agonists at the H₁R (Table 2).

View this table: [in this window] [in a new window]   TABLE 2 H1R Inverse agonist pharmacology of known histaminergic ligands as determined by radioligand binding as well as NF-B and R-SAT functional assays The pKi and pIC50 at the human H1R and their intrinsic activities () are reported. The values are expressed as means ± S.E.M. of separate experiments, each performed in triplicate.

Correlation between Assays. We have reported previously the potencies of a number of histaminergic drugs as H₁R inverse agonists as determined by the NF-B assay (Bakker et al., 2001). Table 2 reports the potencies of many of these histaminergic compounds as determined by this assay as well as the affinities of many of these ligands for the H₁R as determined by radioligand binding experiments. Comparison of the functional potencies of these compounds between assays reveals a close correlation (r² = 0.92; slope = 0.72).

Evaluation of the Functional H₁R Activity of Various Therapeutics Using R-SAT
Examination for H₁R Agonist Activity. We evaluated a library of >130 clinically relevant therapeutic drugs for functional activity at the human H₁R using R-SAT (see Table 3 for a complete list of compounds tested). We controlled for both endogenous receptor- and nonreceptor-mediated effects of the tested drugs on cellular growth by assaying all drugs against cells expressing the -galactosidase marker gene alone, and cells expressing either related or unrelated receptors (e.g., 5-hydroxytryptaime_2A or neurokinin-1 receptors; data not shown). None of the compounds reported herein displayed nonspecific potent amplification or repression of cellular growth when tested in this manner (data not shown).

All compounds were initially screened for H₁R agonist activity. Only three compounds, lisuride, terguride, and methergine, displayed reasonable potency as H₁R agonists. We have recently reported the detailed agonist pharmacology of these compounds (Bakker et al., 2004).

Examination for H₁R Inverse Agonist Activity. After the evaluation of the various CNS drugs for H₁R agonist activity, all compounds were subsequently tested for H₁R inverse agonist activity. In contrast to the finding that only a few compounds display H₁R agonist activity, most of the tested compounds potently inhibited constitutive H₁R activity. Table 3 reports the H₁R inverse agonist potencies of all of these compounds as determined by the R-SAT assays and the inverse agonist behavior of several of the tested antipsychotics, antidepressants, and miscellaneous agents. Of this large data set, only the H₁R inverse agonist potencies of the antipsychotic agents that are listed in this large data set have been reported previously (Weiner et al., 2001). The majority of antipsychotic agents tested possess potent H₁R inverse agonist properties. All behaved as full inverse agonists except for loxapine, risperidone, and haloperidol, and the investigational agent MDL 10097. The dibenzodiazepine-based agents (clozapine, loxapine, clothiapine, olanzapine, and perlapine) were among the most potent, the phenothiazine-based agents (chlorpromazine, thioridazine, mesoridazine, etc.) displayed moderate potencies, and the butyrophenone-based agents (haloperidol, trifluperidol, fluspirilene, moperone, etc.) were among the least potent (Table 3). In addition to the antipsychotics, many antidepressant drugs also display this pharmacological activity. The tricyclic-based agents all display potent H₁R inverse agonism, with observed potencies ranging from 0.25 nM for mirtazepine to 200 nM for desipramine (Table 3). Last, of the various monoaminergic reference compounds tested, only a small number of serotonergic compounds displayed H₁R inverse agonist potencies, whereas the muscarinic and dopaminergic receptor-based compounds tested lack this activity (Table 3).

Examination for Competitive H₁R Antagonists. All compounds lacking intrinsic activity at the H₁R at concentrations up to 10 µM were subsequently tested for their ability at concentrations up to 10 µM to antagonize histamine-induced R-SAT responses. Compounds were tested using agonist-biased assays with a 150 nM final concentration of histamine. We have described the identification of neutral H₁R antagonists in a separate study, and we have shown that both inverse H₁R agonists and neutral H₁R antagonists are able to yield inhibitory actions using such an agonist-biased assay setup (Govoni et al., 2003). Hence, both inverse H₁R agonists and neutral H₁R antagonists can be used as a positive control in these experiments. Herein, we have chosen to use the readily available inverse H₁R agonist mepyramine for this purpose. Screening in this manner failed to identify any compounds that behaved as neutral antagonists of the human H₁R (data not shown). A list of all of the compounds tested in this manner can be found in Table 3.

	Discussion

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That human H₁R antagonists have clinical utility in the treatment of allergic and inflammatory conditions has been appreciated for some time, and antihistamines currently are among the most widely prescribed medications in the world (Woosley, 1996; Zhang et al., 1997; Handley et al., 1998). The development of such agents has been a major focus of drug discovery, and it has yielded a number of widely used antihistamines. These compounds are thought to act primarily by competing with endogenous histamine, blocking histamine-induced H₁R-mediated activation of appropriate second messenger signaling pathways (Zhang et al., 1997). Recent studies have demonstrated that many competitive antagonists, of a wide variety of different receptor types, are actually inverse agonists that possess the intrinsic ability to decrease agonist-independent, constitutive receptor responses (Kenakin, 2001; Seifert and Wenzel-Seifert, 2002). Some classically defined H₁R antagonists have also recently been reclassified as inverse agonists based on the application of functional assays that, unlike radioligand binding techniques, can differentiate competitive antagonists from inverse agonists (Weiner et al., 1999, 2001; Bakker et al., 2000, 2001; Wu et al., 2004; Sakhalkar et al., 2005). In the present study, we set out to determine the functional activity of a large series of clinically useful agents at the human H₁R using the functional, cell-based R-SAT assay. R-SAT assays generate physiologically predictive responses that demonstrate strong correlations to the known in vitro pharmacology of multiple GPCRs, and they are particularly suitable for screening large series of compounds due to the throughput necessary to perform such studies (Weiner et al., 2001; Croston, 2002; Wellendorph et al., 2002).

The development and application of radioligand binding methodologies allowed for the analysis of H₁R affinities of many clinically useful drugs, and it enabled the correlation between high H₁R affinity and the propensity for sedation for brain-penetrating drugs (Sekine et al., 1999; Bakker et al., 2002; Simons, 2002). Validation of the R-SAT-based H₁R pharmacology reported herein is demonstrated by the close correlation between the results obtained in this assay and the previously reported H₁R pharmacology, including rank orders of affinity (Bakker et al., 2000, 2001) and in vitro and in vivo potencies (Sekine et al., 1999) of many histaminergic compounds (Table 2).

The broad functional screening reported herein has demonstrated that all the herein tested H₁R antagonists, despite their various molecular structures, possess negative intrinsic activity and that they are actually H₁R inverse agonists. This observation concurs with our previous observations on H₁R inverse agonism and suggests that perhaps negative intrinsic activity may be necessary for their therapeutic effectiveness.

We also demonstrate a strong correlation between antagonist affinities and potencies of these agents as H₁R inverse agonists in these two assays. Thus, absolute and relative H₁R inverse agonist potencies can be used to predict the propensity of a compound to produce sedation (Sekine et al., 1999; Bakker et al., 2002; Simons, 2002) as well as other H₁R-mediated effects, such as weight gain (Kroeze et al., 2003; Roth and Kroeze, 2006), if it is known that these properties are primarily related to the H₁R effects of the compound and that the drug will enter the CNS. For example, the potent H₁R inverse agonist activity of the antipsychotic perlapine is consistent with its robust sedative effects clinically (Allen and Oswald, 1973; Stille et al., 1973), as is the potent inverse agonist activity of clozapine (The Parkinson Study Group, 1999). Likewise, the high-potency H₁R inverse agonist activity of tricyclic antidepressants is consistent with prior binding affinity data (Richelson, 1978, 2001; Richelson and Nelson, 1984a,b; Cusack et al., 1994; Bymaster et al., 1996). We have, in contrast to prior studies, tested a larger set of clinically useful compounds, and we have found that many serotonergic compounds possess inverse agonist activity at human H₁Rs.

Constitutive, basal, or spontaneous activity of the receptor, in the context of receptor pharmacology, is receptor-mediated signaling in the absence of agonist. It is most commonly seen in systems with high levels of receptor expression where inverse agonists inhibit both basal and agonist-induced receptor signaling. While the detection of constitutive GPCR activity is system-dependent, i.e., dependent on, for instance, receptor and G protein expression levels, it is most commonly seen in systems with high levels of receptor expression where inverse agonists inhibit both basal and agonist-inducing receptor signalling. For the H₁R, we have been able to readily detect constitutive activity as well as the inverse agonistic characteristics of a variety of ligands previously known as H₁R antagonists, when measuring either the accumulation of inositol phosphates (Bakker et al., 2000) or the activation of the transcription factor nuclear factor-B in a reporter-gene assay (Bakker et al., 2001) as well as in R-SAT assays (Weiner et al., 1999; Bakker et al., 2004; this study), when using heterologous expression systems. Because inverse agonists are able to induce a response, they potentially also display physiological activity in the absence of elevated levels of (endogenous) extracellular agonist. Because neutral antagonists and inverse agonists may have physiologically distinct actions in vivo, an H₁R neutral antagonist may differ from existing agents with respect to efficacy, tolerance, and perhaps propensity to induce clinically relevant side effects (Govoni et al., 2003). Constitutive GPCR activity is typically more readily observed in receptor overexpression systems compared with native systems. In line with these observations, to date, there have been no reports directly showing in vivo constitutive activity of the H₁R. The development of high-affinity H₁R ligands that lack intrinsic activity, and the subsequent use of these compounds in in vivo studies will be necessary to fully assess these hypotheses.

We have reported previously on the identification of the neutral H₁R antagonists histabudifen and histapendifen (Govoni et al., 2003). These findings resulted from the screening of a large variety of structurally diverse ligands for their activity at the human H₁R, and although many antagonists were found to possess negative intrinsic activity, only very few ligands failed to display any intrinsic activity at the H₁R. Unfortunately, the affinity of the currently known neutral H₁R antagonists is too poor for the evaluation of their therapeutic efficacy and potential side effects, such as potential induction of weight gain due to antagonizing the action of histamine at the H₁R.

In conclusion, we have screened a large number of CNS drugs for their intrinsic activity at the human H₁R, and we found the majority of these drugs to display pronounced H₁R inverse agonistic properties. Exceptions are the drugs 8R-lisuride and 8R-terguride that we identified to possess H₁R agonistic properties (Bakker et al., 2004) and the drugs that were found not to interact with the H₁R as assessed by their ability to modulate the H₁R-mediated effects of histamine and mepyramine in functional competition experiments. These data may help to understand the propensity of the identified H₁R inverse agonists to induce side effects, including weight gain and sedation, and prompt for the development of high-affinity neutral H₁R antagonist to evaluate their clinical effectiveness and side effects.

	Footnotes

 
R.L. is a recipient of a PIONIER award of the Technology Foundation (Stichting Technische Wetenschappen) of the Netherlands Foundation of Scientific Research (Nederlandse Organisatie voor Wetenschappelijk Onderzoek).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.118869.

ABBREVIATIONS: H₁R, histamine H₁ receptor; CNS, central nervous system; R-SAT, receptor selection and amplification technology; NF-B, nuclear factor-B; DS-121, S-(–)-3-(3-cyanophenyl)-N-n-propyl piperidine; JL-18, 8-methyl-6-(4-methyl-1-piperazinyl)-11H-pyrido[2,3-b][1,4]benzodiazepine; LY 53,857, 6-methyl-1-(methylethyl)-ergoline-8-carboxylic acid 2-hydroxy-1-methylpropyl ester maleate; MDL 10097, (±)-2,3-dimethoxyphenyl-1-[2-(4-piperidine)-methanol]; MK 212, 6-chloro-2-(1-piperazinyl)pyrazine; SB 206553, 5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2,3-f]indole; SCH 12679, N-methyl-1-phenyl-7,8-dimethoxy-2,3,4,5-tetra-hydro-3-benzazepine maleate; SCH 23390, 7-chloro-8-hydroxy-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; SKF 38393, 6-phenyl-4-azabicyclo[5.4.0]undeca-7,9,11-triene-9,10-diol; SKF 83566, (–)-7-bromo-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-3-benzazepine; mirtazepine [Org 3770; (±)-1,2,3,4,10,14b-hexahydro-2-methylpyrazino-[2,1-a]pyrido[2,3-c][2]benzazepine]; DMEM, Dulbecco's modified Eagle's medium; GPCR, G protein-coupled receptor.

¹ Current affiliation: Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany.

² Current affiliation: University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

Address correspondence to: Dr. Rob Leurs, Department of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. E-mail: r.leurs{at}few.vu.nl

	References

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Allen SR and Oswald I (1973) The effects of perlapine on sleep. Psychopharmacologia 32: 1–9.[CrossRef][Medline]

Bakker RA, Schoonus S, Smit MJ, Timmerman H, and Leurs R (2001) Histamine H₁-receptor activation of NF-B: roles for G and G_q/11-subunits in constitutive and agonist-mediated signaling. Mol Pharmacol 60: 1133–1142.[Abstract/Free Full Text]

Bakker RA, Timmerman H, and Leurs R (2002) Histamine receptors: specific ligands, receptor biochemistry, and signal transduction. Clin Allergy Immunol 17: 27–64.[Medline]

Bakker RA, Weiner DM, ter Laak T, Beuming T, Zuiderveld OP, Edelbroek M, Hacksell U, Timmerman H, Brann MR, and Leurs R (2004) 8R-Lisuride is a potent stereospecific histamine H1-receptor partial agonist. Mol Pharmacol 65: 538–549.[Abstract/Free Full Text]

Bakker RA, Wieland K, Timmerman H, and Leurs R (2000) Constitutive activity of the histamine H₁ receptor reveals inverse agonism of histamine H₁ receptor antagonists. Eur J Pharmacol 387: R5–R7.[CrossRef][Medline]

Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.[CrossRef][Medline]

Burstein ES, Spalding TA, and Brann MR (1997) Pharmacology of muscarinic receptor subtypes constitutively activated by G proteins. Mol Pharmacol 51: 312–319.[Abstract/Free Full Text]

Burstein ES, Spalding TA, Bräuner-Osborne H, and Brann MR (1995) Constitutive activation of muscarinic receptors by the G-protein G_q. FEBS Lett 363: 261–263.[CrossRef][Medline]

Bymaster FP, Calligaro DO, Falcone JF, Marsh RD, Moore NA, Tye NC, Seeman P, and Wong DT (1996) Radioreceptor binding profile of the atypical antipsychotic olanzapine. Neuropsychopharmacology 14: 87–96.[CrossRef][Medline]

Croston GE (2002) Functional cell-based uHTS in chemical genomic drug discovery. Trends Biotechnol 20: 110–115.[CrossRef][Medline]

Cusack B, Nelson A, and Richelson E (1994) Binding of antidepressants to human brain receptors: focus on newer generation compounds. Psychopharmacology (Berl) 114: 559–565.[CrossRef][Medline]

Fukui H, Fujimoto K, Mizuguchi H, Sakamoto K, Horio Y, Takai S, Yamada K, and Ito S (1994) Molecular cloning of the human histamine H₁ receptor gene. Biochem Biophys Res Commun 201: 894–901.[CrossRef][Medline]

Govoni M, Bakker RA, van de Wetering I, Smit MJ, Menge WM, Timmerman H, Elz S, Schunack W, and Leurs R (2003) Synthesis and pharmacological identification of neutral histamine H1-receptor antagonists. J Med Chem 46: 5812–5824.[CrossRef][Medline]

Handley DA, Magnetti A, and Higgins AJ (1998) Therapeutic advantages of third generation antihistamines. Exp Opin Investig Drugs 7: 1045–1054.[CrossRef]

Hill SJ, Ganellin CR, Timmerman H, Schwartz JC, Shankley NP, Young JM, Schunack W, Levi R, and Haas HL (1997) International Union of Pharmacology. XIII. Classification of histamine receptors. Pharmacol Rev 49: 253–278.[Abstract/Free Full Text]

Hill SJ and Young M (1978) Antagonism of central histamine H₁ receptors by antipsychotic drugs. Eur J Pharmacol 52: 397–399.[CrossRef][Medline]

Kenakin T (2001) Inverse, protean, and ligand-selective agonism: matters of receptor conformation. FASEB J 15: 598–611.[Abstract/Free Full Text]

Kroeze WK, Hufeisen SJ, Popadak BA, Renock SM, Steinberg S, Ernsberger P, Jayathilake K, Meltzer HY, and Roth BL (2003) H₁-histamine receptor affinity predicts short-term weight gain for typical and atypical antipsychotic drugs. Neuropsychopharmacology 28: 519–526.[CrossRef][Medline]

Leurs R, Rodriguez Pena MS, Bakker RA, Alewijnse AE, and Timmerman H (2000) Constitutive activity of G protein coupled receptors and drug action. Pharm Acta Helv 74: 327–331.[CrossRef][Medline]

Richelson E (1978) Tricyclic antidepressants block histamine H₁ receptors of mouse neuroblastoma cells. Nature 274: 176–177.[CrossRef][Medline]

Richelson E (2001) Pharmacology of antidepressants. Mayo Clin Proc 76: 511–527.[Medline]

Richelson E and Nelson A (1984a) Antagonism by antidepressants of neurotransmitter receptors of normal human brain in vitro. J Pharmacol Exp Ther 230: 94–102.[Abstract/Free Full Text]

Richelson E and Nelson A (1984b) Antagonism by neuroleptics of neurotransmitter receptors of normal human brain in vitro. Eur J Pharmacol 103: 197–204.[CrossRef][Medline]

Richelson E and Souder T (2000) Binding of antipsychotic drugs to human brain receptors focus on newer generation compounds. Life Sci 68: 29–39.[CrossRef][Medline]

Roth BL and Kroeze WK (2006) Screening the receptorome yields validated molecular targets for drug discovery. Curr Pharm Des 12: 1785–1795.[CrossRef][Medline]

Sakhalkar SP, Patterson EB, and Khan MM (2005) Involvement of histamine H1 and H2 receptors in the regulation of STAT-1 phosphorylation: inverse agonism exhibited by the receptor antagonists. Int Immunopharmacol 5: 1299–1309.[CrossRef][Medline]

Seifert R and Wenzel-Seifert K (2002) Constitutive activity of G-protein-coupled receptors: cause of disease and common property of wild-type receptors. Naunyn-Schmiedeberg's Arch Pharmacol 366: 381–416.[CrossRef][Medline]

Sekine Y, Rikihisa T, Ogata H, Echizen H, and Arakawa Y (1999) Correlations between in vitro affinity of antipsychotics to various central neurotransmitter receptors and clinical incidence of their adverse drug reactions. Eur J Clin Pharmacol 55: 583–587.[CrossRef][Medline]

Simons FE (2002) H1-antihistamines in children. Clin Allergy Immunol 17: 437–464.[Medline]

Stille G, Sayers A, Lauener H, and Eichenberger E (1973) 6-(4-Methyl-1-piperazinyl)morphanthridine (Perlapine), a new tricyclic compound with sedative and sleep-promoting properties. A pharmacological study. Psychopharmacologia 28: 325–337.[CrossRef][Medline]

The Parkinson Study Group (1999) Low-dose clozapine for the treatment of drug-induced psychosis in Parkinson's disease. The Parkinson Study Group. N Engl J Med 340: 757–763.[Abstract/Free Full Text]

Tran VT, Chang RS, and Snyder SH (1978) Histamine H1 receptors identified in mammalian brain membranes with [³H]mepyramine. Proc Natl Acad SciUSA 75: 6290–6294.[Abstract/Free Full Text]

Walsh GM, Annunziato L, Frossard N, Knol K, Levander S, Nicolas JM, Taglialatela M, Tharp MD, Tillement JP, and Timmerman H (2001) New insights into the second generation antihistamines. Drugs 61: 207–236.[CrossRef][Medline]

Watanabe T, Yamatodani A, Maeyama K, and Wada H (1990) Pharmacology of -fluoromethylhistidine, a specific inhibitor of histidine decarboxylase. Trends Pharmacol Sci 11: 363–367.[CrossRef][Medline]

Weiner DM, Burstein ES, Nash N, Croston GE, Currier EA, Vanover KE, Harvey SC, Donohue E, Hansen HC, Andersson CM, et al. (2001) 5-Hydroxytryptamine_2A receptor inverse agonists as antipsychotics. J Pharmacol Exp Ther 299: 268–276.[Abstract/Free Full Text]

Weiner DM, Goodman MW, and Brann MR (1999) Constitutive activation of histamine H₁ receptors. Soc Neurosci Abstr 25: 1472.

Wellendorph P, Goodman MW, Burstein ES, Nash NR, Brann MR, and Weiner DM (2002) Molecular cloning and pharmacology of functionally distinct isoforms of the human histamine H₃ receptor. Neuropharmacology 42: 929–940.[CrossRef][Medline]

Woosley RL (1996) Cardiac actions of antihistamines. Annu Rev Pharmacol Toxicol 36: 233–252.[CrossRef][Medline]

Wu RL, Anthes JC, Kreutner W, Harris AG, and West RE, Jr. (2004) Desloratadine inhibits constitutive and histamine-stimulated nuclear factor-kappaB activity consistent with inverse agonism at the histamine H1 receptor. Int Arch Allergy Immunol 135: 313–318.[CrossRef][Medline]

Zhang M-Q, Leurs R, and Timmerman H (1997) Histamine H₁-receptor antagonists, in Burger's Medical Chemistry and Drug Discovery (Wolff ME ed) pp 495–559, John Wiley & Sons, Inc., New York.

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