Abstract
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 Pharmacol65:538–549, 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.
Antagonists of the histamine H1 receptor (H1R) have proven effective in controlling many of the symptoms of the human allergic response. Classical H1R antagonists, known as “first generation” antihistamines, may act as sedatives upon crossing the blood-brain barrier, interacting with H1Rs expressed in the central nervous system (CNS). Moreover, sedation and performance impairment are undesirable and potentially dangerous side effects of first generation antihistamines, and they are a major limitation of their use. Although many of the first generation antihistamines exhibit additional anticholinergic properties that may contribute to their sedative properties, selective H1R antagonists acting in the CNS might be exploited as sleeping aids. Subsequent development of antihistamines focused on H1R antagonists that do not cross the blood-brain barrier, resulting in what are now termed “second generation” antihistamines (Zhang et al., 1997). Another advantage of these second-generation antihistamines is their increased selectivity for H1Rs over other related monoaminergic receptor subtypes (Walsh et al., 2001). In contrast to second generation antihistamines, compounds developed to treat neuropsychiatric disease are specifically designed to enter the CNS and target various monoaminergic G protein-coupled receptors and small molecule reuptake transporters. Radioligand binding studies have demonstrated that these compounds lack target specificity and that they may act at multiple receptor and transporter sites simultaneously (Hill and Young, 1978; Richelson, 1978; Richelson and Nelson, 1984a; Cusack et al., 1994; Richelson and Souder, 2000). It is noteworthy that many of these compounds have been shown to possess high H1R affinity (Tran et al., 1978; Richelson and Nelson, 1984a; Bymaster et al., 1996; Richelson and Souder, 2000). Examples of such molecules include antipsychotic drugs such as clozapine and tricyclic antidepressant drugs such as amitriptyline. Because interactions with H1Rs in brain can produce clinically significant adverse effects, including sedation (Sekine et al., 1999; Bakker et al., 2002; Simons, 2002), and possibly alterations in body weight (Kroeze et al., 2003; Roth and Kroeze, 2006), an improved understanding of the full extent of the H1R-mediated actions of neuropsychiatric drugs as a class may provide critical insight into their clinical profiles.
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-κB 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
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-κB-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 Gαq 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 Gα subunits, and 20 ng/well plasmid DNA encoding β-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-β-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 pNFκB-Luc (125 μg/1 × 107 cells) and either pcDEF3 or pcDEF3hH1 (25 μg/1 × 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
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 α 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 Gαq (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 Gαq compared with receptor alone, ranging from 11- to 26-fold more potent than that observed without Gαq 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 Gαq coexpression experiments. Coexpression of H1Rs and Gαq 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.
Constitutive H1R Activity Is Not Due to Endogenous Histamine
We have reported previously the use of S-(+)-α-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-(+)-α-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, α,of1). Nα-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; α = 0.27 ± 0.04). In contrast, both enantiomers of the H3R-preferring agonist α-methylhistamine displayed only weak partial agonist activity with EC50 values greater than 10 μM, whereas the H3/4 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 Gαq 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 (α =–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 (α =–0.77 ± 0.03; Table 2). The H2R-selective inverse agonists cimetidine and ranitidine, the H3R inverse agonist clobenpropit, and the H3/4 receptor-preferring antagonists thioperamide and iodophenpropit all lacked activity as inverse agonists at the H1R (Table 2).
Correlation between Assays. We have reported previously the potencies of a number of histaminergic drugs as H1R inverse agonists as determined by the NF-κB 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 β-galactosidase marker gene alone, and cells expressing either related or unrelated receptors (e.g., 5-hydroxytryptaime2A 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
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-κB 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
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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).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.118869.
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ABBREVIATIONS: H1R, histamine H1 receptor; CNS, central nervous system; R-SAT, receptor selection and amplification technology; NF-κB, nuclear factor-κB; 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-8β-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.
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↵1 Current affiliation: Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany.
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↵2 Current affiliation: University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
- Received January 2, 2007.
- Accepted March 30, 2007.
- The American Society for Pharmacology and Experimental Therapeutics