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NEUROPHARMACOLOGY

Two Novel and Selective Nonimidazole Histamine H₃ Receptor Antagonists A-304121 and A-317920: I. In Vitro Pharmacological Effects

Neuroscience Research, Global Pharmaceutical Research Division, Abbott Laboratories, Abbott Park, Illinois (T.A.E., K.M.K., T.R.M., C.H.K., D.G.W., B.B.Y., G.B.F., R.F., A.A.H.); Pharmacia Corp., St. Louis, Missouri (L.I.D.); Athersys, Cleveland, Ohio (Y.L.B.); and Northwestern University Medical School, Lake Forest, Illinois (M.W.)

Received November 26, 2002; accepted February 14, 2003.

	Abstract

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Histamine H₃ receptor (H₃R) antagonists enhance neurotransmitter release and are being developed for the treatment of a variety of neurological and cognitive disorders. Many potent histamine H₃R antagonists contain an imidazole moiety that limits receptor selectivity and the tolerability of this class of compounds. Here we present the in vitro pharmacological data for two novel piperazine amide ligands, A-304121 [4-(3-((2R)-2-aminopropanoyl-1-piperazinyl)propoxy)phenyl)cyclopropylmethanone] and A-317920 [N-((1R)-2-(4-(3-(4-(cyclopropylcarbonyl)phenoxy)propyl)-1-piperazinyl)-1-methyl-2-oxo-ethyl-)-2-furamide], and compare them with the imidazole H₃R antagonists ciproxifan, clobenpropit, and thioperamide. Both A-304121 and A-317920 bind potently to recombinant full-length rat H₃R(pK_i values = 8.6 and 9.2, respectively) but have lower potencies for binding the full-length human H₃R (pK_i values = 6.1 and 7.0, respectively). A-304121 and A-317920 are potent antagonists at rat H₃R in reversing R--methylhistamine [(R)--MeHA] inhibition of forskolin-stimulated cAMP formation (pK_b values = 8.0 and 9.1) but weak antagonists at human H₃Rs in cyclase (pK_b values = 6.0 and 6.3) and calcium mobilization (pK_b values = 6.0 and 7.3) assays in cells co-expressing G_qi5-protein. Both compounds potently antagonize native H₃Rs by blocking histamine inhibition of potassium-evoked [³H]histamine release from rat brain cortical synaptosomes (pK_b values = 8.6 and 9.3) and (R)--MeHA reversal of electric field-stimulated guinea pig ileum contractions (pA₂ values = 7.1 and 8.3). A-304121 and A-317920 are also more efficacious inverse agonists in reversing basal guanosine 5'-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPS) binding at the human H₃R (pEC₅₀ values = 5.7 and 7.0) than are the imidazole antagonists. These novel and selective piperazine amides represent useful leads for the development of H₃R antagonist therapeutic agents.

The recent cloning of rat and human histamine H₃Rs (Lovenberg et al., 1999, 2000) has significantly impacted the search for potential therapeutic H₃R antagonists to treat central nervous system disorders. Additionally, the cloning of the H₃R has revealed a number of unique properties associated with this receptor. Multiple splice isoforms for human and rat H₃Rs have been identified that appear to be differentially expressed in the brain (Coge et al., 2001; Drutel et al., 2001). Although no major pharmacological differences have yet been noted for these isoforms using antagonists, agonists do show increased potencies for the short isoform (Wieland et al., 2001). Activation of recombinant H₃Rs has been shown to inhibit adenylate cyclase activity presumably mediated through a G_i/o-protein pathway (Lovenberg et al., 1999; Drutel et al., 2001). The rat H₃R has also been shown to activate mitogen-activated protein kinase and arachidonic acid release in an isoform-selective manner (Drutel et al., 2001). Profound species differences in the antagonist pharmacology of the rat and human H₃Rs have been observed (Ligneau et al., 2000; Lovenberg et al., 2000; Yao et al., 2003) due to variations at amino acids 119 and 122 within the third transmembrane domain of the receptor. Both native and heterologously expressed recombinant H₃Rs are constitutively active (Morisset et al., 2000; Wieland et al., 2001; Rouleau et al., 2002), and several previously characterized H₃R antagonists have subsequently been shown to be inverse agonists, perhaps a desirable quality for therapeutic agents.

A large number of H₃R antagonists have been synthesized since the original discovery of this receptor, but none are yet approved for clinical use. Until very recently, these compounds were primarily imidazole derivatives represented by agents such as thioperamide (Arrang et al., 1987), ciproxifan (Ligneau et al., 1998), clobenpropit (Barnes et al., 1993), and GT-2331 (Tedford et al., 1998). Many of these compounds were originally defined with high affinity for the rat H₃R but were later found to have lower affinity for the human H₃R including thioperamide, ciproxifan, and GT-2331 (Ligneau et al., 2000; Lovenberg et al., 2000; Esbenshade et al., 2001; Ireland-Denny et al., 2001; Yao et al., 2003). Additionally, subsequent studies have shown that as a class, the imidazole H₃R antagonists are not as selective for the human H₃R as originally believed, demonstrating appreciable binding affinities for serotonin 5-HT₃ (Leurs et al., 1995), ₂-adrenergic, and histamine H₄R (Esbenshade et al., 2001; Liu et al., 2001). Not only has clobenpropit been shown to have relatively high binding affinity for the histamine H₄R, but it is also a partial agonist at this receptor (Liu et al., 2001). Potential interaction of imidazole H₃R antagonists with cytochrome P450 enzymes is also of note since metyrapone, a cytochrome P450 inhibitor, markedly improves the specific H₃R binding of radiolabeled thioperamide and clobenpropit (Alves-Rodrigues et al., 1996; Harper et al., 1999). In addition, thioperamide has been shown to bind cytochrome P450 enzymes and inhibit adrenal steroidogenesis (Labella et al., 1992). Interestingly, the imidazole moiety is found in other drug molecules that have been shown to inhibit this important metabolic pathway (Halpert et al., 1994). Thus, it is desirable to synthesize potent and selective non-imidazole H₃R antagonists as potential therapeutic agents in man. Recent reports from our laboratory (Faghih et al., 2002a,b) and from other groups (Ganellin et al., 1998; Walczynski et al., 1999a,b; Linney et al., 2000; Lazewska et al., 2001; Meier et al., 2001) have described the properties of novel nonimidazole H₃R antagonists. Herein, we describe the in vitro pharmacological profile of two non-imidazole, aryloxyalkyl piperazine-based H₃R antagonists, A-304121 [4-(3-((2R)-2-aminopropanoyl-1-piperazinyl)propoxy)phenyl) cyclopropyl-methanone] and A-317920 [N-((1R)-2-(4-(3-(4-(cyclopropyl-carbonyl)phenoxy)propyl)-1-piperazinyl)-1-methyl-2-oxo-ethyl)-2-furamide] (Fig. 1). An accompanying report (Fox et al., 2003) presents the in vivo behavioral data of these two novel compounds.

View larger version (7K): [in this window] [in a new window]   Fig. 1. Chemical structures of A-304121 and A-317920.

	Materials and Methods

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Chemicals. A-304121, A-317920 (Fig. 1), and ciproxifan were synthesized at Abbott Laboratories. [³H]N--methylhistamine, 45 to 90 Ci/mmol, [³H]pyrilamine, 20 to 30 Ci/mmol, [³H]tiotidine, 70 to 90 Ci/mmol, [³H]prazosin, 75 to 80 Ci/mmol, [³H]rauwolscine, 75 Ci/mmol, [³H]histidine, 40 to 60 Ci/mmol, and [³⁵S]GTPS, 1250 Ci/mmol were obtained from PerkinElmer Life Sciences (Boston, MA) and [³H]histamine, 30 to 60 Ci/mmol, and [³H]LY-278584, 60 to 85 Ci/mmol, were purchased from Amersham Biosciences Inc. (Piscataway, NJ). Phentolamine was purchased from Novartis Pharmaceutical Corp. (Basel, Switzerland), (R)--methylhistamine and clobenpropit were purchased from Tocris Cookson Inc. (Bristol, UK), and serotonin and thioperamide were purchased from Sigma-Aldrich (St. Louis, MO).

Animals. Animals for experiments conducted in house were housed in Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) approved facilities at Abbott Laboratories in a temperature-regulated environment with lights on between 6:00 AM and 6:00 PM. Male Sprague-Dawley rats (weighing 200–250 g on arrival) and male Hartley guinea pigs (weighing from 150–200 g on arrival) were supplied by Charles River (Portage, MI). Male beagle dogs were obtained from Marshall Farms (North Rose, NY). The animals were acclimated to laboratory conditions for at least 1 week before testing. All in-house testing was conducted according to protocols approved by Abbott's Institutional Animal Care and Use Committee.

H₃R Cloning and Cell Membrane Preparation. The human histamine H₃R gene was cloned using human thalamus poly(A⁺) RNA (BD Biosciences Clontech, Palo Alto, CA) with reverse transcription-PCR methods using primers designed according to the published human H₃R gene sequences (Lovenberg et al., 1999; GenBank accession number AF140538 [GenBank] ). The full-length (H_3L) human histamine H₃R cDNA encoding 445 amino acids was cloned into the pCIneo expression vector. A partial rat histamine H₃R gene was identified by homology searching using the InCyte Pharmaceutical (Palo Alto, CA) database. This unique clone shared significant homology to the published human histamine H_3LR sequence. RACE (rapid amplification of cDNA ends) was performed with thalamus RNA from Long Evans rat (Charles River Laboratories, Wilmington, MA) using primers designed according to the InCyte clone, and the PCR product was cloned into the pCIneo expression vector.

HEK and C6 cells were grown in Dulbecco's modified Eagle's medium containing high glucose that was supplemented with 10% fetal bovine serum and 20 mM L-glutamine. Transfection of HEK and C6 cells was performed with LipofectAMINE according to the protocol provided by the vendor (Invitrogen, Carlsbad, CA), and cell lines were selected using geneticin. Cells from stable clonal lines were harvested and homogenized in TE buffer (50 mM Tris-HCl, 5 mM EDTA, pH 7.4) using a polytron at 20,000 rpm for 2 x 10 s bursts in the presence of protease inhibitors (1 mM benzamidine, 2 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin; Sigma-Aldrich), followed by centrifugation at 40,000g. The membrane pellets were further purified by repeating the homogenization and centrifugation steps as described above. Final membrane preparations were obtained by re-homogenizing the pellets in 6.25 vol (w/v) of TE buffer and were frozen at –70°C until used.

Human (Analytical Biological Services, Wilmington, DE), rat (Pelfreez, Rogers, AR), dog, or guinea pig brain cerebral cortices expressing H₃R were homogenized in cold TE buffer containing protease inhibitors. The homogenate was centrifuged at 40,000g for 20 min at 4°C. This step was repeated, and the resulting pellet was resuspended in TE buffer in a final volume of 3 times the wet weight of the tissue. Aliquots were frozen at –70°C until needed.

Cells from stable clonal lines expressing the human histamine H₁R (Fukui et al., 1994) or H₂R (Gantz et al., 1991) were harvested and homogenized in TE buffer as described above, and final membrane preparations were obtained by re-homogenizing the pellets in 6.25 vol of TE buffer and frozen at –70°C until used. Membranes were prepared from HEK cells transiently transfected with the pCINeo expression vector harboring the human histamine H₄R (Liu et al., 2001), as described above for the H₃R.

Radioligand Binding Assays. For H₃R binding, membrane preparations were incubated with ³H-labeled N--methylhistamine ([³H]NAMH) (0.5–1.0 nM) in the presence or absence of increasing concentrations (from 5 to 11 concentrations over a 5 log unit range) of ligands for competition binding. The binding reactions were carried out for 30 min at 25°C in a final volume of 0.5 ml of TE buffer. Nonspecific binding was defined with 30 µM thioperamide. Radioligand binding assays for cloned human histamine H₁R and H₂R were performed as described (Esbenshade and Hancock, 2000) using [³H]mepyramine and [³H]tiotidine, respectively. In brief, H₁R membranes were incubated with increasing concentrations of test compound in the presence of 0.7 nM [³H]mepyramine at 25°C for 30 min in a total volume of 0.5 ml of 50 mM sodium/potassium PO₄ buffer, pH 7.4. Nonspecific binding was defined with 2.0 µM promethazine. H₂R membranes were incubated with increasing concentrations of test compound in the presence of 0.6 nM [³H]tiotidine at 25°C for 45 min in a total volume of 0.5 ml of 50 mM sodium/potassium PO₄ buffer, pH 7.4. Nonspecific binding was defined with 100 µM cimetidine. Binding to human histamine H₄R (Liu et al., 2001) transiently expressed in HEK cells was performed essentially as described. Competition binding assays were performed with increasing concentrations of test compound in the presence of 20 nM [³H]histamine incubated at 25°C for 1 h in a total volume of 0.5 ml of 50 mM Tris, 5 mM EDTA, pH 7.4. Nonspecific binding was defined with 0.5 µM clobenpropit. All binding reactions were terminated by filtration under vacuum onto polyethylenimine (0.3%) presoaked Unifilters (PerkinElmer Life Sciences) or Whatman GF/B filters (Whatman, Clifton, NJ) (for human cortex H₃R and human H₄R) followed by three brief washes with 4 ml of ice-cold TE buffer. Bound radiolabel was determined by liquid scintillation counting.

For the binding to ₂-adrenergic receptors, assays were performed using [³H]rauwolscine binding to cloned human _2a and _2c receptors expressed in mouse fibroblast cells (LTK^–) membranes. Competition binding assays were performed with increasing concentrations of test compound in the presence of 200 pM [³H]rauwolscine in 25 mM glycylglycine (pH 7.4), and samples were incubated 120 min at 0°C. All assays were terminated by filtration under vacuum through Unifilter plates. Membranes for 5-HT₃ serotonin binding assays were prepared from rat frontal cortex and 5-HT₃ receptor competition binding assays were performed with increasing concentrations of test compound in the presence of 500 pM [³H]LY278584 in 10 mM HEPES buffer (pH 7.5 at 37°C), and samples were incubated for 2 h at 0°C. Nonspecific binding was defined with 10 µM quipazine. Binding was terminated by filtration under vacuum through Whatman GF/B filters.

For all of the radioligand competition binding assays, IC₅₀ values and Hill slopes were determined by Hill transformation of the data as previously described (Esbenshade and Hancock, 2000) and pK_i values were determined by the generalized Cheng-Prusoff equation (Cheng and Prusoff, 1973). Data are presented as the mean pK_i ± S.E.M. For compounds where the Hill slope was less than 0.8, the data were reanalyzed using GraphPad Prism (GraphPad Software, Inc., San Diego, CA), and the best fit to a one- or two-site binding curve was determined.

Adenylate Cyclase Assay. C6 cells or HEK cells stably expressing the full-length human or rat H_3LR were plated the day before the assay at 75,000 to 100,000 cells per well in a 96-well plate coated with either collagen IV or polyethylenimine. Medium was removed, and cells were incubated with Dulbecco's phosphate-buffered saline with calcium (DPBS; Invitrogen) for 10 min followed by DPBS containing 1 mM 3-isobutyl-1-methylxanthine for 20 min at 37°C with 5% CO₂. Upon removal of buffer, cells were incubated with H₃R antagonists for 2 min before the addition of 30 nM (R)--MeHA. After an additional 5-min incubation, 10 µM forskolin was added to provide a final volume of 150 µl. Cells were hydrolyzed after 10 min by the addition of 20 µl of 1 N HCl and subsequent shaking for 10 min. After the addition of 20 µl of 1 N NaOH, the level of cAMP was determined by scintillation proximity assay (Amersham Biosciences Inc.). Data were normalized to the amount of cAMP produced in control wells and are expressed as the percentage of forskolin-stimulated cAMP response. Experiments were run in triplicate and data were analyzed using GraphPad Prism to obtain IC₅₀ values and Hill slopes. pK_b values were determined by the generalized Cheng-Prusoff equation (Cheng and Prusoff, 1973) and are presented as the mean ± S.E.M.

Measurement of Intracellular Calcium Levels. Functional activity of human H_3LR was determined in a stable HEK-293 cell line coexpressing the receptor and G_q/i5 by measuring agonist-evoked increases in intracellular calcium (Coward et al., 1999). Fluo-4, a calcium-sensitive fluorescent dye, was used as an indicator of intracellular calcium levels. Relative fluorescence was measured in a 96-well format by the fluorometric imaging plate reader (FLIPR; Molecular Devices Corp., Sunnyvale, CA). Confluent cells grown in 96-well black-walled polyethylenimine-treated tissue culture plates were loaded with 8 µM Fluo-4/acetoxymethylester in DPBS at room temperature for 1 to 2 h. Before measuring fluorescence, each plate was washed three times. Increasing concentrations of H₃R antagonists were added at 10-s intervals followed by addition of 30 nM (R)--MeHA 5 min later. Raw fluorescence data were corrected by subtracting fluorescence values just before the addition of test compounds from fluorescence values at all time points. Corrected responses were then measured by selecting peak fluorescence values within the period of drug exposure. Peak response values were then expressed as a percentage of the reference peak response for 30 nM (R)--MeHA in the absence of H₃R antagonists. Experiments were performed in duplicate, and data were analyzed using GraphPad Prism to obtain IC₅₀ values and Hill slopes. pK_b values were determined by the generalized Cheng-Prusoff equation (Cheng and Prusoff, 1973) and are presented as the mean ± S.E.M.

Electric Field-Stimulated (EFS) Guinea Pig Ileum. The modulation of EFS guinea pig ileum by H₃R antagonists was determined as previously described (Ireland-Denny et al., 2001). A 20-cm section of ileum, obtained approximately 10 cm proximal to the ileocecal junction, was removed from male guinea pigs and sectioned into 2-cm segments, cleaned, and placed in warm (37°C) Krebs-Henseleit bicarbonate buffer (0.141 g/l MgSO₄, 0.35 g/l KCl, 0.16 g/l KH₂PO₄, 6.9 g/l NaCl, 2.0 g/l D-glucose, 2.1 g/l NaHCO₃; Sigma-Aldrich) containing 2.6 mM CaCl₂,1 µM mepyramine, and 10 µM ranitidine. One end of the segment was then mounted onto a stationary rod containing parallel platinum electrodes aligned on each side of the tissue, and the other end was connected to a Grass FT03 transducer at a basal preload tension of 1 g. After a 10-min equilibrium period in heated 10-ml tissue baths, the tissues were electrically stimulated (supramaximal voltage 15 V, 0.1 Hz frequency, 0.5 ms duration) and rinsed every 10 min for 1 h. The intensity of the stimulus was then decreased every 5 min by 2 V until the threshold voltage for EFS contraction could be established. The experiment was then performed at a test voltage of 1.5x the observed threshold voltage. The tissues were stimulated for an additional hour at the test voltage (7 to 8 V) before the control agonist (R)--MeHA response curve was determined by cumulatively adding logarithmically increasing doses to the baths. The concentration of the (R)--MeHA necessary to cause a 50% inhibition in the EFS contraction (EC₅₀) was calculated using an Excel-based program, AGANTG (Zielinski and Buckner, 1998), and expressed as the negative logarithm (pD₂). H₃R antagonists were tested by adding various concentrations to the tissue baths 30 min before the generation of (R)--MeHA concentration-response curves. The potency of the antagonists (pA₂) to inhibit the (R)--MeHA response was calculated according to the method of Schild (1947) using AGANTG.

Rat Cerebral Cortical Histamine Release Assay. Functional modulation of histamine release from rat cerebral cortical synaptosomes by histamine H₃R ligands was determined essentially as previously described (Arrang et al., 1985). Freshly dissected rat cerebral cortices were homogenized in cold 4 mM HEPES containing 0.32 M sucrose (pH 7.3) using a Potter-Elvehjem homogenizer with a Teflon pestle and centrifuged at 800g for 10 min to remove debris. Synaptosomes were isolated by centrifugation at 10,000g for 20 min, washed in a modified Krebs-Ringer bicarbonate buffer (0.1 g/l MgCl₂-6H₂O, 0.34 g/l KCl, 0.1 g/l Na₂HPO₄, 0.18 0.34 g/l NaH₂PO₄, 7.0 g/l NaCl, 1.8 g/l D-glucose, 1.26 g/l NaHCO₃; Sigma-Aldrich), and incubated with 1.2 µM [³H]histidine for 30 min at 37°C under a constant stream of 95% O₂/5% CO₂. Synaptosomes were washed extensively by repeated centrifugation to remove unincorporated radioactivity and were subsequently resuspended in the Krebs-Ringer bicarbonate buffer. Aliquots (containing approximately 2 mg of protein) were added to microcentrifuge tubes containing the agonist histamine (1 µM) either alone or together with H₃R antagonists, and the mixture was incubated for 2 min at 37°C in the presence of 5% CO₂ to allow the ligand to bind. Potassium (15 mM final concentration as KCl) was subsequently added, and the samples were incubated for an additional 2 min. Samples were placed on ice and immediately centrifuged (4°C) at 20,000g for 20 min. The supernatant was removed and chromatographic separation of [³H]histidine and [³H]histamine was performed using Amberlite CG-50. Basal release values (obtained in buffer without additional potassium) were subtracted from each sample, and the data were expressed as a percentage of the maximum potassium-stimulated release for each assay. Data were analyzed from duplicate experiments using GraphPad Prism to obtain IC₅₀ values and Hill slopes. pK_b values were determined by the generalized Cheng-Prusoff equation (Cheng and Prusoff, 1973) and are presented as the mean ± S.E.M.

Inverse Agonism: [³⁵S]GTPS Binding Assay. Membranes from HEK cells expressing the human H_3LR were prepared by homogenization in cold buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM EDTA, 10 mM MgCl₂, 1 mM benzamidine, 2 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin. The homogenate was centrifuged at 40,000g for 20 min at 4°C, and the resulting pellet was resuspended in 50 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 10 mM MgCl₂ and homogenized. Glycerol and bovine serum albumin were added to a final concentration of 10% glycerol and 1% bovine serum albumin. Membranes were diluted in GTPS assay buffer (25 mM HEPES, 2.5 mM MgCl₂, and 75 mM NaCl, pH 7.4) and 10 µg of membrane protein was incubated in a 96-well deep-well block in the presence of 5.0 µM unlabeled GDP, approximately 0.5 nM of [³⁵S]GTPS, and increasing concentrations of test compounds. Samples were incubated at 37°C for 20 min and the assays were terminated by the addition of cold buffer (50 mM Tris-HCl, 75 mM NaCl, and 2.5 mM MgCl₂, pH 7.6) and subsequent harvesting onto a Packard Unifilter 96-well GF/B plate followed by extensive washing. Microscint 20 (PerkinElmer Life Sciences) was added to the samples, and bound [³⁵S]GTPS was determined using the Topcount (PerkinElmer Life Sciences). The percentage of [³⁵S]GTPS bound in each sample was calculated as a percentage of that bound to control samples incubated in the absence of the H₃R antagonists (basal). Data were analyzed from experiments performed in triplicate using GraphPad Prism to obtain pEC₅₀ values and Hill slopes.

	Results

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Histamine H₃R Binding. H₃R binding affinities were determined for A-304121, A-317920, thioperamide, ciproxifan, and clobenpropit by determining displacement of specific [³H]NAMH binding from H₃R binding sites in membranes prepared from C6 cells expressing the full-length recombinant rat and human H_3LR as well as brain cortical membranes prepared from rat, human, dog, and guinea pig (Table 1). All five compounds potently bound with pK_i values greater than 8.0 to both the recombinant rat H_3LR as well as rat brain cortical membrane H₃Rs with a fairly similar rank order of potency at the recombinant rat H_3LR (clobenpropit > A-317920 = ciproxifan > A-304121 = thioperamide) and native rat H₃R (clobenpropit > A-317920 = ciproxifan > A-304121 > thioperamide). In contrast, all compounds except clobenpropit (pK_i = 9.4) were markedly less potent at both the recombinant human full-length H_3LR and human brain cortical H₃Rs with pK_i values ranging from 6.1 to 7.2. Again, the compounds demonstrated a similar rank order of potency (clobenpropit > A-317920 = ciproxifan = thioperamide > A-304121) for the recombinant human H_3LR and native H₃R in human brain. The compounds were overall more potent for H₃Rs in dog and guinea pig brain membranes in comparison to human H₃Rs with pK_i values >7 for all compounds. Rank order of potencies for this set of five compounds for the guinea pig brain cortex was clobenpropit > A-317920 = ciproxifan > thioperamide > A-304121 and clobenpropit > A-317920 = ciproxifan = thioperamide > A-304121 for the dog brain cortex. Hill slopes for all the antagonist displacement curves approached unity, indicating the compounds recognized a single H₃R binding site in each membrane preparation. For those membrane preparations where the Hill slopes for A-304121 (guinea pig and dog cortex) and A-317920 (dog cortex) were <0.8, comparison of the nonlinear regression curve fits to one- and two-binding site models revealed that the data were best fit by a one-binding site model.

H₃R Binding Selectivity Profile. The binding affinities of A-304121, A-317920, ciproxifan, thioperamide, and clobenpropit at the three other histamine receptor subtypes (H₁, H₂, H₄) were determined as were binding affinities at other biogenic amine receptors including the rat serotonin 5-HT₃ and human _2a and _2c-adrenergic receptors (Table 2). A-304121 exhibited no binding affinity at concentrations up to 10 µM for any of the other three histamine receptor subtypes (pK_i < 5) and A-317920 likewise had no affinity for either the histamine H₂R or H₄R (pK_i < 5) and low affinity for the histamine H₁R (pK_i = 5.4). Ciproxifan and thioperamide demonstrated no affinity for either the histamine H₁R or H₂R (pK_i values <5) and clobenpropit exhibited low affinity (pK_i values = 5.2 and 5.6, respectively) for these receptors. However, thioperamide and clobenpropit demonstrated appreciable histamine H₄R binding affinity (pK_i values 7.3), whereas the binding affinity of ciproxifan was lower (pK_i value = 5.7). Both A-304121 and A-317920 had no affinity (pK_i values <5) for the _2a-adrenergic and 5-HT₃ receptor binding sites and low affinity (pK_i values = 5.5 and 5.6, respectively) for the _2c-adrenergic receptor. In contrast, clobenpropit, ciproxifan, and thioperamide exhibited varying degrees of higher binding affinities for the serotonin 5-HT₃-(pK_i values = 8.1, 6.5, and 5.6, respectively) and the _2a-(pK_i values = 7.8, 7.4, 6.9, respectively), and _2c-adrenergic (pK_i values = 7.8, 7.2, 6.5, respectively) receptor binding sites with a similar rank order of potency for these receptors of clobenpropit > ciproxifan > thioperamide in parallel with their rat histamine H₃R potencies.

Functional Antagonism at Recombinant H₃Rs. A-304121 and A-317920 inhibited the (R)--MeHA-mediated reversal of forskolin-stimulated cAMP accumulation in both C6 cells expressing the full-length human (Fig. 2, top panel) and rat (Fig. 2, bottom panel) H_3LRs in a concentration-dependent manner. Similar to the results observed in the binding assays, both A-304121 and A-317920 were more potent at the rat H_3LR (pK_b values = 8.0 and 9.1, respectively) than the human H_3LR (pK_b values = 5.3 and 6.5, respectively; Table 3). Clobenpropit and ciproxifan were equipotent as antagonists at the rat H_3LR in the adenylate cyclase assay (Fig. 2, bottom panel) with pK_b values of 9.0 and 9.2, respectively (Table 3), whereas thioperamide was less potent (pK_b = 7.6). Compared with the rat H_3LR potencies, all three imidazole H₃R antagonists were less potent in antagonizing the cAMP response by the human H_3LR (Fig. 2, top panel) with clobenpropit demonstrating the greatest potency (pK_b = 8.2), whereas the potencies of ciproxifan and thioperamide were considerably lower (pK_b values = 6.6 and 6.1, respectively; Table 3). Like those results seen in the adenylate cyclase experiments, A-304121 and A-317920 also inhibited (R)--MeHA-stimulated increases in intracellular calcium in a concentration-dependent manner in HEK cells coexpressing the human histamine H_3LR with the chimeric G_qi5-protein (Fig. 3) with respective pK_b values of 6.0 and 7.3 (Table 3). Clobenpropit more potently antagonized the effects of (R)--MeHA (pK_b value = 8.9) in this assay (Fig. 3) than any of the other H₃R antagonists including ciproxifan and thioperamide (pK_b values = 6.8 for both; Table 3), consistent with its enhanced binding affinity for the human H₃R.

View larger version (29K): [in this window] [in a new window]   Fig. 2. A-304121 and A-317920 block (R)--MeHA reversal of forskolin-stimulated cAMP formation in a concentration-dependent manner in C6 cells stably expressing the full-length human (top panel) and rat (bottom panel) histamine H3LRs. Cells were incubated with increasing concentrations of the H3R antagonists before the addition of 30 nM (R)--MeHA and 10 µM forskolin and cAMP formation determined as described under Materials and Methods. Data are expressed as a percentage of the maximal forskolin-stimulated cAMP response in the absence of H3R antagonist and (R)--MeHA. Results represent the mean ± S.E.M. of 6 to 12 concentration-response curves performed in triplicate.

View larger version (25K): [in this window] [in a new window]   Fig. 3. A-304121 and A-317920 block (R)--MeHA-stimulated increases in intracellular calcium in a concentration-dependent manner in HEK cells stably coexpressing the full-length human histamine H3LR and Gqi5-protein. Cells were incubated with increasing concentrations of the H3R antagonists before the addition of 30 nM (R)--MeHA and fluorescence measured as described under Materials and Methods. Data are expressed as a percentage of the maximal (R)--MeHA-stimulated response in the absence of H3R antagonist. Results represent the mean ± S.E.M. of 5 to 37 concentration-response curves performed in triplicate.

Effects of H₃R Antagonists in Models of Neurotransmitter Release. In the electric field-stimulated guinea pig ileum model, activation of H₃Rs by (R)--MeHA inhibits the electrically evoked release primarily of acetylcholine from nerve terminals that causes contraction of the tissue. Increasing concentrations of A-304121 caused dextral shifts of the concentration-response curves for (R)--MeHA-mediated reversal of electric field-stimulated contractions of guinea pig ileum (Fig. 4, top left panel). Although (R)--MeHA was not able to fully overcome the antagonism of the highest concentration tested of A-304121 (3000 nM), Schild analysis of the data (Fig. 4, top right panel) revealed a pA₂ value of 7.1 (Table 3) with a slope of –1.08, consistent with competitive antagonist activity. A-317920 also behaved as a competitive antagonist but was more potent than A-304121 in this model, producing rightward shifts of the (R)--MeHA concentration-response curves (Fig. 4, bottom left panel) and generating a pA₂ value of 8.25 (Fig. 4, bottom right panel; Table 3) and a slope of –1.0. The rank order of potency for all of the H₃R antagonists tested in this model was clobenpropit > thioperamide = A-317920 = ciproxifan > A-304121.

View larger version (32K): [in this window] [in a new window]   Fig. 4. Schild analysis of A-304121 (top panels) and A-317920 (bottom panels) competitive antagonism of (R)--MeHA-mediated reversal of electric field-stimulated contracted guinea pig ileum. Tissues were incubated with varying concentrations of A-304121 and A-317920 for 30 min before generating the (R)--MeHA concentration-response curves, as described under Materials and Methods. The right panels show the Schild transformations of the shifts in (R)--MeHA concentration-response curves (left panels) for A-304121 and A-317920. Data for the (R)--MeHA concentration-response curves are expressed as a percentage of the maximal electric field-stimulated contractile response in the absence of (R)--MeHA and H3R antagonist. Results represent the mean ± S.E.M. of 15 to 17 (R)--MeHA concentration-response curves for each antagonist.

In the rat brain cortical synaptosome model of neurotransmitter release, activation of H₃Rs inhibits the release of [³H]histamine caused by potassium-stimulated depolarization. Both A-304121 and A-317920 potently antagonized the histamine-mediated reversal of [³H]histamine release from rat synaptosomes (Fig. 5) in a concentration-dependent manner with respective pK_b values of 8.6 and 9.3. All of the H₃R antagonists tested were potent in this model with a rank order of potency of A-317920 > ciproxifan = clobenpropit > A-304121 = thioperamide.

View larger version (29K): [in this window] [in a new window]   Fig. 5. A-304121 and A-317920 block histamine reversal of potassium-stimulated [3H]histamine release in a concentration-dependent manner in rat brain cortical synaptosomes. Synaptosomes were incubated with increasing concentrations of the H3R antagonists before the addition of 1 µM histamine and 15 mM potassium, and [3H]histamine release was determined as described under Materials and Methods. Data are expressed as a percentage of the maximal potassium-stimulated [3H]histamine release in the absence of H3R antagonist and histamine. Results represent the mean ± S.E.M. of two to six concentration-response curves performed in triplicate.

Inverse Agonism: [³⁵S]GTPS Binding. A-304121 reduced basal [³⁵S]GTPS binding in membranes from HEK cells expressing the human H_3LR in a concentration-dependent manner (Fig. 6) with a pEC₅₀ value of 5.7 and a maximal inhibition of 16% from basal (Table 4). A-317920 more potently inhibited basal [³⁵S]GTPS binding than did A-304121 with a pEC₅₀ value of 7 and a maximal inhibition of 21% from basal (Table 4). Although clobenpropit, ciproxifan, and thioperamide were equally or more potent than A-304121 and A-317920 (rank order of potency of clobenpropit > thioperamide > ciproxifan = A-317920 > A-304121), these compounds were of lower inverse agonist intrinsic activity than A-304121 and A-317920 with rank order of intrinsic activity of A-317920 > A-304121 > thioperamide = clobenpropit = ciproxifan.

View larger version (25K): [in this window] [in a new window]   Fig. 6. A-304121 and A-317920 decrease basal [35S]GTPS binding in a concentration-dependent manner in membranes from HEK cells stably expressing the full-length human histamine H3LR. Membranes were incubated with increasing concentrations of the H3R antagonists and [35S]GTPS binding determined as described under Materials and Methods. Data are expressed as a percentage of the basal [35S]GTPS binding in the absence of H3R antagonist. Results represent the mean ± S.E.M. of three to four concentration-response curves performed in triplicate.

View this table: [in this window] [in a new window]   TABLE 4 Inverse agonist properties of A-304121, A-317920, clobenpropit, ciproxifan, and thioperamide at the histamine H3LR as determined by reversing basal [35S]GTPS binding

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Our in vitro and in vivo studies (see accompanying article by Fox et al., 2003) demonstrate that the non-imidazole compounds A-304121 and A-317920 are novel competitive H₃R antagonists. These compounds potently and selectively bind to the rat H₃R with affinities comparable to those of the imidazole H₃R antagonists thioperamide and ciproxifan. Additionally, A-304121 and A-317920 have equal or greater rat or guinea pig H₃R affinities than several other non-imidazole series (Ganellin et al., 1998; Walczynski et al., 1999a,b; Linney et al., 2000; Lazewska et al., 2001; Meier et al., 2001).

Like many of the imidazole H₃R antagonists, A-304121 and A-317920 are potent at rat H₃R, but are considerably less potent at human H₃R. It remains to be seen if this is also true of other non-imidazole H₃R antagonists described previously. The relatively low H₃R affinity of A-304121 is improved by the addition of the furanoyl moiety, creating A-317920 and increasing the potency at the rat H_3LR by 3-fold and at the human H_3LR by 8-fold with a pK_i value of 7.0, similar to that for ciproxifan and thioperamide. It has been shown that amino acids 119 and 122 critically determine the potency of imidazole H₃R antagonists at the human and rat receptors (Ligneau et al., 2000) and are very important in determining the binding potencies of A-304121 and A-317920 (Yao et al., 2003). Mutating the corresponding amino acids in the human H₃R to those amino acids in the rat (T119A and A122V) allows the resulting double mutant human H_3LR to bind to A-304121 and A-317920 with equal affinity as that seen in the wild-type rat H_3LR (Yao et al., 2003). Indeed, A-304121 is thus far in our hands the H₃R antagonist with the greatest potency difference between the rat and human H₃R. Both A-304121 and A-317920 display similar binding affinities for the guinea pig and dog brain H₃Rs that are intermediate between those of rat and human. Interestingly, these two species share the same amino acid 119 (threonine) as human and the same amino acid 122 (valine) as rat, thus perhaps accounting for the pharmacological profile seen in dog and guinea pig. Combining the knowledge about the molecular properties of the H₃R with continued insight into the chemical properties of H₃R antagonists, which contribute to their distinctive binding potencies across species, will allow for the optimization of compounds such as A-304121 and A-317920 with greater human H₃R potency.

Despite the relatively lower affinity of A-304121 and A-317920 for the human H₃R, these non-imidazole H₃R antagonists offer increased H₃R selectivity compared with the more conventional imidazole H₃R antagonists. Both compounds are more selective for the human H₃R versus other biogenic amine receptors than are clobenpropit, ciproxifan, and thioperamide. Indeed, A-304121 and A-317920 have little or no affinity for any of the other three human histamine receptor subtypes (H₁, H₂, and H₄) whereas all three of the imidazole H₃R antagonists tested have considerable binding potencies at the human H₄R. Both clobenpropit and thioperamide possess mid-nanomolar affinity for this receptor, and clobenpropit demonstrated partial agonist activity (Liu et al., 2001). In addition to their demonstrated low affinity for other histamine receptors, A-304121 and A-317920 have little or no affinity for over 80 rodent and human GPCRs and ligand-activated ion channels (data not shown) including those for the biogenic amine serotonin 5-HT₃ and _2a-adrenergic receptors. The one receptor for which either compound exhibited significant binding affinity was the _2c-adrenergic receptor where A-304121 and A-317920 were approximately 4- and 25-fold selective for the human H_3LR, respectively. In contrast, the imidazole H₃R antagonists exhibit little or no selectivity against these receptors. Ciproxifan has equivalent binding affinities for the human histamine H_3LR and human _2a- and _2c-adrenergic receptors and only 5-fold lower affinity for the rat 5-HT₃ receptor. Similarly, thioperamide was only 2- to 5-fold selective for the human H_3LR versus the human _2a- and _2c-adrenergic receptors and 30-fold selective against the rat 5-HT₃ receptor. Because of its higher potency at the human histamine H_3LR, clobenpropit is the most selective of the imidazole H₃R antagonists, approximately 40-fold more potent at the human histamine H_3LR than at the human _2a- and _2c-adrenergic receptors and only 20-fold more potent than at the rat 5-HT₃ receptor. Other imidazole H₃R antagonists such as GT-2331, GT-2016, and iodophenpropit also exhibit this same lack of selectivity for these receptors (Esbenshade et al., 2001). Radiolabeled imidazole H₃R antagonists have also been shown to interact with cytochrome P450 proteins (Alves-Rodrigues et al., 1996; Harper et al., 1999), an effect that can be minimized with the addition of metyrapone, a cytochrome P450 inhibitor. We have synthesized [³H]A-317920, an H₃R-radiolabeled antagonist that exhibits specific binding that is completely displaceable by H₃R agonists and antagonists. In addition, it exhibits low nonspecific binding that is not altered by the addition of metyrapone (data not shown), unlike radiolabeled thioperamide and clobenpropit (Alves-Rodrigues et al., 1996; Harper et al., 1999), suggesting that this series of non-imidazole H₃R antagonists does not interact with cytochrome P450 proteins to an appreciable extent.

A-304121 and A-317920 display well behaved competitive antagonist properties in a variety of tissue and cell-based functional assays. These two novel compounds displayed all of the attributes associated with H₃R antagonists including the blockade of recombinant rat and human H₃R-mediated signaling pathways as well as antagonism of H₃R-mediated neurotransmitter release in two different classical H₃R assay paradigms, H₃R agonist-mediated inhibition of EFS guinea pig ileum contraction and release of [³H]histamine from rat brain synaptosomes. In assays comparing the effect of H₃R antagonists to competitively inhibit (R)--MeHA-induced reversal of forskolin-stimulated cAMP accumulation in C6 cells expressing the rat H_3LR, the compounds exhibited a fairly similar rank order of potency as seen with their binding affinities with A-317920, clobenpropit, and ciproxifan possessing equivalent subnanomolar potencies that were about an order of magnitude greater than those for A-304121 and thioperamide. A similar pharmacological profile for these compounds was seen in the rat brain synaptosome model for the H₃R modulation of [³H]histamine release. In the EFS guinea pig ileum model, A-304121 and A-317920 displayed antagonist potencies intermediate between those in the rat H₃R and human H₃R models, much like that seen in the binding assays. Interestingly, the high degree of selectivity of A-304121 and A-317920 for the H₃R compared with the H₄R would suggest that indeed the EFS guinea pig ileum model is a very appropriate model for determining H₃R antagonist potencies in contrast to suggestions that this tissue may be mediating H₄R effects as well (Leurs et al., 2001). As predicted from their binding affinities, A-304121 and A-317920 are not as potent H₃R antagonists at the human H_3LR in both the adenylate cyclase and FLIPR assays. Again, clobenpropit is the most potent H₃R antagonist in these assay systems with A-317920, ciproxifan, and thioperamide exhibiting comparable affinities and A-304121 showing the lowest affinity. For all of these functional assays, neither A-304121 nor A-317920 displayed any partial or full agonist activity when used alone, unlike results obtained with some imidazole H₃R antagonists such as GT-2331 or proxyfan, which exhibit various degrees of agonism dependent upon the assay system (Esbenshade et al., 2001).

With the cloning of the H₃R and the discovery of the high degree of constitutive activity of the H₃R in both recombinant and native systems, many H₃R antagonists have been subsequently reclassified as inverse agonists because of their ability to reverse basal H₃R activity. As has been previously shown, ciproxifan, thioperamide, and clobenpropit are inverse agonists at the human H_3LR in reducing basal [³⁵S]GTPS binding activity and/or enhancing cAMP formation (Morisset et al., 2000; Wieland et al., 2001). Likewise, A-304121 and A-317920 are also inverse agonists at the human H_3LR and all the compounds tested display comparable potencies as in radioligand binding assays. Surprisingly, both A-304121 and A-317920 appear to be more efficacious as human H_3LR inverse agonists than the three imidazole H₃R antagonists tested with A-317920 reversing the basal level of [³⁵S]GTPS binding to a level over 2-fold greater level than that for the imidazole H₃R antagonists. This suggests that the aryloxyalkyl piperazine pharmacophore may confer a different conformational change on the human H_3LR that decreases the constitutive activity state of this receptor to a lower level than that achieved with the imidazole H₃R antagonists. Since H₃Rs may inhibit neurotransmitter release in the absence of endogenous histamine because of their inherent constitutive activity (Morisset et al., 2000), compounds that demonstrate greater inverse agonist efficacy (negative intrinsic activity) may in turn cause greater enhancement of neurotransmitter release and a potentially greater therapeutic effect. Thus, it is necessary to not only develop compounds that are highly potent and selective for the H₃R, but it is also important to understand the structural properties of such compounds that contribute to their efficacy as inverse agonists in order to develop potent and efficacious H₃R inverse agonists as therapeutic agents.

H₃R antagonists have been proposed as potential therapeutic agents for a variety of central nervous system disorders including attention deficit/hyperactivity disorder, Alzheimer's disease, and schizophrenia because of the regulatory role this receptor has been shown to play in controlling the release of neurotransmitters important in vigilance, attention, and learning in various animal models. However, the full potential of H₃R antagonists has not yet been realized since no potent, selective, and safe H₃R antagonist has been approved for treatment of such disorders even though many H₃R antagonists have been developed since the initial discovery of the H₃R in 1983 (Arrang et al., 1983). We believe that the optimization of non-imidazole H₃R-selective antagonists such as A-304121 and A-317920 with good inverse agonist efficacy will lead to the development of potent, selective, and efficacious human H₃R antagonists that will be important therapeutic agents in the treatment of a variety of neuropsychiatric disorders. Behavioral evidence supporting such a role is provided in the accompanying paper (Fox et al., 2003).

	Acknowledgements

 
We thank Huaquing Liu for the chemical synthesis of A-304121 and A-317920.

	Footnotes

 
Funded by Abbott Laboratories, Inc. Portions of this work were previously presented at the 31st Annual European Histamine Research Society Meeting, May 22–26, 2002 and at the 32nd Annual Society for Neuroscience Meeting, November 2–7, 2002.

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

DOI: 10.1124/jpet.102.047183.

ABBREVIATIONS: GPCR, G-protein-coupled receptor; H₃R, H₃ receptor; GT-2331, (1R,2R)-4-(2-(5,5-dimethylhex-1-ynyl)cyclopropyl)imidazole; A-304121, 4-(3-((2R)-2-aminopropanoyl-1-piperazinyl)propoxy)phenyl)cyclopropylmethanone; A-317920, N-((1R)-2-(4-(3-(4-(cyclopropylcarbonyl)phenoxy)propyl)-1-piperazinyl)-1-methyl-2-oxo-ethyl-)-2-furamide; [³⁵S]GTPS, guanosine 5'-O-(3-[³⁵S]thio)triphosphate; LY-278584, 1-methyl-N-(8-methyl-8-azabicyclo[3.2.1]-oct-3-yl)-1H-indazole-3-carboxamide; PCR, polymerase chain reaction; H_3LR, full-length H₃ receptor; (R)--MeHA, (R)--methylhistamine; FLIPR, fluorometric imaging plate reader; NAMH, N--methylhistamine; HEK, human embryonic kidney; EFS, electric field stimulated; DPBS, Dulbecco's phosphate-buffered saline; 5-HT₃, 5-hydroxytryptamine₃; GT-2016, 5-cyclohexyl-1-(4-imidazol-ylpiperidyl)pentan-1-one.

Address correspondence to: Dr. Timothy A. Esbenshade, Neuroscience Research, Abbott Laboratories, R4MN, AP9A, 100 Abbott Park Road, Abbott Park, IL 60064. E-mail: Tim.Esbenshade{at}Abbott.com

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