Walter Schunack

High constitutive activity of native H3 receptors regulates histamine neurons in brain.

Some G-protein-coupled receptors display ‘constitutive activity’, that is, spontaneous activity in the absence of agonist. This means that a proportion of the receptor population spontaneously undergoes an allosteric transition, leading to a conformation that can bind G proteins. The process has been shown to occur with recombinant receptors expressed at high density, and/or mutated, but also non-mutated recombinant receptors expressed at physiological concentrations. Transgenic mice that express a constitutively active mutant of the β 2-adrenergic receptor display cardiac anomalies; and spontaneous receptor mutations leading to constitutive activity are at the origin of some human diseases. Nevertheless, this process has not previously been found to occur in animals expressing normal levels of receptor. Here we show that two isoforms of the recombinant rat H3 receptor display high constitutive activity. Using drugs that abrogate this activity (‘inverse agonists’) and a drug that opposes both agonists and inverse agonists (‘neutral antagonist’), we show that constitutive activity of native H3 receptors is present in rodent brain and that it controls histaminergic neuron activity in vivo . Inverse agonists may therefore find therapeutic applications, even in the case of diseases involving non-mutated receptors expressed at normal levels.

BF2.649 [1-{3-[3-(4-Chlorophenyl)propoxy]propyl}piperidine, hydrochloride], a nonimidazole inverse agonist/antagonist at the human histamine H3 receptor: Preclinical pharmacology.

Histamine H3 receptor inverse agonists are known to enhance the activity of histaminergic neurons in brain and thereby promote vigilance and cognition. 1-{3-[3-(4-Chlorophenyl)propoxy]propyl}piperidine, hydrochloride (BF2.649) is a novel, potent, and selective nonimidazole inverse agonist at the recombinant human H3 receptor. On the stimulation of guanosine 5′-O-(3-[35S]thio)triphosphate binding to this receptor, BF2.649 behaved as a competitive antagonist with a Ki value of 0.16 nM and as an inverse agonist with an EC50 value of 1.5 nM and an intrinsic activity ∼50% higher than that of ciproxifan. Its in vitro potency was ∼6 times lower at the rodent receptor. In mice, the oral bioavailability coefficient, i.e., the ratio of plasma areas under the curve after oral and i.v. administrations, respectively, was 84%. BF2.649 dose dependently enhanced tele-methylhistamine levels in mouse brain, an index of histaminergic neuron activity, with an ED50 value of 1.6 mg/kg p.o., a response that persisted after repeated administrations for 17 days. In rats, the drug enhanced dopamine and acetylcholine levels in microdialysates of the prefrontal cortex. In cats, it markedly enhanced wakefulness at the expense of sleep states and also enhanced fast cortical rhythms of the electroencephalogram, known to be associated with improved vigilance. On the two-trial object recognition test in mice, a promnesiant effect was shown regarding either scopolamine-induced or natural forgetting. These preclinical data suggest that BF2.649 is a valuable drug candidate to be developed in wakefulness or memory deficits and other cognitive disorders.

Distinct pharmacology of rat and human histamine H3 receptors: role of two amino acids in the third transmembrane domain

Starting from the sequence of the human histamine H3 receptor (hH3R) cDNA, we have cloned the corresponding rat cDNA. Whereas the two deduced proteins show 93.5% overall homology and differ only by five amino acid residues at the level of the transmembrane domains (TMs), some ligands displayed distinct affinities. Thioperamide and ciproxifan were about 10 fold more potent at the rat than at the human receptor, whereas FUB 349 displayed a reverse preference. Histamine, (R)α‐methylhistamine, proxyfan or clobenpropit were nearly equipotent at H3 receptors of both species. The inverse discrimination patterns of ciproxifan and FUB 349 were partially changed by mutation of one amino acid (V122A), and fully abolished by mutation of two amino acids (A119T and V122A), in TM3 of the rH3R located in the vicinity of Asp114 purported to salt‐link the ammonium group of histamine. Therefore, these two residues appear to be responsible for the distinct pharmacology of the H3R in the two species.

BF2.649, A NON-IMIDAZOLE INVERSE AGONIST/ANTAGONIST AT THE HUMAN HISTAMINE H3 RECEPTOR: PRECLINICAL PHARMACOLOGY

Histamine H3 receptor inverse agonists are known to enhance the activity of histaminergic neurons in brain and thereby promote vigilance and cognition. 1-{3-[3-(4-Chlorophenyl)propoxy]propyl}piperidine, hydrochloride (BF2.649) is a novel, potent, and selective nonimidazole inverse agonist at the recombinant human H3 receptor. On the stimulation of guanosine 5′-O-(3-[35S]thio)triphosphate binding to this receptor, BF2.649 behaved as a competitive antagonist with a Ki value of 0.16 nM and as an inverse agonist with an EC50 value of 1.5 nM and an intrinsic activity ∼50% higher than that of ciproxifan. Its in vitro potency was ∼6 times lower at the rodent receptor. In mice, the oral bioavailability coefficient, i.e., the ratio of plasma areas under the curve after oral and i.v. administrations, respectively, was 84%. BF2.649 dose dependently enhanced tele-methylhistamine levels in mouse brain, an index of histaminergic neuron activity, with an ED50 value of 1.6 mg/kg p.o., a response that persisted after repeated administrations for 17 days. In rats, the drug enhanced dopamine and acetylcholine levels in microdialysates of the prefrontal cortex. In cats, it markedly enhanced wakefulness at the expense of sleep states and also enhanced fast cortical rhythms of the electroencephalogram, known to be associated with improved vigilance. On the two-trial object recognition test in mice, a promnesiant effect was shown regarding either scopolamine-induced or natural forgetting. These preclinical data suggest that BF2.649 is a valuable drug candidate to be developed in wakefulness or memory deficits and other cognitive disorders.

Protean agonism at histamine H3 receptors in vitro and in vivo

G protein-coupled receptors (GPCRs) are allosteric proteins that adopt inactive (R) and active (R*) conformations in equilibrium. R* is promoted by agonists or occurs spontaneously, leading to constitutive activity of the receptor. Conversely, inverse agonists promote R and decrease constitutive activity. The existence of another pharmacological entity, referred to as “protean” agonists (after Proteus, the Greek god who could change shape), was assumed on theoretical grounds. It was predicted from the existence of constitutive activity that a same ligand of this class could act either as an agonist or an inverse agonist at the same GPCR. Here, we show that proxyfan, a high-affinity histamine H3-receptor ligand, acts as a protean agonist at recombinant H3 receptors expressed in the same Chinese hamster ovary cells. In support of the physiological relevance of the process, we show that proxyfan also behaves as a protean agonist at native H3 receptors known to display constitutive activity. On neurochemical and behavioral responses in rodents and cats, proxyfan displays a spectrum of activity ranging from full agonism to full inverse agonism. Thus, protean agonism demonstrates the existence of ligand-directed active states LR* different from, and competing with, constitutively active states R* of GPCRs, and defines a pharmacological entity with important therapeutic implications.

Synthesis of Potent Non‐imidazole Histamine H3‐Receptor Antagonists

Histamine has been converted into a non‐imidazole H3‐receptor histamine antagonist by addition of a 4‐phenylbutyl group at the Nα‐position followed by removal of the imidazole ring. The resulting compound, N‐ethyl‐N‐(4‐phenylbutyl)amine, remarkably has a Ki = 1.3 μM as an H3 antagonist. Using this as a lead compound, a novel series of homologous O and S isosteric tertiary amines was synthesised and structure‐activity studies furnished N‐(5‐phenoxypentyl)pyrrolidine (Ki = 0.18 ± 0.10 μM, for [3H]histamine release from rat cerebral cortex synaptosomes) which, more importantly, was active in vivo. Substitution of NO2 into the para position of the phenoxy group gave N‐(5‐p‐nitrophenoxypentyl)pyrrolidine, UCL 1972 (Ki = 39 ± 11 nM), ED50 = 1.1 ± 0.6 mg/kg per os in mice on brain tele‐methylhistamine levels.

Influence of imidazole replacement in different structural classes of histamine H3-receptor antagonists

The reference compounds for histamine H(3)-receptor antagonists carry as a common feature an imidazole moiety substituted in the 4-position. Very recently novel ligands lacking an imidazole ring have been described possessing a N-containing non-aromatic heterocycle instead. In this study we investigated whether imidazole replacement, favourably by a piperidine moiety, is generally applicable to different structural classes of reference compounds, e.g., thioperamide, carboperamide, clobenpropit, FUB 181, ciproxifan, etc. While replacement led to a loss of affinity for many of the compounds, it was successfully applied to some ether derivatives. The piperidine analogues of FUB 181 and ciproxifan, 3-(4-chlorophenyl)propyl 3-piperidinopropyl ether hydrogen oxalate (6) and cyclopropyl 4-(3-piperidinopropyloxy)phenyl methanone hydrogen maleate (7), almost maintained in vitro affinities, pK(i) values of 7.8 and 8.4, respectively, and showed high potency in vivo after p.o. administration (ED(50) values of 1.6 and 0.18 mg/kg, respectively).

Histamine H3 receptor binding sites in rat brain membranes: modulations by guanine nucleotides and divalent cations

The binding of [3H](R)alpha-methylhistamine, a potent and specific agonist at histamine H3 receptors, was investigated with membranes of rat cerebral cortex. In phosphate buffer the specific binding defined with thioperamide, an H3 receptor antagonist, displayed characters of reversibility and saturability with a Bmax of approximately 30 fmol/mg protein. The KD, derived from either dissociation/association rates or saturation kinetics at equilibrium, was approximately 0.5 nM at 25 degrees C. Competition studies indicated that the binding occurred with a stereoselectivity and pharmacological specificity similar to that of functional H3 autoreceptors regulating histamine release in brain slices. However, whereas the potency of antagonists was closely similar in the two assay systems, that of agonists was approximately 10-fold higher in the binding assay. Among antagonists burimamide was the only one to compete with a pseudo-Hill coefficient significantly different from unity (nH = 0.48 +/- 0.03), indicating a possible heterogeneity among binding sites. The presence of 2.6 mM Ca2+, in a modified Krebs-Ringer medium, promoted the conversion of a larger fraction of sites into a low-affinity component with a KD of 16 nM. The presence of guanylnucleotides in the Krebs-Ringer medium with Ca2+ abolished the binding to this low-affinity component whereas in a phosphate buffer only the KD was slightly increased. It is concluded that the H3 receptor, like many other amine receptors, is coupled to its still unidentified effector system via a G-protein and regulated by Ca2+. However, unlike the latter, the H3 receptor is down-regulated by the divalent cation.

Improvement by FUB 181, a novel histamine H3-receptor antagonist, of learning and memory in the elevated plus-maze test in mice

Effects of FUB 181 [3-(4-chlorophenyl)propyl-3-(1H-imidazol-4-yl)propyl ether], a novel histamine H3-receptor antagonist, on a scopolamine-induced learning deficit in the elevated plus-maze test were studied in mice. FUB 181 alone (2.5 and 5 mg/kg, i.p.) ameliorated the scopolamine-induced learning deficit in mice. This effect was antagonized by BP 2.94 (10 mg/kg, i.p.), a prodrug of (R)-—methylhistamine (histamine H3-receptor agonist), and by ketotifen (4 mg/kg, i.p.), a histamine H1-receptor antagonist, both penetrating the blood-brain barrier. However, the ameliorating effect of FUB 181 (2.5 mg/kg) was not antagonized by either terfenadine (10 mg/kg, i.p.), a histamine H1-receptor antagonist with poor penetration of the blood-brain barrier, or zolantidine (20 mg/kg, i.p.), a centrally effective histamine H2-receptor antagonist. In a biochemical study, FUB 181 had no significant effect on either acetylcholine or choline level in mice brain at the doses tested. These findings suggest that FUB 181 increases the release of histamine by blocking presynaptic histamine H3 autoreceptors, and that released histamine in turn activates postsynaptic H1 and H2 receptors, predominantly histamine H1 receptors, and in this fashion improves learning and memory in mice. Our findings also suggest that the histaminergic system may play an important role in learning and memory, and that FUB 181 may be a clinical candidate for the therapy of dementia.

Histamine H3 receptor-mediated inhibition of noradrenaline release in pig retina discs

SummaryDiscs of pig retina were preincubated with 3H-noradrenaline, 3H-dopamine or 3H-serotonin and then superfused. Electrical field stimulation increased the outflow of tritium from discs preincubated with 3H-noradrenaline or 3H-dopamine, but no from discs preincubated with 3H-serotonin. The tritium content at the end of superfusion was similar in discs preincubated with 3H-noradrenaline or 3H-dopamine but about tenfold lower in discs preincubated with 3H-serotonin. The tritium content in discs preincubated with 3H-noradrenaline was markedly reduced when desipramine was present during preincubation but was not affected by selective inhibitors of dopamine and serotonin uptake. The tritium content in discs preincubated with 3Hdopamine and 3H-serotonin, in contrast, was reduced or tended to be reduced by a selective dopamine and serotonin uptake inhibitor, respectively.The electrically evoked overflow of tritium from discs preincubated with 3H-noradrenaline was abolished by tetrodotoxin or omission of Ca2+. In discs superfused with desipramine, the electrically evoked overflow was enhanced by phentolamine but not affected by histamine. When both desipramine and phentolamine were present in the superfusion medium, histamine inhibited the evoked overflow (pIC15 6.85). This effect was mimicked by the histamine H3 receptor agonist R-(−)-α-methylhistamine as well as by its S-(+)-enantiomer (pIC15 7.85 and 5.30, respectively) but not by the H1 receptor agonist 2-(2-thiazolyl)ethylamine and the H2 receptor agonist dimaprit (each 10 μmol/l). The inhibitory effect of histamine was abolished by the H3 receptor antagonist thioperamide 0.32 μmol/l and attenuated by impromidine 3.2 μmol/l but not affected by the H1 receptor antagonist dimetindene 3.2 μmol/l and the H2 receptor antagonist ranitidine 10 μmol/l.The results suggest that, in the pig retina, noradrenaline is taken up into, and released from, noradrenergic neurones (most likely vascular postganglionic sympathetic nerve fibres, less probably tissue-specific noradrenergic neurones of the retina) and that noradrenaline release is subject to modulation via H3 receptors and probably also a-adrenoceptors.