8R-lisuride is a potent stereospecific histamine H1-receptor partial agonist.

The human histamine H1 receptor (H1R) is an important, well characterized target for the development of antagonists to treat allergic conditions. Many neuropsychiatric drugs are known to potently antagonize the H1R, thereby producing some of their side effects. In contrast, the tolerability and potential therapeutic utility of H1R agonism is currently unclear. We have used a cell-based functional assay to evaluate known therapeutics and reference drugs for H1R agonist activity. Our initial functional screen identified three ergot-based compounds possessing heretofore-unknown H1R agonist activity. 8R-lisuride demonstrated potent agonist activity in various assays including receptor selection and amplification technology, inositol phosphate accumulation, and activation of nuclear factor-kappaB with pEC50 values of 8.1, 7.9, and 7.9, respectively, and with varying degrees of efficacy. Based on these assays, 8R-lisuride is the most potent stereospecific partial agonist for the human H1R yet reported. Investigation of the residues involved in histamine and lisuride binding, using H1R mutants and molecular modeling, have revealed that although these ligands are structurally different, the lisuride-binding pocket in the H1R closely corresponds to the histamine-binding pocket. The discovery of a potent stereospecific partial H1R agonist provides a valuable tool to further characterize this important therapeutic target in vitro.

Histamine H1 receptor ligands: part II. Synthesis and in vitro pharmacology of 2-[2-(phenylamino)thiazol-4-yl]ethanamine and 2-(2-benzhydrylthiazol-4-yl)ethanamine derivatives.

New 2-[2-(phenylamino)thiazol-4-yl]ethanamine and 2-(2-benzhydrylthiazol-4-yl)ethanamine derivatives were prepared and tested in vitro as H1 receptor antagonists. The compounds with 2-phenylamino substitution with meta-halide substituents at the phenyl ring, showed weak H1-antagonistic activity (pA2: 4.62-5.04) and this activity was completely lost in the case of meta-methyl substituent (pA2 < 4). When the phenylamino group was replaced by benzhydryl groups of classic antihistamines, the resulting compounds exhibited slightly improved H1-antagonistic activity (at the meta-position pA2: 6.38-6.15; at the para-position pA2: 6.04-5.87).

Non-imidazole histamine H3 ligands, Part 2: New 2-substituted benzothiazoles as histamine H3 antagonists.

New, non-imidazole histamine H3 receptor antagonists were prepared and in vitro tested as H3 receptor antagonists measured as the electrically evoked contraction of the guinea-pig jejunum. The 2-(1-piperidinyl)- and 2-(1-pyrrolidinyl)benzothiazoles show no or very poor activity; 2-[1-(4-amino)piperidinyl]- and 2-(1,2-ethanediamino)- and 2-(1,3-propanediamino)derivatives of benzothiazole possess weak activity at H3 receptors, whereas 2-(4-piperidinyl)benzothiazoles and 2-[1-(4-piperazinyl)]benzothiazoles show moderate to good activity. Lipophilic and not-too-bulky substituents like n-propyl attached to the nitrogen at the piperazine or piperidine ring lead to potent H3 receptor antagonists with pA2 values ranging from 7.0 to 7.2. The structure-activity relationships for different substitution patterns are discussed.

Non-imidazole histamine H3 ligands. Part I. Synthesis of 2-(1-piperazinyl)- and 2-(hexahydro-1H-1,4-diazepin-1-yl)benzothiazole derivatives as H3-antagonists with H1 blocking activities.

New 2-(1-Piperazinyl)- and 2-(hexahydro-1H-1,4-diazepin-1-yl)benzothiazoles were prepared and tested as H1- and H3-receptor antagonists. A number of compounds showed weak H1-antagonistic activity, with pA2 values ranging from 5.5 to 6.1. The simple alkyl substituted, 2-[1-(4-methyl and 4-ethyl)piperazinyl] analogues show increasing, moderate H3-antagonistic activity (pA2 = 6.0, and pA2 = 7.0). The compounds with 4-phenylalkyl substitution, for both the piperazinyl and the hexahydro-1H-1,4-diazepin-1-yl homologues series, regardless of the different physicochemical properties of the para substituents at the phenyl ring, showed weak H3-antagonistic activity with pA2 values ranging from 4.4 to 5.6.

Histamine H1 receptor ligands. Part I. Novel thiazol-4-ylethanamine derivatives: synthesis and in vitro pharmacology.

A series of 2-substituted thiazol-4-ylethanamines have been synthesized and tested for their histaminergic H1-receptor activities. The compounds with 2-phenyl substitution, regardless of the different physicochemical properties of the meta-substituents at the phenyl ring, showed weak H1-agonistic activity with pD2 values ranging from 4.35 to 5.36. When the phenyl group was replaced by a benzyl group, the resulting compounds all exhibited weak H1-antagonistic activity (pA2: 4.14-4.82).

Characterization of the binding site of the histamine H3 receptor. 1. Various approaches to the synthesis of 2-(1H-imidazol-4-yl)cyclopropylamine and histaminergic activity of (1R,2R)- and (1S,2S)-2-(1H-imidazol-4-yl)-cyclopropylamine.

Various approaches to the synthesis of all four stereoisomers of 2-(1H-imidazol-4-yl)cyclopropylamine (cyclopropylhistamine) are described. The rapid and convenient synthesis and resolution of trans-cyclopropylhistamine is reported. The absolute configuration of its enantiomers was determined by single-crystal X-ray crystallographic analysis. The distinct trans-cyclopropylhistamine enantiomers were tested for their activity and affinity on the histamine H3 receptor. (1S,2S)-Cyclopropylhistamine (VUF 5297) acts as an agonist both on the rat cortex (pD2 = 7.1; alpha = 0.75) and on guinea pig jejunum (pD2 = 6.6; alpha = 0.75). Its enantiomer, (1R, 2R)-cyclopropylhistamine (VUF 5296), is about 1 order of magnitude less active. Both enantiomers show weak activity on H1 and H2 receptors. All synthetic attempts to cis-cyclopropylhistamine were unsuccessful. Nevertheless, the results of this study provide an ideal template for molecular modeling studies of histamine H3 receptor ligands.

Pharmacological characterisation of the histamine H3 receptor in the rat hippocampus.

The purpose of this report was to pharmacologically characterise the histamine H3 in the rat hippocampus using radioligand binding studies with the H3 receptor antagonist [125I]iodophenpropit and the H3 receptor mediated inhibition of [3H]noradrenaline release. A dissociation constant of 0.33 nM and a maximal number of binding sites of 125 fmol/mg protein were found for [125I]iodophenpropit. Competition studies showed stereoselectivity for the (R) and (S) enantiomers of alpha-methylhistamine and 10 microM of GTPgammaS shifted the curve of (R)-alpha-methylhistamine rightwards. Up to 1 microM, (R)-alpha-methylhistamine displaced only 30% whereas the tested H3-antagonists displaced 50-60% of the total [125I]iodophenpropit bound. This indicates the presence of an additional non-H3 receptor binding site(s) for [125I]iodophenpropit in the rat hippocampus. This secondary site shows low affinity for H3 agonists, but high affinity for the tested H3 antagonists. Electrically evoked [3H]acetylcholine release was shown in slices of rat hippocampus. No H3 receptor modulation of [3H]acetylcholine release from hippocampal slices was detectable. However, H3 receptor activation inhibited 42% of the electrically-evoked [3H]noradrenaline release in rat hippocampal slices. The inhibition of [3H]noradrenaline release was effectively antagonized by the H3 antagonists thioperamide and burimamide. We describe the pharmacological identification of the histamine H3 receptor in the rat hippocampus and its similarities and differences from the cortical H3 receptor. These studies enable us to investigate changes in density and functionality of the hippocampal H3 receptor under (patho)physiological conditions.

Evaluation of the receptor selectivity of the H3 receptor antagonists, iodophenpropit and thioperamide: an interaction with the 5-HT3 receptor revealed.

1. In the present study we evaluated the receptor selectivity of the potent histamine H3 receptor antagonist, iodophenpropit (IPP) in comparison with the prototype antagonist, thioperamide. 2. IPP proved to be a potent competitive H3 receptor antagonist as measured against (R)-alpha-methylhistamine-induced inhibition of electrically-evoked contractions of the guinea-pig jejunum (pA2 = 9.12 +/- 0.06, Schild slope: 1.0 +/- 0.1, n = 8). In the same assay, thioperamide was slightly less potent (pA2 = 8.9 +/- 0.2). 3. In radioligand binding studies, IPP showed a high affinity for the H3 receptor. Displacement of [125I]-IPP binding to rat cortex membranes by unlabelled IPP resulted in a Ki value of 0.97 +/- 0.06 nM (n = 3). In contrast, IPP showed only a weak affinity for the histamine H1- and H2 receptor. Displacement of [3H]-mepyramine and [125I]-iodoaminopotentidine binding to respectively guinea-pig H1- and human H2 receptors by IPP resulted in Ki values of 1.71 +/- 0.32 microM (n = 3) and 2.28 +/- 0.81 microM (n = 3). For thioperamide the affinities for the H1-, H2- and H3 receptor were respectively > 10 microM, > 10 microM and 4.3 +/- 1.6 nM (n = 7). 4. Testing IPP and thioperamide in 39 different receptor binding assays revealed that IPP showed relatively high affinity for the 5-hydroxytryptamine 5-HT3 receptor (Ki = 11 +/- 1 nM, n = 3), the alpha 2-adrenoceptor (Ki = 120 +/- 5 nM, n = 3) and the sigma receptor (Ki = 170 +/- 70 nM, n = 3). Thioperamide showed relatively high affinity for the 5-HT3 receptor (Ki = 120 +/- 30 nM, n = 3) and the sigma receptor (Ki = 180 +/- 90 nM, n = 3). 5. Due to the low density of histamine H3 receptors in the brain, the interaction of IPP with the 5-HT3-, the alpha 2- and the sigma receptor might interfere with [125I]-IPP binding to rat cortex membranes. Yet, in this preparation [125I]-IPP binding was not influenced by ondansetron, yohimbine or haloperidol. The interaction with the 5-HT3 receptor was not restricted to IPP or thioperamide, but was alsofound with other H3 receptor antagonists. The potent H3 receptor agonist imetit, a compound belongingto the same chemical class of IPP, also interacted with the 5-HT3 receptor (Ki = 240 +/- 40 nM). In contrast,histamine or the H3 receptor agonist, (R)-a-methylhistamine showed no affinity for the 5-HT3 receptor.7 In the guinea-pig isolated ileum, imetit evoked concentration-dependent contractions, resulting in apD2 value of 4.72 +/- 0.03 (n = 9). The contractions were antagonized by ondansetron, yielding a pA2 valueof 7.1 +/- 0.1 (n = 9). Similarly ondansetron antagonized the contractions evoked by the 5-HT3 receptoragonist, 2-methyl-5-HT with a pA2 value of 7.3 +/- 0.1 (n = 4). IPP and thioperamide did not mimic 2-methyl-5-HT but non-competitively inhibited the 2-methyl-5-HT-induced contractions of thispreparation.8 In an in vivo model for 5-HT3 activity, the Von Bezold Jarisch reflex, thioperamide showedantagonism in low dosages, which correlated well with the affinity for the 5-HT3 receptor site. Yet, athigher dosages no further 5-HT3 receptor antagonism was observed. For IPP no 5-HT3 receptor activitycould be observed in vivo.9 In the present study we showed that many H3 receptor compounds, that are regarded as highlyselective (including the prototype drug, thioperamide), also interact with the 5-HT3 receptor, albeit athigher drug concentrations.Keywords.: Histamine H3-receptor; iodophenpropit; thioperamide; receptor selectivity; 5-hydroxytryptamine 5-HT3 receptor;guinea-pig intestine; rat brain; Von Bezold Jarisch reflex

Screening of cattle urine samples for the presence of beta-agonists with a functional test: some preliminary results.

A new procedure based on the measurement of biological effects has been developed for the determination of residues of beta-agonists, such as clenbuterol, in urine. A multi-chamber superfusion apparatus containing isolated trachea strips from guinea pigs was used to detect smooth muscle relaxation induced via beta 2-adrenoceptor activation. The trachea tissue was pre-contracted with metacholine. Urine samples were extracted using a solid-phase column containing reversed-phase and anion-exchange materials. Extracts were introduced into the superfusion apparatus via flow injection. The intensity and response of relaxation are dependent on the type and concentration of the beta-agonist introduced. The sensitivity of the assay for clenbuterol in calf urine is about 1 microgram l-1. This methodology in the present form is especially suitable for survey screening analysis for several types of samples. An extensive validation of the procedure is performed to determine the range of analytes that can be detected, the possibilities of analysing urine samples obtained from mature cattle or other animal species and the influence of cross-reacting substances.

A simple and rapid in vitro test system for the screening of histamine H3 ligands.

A simple and rapid functional test system for the screening of histamine H3 ligands is described. It is based on the inhibitory effect of histamine H3 agonists on electrically-evoked contractile response of isolated guinea pig intestine. Whole jejunum segments are continuously stimulated maximally (15 V) by electrical pulses with a frequency of 0.1 Hz and a duration of 0.5 msec. The resulting twitches are recorded isotonically (1.0 g) and can be completely abolished by atropine (0.1 mcM).

In vitro effect of toluene diisocyanate on beta adrenergic and muscarinic receptor function in lung tissue of the rat.

To investigate the role of pharmacological mechanisms in toluene diisocyanate (TDI) induced occupational asthma, the effects of TDI on rat trachea ring and lung parenchymal strip were studied in vitro. The most prominent effect observed was a stimulation of metacholine (1 microM) induced contraction of the tracheal ring by 1 microM TDI (added in dimethy sulphoxide). The results were less pronounced when TDI was added from a stock solution prepared in water, which is possibly due to (co)polymerisation. It is concluded that the pharmacological effect of TDI may result from an autonomic imbalance between cholinergic and beta-adrenergic neural control.

Effect of inhibitors of enkephalin degradation in the isolated guinea-pig ileum.

Bestatin and high concentration of puromycin increase the depressing effect of [Met] enkephalin on the twitch response of the electrically stimulated guinea-pig ileum. Thiorphan (enkephalinase A inhibitor) is hardly effective, but phelorphan (mercapto-acetyl-Phe-Phe) a newly synthesized enzyme-inhibitor which effectively inhibits the enkephalinase A, enkephalinase B and soluble aminopeptidase activity, potentiates the effect of enkephalin dose-dependently and in low concentrations (0.01-1 microM). Enkephalinase A, though present in these tissues, is not functional under the conditions of the test, because it is inhibited by the physiological buffer itself. These results demonstrate that enkephalinase B and the membrane bound aminopeptidase, but not the soluble aminopeptidase or enkephalinase A hydrolyse enkephalins in the isolated guinea-pig ileum.