Preclinical evaluation of the abuse potential of Pitolisant, a histamine H3 receptor inverse agonist/antagonist compared with Modafinil.

BACKGROUND AND PURPOSE: Pitolisant, a histamine H3 receptor inverse agonist/antagonist is currently under Phase III clinical trials for treatment of excessive daytime sleepiness namely in narcoleptic patients. Its drug abuse potential was investigated using in vivo models in rodents and monkeys and compared with those of Modafinil, a psychostimulant currently used in the same indications. EXPERIMENTAL APPROACH: Effects of Pitolisant on dopamine release in the nucleus accumbens, on spontaneous and cocaine-induced locomotion, locomotor sensitization were monitored. It was also tested in three standard drug abuse tests i.e. conditioned place preference in rats, self-administration in monkeys and cocaine discrimination in mice as well as in a physical dependence model. KEY RESULTS: Pitolisant did not elicit any significant changes in dopaminergic indices in rat nucleus accumbens whereas Modafinil increased dopamine release. In rodents, Pitolisant was without any effect on locomotion and reduced the cocaine-induced hyperlocomotion. In addition, no locomotor sensitization and no conditioned hyperlocomotion were evidenced with this compound in rats whereas significant effects were elicited by Modafinil. Finally, Pitolisant was devoid of any significant effects in the three standard drug abuse tests (including self-administration in monkeys) and in the physical dependence model. CONCLUSIONS AND IMPLICATIONS: No potential drug abuse liability for Pitolisant was evidenced in various in vivo rodent and primate models, whereas the same does not seem so clear in the case of Modafinil.

Ontogeny, regional and cellular distribution of the novel metalloprotease neprilysin 2 in the rat: a comparison with neprilysin and endothelin-converting enzyme-1.

The localisation of the gene transcripts of a recently discovered peptidase, neprilysin 2 (NEP2), was established by in situ hybridisation in rat tissues during development and adulthood. It was compared with those of neprilysin (NEP), a closely related enzyme in terms of sequence homology or substrate specificity, and of endothelin-converting enzyme 1 (ECE-1) which, like the other two, belongs to the M-13 sub-family of zinc-dependent metallopeptidases. The ontogeny of the three enzymes differed markedly, the expression of NEP2 being restricted to developing and differentiating fields of the CNS, whereas NEP and ECE-1 genes were broadly expressed early on in the CNS and periphery. In contrast to the wide expression of NEP and ECE-1 in peripheral adult tissues and in CNS, NEP2 was almost exclusively expressed in selected neuronal populations of the brain and spinal cord. The only exceptions were the intermediate and anterior lobes of the pituitary as well as the choroid plexuses, where NEP2 was also strongly expressed. These localisations as well as those in the hypothalamic nuclei, together with the previously established pattern of cleaved peptides, suggest the involvement of NEP2 in the metabolism of neurohormones of the hypothalamo-pituitary axis.Complementary distributions of NEP and NEP2 mRNAs were observed in a large number of brain areas with, for instance the former being highly expressed in the striatum in which NEP2 transcripts were almost undetectable. In contrast, NEP2 was highly expressed in numerous thalamic, hypothalamic and brainstem nuclei from which NEP was absent. Since both peptidases are able to cleave the same neuropeptides, this pattern may suggest a complementary role in their peptide inactivation functions in the CNS. Finally, ECE-1 mRNAs were generally observed in neuronal populations known to express the pre-proendothelin-1 gene, confirming the function of the metallopeptidase in endothelin-1 generation.

Effects of histamine H3 receptor agonist and antagonist on histamine co-transmitter expression in rat brain.

The histaminergic H3-receptor (H3R) controls histamine synthesis and release in the tuberomamillary nucleus. We evaluated the effects of stimulating or blocking of H(3)R on glutamate-decarboxylase 67 kDa (GAD-67) and galanin mRNA expression, two histamine co-transmitters.After in situ hybridization histochemistry (ISHH), we observed a colocalization of 100% between histidine decarboxylase (HDC) and GAD-67 or H3R and of 80 to 97% with galanin. Adult rats received an H3R agonist ((R)alpha-Methylhistamine) or antagonist (ciproxifan) and were sacrificed 1 or 3 hours later. Treatment effects on HDC, galanin and GAD-67 mRNA were studied by quantitative ISHH on serial sections. Treatment with the H3R agonist known to decrease histamine neuron activity initially reduced HDC and galanin gene expression but an inverse change, presumably reflecting a compensatory mechanism, was observed after 3 h on both markers. In contrast, the H3R antagonist known to activate histamine neurons, had opposite effects on the two markers, suggesting that co-transmitters are submitted to independent control mechanisms. Furthermore, GAD-67 mRNA levels were not significantly modified by these treatments.

Efficacy of transpupillary thermotherapy (TTT) in the treatment of occult subfoveal choroidal neovascularization in age-related macular degeneration.

PURPOSE: To determine the efficacy of transpupillary thermotherapy (TTT) in the treatment of occult subfoveal choroidal neovascularization in patients with age-related macular degeneration (ARMD). METHODS: We conducted a retrospective review of patients with ARMD treated with TTT from June, 1999 through July, 2000 at a retina referral practice. TTT was delivered through a slit-lamp using a modified diode laser at 810 nm wavelength and a spot size of 3 mm delivered at one location for a minimum of 60 seconds duration. Re-treatment was performed at 2-month intervals if indicated. RESULTS: 81 eyes of 77 patients were included in the study. Vision improved greater than one line Snellen in 18 eyes (22%), vision was stable within one line Snellen in 38 (47%), and worsened greater than one line Snellen in 25 (31%). Patients had a mean follow-up of 9 months. The average number of treatments was 1.37 (range 1 to 4). Pretreatment vision was less than or equal to 20/200 in 54% of eyes. CONCLUSIONS: Transpupillary thermotherapy may stabilize visual acuity in a majority of patients with occult subfoveal choroidal neovascularization secondary to ARMD. Proof of therapeutic benefit is best determined by a randomized clinical trial that is currently underway (TTT4CNV).

Distinct pharmacology of rat and human histamine H(3) receptors: role of two amino acids in the third transmembrane domain.

Starting from the sequence of the human histamine H(3) receptor (hH(3)R) 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)alpha-methylhistamine, proxyfan or clobenpropit were nearly equipotent at H(3) 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 rH(3)R located in the vicinity of Asp(114) purported to salt-link the ammonium group of histamine. Therefore, these two residues appear to be responsible for the distinct pharmacology of the H(3)R in the two species.

Novel histamine H(3)-receptor antagonists with carbonyl-substituted 4-(3-(phenoxy)propyl)-1H-imidazole structures like ciproxifan and related compounds.

Novel histamine H(3)-receptor antagonists possessing a 4-(3-(phenoxy)propyl)-1H-imidazole structure generally substituted in the para-position of the phenyl ring have been synthesized according to Mitsunobu or S(N)Ar reactions. With in vitro and in vivo screening for H(3)-receptor antagonist potency, the carbonyl-substituted derivatives proved to be highly active compounds. A number of compounds showed in vitro affinities in the subnanomolar concentration range, and the 4-hexanoyl (10) and 4-acetyl-3-methyl (29) substituted derivatives showed in vivo antagonist potencies of about 0.1 mg/kg after po administration. Many proxifans were also tested for their affinities at other histamine receptor subtypes thereby demonstrating their pronounced H(3)-receptor subtype selectivity. Since the cyclopropyl ketone derivative 14 (ciproxifan) had high affinity in vitro as well as high potency in vivo, it was selected for further studies in monkeys. It showed good oral absorption and long-lasting, dose-dependent plasma levels making it a promising compound for drug development.

New histamine H(3)-receptor ligands of the proxifan series: imoproxifan and other selective antagonists with high oral in vivo potency.

Histamine H(3)-receptor antagonists of the proxifan series are described. The novel compounds possess a 4-(3-(phenoxy)propyl)-1H-imidazole structure and various functional groups, e.g., an oxime moiety, on the phenyl ring. Synthesis of the novel compounds and X-ray crystallography of one highly potent oxime derivative, named imoproxifan (4-(3-(1H-imidazol-4-yl)propyloxy)phenylethanone oxime), are described. Most of the title compounds possess high antagonist potency in histamine H(3)-receptor assays in vitro as well as in vivo in mouse CNS following po administration. Structure-activity relationships are discussed. Imoproxifan displays subnanomolar potency on a functional assay on synaptosomes of rat cerebral cortex (K(i) = 0.26 nM). In vivo, imoproxifan increases the central N(tau)-methylhistamine level with an ED(50) of 0.034 mg/kg po. A receptor profile on several functional in vitro assays was determined for imoproxifan, demonstrating high selectivity toward the histamine H(3) receptor for this promising candidate for further development.

Neprilysin II: A putative novel metalloprotease and its isoforms in CNS and testis.

Metalloproteases of the M13 subfamily, comprising namely neprylisin (NEP) and endothelin-converting enzyme (ECE), are involved in the metabolism of various neuronal and hormonal peptides, and inhibitors thereof have already led to therapeutically useful agents. Using homology cloning, we have identified a new member of this family in rat tissues. It is a glycosylated, type II integral membrane protein of 774 amino acids, containing a zinc-binding consensus motif, highly homologous to NEP and, therefore, designated NEPII. We have characterized multiple splice variants of NEPII mRNA with distinct expression patterns in brain regions, pituitary and testis. In situ hybridization of testis, where levels of the NEPII gene transcript are the highest, reveals a localization within round spermatids. In brain, NEPII is expressed heterogeneously among several neuronal populations and according to a pattern grossly complementary to that of NEP.

Changes in histamine H3 receptor responsiveness in mouse brain.

Changes in various histamine (HA) H3 receptor-mediated responses and H3 receptor binding in brain were investigated in mice receiving single or repeated administration of ciproxifan, a potent brain-penetrating and selective H3 receptor antagonist. Blockade of the H3 autoreceptor was nearly as effective in enhancing levels of tele-methylhistamine (t-MeHA), a major HA metabolite, in brain areas when ciproxifan was administered once either at 7 a.m. or 8 p.m., in spite of the large differences of basal levels at these two phases of the circadian cycle. Blockade after a single ciproxifan administration was, however, followed by a transient decrease in striatal t-MeHA levels, possibly reflecting rapid development of autoreceptor hypersensitivity. Following a 5-day administration of ciproxifan and a 2-day drug-free period, basal t-MeHA levels were significantly decreased (approximately -20%) in three brain areas, and the ED50 values of the drug to enhance t-MeHA levels were increased by 5-15 times without significant change in maximal response, indicating that H3 autoreceptor hypersensitivity had developed. However, in synaptosomes from the cerebral cortex of these animals, the H3 receptor-mediated inhibition of K+-induced [3H]HA release was not significantly modified. Subchronic administration of ciproxifan for 10 days also resulted in an increased binding of [125I]iodoproxyfan to the H3 receptor of striatal and hypothalamic membranes by 40-54%. Hypersensitivity at H3 somatodendritic autoreceptors and at heteroreceptors attributable to an increased number of HA binding sites could account for the various changes observed in this study.

Development of chiral N-alkylcarbamates as new leads for potent and selective H3-receptor antagonists: synthesis, capillary electrophoresis, and in vitro and oral in vivo activity.

Novel carbamates as derivatives of 3-(1H-imidazol-4-yl)propanol with an N-alkyl chain were prepared as histamine H3-receptor antagonists. Branching of the N-alkyl side chain with methyl groups led to chiral compounds which were synthesized stereospecifically by a Mitsunobu protocol adapted Gabriel synthesis. The optical purity of some of the chiral compounds was determined (ee > 95%) by capillary electrophoresis (CE). The investigated compounds showed pronounced to high antagonist activity (Ki values of 4.1-316 nM) in a functional test for histamine H3 receptors on rat cerebral cortex synaptosomes. Similar H3-receptor antagonist activities were observed in a peripheral model on guinea pig ileum. No stereoselective discrimination for the H3 receptor for the chiral antagonists was found with the in vitro assays. All compounds were also screened for central H3-receptor antagonist activity in vivo in mice after po administration. Most compounds were potent agents of the H3-receptor-mediated enhancement of brain Ntau-methylhistamine levels. The enantiomers of the N-2-heptylcarbamate showed a stereoselective differentiation in their pharmacological effect in vivo (ED50 of 0.39 mg/kg for the (S)-derivative vs 1.5 mg/kg for the (R)-derivative) most probably caused by differences in pharmacokinetic parameters. H1- and H2-receptor activities were determined for some of the novel carbamates, demonstrating that they have a highly selective action at the histamine H3 receptor.

Detailed mapping of the histamine H2 receptor and its gene transcripts in guinea-pig brain.

Autoradiographic studies of the distribution of the histamine H2 receptor and its messenger RNAs were performed on serial frontal and a few sagittal sections of guinea-pig brain using [(125)I]iodoaminopotentidine for radioligand binding and a 33P-labelled complementary RNA probe for in situ hybridization, respectively. Both probes were validated by assessing non-specific labelling using non-radioactive competing H2 receptor ligands and a sense probe for binding sites and gene transcripts, respectively. In some areas, e.g., cerebral cortex, hippocampal complex or cerebellum, such studies were completed by identification of neurons expressing the H2 receptor messenger RNAs on emulsion-dipped sections. Nissl-stained sections from comparable levels were used to localize brain structures. In many brain areas, the distribution of the H2 receptor and its messenger RNAs appeared to parallel that known for histaminergic axons. For instance. high levels of both H2 receptor markers were detected in striatal and limbic areas known to receive abundant histaminergic projections. In contrast, in septum, hypothalamic, pontine and several thalamic nuclei, a comparatively low density of both H2 receptor markers was detected, suggesting that histamine actions in these areas are mediated by H1 and/or H3 receptors. Generally, the distribution of H2 receptor messenger RNA correlates well with that of [(125)I]iodoaminopotentidine binding sites, although some differences were observed. In a few regions (e.g., substantia nigra, locus coeruleus) high or moderate densities of binding sites were accompanied by a much more restricted expression of H2 receptor transcripts. Conversely, the mammillary region and the pontine nucleus exhibited higher levels of hybridization than of binding sites. In hippocampus, cerebral and cerebellar cortex there was a selective localization of the H2 receptor messenger RNA in the granule cells of dentate gyrus, pyramidal cells of the Ammon's horn and cerebral cortex, and Purkinje cells of cerebellum, whereas [(125)I]iodoaminopotentidine binding sites were located in layers where the dendritic trees of these messenger RNA-expressing neurons extend. The same discrepancy between messenger RNAs and binding sites suggests that striatonigral endings are endowed with the H2 receptor. The histamine H1 and H2 receptors both appear to be present in several brain areas, in some cases in a way suggesting their potential co-expression by the same neuronal populations, e.g., in granule and pyramidal cells in the hippocampal formation. This co-expression accounts for synergic responses, e.g., on cAMP generation, previously observed upon co-stimulation of both receptor subtypes. The widespread distribution of the H2 receptor, namely in thalamic nuclei or in telencephalic areas such as most layers of the cerebral cortex, together with its excitatory role previously established in electrophysiological studies, support its alleged function in mediating the histamine-driven control of arousal mechanisms. In addition, the detection of H2 receptor expression in brainstem areas from which other monoaminergic pathways involved in the control of states of sleep and wakefulness emanate, e.g., several raphe nuclei, locus coeruleus or substantia innominata, suggests possible interrelationships between all of these systems with highly divergent projections to the thalamus and telencephalon. The present mapping of the H2 receptor and its gene transcripts should facilitate neurochemical, neurophysiological and behavioural studies aimed at clarifying the role of histaminergic systems in brain.

Activation of dopamine D4 receptor inhibits an L-type calcium current in cerebellar granule cells.

The functions of the D4 receptor, a newly cloned D2-like receptor, as well as the identity of cells expressing it, are still poorly defined. Using quantitative polymerase chain reaction we detected the messenger RNA of the D4, but not other D2-like receptor, in cultured granule cells from neonatal rat cerebellum. In these neurons, dopamine reduced high-voltage-activated calcium current, with a pharmacology corresponding to that of the D4 receptor. The response declined from one to three days, when calcium currents were mostly sensitive to nifedipine, to 15 days, when nifedipine-insensitive calcium currents were also present and D4 receptor messenger RNA had declined. The dopamine response was abolished after pretreatment of the cells by pertussis toxin, was potentiated and made irreversible by infusion of guanosine 5'-O-(3-thiotriphosphate) but persisted in the presence of cyclic AMP and isobutylmethylxanthine. These results indicate the presence in the neonatal cerebellum of a functional D4 receptor inhibiting an L-type calcium current, an action involving a Gi/Go protein but independent from adenylate cyclase inhibition.

Responses of anterior pituitary hormones and hypothalamic histamine to blockade of histamine synthesis and to selective activation or inactivation of presynaptic histamine H3 receptors in stressed rats.

The stress-induced release of anterior pituitary hormones and changes in hypothalamic content of histamine (HA) and its metabolite tele-methylHA (t-meHA) were studied in male rats during inhibition of HA synthesis or activation or blockade of HA H3 receptors. Pretreatment with the HA synthesis inhibitor alpha-fluoromethylhistidine (alpha-FMH; 200 micrograms intracerebroventricularly (icv) at -120 min) or the specific H3 receptor agonist R(alpha)methylhistamine (RmHA; 10 mg/kg intraperitoneally (ip) at -180 and -60 min) inhibited by 30-80% the responses of prolactin (PRL), corticotropin (ACTH) and beta-endorphin (beta-END) immunoreactivity to 1, 2.5 or 5 min of restraint stress (p < 0.05-0.01), but had no effect on basal secretion of the hormones. The inhibitory effect of the H3 receptor agonist RmHA (10 mg/kg x 2) on the hormone response to 5 min of restraint stress was prevented by simultaneous ip administration of the H3 receptor antagonist thioperamide. alpha-FMH reduced the hypothalamic content of HA 60% and that of t-meHA 30%, while RmHA had no effect on the HA content. Restraint stress for 5 min did not affect the HA and t-meHA contents, which may be due to the short duration of stress exposure. Pretreatment with the H3 receptor antagonist thioperamide (5 or 10 mg/kg ip at -120 min) had no effect on basal or restraint stress-induced release of PRL, ACTH or beta-END, although the compound increased the hypothalamic content of t-meHA 2-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of acetorphan, an enkephalinase inhibitor, on experimental and acute diarrhoea.

Acetorphan is an orally active inhibitor of enkephalinase (EC 3.4.24.11) with antidiarrhoeal activity in rodents apparently through protection of endogenous enkephalins and a purely antisecretory mechanism. Its antidiarrhoeal activity in man was assessed in an experimental model of cathartic induced secretory diarrhoea as well as in acute diarrhoea of presumed infectious origin. In six healthy volunteers receiving castor oil and pretreated with acetorphan or placebo in a crossover controlled trial, the drug significantly decreased the number and weight of stools passed during 24 hours. About 200 outpatients with severe acute diarrhoea (more than five stools per day) were included in a randomised double blind study of acetorphan against placebo. The significant antidiarrhoeal activity of acetorphan was established using a variety of criteria: (i) the duration of both diarrhoea and treatment were diminished; (ii) no acetorphan treated patient withdrew from the study whereas five dropped out because of worsening in the placebo group; (iii) the frequency of symptoms associated with diarrhoea--for example, abdominal pain or distension, nausea and anorexia--remaining after two weeks was nearly halved; (iv) using visual analogue scales acetorphan treatment was found more effective than placebo by both investigators and patients. There was statistically no significant difference between acetorphan and placebo in respect of side effects, particularly constipation, which often accompanies the antidiarrhoeal activity of mu opioid receptor agonists this difference is attributable to the lack of antipropulsive activity of acetorphan in man. The efficacy and tolerance of acetorphan suggest that enkephalinase inhibition may represent a novel therapeutic approach for the symptomatic management of acute secretory diarrhoea without impairing intestinal transit.

Regulation of histamine release in rat hypothalamus and hippocampus by presynaptic galanin receptors.

The effect of galanin, a peptide present in a subpopulation of histaminergic neurons emanating from the rat posterior hypothalamus, was investigated on K(+)-evoked [3H]histamine release in slices and synaptosomes from rat cerebral cortex, striatum, hippocampus and hypothalamus. Porcine galanin (0.3 microM) significantly inhibited histamine release induced by 25 mM K+ in slices from hypothalamus and hippocampus, but not from cerebral cortex and striatum, i.e., only in regions in which a colocalization of histamine and galanin has been described. The inhibitory effect of galanin was concentration dependent, with an EC50 value of 5.8 +/- 1.9 nM. The maximal inhibition was of 30-40% in hypothalamic and hippocampal slices depolarized with 25 mM K+. The galanin-induced inhibition observed in hypothalamic slices was not prevented in the presence of 0.6 microM tetrodotoxin and also occurred in hippocampal and hypothalamic synaptosomes, strongly suggesting the activation by galanin of presynaptic receptors located upon histaminergic nerve endings. The maximal inhibitory effect of galanin in slices or synaptosomes was lower than that previously reported for histamine acting at H3-autoreceptors, possibly suggesting that not all histaminergic axon terminals, even in the hypothalamus and hippocampus, are endowed with galanin receptors. It increased progressively in hypothalamic and hippocampal synaptosomes as the strength of the depolarizing stimulus was reduced. It is concluded that galanin modulates histamine release via presynaptic receptors, presumably autoreceptors located upon nerve terminals of a subpopulation of cerebral histaminergic neurons.

Dopamine activation of the arachidonic acid cascade as a basis for D1/D2 receptor synergism.

Understanding the actions of the neurotransmitter dopamine in the brain is important in view of its roles in neuropsychiatric illnesses. Dopamine D1 receptors, which stimulate both adenylyl cyclase and phospholipase C, and D2 receptors, which inhibit them, can nevertheless act synergistically to produce many electrophysiological and behavioral responses. Because this functional synergism can occur at the level of single neurons, another, as yet unidentified, signalling pathway activated by dopamine has been hypothesized. We report here that in Chinese hamster ovary (CHO) cells transfected with the D2 receptor complementary DNA, D2 agonists potently enhanced arachidonic acid release, provided that such release has been initiated by stimulating constitutive purinergic receptors or by increasing intracellular Ca2+. In CHO cells expressed D1 receptors, D1 agonists exert no such effect. When D1 and D2 receptors are coexpressed, however, activation of both subtypes results in a marked synergistic potentiation of arachidonic acid release. The numerous actions of arachidonic acid and its metabolites in neuronal signal transduction suggest that facilitation of its release may be implicated in dopaminergic responses, such as feedback inhibition mediated by D2 autoreceptors, and may constitute a molecular basis for D1/D2 receptor synergism.

Interaction of mianserin, amitriptyline and haloperidol with guinea pig cerebral histamine H2 receptors studied with [125I]iodoaminopotentidine.

Tricyclic antidepressants were previously shown to potently inhibit the histamine-induced activation of H2 receptors linked to adenylate cyclase in cerebral membranes, and this effect was postulated to represent the mechanism of their therapeutic activity. However, these drugs were found to be much less potent (up to more than hundred-fold) at H2 receptors mediating cyclic AMP responses in intact cells. We have presently assessed whether this large difference in potency of antidepressants, also observed with haloperidol, results from a modified pharmacological specificity of the H2 receptor according to the medium composition. We have studied the binding of [125I]iodoaminopotentidine (125I-APT) to striatal or hippocampal membranes under various experimental conditions. At equilibrium the Kd of 125I-APT, a highly selective ligand for H2 receptors, was six times higher in a supplemented Tris buffer used for adenylate cyclase assays in cell-free systems than in a Krebs-Ringer medium used in studies with intact cells. The medium composition also variously affected the Ki values of the four compounds studied amitriptyline, mianserin, haloperidol and tiotidine. Whereas the Ki value of amitriptyline was little affected, that of the other compounds was four to five times lower in the supplemented Tris buffer than in the Krebs-Ringer medium. With the exception of tiotidine, the Ki values of other compounds in the binding test performed in this medium, were intermediate between those derived from the antagonism of histamine-induced cyclic AMP responses in membranes and intact cells. These data indicate that the difference between the two test responses is diversely attributable to several factors according to the compounds, the main ones being medium composition and possibly cell disruption.

Naloxone-reversible antidiarrheal effects of enkephalinase inhibitors.

Thiorphan and acetorphan, two potent inhibitors of enkephalinase (EC 3.4.24.11 membrane-metalloendopeptidase) significantly reduced the castor oil-induced diarrhea in rats when administered intravenously (or orally, for acetorphan) but not when administered intracerebroventricularly. These effects were more marked during the 90 min period following the castor oil challenge but were still significant up to 4-8 h after the latter. Acetorphan was about 6 times more potent than thiorphan. The antidiarrheal activity of both compounds was completely prevented in rats receiving naloxone subcutaneously but not intracerebroventricularly (in the case of thiorphan). In contrast to loperamide, a peripherally acting opiate receptor agonist, the enkephalinase inhibitors did not significantly reduce gastrointestinal transit as measured in the charcoal meal test. The antidiarrheal activity of enkephalinase inhibitors therefore seems attributable to protection of endogenous opioids, presumably outside the brain, and to reduction of intestinal secretion rather than transit.

Autoinhibition of histamine synthesis mediated by presynaptic H3-receptors.

The regulation of histamine synthesis was studied on rat brain slices or synaptosomes labeled with L-[3H]histidine. Depolarization by increased extracellular K+ concentration enhanced by about twofold the [3H]histamine formation in slices of cerebral cortex. This stimulation was also observed, although to a lesser extent, in synaptosomes from cerebral cortex and slices from the posterior hypothalamus where most histaminergic cell-bodies are located, suggesting that it may occur in nerve endings as well as in perikarya. In the presence of exogenous histamine in increasing concentrations the K+-induced stimulation was progressively reduced by up to 60-70%. The effect of exogenous histamine appears to be receptor-mediated as shown by its saturable character, high pharmacological specificity and competitive reversal by histamine antagonists. The EC50 value of histamine for synthesis reduction (0.34 +/- 0.03 microM) was similar to its EC50 value for release inhibition known to be mediated by H3-receptors. In addition, whereas mepyramine and tiotidine, two potent antagonists at H1- and H2-receptors, respectively, were poorly effective, the H3-receptor antagonists burimamide and impromidine reversed the histamine effect in an apparently competitive manner. These effects were observed in slices of cerebral cortex or posterior hypothalamus as well as in cortical synaptosomes. Furthermore, even in the absence of added histamine, H3-receptor antagonists enhanced the depolarization-induced stimulation of [3H]histamine synthesis, indicating a participation of released endogenous histamine in the synthesis control process. The potencies of H3-receptor antagonists were similar to those of these agents at presynaptic autoreceptors controlling [3H]histamine release. It is concluded that H3-receptors control not only release but also synthesis of histamine at the level of nerve endings and also, presumably, of perikarya. A relationship between the two regulatory processes, possibly via intracellular calcium, seems likely but remains to be investigated at the molecular level.

Autoregulation of histamine release in brain by presynaptic H3-receptors.

Regulation of histamine release was studied mainly on brain slices prelabeled with L-[3H]-histidine and depolarized by increased extracellular K+ concentration or veratridine in a non-superfused system. The released 3H-labeled amines, isolated by ion-exchange chromatography from a large excess of 3H-labeled precursor consisted by more than 95% of unchanged [3H]histamine. Exogenous histamine reduced the release of neosynthesized [3H]histamine via stimulation of previously characterized H3-receptors whereas it did not modify the 3H-labeled amine release from slices prelabeled with preformed [3H]histamine. The maximal inhibitory effect of exogenous histamine progressively diminished as the strength of the depolarizing stimulus or the external Ca2+ concentration were elevated. On the contrary H3-receptor antagonists like impromidine or burimamide enhanced the depolarization-induced release of [3H]histamine, an effect which was particularly marked when slices were loaded with histamine by preincubation with [3H]histidine in high concentration. These results suggest that the inhibition of [3H]histamine release by exogenous histamine acting via H3-receptor stimulation is mediated by a restricted access of Ca2+ and that its extent is influenced by the degree of autostimulation by endogenous histamine as well as, possibly, by actual internal Ca2+ concentration. In addition the decrease in external Ca2+ concentration shifted rightwards the concentration-response curve to histamine. The autoinhibitory effect of exogenous histamine was found on slices from various regions, known from lesion studies to contain terminals of extrinsic histaminergic neurons. It did not apparently involve interneurones, not being prevented in slices in which the traffic of action potentials was blocked by tetrodotoxin. It also remained unaffected in striatal slices in which the neuronal cell-bodies were selectively destroyed by prior local infusion of kainic acid. Finally exogenous histamine inhibited [3H]histamine release from depolarized synaptosomes of rat cerebral cortex, with an EC50 value similar to that found with slices and was antagonised by impromidine with an apparent Ki value similar to that displayed at H3-receptors. It is concluded that histamine modulates its own release from cerebral neurones by interacting with H3-presynaptic autoreceptors and via mechanisms similar to those previously evidenced on other aminergic systems.