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.

Azole derivatives as histamine H3 receptor antagonists, part 2: C-C and C-S coupled heterocycles.

With a small series of compounds we demonstrated the variability in the core region of the human histamine H(3) receptor (hH(3)R) antagonist structural blueprint by introducing polar azole groups (oxazole, oxadiazole, thiazole and triazole). Additional variations achieved by coupling different residues to the heterocyclic core structure led to further optimisation of in vitro receptor binding of the novel azole derivatives.

Azole derivatives as histamine H3 receptor antagonists, part I: thiazol-2-yl ethers.

Most human histamine H(3) receptor (hH(3)R) antagonists follow a general structural blueprint, containing a basic moiety linked by a spacer to a substituted core element. In this investigation the acceptance of thiazol-2-yl ether moieties in the core region is proved with some ether derivatives showing hH(3)R binding affinities in the nanomolar concentration range. A diversity of structural motifs is used as substituents to enhance the in vitro hH(3)R binding affinity.

Fluorinated non-imidazole histamine H3 receptor antagonists.

Fluorine substituents have become a widespread and important component in drug design and development. Here, the synthesis of fluorine containing compounds and some corresponding precursor molecules are presented for potential isotope labelling as well as their data obtained with in vitro and in vivo screenings. The compounds vary in the basic centres (piperidine or pyrrolidine) and are fluoro substituted in different positions of the basic alicyclic moiety. Pharmacological evaluation resulted in ligands with high affinities at hH(3) receptor in the nanomolar and subnanomolar concentration range and some with high antagonist in vivo potencies.

Potential utility of histamine H3 receptor antagonist pharmacophore in antipsychotics.

Histamine H3 receptor (H3R) antagonists have some antipsychotic properties although the clear molecular mechanism is still unknown. As actually the most effective and less side effective antipsychotics are drugs with multiple targets we have designed typical and atypical neuroleptics with an additional histamine H3 pharmacophore. The 4-(3-piperidinopropoxy)phenyl pharmacophore moiety has been linked to amitriptyline, maprotiline, chlorpromazine, chlorprothixene, fluphenazine, and clozapine. Amide, amine and ester elements have been used generally to maintain or slightly shift affinity at dopamine D(2)-like receptors (D2 and D3), to decrease affinity at histamine H(1) receptors, and to obtain H3R ligands with low nanomolar or subnanomolar affinity. Change of effects at D(1)-like receptors (D1) and (D5) were heterogeneous. With these newly profiled compounds different antipsychotic properties might be achieved.

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 approximately 50% higher than that of ciproxifan. Its in vitro potency was approximately 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.

Search for histamine H3 receptor antagonists with combined inhibitory potency at Ntau-methyltransferase: ether derivatives.

With the recent development of new hybrid compounds having histamine H3 receptor antagonist with combined histamine Ntau-methyltransferase (HMT) inhibitory potency an innovative approach was described in the research of novel lead compounds modulating histaminergic neurotransmission. Several compounds containing an ether moiety derived from the recently published 4-(3-piperidinopropoxy)phenylaminoquinoline derivatives (like FUB 836), were synthesized in this study and tested for their affinity at cloned human histamine H3 (hH3) receptors and on the inhibition of rat HMT. Besides different heterocycles, e.g. nitro- or amino-substituted pyridines, quinolines, benzothiazole or pyrroline, three classes of compounds were produced: heteroaromatic 3-piperidinopropyl ethers, keto- or imino-substituted 4-(3-piperidinopropyl)phenyl ethers and 4-(3-piperidinopropyl)phenyl ethers with substituted (alkyl)aminopyridines. Whereas the (3-piperidinopropoxy)heterocycles showed only moderate activities on both test models, the 4-(3-piperidinopropoxy)phenyl derivatives were identified as potent histamine H3 receptor ligands and/or HMT inhibitors. Ki values up to 0.42 nM were found for the affinity to the hH3 receptor. HMT inhibitory potency was identified with IC50 values about 0.3 microM for the most potent compounds in this series. Comparison of the pyridine-containing derivatives to recently published quinoline analogues showed a decrease in potencies for the pyridines. The dual activity, H3 receptor affinity and HMT inhibition, was moderate to good. For all compounds affinities at hH3 receptors were higher than their inhibitory HMT potencies. The described new histamine H3 receptor antagonists with an ether moiety represent a further promising step in our investigations for a dual activity.

Therapeutic implications of constitutive activity of receptors: the example of the histamine H3 receptor.

Some G-protein-coupled receptors display constitutive activity, that is spontaneous activity in the absence of agonist: a proportion of the receptor population adopts a conformation that can bind and activate G proteins. Whereas this was mainly shown to occur with recombinant or pathologically mutated receptors, the physiological relevance of the process has remained debated. We have adressed this question in the case of the histamine H3 receptor, a presynaptic inhibitory receptor regulating histamine release in brain. Having identified a neutral antagonist and inverse agonists with variable intrinsic activity, we show that the native H3 receptor in brain displays high constitutive activity in vitro and, in vivo, controls the release of endogenous histamine. This implies that inverse agonists with high intrinsic activity should be preferred for therapeutic application as "cognitive enhancers" in several psychiatric disorders.

A detailed mapping of the histamine H(3) receptor and its gene transcripts in rat brain.

The detailed distribution of histamine H(3) receptor mRNAs in rat brain was analyzed by in situ hybridization using a 33P-labelled riboprobe and was combined for the first time with the detailed autoradiographic distribution of the receptor determined in the same animals with [(125)I]iodoproxyfan, a selective radioligand. The signals generated on adjacent brain sections by each probe were quantified and/or rated and were compared in order to identify neuronal populations expressing the receptor. In addition, the cellular localization of the transcripts within various brain structures was analyzed in sections dipped in a photographic emulsion. In the cerebral cortex, the strong mRNA expression in intermediate and deep layers indicates the presence of H(3) receptors on several types of neurons. The binding is dense except in layer V, suggesting that H(3) receptors are located on granule cells and apical dendrites of pyramidal cells. In addition to their localization on monoaminergic afferents, the dense binding in layer IV and strong mRNA expression in thalamic nuclei suggest the presence of heteroreceptors on thalamocortical projections. In the hippocampus, the strong mRNA expression but low binding in pyramidal layers of the CA1 and ventral CA3 fields suggest that H(3) receptors are abundant on efferent projections of pyramidal cells. In the dentate gyrus, some binding sites in the molecular layer may correspond to H(3) receptors synthesized in granule cells and coexpressed with H(1) and H(2) receptors in their dendrites. In the basal ganglia, H(3) receptors are highly expressed in the striatal complex and olfactory tubercles but not in islands of Calleja. Some of the striatal binding sites may correspond to presynaptic receptors present on afferents. The mRNAs in cortical layer V may encode for heteroreceptors on corticostriatal neurons. The presence of mRNAs in the substantia nigra pars compacta suggests that H(3) receptors are located upon nigrostriatal afferents. However, the absence of any signal in the ventral tegmental area indicates that some but not all dopaminergic neurons express H(3) receptors. In addition, the homogeneous mRNA expression within the caudate putamen and nucleus accumbens suggests that many striatal H(3) receptors are present on medium-sized, spiny projection neurons of both the direct and indirect movement pathways. In agreement, a dense binding, but low mRNA expression, is observed in external and internal pallidum and in substantia nigra pars reticulata. In the amygdala, the dense binding and mRNA expression indicate the presence of receptors on both afferents and projections. In the thalamus, the binding in some association nuclei may correspond to receptors present on neurons emanating from the deep cortical layers that strongly express the mRNAs, as well as receptors on the visual systems. However, the low binding and high mRNA expression in most nuclei indicate that many receptors are present upon thalamic projections. In the hypothalamus, the mRNA expression parallels the density of binding sites and is the highest in the tuberomammillary nucleus. Further investigation is needed to know if the dense binding and mRNA expression observed in other nuclei such as the paraventricular, ventromedial and medial tuberal nuclei correspond to pre- and/or postsynaptic receptors. mRNAs are also observed in several areas projecting to the tuberomammillary nucleus, such as the ventrolateral preoptic nucleus. In the lower brainstem, the high mRNA expression and very low binding in the locus coeruleus and raphe nuclei indicate that presynaptic rather than somatodendritic receptors regulate noradrenaline and serotonin release, respectively. A similar pattern in vestibular nuclei suggests that receptors located on projections account for the anti-vertigo properties of H(3) receptor antagonists. In the cerebellum, binding is hardly detectable but a strong mRNA expression is found in most, if not all, Purkinje cells as well as in several central cerebellar nuclei, suggesting the presence of H(3) receptors on efferent projections. The present study reports the first detailed quantification and/or rating of H(3) receptor mRNAs in the brain. The comparison, performed in the same animals, with the distribution of the H(3) receptor protein provides evidence for the presence of H(3) receptors on many neuronal perikarya, dendrites and projections. Although some localizations, mainly as auto- or heteroreceptors, are consistent with previous functional studies, the physiological role, if any, of most of these presynaptic or postsynaptic receptors remains to be established.

Histamine H3-receptor-mediated [35S]GTP gamma[S] binding: evidence for constitutive activity of the recombinant and native rat and human H3 receptors.

Constitutive activity of the recombinant and native rat and human H(3) receptors (H(3)Rs) was studied using H(3)R-mediated [(35)S]GTPgamma[S] binding and [(3)H]-arachidonic acid release. Ciproxifan, an inverse agonist at the rat H(3)R (rH(3)R), decreased [(3)H]arachidonic acid release from CHO cells expressing moderate densities (approximately 200 - 300 fmol mg(-1) protein) of the human H(3)R (hH(3)R). This effect occurred with the same magnitude than at the rH(3)R. The expression of the hH(3)R was associated with an increase in [(35)S]GTPgamma[S] binding to membranes of CHO cells. Ciproxifan decreased [(35)S]GTPgamma[S] binding to membranes of CHO (hH(3)R) cells. Both effects were correlated to receptor density and revealed that constitutive activity of the hH(3)R, although lower than that of the rH(3)R in this assay, was again observed at physiological densities (<500 fmol mg(-1) protein). Ciproxifan was less potent at the human than the rat receptor, not only as an antagonist (K(i)=45 nM), but also as an inverse agonist (EC(50)=15 nM). Constitutive activity of the hH(3)R was also evidenced using inhibition of [(35)S]GTPgamma[S] binding by unlabelled GTPgammaS. The expression of the hH(3)R generated a high affinity binding for GTPgammaS which was increased by imetit, but partially decreased by ciproxifan, therefore acting as a partial inverse agonist. [(35)S]GTPgamma[S] binding to rat brain membranes was decreased in several regions by thioperamide, ciproxifan and FUB 465, three inverse agonists at the H(3)R, whose effects were blocked by proxyfan, a neutral antagonist. [(35)S]GTPgamma[S] binding was also decreased by an A(1)-adenosine receptor inverse agonist, but remained unchanged in the presence of inverse agonists at D(2)/D(3) dopamine, H(1) and H(2) histamine, alpha(2)-adrenergic and delta opioid receptors. In conclusion, the present study shows that the recombinant rat and human H(3) receptors expressed at physiological densities display constitutive activity and suggests that constitutive activity of native H(3)Rs is one of the highest among G-protein-coupled receptors present in rat brain.

Application of genomics to drug design: the example of the histamine H3 receptor.

The histamine H(3) receptor was characterized in the 1980s as an autoreceptor regulating histamine release in brain. Since then, selective drugs have been designed, many of them displaying a high potency in vivo, and used in many studies to delineate the implications of cerebral histaminergic systems in physiological functions such as arousal or cognitive functions. The recent cloning of the H(3) receptor, more than 15 years later, has allowed to start molecular studies that led to important findings for optimization of drug design. In agreement some ligands display distinct affinities for the recombinant rat and human H(3) receptors, a difference that we assign to two amino acids in the third transmembrane domain. In addition, H(3) autoreceptors present in the brain display high constitutive activity including in vivo. As a consequence, inverse agonists enhance histamine neuron activity and constitute a novel potential therapeutic approach to schizophrenia and Alzheimer's disease.

Influence of imidazole replacement in different structural classes of histamine H(3)-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).

Different antagonist binding properties of human and rat histamine H3 receptors.

Different histamine H3-receptor antagonists have been tested in displacement studies at human and rat H3 receptors in stably transfected cells. Based on an actual rhodopsin structure, models for receptor antagonist interaction were developed for receptors of both species. Similarities and discrepancies in binding profiles can be explained, but not quantified by hydrophilic interactions with Asp114 and an important lipophilic binding pocket modified by two nearby amino acids.

Benzophenone derivatives and related compounds as potent histamine H3-receptor antagonists and potential PET/SPECT ligands.

Para-substituted aromatic ethers with benzophenone or related structural elements and a 3-(1H-imidazol-4-yl)propyloxy moiety were prepared by Mitsunobu-type ether synthesis or SNAr reaction. Most of the title compounds possess high antagonist potency in histamine H3-receptor assays in vitro as well as in vivo in mouse CNS following oral administration. After defining 4-(3-(1H-imidazol-4-yl)propyloxy)phenyl phenyl methanone as a new lead, structure-activity relationships were investigated for this new class of compounds. Substitution of the meta'-position of the benzophenone moiety with halogen atoms (e.g., iodine, fluorine) led to compounds with high antagonist potency in vitro as well as in vivo (Ki = 9.3 and 4.3 nM, ED50 = 0.7 and 0.47 mg/kg p.o., 18 and 12, respectively). A receptor profile of several functional in vitro assays for several biogenic amine receptors for the meta'-iodinated derivative demonstrated high selectivity toward the histamine H3 receptor.

The rat H3 receptor: gene organization and multiple isoforms.

Genomic DNA analysis revealed that the coding region of the rat histamine H3 receptor comprises three exons interrupted by two introns of approximately 1 kb each. Several H3 receptor mRNA variants were identified by PCR and cDNA cloning and sequencing. Four variants generated by pseudo-intron retention/deletion at the level of the third intracellular loop were designated H3(445), H3(413), H3(410), and H3(397), according to the length of their deduced amino acid sequence and display differential tissue expression. When expressed in CHO-K1 or Cos-1 cells, the H3(445), H3(413), and H3(397) were found to generate specific 125I iodoproxyfan binding of similar pharmacological profile. In addition, we identified two short variants, termed H3(nf1) and H3(nf2), which correspond to frame shift and stop codon interposition, respectively, and are presumably nonfunctional, among which H3(nf2) displays brain expression similar to that of the longer isoforms.

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 beta2-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.