Hans O. Kalkman

Molecular pharmacology of 5-HT1 and 5-HT2 recognition sites in rat and pig brain membranes: Radioligand binding studies with [3H]5-HT, [3H]8-OH-DPAT, (−)[125I]iodocyanopindolol, [3H]mesulergine and [3H]Ketanserin☆

The pharmacological characteristics of the binding of [3H]8-OH-DPAT ([3H]8-hydroxy-2(di-n-propylamino)tetralin, [125I]CYP ((-)[125I]iodocyanopindolol) (in the presence of 30 microM (-)isoprenaline) and [3H]mesulergine to 5-HT1 recognition sites were studied in rat and pig brain membranes. [3H]8-OH-DPAT bound in rat and pig cortex to the 5-HT1A recognition site characterized by high affinity for 5-CT (5-carboxamido-tryptamine), 8-OH-DPAT, 5-HT (5-hydroxytryptamine, serotonin) and low affinity for pirenperone, ketanserin and mesulergine. [125I]CYP bound in rat but not in pig cortex to the 5-HT1B site which shows high affinity for (-)21-009 (4[3-ter-butyl-amino-2-hydroxy-propoxy]indol-2-carbonic acid isopropyl ester), (+/-)ICYP (3-I-cyanopindolol), 5-HT, RU 24969 (5-methoxy-3-[1,2,3,6-tetrahydropyridon-4-yl]1H-indole) and low affinity for 8-OH-DPAT, mesulergine and pirenperone. [3H]Mesulergine bound in pig choroid plexus and in rat cortex (besides binding to 5-HT2 sites in rat cortex) to the 5-HT1C recognition site characterized by high affinity for metergoline, mesulergine, 5-HT and methergine and low affinity for (-)21-009, ICYP, 8-OH-DPAT and spiroperidol. The pharmacological profile of 5-HT1A sites in rat and pig cortex appears to be identical; 5-HT1C sites in pig choroid plexus and rat cortex show no differences. In contrast, it was not possible to label 5-HT1B sites with [125I]CYP in pig brain membranes indicating that like 5-HT2 receptors, 5-HT1 recognition sites show species differences. The pharmacological profiles of the three 5-HT1 recognition sites are clearly different from one another. Furthermore, the pharmacological profile of each individual 5-HT1 recognition site is also different from that of the 5-HT2 receptors labelled with [3H]ketanserin in rat cortex membranes although some similarities exist between 5-HT2 and 5-HT1C sites. Finally, the beta-adrenoceptor antagonist (-)21-009 which has different affinities for 5-HT1A, 5-HT1B and 5-HT1C recognition sites, yielded triphasic competition curves for [3H]5-HT binding in rat cortex membranes providing evidence that [3H]5-HT labels three distinct 5-HT1 sites in these membranes.

Characterization of the 5-HTIB recognition site in rat brain: Binding studies with (−)[125I]Iodocyanopindolol

(-)[125I]Iodocyanopindolol ([125I]CYP) labels rat brain membrane sites which display high affinity for several serotonergic and beta-adrenergic compounds. The binding of [125I]CYP to these serotonergic recognition sites was evaluated in the presence of 30 microM (-)isoprenaline in order to suppress binding to beta-adrenoceptors. [125I]CYP binds in rat cortex membranes rapidly, reversibly and stereoselectively to a finite number of recognition sites: Bmax = 180 fmol/mg, KD = 230 pM. Similar affinity values of [125I]CYP were obtained in membranes from rat hippocampus and striatum. Kinetic, saturation and competition experiments suggest that under these conditions [125I]CYP binds to a single serotonergic recognition site named 5-HT1B. The pharmacological profile of 5-HT1B sites is characteristic of a 5-HT1 binding site and shows the following rank order of affinity for agonists: RU 24969, (5-methoxy-3-[1,2,3,6-tetrahydropyridin-4-yl]1H-indole) greater than 5-CT, (5-carboxamidotryptamine) greater than 5-HT, (5-hydroxytryptamine, serotonin) greater than 5-OCH3-T, (5-methoxytryptamine) much greater than 2-CH3-5-HT, (2-methylserotonin) greater than 8-OH-DPAT, (8-hydroxy-2-(di-n-pro-pylamino)-tetralin). The rank order of affinity for antagonists is: (+/-)ICYP, ((+/- )-3-I-cyano-pindolol) greater than (-)21-009, (4-[3-ter-butyl-amino-2-hydroxy-propoxy]-indol-2-carbonic acid isopropyl ester) greater than (+)21-009 greater than (-)propranolol greater than metitepin greater than (-)pindolol much greater than ketanserin greater than spiroperidol greater than mesulergine. 5-HT1B recognition sites display low affinity for selective beta 1- and beta 2-adrenoceptor antagonists, e.g. atenolol, betaxolol, ICI 89-406 and ICI 118-551. The low affinity of 5-HT1B recognition sites for some 5-HT1A, 5-HT1C and 5-HT2 selective compounds (e.g. 8-OH-DPAT, mesulergine, ketanserin) suggests that 5-HT1B recognition sites are pharmacologically different from 5-HT1A, 5-HT1C and 5-HT2 recognition sites.

Translocator protein (18 kD) as target for anxiolytics without benzodiazepine-like side effects.

Keeping Calm Benzodiazepines are the most prescribed anxiolytics and are used by a broad population. However, benzodiazepines can cause unwanted side effects, including sedation, development of tolerance, and withdrawal symptoms after long-term administration. Rupprecht et al. (p. 490; published online 18 June) now find that a translocator protein (18-kD) ligand, XBD173, is a fast-acting anxiolytic agent, both in animals and humans, which lacks the unwanted side effects of benzodiazepines and provides a promising target for novel clinically effective anxiolytic drugs. Possible drug alternative for rapid treatment of anxiety disorders could replace benzodiazepines. Most antianxiety drugs (anxiolytics) work by modulating neurotransmitters in the brain. Benzodiazepines are fast and effective anxiolytic drugs; however, their long-term use is limited by the development of tolerance and withdrawal symptoms. Ligands of the translocator protein [18 kilodaltons (kD)] may promote the synthesis of endogenous neurosteroids, which also exert anxiolytic effects in animal models. Here, we found that the translocator protein (18 kD) ligand XBD173 enhanced γ-aminobutyric acid–mediated neurotransmission and counteracted induced panic attacks in rodents in the absence of sedation and tolerance development. XBD173 also exerted antipanic activity in humans and, in contrast to benzodiazepines, did not cause sedation or withdrawal symptoms. Thus, translocator protein (18 kD) ligands are promising candidates for fast-acting anxiolytic drugs with less severe side effects than benzodiazepines.

α2C-Adrenoceptor blockade by clozapine and other antipsychotic drugs

The noradrenergic system may play a role in antipsychotic modulation of schizophrenia symptoms. Therefore, the antagonistic potencies of the antipsychotics clozapine, chlorpromazine, risperidone, olanzapine, haloperidol, quetiapine, ziprasidone, iloperidone and aripiprazole were quantified using cell lines expressing the recombinant human alpha(2C)-adrenoceptor, alpha(2A)-adrenoceptor, or dopamine D(2L) receptor. The alpha(2)-adrenoceptor antagonists, yohimbine and idazoxan, were also tested. Alterations in cAMP were measured as changes in luminescence. In the alpha(2A)-adrenoceptor cell line, the agonist 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline (UK14,304) induced a concentration-dependent increase in luminescence. In cell lines expressing alpha(2C) and D(2L) receptors, agonists induced a concentration-dependent reduction in luminescence. Yohimbine and idazoxan were the most potent alpha(2A)-adrenoceptor antagonists, yohimbine and iloperidone were the most potent alpha(2C)-adrenoceptor antagonists, and haloperidol and olanzapine were the most potent dopamine D(2) receptor antagonists. Clozapine had the highest alpha(2C)/D(2) selectivity, and iloperidone the highest alpha(2C)/alpha(2A) ratio. It is hypothesised that alpha(2C)-adrenoceptor blockade contributes to improvement of cognitive function.

A review of the evidence for the canonical Wnt pathway in autism spectrum disorders

Microdeletion and microduplication copy number variations are found in patients with autism spectrum disorder and in a number of cases they include genes that are involved in the canonical Wnt signaling pathway (for example, FZD9, BCL9 or CDH8). Association studies investigating WNT2, DISC1, MET, DOCK4 or AHI1 also provide evidence that the canonical Wnt pathway might be affected in autism. Prenatal medication with sodium-valproate or antidepressant drugs increases autism risk. In animal studies, it has been found that these medications promote Wnt signaling, including among others an increase in Wnt2 gene expression. Notably, the available genetic information indicates that not only canonical Wnt pathway activation, but also inhibition seems to increase autism risk. The canonical Wnt pathway plays a role in dendrite growth and suboptimal activity negatively affects the dendritic arbor. In principle, this provides a logical explanation as to why both hypo- and hyperactivity may generate a similar set of behavioral and cognitive symptoms. However, without a validated biomarker to stratify for deviant canonical Wnt pathway activity, it is probably too dangerous to treat patients with compounds that modify pathway activity.

Is migraine prophylactic activity caused by 5-HT2B or 5-HT2C receptor blockade?

It has been suggested that activation of 5-HT2C receptors is involved in the initiation of a migraine attack. The 5-HT2C receptor and the newly cloned rat fundus 5-HT2B receptor show close pharmacological and structural resemblance. Antagonist pA2 values from the rat stomach and pKD values from a 5-HT2C receptor binding assay correlated both highly significantly (p < 0.005) with the daily dose of eight migraine prophylactic compounds. Although the small difference in antimigraine potency between the enantiomers of propranolol agrees with the lack of stereo-selectivity found on the rat fundus 5-HT2B receptor but not with the 5-HT2C receptor, the evidence available does not allow one to distinguish between 5-HT2C and 5-HT2B receptor blockade as possible mechanisms for prophylactic activity.

The role of α2-adrenoceptor antagonism in the anti-cataleptic properties of the atypical neuroleptic agent, clozapine, in the rat

1 The mechanism underlying the anticataleptic properties of the atypical neuroleptic agent, clozapine, has been investigated in the rat. 2 The close structural analogues of clozapine, loxapine (0.1 mg kg−1 s.c.) and iso‐clozapine (1 and 3 mg kg−1 s.c.) induced catalepsy in rats. In contrast, clozapine and the regio‐isomer of loxapine, iso‐loxapine (up to 10 mg kg−1 s.c.) did not produce catalepsy, but at a dose of 1 mg kg−1 significantly inhibited catalepsy induced by loxapine (0.3 mg kg−1 s.c.). 3 Radioligand binding assays showed that cataleptogenic potential was most clearly predicted by the D2/5‐HT1A, D2/5‐HT1B/1D and D2/α2‐receptor affinity (KD) ratios: i.e. 30–100‐fold higher ratios were calculated for loxapine and iso‐clozapine, whereas the ratios were less than 1 for clozapine and iso‐loxapine. The ratios of affinities for D2 to 5‐HT2A, 5‐HT2C or D1 did not reflect the grouping of cataleptic and non‐cataleptic compounds. 4 Co‐treatment with the α2‐adrenoceptor antagonists, yohimbine (1–10 mg kg−1 s.c.), RX 821002 (1–10 mg kg−1 s.c.) and MK‐912 (0.3 and 1 mg kg−1 s.c.) dose‐dependently inhibited the cataleptic response to loxapine (0.3 mg kg−1). Yohimbine (1–10 mg kg−1 s.c.) also dose‐dependently inhibited the cateleptic response to haloperidol (0.3 mg kg−1 s.c.). The α2‐adrenoceptor antagonists had no effect per se. 5 Neither yohimbine (10 mg kg−1) nor RX821002 (3 mg kg−1) altered the cataleptic response to the D1 receptor antagonist, SCH 23390 (1 mg kg−1 s.c.), while, like clozapine, both compounds abolished the response to the 5‐HT2A receptor antagonist, MDL 100,151 (3 mg kg−1 s.c.). 6 The present data strongly implicate α2‐adrenoceptor blockade in the anticataleptic properties of clozapine and suggest that its lack of extrapyramidal side effects in the clinic may also be a consequence of this property.

Altered growth factor signaling pathways as the basis of aberrant stem cell maturation in schizophrenia.

In recent years evidence has accumulated that the activity of the signaling cascades of Neuregulin-1, Wnt, TGF-beta, BDNF-p75 and DISC1 is different between control subjects and patients with schizophrenia. These pathways are involved in embryonic and adult neurogenesis and neuronal maturation. A review of the clinical data indicates that in schizophrenia the Wnt pathway is most likely hypoactive, whereas the Nrg1-ErbB4, the TGF-beta- and the BDNF-p75-pathways are hyperactive. Haplo-insuffiency of the DISC1 gene is currently the best established schizophrenia risk factor. Preclinical experiments indicate that suppression of DISC1 signaling leads to accelerated dendrite development in neuronal stem cells, accelerated migration and aberrant integration into the neuronal network. Other preclinical experiments show that increasing NRG1-, BDNF- and TGF-beta signaling and decreasing Wnt signaling, also promotes adult neuronal differentiation and migration. Thus deviations in these pathways detected in schizophrenia could contribute to premature neuronal differentiation, accelerated migration and inappropriate insertion into the neuronal network. Initial clinical findings are confirmatory: neuronal stem cells isolated from nasal biopsies from schizophrenia patients display signs of accelerated development, whilst increased erosion of telomeres and bone age provide further support for accelerated cell maturation in schizophrenia.

Receptor profile of P88-8991 and P95-12113, metabolites of the novel antipsychotic iloperidone

Iloperidone is a novel atypical antipsychotic compound currently under clinical development for the treatment of psychotic disorders. In radioligand binding studies, iloperidone binds with high affinity to serotonin (5-HT) 5-HT2A and noradrenaline alpha1 and alpha2C receptors [Neuropsychopharmacology (2001) 25, 904-914]. The human metabolism of iloperidone generates two major metabolites, P88-8991 and P95-12113. The aim of this study was to compare the receptor affinity profile of P88-8991 and P95-12113 with that of the parent compound. The receptor affinity profile of P88-8991 is comparable to that of iloperidone. This metabolite binds to the following monoamine receptors (pKi values in nM): serotonin 5-HT2A receptors (9.56), adrenergic alpha1 (8.08) and alpha2C (7.79) receptors, and D2A receptors (7.80). Lower affinity is seen for other dopamine, serotonin, alpha2-adrenergic and histamine H1 receptors. In contrast, P95-12113 shows affinity for 5-HT2A receptors (pKi 8.15; which is 60-fold lower than that of iloperidone), adrenergic alpha1 (7.67), alpha2C (7.32) and alpha2B (7.08) receptors. Given this affinity profile, and the observation that P95-12113 does not readily cross the blood-brain barrier, it is unlikely that this metabolite contributes to the therapeutic effect of iloperidone in patients with schizophrenia. However, the comparable receptor binding profile of P88-8991 indicates that it is likely to contribute to the clinical profile of iloperidone.

Zacopride and BRL 24924 induce an increase in EEG‐energy in rats

1 The substituted benzamides, zacopride and BRL 24924 induced dose‐dependent increases of the total EEG‐energy of rats when applied intracerebroventricularly (i.c.v.) with ED50 values of 8.0 ± 0.6 and 3.6 ± 0.9 μg, respectively. Not only the energy of the low frequency hippocampal theta rhythm but also that of the other frequency bands was increased. 2 In contrast to i.c.v. application intraperitoneal administration of zacopride or BRL 24924 (1 and 10 mg kg−1) did not lead to an increase in EEG‐energy. 3 The increase in EEG‐energy induced by zacopride (10 μg, i.c.v.) was blocked by ICS 205–930 (1 μg, i.c.v.). Neither the 5‐HT3 receptor agonist 2‐methyl‐5‐hydroxytryptamine (30 μg, i.c.v.) nor the selective 5‐HT3 receptor antagonist MDL 72222 (30 μg, i.c.v.) had any effect upon rat EEG. 4 Scopolamine (0.01 μg and 0.1 μg, i.c.v.) dose‐dependently antagonized the effect of zacopride (10 μg, i.c.v.). 5 An agonist action of zacopride and BRL 24924 and inhibition of these effects by ICS 205–930 but not by MDL 72222 was recently described in isolated colliculi neurones from neonatal mice. The receptor involved was described as ‘5‐HT4’. The present results indicate that the central effects of zacopride and BRL 24924 may be due to activation of such a 5‐HT receptor.