Dominik Feuerbach

The selective nicotinic acetylcholine receptor α7 agonist JN403 is active in animal models of cognition, sensory gating, epilepsy and pain

Several lines of evidence suggest that the nicotinic acetylcholine receptor alpha7 (nAChR alpha7) is involved in central nervous system disorders like schizophrenia and Alzheimers disease as well as in inflammatory disorders like sepsis and pancreatitis. The present article describes the in vivo effects of JN403, a compound recently characterized to be a potent and selective partial nAChR alpha7 agonist. JN403 rapidly penetrates into the brain after i.v. and after p.o. administration in mice and rats. In the social recognition test in mice JN403 facilitates learning/memory performance over a broad dose range. JN403 shows anxiolytic-like properties in the social exploration model in rats and the effects are retained after a 6h pre-treatment period and after subchronic administration. The effect on sensory inhibition was investigated in DBA/2 mice, a strain with reduced sensory inhibition under standard experimental conditions. Systemic administration of JN403 restores sensory gating in DBA/2 mice, both in anaesthetized and awake animals. Furthermore, JN403 shows anticonvulsant potential in the audiogenic seizure paradigm in DBA/2 mice. In the two models of permanent pain tested, JN403 produces a significant reversal of mechanical hyperalgesia. The onset was fast and the duration lasted for about 6h. Altogether, the present set of data suggests that nAChR alpha7 agonists, like JN403 may be beneficial for improving learning/memory performance, restoring sensory gating deficits, and alleviating pain, epileptic seizures and conditions of anxiety.

Distribution and characterisation of somatostatin receptor mRNA and binding sites in the brain and periphery

The distribution and nature of (somatostatin) SRIF receptors and receptor mRNAs was studied in the brain and periphery of various laboratory animals using in situ hybridisation, autoradiography and radioligand binding. The messenger RNA (mRNA) expression of SRIF receptors msst1, msst2, msst3, msst4 and msst5 was studied in the adult mouse brain by in situ hybridisation histochemistry using specific oligonucleotide probes and compared to that of adult rats. As observed in rat brain, sst3 receptor mRNA is prominently expressed across the mouse brain, although equivalent binding has not yet been identified in situ. Sst1 and sst2 receptor mRNA expression, was prominent and again comparable to that observed in rat brain, whereas sst4 and especially sst5 receptor mRNA show comparatively low levels, although the former appears to be widely distributed while the latter could only be identified in a few nuclei. Altogether, the data are compatible with current knowledge, i.e. sst1 and sst2 receptor mRNA is prominent (both receptors have been functionally identified in the brain and for sst2 in the periphery), sst3 mRNA is highly expressed but in the absence of any functional correlate remains elusive. The expression of sst4 mRNA is comparatively low (especially when compared to what is seen in the lung, where high densities of sst4 receptors are present) and it remains to be seen whether sst5 receptor mRNA, which is confined to a few nuclei, will play a role in the brain, keeping in mind that high levels are found in the pituitary. Radioligand binding studies were performed in CCL39 cells expressing the five human recombinant receptors and compared to binding in membranes of rat cerebral cortex with [125I]Tyr11-SRIF14 which in the presence of 120 mM labels primarily sst1 receptor as suggested by the better correlation hsst1 and similar rank order of potency. The profile of [125I]Tyr3-octreotide labelled sites in rat cortex correlates better with recombinant sst2 than sst3 or sst5 binding profiles. Finally, [125I]LTT-SRIF28-labelled sites in rat lung express a sst4 receptor profile in agreement with previous findings. SRIF receptor autoradiography was performed in the brain and peripheral tissue of rat and/or guinea-pig using a number of ligands known to label recombinant SRIF receptors: [125I]LTT-SRIF28, [125I]CGP 23996, [125I]Tyr10-CST, or [125I]Tyr3-octreotide. Although, [125I]Tyr10-CST has been shown to label all five recombinant SRIF receptors, it is apparent that this radioligand is not useful for autoradiographic studies. By contrast, the other three ligands show good signal to noise ratios in rat or guinea-pig brain, rat lung, rat pancreas, or guinea-pig ileum. In most tissues, [125I]Tyr3-octreotide represents a prominent part of the binding (when compared to [125I]LTT-SRIF28 and [125I]CGP 23996), suggesting that sst2 receptors are strongly expressed in most tissues; it is only in rat lung that [125I]LTT-SRIF28 and [125I]CGP 23996 show marked binding, whereas [125I]Tyr3-octreotide does apparently label no sites, in agreement with the sole presence of sst4 receptors in this tissue.

Pharmacological profile of somatostatin and cortistatin receptors

Somatostatin (SRIF) and cortistatin (CST) are two endogenous peptides with high sequence similarities that act as hormones/neurotransmitters both in the CNS and the periphery; their genes although distinct result from gene duplication. Their receptors appear to be common, since the five known SRIF receptors (sst1-sst5) have similar subnanomolar affinity for SRIF and CST, whether the short (SRIF-14, CST-14, CST-17) or the long versions (SRIF-28, CST-29) of the peptides. Whether CST targets specific receptors not shared by SRIF, is still debated: MrgX2 has been described as a selective CST receptor, with submicromolar affinity for CST but devoid of affinity for SRIF; however the distribution of CST and MrgX2 is largely different, and there is no MrgX2 in rodents. A similar situation arises with the GHS receptor GHS-R1a, which displays some preferential affinity for CST over SRIF, but for which there is no evidence that it is activated by CST in vivo. In both cases, one may argue that submicromolar affinity is not the norm of a GPCR for its endogenous neuropeptide. On the other hand, all receptors known to bind SRIF have similar high affinity for CST and both peptides act as potent agonists at the sst1-sst5 receptors, whichever transduction pathway is considered. In addition, [(125)I][Tyr(10)]CST(14) labels sst1-sst5 receptors with subnanomolar affinity, and [(125)I][Tyr(10)]CST(14) binding in the brain is overlapping with that of [(125)I][Tyr(0)]SRIF(14). The functional differences reported that distinguish CST from SRIF, have not been explained convincingly and may relate to ligand-driven transductional selectivity, and other complicating factors such as receptor dimerisation, (homo or heterodimerisation), and/or the influence of accessory proteins (GIPs, RAMPS), which remain to be studied in more detail.

Modulatory effects of α7 nAChRs on the immune system and its relevance for CNS disorders

The clinical development of selective alpha-7 nicotinic acetylcholine receptor (α7 nAChR) agonists has hitherto been focused on disorders characterized by cognitive deficits (e.g., Alzheimer’s disease, schizophrenia). However, α7 nAChRs are also widely expressed by cells of the immune system and by cells with a secondary role in pathogen defense. Activation of α7 nAChRs leads to an anti-inflammatory effect. Since sterile inflammation is a frequently observed phenomenon in both psychiatric disorders (e.g., schizophrenia, melancholic and bipolar depression) and neurological disorders (e.g., Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis), α7 nAChR agonists might show beneficial effects in these central nervous system disorders. In the current review, we summarize information on receptor expression, the intracellular signaling pathways they modulate and reasons for receptor dysfunction. Information from tobacco smoking, vagus nerve stimulation, and cholinesterase inhibition is used to evaluate the therapeutic potential of selective α7 nAChR agonists in these inflammation-related disorders.

Novel Positive Allosteric Modulators of the Human α7 Nicotinic Acetylcholine Receptor

The pharmacological activity of a series of novel amide derivatives was characterized on several nicotinic acetylcholine receptors (AChRs). Ca(2+) influx results indicate that these compounds are not agonists of the human (h) α4β2, α3β4, α7, and α1β1γδ AChRs; compounds 2-4 are specific positive allosteric modulators (PAMs) of hα7 AChRs, whereas compounds 1-4, 7, and 12 are noncompetitive antagonists of the other AChRs. Radioligand binding results indicate that PAMs do not inhibit binding of [(3)H]methyllycaconitine but enhance binding of [(3)H]epibatidine to hα7 AChRs, indicating that these compounds do not directly, but allosterically, interact with the hα7 agonist sites. Additional competition binding results indicate that the antagonistic action mediated by these compounds is produced by direct interaction with neither the phencyclidine site in the Torpedo AChR ion channel nor the imipramine and the agonist sites in the hα4β2 and hα3β4 AChRs. Molecular dynamics and docking results suggest that the binding site for compounds 2-4 is mainly located in the inner β-sheet of the hα7-α7 interface, ∼12 Å from the agonist locus. Hydrogen bond interactions between the amide group of the PAMs and the hα7 AChR binding site are found to be critical for their activity. The dual PAM and antagonistic activities elicited by compounds 2-4 might be therapeutically important.

Functional characterization of the novel antipsychotic iloperidone at human D2, D3, α2C, 5-HT6, and 5-HT1A receptors

Iloperidone has demonstrated an interesting monoamine receptor profile in radioligand binding studies, with nanomolar affinity for certain noradrenaline, dopamine, and serotonin receptors. In this study, the agonist/antagonist activity of iloperidone was determined in cell lines expressing recombinant human D2A, D3, α2C, 5-HT1A, or 5-HT6 receptors. With the exception of 5-HT6 receptors, these receptors are negatively coupled to cyclase. Thus, after stimulation with forskolin, the agonists dopamine (at D2A and D3), noradrenaline (at α2C), or 8-OH-DPAT (at 5-HT1A) induced a reduction in cAMP accumulation. Conversely, activation of the 5-HT6 receptor by 5-HT led to an increase in cAMP accumulation. Iloperidone alone was devoid of significant agonist activity but inhibited the agonist response in all 5 cell lines in a surmountable and concentration-dependent fashion. Iloperidone was most potent at D3 receptors (pKB 8.59 ± 0.20; n = 6), followed by α2C (pKB 7.83 ± 0.06; n = 15), 5-HT1A (pKB 7.69 ± 0.18; n = 10), D2A (pKB 7.53 ± 0.04; n = 11) and 5-HT6 (pKB 7.11 ± 0.08; n = 11) receptors.

Expression of the cell-adhesion molecule VCAM-1 by stromal cells is necessary for osteoclastogenesis

Osteoblastic cells have been shown to be involved in osteoclast formation through cell to cell contacts. This study was designed to examine the possible function of vascular cell adhesion molecule 1 (VCAM‐1) during osteoclastogenesis. As a source for stromal cells we used the recently established mouse bone marrow stromal cell line mBMS‐B1 which has the ability to support osteoclastogenesis when used in co‐culture with a crude spleen cell suspension. mBMS‐B1 cells express a single ∼3.9 kb VCAM‐1 mRNA species. Expression was low under basal culture conditions and a 5–10‐fold increase was observed in the presence of 1,25(OH)2D3. Cell surface expression of VCAM‐1 examined by FACS analysis was increased about 2‐fold after 1,25(OH)2D3 treatment. Immunoprecipitation of cell surface expressed VCAM‐1 or total VCAM‐1 protein using the anti‐VCAM‐1 monoclonal antibody MK2.7 resulted in a single ∼110 kDa protein on SDS‐PAGE. Induction by 1,25(OH)2D3 was about 2–5‐fold on day 3. The stromal cell–osteoclast precursor cell interaction was investigated in a co‐culture of the mBMS‐B1 and mouse spleen cells in the presence of 1,25(OH)2D3. The monoclonal antibody MK2.7 which is known to block hemopoietic‐stromal cell recognition inhibited the formation of osteoclasts when added to the co‐culture at day 2 but not day 4. These data suggest that VCAM‐1 is involved in the interaction between stromal cells and osteoclastic precursor cells during osteoclastogenesis presumably most important during early stages of the formation of osteoclasts.

Compensatory changes in the hippocampus of somatostatin knockout mice: upregulation of somatostatin receptor 2 and its function in the control of bursting activity and synaptic transmission

Somatostatin‐14 (SRIF) co‐localizes with γ‐aminobutyric acid (GABA) in the hippocampus and regulates neuronal excitability. A role of SRIF in the control of seizures has been proposed, although its exact contribution requires some clarification. In particular, SRIF knockout (KO) mice do not exhibit spontaneous seizures, indicating that compensatory changes may occur in KO. In the KO hippocampus, we examined whether specific SRIF receptors and/or the cognate peptide cortistatin‐14 (CST) compensate for the absence of SRIF. We found increased levels of both sst2 receptors (sst2) and CST, and we explored the functional consequences of sst2 compensation on bursting activity and synaptic responses in hippocampal slices. Bursting was decreased by SRIF in wild‐type (WT) mice, but it was not affected by either CST or sst2 agonist and antagonist. sst4 agonist increased bursting frequency in either WT or KO. In WT, but not in KO, its effects were blocked by agonizing or antagonizing sst2, suggesting that sst2 and sst4 are functionally coupled in the WT hippocampus. Bursting was reduced in KO as compared with WT and was increased upon application of sst2 antagonist, while SRIF, CST and sst2 agonist had no effect. At the synaptic level, we observed that in WT, SRIF decreased excitatory postsynaptic potentials which were, in contrast, increased by sst2 antagonist in KO. We conclude that sst2 compensates for SRIF absence and that its upregulation is responsible for reduced bursting and decreased excitatory transmission in KO mice. We suggest that a critical density of sst2 is needed to control hippocampal activity.

Cloning, expression and pharmacological characterisation of the mouse somatostatin sst5 receptor

Abstract The mouse somatostatin (somatotropin release inhibiting factor, SRIF) sst 5 receptor coding sequence was cloned from a mouse BALB/c genomic library. It shows 97% and 81% homology with the corresponding rat and human receptors, respectively. The msst 5 receptor messenger RNA (mRNA) is present at low levels in the adult mouse brain, with significant expression in a few nuclei only, e.g. in the septum (lateral septal nuclei) or the amygdala (medial amygdaloid nucleus); very few signals were observed in the mesencephalon, metencephalon, and myelencephalon (except the dorsal motor nucleus of the vagus nerve). The msst 5 receptor was stably expressed in the hamster fibroblast cell line CCL39-SRE-Luci, which harbours the luciferase reporter gene driven by the serum responsive element. [ 125 I]LTT-SRIF-28 ([Leu 8 , D-Trp 22 , 125 I-Tyr 25 ]-SRIF-28), [ 125 I]Tyr 10 -CST, [ 125 I]CGP 23996, and [ 125 I]Tyr 3 -octreotide labelled msst 5 receptors with high affinity (pK d values: 11.0, 10.15, 9.75 and 9.43) and in a saturable manner, but defined different Bmax values: 697, 495, 540 and 144 fmoles/mg, respectively. [ 125 I]LTT-SRIF-28-labelled sites displayed the following rank order: SRIF-28> rCST-14> somatuline > CGP-23996= SRIF-14= octreotide, whereas [ 125 I]Tyr 3 -octreotide-labelled sites displayed a different profile: octreotide > SRIF-28> rCST-14= somatuline > SRIF-14> CGP-23996. The pharmacological profiles determined with [ 125 I]LTT-SRIF-28, [ 125 I]CGP 23996 and [ 125 I]Tyr 10 -CST correlated highly significantly (r 2 =0.88–0.99), whereas [ 125 I]Tyr 3 -octreotide binding was rather divergent (r 2 =0.77). Also, human and mouse sst 5 receptor profiles are very different, e.g. r 2 =0.385 for [ 125 I]Tyr 10 -CST and r 2 =0.323 for [ 125 I]LTT-SRIF-28-labelled sites. Somatostatin induces expression of luciferase reporter gene in CCL39-SRE-Luci cells. The profile was consistent with a msst 5 receptor-mediated effect although apparent potency in the luciferase assay was much reduced compared to radioligand binding data: Octreotide = SRIF-28> rCST-14= SRIF-14= CGP-23996. Octreotide, SRIF-28, BIM23052 and D Tyr Cyanamid 154806 behaved as full or nearly full agonists in comparison to SRIF-14, whereas the other compounds had relative efficacies of 40 to 70%. The present study shows that agonists radioligands define apparently different receptor populations in terms of number of sites and pharmacological profile in cells expressing a single recombinant receptor. These variations suggest that the conformation of the ligand receptor complex may vary depending on the agonist. Further, the msst 5 receptor, although primarily coupled to Gi/Go proteins, is able to stimulate luciferase gene expression driven by the serum responsive element. Finally, it is suggested that putative sst 2 selective agonists e.g. octreotide, RC160 or BIM23027 show similar or higher potency at msst 5 receptors than SRIF-14.

JN403, in vitro characterization of a novel nicotinic acetylcholine receptor α7 selective agonist

This report describes the in vitro features of a novel selective nicotinic acetylcholine receptor (nAChR) alpha7 agonist, JN403, (S)-(1-Aza-bicyclo[2.2.2]oct-3-yl)-carbamic acid (S)-1-(2-fluoro-phenyl)-ethyl ester. JN403 was evaluated in a number of in vitro systems of different species, at recombinant receptors using radioligand binding, signal transduction and electrophysiological studies. When using [(125)I] alpha-bungarotoxin (alpha-BTX) as a radioligand, JN403 has high affinity for human recombinant nAChR alpha7 (pK(D)=6.7). Functionally, JN403 is a partial and potent agonist at human nAChR alpha7. The compound stimulates calcium influx in GH3 cells recombinantly expressing the human nAChR with an pEC(50) of 7.0 and an E(max) of 85% (compared to the full agonist epibatidine). In Xenopus oocytes expressing human nAChR alpha7 JN403 induces inward currents with an pEC(50) of 5.7 and an E(max) of 55%. In both recombinant systems JN403 is a partial agonist and the agonistic effects are blocked after pre-administration of methyllycaconitine (MLA, 100nM), a nAChR alpha7 antagonist. In functional calcium influx assays, JN403 displays a significantly lower potency for other subtypes of human nAChRs like alpha4beta2, alpha3beta4, alpha1beta1gammadelta as well as 5HT(3) receptors when tested functionally as an antagonist (pIC(50)<4.8) and is devoid of agonistic activity (pEC(50)<4). Similarly, JN403 shows low binding activity at a wide panel of neurotransmitter receptors. Thus, JN403 is a potent and selective nAChR alpha7 agonist and will be a useful tool for the characterization of nAChR alpha7 mediated effects both in vitro and in vivo.