Frank Boess

Characterization of Ro 04‐6790 and Ro 63‐0563: potent and selective antagonists at human and rat 5‐HT6 receptors

This study describes the in vitro characterization of two potent and selective 5‐HT6 receptor antagonists at the rat and human recombinant 5‐HT6 receptor. In binding assays with [3H]‐LSD, 4‐amino‐N‐(2,6 bis‐methylamino‐pyrimidin‐4‐yl)‐benzene sulphonamide (Ro 04‐6790) and 4‐amino‐N‐(2,6 bis‐methylamino‐pyridin‐4‐yl)‐benzene sulphonamide (Ro 63‐0563) had mean pKi values ±s.e.mean at the rat 5‐HT6 receptor of 7.35±0.04 and 7.83±0.01, respectively and pKi values at the human 5‐HT6 receptor of 7.26±0.06 and 7.91±0.02, respectively. Both compounds were found to be over 100 fold selective for the 5‐HT6 receptor compared to 23 (Ro 04‐6790) and 69 (Ro 63‐0563) other receptor binding sites. In functional studies, neither compound had any significant effect on basal levels of cyclicAMP accumulation in Hela cells stably expressing the human 5‐HT6 receptor, suggesting that the compounds are neither agonists nor inverse agonists at the 5‐HT6 receptor. However, both Ro 04‐6790 and Ro 63‐0563 behaved as competitive antagonists with mean ±s.e.mean pA2 values of 6.75±0.07 and 7.10±0.09, respectively. In rats habituated to observation cages, Ro 04‐6790 produced a behavioural syndrome similar to that seen following treatment with antisense oligonucleotides designed to reduce the expression of 5‐HT6 receptors. This behavioural syndrome consisted of stretching, yawning and chewing. Ro 04‐6790 and Ro 63‐0563 represent valuable pharmacological tools for the identification of 5‐HT6 receptors in natural tissues and the study of their physiological function.

Investigation of stretching behaviour induced by the selective 5‐HT6 receptor antagonist, Ro 04–6790, in rats

The present study examined the effects of the selective 5‐HT6 receptor antagonist 4‐amino‐N‐(2, 6 bis‐methylamino‐pyrimidin‐4‐yl)‐benzene sulphonamide (Ro 04–6790) on locomotor activity and unconditioned behaviour in male Sprague Dawley rats (230–300 g). In non‐quantified behavioural observations, animals treated with Ro 04–6790 (3, 10 or 30 mg kg−1, i.p) showed no overt behavioural signs except a dose‐dependent reduction in locomotor activity and a behavioural syndrome of stretching, yawning and chewing. The latter behaviour was most pronounced between 30 and 90 min following the administration of Ro 04–6790. Detailed analysis of the stretching and yawning behaviour showed that Ro 04–6790 (3, 10 or 30 mg kg−1, i.p.) dose‐dependently induced stretching. The number of stretches observed following treatment with either Ro 04–6790 (10 mg kg−1 i.p.) or Ro‐04‐6790 (30 mg kg−1, i.p.) was significantly greater than that observed in saline‐treated rats. The yawning behaviour, however, was not dose‐dependent nor was the number of yawns in any of the drug treated groups significantly greater than in those treated with saline. Pretreatment (30 min) with the non‐selective muscarinic antagonists scopolamine (0.1, 0.3 or 1 mg kg−1, i.p.) and atropine (0.3, 1 or 3 mg kg−1, s.c.) but not methylatropine (1, 3 or 10 mg kg−1, s.c) significantly inhibited stretching induced by Ro 04–6790 (30 mg kg−1, i.p.). The dopamine D2‐like receptor antagonist, haloperidol (0.03, 0.1 or 0.3 mg kg−1, s.c.) given at the same time as Ro 04–6790 (30 mg kg−1, i.p.) had no effect on the stretching induced by the 5‐HT6 antagonist. These data suggest that systemic injection of the 5‐HT6 antagonist, Ro 04–6790, produces a stretching behaviour that appears to be mediated by an increase in cholinergic neurotransmission in the CNS and which could be a useful functional correlate for 5‐HT6 receptor blockade. There is no evidence for dopamine D2‐like receptor involvement in this behaviour.

Functional and Radioligand Binding Characterization of Rat 5-HT6 Receptors Stably Expressed in HEK293 Cells

We have stably expressed the rat 5-HT6 receptor in HEK293 cells at a density of > 2 pmol/mg protein, as determined in equilibrium binding studies with [3H]-LSD and [3H]-5-HT and compared the affinity of a range of compounds in competition binding experiments with either [3H]-LSD or [3H]-5-HT as radioligand. A variety of tryptamine derivatives were tested and showed a significantly higher affinity when the 5-HT6 receptor was labelled with [3H]-5-HT, whereas ergoline compounds and several antagonists had higher affinities when [3H]-LSD was used as radioligand. Subsequently we examined the ability of LSD, 5-HT and a number of tryptamine derivatives to stimulate cAMP accumulation in order to determine their agonist potency and efficacy. We observed the following rank order of potency: LSD > omega-N-methyl-5-HT approximately bufotenine approximately 5- methoxytryptamine > 5-HT > 2-methyl-5-HT approximately 5-benzyloxytryptamine approximately tryptamine > 5-carboxamidotryptamine > > 5-HTQ. LSD, lisuride, 2-methyl-5-HT, tryptamine and 5-benzyloxytryptamine behaved as partial agonists relative to 5-HT. The rank order of potency of the tryptamine compounds correlated well with their affinities determined in binding assays. In addition, we have tested a number of antagonists in this system (rank order of potency: methiothepin, clozapine, mianserin and ritanserin). This characterization of the pharmacological properties of recombinant 5-HT6 receptor will facilitate the identification of 5-HT6 receptor-mediated responses in physiological systems.

Correlation between 5-HT7 receptor affinity and protection against sound-induced seizures in DBA/2J mice

Audiogenic seizures can be induced in DBA/2J mice following intense auditory stimulation. A number of neurotransmitters, including 5-hydroxytryptamine (5-HT), are believed to be involved in mediating this effect since it has been shown previously that depletion of 5-HT or blockade of 5-HT receptors protects DBA/2J mice from these audiogenic seizures. The present study was undertaken to determine whether antagonism of the newly identified 5-HT7 receptor may protect DBA/2J mice from audiogenic seizures by attempting to correlate in vivo potency of compounds with their affinity at the 5-HT7 receptor. All compounds used in the correlation were shown to be antagonists at the 5-HT7 receptor and a statistically significant correlation was observed between 5-HT7 affinity and doses for half-maximal response (ED50) for protection of DBA/2J mice from sound-induced seizures (r = 0.80; P < 0.05). No significant correlation was observed between in vivo activity and affinity at either 5-HT1A, 5-HT2A or 5-HT2C receptors. It is also unlikely that interactions between the 5-ht5 receptor will protect DBA/2J mice from audiogenic seizures since metergoline and mesulergine which are both active in this in vivo model have no affinity for the 5-ht5 receptor. There are similarities between the pharmacology of the 5-HT7 receptor and that of the 5-HT1A receptor, however the correlation between the in vivo potency in DBA/2J mice and 5-HT1A affinity was not significant. Furthermore, the 5-HT1A receptor antagonist WAY 100135 did not protect DBA/2J mice from audiogenic seizures at doses that antagonise 5-HT1A receptor-mediated effects in mice. These data suggest that antagonism of 5-HT7 receptors may protect against audiogenic seizures in DBA/2J mice although a definitive conclusion must await studies with selective 5-HT7 antagonists.

Identification of Residues in Transmembrane Regions III and VI that Contribute to the Ligand Binding Site of the Serotonin 5‐HT6 Receptor

Abstract: We have examined the ligand binding site of the serotonin 5‐HT6 receptor using site‐directed mutagenesis. Replacing the highly conserved Asp106 in transmembrane region III by asparagine eliminated d‐[3H]lysergic acid diethylamide ([3H]LSD) binding to the mutant receptor transiently expressed in HEK293 cells. The potency of 5‐HT and LSD to stimulate adenylyl cyclase was reduced by 3,600‐ and 500‐fold, respectively, suggesting that an ionic interaction between the positively charged amino group of 5‐HT and D106 is essential for high‐affinity binding and important for receptor activation. In addition, basal cyclic AMP levels in cells expressing this mutant were increased. Mutation of a tryptophan residue one helix turn toward the extracellular side of transmembrane region III (Trp102) to phenylalanine produced significant changes in the binding affinity and potency of several ligands, consistent with a role of this residue in the formation of the ligand binding site. The exchange of two neighboring residues in the carboxy‐terminal half of transmembrane region VI (Ala287 and Asn288) for leucine and serine resulted in a mutant receptor with increased affinities (seven‐ to 30‐fold) for sumatriptan and several ergopeptine ligands. The identification of these interactions will help to improve models of the 5‐HT6 receptor ligand binding site.

Stimulation of muscarinic acetylcholine receptors increases synaptosomal free calcium concentration by protein kinase-dependent opening of L-type calcium channels.

Abstract: In synaptosomes prepared from rat cerebral cortex, free cytosolic calcium concentration ([Ca2+]i) was measured using the fluorescent dye fura‐2, Incubation of fura‐2‐loaded synaptosomes with carbachol increased [Ca2+]i in a dose‐dependent manner (1–1,000 μM), with a maximum response of 22 × 2% at approximately 100 μM and an EC50 (calculated concentration producing 50% of the maximum response) of 30 μM The effect of carbachol (100 μM) on [Ca2+], was antagonised by atropine, but not by hexamethonium (10 μM). The calculated concentration of atropine needed for 50% inhibition (IC50) was 260 nM. The rise in [Ca2+]i produced by carbachol was reduced in the absence of extrasynaptosomal Ca2+ and effectively blocked by the L‐type calcium channel blocker nifedipine (with an IC50 of 29 nM). The response to carbachol was reduced if the synaptosomes were preincubated with the protein kinase inhibitors H7 [1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine] (from 17% in the solvent control to 4%) and staurosporine (from 20% in the solvent control to 3%). These results show that stimulation of muscarinic acetylcholine receptors in synaptosomes increases [Ca2+]i by protein kinase‐dependent activation of 1,4‐dihydropyridinesensitive calcium channels.

The 5-hydroxytryptamine 6 receptor: localisation and function

The application of molecular biological techniques has revealed the existence of gene products encoding several novel putative receptors for the neurotransmitter 5-hydroxytryptamine (5-HT). For most of these novel receptors, no prior pharmacological or functional data existed, and the present challenges to pharmacologists are to identify tools to study these gene products, to determine whether or not they function as endogenous receptors and to determine potential therapeutic uses of ligands for these receptors. Here, we review work detailing the cloning and characterisation of the 5-HT6 receptor, its distribution and evidence for functional responses mediated by naturally-occurring 5-HT6 receptors.

Evaluation of smartphone-based testing to generate exploratory outcome measures in a phase 1 Parkinson's disease clinical trial: Remote PD Testing with Smartphones

Background: Ubiquitous digital technologies such as smartphone sensors promise to fundamentally change biomedical research and treatment monitoring in neurological diseases such as PD, creating a new domain of digital biomarkers.

Safety and Tolerability of Multiple Ascending Doses of PRX002/RG7935, an Anti–α-Synuclein Monoclonal Antibody, in Patients With Parkinson Disease: A Randomized Clinical Trial

Importance Aggregated &agr;-synuclein is believed to be central to the pathogenesis of Parkinson disease (PD). PRX002/RG7935 (PRX002) is a humanized monoclonal antibody designed to target aggregated forms of &agr;-synuclein, thereby inhibiting neuron-to-neuron transfer of presumed pathogenic forms of &agr;-synuclein, potentially resulting in neuronal protection and slowing disease progression. Objective To evaluate the safety and tolerability of multiple intravenous infusions of PRX002 in patients with idiopathic PD. Design, Setting, and Participants Multicenter, randomized, double-blind, placebo-controlled, multiple ascending-dose trial at 8 US study centers from July 2014 to September 2016. Eligible participants were aged 40 to 80 years with mild to moderate idiopathic PD (Hoehn and Yahr stages 1-3). Interventions Participants were enrolled into 6 ascending-dose cohorts and randomly assigned to receive PRX002 (0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10 mg/kg, 30 mg/kg, or 60 mg/kg) or placebo. Participants received 3 intravenous infusions every 4 weeks of PRX002 or placebo and were monitored during a 24-week observational period. Main Outcomes and Measures Safety and tolerability assessments included physical and neurological examinations, laboratory tests, vital signs, and adverse events. Pharmacokinetic parameters included maximum PRX002 concentration, area under the curve, and half-life. Results Of the 80 participants, most were white (97.5%; n = 78) and male (80%; n = 64); median (SD) age was 58 (8.4) years. PRX002 was generally safe and well tolerated; no serious or severe PRX002-related treatment-emergent adverse events (TEAEs) were reported. The TEAEs experienced by at least 5% of patients receiving PRX002, irrespective of relatedness to study drug, were constipation (9.1%; n = 5), infusion reaction (7.3%; n = 4), diarrhea (5.5%; n = 3), headache (5.5%; n = 3), peripheral edema (5.5%; n = 3), post–lumbar puncture syndrome (5.5%; n = 3), and upper respiratory tract infection (5.5%; n = 3). No antidrug antibodies were detected. Serum PRX002 levels increased in an approximately dose-proportional manner; mean terminal elimination half-life was similar across all doses (10.2 days). Rapid dose- and time-dependent mean reductions from baseline vs placebo in free serum &agr;-synuclein levels of up to 97% were seen after a single infusion at the highest dose (F78,284 = 1.66; P = .002), with similar reductions after 2 additional infusions. Mean cerebrospinal fluid PRX002 concentration increased with PRX002 dose and was approximately 0.3% relative to serum across all dose cohorts. Conclusions and Relevance Single and multiple doses of PRX002 were generally safe and well tolerated and resulted in robust binding of peripheral &agr;-synuclein and dose-dependent increases of PRX002 in cerebrospinal fluid, reaching cerebrospinal fluid concentrations that may be expected to engage extracellular aggregated &agr;-synuclein in the brain. Findings support the design of an ongoing phase 2 clinical study (NCT03100149). Trial Registration ClinicalTrials.gov Identifier: NCT02157714

Progressive Decline in Gray and White Matter Integrity in de novo Parkinson’s Disease: An Analysis of Longitudinal Parkinson Progression Markers Initiative Diffusion Tensor Imaging Data

Background: Progressive neuronal loss in neurodegenerative diseases such as Parkinson’s disease (PD) is associated with progressive degeneration of associated white matter tracts as measured by diffusion tensor imaging (DTI). These findings may have diagnostic and functional implications but their value in de novo PD remains unknown. Here we analyzed longitudinal DTI data from Parkinson’s Progression Markers Initiative de novo PD patients for changes over time relative to healthy control (HC) participants. Methods: Baseline and 1-year follow-up DTI MRI data from 71 PD patients and 45 HC PPMI participants were included in the analyses. Whole-brain fractional anisotropy (FA) and mean diffusivity (MD) images were compared for baseline group differences and group–by–time interactions. Baseline and 1-year changes in DTI values were correlated with changes in DTI measures and symptom severity, respectively. Results: At baseline, PD patients showed significantly increased FA in brainstem, cerebellar, anterior corpus callosal, inferior frontal and inferior fronto-occipital white matter and increased MD in primary sensorimotor and supplementary motor regions. Over 1 year PD patients showed a significantly stronger decline in FA compared to HC in the optic radiation and corpus callosum and parietal, occipital, posterior temporal, posterior thalamic, and vermis gray matter. Significant increases in MD were observed in white matter of the midbrain, optic radiation and corpus callosum, while gray matter of prefrontal, insular and posterior thalamic regions. Baseline brainstem FA white matter (WM) values predicted 1-year changes in FA white matter and MD gray matter values. White but not gray matter changes in both FA and MD were significantly associated with changes in symptom severity. Conclusion: Significant gray and white matter DTI alterations are observable at the time of PD diagnosis and expand in the first year of de novo PD to other cortical and white matter regions. This pattern of DTI changes is in line with preclinical and neuroanatomical studies suggesting that the increased spatial spread of alpha-synuclein neuropathology is the key mechanism of PD progression. Taken together, these findings suggest that DTI may serve as a sensitive biomarker of disease progression in early-stage PD.