At-Home Administration of Gantenerumab by Care Partners to People with Early Alzheimer's Disease: Feasibility, Safety and Pharmacodynamic Impact.

BACKGROUND: Monoclonal antibodies that target amyloid-beta and remove amyloid plaques can slow cognitive and functional decline in early Alzheimer's disease. Gantenerumab is a subcutaneously administered fully-human anti-amyloid-beta monoclonal antibody with highest affinity for aggregated amyloid-beta. Since the phase 3 GRADUATE trials did not meet the primary endpoint (change from baseline to Week 116 in Clinical Dementia Rating scale - Sum of Boxes), development of gantenerumab in sporadic Alzheimer's disease was stopped and all ongoing trials were terminated early due to sponsor decision. Subcutaneous administration at the clinic or at home by care partner would be an important option for other therapies in this class in order to increase flexibility and reduce overall burden. The insights obtained from the experience with gantenerumab home administration by care partner in the phase 2 GRADUATION trial will serve to guide the ongoing efforts with other anti-amyloid-beta antibodies. OBJECTIVES: To evaluate the pharmacodynamic effects on brain amyloid load of once weekly subcutaneous administration of gantenerumab and the safety and feasibility of home administration by care partners. DESIGN: Phase 2, open-label, single arm study. SETTING: Multicenter trial conducted in 33 sites in 8 countries from November 2020 to March 2023. PARTICIPANTS: Participants aged 50 to 90 with early symptomatic Alzheimer's disease (mild cognitive impairment/mild dementia due to Alzheimer's disease), and evidence of amyloid positron emission tomography positivity. INTERVENTION: Participants could receive up to 255 mg gantenerumab once-weekly, administered subcutaneously at site or at home by healthcare professionals or non-healthcare-professional care partners. MEASUREMENTS: The primary endpoint was the change from baseline to Week 52 and to Week 104 in brain amyloid load as measured by PET centiloid levels. The secondary endpoints were responses to the home administration questionnaire, plasma concentrations and safety. RESULTS: The overall number of participants enrolled was 192, with a mean (standard deviation) amyloid PET load at baseline of 101.80 (29.80) centiloids. At the time of early study termination by sponsor, 149 participants had valid Week 52 amyloid PET data (primary endpoint), and 12 participants had an early termination PET within the pre-defined time range of Week 104. The mean change in amyloid PET from baseline to Week 52 and Week 104 was -26.19 centiloids (range: -75.6-15.8; n=149) and -35.48 centiloids (range: -63.2--7.0; n=12), respectively. Responses to the home administration questionnaire at Week 52 (n=148) indicated that the majority of care partners (88-97%) considered administration of study drug at home easy (30.4%) or very easy (57.4%), and convenient (25.7%) or very convenient (70.9%). Care partners felt confident (31.1%) or very confident (62.2%) and satisfied (29.7%) or very satisfied (64.9%) with giving the injection at home. Responses by care partners at Week 36 (n=72), Week 76 (n=126) and Week 104 (n=29) and participant (patient) assessment of convenience and satisfaction at these time points were similar. There were no new safety findings associated with gantenerumab administered subcutaneously once weekly at 255 mg or safety issues associated with at-home injections by non-healthcare professional care partners. CONCLUSIONS: Once-weekly subcutaneous home administration of the anti-amyloid-beta antibody gantenerumab by non-healthcare-professional care partners to participants with early Alzheimer's disease was feasible, safe, well tolerated, and considered as a convenient option by both the care partners and participants with Alzheimer's disease. Although gantenerumab's development has been stopped due to lack of efficacy, this approach has the potential to reduce the frequency of hospital/outpatient clinic visits required for treatment with other anti-amyloid-ß antibodies and can increase flexibility of drug administration for people living with Alzheimer's disease and their families.

Continuation and effectiveness of tadalafil once daily during a 6-month observational study in erectile dysfunction: the EDATE study.

AIMS: This was the first observational study evaluating treatment continuation, effectiveness and tolerability of tadalafil 5 mg once daily (TAD-OaD) in patients who chose and paid for treatment of erectile dysfunction (ED) in routine clinical practice. METHODS: Men ≥ 18 years with ED, treated previously with phosphodiesterase type 5 (PDE5)-inhibitor on-demand (PRN) or treatment-naïve, were enrolled at 59 sites. For patients prescribed TAD-OaD at baseline (T1), change in erectile function (IIEF-EF and GAQ) was documented after 1-3 (T2) and 4-6 (T3) months. The primary outcome was the probability to switch/discontinue from TAD-OaD, estimated by Kaplan-Meier (KM) product-limit method. Changes in IIEF-EF were evaluated using a mixed model for repeated measures adjusting for patient baseline characteristics. RESULTS: Of 975 men enrolled (median age 56.8 years, 33.7% with previous PDE5-inhibitor use), 778 were prescribed TAD-OaD, 135 TAD-PRN and 62 sildenafil or vardenafil PRN. During the 6-month longitudinal observation, 107 patients (13.8% of 778) switched or discontinued TAD-OaD-treatment. KM-rates (95%CI) for continuing TAD-OaD at 2, 4 and 6 months were 94.0% (92.3, 95.7), 88.3% (85.9, 90.6) and 86.3% (83.7, 88.9), respectively. The 25th percentile of time to switch/discontinuation of TAD-OaD was estimated as 31.1 weeks (lower 95%CI 30.3 weeks). At T3, IIEF-EF scores had increased by 7.1 (LSmean; 95%CI 5.8, 8.5) points; 91.3% of patients reported improved erections. The most frequently reported AE was headache (10 patients; 1.3%); no new/unexpected safety signals were observed. CONCLUSION: Under routine conditions, and when patients were involved in treatment decision-making, more than 86% of men starting/switching to tadalafil once daily (OaD) at baseline continued tadalafil OaD treatment for ≥ 6 months.

Psychosocial outcomes after initial treatment of erectile dysfunction with tadalafil once daily, tadalafil on demand or sildenafil citrate on demand: results from a randomized, open-label study.

Initiation of ED treatment with a particular PDE5I may influence treatment-adherence and other outcomes. In this multicenter, open-label study, men with ED, naïve to PDE5I, were randomized to tadalafil 5 mg once-a-day (OaD; N=257), 10 mg on demand (PRN; N = 252) or sildenafil-citrate (sildenafil) 50 mg PRN (N = 261) for 8 weeks (dose adjustments allowed), followed by 16 weeks of pragmatic treatment (switching between PDE5I allowed). Primary outcomes (treatment-adherence) were reported previously. Here, we report effects on: Psychological and Interpersonal Relationship Scales, Self-Esteem and Relationship (SEAR) questionnaire, ED Inventory of Treatment Satisfaction (EDITS), International Index of Erectile Function (IIEF), Sexual Encounter Profile (SEP) and Global Assessment Questions (GAQ). Mixed-model for repeated measures and analysis of covariance were used to analyze changes from baseline; GAQ-responses were evaluated by logistic regression. Analyses were adjusted for treatment, country, ED-severity, baseline and baseline-by-treatment interaction. Patients randomized to tadalafil OaD or PRN reported greater improvement (least-square mean (s.e.) change) in Sexual Self-Confidence (OaD +0.90 (0.048), PRN +0.93 (0.050), vs +0.73 (0.049); P=0.006 and P=0.001) and Spontaneity (OaD +0.11 (0.035), PRN +0.13 (0.035), vs +0.02 (0.035); P = 0.044 and P = 0.010) compared with sildenafil. Improvements in GAQ and SEP responses, IIEF-EF, orgasmic function, sexual desire, overall satisfaction domains, SEAR and EDITS scores did not differ significantly between treatment groups.

Importance of phenylalanine 107 in agonist recognition by the 5-hydroxytryptamine(3A) receptor.

The 5-hydroxytryptamine (5-HT)(3) receptor is a member of the ligand-gated ion channel receptor family with significant homology to the nicotinic acetylcholine, gamma-aminobutyric acid(A), and glycine receptors. In this receptor class, the agonist binding site is formed by parts of the extracellular amino-terminal region. This study examines the effects of altering phenylalanine 107 (F107) of the 5-HT(3AL) subunit, obtained from NG108-15 cells, using site-directed mutagenesis. The wild-type (WT) and mutant receptors were expressed in HEK 293 cells and characterized using both whole-cell patch-clamp and radioligand binding. The tyrosine mutant F107Y exhibits a significantly lower affinity for the agonist 5-HT (K(i) = 203 versus 15.6 nM) and an increase of similar magnitude in the EC(50) value (10.6 versus 1.2 microM) compared with WT. The activation kinetics of the maximal currents generated by 5-HT with this mutant were markedly slower than those of the WT receptor, but application of supramaximal concentrations of the agonist markedly decreased the time to half-peak. The asparagine mutant F107N displayed a significantly higher affinity for 5-HT than the WT receptor (1.62 versus 15.6 nM), which was mirrored in direction and magnitude by changes in the EC(50) value for this agonist (0.2 versus 1.2 microM). In contrast to the WT receptor, the mutant F107N was activated by acetylcholine (EC(50) = 260 microM). The response to acetylcholine was blocked by the 5-HT(3) receptor antagonist renzapride with a similar IC(50) value as that determined against currents generated by 5-HT in the WT receptor. These data suggest that F107 is an important determinant of agonist recognition at the 5-HT(3) receptor.

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

1. 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). 2. 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. 3. 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. 4. 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.). 5. 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. 6. 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.

Identification of residues in transmembrane regions III and VI that contribute to the ligand binding site of the serotonin 5-HT6 receptor.

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.

The 5-hydroxytryptamine6 receptor-selective radioligand [3H]Ro 63-0563 labels 5-hydroxytryptamine receptor binding sites in rat and porcine striatum.

Ro 63-0563 [4-amino-N-(2,6 bis-methylamino-pyridin-4-yl)-benzene sulfonamide] is a high affinity 5-hydroxytryptamine6 (HT6) receptor antagonist with more than 100-fold selectivity for the 5-HT6 receptor compared with 69 other receptors and binding sites. The present study describes the properties of [3H]Ro 63-0563, the first selective 5-HT6 receptor radioligand. Specific binding of [3H]Ro 63-0563 (nonspecific binding defined in the presence of 10 microM methiothepin) to recombinant rat and human 5-HT6 receptors was saturable, rapid, and reversible with equilibrium dissociation constants (Kd) of 6.8 nM and 4.96 nM, respectively. The pharmacological profile of the rat 5-HT6 receptor labeled with [3H]Ro 63-0563 (methiothepin > D-lysergic acid diethylamide > clozapine approximately Ro 63-0563 > lisuride > ergotamine approximately Ro 04-6790 > 5-HT > amitriptyline approximately metergoline approximately mianserin approximately ritanserin > methysergide > mesulergine) was similar to that obtained by using either [3H]D-lysergic acide diethylamide or [3H]5-HT as radioligand. In equilibrium binding studies with rat striatal membranes, [3H]Ro 63-0563 labeled a single binding site with Kd and Bmax values of 11. 7 nM and 175 fmol/mg protein, respectively. In porcine striatal membranes, [3H]Ro 63-0563 also labeled a single binding site with Kd and Bmax values of 8.0 nM and 130 fmol/mg protein, respectively. The affinities of 14 5-HT6 receptor ligands at this binding site were similar to those found for the recombinant rat and human 5-HT6 receptor, which suggested the presence of 5-HT6 receptors in porcine striatum.

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

1. 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. 2. 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. 3 .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. 4. 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. 5. 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. 6. 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.

Involvement of 5-HT6 receptors in nigro-striatal function in rodents.

4-Amino-N-(2,4 bis-methylamino-pyrimidin-4-yl) benzene sulphonamide (Ro 04-6790) is a potent, selective and competitive antagonist for the 5-HT6 receptor which can be detected in the cerebro-spinal fluid (CSF) of rats following intraperitoneal administration. Since 5-HT6 receptor mRNA and 5-HT6 receptor-like immunoreactivity have been shown to be present in the striatum, the purpose of the present study was to evaluate the effect of 5-HT6 receptor antagonism on haloperidol- and SCH 23390-induced catalepsy in mice and on the turning behaviour of rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle. Ro 04-6790 (3, 10 and 30 mg kg(-1) i.p.) did not induce catalepsy and had no effect on catalepsy induced by either haloperidol or SCH 23390. Ro 04-6790 (3, 10 and 30 mg kg(-1) i.p.) did not itself induce rotational behaviour in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle nor did it affect the rotational behaviour induced by either L-Dopa or amphetamine. 5-HT6 receptor antagonism inhibited the rotational behaviour of 6-OHDA lesioned rats induced by treatment with the muscarinic antagonists scopolamine and atropine. The data support earlier conclusions from experiments with antisense oligonucleotides that the 5-HT6 receptor is involved in the control of acetylcholine neurotransmission in the rat brain.

The putative 5-ht6 receptor: localization and function.

Until recently, the majority of actions of the neurotransmitter 5-hydroxytryptamine (5-HT) were generally believed to be mediated by members of the 5-HT1, 5-HT2, 5-HT3 and 5-HT4 receptor families. The application of molecular cloning techniques has revealed the existence of gene products encoding several novel, putative 5-HT receptors for which little or no prior pharmacological or functional data presently exist. The present challenge to pharmacologists is to determine the physiological relevance of these gene products, establish whether or not they function as endogenous receptors, find selective agents and determine potential therapeutic uses of these compounds. Here we review work detailing the cloning and characterization of the recombinant 5-ht6 receptor, its distribution and evidence for functional responses mediated by naturally occurring 5-ht6 receptors.

Interaction of tryptamine and ergoline compounds with threonine 196 in the ligand binding site of the 5-hydroxytryptamine6 receptor.

We examined the ligand-binding site of the 5-hydroxytryptamine6 (5-HT6) receptor using site-directed mutagenesis. Interactions with residues in two characteristic positions of trans-membrane region V are important for ligand binding in several bioamine receptors. In the 5-HT6 receptor, one of these residues is a threonine (Thr196), whereas in most other mammalian 5-HT receptors, the corresponding residue is alanine. After transient expression in human embryonic kidney 293 cells, we determined the effects of the mutation T196A on [3H]d-lysergic acid diethylamide (LSD) binding and adenylyl cyclase stimulation. This mutation produced a receptor with a 10-fold reduced affinity for [3H]LSD and a 6-fold reduced affinity for 5-HT. The potency of both LSD and 5-HT for stimulation of adenylyl cyclase was also reduced by 18- and 7-fold, respectively. The affinity of other N1-unsubstituted ergolines (e.g., ergotamine, lisuride) was reduced 10-30 fold, whereas the affinity of N1-methylated ergolines (e.g., metergoline, methysergide, mesulergine) and other ligands, such as methiothepine, clozapine, ritanserin, amitriptyline, and mainserin, changed very little or increased. This indicates that in wild-type 5-HT6 receptor, Thr196 interacts with the N1 of N1-unsubstituted ergolines and tryptamines, probably forming a hydrogen bond. Based on molecular modeling, a serine residue in transmembrane region IV of the 5-HT2A receptor has previously been proposed to interact with the N1-position of 5-HT. When the corresponding residue of the 5-HT6 receptor (Ala154) was converted to serine, no change in the affinity of twelve 5-HT6 receptor ligands or in the potency of 5-HT and LSD could be detected, suggesting that this position does not contribute to the ligand binding site of the 5-HT6 receptor.

Analysis of the ligand binding site of the 5-HT3 receptor using site directed mutagenesis: importance of glutamate 106.

The 5-HT3 receptor is a ligand-gated ion channel with significant structural similarity to the nicotinic acetylcholine receptor. Several regions that form the ligand binding site in the nicotinic acetylcholine receptor are partially conserved in the 5-HT3 receptor, presumably reflecting the conserved signal transduction mechanism. Specific amino acid differences in these regions may account for their distinct ligand recognition properties. Using site-directed mutagenesis, we have replaced one of these residues, glutamate 106 (E106), with aspartate (D), asparagine (N), alanine (A) or glutamine (Q) and characterized the ligand-binding and electrophysiological properties of the mutant receptors after transient expression in HEK-293 cells. The affinity for the selective 5-HT3 receptor antagonist [3H]GR65630 was decreased 14-fold in the mutant E106D (Kd = 3.69 +/- 0.32 nM) when compared to wildtype (WT, E106) 5-HT3 receptor (0.27 +/- 0.03 nM), while the affinity for E106N was unchanged (0.42 +/- 0.07 nM, means +/- SEM, n = 3-10). Decreased affinities for both E106D and E106N were observed for the antagonists granisetron, ondansetron and renzapride and for the agonists 5-HT (130- and 30-fold) and 2-methyl-5-HT (250- and 20-fold), respectively. Both mutants still formed 5-HT-activatable ion channels, but the high Hill coefficient of the concentration effect curves in wildtype (2.0) was decreased to unity in both cases. The EC50 of 5-HT was increased seven-fold in E106N (8.7 microM) when compared to wildtype (1.2 microM), but unchanged in E106D, and the potency of the antagonist ondansetron for both mutants was decreased. E106A and E106Q expressed poorly preventing a detailed characterization. These data suggest that E106 contributes to the ligand-binding site of the 5-HT3 receptor and may form an ionic or hydrogen bond interaction with the primary ammonium group of 5-HT.

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.

Ultrastructure of the 5-hydroxytryptamine3 receptor.

We have determined the ultrastructure of 5-hydroxytryptamine3 (5-HT3) serotonin receptors purified from NG108-15 mouse neuroblastoma x rat glioma cells by electron microscopic examination of receptor particles embedded in uranyl acetate stain and metal replicas of rapidly frozen receptors. The 5-HT3 receptor can be modelled as a cylinder 11 nm in length and 8 nm in diameter with a closed end and a central cavity 3 nm in diameter. Analysis of the rotational symmetry of single receptor particles indicates that the 5-HT3 receptor is composed of five subunits arranged symmetrically around a central cavity. Together with evidence obtained for related proteins in other studies using ultrastructural, biochemical, or electrophysiological methods, our observations suggest that all members of the ligand-gated ion channel superfamily may possess a pentameric quaternary structure.

Molecular properties of 5-hydroxytryptamine3 receptor-type binding sites purified from NG108-15 cells.

5-Hydroxytryptamine3 (5-HT3) receptor-type binding sites were solubilised from NG108-15 mouse neuroblastoma x rat glioma hybrid cells using five different detergents [n-octyl-beta-D-glucoside, Triton X-100, 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS), sodium cholate, and deoxycholate] and the solubilisation efficiencies compared. The equilibrium binding, kinetic properties, and pharmacological profile of solubilised binding sites were similar to those of 5-HT3 receptor-type binding sites (5-HT3R) in membrane preparations determined using [3H]GR65630. The solubilised binding sites were purified using an affinity column constructed by coupling the high-affinity antagonist GR119566X to an Affi-Gel 15 resin. The affinity of purified 5-HT3R for [3H]-GR65630 was reduced threefold compared to the crude soluble preparation, but the pharmacological profile was similar. The sedimentation coefficient of the purified protein (11S, detergent: CHAPS) was determined by sucrose density gradient centrifugation. The apparent molecular mass of the detergent/binding site complex (370 kDa) was determined by size exclusion chromatography in the presence of n-dodecyl-beta-D-maltoside. Gel electrophoresis of the purified protein revealed bands at apparent molecular masses of 36, 40, 50, and 76 kDa. Electron microscopy of the negatively stained purified protein showed the presence of round particles of 8-9 nm diameter with a 2-nm stained pit in the centre, closely resembling the doughnut shapes described for nicotinic acetylcholine receptors.

5-HT3 receptors in NG108-15 neuroblastoma x glioma cells: effect of the novel agonist 1-(m-chlorophenyl)-biguanide.

The effect of the novel agonist, 1-(m-chlorophenyl)-biguanide (mCPBG) was examined on 5-HT3 receptors in NG108-15 mouse neuroblastoma x rat glioma hybrid cells, using whole-cell voltage-clamp and radioligand binding on intact cells. Electrophysiological studies showed that mCPBG is a partial agonist, with an EC50 of 3.1 microM. Displacement of the selective 5-HT3 receptor antagonist [3H]GR65630 by mCPBG revealed a Ki of 14.2 nM. The study suggests that mCPBG may have a high affinity for desensitized 5-HT3 receptors and also revealed some differences between 5-HT3 receptors in NG108-15 and N1E-115 cells.