Effects of sarizotan in animal models of ADHD: challenging pharmacokinetic-pharmacodynamic relationships.

Sarizotan 1-[(2R)-3,4-dihydro-2H-chromen-2-yl]-N-[[5-(4-fluorophenyl) pyridin-3-yl]methyl] methenamine, showed an in vivo pharmaco-EEG profile resembling that of methylphenidate which is used in attention deficit/hyperactivity disorder (ADHD). In turn, we tested sarizotan against impulsivity in juvenile rats measuring the choice for large delayed vs. a small immediate reward in a T-maze and obtained encouraging results starting at 0.03 mg/kg (plasma levels of ~11 nM). Results from rats treated neonatally with 6-hydroxydopamine (6-OHDA), also supported anti-ADHD activity although starting at 0.3 mg/kg. However, microdialysis studies revealed that free brain concentration of sarizotan at active doses were below its affinity for 5-HT1A receptors, the assumed primary target. In contrast, electrophysiological experiments in mid-brain Raphé serotonergic cells paralleled by plasma sampling showed that there was ~60% inhibition of firing rate­indicating significant activation of 5-HT1A receptors­at a plasma concentration of 76 nM. In line with this, we observed that sarizotan concentrations in brain homogenates were similar to total blood levels but over 500 fold higher than free extracellular fluid (ECF) concentrations as measured using brain microdialysis. These data suggest that sarizotan may have potential anti-ADHD effects at low doses free of the previously reported side-effects. Moreover, in this case a classical pharmacokinetic-pharmacodynamic relationship based on free brain concentrations seems to be less appropriate than target engagement pharmacodynamic readouts.

Using cholinergic M1 receptor positive allosteric modulators to improve memory via enhancement of brain cholinergic communication.

Benzylquinolone carboxylic acid (BQCA) is a recently described cholinergic muscarinic M(1) receptor positive allosteric modulator having potential as cognitive enhancer in dementia. The present study focused on the characterisation of BQCA's mode of action in relation to positive effects on memory and side-effects in an animal model. To get insight into this mode of action, in vitro receptor potency/left shift experiments in cells stably expressing the rat's M(1) receptor were performed. They revealed an inflection point value of BQCA corresponding to 306nM, and potentiation of the agonist response up to 47-fold in presence of 10µM of BQCA. In vivo, brain microdialysis showed a maximal brain level of 270nM, 40min after i.p. administration at 10mg/kg. Based on in vitro data obtained with this dose, it can be concluded that BQCA reaches brain levels which should potentiate the agonist response about 4-fold. Behavioural data confirmed that BQCA used at 10mg/kg attenuated scopolamine-induced memory deficit in a spontaneous alternation task. Moreover, BQCA showed no side effect at 10mg/kg and above in spontaneous locomotion and salivation tests. The profile of BQCA observed in the present study displays a clear advantage over the M(1)-M(3) agonist cevimeline. The present data show the therapeutic potential of the M(1) receptor positive allosteric modulator BQCA for the treatment of memory deficits observed in Alzheimer's disease.

Effect of 5-HT5A antagonists in animal models of schizophrenia, anxiety and depression.

The few available data on the pharmacological effect of 5-HT5A receptors suggest that antagonists may have anxiolytic, antidepressant and antipsychotic activity. The aim of our study was to verify these suggestions in relevant animal models. Two 5-HT5A antagonist ligands, SB-699551-A (N-[2-(dimethylamino)ethyl]-N-[[4'-[[(2-phenylethyl)amino]methyl][1,1'-biphenyl]-4-yl]methyl]cyclopentanepropanamide dihydrochloride) (3-60 mg/kg, intraperitoneally) and A-843277 (N-(2,6-dimethoxybenzyl)-N'[4-(4-fluorophenyl)thiazol-2-yl]guanidine) (3-30 mg/kg, intraperitoneally), were examined in the open-field test, in a foot-shock-induced ultrasonic vocalization test, in the forced swim test (FST) and in the amphetamine-induced and phencyclidine-induced hyperlocomotion tests to examine their effect on general behavioural patterns, and their anxiolytic-like, antidepressant-like and antipsychotic-like properties, respectively. In the open-field test, SB-699551-A induced sedation and A-843277 induced writhing. In the ultrasonic vocalization test, SB-699551-A reduced vocalizations, whereas A-843277 was ineffective. In the FST, SB-699551-A was ineffective and A-843277 reduced immobility, but only at the highest dose. In the amphetamine-induced and phencyclidine-induced hyperlocomotion test, both compounds were ineffective. SB-699551-A showed an anxiolytic-like property in the ultrasonic vocalization test; however, this compound has a sedative effect. A-843277 showed an antidepressant-like property in the FST, but its immobility-reducing effect may also be a consequence of abdominal irritation. Consequently, further investigations are required to define the therapeutic potential of 5-HT5A receptor ligands in anxiety, depression and schizophrenia models.

Potency, voltage-dependency, agonist concentration-dependency, blocking kinetics and partial untrapping of the uncompetitive N-methyl-D-aspartate (NMDA) channel blocker memantine at human NMDA (GluN1/GluN2A) receptors.

Both the clinical tolerability and the symptomatic effects of memantine in the treatment of Alzheimer's disease have been attributed to its moderate affinity (IC(50) around 1 microM at -70 mV) for NMDA receptor channels and associated fast, double exponential blocking/unblocking kinetics and strong voltage-dependency. Most of these biophysical data have been obtained from rodent receptors. Some substances show large species-specific differences, so using human rather than rodent receptors and tissue may highlight important differences in the effects of drugs. In the present study we compared the potency of memantine, ketamine and (+)MK-801 in binding to NMDA receptors in post-mortem human cortical tissue and to antagonize intracellular Ca(2+) responses of human GluN1/GluN2A receptors expressed in HEK-293 cells. In addition, the biophysical properties of memantine and ketamine were compared using patch clamp recordings from these cells. Memantine was confirmed to be a moderate affinity (IC(50) at -70 mV of 0.79+/-0.02 microM, Hill=0.92+/-0.02), strongly voltage-dependent (delta=0.90+/-0.09) uncompetitive antagonist of human GluN1/GluN2A receptors. Moreover, the rapid double exponential blocking kinetics (e.g. at 10 microM - onset tau(fast)=273+/-25 ms (weight 69%), onset tau(slow)=2756+/-296 ms, offset tau(fast)=415+/-82 ms (weight 38%) offset tau(slow)=5107+/-1204 ms) and partial untrapping (around 20%) previously reported for memantine on rodent receptors were confirmed for human receptors. Ketamine showed similar potency (IC(50) at -70 mV of 0.71+/-0.03 microM, Hill=0.84+/-0.02) but somewhat less pronounced voltage-dependency (delta=0.79+/-0.04), slower, single exponential kinetics (ketamine: k(on)=0.15+/-0.05 x 10(6)M(-1)s(-1), k(off)=0.22+/-0.05 s(-1)c.f. memantine following normalization k(on)=0.32+/-0.11 x 10(6)M(-1)s(-1), k(off)=0.53+/-0.10s(-1)) and was fully trapped. The present data closely match previously reported data from studies in rodent receptors and suggest that the proposed mechanism of action of memantine in Alzheimer's disease as a fast, voltage-dependent open-channel blocker of NMDA receptors can be confirmed for human NMDA receptors.

Blocking kinetics of memantine on NR1a/2A receptors recorded in inside-out and outside-out patches from Xenopus oocytes.

Previous experiments on primary cultures of hippocampal/cortical neurones revealed that the block and unblock of N-Methyl-D-Aspartate (NMDA) receptor channels by memantine showed double exponential kinetics and that the offset kinetics following a voltage-step were much faster than following a concentration jump. There are, however, two major problems when using such cultured primary neurones for these experiments (1) the almost certain expression of heterogeneous NMDA receptor subunits which could underlie double exponential kinetics due to different potencies at receptor subtypes and (2) slow space- and concentration-clamp due to neuronal morphology which could mask even faster kinetics. Therefore, we performed similar experiments with Xenopus oocytes exclusively expressing one NMDA receptor type (NR1a/2A) at high levels which allowed recordings from membrane patches with large currents. The use of inside-out patches for voltage-step and outside-out patches in combination with a piezo driven fast application system largely negated potential space- and concentration-clamp problems. Block and unblock of the NMDA receptor by memantine after both voltage jump and concentration jumps showed triple exponential kinetics. The fast onset kinetics of NMDA receptor channel block following both concentration-clamp and voltage jumps from +70 to -70 mV were similar. In contrast, offset kinetics after a voltage-step from -70 to +70 mV were much faster than following a concentration jump at the holding potential of -70 mV. These results provide further support for the hypothesis that rapid relief of block via strong synaptic membrane depolarisation underlies the good therapeutic profile of memantine.

Memantine does not show intracellular block of the NMDA receptor channel.

Mg2+ is known to gain access to the NMDA receptor channel from both the extra- and intracellular compartments. Memantine, being an amphiphilic substance, reaches intracellular concentrations of approximately 30 microM, which are much higher than therapeutic extracellular concentrations ( approximately 1 microM). We therefore investigated whether memantine can also block the NMDA receptor channel from the intracellular compartment. NR1a/NR2A receptors were expressed in Xenopus oocytes and in classical two electrode voltage-clamp recordings, voltage-ramps from -100 to +100 mV confirmed moderate inward rectification of NR1a/NR2A receptor control responses at positive membrane potentials above +40 mV. Patch clamp recordings from these same cells (applying 100 microM glutamate and 1 mM Mg2+) revealed similar rectification at positive potentials in cell-attached mode which disappeared after pulling an inside-out patch. Application of 1 mM Mg2+ to the intracellular side of the receptor re-introduced the rectification seen in cell-attached mode, and 5 mM Mg2+ produced much more pronounced block. In contrast, 30 microM memantine was completely unable to block the NMDA receptor from the intracellular compartment. In conclusion, intracellular block of the NMDA receptor, as reported for Mg2+, is not of significance for the therapeutic effects of memantine.

Positive and negative modulation of group I metabotropic glutamate receptors.

A discriminating pharmacophore model for noncompetitive metabotropic glutamate receptor antagonists of subtype 1 (mGluR1) was developed that facilitated the discovery of moderately active mGluR1 antagonists. One scaffold was selected for the design of several focused libraries where different substitution patterns were introduced. This approach facilitated the discovery of potent mGluR1 antagonists, as well as positive and negative mGluR5 modulators, because both receptor subtypes share similar binding pockets. For mGluR1 antagonists, a homology model of the mGlu1 receptor was established, and a putative binding mode within the receptor's transmembrane domain was visualized.

Water-soluble rylene dyes as high-performance colorants for the staining of cells.

The synthesis of perylene and terrylene chromophores carrying a single poly(ethylene oxide) chain is presented. These chromophores reveal a strong solvatochromic behavior: High fluorescence in nonpolar solvents and weak fluorescence in polar solvents which is mainly attributed to aggregation. Therefore, such chromophores are attractive candidates as sensitive fluorescent probes reflecting the polarity of their environment. In particular, their suitability for the staining of cellular membranes is presented in detail.

Block of AMPA receptor desensitization by a point mutation outside the ligand-binding domain.

Desensitization of ionotropic glutamate receptors (GluRs), specifically the AMPA receptor subtype, shapes the postsynaptic response at certain synapses in the brain. All known mechanisms that alter desensitization, either pharmacological or mutational, are associated with the ligand-binding domain. Here we report that substitution of a conserved positively charged arginine (R) with a negatively charged glutamate in the linker between the pore-forming M3 segment and the S2 lobe, a region outside the ligand-binding domain, blocks desensitization in homomeric AMPA receptors composed of GluR-B(i) subunits. A charge-reversing substitution of a glutamate adjacent to this conserved R enhanced desensitization, consistent with these effects attributable to electrostatics. Homologous substitutions of the conserved R in GluR-B(o), GluR-A(i) and the kainate receptor GluR-6 subunits produced comparable but less visible effects on desensitization. Subunit specificity was also apparent for accessibility of substituted cysteines in the M3-S2 linker, suggesting that this part of the channel is not structurally identical in different GluRs. Additionally, reactivity with a sulfhydryl-specific reagent was state dependent, suggesting that the conformations of the nonconducting closed and desensitized states are different at the level of the M3-S2 linker. Our results therefore represent the first identification of elements outside the ligand-binding domain affecting desensitization in non-NMDA receptor channels and suggest that electrostatic interactions involving charged residues in the M3-S2 linker influence channel gating in a subunit- and subtype-specific manner.

Extracellular vestibule determinants of Ca2+ influx in Ca2+-permeable AMPA receptor channels.

At certain synapses in the brain, Ca2+-permeable AMPA receptor (AMPAR) channels represent an important pathway for synaptically controlled Ca2+ entry. However, the molecular determinants of this Ca2+ influx are poorly defined. In NMDA receptor (NMDAR) channels, where the influx is much greater, the extracellular vestibule, specifically the M3 segment and regions C-terminal to it in the NR1 subunit, contains elements critical to their high Ca2+ influx under physiological conditions. We therefore investigated the contribution of homologous positions in AMPAR as well as kainate receptor (KAR) subunits to the process of Ca2+ influx. Substitutions of a conserved asparagine (N) in M3 of AMPAR GluR-B(Q) channels strongly attenuated Ca2+ permeability measured using reversal potentials under biionic conditions and fractional Ca2+ currents recorded under physiological conditions. Hence, as in NMDAR channels, the conserved N makes a significant contribution to Ca2+ influx in AMPAR channels. In addition, C-terminal to M3, substitutions of negatively (glutamate, E) or positively (arginine, R) charged residues also altered Ca2+ influx. However, in contrast to charged residues occupying homologous positions in NMDAR channels, these effects were about equal and opposite suggesting that this ER in AMPARs does not contribute significantly to the mechanism of Ca2+ influx. Opposite charge substitutions of two negative residues C-terminal to M3 in KAR GluR-6(Q) subunits had no effect on Ca2+ permeability. We conclude that the different contribution of residues C-terminal to M3 to Ca2+ permeation in NMDAR and non-NMDAR channels reflects a different positioning of these residues relative to the tip of the M2 loop.

Voltage and concentration dependence of Ca(2+) permeability in recombinant glutamate receptor subtypes.

The channels associated with glutamate receptor (GluR) subtypes, namely N-methyl-D-aspartate receptors (NMDARs), and Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) and kainate receptors (KARs), are to varying degrees permeable to Ca(2+). To compare the mechanism of Ca(2+) influx, we measured Ca(2+) permeability relative to that of Na(+) (P(Ca)/P(Na)) using fractional Ca(2+) currents (P(f)) and reversal potential measurements over a wide voltage and Ca(2+) concentration range in recombinant NMDAR NR1-NR2A, AMPAR GluR-A(Q) and KAR GluR-6(Q) channels. For NR1-NR2A channels, P(Ca)/P(Na) derived from P(f) measurements was voltage independent but showed a weak concentration dependence. A stronger concentration dependence was found when P(Ca)/P(Na) was derived from changes in reversal potentials on going from a Na(+) reference solution to a solution with Ca(2+) as the only permeant ion ('biionic' condition). In contrast, P(Ca)/P(Na) was concentration independent when derived from changes in reversal potentials on going from a Na(+) reference solution to the same solution with added Ca(2+) ('high monovalent' condition). For GluR-A(Q) channels, P(Ca)/P(Na) derived from all three approaches was concentration independent, and for the reversal potential-based approaches were of comparable magnitude. Their most distinctive property was that P(Ca)/P(Na) derived from P(f) measurements was strongly voltage dependent. For GluR-6(Q) channels, P(Ca)/P(Na) derived from P(f) measurements was weakly voltage dependent. On the other hand, P(Ca)/P(Na) derived from all three approaches was the most strongly concentration dependent of any GluR subtype and, except for low Ca(2+) concentrations, the values were of comparable magnitude. Thus, the three Ca(2+)-permeable GluR subtypes showed unique patterns of Ca(2+) permeability, indicating that distinct biophysical and molecular events underlie Ca(2+) influx in each subtype.