Lise T. Brennum

Escitalopram, the S-(+)-enantiomer of citalopram, is a selective serotonin reuptake inhibitor with potent effects in animal models predictive of antidepressant and anxiolytic activities

ObjectiveThe pharmacological profile of escitalopram, the S-(+)-enantiomer of citalopram, was studied and compared with citalopram and the R-(−)-enantiomer, R-citalopram.MethodsInhibition of the serotonin transporter (5-HTT) was studied in COS-1 cells expressing the human 5-HTT (h-5-HTT) and in rat brain synaptosomes. In vitro selectivity was studied relative to noradrenaline transporter (NAT) and dopamine transporter (DAT) function in rat brain synaptosomes, and affinities for other binding sites were determined. In vivo 5-HT activity was measured as inhibition of neuronal firing rate in rat dorsal raphe nucleus (DRN) and enhancement of 5-hydroxytryptophan (5-HTP)-induced behaviour (mouse and rat). Furthermore, studies were conducted in models of antidepressant (mouse forced-swim test), anxiolytic [foot-shock-induced ultrasonic vocalization (USV) in adult rats and mouse black and white box] and anti-aggressive activity (socially isolated mice).ResultsEscitalopram inhibited 5-HTT functions approximately 2 times more potently than citalopram and at least 40 times more potently than R-citalopram. Escitalopram showed insignificant activity at other monoamine transporters and 144 other binding sites. Escitalopram inhibited 5-HT neuronal firing in DRN and potentiated 5-HTP-induced behaviours more potently than citalopram; R-citalopram was inactive. Escitalopram and citalopram, but not R-citalopram, reduced forced-swimming-induced immobility and facilitated exploratory behaviour in the black and white box. Escitalopram and citalopram inhibited USV potently; R-citalopram was several times less potent. Escitalopram, citalopram and R-citalopram inhibited aggressive behaviour weakly. Escitalopram and citalopram had very potent anti-aggressive effects when co-administered with l-5-HTP.ConclusionEscitalopram is a very selective 5-HT reuptake inhibitor. It is more potent than its racemate citalopram and is effective in animal models predictive of antidepressant and anxiolytic activities.

Pharmacological effects of Lu AA21004: a novel multimodal compound for the treatment of major depressive disorder.

1-[2-(2,4-Dimethylphenyl-sulfanyl)-phenyl]-piperazine (Lu AA21004) is a human (h) serotonin (5-HT)3A receptor antagonist (Ki = 3.7 nM), h5-HT7 receptor antagonist (Ki = 19 nM), h5-HT1B receptor partial agonist (Ki = 33 nM), h5-HT1A receptor agonist (Ki = 15 nM), and a human 5-HT transporter (SERT) inhibitor (Ki = 1.6 nM) (J Med Chem 54:3206–3221, 2011). Here, we confirm that Lu AA21004 is a partial h5-HT1B receptor agonist [EC50 = 460 nM, intrinsic activity = 22%] using a whole-cell cAMP-based assay and demonstrate that Lu AA21004 is a rat (r) 5-HT7 receptor antagonist (Ki = 200 nM and IC50 = 2080 nM). In vivo, Lu AA21004 occupies the r5-HT1B receptor and rSERT (ED50 = 3.2 and 0.4 mg/kg, respectively) after subcutaneous administration and is a 5-HT3 receptor antagonist in the Bezold-Jarisch reflex assay (ED50 = 0.11 mg/kg s.c.). In rat microdialysis experiments, Lu AA21004 (2.5–10.0 mg/kg s.c.) increased extracellular 5-HT, dopamine, and noradrenaline in the medial prefrontal cortex and ventral hippocampus. Lu AA21004 (5 mg/kg per day for 3 days; minipump subcutaneously), corresponding to 41% rSERT occupancy, significantly increased extracellular 5-HT in the ventral hippocampus. Furthermore, the 5-HT3 receptor antagonist, ondansetron, potentiated the increase in extracellular levels of 5-HT induced by citalopram. Lu AA21004 has antidepressant- and anxiolytic-like effects in the rat forced swim (Flinders Sensitive Line) and social interaction and conditioned fear tests (minimal effective doses: 7.8, 2.0, and 3.9 mg/kg). In conclusion, Lu AA21004 mediates its pharmacological effects via two pharmacological modalities: SERT inhibition and 5-HT receptor modulation. In vivo, this results in enhanced release of several neurotransmitters and antidepressant- and anxiolytic-like profiles at doses for which targets in addition to the SERT are occupied. The multimodal activity profile of Lu AA21004 is distinct from that of current antidepressants.

R-citalopram functionally antagonises escitalopram in vivo and in vitro: evidence for kinetic interaction at the serotonin transporter

Clinical observations with the selective serotonin reuptake inhibitor (SSRI), S‐citalopram, indicate that S‐citalopram is more efficacious and produces earlier symptom relief than RS‐citalopram. Since R‐citalopram is at least 20‐fold weaker than S‐citalopram as inhibitor of the 5‐HT transporter (SERT) in preclinical studies, the clinical data suggest an unexpected antagonistic interaction between the two enantiomers. We therefore characterised the interaction of R‐ and S‐citalopram with the SERT in in vivo and in vitro assays. In both behavioural (potentiation of 5‐hydroxytryptophan (5‐HTP)‐induced behaviour) and electrophysiological studies (inhibition of 5‐HT‐elicited ion currents in Xenopus oocytes expressing the human SERT (hSERT) R‐citalopram inhibited the effects of S‐citalopram in a dose‐dependent manner. With S‐citalopram : R‐citalopram ratios of 1 : 2 and 1 : 4, 5‐HTP potentiation was significantly smaller than with S‐citalopram alone. emsp;R‐citalopram did not antagonise the effects of another SSRI (fluoxetine) in either behavioural or electrophysiological studies. In oocytes, inhibition of hSERT‐mediated currents by R‐citalopram was almost completely reversible and characterised by fast on‐ and off‐sets of action. In contrast, the off‐set for S‐citalopram was 35‐fold slower than for R‐citalopram. Kinetic analysis of the oocyte experiments suggests that S‐citalopram binding to SERT induces a long‐lasting, inhibited state of the transporter and that coapplication of R‐citalopram partially relieves SERT of this persistent inhibition. We propose that the kinetic interaction of R‐ and S‐citalopram with SERT is a critical factor contributing to the antagonistic effects of R‐citalopram on S‐citalopram in vitro and in vivo.

Using pharmacokinetic-pharmacodynamic modelling as a tool for prediction of therapeutic effective plasma levels of antipsychotics

In the rat, selective suppression of conditioned avoidance response has been widely reported as a test with high predictive validity for antipsychotic efficacy. Recent studies have shown that the relationship between dopamine D2 receptor occupancy and the suppression of conditioned avoidance response behaviour correlates well with the relationship between human dopamine D2 receptor occupancy and clinical effect. The aim of the present study was to evaluate how pharmacokinetic/pharmacodynamic (PK/PD) predictions of therapeutic effective steady-state plasma levels by means of conditioned avoidance response behaviour in rodents, correlate with clinically relevant plasma exposure for the classical antipsychotic drug haloperidol and four second generation antipsychotics: sertindole, clozapine, risperidone and olanzapine, including selected metabolites. In order to confirm the validity of the present conditioned avoidance response procedure, in vivo striatal dopamine D2 receptor occupancy was determined in parallel using 3H-raclopride as the radioligand. The PK/PD relationship was established by modelling the time-response and time-plasma concentration data. We found the order of dopamine D2 receptor occupancy required to suppress conditioned avoidance response behaviour according to EC50 measurements to be sertindole (+dehydrosertindole)=dehydrosertindole=paliperidone (the metabolite of risperidone)=haloperidol=olanzapine>risperidone>>clozapine. Overall, a good agreement was observed between the rat dopamine D2 receptor occupancy levels providing 50% response in the conditioned avoidance response test and the dopamine D2 receptor occupancy levels reported from responding schizophrenic patients treated with antipsychotics. Predictions of therapeutically effective steady-state levels for sertindole (+dehydrosertindole) and olanzapine were 3-4-fold too high whereas for haloperidol, clozapine and risperidone the predicted steady-state EC50 in conditioned avoidance responding rats correlated well with the therapeutically effective plasma levels observed in patients. Accordingly, the proposed PK/PD model may act as a guide for determining effective plasma concentrations of potential antipsychotics in the clinical setting and thereby accelerating the overall drug development process.

Oral tremor induced by the muscarinic agonist pilocarpine is suppressed by the adenosine A2A antagonists MSX-3 and SCH58261, but not the adenosine A1 antagonist DPCPX

Tremulous jaw movements in rats, which can be induced by dopamine (DA) antagonists, DA depletion, and cholinomimetics, have served as a useful model for studies of tremor. Although adenosine A(2A) antagonists can reduce the tremulous jaw movements induced by DA antagonists and DA depletion, there are conflicting reports about the interaction between adenosine antagonists and cholinomimetic drugs. The present studies investigated the ability of adenosine antagonists to reverse the tremorogenic effect of the muscarinic agonist pilocarpine. While the adenosine A(2A) antagonist MSX-3 was incapable of reversing the tremulous jaw movements induced by the 4.0mg/kg dose of pilocarpine, both MSX-3 and the adenosine A(2A) antagonist SCH58261 reversed the tremulous jaw movements elicited by 0.5mg/kg pilocarpine. Systemic administration of the adenosine A(1) antagonist DPCPX failed to reverse the tremulous jaw movements induced by either an acute 0.5mg/kg dose of the cholinomimetic pilocarpine or the DA D2 antagonist pimozide, indicating that the tremorolytic effects of adenosine antagonists may be receptor subtype specific. Behaviorally active doses of MSX-3 and SCH 58261 showed substantial in vivo occupancy of A(2A) receptors, but DPCPX did not. The results of these studies support the use of adenosine A(2A) antagonists for the treatment of tremor.

Prediction of clinical response based on pharmacokinetic/pharmacodynamic models of 5‐hydroxytryptamine reuptake inhibitors in mice

Bridging the gap between preclinical research and clinical trials is vital for drug development. Predicting clinically relevant steady‐state drug concentrations (Css) in serum from preclinical animal models may facilitate this transition. Here we used a pharmacokinetic/pharmacodynamic (PK/PD) modelling approach to evaluate the predictive validity of 5‐hydroxytryptamine (5‐HT; serotonin) transporter (SERT) occupancy and 5‐hydroxytryptophan (5‐HTP)‐potentiated behavioral syndrome induced by 5‐HT reuptake inhibitor (SRI) antidepressants in mice.

Depression and poor sleep: the effect of monoaminergic antidepressants in a pre-clinical model in rats.

The effects of five antidepressants (escitalopram, paroxetine, duloxetine, venlafaxine, and reboxetine) on the sleep architecture were investigated in freely moving rats in the light phase of a 12:12 h light:dark cycle following a single i.p. dose of antidepressant. Overall, paroxetine and escitalopram exhibited the least sleep disruptive profiles, whereas duloxetine, venlafaxine, and reboxetine increased the time spent awake and suppressed paradoxical sleep. Analysis of the EEG at 1 h intervals revealed only subtle differences from the overall picture. The effect of venlafaxine on disruption of sleep architecture could not be readily explained by its in vitro serotonin (5-HT) and noradrenaline (NA) reuptake inhibitory potencies. In vivo microdialysis experiments in the ventral hippocampus of freely moving rats revealed that venlafaxine affected the 5-HT and NA systems equally at the doses tested. Duloxetine (7.7 mg/kg) induced maximal blockade of the 5-HT transporter and duloxetine 7.7 mg/kg also modulated the noradrenaline system. Thus, in this animal model, the enhancement of noradrenergic activity is more disruptive on the sleep architecture than enhancement of serotonergic activity.

Selectivity of 3H‐MADAM binding to 5‐hydroxytryptamine transporters in vitro and in vivo in mice; correlation with behavioural effects

Binding of the novel radioligand 3H‐2‐(2‐dimethylaminomethyl‐phenylsulphanyl)‐5‐methyl‐phenylamine (3H‐MADAM) to the serotonin transporter (SERT) was used to characterise a range of selective serotonin re‐uptake inhibitors (SSRIs) in vitro and in vivo. 3H‐MADAM bound with high affinity in a saturable manner to both human SERT expressed in CHO cells (Kd=0.20 nM (pKd=9.74±0.12), Bmax=35±4 fmol mg−1 protein) and mouse cerebral cortex membranes (Kd=0.21 nM (pKd=9.66±0.10), Bmax=50±24 fmol mg−1 protein). Binding of 3H‐MADAM was highly selective for SERT in vitro as demonstrated by the in vitro profile of MADAM tested at 75 different receptors, ion channels and transporters. This was further substantiated by the pharmacological profile of the binding. Hence, the binding of 3H‐MADAM was potently inhibited by SSRIs but not by selective inhibitors of noradrenaline transport and dopamine transport. Likewise, a 5‐HT2A/2C receptor antagonist did not inhibit 3H‐MADAM binding. 3H‐MADAM binding in vivo was inhibited only by compounds which also inhibited the binding of 3H‐MADAM in vitro (the SSRIs, mixed SERT/noradrenaline transport inhibitors and clomipramine), confirming the selectivity of 3H‐MADAM for SERT also in vivo. Moreover, compounds effective in inhibiting 3H‐MADAM binding were the only ones found to be active in the mouse 5‐HTP potentiation test confirming the model as a behavioural correlate to in vivo 5‐HT uptake. Finally, it was found that a SERT occupancy of 85–95% was necessary to produce 50% of the maximum behavioural response (ED50).

Discovery and Development of 11C-Lu AE92686 as a Radioligand for PET Imaging of Phosphodiesterase10A in the Brain

Phosphodiesterase 10A (PDE10A) plays a key role in the regulation of brain striatal signaling, and several pharmaceutical companies currently investigate PDE10A inhibitors in clinical trials for various central nervous system diseases. A PDE10A PET ligand may provide evidence that a clinical drug candidate reaches and binds to the target. Here we describe the successful discovery and initial validation of the novel radiolabeled PDE10A ligand 5,8-dimethyl-2-[2-((1-11C-methyl)-4-phenyl-1H-imidazol-2-yl)-ethyl]-[1,2,4]triazolo[1,5-a]pyridine (11C-Lu AE92686) and its tritiated analog 3H-Lu AE92686. Methods: Initial in vitro experiments suggested Lu AE92686 as a promising radioligand, and the corresponding tritiated and 11C-labeled compounds were synthesized. 3H-Lu AE92686 was evaluated as a ligand for in vivo occupancy studies in mice and rats, and 11C-Lu AE92686 was evaluated as a PET tracer candidate in cynomolgus monkeys and in humans. Results: 11C-Lu AE92686 displayed high specificity and selectivity for PDE10A-expressing regions in the brain of cynomolgus monkeys and humans. Similar results were found in rodents using 3H-Lu AE92686. The binding of 11C-Lu AE92686 and 3H-Lu AE92686 to striatum was completely and dose-dependently blocked by the structurally different PDE10A inhibitor 2-[4-(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)-phenoxymethyl]-quinoline (MP-10) in rodents and in monkeys. In all species, specific binding of the radioligand was seen in the striatum but not in the cerebellum, supporting the use of the cerebellum as a reference region. The binding potentials (BPND) of 11C-Lu AE92686 in the striatum of both cynomolgus monkeys and humans were evaluated by the simplified reference tissue model with the cerebellum as the reference tissue, and BPND was found to be high and reproducible—that is, BPNDs were 6.5 ± 0.3 (n = 3) and 7.5 ± 1.0 (n = 12) in monkeys and humans, respectively. Conclusion: Rodent, monkey, and human tests of labeled Lu AE92686 suggest that 11C-Lu AE92686 has great potential as a human PET tracer for the PDE10A enzyme.

Short and efficient syntheses of analogues of WAY-100635: new and potent 5-HT1A receptor antagonists.

Simple syntheses of four new and potent analogues of the 5-HT1A receptor ligand, WAY-100635 are described, namely the 6-(pyridinyl)-bromo-, the 6-(pyridinyl)-fluoro-, the pyrimidine- and the 5-(pyridinyl)-bromo-analogues. The first three analogues were obtained by aromatic nucleophilic substitution of the 2,6-dihalogenopyridine (activated or not as an N-oxide) or of the 2-chloropyrimidine with the corresponding amine nucleophile as a key step. The fourth analogue, the 5-(pyridinyl)-bromo-analogue, was synthesized from the 2-amino-5-bromopyridine via a progressive elongation of the skeleton. The four compounds described are all full antagonists and show good in vitro binding affinities (Ki).