Search

Here we describe for the first time the distinctive pharmacological profile for (3S)-3-(2,3-difluorophenyl)-3-methoxypyrrolidine (IRL752), a new phenyl-pyrrolidine derivative with regioselective central nervous system transmission-enhancing properties. IRL752 (3.7-150 µmol/kg, s.c.) was characterized through extensive in vivo studies using behavioral, tissue neurochemical, and gene expression as well as microdialysis methods. Behaviorally, the compound normalized tetrabenazine-induced hypoactivity, whereas it was unable to stimulate basal locomotion in normal animals or either accentuate or reverse hyperactivity induced by amphetamine or MK-801. IRL752 induced but minor changes in monoaminergic tissue neurochemistry across noradrenaline (NA)- and dopamine (DA)-dominated brain regions. The expression of neuronal activity-, plasticity-, and cognition-related immediate early genes (IEGs), however, increased by 1.5-fold to 2-fold. Furthermore, IRL752 dose-dependently enhanced cortical catecholamine dialysate output to 600%-750% above baseline, whereas striatal DA remained unaltered, and NA rose to ∼250%; cortical and hippocampal dialysate acetylcholine (ACh) increased to ∼250% and 190% above corresponding baseline, respectively. In line with this cortically preferential transmission-promoting action, the drug was also procognitive in the novel object recognition and reversal learning tests. In vitro neurotarget affinity and functional data coupled to drug exposure support the hypothesis that 5-hydroxytryptamine 7 receptor and α2(C)-adrenoceptor antagonism are key contributors to the in vivo efficacy and original profile of IRL752. The cortical-preferring facilitatory impact on catecholamine (and ACh) neurotransmission, along with effects on IEG expression and cognition-enhancing features, are in line with the potential clinical usefulness of IRL752 in conditions wherein these aspects may be dysregulated, such as in axial motor and cognitive deficits in Parkinson disease. SIGNIFICANCE STATEMENT: This report describes the distinctive preclinical profile of (3S)-3-(2,3-difluorophenyl)-3-methoxypyrrolidine (IRL752). Its in vivo neurochemical, behavioral, microdialysis, and gene expression properties are consistent with a cortically regioselective facilitatory impact on catecholaminergic and cholinergic neurotransmission accompanied by cognitive impairment-reversing features. The pharmacological characteristics of IRL752 are in line with the clinical usefulness of IRL752 in conditions wherein these aspects may be dysregulated, such as in axial motor and cognitive deficits in Parkinson disease.

IRL790 ([2-(3-fluoro-5-methanesulfonylphenoxy)ethyl](propyl)amine, mesdopetam) is a novel compound in development for the clinical management of motor and psychiatric disabilities in Parkinson disease. The discovery of IRL790 was made applying a systems pharmacology approach based on in vivo response profiling. The chemical design idea was to develop a new type of DA D3/D2 receptor type antagonist built on agonist rather than antagonist structural motifs. We hypothesized that such a dopamine antagonist with physicochemical properties similar to agonists would exert antidyskinetic and antipsychotic effects in states of dysregulated dopaminergic signaling while having little negative impact on physiologic dopamine transmission and, hence, minimal liability for side effects related to dopamine-dependent functions. At the level of in vivo pharmacology, IRL790 displays balancing effects on aberrant motor phenotypes, reducing l-DOPA-induced dyskinesias in the rodent 6-hydroxydopamine lesion model and reducing psychostimulant-induced locomotor hyperactivity elicited by pretreatment with either d-amphetamine or dizocilpine, without negatively impacting normal motor performance. Thus, IRL790 has the ability to normalize the behavioral phenotype in hyperdopaminergic as well as hypoglutamatergic states. Neurochemical and immediate early gene (IEG) response profiles suggest modulation of DA neurotransmission, with some features, such as increased DA metabolites and extracellular DA, shared by atypical antipsychotics and others, such as increased frontal cortex IEGs, unique to IRL790. IRL790 also increases extracellular levels of acetylcholine in the prefrontal cortex and ventral hippocampus. At the receptor level, IRL790 appears to act as a preferential DA D3 receptor antagonist. Computational docking studies support preferential affinity at D3 receptors with an agonist-like binding mode. SIGNIFICANCE STATEMENT: This paper reports preclinical pharmacology along with molecular modeling results on IRL790, a novel compound in clinical development for the treatment of motor and psychiatric complications in advanced Parkinson disease. IRL790 is active in models of perturbed dopaminergic and glutamatergic signaling, including rodent 6-hydroxydopamine l-DOPA-induced dyskinesias and psychostimulant-induced hyperactivity, in a dose range that does not impair normal behavior. This effect profile is attributed to interactions at dopamine D2/D3 receptors, with a 6- to 8-fold preference for the D3 subtype.

The most common approach to in vivo pharmacokinetic and pharmacodynamic analyses involves sequential analysis of the plasma concentration - and response - time data, such that the plasma kinetic model provides an independent function, driving the dynamics. However, in situations when plasma sampling may jeopardize the effect measurements or is scarce, nonexistent, or unlinked to the effect (e.g., in intensive care units, pediatric or frail elderly populations, or drug discovery), focusing on the response-time course alone may be an adequate alternative for pharmacodynamic analyses. Response-time data inherently contain useful information about the turnover characteristics of response (target turnover rate, half-life of response), as well as the drug’s biophase kinetics (biophase availability, absorption half-life, and disposition half-life) pharmacodynamic properties (potency, efficacy). The use of pharmacodynamic time-response data circumvents the need for a direct assay method for the drug and has the additional advantage of being applicable to cases of local drug administration close to its intended targets in the immediate vicinity of target, or when target precedes systemic plasma concentrations. This review exemplifies the potential of biophase functions in pharmacodynamic analyses in both preclinical and clinical studies, with the purpose of characterizing response data and optimizing subsequent study protocols. This article illustrates crucial determinants to the success of modeling dose-response-time (DRT) data, such as the dose selection, repeated dosing, and different input rates and routes. Finally, a literature search was also performed to gauge how frequently this technique has been applied in preclinical and clinical studies. This review highlights situations in which DRT should be carefully scrutinized and discusses future perspectives of the field.

The in vitro affinity of a compound for its target is an important feature in drug discovery, but what remains is how predictive in vitro properties are of in vivo therapeutic drug exposure. We assessed the relationship between in vitro potency and clinically efficacious concentrations for marketed small molecule drugs (n = 164) and how they may differ depending on therapeutic indication, mode of action, receptor type, target localization, and function. Approximately 70% of compounds had a therapeutic unbound plasma exposure lower than in vitro potency; the median ratio of exposure in relation to in vitro potency was 0.32, and 80% had ratios within the range of 0.007 to 8.7. We identified differences in the in vivo-to-in vitro potency ratio between indications, mode of action, target type, and matrix localization, and whether or not the drugs had active metabolites. The in vitro-assay variability contributions appeared to be the smallest; within the same drug target and mode of action the within-variability was slightly broader; but both were substantially less compared with the overall distribution of ratios. These data suggest that in vitro potency conditions, estimated in vivo potency, required level of receptor occupancy, and target turnover are key components for further understanding the link between clinical drug exposure and in vitro potency.

Translation across species and from in vitro to in vivo is a central tenet in drug discovery pharmacology. Successful implementation requires proper assessment of both in vivo potency and efficacy. This notwithstanding, in vivo data is typically defined mostly in terms of ligand-to-target binding affinity, similar to in vitro studies. As in vivo potency and efficacy involve a combination not only of drug, but also partitioning, target, and drug-target-complex events and processes, ignoring some of the central differences between in vivo and in vitro may result in serious miscalculations of in vivo efficacious exposure for translational predictions. We compare potency measures derived from two basic pharmacodynamic model situations: A 'closed' in vitro system defining target binding of a ligand when both concentrations remain essentially static, and an 'open' in vivo system where target turnover dynamics and elimination of the drug-target complex are also included. Corresponding equilibrium (steady-state) expressions in the central pharmacokinetic compartment are derived and presented. Three representative variants of 'open' in vivo systems are discussed, showing relationships for ligand-target complex and ligand for each of the systems and graphically illustrating corresponding shapes. The examples include i) two ligands competing for one target, ii) two targets competing for one ligand (/drug), and iii) target-ligand (/drug) interactions in a peripheral PK compartment. The expanded in vivo potency EC50 expression emphasises the contribution from target-related biology parameters that need accounting for, and particularly that 'closed' system (in vitro) properties should not be first choice when ranking compounds in vivo ('open' system).

Potency is a central parameter in pharmacological and biochemical sciences, as well as in drug discovery and development endeavors. It is however typically defined in terms only of ligand to target binding affinity also in in vivo experimentation, thus in a manner analogous to in in vitro studies. As in vivo potency is in fact a conglomerate of events involving ligand, target, and target-ligand complex processes, overlooking some of the fundamental differences between in vivo and in vitro may result in serious mispredictions of in vivo efficacious dose and exposure. The analysis presented in this paper compares potency measures derived from three model situations. Model A represents the closed in vitro system, defining target binding of a ligand when total target and ligand concentrations remain static and constant. Model B describes an open in vivo system with ligand input and clearance (Cl(L)), adding in parallel to the turnover (ksyn, kdeg) of the target. Model C further adds to the open in vivo system in Model B also the elimination of the target-ligand complex (ke(RL)) via a first-order process. We formulate corresponding equations of the equilibrium (steady-state) relationships between target and ligand, and complex and ligand for each of the three model systems and graphically illustrate the resulting simulations. These equilibrium relationships demonstrate the relative impact of target and target-ligand complex turnover, and are easier to interpret than the more commonly used ligand-, target- and complex concentration-time courses. A new potency expression, labeled L50, is then derived. L50 is the ligand concentration at half-maximal target and complex concentrations and is an amalgamation of target turnover, target-ligand binding and complex elimination parameters estimated from concentration-time data. L50 is then compared to the dissociation constant Kd (target-ligand binding affinity), the conventional Black & Leff potency estimate EC50, and the derived Michaelis-Menten parameter Km (target-ligand binding and complex removal) across a set of literature data. It is evident from a comparison between parameters derived from in vitro vs. in vivo experiments that L50 can be either numerically greater or smaller than the Kd (or Km) parameter, primarily depending on the ratio of kdeg-to-ke(RL). Contrasting the limit values of target R and target-ligand complex RL for ligand concentrations approaching infinity demonstrates that the outcome of the three models differs to a great extent. Based on the analysis we propose that a better understanding of in vivo pharmacological potency requires simultaneous assessment of the impact of its underlying determinants in the open system setting. We propose that L50 will be a useful parameter guiding predictions of the effective concentration range, for translational purposes, and assessment of in vivo target occupancy/suppression by ligand, since it also encompasses target turnover - in turn also subject to influence by pathophysiology and drug treatment. Different compounds may have similar binding affinity for a target in vitro (same Kd), but vastly different potencies in vivo. L50 points to what parameters need to be taken into account, and particularly that closed-system (in vitro) parameters should not be first choice when ranking compounds in vivo (open system).

There is a medical need for safe and efficacious anti-obesity drugs with acceptable side effect profiles. To mitigate the challenge posed by translating target interaction across species and balancing beneficial vs. adverse effects, a positron emission tomography (PET) approach could help guide clinical dose optimization. Thus, as part of a compound differentiation effort, three novel selective CB1 receptor (CB1R) antagonists, developed by AstraZeneca (AZ) for the treatment of obesity, were compared with two clinically tested reference compounds, rimonabant and taranabant, with regard to receptor occupancy relative to dose and exposure. A total of 42 PET measurements were performed in 6 non-human primates using the novel CB1R antagonist radioligand [(11)C]SD5024. The AZ CB1R antagonists bound in a saturable manner to brain CB1R with in vivo affinities similar to that of rimonabant and taranabant, compounds with proven weight loss efficacy in clinical trials. Interestingly, it was found that exposures corresponding to those needed for optimal clinical efficacy of rimonabant and taranabant resulted in a CB1R occupancy typically around ∼20-30%, thus much lower than what would be expected for classical G-protein coupled receptor (GPCR) antagonists in other therapeutic contexts. These findings are also discussed in relation to emerging literature on the potential usefulness of 'neutral' vs. 'classical' CB1R (inverse agonist) antagonists. The study additionally highlighted the usefulness of the radioligand [(11)C]SD5024 as a specific tracer for CB1R in the primate brain, though an arterial input function would ideally be required in future studies to further assure accurate quantitative analysis of specific binding.

Our article in this journal some 15 years ago focussed on the role of serotonin (5-HT) autoreceptors in the mechanism of action of antidepressant drugs. Specifically in this regard, the results were summarised of rat microdialysis studies carried out to examine: (a) the relative importance of 5-HT1A and 5-HT1B autoreceptors, including (b) possible regional variation, and (c) potential changes in autoreceptor responsiveness following chronic selective serotonin reuptake inhibitor administration. In the present reflection piece, I recap some of the key findings against a brief background and provide an account of their bearing within the context of subsequent endeavours in the antidepressant drug research and development field. I conclude by shortly commenting on selected topics relevant to novel, interesting advances and avenues for future research.

The implication of the cannabinoid receptor 1 (CB(1) receptor) in several pathophysiological states has sparked the development of selective antagonists. Here we compare binding of the antagonists [(3) H]-AZ12491187, [(3) H]-taranabant and [(3) H]-rimonabant to intact human embryonic kidney cells stably expressing recombinant human CB(1) receptors (CB1r cells). Unlabelled ligands decreased the total binding of the three radioligands with closely the same order of potency: i.e. AZ12288553∼AZ12491187∼taranabant>rimonabant. Nondisplaceable (i.e. nonspecific) binding to the CB1r cells was the same as total binding to the wells containing untransfected cells and it was more pronounced for [(3) H]-AZ12491187 and [(3) H]-rimonabant than for [(3) H]-taranabant. [(3) H]-Rimonabant and (to a lesser extent) [(3) H]-AZ12491187 were also prone to bind nonspecifically to the walls of the wells. Compared to the other radioligands, [(3) H]-rimonabant displayed lower potency for the CB(1) receptors in saturation binding studies and faster association and dissociation in kinetic experiments. When dissociated, the three radioligands also showed prominent rebinding to the cells in medium only. This could be relieved by the presence of excess of unlabelled ligand and of bovine serum albumin (BSA) but a combination thereof was most efficient. The long 'residence time' of AZ12491187 at the CB(1) receptor (because of slow dissociation and prominent rebinding) and its pronounced incorporation into the membranes of the cells could contribute to long-lasting in vivo CB(1) receptor blockade.

5-Hydroxytryptamine 1A (5-HT(1A)) receptors have been suggested as a target for the treatment of irritable bowel syndrome (IBS). A recent clinical trial investigating the efficacy of the selective 5-HT(1A) antagonist AZD7371 [3(R)-(N,N-dicyclobutylamino)-8-fluoro-3,4-dihydro-2H-1-benzopyran-5-carboxamide (R,R)-tartrate monohydrate] showed no symptomatic improvement in IBS patients. We characterized the mechanisms mediating potential analgesic effects of AZD7371 in a model of colorectal distension (CRD)-induced visceral pain in rats to understand its mechanism of action and the lack of clinical efficacy. Visceromotor and cardiovascular responses (telemetry) were assessed in conscious rats during noxious CRD (80 mm Hg). Effects of AZD7371 (3-300 nmol/kg i.v.; 1-30 micromol/kg p.o.) and a reference 5-HT(1A) antagonist, WAY-100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridyl)cyclohexanecarboxamide maleate salt; 3-300 nmol/kg i.v.), were assessed. Effects of intracerebroventricular AZD7371 were also evaluated. Intravenous AZD7371 or WAY-100635 and oral AZD7371 dose-dependently inhibited visceromotor responses to CRD (ED(50), 203, 231, and 14 micromol/kg, respectively). In telemetrized rats, oral AZD7371 inhibited visceromotor responses to CRD without affecting the concomitant hypertensive and tachycardic responses. Intracerebroventricular AZD7371 did not affect visceromotor responses, whereas it inhibited micturition. None of the doses tested induced visible gross side effects. AZD7371, likely acting at a spinal site, inhibited the visceromotor but not the cardiovascular responses to visceral pain in the CRD model in rats. Although agents effective on multiple pain-related readouts in the CRD model (e.g., pregabalin or clonidine) alleviate IBS symptoms, AZD7371, which is effective on only one pain-related pseudoaffective readout, does not. Data from preclinical CRD models of visceral pain need to be interpreted cautiously as it relates to their clinical translational value.

Background: The endocannabinoid system functions through two well characterized receptor systems, the CB1 and CB2 receptors. Work by a number of groups in recent years has provided evidence that the system is more complicated and additional receptor types should exist to explain ligand activity in a number of physiological processes.

Given the complexity of pharmacological challenge experiments, it is perhaps not surprising that design and analysis, and in turn interpretation and communication of results from a quantitative point of view, is often suboptimal. Here we report an inventory of common designs sampled from anti-inflammatory, respiratory and metabolic disease drug discovery studies, all of which are based on animal models of disease involving pharmacological and/or patho/physiological interaction challenges. The corresponding data are modeled and analyzed quantitatively, the merits of the respective approach discussed and inferences made with respect to future design improvements. Although our analysis is limited to these disease model examples, the challenge approach is generally applicable to the vast majority of pharmacological intervention studies. In the present five Case Studies results from pharmacodynamic effect models from different therapeutic areas were explored and analyzed according to five typical designs. Plasma exposures of test compounds were assayed by either liquid chromatography/mass spectrometry or ligand binding assays. To describe how drug intervention can regulate diverse processes, turnover models of test compound-challenger interaction, transduction processes, and biophase time courses were applied for biomarker response in eosinophil count, IL6 response, paw-swelling, TNFα response and glucose turnover in vivo. Case Study 1 shows results from intratracheal administration of Sephadex, which is a glucocorticoid-sensitive model of airway inflammation in rats. Eosinophils in bronchoalveolar fluid were obtained at different time points via destructive sampling and then regressed by the mixed-effects modeling. A biophase function of the Sephadex time course was inferred from the modeled eosinophil time courses. In Case Study 2, a mouse model showed that the time course of cytokine-induced IL1β challenge was altered with or without drug intervention. Anakinra reversed the IL1β induced cytokine IL6 response in a dose-dependent manner. This Case Study contained time courses of test compound (drug), challenger (IL1β) and cytokine response (IL6), which resulted in high parameter precision. Case Study 3 illustrates collagen-induced arthritis progression in the rat. Swelling scores (based on severity of hind paw swelling) were used to describe arthritis progression after the challenge and the inhibitory effect of two doses of an orally administered test compound. In Case Study 4, a cynomolgus monkey model for lipopolysaccharide LPS-induced TNFα synthesis and/or release was investigated. This model provides integrated information on pharmacokinetics and in vivo potency of the test compounds. Case Study 5 contains data from an oral glucose tolerance test in rats, where the challenger is the same as the pharmacodynamic response biomarker (glucose). It is therefore convenient to model the extra input of glucose simultaneously with baseline data and during intervention of a glucose-lowering compound at different dose levels. Typically time-series analyses of challenger- and biomarker-time data are necessary if an accurate and precise estimate of the pharmacodynamic properties of a test compound is sought. Erosion of data, resulting in the single-point assessment of drug action after a challenge test, should generally be avoided. This is particularly relevant for situations where one expects time-curve shifts, tolerance/rebound, impact of disease, or hormetic concentration-response relationships to occur.

Some clinical reports suggest that tricyclic antidepressants which block both noradrenaline and serotonin (5-HT) reuptake (SNRIs) are more effective than selective 5-HT reuptake inhibitors (SSRIs) in treating severe depression. Moreover, one neurochemical study reported larger increases in extracellular 5-HT in rat frontal cortex in response to the tricyclic antidepressant imipramine compared to the SSRI fluoxetine. However, imipramine, which blocks both 5-HT and noradrenaline reuptake, also binds with relatively high affinity to receptors for noradrenaline, histamine and acetylcholine. Thus, to test the hypothesis that compounds that inhibit both 5-HT and noradrenaline reuptake produce larger increases in 5-HT efflux, we compared the effects of acute systemic administration of several SNRIs and SSRIs. Extracellular 5-HT was measured using microdialysis probes implanted in the diencephalon and frontal cortex of unanesthetized rats. We tested the SSRIs paroxetine (0.3-10 mg/kg), citalopram (10-20 mg/kg) and fluoxetine (10 mg/kg), the nonselective tricyclic antidepressant imipramine (20 mg/kg) and the more selective SNRIs duloxetine (3-30 mg/kg) and venlafaxine (30-50 mg/kg). During the lights-off period, paroxetine and duloxetine increased 5-HT in the diencephalon approximately 300 and approximately 200%, respectively. During the lights-on period, paroxetine and duloxetine each increased 5-HT approximately 400% in the diencephalon. In the frontal cortex, both paroxetine and duloxetine increased 5-HT approximately 200%. Citalopram and venlafaxine each increased 5-HT in the diencephalon approximately 300%. Fluoxetine and imipramine increased 5-HT in the diencephalon by approximately 125 and approximately 80%, respectively. Thus, these results do not support the hypothesis that compared to SSRIs, compounds which inhibit both 5-HT and noradrenaline reuptake have a larger acute effect on extracellular 5-HT.

Patients with chronic hepatic encephalopathy (HE) may present affective symptoms and antidepressant drug treatment in this condition is not uncommon. The present microdialysis study investigated treatment with the chronic antidepressant venlafaxine (VEN) in experimental HE with regard to tentative changes in pharmacokinetic and/or pharmacodynamic parameters. Three weeks after portacaval shunt (PCS) or sham operation in rats, VEN (10 mg/kg daily) was administered by implanted osmotic minipumps. VEN treatment for 14 days resulted in higher concentrations of VEN in PCS rats than in sham controls in serum and brain compartments, and the VEN levels in serum and brain were strongly inter-correlated. The serum N-desmethylvenlafaxine concentration did not differ between the groups, but correlated with the serum VEN levels. The other VEN metabolites were below the quantification limits. VEN treatment for 9-12 days significantly stimulated locomotion and rearing in the open field in sham controls, but failed to do so in the PCS rats. The concentrations of noradrenaline, dopamine, 5-HT, and 5-HIAA in neocortical dialysates were higher in PCS than in sham rats after 14 days of VEN treatment, but the elevations reached statistical significance only in the case of dopamine and 5-HIAA. In summary, there were significant pharmacokinetic and pharmacodynamic alterations in rats with experimental HE as compared to controls. The described experimental HE model may be useful for continued pharmacokinetic/pharmacodynamic interaction studies to unravel the pathophysiological consequences of HE on the CNS.

Energy intake (EI) is a pivotal biomarker used in quantification approaches to metabolic disease processes such as obesity, diabetes, and growth disorders. Eating behavior is however under both short-term and long-term control. This control system manifests itself as tolerance and rebound phenomena in EI, when challenged by drug treatment or diet restriction. The paper describes a model with the capability to capture physiological counter-regulatory feedback actions triggered by energy imbalances. This feedback is general as it handles tolerance to both increases and decreases in EI, and works in both acute and chronic settings. A drug mechanism function inhibits (or stimulates) EI. The deviation of EI relative to a reference level (set-point) serves as input to a non-linear appetite control signal which in turn impacts EI in parallel to the drug intervention. Three examples demonstrate the potential usefulness of the model in both acute and chronic dosing situations. The model shifts the predicted concentration-response relationship rightwardly at lower concentrations, in contrast to models that do not handle functional adaptation. A fourth example further shows that the model may qualitatively explain differences in rate and extent of adaptation in observed EI and its concomitants in both rodents and humans.

Rats were administered venlafaxine (10 mg/kg per day) for 14 days by using subcutaneously implanted osmotic minipumps. The present study assessed the distribution of VEN in different compartments, whether the VEN concentration in the compartments correlated, the effect of VEN on dialysate monoamine levels and on the spontaneous open-field behavior, and possible relations between the pharmacokinetic and pharmacodynamic parameters. The venlafaxine level in serum after sustained treatment was about 25% of the concentration in brain parenchyma and much higher than in brain dialysate. There was a clear correlation between venlafaxine concentrations in blood and brain compartments. The sustained venlafaxine challenge resulted in higher neocortical concentration of serotonin and noradrenaline, lower 5-hydroxyindole-3-acetic acid levels and increased locomotor activity in the central part of the test arena as compared to controls. No correlations were found between the venlafaxine concentration and brain monoamine parameters or the open-field behaviors. We conclude that, although species differences in pharmacokinetic properties for venlafaxine between rat and man exist, the pharmacokinetic correlations found after sustained treatment add information to the in vivo nature of the drug. Also, more studies like the present need to be performed to find the pharmacokinetic/pharmacodynamic interrelations for drugs like VEN.

THE NMDA receptor antagonist phencyclidine (PCP) has low micromolar affinity for the 5-HT reuptake site, but it is uncertain whether PCP blocks 5-HT reuptake when given systemically to rats in behaviourally stimulating doses. We here report for the first time that systemically administered PCP (5 mg/kg, s.c.) increases extracellular 5-HT levels in the rat medial prefrontal cortex (to 322%) and dorsal hippocampus (to 233%). Increases were found also when citalopram (1 microM) was included in the perfusion medium (to 184 and 180%, respectively). Extracellular 5-HIAA concentrations increased during both conditions, and extracellular GABA decreased in the dorsal hippocampus. It is concluded that systemic PCP treatment elevates extracellular 5-HT levels, probably through mechanisms other than a blockade of 5-HT reuptake.

Clozapine and the novel putative, antipsychotic S 16924 ((1-(benzodioxane-5-yl)-3-[3-(4-fluorophenacyl)pyrrolidine]-1-o xapropane HCl) share significant affinity for alpha1-adrenoceptors and 5-HT1A autoreceptors in vitro and display an 'atypical' behavioural profile in in vivo models used for detecting potential neuroleptic effects. In the present study, in vivo microdialysis was used to examine the effect of clozapine and S 16924 on 5-HT overflow in the rat ventral hippocampus, and to assess the relative role of putative alpha1-adrenoceptor antagonist and 5-HT1A autoreceptor agonist properties of the drugs in this regard. S 16924 (0.1-3 mg/kg, s.c.) reduced dialysate 5-HT in a dose- and time-dependent fashion by maximally approximately 70% from baseline 40-60 min after injection. Clozapine (0.1-10 mg/kg, s.c.) reduced 5-HT overflow in the same manner, with a maximum effect of approximately 60% from baseline, obtained after 60-80 min. The 5-HT decrease elicited by S 16924 (1.0 mg/kg, s.c.) was significantly, though only partially, antagonized by pretreatment with the selective 5-HT1A receptor antagonist WAY 100635 (0.3 mg/kg, s.c.). The selective alpha1-adrenoceptor agonist cirazoline (0.02 mg/kg, i.p.) alone did not significantly attenuate the effect of S 16924 (1.0 mg/kg, s.c.) on 5-HT overflow. Combined treatment with both WAY 100635 and cirazoline, however, totally reversed the 5-HT-suppressing effect of S 16924 (1.0 mg/kg, s.c.). By comparison, when given separately, neither WAY 100635 (0.3 mg/kg, s.c.) nor cirazoline (0.02 mg/kg, i.p.) antagonized the clozapine (0.3 mg/kg, s.c.)-induced decrease of 5-HT in ventral hippocampus dialysates. In the presence of both WAY 100635 and cirazoline, the response to this dose of clozapine was however significantly, though modestly, attenuated. In contrast, the WAY 100635/cirazoline combination failed to antagonise the 5-HT decrease resulting from a higher dose (3.0 mg/kg, s.c.) of clozapine. We conclude that both alpha1-adrenoceptor antagonist and 5-HT1A receptor agonist properties of clozapine and S 16924 contribute to the 5-HT release-reducing action of these drugs. Whereas these factors apparently explain the effect of S 16924 fully, additional mechanism(s) appear to be involved in the case of clozapine. With regard to the interplay between alpha1-adrenoceptor and 5-HT1A (auto)receptor mechanisms in the control of 5-HT release in the rat forebrain, the present data suggest that an excitation mediated by the former is outweighed by the simultaneous activation of the latter-inhibitory-receptors.

Ammonia has been shown to cause release of neurotransmitters such as serotonin (5-hydroxytryptamine; 5-HT) from synaptosomal preparations in vitro. In the present study, frontal neocortical extracellular levels of 5-HT and its major metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA), were determined in vivo by the use of microdialysis in portacaval shunted (PCS) rats, an experimental model of chronic hepatic encephalopathy (HE), prior to and after an acute coma-inducing administration of ammonium acetate (NH4Ac; 5.2 mmol/kg, i.p.). PCS rats displayed elevated (P0.01) 5-HIAA but unaltered 5-HT extracellular levels compared with controls, supporting the contention of an increased neocortical 5-HT metabolism but unaltered neuronal 5-HT output in chronic HE. However, a transient elevation of extracellular 5-HT levels was observed when PCS-NH4Ac rats were in coma. Increased brain ammonia may thus augment neuronal 5-HT release in chronic HE, which in turn could be a causative for precipitation of more severe stages of HE.

Recent open clinical studies suggest that pindolol and buspirone may enhance the efficacy and/or shorten the latency to antidepressant action of selective serotonin reuptake inhibitors (SSRI) in unipolar major depressive disorder. The present investigation addressed the possibility that these agents share the ability to enhance the extracellular 5-hydroxytryptamine (5-HT)-elevating response to the SSRI citalopram. For the purpose, in vivo microdialysis in the rat ventral hippocampus was employed. (-)-Pindolol (8 mg/kg s.c.) augmented the citalopram (5 mg/kg s.c.)-induced rise of extracellular 5-HT levels, whereas buspirone (5 mg/kg s.c.) failed to do so. This effect of (-)-pindolol probably reflects its ability to block 5-HT1A autoreceptors, thereby abating the citalopram-induced indirect activation of these sites (secondary to the inhibition of 5-HT reuptake and elevation of extracellular 5-HT in the midbrain raphe). The lack of effect of buspirone in this model indicates that the clinically observed antidepressant augmentation action of buspirone is not mediated indirectly, via enhanced extracellular levels of 5-HT.

The inhibition of serotonin (5-HT) release produced by antidepressants varying in relative selectivity for blocking uptake of 5-HT and noradrenaline (NA) was compared. Release was measured by microdialysis in anesthetized rats with nerve terminal 5-HT uptake inhibited by local infusion of citalopram (1 μM) through a dialysis probe in hippocampus. With 5-HT uptake first blocked in hippocampus, systemic injection of uptake inhibitors produced decreases in dialysate 5-HT, presumably due to autoreceptor stimulation in the raphe. The largest decreases (about 60–70%) in 5-HT were produced by the selective 5-HT uptake inhibitors sertraline, paroxetine and citalopram. Nonselective blockers caused less suppression of release. Thus, the maximum decrease in 5-HT was 35% after clomipramine, a less selective 5-HT uptake inhibitor, and ⩽ 30% after the nonselective 5-HT/NA uptake blockers imipramine and amitriptyline, 5-HT was not decreased after maprotiline, a selective NA uptake blocker. Pretreatment with (+)WAY 100135 to block 5-HT 1A autoreceptors, abolished the inhibition of 5-HT release produced by systemic sertraline, clomipramine and imipramine. One explanation for the difference between selective and nonselective inhibitors with respect to central 5-HT release, is the excitatory effect of ( α 1 ) adrenergic receptor stimulation on 5-HT neuronal discharge. However, pretreatment with α -methyl- p -tyrosine to deplete NA, did not influence the inhibition of 5-HT release produced by imipramine.

The clinical efficacy of antidepressants that block serotonin (5-hydroxytryptamine, 5-HT) reuptake may be restrained by indirect activation of autoreceptors. In vivo microdialysis in rat hippocampus was used to assess the release-inhibitory properties of the 5-HT reuptake inhibitors citalopram and paroxetine. When reuptake was first blocked by infusing citalopram into the hippocampus, systemic administration of citalopram or paroxetine resulted in a 50–70% decrease in hippocampal 5-HT overflow. This presumably reflected the inhibition of 5-HT release subsequent to reuptake blockade in the raphe nuclei and, in turn, activation of somatodendritic autoreceptors. In support, pretreatment with (±)-pindolol or(+)-WAY100135 ((+)-N-tert-butyl-3-(4- (2-methoxyphenyl)piperazine-1-yl)-2-phenylpropanamide dihydrochloride), to block 5-HT1A autoreceptors, abolished the decrease in 5-HT produced by systemic injection of the uptake blockers.

The two diphenylbutylpiperazinepyridinyl derivatives, FG5865 and FG5893, have a unique receptor binding profile in that they show very high and essentially equipotent affinities for both 5-HT 1A and 5-HT 2 receptors. The present report describes the acute effects of FG5865 and FG5893 on presynaptic 5-hydroxytryptamine (5-HT) neuronal function in the rat CNS, using established ex vivo and in vivo neurochemical techniques. Post-mortem measurements of tissue levels of 5-HT, its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), and of the formation of 5-hydroxytryptophan (5-HTP; after inhibition of aromatic amino acid decarboxylase by NSD 1015) showed that FG5865 (0.1–20 mg/kg, s.c.) and FG5893 (0.1–20 mg/kg, s.c.) dose dependently decreased the synthesis and the metabolism/turnover of 5-HT - this to an extent comparable to the reference 5-HT 1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin. Reserpine (5 mg/kg, s.c.) pretreatment did not prevent the FG5893-induced decrease of 5-HT synthesis rate. In contrast, about 25–50 times higher doses of FG5865 were required to produce a comparable decrease in brain 5-HT synthesis in reserpinized vs. non-pretreated rats. In in vivo microdialysis experiments, both FG5865 (0.1–3.0 mg/kg, s.c) and FG5893 (0.3–1.0 mg/kg, s.c.) caused a marked and dose-dependent decrease of 5-HT release in the ventral hippocampus. Pretreatment with the 5-HT 1A receptor antagonist, (±)-pindolol (8 mg/kg, s.c.), abolished the FG5865 (0.3 mg/kg, s.c.)-induced reduction of 5-HT release, and (−)-pindolol (8 mg/kg, s.c.) similarly reversed the FG5893 (0.3 mg/kg, s.c.)-induced decrease. Local infusion of FG5865 into the ventral hippocampus (10 μM, 20-min pulse) resulted in a rapid and transient elevation of the 5-HT output, an effect that was independent of extracellular Ca 2+ , FG5893, on the other hand, did not affect the 5-HT release upon local administration. The results demonstrate that FG5865 and FG5893 potently affect a range of neurochemical indices of rat brain 5-HT neuronal activity in vivo, in a way consistent with indirect (FG5865) and direct (FG5865 and FG5893) stimulation of the 5-HT 1A autoreceptors in the raphe nuclei.

The present study addressed the possibility that disinhibition of serotonin (5-HT) autoreceptor-mediated negative feedback might potentiate the elevation of nerve terminal 5-HT output induced by selective 5-HT reuptake blockade. To this end, rats were given citalopram and the 5-HT autoreceptor-blocking agents (S)-UH-301 (5-HT1A) and (-)-penbutolol (5-HT1A/1B), and the effect on extracellular 5-HT in the ventral hippocampus was monitored by means of in vivo microdialysis. Citalopram (5 mg/kg, s.c.) approximately doubled the 5-HT output, a response that was markedly augmented by (S)-UH-301 (3 mg/kg, s.c.) and (-)-penbutolol (8 mg/kg, s.c.) and by combined treatment with (S)-UH-301 (3 mg/kg, s.c.) plus (-)-penbutolol (1 microM; via the dialysis perfusion medium), but not by (-)-penbutolol (1 microM) alone. These findings provide evidence that 5-HT, in particular 5-HT1A, autoreceptor-mediated negative feedback mechanisms are pivotal in determining the nerve terminal 5-HT output level after 5-HT reuptake inhibition. These findings have important implications for the interplay between different processes controlling 5-HT transmission in vivo and might possibly offer a lead toward novel, therapeutically exploitable principles.

Racemic 5-methoxy-2-methyl-2-dipropylaminotetralin (3) has been prepared by a short synthetic route, in which the N,N-dipropyliminium perchlorate of 5-methoxy-2-tetralone (4) is a key intermediate. Racemic 3 was resolved by crystallization of the corresponding diastereomeric di-p-toluoyltartrates. The enantiomeric excess (%ee) of the phenolic derivatives of (+)-(R)- and (−)-(S)-3 [(+)-(R)- and (−)-(S)-2] was determined by 1HNMR spectroscopic analysis of the corresponding diastereomeric (−)-(R)-1,1′-binaphthyl-2,2′-diylphosphoric acid salts utilizing 13C satellites. X-ray crystallography established the absolute configuration of (−)-(S)-2 · HCl. The enantiomers of 2 were tested for hippocampal output of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, and dihydroxyphenylacetic acid in rats by use of in vivo microdialysis. The (−)-(S)-enantiomer appeared to affect 5-HT-turnover, whereas (+)-(R)-2 was inactive. Results obtained provide support for the previously reported hypothesis that the inactivity of (−)-(S)-2 at central DA receptors is caused by the steric bulk of the C(2)-methyl group. This makes it possible to define a “DA D2 receptor essential volume.” © 1993 Wiley-Liss, Inc.

Brain 5-HT1A and 5-HT1B receptors are important targets for drug-induced modulation of 5-HT function in vivo. However, very few compounds are available that are effective antagonists at 5-HT1 receptors, thus hampering the progress of fundamental as well as clinical research in this area. The present study assessed the usefulness of the beta-adrenolytic agent (-)-penbutolol (and its (+)-counterpart) as a 5-HT1A receptor-blocking agent. The compound was found to counteract, in a stereospecific fashion, not only the behavioural and hypothermic but also the in vivo 5-HT synthesis/turnover-reducing effects of the specific 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). These findings indicate that (-)-penbutolol is an antagonist at both postsynaptic receptors and somatodendritic autoreceptors of the 5-HT1A subtype. Thus, (-)-penbutolol represents a useful addition to the array of pharmacological tools available for the study of central 5-HT1 receptor-mediated functions.

It has recently been suggested that central 5-HT1A autoreceptors are already desensitised after single-dose 5-HT1A agonist treatment. In turn, this would lead to an attenuated feedback suppression of transmitter release from 5-HT neurones, and thus to enhanced 5-HT synaptic transmission. In the present study in vivo brain microdialysis techniques were used in an attempt to test this hypothesis. The results show that single-dose pretreatment with the reference 5-HT1A receptor agonist 8-hydroxy-2-(din-propylamino)tetralin, 8-OH-DPAT, (i) did not significantly alter the baseline output of 5-HT in the rat ventral hippocampus 24 h later, and (ii) did not alter the release-reducing response to 5-HT1A agonist (8-OH-DPAT, ipsapirone or BMY 7378) challenge under the same conditions. These observations indicate that the functional responsiveness of the 5-HT release-controlling 5-HT1A autoreceptors is maintained after bolus 8-OH-DPAT pretreatment. When related to the acute 8-OH-DPAT-induced reduction in raphe 5-HT1A radioligand binding density recently reported by others, the present results are consistent with a large functional overcapacity of this 5-HT1A receptor population. The mechanism by which 5-HT1A receptor-mediated hypothermia and hyperphagia are rapidly attenuated by a previous large single dose of a 5-HT1A receptor agonist remains to be explained.

Biodegradable controlled-release microsphere systems made with the biocompatible biodegradable polyester excipient poly [DL lactide-co-glycolide] constitute an exciting new technology for drug delivery to the central nervous system (CNS). The present study describes functional observations indicating that implantation of dopamine (DA) microspheres encapsulated within two different polymer excipients into denervated- striatal tissue assures a prolonged release of the transmitterin vivo. Moreover, in this regard, the results show that there were clear cut temporal differences in the effect of the two DA microsphere formulations compared in this study, probably reflecting variations in the actual composition (i.e., lactide to glycolide ratio) of the two copolymer excipients examined. This technology has considerable potential for basic research with possible clinical application.

The effects of MDL 73005EF (8-[2-(2,3-dihydro-1,4-benzodioxin-2-yl)methylamino]-8- azaspiro[4,5]decan-7,9-dione methyl sulphonate), a novel selective 5-HT1A receptor ligand with putative anxiolytic properties, were explored using models of central pre- and postsynaptic 5-HT1A receptor function in the male rat. MDL 73005EF dose dependently decreased the hippocampal 5-HT output measured by in vivo microdialysis in chloral hydrate-anaesthetised rats and this response was antagonised by the 5-HT1A/B receptor antagonist, pindolol. Local administration of MDL 73005EF had no effect on the hippocampal 5-HT output. MDL 73005EF failed to alter basal plasma adrenocorticotropin (ACTH) levels but, in common with pindolol, attenuated the ACTH response to the 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). In contrast to 8-OH-DPAT, MDL 73005EF significantly increased plasma prolactin but apparently not through a 5-HT receptor-mediated mechanism. The results indicate that MDL 73005EF possesses mixed 5-HT1A receptor agonist/antagonist properties, acting as an agonist at presynaptic 5-HT1A receptors controlling 5-HT release and as an antagonist at postsynaptic 5-HT1A receptors mediating ACTH release.

Recent results in studies of interactions between dopamine receptors and stereoselec­ tive agonists and antagonists are reviewed. Also discussed are the stereoselectivity of serotonergic 5-HT1A-receptor agonists and some of the difficulties associated with modeling the interaction between ligands and receptors on a molecular level.

The receptors mediating the hemodynamic responses to cannabinoids are not clearly defined due to the multifarious pharmacology of many commonly used cannabinoid ligands. While both CB1 and TRPV1 receptors are implicated, G protein-coupled receptor 55 (GPR55) may also mediate some of the hemodynamic effects of several atypical cannabinoid ligands. The present studies attempted to unravel the pharmacology underlying the in vivo hemodynamic responses to ACEA (CB1 agonist), O-1602 (GPR55 agonist), AM251 (CB1 antagonist), and cannabidiol (CBD; GPR55 antagonist). Agonist and antagonist profiles of each ligand were determined by ligand-induced GTPγS binding in membrane preparations expressing rat and mouse CB1 and GPR55 receptors. Blood pressure responses to ACEA and O-1602 were recorded in anesthetized and conscious mice (wild type, CB1 (-/-) and GPR55(-/-)) and rats in the absence and presence of AM251 and CBD. ACEA demonstrated GTPγS activation at both receptors, while O-1602 only activated GPR55. AM251 exhibited antagonist activity at CB1 and agonist activity at GPR55, while CBD demonstrated selective antagonist activity at GPR55. The depressor response to ACEA was blocked by AM251 and attenuated by CBD, while O-1602 did not induce a depressor response. AM251 caused a depressor response that was absent in GPR55(-/-) mice but enhanced by CBD, while CBD caused a small vasodepressor response that persisted in GPR55(-/-) mice. Our findings show that assessment of the pharmacological profile of receptor activation by cannabinoid ligands in in vitro studies alongside in vivo functional studies is essential to understand the role of cannabinoids in hemodynamic control.

Patients with chronic hepatic encephalopathy display monoaminergic perturbations together with affective symptoms. Thus, these patients belong to a group with a probability of receiving antidepressant drug treatment. The liver impairment may result in pharmacokinetic alterations of the antidepressant drug, which in turn may affect the already perturbed monoaminergic function. Venlafaxine (VEN) was administered as a single subcutaneous challenge to portacaval shunted (experimental hepatic encephalopathy model) rats (5 mg/kg) and sham-operated rats (5 and 10 mg/kg). The aims were to investigate whether a reduced dose in portacaval shunted rats resulted in higher concentrations of VEN and serotonin as compared to control rats, which had been demonstrated earlier when the rats received the same dose (10 mg/kg). A 50% reduction of the dose of VEN administered to portacaval shunted rats resulted in elevated levels of VEN in serum, brain parenchyma, and brain dialysate about 300 minutes after the injection. The VEN challenge increased the serotonin and noradrenaline concentrations in dialysate in portacaval shunted rats and both sham groups, but the three VEN groups did not differ in any major way in serotonin and noradrenaline output. Therefore, when the dose of VEN administered to experimental hepatic encephalopathy was reduced 50% as compared to control rats, mainly pharmacokinetic, and possibly also monoaminergic, alterations were observed.

In the present study, effects of citalopram (CIT) on brain 5-hydroxytryptamine (5-HT) release in experimental chronic hepatic encephalopathy (HE) were investigated. Neocortical administration of CIT (1.0 microM) increased the brain 5-HT output to a similar extent in portacaval shunted (PCS) rats and sham-operated controls, indicating that a previous described mismatch between increased 5-HT turnover and unchanged release in PCS rats is not explained by an accelerated brain 5-HT reuptake. Subsequent systemic administration of CIT (5 mg/kg subcutaneously) resulted in a more pronounced attenuation of the brain 5-HT release in PCS rats than in sham-operated controls, possibly indicating a higher susceptibility to indirect mid-brain 5-HT1A autoreceptor activation in experimental portal-systemic encephalopathy (PSE). A KCl (60 mM) challenge in the presence of locally administered CIT (1 microM) induced a more marked neocortical 5-HT response in PCS rats than in sham-operated controls, confirming previous results of a higher than normal amount of 5-HT available for depolarization-induced release in PCS rats. Although the pharmacodynamic parameters in this study was investigated for CIT, the likelihood of a parallel pharmacokinetic alteration existing for this drug in the PCS condition also was indicated. It is thus suggested that otherwise generally safe central nervous system 5-HT-active drugs may represent a potential hazard in patients with liver failure with or without PSE.

In vivo microdialysis in rat ventral hippocampus was used (i) to verify the importance of 5-HT1A autoreceptors in the raphe as targets for drugs that enhance the citalopram-induced elevation of forebrain 5-hydroxytryptamine (5-HT), and (ii) to further examine the specificity of (−)-penbutolol in this regard. The selective 5-HT1A receptor antagonist WAY100635 (s.c., or intra-raphe) or the mixed 5-HT 1A 1B β- adrenoceptor antagonist (−)-penbutolol (s.c.), potentiated the citalopram-induced 5-HT rise, whereas local ‘reverse’ dialysis of WAY100635 into the ventral hippocampus did not. Furthermore, the (−)-penbutolol-induced augmentation proved stereoselective and not mediated by β-adrenoceptors (no effect of s.c. (+)-penbutolol, or β1- and β2-adrenoceptor blockers (betaxolol, ICI118.551)). These data provide direct evidence that increased stimulation of 5-HT1A autoreceptors in the midbrain raphe impedes the effect of citalopram on forebrain extracellular 5-HT, whereas neither postsynaptic 5-HT1A receptors nor β-adrenoceptors appear to be involved.

Rats were administered the selective serotonin (5-HT) uptake blocker citalopram or saline for 14 days to determine if prolonged treatment would lead to changes in extracellular 5-HT or autoreceptor sensitivity. One day after drug withdrawal, dialysis probes were implanted in the frontal cortex and dorsal hippocampus. Dialysis experiments were carried out using chloral hydrate anesthetized rats. The experimental protocol comprised the administration of three consecutive drug challenges: (1) After stable baseline levels were obtained, citalopram was infused through the dialysis probes to locally block uptake in the forebrain. (2) Subsequently, a 5-HT1B receptor agonist (RU24969 or CP93,129) was infused through the probe to test for changes in terminal autoreceptor sensitivity. (3) Last, citalopram was administered systemically to test the effect of indirect activation of somatodendritic autoreceptors. Under these conditions, with uptake already blocked locally in the forebrain, systemic citalopram produces a decrease in extracellular 5-HT, an effect that can be inhibited by pretreatment with antagonists of 5-HT1A receptors. The results indicate that during local infusion of citalopram extracellular 5-HT was significantly higher in the dorsal hippocampus of the chronic citalopram as compared to saline treatment group. This difference persisted throughout the full time course of the experiment. However, the decreases in 5-HT levels produced by local infusion of a 5-HT1B receptor agonist or after systemic citalopram administration were not significantly different between the chronic citalopram and saline treated groups. There were no significant differences between chronic citalopram and saline treated animals in frontal cortex. These results suggest that prolonged inhibition of 5-HT uptake may produce a selective change in the regulation of release from median raphe 5-HT neurons, but this change could not be clearly linked to a change in nerve terminal or somatodendritic autoreceptor sensitivity.

The present study utilized in vivo microdialysis to investigate the importance of 5-HT1A autoreceptors and alpha 1-adrenoceptors in the decreased 5-HT release obtained following administration of the mixed 5-HT1A autoreceptor partial agonist/alpha 1-adrenoceptor antagonist BMY7378, the selective 5-HT1A receptor agonist 8-OH-DPAT and the alpha 1-adrenoceptor antagonist prazosin. BMY7378 (0.25 mg/kg, s.c.), 8-OH-DPAT (0.025 mg/kg, s.c.) and prazosin (0.1-1.0 mg/kg, s.c.) all suppressed ventral hippocampal 5-HT efflux. The BMY7378- and 8-OH-DPAT-induced inhibition of 5-HT release were reversed by a 40 min pre-treatment with either (+/-)pindolol (8 mg/kg, s.c.) or WAY-100635 (0.3 mg/kg, s.c.), to block 5-HT1A autoreceptors. Neitehr of these antagonists altered the prazosin-induced (0.3 mg/kg, s.c.) 5-HT disease.(i) confirm that both an alpha 1-adrenoceptor antagonist (prazosin) and 5-HT1A autoreceptor stimulants (BMY7378 and 8-OH-DPAT) may reduce cerebral 5-HT release; (ii) support that the BMY7378-induced decrease in 5-HT release results from 5-HT1A autoreceptor agonism, rather than alpha 1-adrenoceptor blockade; and (iii) argue against "physiological" antagonism (i.e. via blockade of beta-adrenoceptors, 5-HT1B receptors or some other mechanism) as an explanation for the reversal by pindolol of 5-HT1A autoreceptor agonist-induced suppression of 5-HT release. These data support the usefulness of pindolol, as well as the more specific compound WAY-100635, to block 5-HT1A autoreceptors.

The effect of local injection of pertussis toxin (PTX) into the ventral tegmental area (VTA) on acoustic startle in rats was investigated. The PTX treatment caused only minor effects of its own on the acoustic startle response (ASR) or prepulse inhibition (PPI) of acoustic startle. However, systemic treatment with the indirect DA receptor agonist, amphetamine (2 mg/kg, SC) caused a significant increase in ASR magnitude and a significant disruption of PPI in PTX-treated rats while no such effects were observed in sham-treated rats. Treatment with the direct DA receptor agonist, apomorphine (2 mg/kg, SC), caused a significant disruption of PPI, an effect that was observed in both PTX- and sham-treated rats. Treatment with the 5-HT1A receptor agonist, 8-OH-DPAT (0.5 mg/kg, SC), did not affect PPI in either group but caused a marked increase in ASR magnitude in sham-treated rats. Interestingly, this effect was blocked in PTX-treated rats. The present results suggest that local injection of PTX into the VTA causes an increased sensitivity to the behavioural effects of psychostimulants on acoustic startle and may also suggest that intact midbrain 5-HT1A receptors are essential for the effect of 5-HT1A agonists on acoustic startle.

To gain further insight into the operation of 5-HT autoreceptor-mediated feedback control of 5-HT biosynthesis in serotonergic nerve terminal areas, the effect of the 5-HT1B and the 5-HT1A receptor agonists, TFMPP and 8-OH-DPAT, respectively, were investigated in the rat central nervous system (CNS) using in vivo and in vitro neurochemical approaches. TFMPP suppressed 5-HT synthesis (5-HTP accumulation after decarboxylase inhibition) both in vivo and in vitro. In vivo, the 5-HT synthesis-suppressing effect of the drug (3.0 mg/kg, s.c.) proved resistant to either acute hemitransection or reserpine (5 mg/kg, i.p.; 90 min before) pretreatment. In vitro, in cortical, hippocampal and striatal slice preparations, TFMPP (0.1-10 microM) decreased 5-HT synthesis under basal and stimulated (30 mM K+) conditions, an effect which was unaltered by prior in vivo reserpine-induced 5-HT depletion but was attenuated in the presence of 5-HT1B receptor antagonists such as methiothepin, cyanopindolol or propranolol. The 8-OH-DPAT (0.1 mg/kg, s.c.)-induced decrease of 5-HT synthesis in vivo was abolished by hemitransection but resistant to acute reserpine pretreatment; 8-OH-DPAT (10 microM) did not decrease 5-HT synthesis in vitro. In conclusion, the present study confirms the importance of 5-HT autoreceptors in the feedback control of nerve terminal 5-HT biosynthesis. Specifically, our data indicate: (1) that the reduction of rat brain 5-HT synthesis after TFMPP is mediated by 5-HT1B autoreceptors located on the serotonergic axon terminals, and (2) that the effect is directly mediated and occurs independently of 5-HT neuronal firing and intact monoamine stores.

The main neurochemical characteristic of Parkinson’s disease (PD) is a marked lesion of the nigro-striatal dopamine pathway. In attempts to provide dopamine replacement therapy to Parkinson’s patients, the current medication is L-DOPA (Birkmayer and Hornykiewicz, 1961). Dopamine (DA) itself cannot be taken orally because it will not reach the brain. Unfortunately, L-DOPA can cause serious adverse reactions and its effectiveness decreases with time. For these reasons, there has been an increasing demand for and interest in novel techniques for site-directed delivery of substances into the central nervous system (CNS) (Stahl, 1984).

(R)-11-Hydroxyaporphine (2) and (R)-11-hydroxy-10-methylaporphine (3) were synthesized from natural morphine by using new, short, and efficient synthetic sequences. The dopaminergic and serotonergic effects of 2 and 3 were evaluated by use of in vitro and in vivo test systems. The results indicate that 3 is a potent, selective, and efficacious 5-HT1A receptor agonist. In contrast, 2 is a partial 5-HT1A receptor agonist of low potency which has affinity also for central D1 and D2A receptors. The differences in pharmacological profiles were rationalized by modeling of ligand-receptor interactions using homology-based receptor models of the 5-HT1A and D2A receptor binding site. The selective and pronounced serotonergic effects of 3 appear to be due to the C10-methyl group, which is accommodated by a lipophilic pocket in the 5-HT1A receptor. In contrast, the C10-methyl group of 3 is not accommodated by the binding site model of the D2A receptor.

Biodegradable controlled-release microsphere systems made with the biocompatible biodegradable polyester excipient poly(DL-lactide-co-glycolide) constitute an exciting new technology for drug delivery to the central nervous system (CNS). Implantable controlled-release microspheres containing dopamine (DA) or norepinephrine (NE) provide a novel means to compare DA- or NE -induced restitution of function in unilateral 6-hydroxydopamine lesioned rats. A suspension of 3 microL of DA- or NE-containing microspheres or empty microspheres was implanted in 2 sites of the DA denervated striatum of rats previously unilaterally lesioned with 6-hydroxydopamine. Contralateral-rotational behavior induced by apomorphine was used as an index of lesion success and, following implantation of the microspheres, also as an index of functional recovery. Interestingly, both DA- and NE-microsphere-implanted rats displayed a 30-50% reduction in the number of apomorphine-induced rotations up to 8 wk postimplantation. Rats implanted with empty microspheres did not demonstrate significant changes in contralateral rotational behavior. Behavioral studies following implantation of a mixture of DA and NE microspheres revealed an 80% decrease in the number of apomorphine induced rotations up to 4 wk. On conclusion of the studies, immunocytochemical examination revealed growth of DA and tyrosine hydroxylase immunoreactive fibers in the striatum of DA and NE microsphere-implanted rats. Functional behavior appeared to correlate with the degree of fiber growth. Preliminary electron microscopic studies showed signs of axonal sprouting in the vicinity of the implanted microspheres. No growth was noted in rats implanted with empty microspheres. This report reviews the abilities of both microencapsulated NE and DA to assure functional recovery and to promote DA fiber (re)growth in parkinsonian rats. This novel means to deliver these substances to the central nervous system could be of therapeutic usefulness in Parkinson's disease.

Electrophysiological studies suggest that 5-HT autoreceptor desensitization may be responsible for the delayed clinical efficacy of some antidepressant drugs, such as selective 5-HT reuptake inhibitors (SSRI) and certain MAO inhibitors (MAOI). In the present study we have used in vivo microdialysis to test this hypothesis. Rats were treated for 2 weeks with the antidepressant SSRI citalopram (5 mg/kg, s.c., b.i.d.). After 24 hr withdrawal, dialysis probes were implanted in the dorsal hippocampus (DH) and the frontal cortex (FCx). The rats then received as acute challenge, a 5-HT1A autoreceptor-active dose of the reference 5-HT1A agonist 8-OH-DPAT (0.025 mg/kg s.c.). The 8-OH-DPAT-induced changes in dialysate 5-HT from the DH and the FCx were monitored and taken as an index of autoreceptor sensitivity. Chronic citalopram and control animals responded similarly to 8-OH-DPAT with a drop of 5-HT of about 50-65%; no significant difference between the chronic citalopram and control groups were obtained, either in the DH or in the FCx. These data suggest that cell body 5-HT1A autoreceptors do not desensitize in response to repeated administration with antidepressant SSRI drugs such as citalopram.

1. Electrophysiological measurements of 5-HT neuronal activity report that repeated administration of 5-HT1A receptor agonists leads to desensitization of the 5-HT1A autoreceptor but this has not yet been detected in measurements of brain 5-HT synthesis or metabolism. Here we have determined the effect of repeated administration of 5-HT1A receptor agonists on brain 5-HT release using microdialysis. 2. Acute administration of the 5-HT1A receptor agonists buspirone (0.1–5 mg/kg s.c.) and ipsapirone (0.03–3 mg/kg s.c.) caused a dose-dependent decrease in 5-HT output in ventral hippocampus of the chloral hydrate anaesthetized rat. 3. The 5-HT response to buspirone (0.1 and 0.5 mg/kg s.c.) and ipsapirone (0.3 mg/kg s.c.) was significantly inhibited by pre-treatment with the 5-HT1/β-adrenoceptor antagonist pindolol (8–16 mg/kg s.c.). The 5-HT response to buspirone (0.1 mg/kg s.c.) and ipsapirone (0.3 mg/kg s.c.) was not blocked by pretreatment with a combination of the β1 and β2-adrenoceptor antagonists metoprolol and ICI 118,551 (4 mg/kg s.c.). 4. The effect of an acute challenge of buspirone (0.5 mg/kg s.c.) on 5-HT output in ventral hippocampus was not attenuated in rats treated twice daily for 14 days with 0.5 or 5 mg/kg s.c. buspirone compared to saline-injected controls. Similarly, the decrease in 5-HT induced by an acute challenge of ipsapirone (0.5 mg/kg s.c.) was not attenuated in rats treated twice daily for 14 days with 5 mg/kg s.c. ipsapirone. 5. In further experiments it was shown that the decrease in 5-HT induced in both ventral hippocampus and striatum by an acute challenge of the selective 5-HT1A receptor agonist 8-OH-DPAT (0.025 mg/kg s.c.), was not attenuated in rats treated twice daily for 14 days with 1 mg/kg s.c. 8-OH-DPAT. 6. Basal levels of 5-HT in hippocampal and striatal microdialysates of animals treated repeatedly with the 5-HT1A receptor agonists were not consistently altered relative to treatment controls. 7. In agreement with earlier studies measuring regional brain 5-HT synthesis and metabolism, the present microdialysis measurements of 5-HT release indicate that the inhibitory effect of 5-HT1A receptor agonists on presynaptic 5-HT function is maintained in rats treated repeatedly with the same drugs.

The effects of serotoninergic drugs on dopaminergic neurotransmission in the substantia nigra, the striatum and the limbic forebrain of rat have been investigated. The accumulation of 3-methoxytyramine (3-MT) following inhibition of monoamine oxidase with pargyline was used as an indirect measure of dopamine (DA) activity in vivo. The effects of the following serotoninergic drugs were tested: the 5-HT1A receptor agonist 8-OH-DPAT, the 5-HT1B receptor agonist trifluoromethyl-phenylpiperazine (TFMPP), CGS 12066B and RU 24969, the 5-HT1A/1B antagonist (±)pindolol, the 5-HT2/1C receptor antagonist ritanserin, the 5-HT2/1C receptor agonist DL-1-(2,5-dimethoxy-4-iodo-phenyl)-2-aminopropane (DOI), the 5-HT3 receptor antagonist BRL 43 694, the unselective 5-HT1 receptor antagonist methiothepin, and carbidopa+L-5-hydroxytryptophan (L5-HTP) to achieve a general, unselective stimulation of multiple 5-HT receptors. In the substantia nigra, carbidopa + 5-HTP treatment increased the 3-MT accumulation by 26% and decreased the DA concentration to 67% of controls, tentatively suggesting a 5-HTP-induced displacement of nigral DA. A minor, non dose-related reduction in nigral 3-MT was seen after the 5-HT1A receptor agonist 8-OH-DPAT. None of the other serotonin receptor acting drugs induced any pronounced effect on the nigral 3-MT accumulation. Taken together, the findings provide little support for the idea that one single 5-HT1 receptor subtype serves a modulatory function on DA activity in the substantia nigra. In the striatum and the limbic forebrain, trifluoromethylphenylpiperazine dose-dependently increased the 3-MT accumulation to maximally 200%–220% of controls. In the limbic forebrain also the highest dose of RU 24 969 (15 mg/kg) increased the 3-MT accumulation (78%), whereas in the striatum the lowest does of the drug (1.5 mg/kg) decreased it by 30%. The trifluoromethylphenylpiperazine-induced stimulation of 3-MT accumulation was not blocked by ritanserin. In the limbic forebrain, also DL-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane and carbidopa+Lr5-HTP treatment increased the 3-MT concentrations to 120% and 150% of controls, respectively. Paradoxically, methiothepin also induced an increase of the 3-MT accumulation in these brain regions, probably due to its DA receptor antagonism. None of the other serotoninergic drugs induced any pronounced effects on the 3-MT accumulation in these brain parts. The results may overall support the hypothesis that 5-HT1 does modulate the DA activity in the striatum and limbic forebrain, tentatively via 5-HT1B receptors in the striatum and 5-HT1B and 5-HT2 or 5-HT1C receptors in the limbic forebrain. It may be speculated therefore, that clinical application of 5-HT1 receptor-modulating drugs to influence central dopaminergic activity might be of therapeutical benefit, for example, in motor disorders like Parkinson's disease.

The putative existence of a functional alpha 2-adrenoceptor-mediated modulation of 5-HT release in vivo from serotonergic neuronal terminals in the ventral hippocampus was investigated using intracerebral microdialysis in chloral hydrate-anaesthetised rats. The alpha 2-adrenoceptor agonist clonidine (0.01-0.3 mg/kg, SC) dose-dependently decreased the 5-HT output. The response to clonidine was antagonized by systemic or local administration of the alpha 2-adrenoceptor antagonist idazoxan (0.1 mg/kg, SC, or 10 mumol/l, via the dialysis perfusion medium). Similarly, the 5-HT release-suppressing response to the thiazole alpha 2-adrenoceptor agonist jingsongling (0.1 mg/kg, SC) was blocked by idazoxan (0.1 mg/kg, SC). The mixed beta-adrenoceptor/5-HT1 receptor antagonist pindolol (8.0 mg/kg, SC) did not affect the clonidine-induced reduction of 5-HT release. Tyrosine hydroxylase inhibition by means of alpha-methyl-para-tyrosine (alpha-MT; 250 mg/kg, IP) caused a drastic reduction (greater than 80%) in dialysate 3,4-dihydroxyphenyl acetic acid (DOPAC) output but did not affect the 5-HT output per se over 3 h post-injection. Nor did the alpha-MT pretreatment prevent, but instead significantly enhanced, the 5-HT release-suppressing effect of clonidine. The results demonstrate that the release of 5-HT from serotonergic nerve terminals in rat ventral hippocampus in vivo is modulated by alpha 2-adrenoceptors, probably both by heteroreceptors on the axon terminals of the serotonergic neurones and by other alpha 2-adrenoceptor sites situated pre- and/or postsynaptic to the noradrenergic terminals. Our results also suggest that while functionally operative, these sites may receive little physiological tone, at least in chloral hydrate-anaesthetised rats.