A hidden Markov model to assess drug-induced sleep fragmentation in the telemetered rat.

Drug-induced sleep fragmentation can cause sleep disturbances either via their intended pharmacological action or as a side effect. Examples of disturbances include excessive daytime sleepiness, insomnia and nightmares. Developing drugs without these side effects requires insight into the mechanisms leading to sleep disturbance. The characterization of the circadian sleep pattern by EEG following drug exposure has improved our understanding of these mechanisms and their translatability across species. The EEG shows frequent transitions between specific sleep states leading to multiple correlated sojourns in these states. We have developed a Markov model to consider the high correlation in the data and quantitatively compared sleep disturbance in telemetered rats induced by methylphenidate, which is known to disturb sleep, and of a new chemical entity (NCE). It was assumed that these drugs could either accelerate or decelerate the transitions between the sleep states. The difference in sleep disturbance of methylphenidate and the NCE were quantitated and different mechanisms of action on rebound sleep were identified. The estimated effect showed that both compounds induce sleep fragmentation with methylphenidate being fivefold more potent compared to the NCE.

Bundling arrows: improving translational CNS drug development by integrated PK/PD-metabolomics.

INTRODUCTION: Diseases of the Central Nervous System (CNS) affect millions of people worldwide, with the number of people affected quickly growing. Unfortunately, the successful development of CNS-acting drugs is less than 10%, and this is attributed to the complexity of the CNS, unexpected side effects, difficulties in penetrating the blood-brain barrier and lack of biomarkers. Areas covered: Herein, the authors first review how pharmacokinetic/pharmacodynamic (PK/PD) models are designed to predict the dose-dependent time course of effect, and how they are used to translate drug effects from animal to man. Then, the authors discuss how pharmacometabolomics gives insight into system-wide pharmacological effects and why it is a promising method to study interspecies differences. Finally, the authors advocate the application of PK/PD-metabolomics modeling to advance translational CNS drug development by discussing its opportunities and challenges. Expert opinion: It is envisioned that PK/PD-metabolomics will increase our understanding of CNS drug effects and improve translational CNS drug development, thereby increasing success rates. The successful future development of this concept will require multi-level and longitudinal biomarker evaluation over a large dose range, multi-tissue biomarker evaluation, and the generation of a proof of principle by application to multiple CNS drugs in multiple species.

The influence of drug distribution and drug-target binding on target occupancy: The rate-limiting step approximation.

The influence of drug-target binding kinetics on target occupancy can be influenced by drug distribution and diffusion around the target, often referred to as "rebinding" or "diffusion-limited binding". This gives rise to a decreased decline of the drug-target complex concentration as a result of a locally higher drug concentration that arises around the target, which leads to prolonged target exposure to the drug. This phenomenon has been approximated by the steady-state approximation, assuming a steady-state concentration around the target. Recently, a rate-limiting step approximation of drug distribution and drug-target binding has been published. However, a comparison between both approaches has not been made so far. In this study, the rate-limiting step approximation has been rewritten into the same mathematical format as the steady-state approximation in order to compare the performance of both approaches for the investigation of the influence of drug-target binding kinetics on target occupancy. While both approximations clearly indicated the importance of kon and high target concentrations, it was shown that the rate-limiting step approximation is more accurate than the steady-state approximation, especially when dissociation is fast compared to association and distribution out of the binding compartment. It is therefore concluded that the new rate-limiting step approximation is to be preferred for assessing the influence of binding kinetics on local target site concentrations and target occupancy.

Multivariate pharmacokinetic/pharmacodynamic (PKPD) analysis with metabolomics shows multiple effects of remoxipride in rats.

The study of central nervous system (CNS) pharmacology is limited by a lack of drug effect biomarkers. Pharmacometabolomics is a promising new tool to identify multiple molecular responses upon drug treatment. However, the pharmacodynamics is typically not evaluated in metabolomics studies, although being important properties of biomarkers. In this study we integrated pharmacometabolomics with pharmacokinetic/pharmacodynamic (PKPD) modeling to identify and quantify the multiple endogenous metabolite dose-response relations for the dopamine D2 antagonist remoxipride. Remoxipride (vehicle, 0.7 or 3.5mg/kg) was administered to rats. Endogenous metabolites were analyzed in plasma using a biogenic amine platform and PKPD models were derived for each single metabolite. These models were clustered on basis of proximity between their PKPD parameter estimates, and PKPD models were subsequently fitted for the individual clusters. Finally, the metabolites were evaluated for being significantly affected by remoxipride. In total 44 metabolites were detected in plasma, many of them showing a dose dependent decrease from baseline. We identified 6 different clusters with different time and dose dependent responses and 18 metabolites were revealed as potential biomarker. The glycine, serine and threonine pathway was associated with remoxipride pharmacology, as well as the brain uptake of the dopamine and serotonin precursors. This is the first time that pharmacometabolomics and PKPD modeling were integrated. The resulting PKPD cluster model described diverse pharmacometabolomics responses and provided a further understanding of remoxipride pharmacodynamics. Future research should focus on the simultaneous pharmacometabolomics analysis in brain and plasma to increase the interpretability of these responses.

Methodologies for Quantitative Systems Pharmacology (QSP) Models: Design and Estimation.

With the increased interest in the application of quantitative systems pharmacology (QSP) models within medicine research and development, there is an increasing need to formalize model development and verification aspects. In February 2016, a workshop was held at Roche Pharma Research and Early Development to focus discussions on two critical methodological aspects of QSP model development: optimal structural granularity and parameter estimation. We here report in a perspective article a summary of presentations and discussions.

Advanced Methods for Dose and Regimen Finding During Drug Development: Summary of the EMA/EFPIA Workshop on Dose Finding (London 4-5 December 2014).

Inadequate dose selection for confirmatory trials is currently still one of the most challenging issues in drug development, as illustrated by high rates of late-stage attritions in clinical development and postmarketing commitments required by regulatory institutions. In an effort to shift the current paradigm in dose and regimen selection and highlight the availability and usefulness of well-established and regulatory-acceptable methods, the European Medicines Agency (EMA) in collaboration with the European Federation of Pharmaceutical Industries Association (EFPIA) hosted a multistakeholder workshop on dose finding (London 4-5 December 2014). Some methodologies that could constitute a toolkit for drug developers and regulators were presented. These methods are described in the present report: they include five advanced methods for data analysis (empirical regression models, pharmacometrics models, quantitative systems pharmacology models, MCP-Mod, and model averaging) and three methods for study design optimization (Fisher information matrix (FIM)-based methods, clinical trial simulations, and adaptive studies). Pairwise comparisons were also discussed during the workshop; however, mostly for historical reasons. This paper discusses the added value and limitations of these methods as well as challenges for their implementation. Some applications in different therapeutic areas are also summarized, in line with the discussions at the workshop. There was agreement at the workshop on the fact that selection of dose for phase III is an estimation problem and should not be addressed via hypothesis testing. Dose selection for phase III trials should be informed by well-designed dose-finding studies; however, the specific choice of method(s) will depend on several aspects and it is not possible to recommend a generalized decision tree. There are many valuable methods available, the methods are not mutually exclusive, and they should be used in conjunction to ensure a scientifically rigorous understanding of the dosing rationale.

A Six-Stage Workflow for Robust Application of Systems Pharmacology.

Quantitative and systems pharmacology (QSP) is increasingly being applied in pharmaceutical research and development. One factor critical to the ultimate success of QSP is the establishment of commonly accepted language, technical criteria, and workflows. We propose an integrated workflow that bridges conceptual objectives with underlying technical detail to support the execution, communication, and evaluation of QSP projects.

Understanding the Behavior of Systems Pharmacology Models Using Mathematical Analysis of Differential Equations: Prolactin Modeling as a Case Study.

In this tutorial, we introduce basic concepts in dynamical systems analysis, such as phase-planes, stability, and bifurcation theory, useful for dissecting the behavior of complex and nonlinear models. A precursor-pool model with positive feedback is used to demonstrate the power of mathematical analysis. This model is nonlinear and exhibits multiple steady states, the stability of which is analyzed. The analysis offers insight into model behavior and suggests useful parameter regions, which simulations alone could not.

A Tutorial on Target-Mediated Drug Disposition (TMDD) Models.

Target-mediated drug disposition (TMDD) is the phenomenon in which a drug binds with high affinity to its pharmacological target site (such as a receptor) to such an extent that this affects its pharmacokinetic characteristics.1 The aim of this Tutorial is to provide an introductory guide to the mathematical aspects of TMDD models for pharmaceutical researchers. Examples of Berkeley Madonna2 code for some models discussed in this Tutorial are provided in the Supplementary Materials.

Inter-study variability of preclinical in vivo safety studies and translational exposure-QTc relationships--a PKPD meta-analysis.

BACKGROUND AND PURPOSE: Preclinical cardiovascular safety studies (CVS) have been compared between facilities with respect to their sensitivity to detect drug-induced QTc prolongation (ΔQTc). Little is known about the consistency of quantitative ΔQTc predictions that are relevant for translation to humans. EXPERIMENTAL APPROACH: We derived typical ΔQTc predictions at therapeutic exposure (ΔQTcTHER ) with 95% confidence intervals (95%CI) for 3 Kv 11.1 (hERG) channel blockers (moxifloxacin, dofetilide and sotalol) from a total of 14 CVS with variable designs in the conscious dog. Population pharmacokinetic-pharmacodynamic (PKPD) analysis of each study was followed by a meta-analysis (pooling 2-6 studies including 10-32 dogs per compound) to derive meta-predictions of typical ΔQTcTHER . Meta-predictions were used as a reference to evaluate the consistency of study predictions and to relate results to those found in the clinical literature. KEY RESULTS: The 95%CIs of study-predicted ΔQTcTHER comprised in 13 out of 14 cases the meta-prediction. Overall inter-study variability (mean deviation from meta-prediction at upper level of therapeutic exposure) was 30% (range: 1-69%). Meta-ΔQTcTHER predictions for moxifloxacin, dofetilide and sotalol overlapped with reported clinical QTc prolongation when expressed as %-prolongation from baseline. CONCLUSIONS AND IMPLICATIONS: Consistent exposure-ΔQTc predictions were obtained from single preclinical dog studies of highly variable designs by systematic PKPD analysis, which is suitable for translational purposes. The good preclinical-clinical pharmacodynamic correlations obtained suggest that such an analysis should be more routinely applied to increase the informative and predictive value of results obtained from animal experiments.

Analysis of the effects of alpha 1-adrenoceptor antagonists on noradrenaline-mediated contraction of rat small mesenteric artery.

1. In this study, we examined the interaction between noradrenaline (NA) and phenylephrine (PE) with seven antagonists (prazosin, tamsulosin, phentolamine, WB-4101, 5-methylurapidil, spiperone and HV 723) in an attempt to characterize the alpha 1-adrenoceptor population of the rat isolated small mesenteric artery (SMA) preparation. 2. Six of the seven antagonists investigated produced concentration-dependent, parallel, rightward shift of the NA concentration-effect (E/[A]) curves. The exception was tamsulosin, which produced significant decrease of the upper asymptote. In the case of 5-methylurapidil and HV723, the Schild plot slope parameters were not significantly different from unity over the range of concentrations used. However, the Schild plot slopes obtained for the other antagonists were all significantly greater than unity, inconsistent with expectations for simple competitive antagonism. 3. HV723, prazosin and tamsulosin were also tested using PE as an agonist. All three antagonists produced concentration-dependent, parallel, rightward shifts of the PE curves and Schild analysis yielded slope parameters not significantly different from unity. The pKB estimates obtained for tamsulosin and prazosin were not significantly different from the pA2 values obtained when NA was used as agonist. In the case of HV723, the 95% confidence intervals for the pKB values yielded with NA and PE did not overlap (pKB = 8.80-9.13 and 8.15-8.77 for NA and PE, respectively). 4. In the absence of evidence to indicate that the steep Schild plots were due to failure to satisfy the basic criteria for quantitative analysis in a one-receptor system, we considered the possibility that the complexity was caused by an action of NA at inhibitory D1 receptors. The selective D1 receptor antagonists, SCH-23390 (10 nM), had no significant effect on the NA E/[A] control curve, but the apparent potency of 100 nM prazosin was reduced by approximately 3.5 fold. 5. This study indicates that the steep Schild plots obtained from the interaction between NA and alpha 1-adrenoceptor antagonists were due to the simultaneous activation of inhibitory D1 receptors by NA. Notwithstanding this complexity, our explanatory model of the system (see Appendix) suggests that the antagonist affinity values estimated in the absence of D1 receptor block were not significantly affected by this other action of NA. The low affinity estimate obtained for prazosin suggests that the pharmacologically-defined alpha IL-subtype operates in the SMA.

Characterization of receptors mediating contraction of the rat isolated small mesenteric artery and aorta to arginine vasopressin and oxytocin.

1. The exact nature of the receptor subtype(s) involved in the action of arg-vasopressin (AVP) on the rat aorta and small mesenteric artery (SMA) is controversial. Therefore, we have studied the effects of the selective V1A receptor antagonists, OPC 21268 and SR 49059, and the oxytocin (OT) receptor antagonist, atosiban, on the AVP- and OT-induced contractions of the two vessels. 2. AVP and OT displayed similar intrinsic activities in the rat aorta and SMA, but AVP was approximately 130 fold and approximately 500 fold more potent than OT, respectively. In the rat aorta, Hill slopes (nH) were similar for OT and AVP. However, in rat SMA, the OT concentration-effect (E/[A]) curve was significantly steeper than the AVP E/[A] curve (nH, = 3.3+/-0.20, 2.3+/-0.15; P<0.001). 3. In the aorta OPC 21268, SR 49059 and atosiban competitively antagonized the AVP and OT E/[A] curves. Except for atosiban and SR 49059 against AVP, competitive antagonism was also observed in the SMA. Atosiban caused concentration-dependent steepening of the AVP E/[A] curve, whereas SR 49059 decreased the upper asymptote. 4. Schild analysis yielded affinities indicative of V1A receptor involvement in both vessels: pKB/ pA2=9.20 9.48, 7.56 7.71 and 6.19 6.48 for SR 49059, OPC 21268 and atosiban, respectively. 5. Neither AVP nor OT relaxed U46619 pre-contracted aorta or SMA in the presence of SR 49059, suggesting no interference of a vasodilatory component. 6. Despite predominant involvement of V1A receptors in both vessels, the different Hill slopes of AVP and OT E/[A] curves as well as the steepening of the AVP E/[A] curves by atosiban are indicative of receptor heterogeneity in the rat SMA.

Analysis of alpha 1L-adrenoceptor pharmacology in rat small mesenteric artery.

1. To illuminate the controversy on alpha 1A- or alpha 1L-adrenoceptor involvement in noradrenaline-mediated contractions of rat small mesenteric artery (SMA), we have studied the effects of subtype-selective alpha 1-adrenoceptor agonists and antagonists under different experimental conditions. 2. The agonist potency order in rat SMA was: A61603 >> SKF89748-A > cirazoline > noradrenaline > ST-587 > methoxamine. Prazosin antagonized all agonists with a low potency (pA2: 8.29-8.80) indicating the involvement of alpha 1L-rather than alpha 1A-adrenoceptors. 3. The putative alpha 1L-adrenoceptor antagonist JTH-601, but not the alpha 1B-adrenoceptor antagonist chloroethylclonidine (10 microM) antagonized noradrenaline-induced contractions of SMA. The potency of the selective alpha 1D-adrenoceptor antagonist BMY 7378 against noradrenaline (pA2 = 6.16 +/- 0.13) and of the selective alpha 1A-adrenoceptor antagonist RS-17053 against noradrenaline (pKB = 8.35 +/- 0.10) and against the selective alpha 1A-adrenoceptor agonist A-61603 (pKB = 8.40 +/- 0.09) were too low to account for alpha 1D- and alpha 1A-adrenoceptor involvement. 4. The potency of RS-17053 (pKB/pA2's = 7.72-8.46) was not affected by lowering temperature, changing experimental protocol or inducing myogenic tone via KCl or U46619. 5. Selective protection of a putative alpha 1A-adrenoceptor population against the irreversible action of phenoxybenzamine also failed to increase the potency of RS-17053 (pA2 = 8.25 +/- 0.06 against A61603). 6. Combined concentration-ratio analysis demonstrated that tamsulosin, which does not discriminate between alpha 1A- and alpha 1L-adrenoceptors, and RS-17053 competed for binding at the same site in the SMA. 7. In summary, data obtained in our experiments in rat SMA indicate that the alpha 1-adrenoceptor mediating noradrenaline-induced contraction displays a distinct alpha 1L-adrenoceptor pharmacology. This study does not provide evidence for the hypothesis that alpha 1L-adrenoceptors represent an affinity state of the alpha 1A-adrenoceptor in functional assays. Furthermore, there is no co-existing alpha 1A-adrenoceptor in the SMA.

Analysis of the activity of alpha 1-adrenoceptor antagonists in rat aorta.

1. In this study, the effect of seven alpha 1-adrenoceptor antagonists (tamsulosin, phentolamine, prazosin, WB-4101, 5-methylurapidil, spiperone and HV723) have been examined on the contractile response to noradrenaline (NA) and phenylephrine (PE) in rat isolated aorta. 2. NA and PE, when administered using a cumulative dosing schedule, both produced concentration-dependent contraction of aortic rings. It was possible to fit the individual concentration-effect (E/[A]) curve data to the Hill equation to provide estimates of the curve midpoint location (p[A]50 = 7.74 +/- 0.10 and 7.14 +/- 0.18), midpoint slope (nH = 0.82 +/- 0.03 and 0.99 +/- 0.10) and upper asymptote (alpha = 3.2 +/- 0.3 and 3.1 +/- 0.2 g) parameters for NA and PE, respectively. However, the Hill equation provided a better fit to the E/[A] curve data obtained with another contractile agent, 5-hydroxytryptamine (5-HT) (p[A50] = 6.09 +/- 0.08, nH = 1.49 +/- 0.09, alpha = 2.6 +/- 0.3 g), as judged by calculation of the mean sum of squares of the differences between the observed and predicted values. 3. All of the antagonists investigated produced concentration-dependent inhibition of the contractile responses of the aorta to NA and PE. Although no significant effects on the upper asymptotes of the E/[A] curves of any of the antagonists tested were detected, only tamsulosin and 5-methylurapidil did not have a significant effect on the slope (nH) of the NA and PE E/[A] curves. The other antagonists produced significant steepening of the curves obtained with NA and/or PE. 4. Notwithstanding the fact that one of the basic criteria for simple competitive antagonism at a single receptor class was not always satisfied, the individual log [A]50 values estimated in the absence and presence of antagonist within each experiment were fitted to the competitive model. The Schild plot slope parameters for the antagonism of NA and PE by phentolamine and HV723 were found to be significantly less than unity. The Schild plot slope parameters for the other antagonists were not significantly different from unity. 5. In the absence of evidence to suggest that the deviations from simple competitive antagonism were due to failure to satisfy basic experimental conditions for quantitative analysis, an attempt was made to see whether the data could be accounted for by an existing two-receptor model (Furchgott, 1981). The goodness-of-fit obtained with the two-receptor model was significantly better than that obtained with the one-receptor model. Furthermore, with the exception of the data obtained with phentolamine, the pKB estimates for the two receptors were independent of whether NA or PE was used as agonist. 6. To determine which alpha 1-adrenoceptor subtypes may be associated with those defined by the two receptor model, the mean pKB estimates obtained from the two-receptor model fit were compared with affinities measured by Laz et al. (1994) for rat cloned alpha 1-adrenoceptor subtypes expressed in COS-7 cells. The sum of squared differences of the data points from the line of identity was smallest for both pKB1 and pKB2 in the case of the alpha 1a/d-adrenoceptor (now referred to as alpha 1d-adrenoceptor; Hieble et al., 1995). Therefore, the complexity exposed in this study may be due to the expression of closely-related forms of the alpha 1d-adrenoceptor. However, relatively good matches were also found between pKB1 and alpha 1c and between pKB2 and alpha 1b. Therefore, on the basis of these data, it is not possible to rule out the involvement of all three alpha 1-adrenoceptors. The conflicting reports concerning the characteristics of the alpha 1-adrenoceptor population mediating contraction of the rat aorta may, at least in part, be due to the lack of highly selective ligands and to between-assay variation in the expression of multiple alpha 1-adrenoceptors.

Exposure and characterization of the action of noradrenaline at dopamine receptors mediating endothelium-independent relaxation of rat isolated small mesenteric arteries.

1. Previously, we reported that noradrenaline (NA), in addition to its alpha 1-adrenoceptor-mediated contractile effect, may relax the rat small mesenteric artery (SMA) in order to account for steep Schild plots obtained with compounds classified as alpha 1-adrenoceptor antagonists. In this study, a relaxant action of NA has been exposed in the rat isolated, endothelium-denuded SMA precontracted by the thromboxane A2-mimetic, U46619. 2. NA, but not the selective alpha 2-adrenoceptor agonist, UK14304, produced concentration-dependent contraction of the SMA (pEC50 = 5.7 +/- 0.1). After precontraction with 0.1 microM U46619, 10 nM-30 microM NA produced a further contraction (pEC50 = 6.1 +/- 0.2), while higher concentrations of NA produced small, but significant, relaxant responses. 3. In the presence of 1 microM prazosin, 0.1-30 microM NA produced concentration dependent relaxation (pIC50 = 5.9 +/- 0.1) after precontraction with 0.1 microM U46619. The NA relaxation concentration-effect curve was completely inhibited by 1 microM of the beta 1/beta 2-adrenoceptor antagonist, timolol. However, when the concentration of prazosin was increased by 10 fold (10 microM), NA once again produced concentration-dependent relaxation (pIC50 = 4.5 +/- 0.2). This relaxation concentration-effect curve was not blocked by a 10 fold higher concentration of timolol (10 microM), nor by the presence of idazoxan (10 microM), cyanopindolol (10 microM), NG-nitro-L-arginine methyl ester (L-NAME, 100 microM), indomethacin (10 microM) or sulpiride (1 microM). However, haloperidol (10 microM) and (+/-)-SCH-23390 (10 nM) produced significant inhibition of the relaxation, suggesting the involvement of dopamine D1 receptors. 4. Dopamine also produced concentration-dependent relaxation following U46619 precontraction (pIC50 = 5.4 +/- 0.1) which was significantly inhibited by haloperidol and (+)-SCH-23390. Pretreatment with 10 microM phenoxybenzamine for 60 min produced a significant inhibition of the dopamine and NA relaxation curves and application of the operational model of agonism yielded estimates of the affinity (pKA = 5.3 +/- 0.2 and 4.4 +/- 0.2) and efficacy (log gamma = 0.06 +/- 0.11 and 0.01 +/- 0.10) for dopamine and NA, respectively, at D1 receptors. 5. HV723 (0.1 and 1 microM), a ligand that yielded a Schild plot slope parameter of unity as an antagonist of NA in the contractile assay, produced concentration-dependent inhibition of the NA-mediated relaxation (pA2 approximately 8). 6. The results of this study indicate that NA can activate D1 receptors mediating relaxation in the rat SMA at concentrations which were encountered in our previous receptor classification experiments using competitive alpha 1-adrenoceptor antagonists.

Analysis of alpha1-adrenoceptors in rabbit lower urinary tract and mesenteric artery.

In this study, we have investigated the effects of a series of alpha1-adrenoceptor antagonists on the phenylephrine-mediated contractions of rabbit isolated prostate, urethra, trigone and mesenteric artery. With the exception of RS-17053 (N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-alpha,alpha-dim ethyl-1 H-indole-3-ethanamine hydrochloride), the antagonists displayed the lowest potency in the urethra. Catecholamine uptake1 and uptake2 appeared not to be the cause for the low pK(B)/pA2 values obtained in the urethra because cocaine and corticosterone had no effect on the potency of phenylephrine in this tissue. The low potencies displayed by prazosin. RS-17053 and HV723 (alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)amino )propyl)benzene-acetonitrile fumarate) suggest that the functional receptors in all four tissues belong to the alpha(1L)-adrenoceptor class. Whether or not the significant between-tissue differences in antagonist potencies are due to heterogeneity of this receptor class remains to be elucidated.

The alpha 1-adrenoceptor agonist, SDZ NVI-085, behaves as a potent, competitive antagonist of 5-hydroxytryptamine-induced contraction of rat aorta.

In this study we investigated the actions of SDZ NVI-085 ((-)-(4aR,10aR)-3,4,4a,5,10,10a-hexahydro-6-methoxy-4-methyl -9-(methylthio)-2H- naphth[2,3-b)-1,4-oxazine hydrogen malonate), previously classified and employed as a selective alpha 1-adrenoceptor agonist, in the rat isolated aorta assay. It was shown that, in addition to its alpha 1-adrenoceptor agonistic action, SDZ NVI-085 behaves as a competitive antagonist of 5-hydroxytryptamine (5-HT)-induced contraction of rat aorta (pKB = 8.13 +/- 0.08). The structurally related alpha 1-adrenoceptor agonist, SK & F 89748-A (l-1,2,3,4-tetrahydro-8-methoxy-5-(methylthio)-2-naphthalenamine hydrochloride), produced inhibition of the 5-HT response only at the highest concentration tested (1 microM) with an associated pA2 value of 6.0 +/- 0.1. These findings suggest that the affinity of SDZ NVI-085 for 5-HT2 receptors is considerably higher than for alpha 1-adrenoceptors, which may have implications for its use as a pharmacological tool.

Functional characterisation of the pharmacological profile of the putative alpha1B-adrenoceptor antagonist, (+)-cyclazosin.

We studied the functional pharmacological profile of (+)-cyclazosin, which has been characterised as a selective, high-affinity (pKi = 9.68) alpha1B-adrenoceptor ligand in binding experiments with rat liver membranes. The pKa/pA2 values for antagonism of contractions mediated via alpha1A/L-adrenoceptors of rat small mesenteric artery, alpha1B-adrenoceptors of rat aorta and alpha1B-adrenoceptors of rat spleen were 7.78 +/- 0.04, 6.86 +/- 0.07 and 7.96 +/- 0.08, respectively. Furthermore, in mouse spleen, which is also regarded as an alpha1B-adrenoceptor preparation, (+)-cyclazosin displayed low potency and did not act as a competitive antagonist. Thus, in contrast with results obtained in radioligand binding experiments, (+)-cyclazosin does not behave as a selective alpha1B-adrenoceptor antagonist in functional tissues. Whether this discrepancy has consequences for the classification of alpha1-adrenoceptors requires further investigation.

Benextramine acts as an irreversible noncompetitive antagonist of U46619-mediated contraction of the rat small mesenteric artery.

We have studied the effects of benextramine on the U46619 (11 alpha,9 alpha-epoxymethano-15S-hydroxy-prosta-5Z,13E-dienoic acid)-mediated contraction of the rat isolated small mesenteric artery. U46619 (10 nM-10 microM) produced a concentration-dependent contraction of the small mesenteric artery. The selective prostanoid TP receptor antagonist, SQ 30,741 ([1S-[1 alpha,2 alpha(5Z),3 alpha,4 alpha]]-7- [[[[[(oxaheptyl)amino]acetyl]amino]-methyl]-7-oxabicyclo[2.2.1]hep t-2-yl]-5- heptenoic acid; 1 microM), produced a parallel, rightward shift of the U46619 curve with an associated pA2 value of 7.43 +/- 0.09. Treatment of tissues with 100 microM benextramine depressed the maximum response to U46619 in a time-dependent manner. However, neither SQ 30,741 (10 microM) nor U46619 (10 microM) incubation significantly protected against this effect. Thus, benextramine acts as an irreversible noncompetitive antagonist of U46619. The mechanism of this action is not yet clear.

Characterisation of the 5-HT receptor potentiating neurotransmission in rabbit bladder.

We have investigated the effects of 5-hydroxytryptamine (5-HT) on electrically induced contractions of rabbit isolated bladder. Electrical field stimulation evoked twitch contractions which were potentiated by 5-HT (0.3-10 microM). The potentiating effect of 5-HT was inhibited by ondansetron (pA2 9.2) and granisetron (pA2 9.1) but not by methysergide or SB 204070 ((1-butyl-4-piperidinyl)methyl 8-amino-7-chloro-1,4-benzodioxan-5-carboxylate hydrochloride). This suggests that the potentiating effect of micromolar concentrations of 5-HT on neuromuscular transmission in rabbit isolated bladder is mediated by 5-HT3 receptors. The receptors involved in the response to lower concentrations of 5-HT, observed in some tissues, remain to be characterised.