Validation of an LC-MS/MS Method to Quantify the New TRPC6 Inhibitor SH045 (Larixyl N-methylcarbamate) and Its Application in an Exploratory Pharmacokinetic Study in Mice

TRPC6 (transient receptor potential cation channels; canonical subfamily C, member 6) is widespread localized in mammalian tissues like kidney and lung and associated with progressive proteinuria and pathophysiological pulmonary alterations, e.g., reperfusion edema or lung fibrosis. However, the understanding of TRPC6 channelopathies is still at the beginning stages. Recently, by chemical diversification of (+)-larixol originating from Larix decidua resin traditionally used for inhalation, its methylcarbamate congener, named SH045, was obtained and identified in functional assays as a highly potent, subtype-selective inhibitor of TRPC6. To pave the way for use of SH045 in animal disease models, this study aimed at developing a capable bioanalytical method and to provide exploratory pharmacokinetic data for this promising derivative. According to international guidelines, a robust and selective LC-MS/MS method based on MRM detection in positive ion mode was established and validated for quantification of SH045 in mice plasma, whereby linearity and accuracy were demonstrated for the range of 2–1600 ng/mL. Applying this method, the plasma concentration time course of SH045 following single intraperitoneal administration (20 mg/kg body weight) revealed a short half-life of 1.3 h. However, the pharmacological profile of SH045 is promising, as five hours after administration, plasma levels still remained sufficiently higher than published low nanomolar IC50 values. Summarizing, the LC-MS/MS method and exploratory pharmacokinetic data provide essential prerequisites for experimental pharmacological TRPC6 modulation and translational treatment of TRPC6 channelopathies

Predicting beneficial effects of atomoxetine and citalopram on response inhibition in Parkinson's disease with clinical and neuroimaging measures.

Recent studies indicate that selective noradrenergic (atomoxetine) and serotonergic (citalopram) reuptake inhibitors may improve response inhibition in selected patients with Parkinson's disease, restoring behavioral performance and brain activity. We reassessed the behavioral efficacy of these drugs in a larger cohort and developed predictive models to identify patient responders. We used a double-blind randomized three-way crossover design to investigate stopping efficiency in 34 patients with idiopathic Parkinson's disease after 40 mg atomoxetine, 30 mg citalopram, or placebo. Diffusion-weighted and functional imaging measured microstructural properties and regional brain activations, respectively. We confirmed that Parkinson's disease impairs response inhibition. Overall, drug effects on response inhibition varied substantially across patients at both behavioral and brain activity levels. We therefore built binary classifiers with leave-one-out cross-validation (LOOCV) to predict patients' responses in terms of improved stopping efficiency. We identified two optimal models: (1) a "clinical" model that predicted the response of an individual patient with 77-79% accuracy for atomoxetine and citalopram, using clinically available information including age, cognitive status, and levodopa equivalent dose, and a simple diffusion-weighted imaging scan; and (2) a "mechanistic" model that explained the behavioral response with 85% accuracy for each drug, using drug-induced changes of brain activations in the striatum and presupplementary motor area from functional imaging. These data support growing evidence for the role of noradrenaline and serotonin in inhibitory control. Although noradrenergic and serotonergic drugs have highly variable effects in patients with Parkinson's disease, the individual patient's response to each drug can be predicted using a pattern of clinical and neuroimaging features.The BCNI is supported by the Wellcome Trust and Medical Research Council. We are grateful to Dr Gordon Logan for advice on stop-signal reaction time estimation and to Dr Marta Correia for advice on diffusion-weighted imaging data analysis. Conflict of interest: Prof. Sahakian has received grants from Janssen/J&J, personal fees from Cambridge Cognition, personal fees from Lundbeck, and personal fees from Servier, outside the submitted work. Prof. Robbins has received personal fees and royalties from Cambridge Cognition, personal fees and grants from Eli Lilly Inc, personal fees and grants from Lundbeck, grants from GSK, personal fees from Teva Pharmaceuticals, personal fees from Shire Pharmaceuticals, grants from Medical Research Council, editorial honorarium from Springer Verlag Germany, and personal fees from Chempartners, outside the submitted work. Prof. Rowe has received grant funding from AZ-Medimmune unrelated to the current work. Dr Housden is an employee of Cambridge Cognition. Other authors reported no biomedical financial interests or potential conflict of interest.This is the final version of the article. It was first available from Wiley via http://dx.doi.org/10.1002/hbm.2308

A positron emission tomography study of nigro-striatal dopaminergic mechanisms underlying attention: implications for ADHD and its treatment.

Through the combined use of (18)F-fallypride positron emission tomography and magnetic resonance imaging this study examined the neural mechanisms underlying the attentional deficits associated with attention deficit/hyperactivity disorder and their potential reversal with a single therapeutic dose of methylphenidate. Sixteen adult patients with attention deficit/hyperactivity disorder and 16 matched healthy control subjects were positron emission tomography and magnetic resonance imaging scanned and tested on a computerized sustained attention task after oral methylphenidate (0.5 mg/kg) and placebo administration in a within-subject, double-blind, cross-over design. Although patients with attention deficit/hyperactivity disorder as a group showed significant attentional deficits and reduced grey matter volume in fronto-striato-cerebellar and limbic networks, they had equivalent D2/D3 receptor availability and equivalent increases in endogenous dopamine after methylphenidate treatment to that observed in healthy control subjects. However, poor attentional performers drawn from both the attention deficit/hyperactivity disorder and the control groups had significantly reduced left caudate dopamine activity. Methylphenidate significantly increased dopamine levels in all nigro-striatal regions, thereby normalizing dopamine levels in the left caudate in low performers. Behaviourally, methylphenidate improved sustained attention in a baseline performance-dependent manner, irrespective of diagnosis. This finding was accompanied by an equally performance-dependent effect of the drug on dopamine release in the midbrain, whereby low performers showed reduced dopamine release in this region. Collectively, these findings support a dimensional model of attentional deficits and underlying nigro-striatal dopaminergic mechanisms of attention deficit/hyperactivity disorder that extends into the healthy population. Moreover, they confer midbrain dopamine autoreceptors a hitherto neglected role in the therapeutic effects of oral methylphenidate in attention deficit/hyperactivity disorder. The absence of significant case-control differences in D2/D3 receptor availability (despite the observed relationships between dopamine activity and attention) suggests that dopamine dysregulation per se is unlikely to be the primary cause underlying attention deficit/hyperactivity disorder pathology in adults. This conclusion is reinforced by evidence of neuroanatomical changes in the same set of patients with attention deficit/hyperactivity disorder

Atomoxetine restores the response inhibition network in Parkinson's disease

Parkinson's disease impairs the inhibition of responses, and whilst impulsivity is mild for some patients, severe impulse control disorders affect ∼10% of cases. Based on preclinical models we proposed that noradrenergic denervation contributes to the impairment of response inhibition, via changes in the prefrontal cortex and its subcortical connections. Previous work in Parkinson's disease found that the selective noradrenaline reuptake inhibitor atomoxetine could improve response inhibition, gambling decisions and reflection impulsivity. Here we tested the hypotheses that atomoxetine can restore functional brain networks for response inhibition in Parkinson's disease, and that both structural and functional connectivity determine the behavioural effect. In a randomized, double-blind placebo-controlled crossover study, 19 patients with mild-to-moderate idiopathic Parkinson's disease underwent functional magnetic resonance imaging during a stop-signal task, while on their usual dopaminergic therapy. Patients received 40 mg atomoxetine or placebo, orally. This regimen anticipates that noradrenergic therapies for behavioural symptoms would be adjunctive to, not a replacement for, dopaminergic therapy. Twenty matched control participants provided normative data. Arterial spin labelling identified no significant changes in regional perfusion. We assessed functional interactions between key frontal and subcortical brain areas for response inhibition, by comparing 20 dynamic causal models of the response inhibition network, inverted to the functional magnetic resonance imaging data and compared using random effects model selection. We found that the normal interaction between pre-supplementary motor cortex and the inferior frontal gyrus was absent in Parkinson's disease patients on placebo (despite dopaminergic therapy), but this connection was restored by atomoxetine. The behavioural change in response inhibition (improvement indicated by reduced stop-signal reaction time) following atomoxetine correlated with structural connectivity as measured by the fractional anisotropy in the white matter underlying the inferior frontal gyrus. Using multiple regression models, we examined the factors that influenced the individual differences in the response to atomoxetine: the reduction in stop-signal reaction time correlated with structural connectivity and baseline performance, while disease severity and drug plasma level predicted the change in fronto-striatal effective connectivity following atomoxetine. These results suggest that (i) atomoxetine increases sensitivity of the inferior frontal gyrus to afferent inputs from the pre-supplementary motor cortex; (ii) atomoxetine can enhance downstream modulation of frontal-subcortical connections for response inhibition; and (iii) the behavioural consequences of treatment are dependent on fronto-striatal structural connections. The individual differences in behavioural responses to atomoxetine highlight the need for patient stratification in future clinical trials of noradrenergic therapies for Parkinson's disease.This work was primarily funded by the Wellcome Trust (103838) with additional support from the Medical Research Council (MC-A060-5PQ30, and RG62761), the NIHR Cambridge Biomedical Research Centre, Parkinson's UK and the James F McDonnell Foundation. The BCNI is supported by the Wellcome Trust and Medical Research Council

P2 receptors are involved in the mediation of motivation-related behavior

The importance of purinergic signaling in the intact mesolimbic–mesocortical circuit of the brain of freely moving rats is reviewed. In the rat, an endogenous ADP/ATPergic tone reinforces the release of dopamine from the axon terminals in the nucleus accumbens as well as from the somatodendritic region of these neurons in the ventral tegmental area, as well as the release of glutamate, probably via P2Y1 receptor stimulation. Similar mechanisms may regulate the release of glutamate in both areas of the brain. Dopamine and glutamate determine in concert the activity of the accumbal GABAergic, medium-size spiny neurons thought to act as an interface between the limbic cortex and the extrapyramidal motor system. These neurons project to the pallidal and mesencephalic areas, thereby mediating the behavioral reaction of the animal in response to a motivation-related stimulus. There is evidence that extracellular ADP/ATP promotes goal-directed behavior, e.g., intention and feeding, via dopamine, probably via P2Y1 receptor stimulation. Accumbal P2 receptor-mediated glutamatergic mechanisms seem to counteract the dopaminergic effects on behavior. Furthermore, adaptive changes of motivation-related behavior, e.g., by chronic succession of starvation and feeding or by repeated amphetamine administration, are accompanied by changes in the expression of the P2Y1 receptor, thought to modulate the sensitivity of the animal to respond to certain stimuli

Key Learning Outcomes for Clinical Pharmacology and Therapeutics Education in Europe: A Modified Delphi Study.

Harmonizing clinical pharmacology and therapeutics (CPT) education in Europe is necessary to ensure that the prescribing competency of future doctors is of a uniform high standard. As there are currently no uniform requirements, our aim was to achieve consensus on key learning outcomes for undergraduate CPT education in Europe. We used a modified Delphi method consisting of three questionnaire rounds and a panel meeting. A total of 129 experts from 27 European countries were asked to rate 307 learning outcomes. In all, 92 experts (71%) completed all three questionnaire rounds, and 33 experts (26%) attended the meeting. 232 learning outcomes from the original list, 15 newly suggested and 5 rephrased outcomes were included. These 252 learning outcomes should be included in undergraduate CPT curricula to ensure that European graduates are able to prescribe safely and effectively. We provide a blueprint of a European core curriculum describing when and how the learning outcomes might be acquired

Characterization of Drug Release from Mesoporous SiO2-Based Membranes with Variable Pore Structure and Geometry

Transdermal drug delivery systems (TDDSs) play important roles in therapy due to distinct advantages over other forms and types of drug application. While common TDDS patches mainly consist of polymeric matrices so far, inorganic carriers show numerous advantages such as high mechanical stability, possible re-use and re-loading of drugs, and a broad chemical compatibility with therapeutically relevant compounds and chemical enhancers. Mesoporous glasses can be prepared in different monolithic shapes, and offer a particularly wide range of possible pore volumes, pore diameters, and specific surface areas. Further, they show high loading capacities and favorable physical, technical, and biological properties. Here, we explored for the first time monolithic SiO2-based carriers as sustained release systems of therapeutic drugs. In an ideally stirred vessel as model system, we systematically analyzed the influence of pore diameter, pore volume, and the dimensions of glass monoliths on the loading and sustained release of different drugs, including anastrozole, xylazine, imiquimod, levetiracetam, and flunixin. Through multilinear regression, we calculated the influence of different parameters on drug loading and diffusion coefficients. The systematic variation of the mesoporous glass properties revealed pore volumes and drug loading concentrations, but not pore diameter or pore surface area as important parameters of drug loading and release kinetics. Other relevant effectors include the occurrence of lateral diffusion within the carrier and drug-specific properties such as adsorption. The structure–property relationships derived from our data will allow further fine-tuning of the systems according to their desired properties as TDDS, thus guiding towards optimal systems for their use in transdermal drug applications

Determination of Atomoxetine or Escitalopram in human plasma by HPLC. Applications in Neuroscience Research Studies

Background: Atomoxetine and escitalopram are potent and selective drugs approved for noradrenergic or serotonergic modulation of neuronal networks in attention-deficit hyperactivity disorder (ADHD) or depression, respectively. High-performance liquid chromatography (HPLC) methods still play an important role in the Therapeutic Drug Monitoring (TDM) of psychopharmacological drugs, and coupled with tandem mass spectrometry are the gold standard for the quantification of drugs in biological matrices, but not available everywhere. The aim of this work was to develop and validate a HPLC method for neuroscientific studies using atomoxetine or escitalopram as a test drug. Methods: A HPLC method from routine TDM determination of atomoxetine or citalopram in plasma was adapted and validated for use in neuroscientific research. Using photo diode array detection with UV absorption at 205 nm, the variation of internal standard within one chromatographic method enables separate drug monitoring for concentration-controlled explorative studies in healthy humans and patients with Parkinson’s disease. Results: The method described here was found to be linear in the range of 0.002 - 1.4 mg/L for atomoxetine and 0.0012 - 0.197 mg/L for escitalopram, with overall mean intra-day and inter-day imprecision and accuracy bias < 10% for both drugs. The method was successfully applied in concentration-controlled neuroimaging studies in populations of healthy humans and patients with Parkinson’s disease. Conclusion: A simple, sensitive, robust HPLC method capable of monitoring escitalopram and atomoxetine is presented and validated, as a useful tool for drug monitoring and the study of pharmacokinetics in neuroscientific study applications