Seth C. Hopkins

Translational aspects of pharmacological research into anxiety disorders: The stress-induced hyperthermia (SIH) paradigm

In anxiety research, the search for models with sufficient clinical predictive validity to support the translation of animal studies on anxiolytic drugs to clinical research is often challenging. This review describes the stress-induced hyperthermia (SIH) paradigm, a model that studies the activation of the autonomic nervous system in response to stress by measuring body temperature. The reproducible and robust SIH response, combined with ease of testing, make the SIH paradigm very suitable for drug screening. We will review the current knowledge on the neurobiology of the SIH response, discuss the role of GABA(A) and serotonin (5-HT) pharmacology, as well as how the SIH response relates to infectious fever. Furthermore, we will present novel data on the SIH response variance across different mice and their sensitivity to anxiolytic drugs. The SIH response is an autonomic stress response that can be successfully studied at the level of its physiology, pharmacology, neurobiology and genetics and possesses excellent animal-to-human translational properties.

Dissociating anxiolytic and sedative effects of GABAAergic drugs using temperature and locomotor responses to acute stress

RationaleThe stress-induced hyperthermia (SIH) model is an anxiety model that uses the transient rise in body temperature in response to acute stress. Benzodiazepines produce anxiolytic as well as sedative side effects through nonselective binding to GABAA receptor subunits. The GABAA receptor α1 subunit is associated with sedation, whereas the GABAA receptor α2 and α3 subunits are involved in anxiolytic effects.ObjectivesWe therefore examined the effects of (non)subunit-selective GABAA receptor agonists on temperature and locomotor responses to novel cage stress.ResultsUsing telemetric monitoring of temperature and locomotor activity, we found that nonsubunit-selective GABAA receptor agonist diazepam as well as the α3 subunit-selective receptor agonist TP003 dose-dependently attenuated SIH and locomotor responses. Administration of GABAA receptor α1-selective agonist zolpidem resulted in profound hypothermia and locomotor sedation. The GABAA receptor α1-selective antagonist βCCt antagonized the hypothermia, but did not reverse the SIH response attenuation caused by diazepam and zolpidem. These results suggest an important regulating role for the α1 subunit in thermoregulation and sedation. Ligands of extrasynaptic GABAA receptors such as alcohol and nonbenzodiazepine THIP attenuated the SIH response only at high doses.ConclusionsThe present study confirms a putative role for the GABAA receptor α1 subunit in hypothermia and sedation and supports a role for α2/3 subunit GABAA receptor agonists in anxiety processes. In conclusion, we show that home cage temperature and locomotor responses to novel home cage stress provide an excellent tool to assess both anxiolytic and sedative effects of various (subunit-selective) GABAAergic compounds.

Pharmacodynamic Effects of a d-Amino Acid Oxidase Inhibitor Indicate a Spinal Site of Action in Rat Models of Neuropathic Pain

Inhibition of d-amino acid oxidase (DAAO) activity is a potential target for the treatment of chronic pain. Here we characterized the effects of systemic administration of the DAAO inhibitor 4H-furo[3,2-b]pyrrole-5-carboxylic acid (SUN) in rat models of neuropathic and inflammatory pain. Oral administration of SUN dose dependently attenuated tactile allodynia induced by ligation of the L5 spinal nerve (SNL) and similarly reversed thermal hyperalgesia produced by chronic constriction injury. In addition, SUN was efficacious against complete Freund’s adjuvant-induced thermal hyperalgesia. In these models, maximal reversal of pain-related behaviors corresponded with maximum rates of increase in brain and plasma d-serine concentrations, indicative of full inhibition of DAAO activity. To investigate the possible site(s) of action, we recorded spontaneous nerve activity and mechanically evoked responses of central spinal cord dorsal horn neurons and compared these with spontaneous activity of peripheral dorsal root filaments in anesthetized SNL model animals. Oral SUN reduced spontaneous activity in both central and peripheral recordings at doses and pretreatment times that corresponded to reduced mechanical allodynia in behavioral experiments. After intravenous administration of SUN, the onset of action for this central effect was rapid (maximal effects within 30 minutes), but was abolished by severing afferent inputs to the dorsal horn. Overall, these results indicate that inhibition of DAAO in peripheral afferent spinal circuits reduced spontaneous neuronal activity to attenuate pain-related behaviors in rat models of neuropathic and inflammatory pain.

Dasotraline for the Treatment of Attention-Deficit/Hyperactivity Disorder: A Randomized, Double-Blind, Placebo-Controlled, Proof-of-Concept Trial in Adults

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by symptoms of inattention, hyperactivity, and impulsivity associated with clinically significant impairment in functioning. ADHD has an early onset, but frequently persists, with a prevalence estimate of 4% in adults. Dasotraline is a novel compound that is a potent inhibitor of dopamine and norepinephrine transporters that achieves stable plasma concentrations with once-daily dosing. In this study, adult outpatients meeting DSM-IV-TR criteria for ADHD were randomized to 4 weeks of double-blind, once-daily treatment with dasotraline 4 and 8 mg/day or placebo. The primary efficacy end point was change from baseline at week 4 in the ADHD Rating Scale, Version IV (ADHD RS-IV) total score. Secondary efficacy end points included the Clinical Global Impression, Severity (CGI-S) scale, modified for ADHD symptoms. Least squares (LS) mean improvements at week 4 in ADHD RS-IV total score were significantly greater for dasotraline 8 mg/day vs placebo (−13.9 vs −9.7; P=0.019), and nonsignificantly greater for 4 mg/day (−12.4; P=0.076). The LS mean improvements in modified CGI-S were significantly greater at week 4 for dasotraline 8 mg/day vs placebo (−1.1 vs −0.7; P=0.013), and for 4 mg/day vs placebo (−1.1 vs −0.7; P=0.021). The most frequent adverse events reported were insomnia, decreased appetite, nausea, and dry mouth. Discontinuations due to treatment-emergent adverse events were 10.3% and 27.8% of patients in 4 and 8 mg/day treatment groups, respectively. This study provides preliminary evidence that once-daily dosing with dasotraline, a long-acting, dual monoamine reuptake inhibitor, may be a safe and efficacious treatment for adult ADHD.

Discriminative stimulus properties of GABAA receptor positive allosteric modulators TPA023, ocinaplon and NG2-73 in rats trained to discriminate chlordiazepoxide or zolpidem.

There is increased understanding that distinct GABA(A) receptor subtypes mediate different effects of classical benzodiazepines. Here, we aimed to define the contributions of α(1)-containing subtypes of the subtype-selective GABA(A) receptor positive allosteric modulators TPA023, ocinaplon, and NG2-73 using drug discrimination. We characterized these compounds with defined subunit preferences in rats that were trained to discriminate either the non-selective benzodiazepine chlordiazepoxide (CDP, 5.0 mg/kg) or the α(1)-selective drug zolpidem (1.5 mg/kg). In short, CDP but not zolpidem generalized to the CDP cue. In contrast, zolpidem-trained rats showed opposite effects and generalized to zolpidem but not to CDP, while the response rate reducing effects of both ligands were comparable. Moreover, TPA023, lacking efficacy at the GABA(A) receptor α(1) subunit, occasioned dose-dependent CDP-appropriate responding but generalized only to around 10% to zolpidem. Both ocinaplon and NG2-73 completely generalized to both the CDP and zolpidem cue. Overall, our data confirm and extend the previous findings in rats that compounds that lack efficacy at α(1)-containing GABA(A) receptors generalize to CDP, whereas the opposite holds true for α(1)-preferential compounds, which generalize to the α(1)-selective positive allosteric modulator zolpidem. Also, our data support the hypothesis that subtle in vitro differences in α subunit efficacy and/or affinity may eventually have large consequences in vivo. Together, our data demonstrate a reliable in vivo method to determine the contribution of the subtype-selective mechanism(s) of action for novel and subtype-selective GABA(A) receptor positive allosteric modulators, suggesting that a complex activation of multiple α subunits accounts for drug discrimination between non-selective and selective GABA(A) receptor ligands.

Effects of D-amino acid oxidase inhibition on memory performance and long-term potentiation in vivo.

N‐methyl‐d‐aspartate receptor (NMDAR) activation can initiate changes in synaptic strength, evident as long‐term potentiation (LTP), and is a key molecular correlate of memory formation. Inhibition of d‐amino acid oxidase (DAAO) may increase NMDAR activity by regulating d‐serine concentrations, but which neuronal and behavioral effects are influenced by DAAO inhibition remain elusive. In anesthetized rats, extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded before and after a theta frequency burst stimulation (TBS) of the Schaffer collateral pathway of the CA1 region in the hippocampus. Memory performance was assessed after training with tests of contextual fear conditioning (FC, mice) and novel object recognition (NOR, rats). Oral administration of 3, 10, and 30 mg/kg 4H‐furo[3,2‐b]pyrrole‐5‐carboxylic acid (SUN) produced dose‐related and steady increases of cerebellum d‐serine in rats and mice, indicative of lasting inhibition of central DAAO. SUN administered 2 h prior to training improved contextual fear conditioning in mice and novel object recognition memory in rats when tested 24 h after training. In anesthetized rats, LTP was established proportional to the number of TBS trains. d‐cycloserine (DCS) was used to identify a submaximal level of LTP (5× TBS) that responded to NMDA receptor activation; SUN administered at 10 mg/kg 3–4 h prior to testing similarly increased in vivo LTP levels compared to vehicle control animals. Interestingly, in vivo administration of DCS also increased brain d‐serine concentrations. These results indicate that DAAO inhibition increased NMDAR‐related synaptic plasticity during phases of post training memory consolidation to improve memory performance in hippocampal‐dependent behavioral tests.

Catechol-O-methyltransferase genotype as modifier of superior responses to venlafaxine treatment in major depressive disorder.

Responses to venlafaxine treatment in major depressive disorder were stratified by COMT genotypes (Val158Met, rs4680) in a randomized, double-blind, placebo-controlled clinical trial. Improvements in depression scores among subjects with Val/Val genotypes were larger than those in Met/Met genotypes, suggesting that venlafaxine may alter noradrenergic flux differentially according to COMT activity.

In vivo saturation binding of GABA‐A receptor ligands to estimate receptor occupancy using liquid chromatography/tandem mass spectrometry

Typically, the dose‐occupancy curves for GABA‐A receptor ligands are determined using in vivo binding of [3H]flumazenil. This study describes in vivo binding experiments without the use of tracer ligands. Bound and free fractions were measured directly using a highly sensitive LC/MS/MS detection method after in vivo administration of the GABA‐A ligands zolpidem, (RS)‐zopiclone, L‐838417 and flumazenil, to demonstrate affinity and saturation of the filter‐retained, membrane‐bound fraction. The in vivo binding of flumazenil and L‐838417 both saturated around 200 nm, at a similar level to the specific binding of (S)‐zopiclone after doses of the racemic zopiclone, using (R)‐zopiclone to estimate non‐specific binding. This saturable component represented an estimate of benzodiazepine binding sites available on GABA‐A receptors in vivo (200 nm). Dose‐occupancy curves were constructed to estimate the dose required to achieve 50% occupancy and matched estimates obtained with tracer methods. In contrast to tracer methods, this method is uniquely suitable to the demonstration of stereoselective binding of the (S)‐isomer in vivo after doses of racemic zopiclone. These results demonstrate that the LC/MS/MS measurements of total drug concentrations typically used in early drug development can be adapted to provide information about receptor occupancy in vivo. Copyright

Assessment of human abuse potential of dasotraline compared to methylphenidate and placebo in recreational stimulant users

AIMS The aim of this study was to evaluate the abuse potential of dasotraline, a novel dopamine and norepinephrine reuptake inhibitor with slow absorption (tmax, 10-12h) and elimination (t1/2=47-77 h) that is in development for the treatment of attention deficit hyperactivity disorder (ADHD). METHODS Recreational stimulant users (N=48) who had specific experience with cocaine, and who were able to distinguish methylphenidate (60 mg) versus placebo in a qualification session, were randomized, in a 6-period, double-blind, crossover design, to receive single doses of dasotraline 8 mg, 16 mg, and 36 mg, methylphenidate (MPH) 40 mg and 80 mg, and placebo. The primary endpoint was the Drug Liking Visual Analog Scale (VAS) score at the time of peak effect (Emax). RESULTS There were no significant differences between the 3 doses of dasotraline and placebo on the drug liking VAS at Emax, and on most secondary endpoints. Both doses of MPH had significantly higher VAS-drug liking scores at Emax relative to both placebo (P<0.001 for all comparisons) and dasotraline 8 mg (P<0.001), 16 mg (P<0.001) and 36 mg (P<0.01). The increase in heart rate for MPH and dasotraline 36 mg showed a time-course that closely matched subject-rated measures such as Any Effects VAS. CONCLUSIONS In this study, dasotraline was found to have low potential for abuse, which may be, in part, related to its established pharmacokinetics (PK) profile, which is characterized by slow absorption and gradual elimination.

Differences in memory function between 5-HT1A receptor genotypes in patients with major depressive disorder

BACKGROUND While extensive literature on the role of the serotonin receptor 1A (5-HT1A-R) in cognition exists, the findings are largely from animal studies. There has been little research conducted into 5-HT1A-R genotypes and cognitive function in humans. This article evaluates the role of 5-HT1A-R genotypes on the profile of cognitive function in patients with major depressive disorder (MDD). METHODS The study sample was 455 MDD patients aged between 18 and 55 years. They had enrolled into a clinical trial and were tested prior to dosing on the baseline study day using the CDR System, an integrated set of 3 attention tests, 2 working memory tests, and 4 episodic memory tests. 5-HT1A-R genotyping for (SNP ID rs6295) had been conducted during the study screening period. RESULTS Validated factor scores were derived from the 9 tests. It was found that patients with the C/C genotype for the C(1019)G polymorphism of the 5-HT1A-R were significantly superior in retaining and retrieving information, in both working and episodic memory, than those with either the C/G or the G/G genotypes. No differences were found in measures of attention or in the speed of retrieval of information from memory. CONCLUSIONS This is, to our knowledge, the first relationship found between objective tests of cognitive function and 5-HT1A-R genotypes in MDD.