Stimulation of angiotensin AT2 receptors by the non-peptide agonist,Compound 21, evokes vasodepressor effects in conscious spontaneously hypertensive rats

Background and purpose:

Angiotensin type 2 receptor (AT₂ receptor) stimulation evokes vasodilator effects in vitro and in vivo that oppose the vasoconstrictor effects of angiotensin type 1 receptors (AT₁ receptors). Recently, a novel non-peptide AT₂ receptor agonist, Compound 21, was described, which exhibited high AT₂ receptor selectivity.

Experimental approach:

Functional cardiovascular effects of the drug candidate Compound 21 were assessed, using mouse isolated aorta and rat mesenteric arteries in vitro and in conscious spontaneously hypertensive rats (SHR).

Key results:

Compound 21 evoked dose-dependent vasorelaxations in aortic and mesenteric vessels, abolished by the AT₂ receptor antagonist, PD123319. In vivo, Compound 21 administered alone, at doses ranging from 50 to 1000 ng·kg⁻¹·min⁻¹ over 4 h did not decrease blood pressure in conscious normotensive Wistar-Kyoto rats or SHR. However, when given in combination with the AT₁ receptor antagonist, candesartan, Compound 21 (300 ng·kg⁻¹·min⁻¹) lowered blood pressure in SHR only. Further analysis in separate groups of conscious SHR revealed that, at a sixfold lower dose, Compound 21 (50 ng·kg⁻¹·min⁻¹) still evoked a significant depressor response in adult SHR (∼30 mmHg) when combined with different doses of candesartan (0.01 or 0.1 mg·kg⁻¹). Moreover, the Compound 21-evoked depressor effect was abolished when co-infused (50 µg·kg⁻¹·min⁻¹ for 2 h) with the AT₂ receptor antagonist PD123319.

Conclusion and implications:

Collectively, our results indicate that acute administration of Compound 21 evoked blood pressure reductions via AT₂ receptor stimulation. Thus Compound 21 can be considered an excellent drug candidate for further study of AT₂ receptor function in cardiovascular disease.     

BGC20-1531, a novel,potent and selective prostanoid EP4 receptor antagonist: a putative new treatment for migraine headache

Background and purpose:

Prostanoid EP₄ receptor antagonists may have therapeutic utility in the treatment of migraine since EP₄ receptors have been shown to be involved in prostaglandin (PG)E₂-induced cerebral vascular dilatation, which may be an important contributor to migraine pain. This study reports the pharmacological characterization of BGC20-1531, a novel EP₄ receptor antagonist.

Experimental approach:

BGC20-1531 was characterized in radioligand binding and in vitro functional assays employing recombinant and native EP₄ receptors. Changes in canine carotid haemodynamics were used to assess the pharmacodynamic profile of BGC20-1531 in vivo.

Key results:

BGC20-1531 exhibited high affinity at recombinant human EP₄ receptors expressed in cell lines (pK_B 7.6) and native EP₄ receptors in human cerebral and meningeal artery (pK_B 7.6–7.8) but showed no appreciable affinity at a wide range of other receptors (including other prostanoid receptors), channels, transporters and enzymes (pKi < 5). BGC20-1531 competitively antagonized PGE₂-induced vasodilatation of human middle cerebral (pK_B 7.8) and meningeal (pK_B 7.6) arteries in vitro, but had no effect on responses induced by PGE₂ on coronary, pulmonary or renal arteries in vitro. BGC20-1531 (1–10 mg·kg⁻¹ i.v.) caused a dose-dependent antagonism of the PGE₂-induced increase in canine carotid blood flow in vivo.

Conclusions and implications:

BGC20-1531 is a potent and selective antagonist at EP₄ receptors in vitro and in vivo, with the potential to alleviate the symptoms of migraine that result from cerebral vasodilatation. BGC20-1531 is currently in clinical development for the treatment of migraine headache.     

In vivo and in vitro pharmacological characterization of SVT-40776, a novel M3 muscarinic receptor antagonist,for the treatment of overactive bladder

Background and purpose:

Highly selective M₃ muscarinic receptor antagonists may represent a better treatment for overactive bladder syndrome, diminishing side effects. Cardiac side effects of non-selective antimuscarinics have been associated with activity at M₂ receptors as these receptors are mainly responsible for muscarinic receptor-dependent bradycardia. We have investigated a novel antimuscarinic, SVT-40776, highly selective for M₃ over M₂ receptors (Ki = 0.19 nmol·L⁻¹ for M₃ receptor affinity). This study reports the functional activity of SVT-40776 in the bladder, relative to its activity in atria.

Experimental approach:

In vitro and ex vivo (oral dosing) inhibition of mouse detrusor and atrial contractile responses to carbachol were used to study the functional activity of SVT-40776. The in vivo efficacy of SVT-40776 was characterized by suppression of isovolumetric spontaneous bladder contractions in anaesthetized guinea pigs after intravenous administration.

Key results:

SVT-40776 was the most potent in inhibiting carbachol-induced bladder contractions of the anti-cholinergic agents tested, without affecting atrial contractions over the same range of concentrations. SVT-40776 exhibited the highest urinary versus cardiac selectivity (199-fold). In the guinea pig in vivo model, SVT-40776 inhibited 25% of spontaneous bladder contractions at a very low dose (6.97 µg·kg⁻¹ i.v), without affecting arterial blood pressure.

Conclusions and implications:

SVT-40776 is a potent inhibitor of M₃ receptor-related detrusor contractile activity. The absence of effects on isolated atria preparations represents an interesting characteristic and suggests that SVT-40776 may lack unwanted cardiac effects; a feature especially relevant in a compound intended to treat mainly elderly patients.British Journal of Pharmacology (2009) doi:10.1111/j.1476-5381.2008.00082.x     

Antidepressant-like effects of cannabidiol in mice: possible involvement of 5-HT1A receptors

Background and purpose:

Cannabidiol (CBD) is a non-psychotomimetic compound from Cannabis sativa that induces anxiolytic- and antipsychotic-like effects in animal models. Effects of CBD may be mediated by the activation of 5-HT_1A receptors. As 5-HT_1A receptor activation may induce antidepressant-like effects, the aim of this work was to test the hypothesis that CBD would have antidepressant-like activity in mice as assessed by the forced swimming test. We also investigated if these responses depended on the activation of 5-HT_1A receptors and on hippocampal expression of brain-derived neurotrophic factor (BDNF).

Experimental approach:

Male Swiss mice were given (i.p.) CBD (3, 10, 30, 100 mg·kg⁻¹), imipramine (30 mg·kg⁻¹) or vehicle and were submitted to the forced swimming test or to an open field arena, 30 min later. An additional group received WAY100635 (0.1 mg·kg⁻¹, i.p.), a 5-HT_1A receptor antagonist, before CBD (30 mg·kg⁻¹) and assessment by the forced swimming test. BDNF protein levels were measured in the hippocampus of another group of mice treated with CBD (30 mg·kg⁻¹) and submitted to the forced swimming test.

Key results:

CBD (30 mg·kg⁻¹) treatment reduced immobility time in the forced swimming test, as did the prototype antidepressant imipramine, without changing exploratory behaviour in the open field arena. WAY100635 pretreatment blocked CBD-induced effect in the forced swimming test. CBD (30 mg·kg⁻¹) treatment did not change hippocampal BDNF levels.

Conclusion and implications:

CBD induces antidepressant-like effects comparable to those of imipramine. These effects of CBD were probably mediated by activation of 5-HT_1A receptors.     

Cannabidiolic acid prevents vomiting in Suncus murinus and nausea-induced behaviour in rats by enhancing 5-HT1A receptor activation

Background and Purpose

To evaluate the ability of cannabidiolic acid (CBDA) to reduce nausea and vomiting and enhance 5-HT_1A receptor activation in animal models.

Experimental Approach

We investigated the effect of CBDA on (i) lithium chloride (LiCl)-induced conditioned gaping to a flavour (nausea-induced behaviour) or a context (model of anticipatory nausea) in rats; (ii) saccharin palatability in rats; (iii) motion-, LiCl- or cisplatin-induced vomiting in house musk shrews (Suncus murinus); and (iv) rat brainstem 5-HT_1A receptor activation by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and mouse whole brain CB₁ receptor activation by CP55940, using [³⁵S]GTPγS-binding assays.

Key Results

In shrews, CBDA (0.1 and/or 0.5 mg·kg⁻¹ i.p.) reduced toxin- and motion-induced vomiting, and increased the onset latency of the first motion-induced emetic episode. In rats, CBDA (0.01 and 0.1 mg·kg⁻¹ i.p.) suppressed LiCl- and context-induced conditioned gaping, effects that were blocked by the 5-HT_1A receptor antagonist, WAY100635 (0.1 mg·kg⁻¹ i.p.), and, at 0.01 mg·kg⁻¹ i.p., enhanced saccharin palatability. CBDA-induced suppression of LiCl-induced conditioned gaping was unaffected by the CB₁ receptor antagonist, SR141716A (1 mg·kg⁻¹ i.p.). In vitro, CBDA (0.1–100 nM) increased the E_max of 8-OH-DPAT.

Conclusions and Implications

Compared with cannabidiol, CBDA displays significantly greater potency at inhibiting vomiting in shrews and nausea in rats, and at enhancing 5-HT_1A receptor activation, an action that accounts for its ability to attenuate conditioned gaping in rats. Consequently, CBDA shows promise as a treatment for nausea and vomiting, including anticipatory nausea for which no specific therapy is currently available.     

Activation of EP4 receptors prevents endotoxin-induced neutrophil infiltration into the airways and enhances microvascular barrier function

Background and Purpose

Pulmonary vascular dysfunction is a key event in acute lung injury. We recently demonstrated that PGE₂, via activation of E-prostanoid (EP)₄ receptors, strongly enhances microvascular barrier function in vitro. The aim of this study was to investigate the beneficial effects of concomitant EP₄ receptor activation in murine models of acute pulmonary inflammation.

Experimental Approach

Pulmonary inflammation in male BALB/c mice was induced by LPS (20 μg per mouse intranasally) or oleic acid (0.15 μL·g^-1, i.v^.). In-vitro, endothelial barrier function was determined by measuring electrical impedance.

Key Results

PGE₂ activation of EP₄ receptors reduced neutrophil infiltration, pulmonary vascular leakage and TNF-α concentration in bronchoalveolar lavage fluid from LPS-induced pulmonary inflammation. Similarly, pulmonary vascular hyperpermeability induced by oleic acid was counteracted by EP₄ receptor activation. In lung function assays, the EP₄ agonist ONO AE1-329 restored the increased resistance and reduced compliance upon methacholine challenge in mice treated with LPS or oleic acid. In agreement with these findings, EP₄ receptor activation increased the in vitro vascular barrier function of human and mouse pulmonary microvascular endothelial cells and diminished the barrier disruption induced by LPS. The EP₂ agonist ONO AE1-259 likewise reversed LPS-induced lung dysfunction without enhancing vascular barrier function.

Conclusion and Implications

Our results show that activation of the EP₄ receptor strengthens the microvascular barrier function and thereby ameliorates the pathology of acute lung inflammation, including neutrophil infiltration, vascular oedema formation and airway dysfunction. This suggests a potential benefit for EP₄ agonists in acute pulmonary inflammation.     

The antipsychotic-like effects of positive allosteric modulators of metabotropic glutamate mGlu4 receptors in rodents

Background and Purpose

Because agonists at metabotropic glutamate receptors exert beneficial effects in schizophrenia, we have assessed the actions of Lu AF21934 and Lu AF32615, two chemically distinct, selective and brain-penetrant positive allosteric modulators (PAMs) of the mGlu₄ receptor, in several tests reflecting positive, negative and cognitive symptoms of schizophrenia in rodents.

Experimental Approach

Hyperactivity induced by MK-801 or amphetamine and head twitches induced by 2,5-dimethoxy-4-iodoamphetamine (DOI) in mice were used as models for positive symptoms. Disruption of social interaction and spatial delayed alternation tests induced by MK-801 in rats were used as models for negative and cognitive symptoms of schizophrenia, respectively.

Key Results

Lu AF21934 (0.1–5 mg·kg⁻¹) and Lu AF32615 (2–10 mg·kg⁻¹) dose-dependently inhibited hyperactivity induced by MK-801 or amphetamine. They also antagonized head twitches and increased frequency of spontaneous excitatory postsynaptic currents (EPSCs) in brain slices, induced by DOI. In mice lacking the mGlu₄ receptor (mGlu₄^−/−) mice, Lu AF21934 did not antagonize DOI-induced head twitches. MK-801-induced disruption in the social interaction test was decreased by Lu AF21934 at 0.5 mg·kg⁻¹ and by Lu AF32615 at 10 mg·kg⁻¹. In the delayed spatial alternation test, Lu AF21934 was active at 1 and 2 mg·kg⁻¹, while Lu AF32615 was active at 10 mg·kg⁻¹.

Conclusions and Implications

We propose that activation by PAMs of the mGlu₄ receptor is a promising approach to the discovery of novel antipsychotic drugs.     

Potential anxiolytic- and antidepressant-like effects of salvinorin A,the main active ingredient of Salvia divinorum,in rodents

Background and purpose:

Drugs targeting brain κ-opioid receptors produce profound alterations in mood. In the present study we investigated the possible anxiolytic- and antidepressant-like effects of the κ-opioid receptor agonist salvinorin A, the main active ingredient of Salvia divinorum, in rats and mice.

Experimental approach:

Experiments were performed on male Sprague-Dawley rats or male Albino Swiss mice. The anxiolytic-like effects were tested by using the elevated plus maze, in rats. The antidepressant-like effect was estimated through the forced swim (rats) and the tail suspension (mice) test. κ-Opioid receptor involvement was investigated pretreating animals with the κ-opioid receptor antagonist, nor-binaltorphimine (1 or 10 mg·kg⁻¹), while direct or indirect activity at CB₁ cannabinoid receptors was evaluated with the CB₁ cannabinoid receptor antagonist, N-(piperidin-1-yl) -5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251, 0.5 or 3 mg·kg⁻¹), binding to striatal membranes of naïve rats and assay of fatty acid amide hydrolase in prefrontal cortex, hippocampus and amygdala.

Key results:

Salvinorin A, given s.c. (0.001–1000 µg·kg⁻¹), exhibited both anxiolytic- and antidepressant-like effects that were prevented by nor-binaltorphimine or AM251 (0.5 or 3 mg·kg⁻¹). Salvinorin A reduced fatty acid amide hydrolase activity in amygdala but had very weak affinity for cannabinoid CB₁ receptors.

Conclusions and implications:

The anxiolytic- and antidepressant-like effects of Salvinorin A are mediated by both κ-opioid and endocannabinoid systems and may partly explain the subjective symptoms reported by recreational users of S. divinorum.     

Central 5-HT1A receptor-mediated modulation of heart rate dynamics and its adjustment by conditioned and unconditioned fear in mice

BACKGROUND AND PURPOSE

The beat-by-beat fluctuation (dynamics) of heart rate (HR) depends on centrally mediated control of the autonomic nervous system (ANS) reflecting the physiological state of an organism. 5-HT_1A receptors are implicated in affective disorders,associated with ANS dysregulation which increases cardiac risk but their role in autonomic HR regulation under physiological conditions is insufficiently characterized.

EXPERIMENTAL APPROACH

The effects of subcutaneously administered 5-HT_1A receptor ligands on HR dynamics were investigated in C57BL/6 mice during stress-free conditions and emotional challenge (recall of fear conditioned to an auditory stimulus and novelty exposure) using time domain and non-linear HR analyses.

KEY RESULTS

Pre-training treatment with of 8-OH-DPAT (0.5 mg·kg⁻¹, s.c.) prevented conditioned tachycardia in the retention test indicating impaired fear memory. Pretest 5-HT_1A receptor activation by 8-OH-DPAT (0.5 but not 0.1 and 0.02 mg·kg⁻¹) caused bradycardia and increased HR variability. 8-OH-DPAT (0.5 mg·kg⁻¹) lowered the unconditioned and conditioned tachycardia from ∼750 to ∼550 bpm, without changing the conditioned HR response to the sound. 8-OH-DPAT induced profound QT prolongation and bradyarrhythmic episodes. Non-linear analysis indicated a pathological state of HR dynamics after 8-OH-DPAT (0.5 mg·kg⁻¹) with ANS hyperactivation impairing HR adaptability. The 5-HT_1A receptor antagonist WAY-100635 (0.03 mg·kg⁻¹) blocked these effects of 8-OH-DPAT.

CONCLUSIONS AND IMPLICATIONS

Pre-training 5-HT_1A receptor activation by 8-OH-DPAT (0.5 mg·kg⁻¹) impaired memory of conditioned auditory fear based on an attenuated HR increase, whereas pretest administration did not prevent the fear-conditioned HR increase but induced pathological HR dynamics through central ANS dysregulation with cardiac effects similar to acute SSRI overdose.     

ADX71441, a novel,potent and selective positive allosteric modulator of the GABAB receptor,shows efficacy in rodent models of overactive bladder

Background and Purpose

The GABA_B receptor agonist baclofen reduces urethral resistance and detrusor overactivity in patients with spasticity. However, baclofen''s side effects limit its use for the treatment of overactive bladder (OAB). Here, we tested a novel GABA_B positive allosteric modulator (PAM) {"type":"entrez-protein","attrs":{"text":"ADX71441","term_id":"323467756","term_text":"ADX71441"}}ADX71441 in models of OAB in mice and guinea pigs.

Experimental Approach

Mice were left untreated or given (p.o.) vehicle (1= CMC), {"type":"entrez-protein","attrs":{"text":"ADX71441","term_id":"323467756","term_text":"ADX71441"}}ADX71441 (1, 3, 10 mg kg⁻¹) or oxybutynin (100 mg kg⁻¹; Experiment 1) or vehicle (1= CMC), baclofen (1, 3, 6 mg kg⁻¹) or oxybutynin (Experiment 2). Treated mice were then overhydrated with water, challenged with furosemide, before being placed into micturition chambers and monitored for urinary parameters. In anaesthetized guinea pigs, intravesical infusion of acetic acid was used to induce OAB and the effects of {"type":"entrez-protein","attrs":{"text":"ADX71441","term_id":"323467756","term_text":"ADX71441"}}ADX71441 (1, 3 mg kg⁻¹) or baclofen (1 mg kg⁻¹), administered i.v., on cystometric parameters were monitored.

Key Results

In mice, 10 mg kg⁻¹ {"type":"entrez-protein","attrs":{"text":"ADX71441","term_id":"323467756","term_text":"ADX71441"}}ADX71441 increased urinary latencies, reduced the number of urinary events and the total and average urinary volumes. In guinea pigs, {"type":"entrez-protein","attrs":{"text":"ADX71441","term_id":"323467756","term_text":"ADX71441"}}ADX71441 (1 and 3 mg kg⁻¹) increased the intercontraction interval (ICI) and bladder capacity (BC), and reduced micturition frequency (MF) compared to vehicle. At 3 mg kg⁻¹ {"type":"entrez-protein","attrs":{"text":"ADX71441","term_id":"323467756","term_text":"ADX71441"}}ADX71441 completely inhibited the micturition reflex and induced overflow incontinence in five out of 10 animals. Baclofen slightly increased ICI and BC and reduced MF.

Conclusion and Implications

Our findings demonstrate, for the first time, that a GABA_B PAM has potential as a novel approach for the treatment of OAB.     

Intermolecular cross-talk between the prostaglandin E2 receptor (EP)3 subtype and thromboxane A2 receptor signalling in human erythroleukaemic cells

Background and purpose:

In previous studies investigating cross-talk of signalling between prostaglandin (PG)E₂ receptor (EP) and the TPα and TPβ isoforms of the human thromboxane (TX)A₂ receptor (TP), 17-phenyl trinor PGE₂-induced desensitization of TP receptor signalling through activation of the AH6809 and SC19220-sensitive EP₁ subtype of the EP receptor family, in a cell-specific manner. Here, we sought to further investigate that cross-talk in human erythroleukaemic (HEL) 92.1.7 cells.

Experimental approach:

Specificity of 17-phenyl trinor PGE₂ signalling and its possible cross-talk with signalling by TPα/TPβ receptors endogenously expressed in HEL cells was examined through assessment of agonist-induced inositol 1,4,5-trisphosphate (IP)₃ generation and intracellular calcium ([Ca²⁺]_i) mobilization.

Key results:

While 17-Phenyl trinor PGE₂ led to activation of phospholipase (PL)Cβ to yield increases in IP₃ generation and [Ca²⁺]_i, it did not desensitize but rather augmented that signalling in response to subsequent stimulation with the TXA₂ mimetic U46619. Furthermore, the augmentation was reciprocal. Signalling by 17-phenyl trinor PGE₂ was found to occur through AH6809- and SC19920-insensitive, Pertussis toxin-sensitive, G_i/G_βγ-dependent activation of PLCβ. Further pharmacological investigation using selective EP receptor subtype agonists and antagonists confirmed that 17-phenyl trinor PGE₂-mediated signalling and reciprocal cross-talk with the TP receptors occurred through the EP₃, rather than the EP₁, EP₂ or EP₄ receptor subtype in HEL cells.

Conclusions and Implications:

The EP₁ and EP₃ subtypes of the EP receptor family mediated intermolecular cross-talk to differentially regulate TP receptor-mediated signalling whereby activation of EP₁ receptors impaired or desensitized, while that of EP₃ receptors augmented signalling through TPα/TPβ receptors, in a cell type-specific manner.     

Tetrahydrocannabinolic acid reduces nausea-induced conditioned gaping in rats and vomiting in Suncus murinus

BACKGROUND AND PURPOSE

We evaluated the anti-emetic and anti-nausea properties of the acid precursor of Δ⁹-tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THCA), and determined its mechanism of action in these animal models.

EXPERIMENTAL APPROACH

We investigated the effect of THCA on lithium chloride- (LiCl) induced conditioned gaping (nausea-induced behaviour) to a flavour, and context (a model of anticipatory nausea) in rats, and on LiCl-induced vomiting in Suncus murinus. Furthermore, we investigated THCA''s ability to induce hypothermia and suppress locomotion [rodent tasks to assess cannabinoid₁ (CB₁) receptor agonist-like activity], and measured plasma and brain THCA and THC levels. We also determined whether THCA''s effect could be blocked by pretreatment with SR141716 (SR, a CB₁ receptor antagonist).

KEY RESULTS

In rats, THCA (0.05 and/or 0.5 mg·kg⁻¹) suppressed LiCl-induced conditioned gaping to a flavour and context; the latter effect blocked by the CB₁ receptor antagonist, SR, but not by the 5-hydroxytryptamine-1A receptor antagonist, WAY100635. In S. murinus, THCA (0.05 and 0.5 mg·kg⁻¹) reduced LiCl-induced vomiting, an effect that was reversed with SR. A comparatively low dose of THC (0.05 mg·kg⁻¹) did not suppress conditioned gaping to a LiCl-paired flavour or context. THCA did not induce hypothermia or reduce locomotion, indicating non-CB₁ agonist-like effects. THCA, but not THC was detected in plasma samples.

CONCLUSIONS AND IMPLICATIONS

THCA potently reduced conditioned gaping in rats and vomiting in S. murinus, effects that were blocked by SR. These data suggest that THCA may be a more potent alternative to THC in the treatment of nausea and vomiting.     

ADN-1184 a monoaminergic ligand with 5-HT6/7 receptor antagonist activity: pharmacological profile and potential therapeutic utility

Background and Purpose

Many dementia patients exhibit behavioural and psychological symptoms (BPSD) that include psychosis, aggressivity, depression and anxiety. Antipsychotic drugs are frequently prescribed but fail to significantly attenuate mood deficits, may interfere with cognitive function and are associated with motor and cardiac side effects, which are problematic in elderly patients. A need therefore exists for drugs that are better suited for the treatment of BPSD.

Experimental Approach

We used in vitro cellular and in vivo behavioural tests to characterize ADN-1184, a novel arylsulfonamide ligand with potential utility for treatment of BPSD.

Key Results

ADN-1184 exhibits substantial 5-HT₆/5-HT₇/5-HT_2A/D₂ receptor affinity and antagonist properties in vitro. In tests of antipsychotic-like activity, it reversed MK-801-induced hyperactivity and stereotypies and inhibited conditioned avoidance response (MED = 3 mg·kg⁻¹ i.p.). Remarkably, ADN-1184 also reduced immobility time in the forced swim test at low doses (0.3 and 1 mg·kg⁻¹ i.p.; higher doses were not significantly active). Notably, up to 30 mg·kg⁻¹ ADN-1184 did not impair memory performance in the passive avoidance test or elicit significant catalepsy and only modestly inhibited spontaneous locomotor activity (MED = 30 mg·kg⁻¹ i.p.).

Conclusions and Implications

ADN-1184 combines antipsychotic-like with antidepressant-like properties without interfering with memory function or locomotion. This profile is better than that of commonly used atypical antipsychotics tested under the same conditions and suggests that it is feasible to identify drugs that improve BPSD, without exacerbating cognitive deficit or movement impairment, which are of particular concern in patients with dementia.     

Pharmacological characterization of a novel centrally permeable P2X7 receptor antagonist: JNJ-47965567

BACKGROUND AND PURPOSE

An increasing body of evidence suggests that the purinergic receptor P2X, ligand-gated ion channel, 7 (P2X7) in the CNS may play a key role in neuropsychiatry, neurodegeneration and chronic pain. In this study, we characterized JNJ-47965567, a centrally permeable, high-affinity, selective P2X7 antagonist.

EXPERIMENTAL APPROACH

We have used a combination of in vitro assays (calcium flux, radioligand binding, electrophysiology, IL-1β release) in both recombinant and native systems. Target engagement of JNJ-47965567 was demonstrated by ex vivo receptor binding autoradiography and in vivo blockade of Bz-ATP induced IL-1β release in the rat brain. Finally, the efficacy of JNJ-47965567 was tested in standard models of depression, mania and neuropathic pain.

KEY RESULTS

JNJ-47965567 is potent high affinity (pK_i 7.9 ± 0.07), selective human P2X7 antagonist, with no significant observed speciation. In native systems, the potency of the compound to attenuate IL-1β release was 6.7 ± 0.07 (human blood), 7.5 ± 0.07 (human monocytes) and 7.1 ± 0.1 (rat microglia). JNJ-47965567 exhibited target engagement in rat brain, with a brain EC₅₀ of 78 ± 19 ng·mL⁻¹ (P2X7 receptor autoradiography) and functional block of Bz-ATP induced IL-1β release. JNJ-47965567 (30 mg·kg⁻¹) attenuated amphetamine-induced hyperactivity and exhibited modest, yet significant efficacy in the rat model of neuropathic pain. No efficacy was observed in forced swim test.

Conclusion and Implications

JNJ-47965567 is centrally permeable, high affinity P2X7 antagonist that can be used to probe the role of central P2X7 in rodent models of CNS pathophysiology.     

Inhibition of colonic motility and defecation by RS-127445 suggests an involvement of the 5-HT2B receptor in rodent large bowel physiology

Background:

5-HT_2B receptors are localized within the myenteric nervous system, but their functions on motor/sensory neurons are unclear. To explore the role of these receptors, we further characterized the 5-HT_2B receptor antagonist RS-127445 and studied its effects on peristalsis and defecation.

Experimental approach:

Although reported as a selective 5-HT_2B receptor antagonist, any interactions of RS-127445 with 5-HT₄ receptors are unknown; this was examined using the recombinant receptor and Biomolecular Interaction Detection technology. Mouse isolated colon was mounted in tissue baths for isometric recording of neuronal contractions evoked by electrical field stimulation (EFS), or under an intraluminal pressure gradient to induce peristalsis; the effects of RS-127445 on EFS-induced and on peristaltic contractions were measured. Faecal output of rats in grid-bottom cages was measured over 3 h following i.p. RS-127445 and separately, validation of the effective doses was achieved by determining the free, unbound fraction of RS-127445 in blood and brain.

Key results:

RS-127445 (up to 1 µmol·L⁻¹) did not interact with the 5-HT₄ receptor. RS-127445 (0.001–1 µmol·L⁻¹) did not affect EFS-induced contractions of the colon, although at 10 µmol·L⁻¹ the contractions were reduced (to 36 ± 8% of control, n= 4). RS-127445 (0.1–10 µmol·L⁻¹) concentration-dependently reduced peristaltic frequency (n= 4). RS-127445 (1–30 mg·kg⁻¹), dose-dependently reduced faecal output, reaching significance at 10 and 30 mg·kg⁻¹ (n= 6–11). In blood and brain, >98% of RS-127445 was protein-bound.

Conclusions and implications:

High-protein binding of RS-127445 indicates that relatively high doses are required for efficacy. The results suggest that 5-HT_2B receptors tonically regulate colonic motility.     

Identification of an additional supraspinal component to the analgesic mechanism of action of buprenorphine

Background and purpose:

Buprenorphine displays attributes of opioids, but also some features distinct from them. We examined spinal and supraspinal signal transduction of buprenorphine-induced anti-nociception in mice compared with morphine and fentanyl.

Experimental approach:

The opioid receptor antagonist naloxone, Pertussis toxin (PTX), G_z protein antisense and nociceptin/orphanin-FQ receptor agonist nociceptin, and antagonist, JTC-801, were injected supraspinally (intracerebroventricular) and spinally (intrathecal). Also the cell-permeable Ser/Thr protein phosphatase inhibitor okadaic acid was given supraspinally.

Key results:

Spinal naloxone (20 µg) or PTX (1 µg) attenuated morphine, fentanyl and buprenorphine (s.c.) anti-nociception. Supraspinal naloxone or PTX attenuated morphine and fentanyl, but not buprenorphine anti-nociception. Spinal G_z protein antisense did not alter buprenorphine, morphine or fentanyl anti-nociception and supraspinal G_z-antisense did not alter morphine or fentanyl anti-nociception. However, supraspinal G_z-antisense (not random sense) reduced buprenorphine anti-nociception. Peripheral JTC-801 (1 mg·kg⁻¹, i.p.) enhanced the ascending (3 mg·kg⁻¹) and descending (30 mg·kg⁻¹) portions of buprenorphine''s dose–response curve, but only spinal, not supraspinal, nociceptin (10 nmol·L⁻¹) enhanced buprenorphine anti-nociception. Intracereboventricular okadaic acid (0.001–10 pg) produced a biphasic low-dose attenuation, high-dose enhancement of buprenorphine(3 or 30 mg·kg⁻¹, s.c.) anti-nociception, but did not affect morphine or fentanyl anti-nociception.

Conclusions and implications:

Buprenorphine has an opioid component to its supraspinal mechanism of analgesic action. Our present results reveal an additional supraspinal component insensitive to naloxone, PTX and nociceptin/orphanin-FQ, but involving G_z protein and Ser/Thr protein phosphatase. These data might help explain the unique preclinical and clinical profiles of buprenorphine.     

Cannabidivarin-rich cannabis extracts are anticonvulsant in mouse and rat via a CB1 receptor-independent mechanism

BACKGROUND AND PURPOSE

Epilepsy is the most prevalent neurological disease and is characterized by recurrent seizures. Here, we investigate (i) the anticonvulsant profiles of cannabis-derived botanical drug substances (BDSs) rich in cannabidivarin (CBDV) and containing cannabidiol (CBD) in acute in vivo seizure models and (ii) the binding of CBDV BDSs and their components at cannabinoid CB₁ receptors.

EXPERIMENTAL APPROACH

The anticonvulsant profiles of two CBDV BDSs (50–422 mg·kg⁻¹) were evaluated in three animal models of acute seizure. Purified CBDV and CBD were also evaluated in an isobolographic study to evaluate potential pharmacological interactions. CBDV BDS effects on motor function were also investigated using static beam and grip strength assays. Binding of CBDV BDSs to cannabinoid CB₁ receptors was evaluated using displacement binding assays.

KEY RESULTS

CBDV BDSs exerted significant anticonvulsant effects in the pentylenetetrazole (≥100 mg·kg⁻¹) and audiogenic seizure models (≥87 mg·kg⁻¹), and suppressed pilocarpine-induced convulsions (≥100 mg·kg⁻¹). The isobolographic study revealed that the anticonvulsant effects of purified CBDV and CBD were linearly additive when co-administered. Some motor effects of CBDV BDSs were observed on static beam performance; no effects on grip strength were found. The Δ⁹-tetrahydrocannabinol and Δ⁹-tetrahydrocannabivarin content of CBDV BDS accounted for its greater affinity for CB₁ cannabinoid receptors than purified CBDV.

CONCLUSIONS AND IMPLICATIONS

CBDV BDSs exerted significant anticonvulsant effects in three models of seizure that were not mediated by the CB₁ cannabinoid receptor and were of comparable efficacy with purified CBDV. These findings strongly support the further clinical development of CBDV BDSs for the treatment of epilepsy.     

Population pharmacokinetics and dosing optimization of teicoplanin in children with malignant haematological disease

Aim

Children with haematological malignancy represent an identified subgroup of the paediatric population with specific pharmacokinetic parameters. In these patients, inadequate empirical antibacterial therapy may result in infection-related morbidity and increased mortality, making optimization of the dosing regimen essential. As paediatric data are limited, our aim was to evaluate the population pharmacokinetics of teicoplanin in order to define the appropriate dosing regimen in this high risk population.

Methods

The current dose of teicoplanin was evaluated in children with haematological malignancy. Population pharmacokinetics of teicoplanin were analyzed using nonmem software. The dosing regimen was optimized based on the final model.

Results

Eighty-five children (age range 0.5 to 16.9 years) were included. Therapeutic drug monitoring and opportunistic samples (n = 143) were available for analysis. With the current recommended dose of 10 mg kg^–1 day^–1, 41 children (48%) had sub-therapeutic steady-state trough concentrations (C_ss,min<10 mg l^–1). A two compartment pharmacokinetic model with first order elimination was developed. Systematic covariate analysis identified that bodyweight (size) and creatinine clearance significantly influenced teicoplanin clearance. The model was validated internally. Its predictive performance was further confirmed in an external validation. In order to reach the target AUC of 750 mg l^–1 h 18 mg kg^–1 was required for infants, 14 mg kg^–1 for children and 12 mg kg^–1 for adolescents. A patient-tailored dose regimen was further developed and reduced variability in AUC and C_ss,min values compared with the mg kg^–1 basis dose, making the modelling approach an important tool for dosing individualization.

Conclusions

This first population pharmacokinetic study of teicoplanin in children with haematological malignancy provided evidence-based support to individualize teicoplanin therapy in this vulnerable population.     

Activation of P2Y6 receptors increases the voiding frequency in anaesthetized rats by releasing ATP from the bladder urothelium

BACKGROUND AND PURPOSE

Despite the abundant expression of the UDP-sensitive P2Y₆ receptor in urothelial cells and sub-urothelial myofibroblasts its role in the control of bladder function is not well understood.

EXPERIMENTAL APPROACH

We compared the effects of UDP and of the selective P2Y₆ receptor agonist, PSB0474, on bladder urodynamics in anaesthetized rats; the voided fluid was tested for ATP bioluminescence. The isolated urinary bladder was used for in vitro myographic recordings and [³H]-ACh overflow experiments.

KEY RESULTS

Instillation of UDP or PSB0474 into the bladder increased the voiding frequency (VF) without affecting the amplitude (A) and the duration (Δt) of bladder contractions; an effect blocked by the P2Y₆ receptor antagonist, MRS2578. Effects mediated by urothelial P2Y₆ receptors required extrinsic neuronal circuitry as they were not detected in the isolated bladder. UDP-induced bladder hyperactvity was also prevented by blocking P2X3 and P2Y₁ receptors, respectively, with A317491 and MRS2179 applied i.v.. UDP decreased [³H]-ACh release from stimulated bladder strips with urothelium, but not in its absence. Inhibitory effects of UDP were converted into facilitation by the P2Y₁ receptor antagonist, MRS2179. The P2Y₆ receptor agonist increased threefold ATP levels in the voided fluid.

CONCLUSIONS AND IMPLICATIONS

Activation of P2Y₆ receptors increased the voiding frequency indirectly by releasing ATP from the urothelium and activation of P2X3 receptors on sub-urothelial nerve afferents. Bladder hyperactivity may be partly reversed following ATP hydrolysis to ADP by E-NTPDases, thereby decreasing ACh release from cholinergic nerves expressing P2Y₁ receptors.     

Antidepressant-like effects of a novel 5-HT3 receptor antagonist 6z in acute and chronic murine models of depression

Aim:

To investigate the antidepressant-like effects of a novel 5-HT₃ receptor antagonist N-(benzo[d]thiazol-2-yl)-3-methoxyquinoxalin-2-carboxamide (6z) in acute and chronic murine models of depression.

Methods:

5-HT₃ receptor antagonism was examined in guinea pig ileum in vitro. A tail suspension test (TST) was used as acute depression model to evaluate the antidepressant-like behavior in mice treated with 6z (0.5–2 mg/kg, ip). In chronic depression model, mice were exposed to a 4-week chronic unpredictable stress (CUS) protocol, and treated with 6z (0.5–2 mg·kg⁻¹·d⁻¹, po) or a positive drug fluoxetine (10 mg·kg⁻¹·d⁻¹, po) in the last 2 weeks, followed by behavioral and biochemical assessments.

Results:

The 5-HT₃ receptor antagonism of 6z (pA₂=7.4) in guinea pig ileum was more potent than that of a standard 5-HT₃ receptor antagonist ondansetron (pA₂=6.9). In acute depression model, 6z administration significantly decreased the immobility duration. In chronic depression model, 6z administration reversed CUS-induced depressive-like behavior, as evidenced by increased immobility duration in the forced swim test and sucrose preference in the sucrose preference test. Furthermore, chronic administration of 6z prevented CUS-induced brain oxidative stress, with significant reduction of pro-oxidant markers and elevation of antioxidant enzyme activity. Moreover, chronic administration of 6z attenuated CUS-induced hypothalamic-pituitary-adrenal axis hyperactivity, as shown by reduced plasma corticosterone levels. Similar results were observed in the fluoxetine-treated group.

Conclusion:

6z is a novel 5-HT₃ receptor antagonist with potential antidepressant-like activities, which may be related to modulating hypothalamic-pituitary-adrenal axis and attenuating brain oxidative damage.