Critical evaluation of P2X7 receptor antagonists in selected seizure models

The ATP-gated P2X7 receptor (P2X7R) is a non-selective cation channel which senses high extracellular ATP concentrations and has been suggested as a target for the treatment of neuroinflammation and neurodegenerative diseases. The use of P2X7R antagonists may therefore be a viable approach for treating CNS pathologies, including epileptic disorders. Recent studies showed anticonvulsant potential of P2X7R antagonists in certain animal models. To extend this work, we tested three CNS-permeable P2X7R blocker (Brilliant Blue G, AFC-5128, JNJ-47965567) and a natural compound derivative (tanshinone IIA sulfonate) in four well-characterized animal seizure models. In the maximal electroshock seizure threshold test and the pentylenetetrazol (PTZ) seizure threshold test in mice, none of the four compounds demonstrated anticonvulsant effects when given alone. Notably, in combination with carbamazepine, both AFC-5128 and JNJ-47965567 increased the threshold in the maximal electroshock seizure test. In the PTZ-kindling model in rats, useful for testing antiepileptogenic activities, Brilliant Blue G and tanshinone exhibited a moderate retarding effect, whereas the potent P2X7R blocker AFC-5128 and JNJ-47965567 showed a significant and long-lasting delay in kindling development. In fully kindled rats, the investigated compounds revealed modest effects to reduce the mean seizure stage. Furthermore, AFC-5128- and JNJ-47965567-treated animals displayed strongly reduced Iba 1 and GFAP immunoreactivity in the hippocampal CA3 region. In summary, our results show that P2X7R antagonists possess no remarkable anticonvulsant effects in the used acute screening tests, but can attenuate chemically-induced kindling. Further studies would be of interest to support the concept that P2X7R signalling plays a crucial role in the pathogenesis of epileptic disorders

Transient P2X7 Receptor Antagonism Produces Lasting Reductions in Spontaneous Seizures and Gliosis in Experimental Temporal Lobe Epilepsy

Neuroinflammation is thought to contribute to the pathogenesis and maintenance of temporal lobe epilepsy, but the underlying cell and molecular mechanisms are not fully understood. The P2X7 receptor is an ionotropic receptor predominantly expressed on the surface of microglia, although neuronal expression has also been reported. The receptor is activated by the release of ATP from intracellular sources that occurs during neurodegeneration, leading to microglial activation and inflammasome-mediated interleukin 1β release that contributes to neuroinflammation. Using a reporter mouse in which green fluorescent protein is induced in response to the transcription of P2rx7, we show that expression of the receptor is selectively increased in CA1 pyramidal and dentate granule neurons, as well as in microglia in mice that developed epilepsy after intra-amygdala kainic acid-induced status epilepticus. P2X7 receptor levels were increased in hippocampal subfields in the mice and in resected hippocampus from patients with pharmacoresistant temporal lobe epilepsy. Cells transcribing P2rx7 in hippocampal slices from epileptic mice displayed enhanced agonist-evoked P2X7 receptor currents, and synaptosomes from these animals showed increased P2X7 receptor levels and altered calcium responses. A 5 d treatment of epileptic mice with systemic injections of the centrally available, potent, and specific P2X7 receptor antagonist JNJ-47965567 (30 mg/kg) significantly reduced spontaneous seizures during continuous video-EEG monitoring that persisted beyond the time of drug presence in the brain. Hippocampal sections from JNJ-47965567-treated animals obtained >5 d after treatment ceased displayed strongly reduced microgliosis and astrogliosis. The present study suggests that targeting the P2X7 receptor has anticonvulsant and possibly disease-modifying effects in experimental epilepsy. SIGNIFICANCE STATEMENT: Temporal lobe epilepsy is the most common and drug-resistant form of epilepsy in adults. Neuroinflammation is implicated as a pathomechanism, but the upstream mechanisms driving gliosis and how important this is for seizures remain unclear. In our study, we show that the ATP-gated P2X7 receptor is upregulated in experimental epilepsy and resected hippocampus from epilepsy patients. Targeting the receptor with a new centrally available antagonist, JNJ-47965567, suppressed epileptic seizures well beyond the time of treatment and reduced underlying gliosis in the hippocampus. The findings suggest a potential disease-modifying treatment for epilepsy based on targeting the P2X7 receptor.</p

Selective Inhibition of Orexin-2 Receptors Prevents Stress-Induced ACTH Release in Mice

Orexins peptides exert a prominent role in arousal-related processes including stress responding, by activating orexin-1 (OX1R) and orexin-2 (OX2R) receptors located widely throughout the brain. Stress or orexin administration stimulates hyperarousal, adrenocorticotropic hormone (ACTH) and corticosterone release, and selective OX1R blockade can attenuate several stress-induced behavioral and cardiovascular responses but not the hypothalamic-pituitary-adrenal (HPA) axis activation. As opposed to OX1R, OX2R are preferentially expressed in the paraventricular hypothalamic nucleus which is involved in the HPA axis regulation. In the present study, we investigated the effects of a psychological stress elicited by cage exchange (CE) on ACTH release in two murine models (genetic and pharmacological) of selective OX2R inhibition. CE-induced stress produced a significant increase in ACTH serum levels. Mice lacking the OX2R exhibited a blunted stress response. Stress-induced ACTH release was absent in mice pre-treated with the selective OX2R antagonist JNJ-42847922 (30 mg/kg po), whereas pre-treatment with the dual OX1/2R antagonist SB-649868 (30 mg/kg po) only partially attenuated the increase of ACTH. To assess whether the intrinsic and distinct sleep-promoting properties of each antagonist could account for the differential stress response, a separate group of mice implanted with electrodes for standard sleep recording were orally dosed with JNJ-42847922 or SB-649868 during the light phase. While both compounds reduced the latency to non-rapid eye movement (NREM) sleep without affecting its duration, a prevalent REM-sleep promoting effect was observed only in mice treated with the dual OX1/2R antagonist. These data indicate that in a psychological stress model, genetic or pharmacological inhibition of OX2R markedly attenuated stress-induced ACTH secretion, as a separately mediated effect from the NREM sleep induction of OX2R antagonism

Molecular determinants of ASIC1 modulation by divalent cations

Abstract Acid-sensing ion channels (ASICs) are proton-gated cation channels widely expressed in the nervous system. ASIC gating is modulated by divalent cations as well as small molecules; however, the molecular determinants of gating modulation by divalent cations are not well understood. Previously, we identified two small molecules that bind to ASIC1a at a novel site in the acidic pocket and modulate ASIC1 gating in a manner broadly resembling divalent cations, raising the possibility that these small molecules may help to illuminate the molecular determinants of gating modulation by divalent cations. Here, we examined how these two groups of modulators might interact as well as mutational effects on ASIC1a gating and its modulation by divalent cations. Our results indicate that binding of divalent cations to an acidic pocket site plays a key role in gating modulation of the channel

Critical evaluation of P2X7 receptor antagonists in selected seizure models

The ATP-gated P2X7 receptor (P2X7R) is a non-selective cation channel which senses high extracellular ATP concentrations and has been suggested as a target for the treatment of neuroinflammation and neurodegenerative diseases. The use of P2X7R antagonists may therefore be a viable approach for treating CNS pathologies, including epileptic disorders. Recent studies showed anticonvulsant potential of P2X7R antagonists in certain animal models. To extend this work, we tested three CNS-permeable P2X7R blocker (Brilliant Blue G, AFC-5128, JNJ-47965567) and a natural compound derivative (tanshinone IIA sulfonate) in four well-characterized animal seizure models. In the maximal electroshock seizure threshold test and the pentylenetetrazol (PTZ) seizure threshold test in mice, none of the four compounds demonstrated anticonvulsant effects when given alone. Notably, in combination with carbamazepine, both AFC-5128 and JNJ-47965567 increased the threshold in the maximal electroshock seizure test. In the PTZ-kindling model in rats, useful for testing antiepileptogenic activities, Brilliant Blue G and tanshinone exhibited a moderate retarding effect, whereas the potent P2X7R blocker AFC-5128 and JNJ-47965567 showed a significant and long-lasting delay in kindling development. In fully kindled rats, the investigated compounds revealed modest effects to reduce the mean seizure stage. Furthermore, AFC-5128- and JNJ-47965567-treated animals displayed strongly reduced Iba 1 and GFAP immunoreactivity in the hippocampal CA3 region. In summary, our results show that P2X7R antagonists possess no remarkable anticonvulsant effects in the used acute screening tests, but can attenuate chemically-induced kindling. Further studies would be of interest to support the concept that P2X7R signalling plays a crucial role in the pathogenesis of epileptic disorders

Critical evaluation of P2X7 receptor antagonists in selected seizure models

The ATP-gated P2X7 receptor (P2X7R) is a non-selective cation channel which senses high extracellular ATP concentrations and has been suggested as a target for the treatment of neuroinflammation and neurodegenerative diseases. The use of P2X7R antagonists may therefore be a viable approach for treating CNS pathologies, including epileptic disorders. Recent studies showed anticonvulsant potential of P2X7R antagonists in certain animal models. To extend this work, we tested three CNS-permeable P2X7R blocker (Brilliant Blue G, AFC-5128, JNJ-47965567) and a natural compound derivative (tanshinone IIA sulfonate) in four well-characterized animal seizure models. In the maximal electroshock seizure threshold test and the pentylenetetrazol (PTZ) seizure threshold test in mice, none of the four compounds demonstrated anticonvulsant effects when given alone. Notably, in combination with carbamazepine, both AFC-5128 and JNJ-47965567 increased the threshold in the maximal electroshock seizure test. In the PTZ-kindling model in rats, useful for testing antiepileptogenic activities, Brilliant Blue G and tanshinone exhibited a moderate retarding effect, whereas the potent P2X7R blocker AFC-5128 and JNJ-47965567 showed a significant and long-lasting delay in kindling development. In fully kindled rats, the investigated compounds revealed modest effects to reduce the mean seizure stage. Furthermore, AFC-5128- and JNJ-47965567-treated animals displayed strongly reduced Iba 1 and GFAP immunoreactivity in the hippocampal CA3 region. In summary, our results show that P2X7R antagonists possess no remarkable anticonvulsant effects in the used acute screening tests, but can attenuate chemically-induced kindling. Further studies would be of interest to support the concept that P2X7R signalling plays a crucial role in the pathogenesis of epileptic disorders

Blockade of the brain histamine H3 receptor by JNJ-39220675: preclinical PET studies with [11C]GSK189254 in anesthetized baboon

RATIONALE: The preclinical characterization of a series of aryloxypyridine amides has identified JNJ-39220675 ((4-cyclobutyl-1,4-diazepan-1-yl)(6-(4-fluorophenoxy)pyridin-3-yl)methanone) as a high-affinity histamine H(3) receptor antagonist and a candidate for further drug development particularly in the treatment of alcohol-related behaviors. OBJECTIVE: This study measured brain histamine H(3) receptor blockade by JNJ-39220675 (1 mg/kg) in the female baboon. METHODS: Positron emission tomography imaging and [(11)C]GSK189254, a reversible high-affinity radiotracer with specificity for the histamine H(3) receptor, was used to measure histamine H(3) receptor availability at baseline and after i.v. and oral administration of JNJ-39220675 (1 mg/kg) in the anesthetized baboon. Histamine H(3) receptor availability was estimated as the total distribution volume (V(T)) in brain regions. The sensitivity of [(11)C]GSK189254 binding to injected mass and carryover effects was determined. RESULTS: JNJ-39220675 produces robust (ca. 90 %) blockade of [(11)C]GSK189254 binding after i.v. and oral administration. After oral administration of JNJ-39220675 (1 mg/kg), the fractional receptor occupancy was >0.9 at 90 min with a slight increase from 90 to 240 min. Similar to prior studies in humans, V(T) was highly sensitive to the mass of GSK189254 with ED(50) estimated to be 0.16 μg/kg. CONCLUSIONS: The robust blockade of binding of [(11)C]GSK189254 by JNJ-39220675 demonstrates that this compound readily penetrates the blood–brain barrier and occupies the histamine H(3) receptor after oral administration at low plasma concentrations (∼1 ng/cc) supporting further drug development for alcohol addiction and other disorders. This study corroborates prior reports of the high sensitivity of [(11)C]GSK189254 to injected mass at doses >0.1 μg/kg

Clinical qualification of [18F] JNJ-64413739, a novel candidate PET ligand for the P2X7 receptor

Introduction: The P2X7 purinergic receptor is a membrane bound, ATP-gated cation channel embedded in the cell membrane. In the CNS, P2X7 receptors have been identified on several cell types with the most abundant expression on microglia. Activation of the P2X7 promotes the NLRP3 inflammasome assembly and release of neuroactive cytokines such as IL1β. Over-activation of this pathway is hypothesized to play a role in the pathophysiology of mood disorders. Selective, brain penetrant inhibitors of P2X7 have been developed as candidate therapeutics. A site-specific PET ligand for the P2X7 receptor could provide evidence of target engagement and assist in clinical dose selection. [18F] JNJ-64413739, a selective P2X7 inhibitor, has been extensively evaluated in preclinical models as a candidate PET ligand. We now report the clinical qualification of the ligand in healthy adult males including dosimetry, brain distribution, test-retest (TRT) variability, and blocking by pharmacological doses of the P2X7 inhibitor JNJ-54175446. Methods: For the radiation dosimetry 3 healthy male subjects were given a dose of ~185 MBq [18F] JNJ-64413739 and underwent 10 consecutive whole body PET/CT scans (Siemens Biograph) to assess the tracer biodistribution for approximately 5 hours post injection. Delineation of source organs allowed estimation of the normalized cumulated activity and to calculate the corresponding organ doses and effective dose (ED) with OLINDA. To evaluate the tracer distribution in the brain, 5 healthy male subjects were given 115-196 MBq [18F] JNJ-64413739 and completed a 120-minute dynamic PET scan (PETMR GE Signa) combined with arterial blood sampling and tracer metabolite analysis. 1 and 2 tissue compartment models (1-2TCM) and Logan graphical analysis (LGA) were evaluated to quantify tracer kinetics and calculate regional volumes of distribution (VT) (PMOD v.3.7). Retest scans were acquired for 3 of the 5 subjects (interscan interval 26-44 days). As part of a PET dose occupancy study, a subject underwent a baseline and post dose [18F] JNJ-64413739 PETMR scan. The latter was performed after oral dosing of JNJ-54175446 predicted to saturate binding based on preclinical PET. Occupancy was estimated by analyzing baseline and post dose VT values using a Lassen plot. Results: Average ED was 22 ± 1 µSv/MBq with the highest organ absorbed doses for gall bladder, urinary bladder, small and large intestine. 2TCM provide to be the most appropriate kinetic model to estimate regional VT with very similar estimates for LGA. Regional VT values were similar across all brain volumes with slightly higher values in thalamus, striatum, and brain stem. Inter subject variability of VT was relatively high with a composite cortical region of interest VT showing an approximate 3-fold range across subjects. Average regional TRT was 12.7 ± 8.4% by 2TCM and 13.5 ± 8.0 % by LGA while the acquisition time could be reduced to 90 minutes with only limited increase in bias and TRT variability. A single oral dose of JNJ-54175446 resulted in 78% occupancy across all brain regions and no reference region could be identified. Tracer metabolism was not altered by pretreatment with JNJ-54175446. Conclusions: [18F] JNJ-64413739 appears to be a suitable PET ligand for quantitation of P2X7 receptors in human brain. As such it can be used for testing target engagement by P2X7 inhibitors, assist in guiding dose selection of JNJ-54175446 for Phase 2 clinical trials, and provide insight into the expression of P2X7 receptors in health and disease.status: submitte

Chemical structure of the four investigated compounds.

<p>Chemical structure of the four investigated compounds.</p

PTZ-induced kindling in rats. Influence of the four investigated compounds on the development of PTZ-kindling.

<p><b>(a)</b><i>First series</i>: Tanshinone IIA SO₃Na (TIIAS, 30 mg/kg) and Brilliant Blue G (BBG, 50 mg/kg) were administered i.p. 45 min before PTZ with vehicle (20% PEG 400). <b>(b)</b><i>Second series</i>: AFC-5128 (AFC, 30 mg/kg) was given i.p. 45 min before PTZ with vehicle (DMA/β-CD). <b>(c)</b> <i>Third series</i>: JNJ-47965567 (JNJ, 15 mg/kg) was given s.c. 30 min before PTZ with vehicle (SBE-β-CD). The control groups (n = 12) received the corresponding vehicle alone. In addition, in the third series a further control group pre-treated with 0.9% NaCl-solution was included. PTZ (35 mg/kg i.p.) was injected once every 48 h (three times a week on Monday, Wednesday and Friday) for 20 successive sessions. After 20 PTZ-injections and an 8-day interruption of the kindling procedure, the rats were further kindled with only vehicle pre-treatment instead of the compounds (21^st to 25^th PTZ-injection). Values shown represent mean seizure stages. In order to keep the curves clear from the others, error bars (± SEM) were indicated only in Fig 4b. (SEM in all line graphs was not higher than ± 0.4.) Significance level: *P <0.05 (two-way repeated measures ANOVA, Holm-Sidak post hoc test).</p