Pharmacological inhibition of the inflammatory receptor CCR2 relieves the early deleterious consequences of status epilepticus.

Generalized status epilepticus (SE) triggers a robust neuroinflammatory response involving reactive astrocytosis, activation of brain-resident microglia, and brain infiltration of CCR2+ monocytes. Multiple lines of evidence indicate that quenching SE-induced neuroinflammation can alleviate the adverse consequences of SE, including neuronal damage and cognitive impairments. Our recent findings show that blocking monocyte brain entry after SE, via global Ccr2 KO, rescues several SE-induced adverse effects including blood-brain barrier (BBB) erosion, microgliosis and neuronal damage while enhancing weight regain. The goals of the present study were to determine if CCR2 antagonism with a small molecule after SE replicates the effects of the CCR2 knockout. Male Ccr2+/rfp heterozygous mice were subject to intraperitoneal injection of kainic acid, scored for seizure severity, weight recovery, and nest building capability. Surviving mice were randomized into CCR2 antagonist and vehicle groups. The CCR2 antagonist, or vehicle, was administered 24- and 48-h post-SE via oral gavage, and mice were sacrificed three days post-SE. Mice subject to the CCR2 antagonist displayed faster weight recovery between one- and three-days post-SE and modestly enhanced ability to build a nest on the third day after SE when compared to vehicle-treated controls. CCR2 antagonism limited monocyte recruitment to the hippocampus and reduced numbers of Iba1+ macrophages. The mRNA levels of inflammatory mediators were depressed by 47%, and glial markers were reduced by 30% in mice treated with the CCR2 antagonist compared to controls. Astrocytosis was reduced in four brain regions. Neuroprotection was observed in the hippocampus, and erosion of the BBB was lessened in mice subject to the antagonist. Our findings provide proof-of-concept that brief CCR2 antagonism beginning one day after SE can alleviate multiple adverse SE-induced effects, including functional impairment, and identify circulating CCR2+ monocytes as a viable therapeutic target.

Novel GluN2B-Selective NMDA Receptor Negative Allosteric Modulator Possesses Intrinsic Analgesic Properties and Enhances Analgesia of Morphine in a Rodent Tail Flick Pain Model.

Many cases of accidental death associated with drug overdose are due to chronic opioid use, tolerance, and addiction. Analgesic tolerance is characterized by a decreased response to the analgesic effects of opioids, requiring increasingly higher doses to maintain the desired level of pain relief. Overactivation of GluN2B-containing N-methyl-d-Aspartate receptors is thought to play a key role in mechanisms underlying cellular adaptation that takes place in the development of analgesic tolerance. Herein, we describe a novel GluN2B-selective negative allosteric modulator, EU93-108, that shows high potency and brain penetrance. We describe the structural basis for binding at atomic resolution. This compound possesses intrinsic analgesic properties in the rodent tail immersion test. EU93-108 has an acute and significant anodyne effect, whereby morphine when combined with EU93-108 produces a higher tail flick latency compared to that of morphine alone. These data suggest that engagement of GluN2B as a target has utility in the treatment of pain, and EU93-108 could serve as an appropriate tool compound to interrogate this hypothesis. Future structure-activity relationship work around this scaffold could give rise to compounds that can be co-administered with opioids to diminish the onset of tolerance due to chronic opioid use, thereby modifying their utility.

Phase 1 Clinical Results for NP10679, a pH-sensitive GluN2B-selective N-methyl-d-aspartate Receptor Inhibitor.

NP10679 is a context-dependent and subunit-selective negative allosteric modulator of N-methyl-d-aspartate (NMDA) receptors. It is a more potent inhibitor of GluN2B-containing NMDA receptors at the acidic levels of extracellular pH (eg, 6.9) found in the penumbral regions associated with cerebral ischemia than at physiological pH. This property allows NP10679 to act selectively in ischemic tissue while minimizing the nonselective blockade of NMDA receptors in healthy brain, thereby reducing on-target adverse effects. We report the results of a first-in-human pharmacokinetic and safety phase 1 clinical trial in healthy volunteers receiving single or multiple doses of NP10679 (NCT04007263). We found that NP10679 was well-tolerated and with a half-life of 20 hours, which is amenable to once per day dosing. The only notable side effect in this clinical trial was modest somnolence at higher doses, atypical in that the subject could easily be aroused. The overall results suggest that NP10679 is a candidate for further development for use in acute brain injury, such as ischemic stroke or aneurysmal subarachnoid hemorrhage, as well as for use in neuropsychiatric indications.

An Agonist Dependent Allosteric Antagonist of Prostaglandin EP2 Receptors.

All reported prostaglandin EP2 receptor antagonists have a purely orthosteric, competitive mode of action. Herein, we report the characterization of compound 1 (pubchem CID 664888) as the first EP2 antagonist that features a reversible, agonist dependent allosteric mode of action. Compound 1 displayed an unsurmountable inhibition of cAMP accumulation stimulated by different EP2 agonists in C6 glioma cells overexpressing human EP2 (C6G-hEP2). The degree of reduction of agonist potency and efficacy depended on the agonist employed. Negative allosteric modulation was not observed in C6G cells overexpressing human EP4, IP, or DP1 receptors. Moreover, in the murine microglial cell line that stably expresses human EP2 receptors (BV2-hEP2), compound 1 reduced the EP2 agonist-induced elevation of interleukin 6 (IL-6), IL-1ß, and hEP2 mRNA levels and increased that of tumor necrosis factor (TNF)-α. Compound 1 was docked into a homology model of hEP2. The predicted binding site on the cytoplasmic receptor surface was similar to that of allosteric inhibitors of the ß2-adrenergic, CC chemokine receptor 9 (CCR9), and CC chemokine receptor 2 (CCR2) receptors, which supports the notion of a conserved G-protein-coupled receptor (GPCR) binding pocket for allosteric inhibitors. As the first agonist dependent negative allosteric modulator of EP2 receptor, the structure of this compound may provide a basis for developing improved allosteric modulators of EP2 receptors.

Potent, Selective, Water Soluble, Brain-Permeable EP2 Receptor Antagonist for Use in Central Nervous System Disease Models.

Activation of prostanoid EP2 receptor exacerbates neuroinflammatory and neurodegenerative pathology in central nervous system diseases such as epilepsy, Alzheimer's disease, and cerebral aneurysms. A selective and brain-permeable EP2 antagonist will be useful to attenuate the inflammatory consequences of EP2 activation and to reduce the severity of these chronic diseases. We recently developed a brain-permeable EP2 antagonist 1 (TG6-10-1), which displayed anti-inflammatory and neuroprotective actions in rodent models of status epilepticus. However, this compound exhibited moderate selectivity to EP2, a short plasma half-life in rodents (1.7 h) and low aqueous solubility (27 µM), limiting its use in animal models of chronic disease. With lead-optimization studies, we have developed several novel EP2 antagonists with improved water solubility, brain penetration, high EP2 potency, and selectivity. These novel inhibitors suppress inflammatory gene expression induced by EP2 receptor activation in a microglial cell line, reinforcing the use of EP2 antagonists as anti-inflammatory agents.

Inhibition of the prostaglandin EP2 receptor prevents long-term cognitive impairment in a model of systemic inflammation.

Long-term cognitive and affective impairments are common problems in the survivors of sepsis, which weakens their vocational and daily life ability. Neuroinflammation has been reported to exert a key role in the development of cognitive deficit in different disorders including epilepsy, Alzheimer's disease (AD) and stroke. Mice treated with lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, show a robust but short-lived neuroinflammation and develop long-term memory and affective problems. In this study, we test the hypothesis that pharmacological blockade of the EP2 receptor for prostaglandin E2 reduces neuroinflammation and prevents long-term affective and memory deficits in a mouse model of LPS-induced, sepsis-associated encephalopathy (SAE). Our results show that an EP2 antagonist, TG6-10-1, promotes the recovery of body weight, mitigates neuroinflammation as judged by inflammatory cytokines and microgliosis, prevents the loss of synaptic proteins, and ameliorates depression-like behavior in the sucrose preference test as well as memory loss in the novel object recognition test. Our results point to a new avenue to ameliorate neuroinflammation and long-term affective and cognition problems of sepsis survivors.

Suppressing pro-inflammatory prostaglandin signaling attenuates excitotoxicity-associated neuronal inflammation and injury.

Glutamate receptor-mediated excitotoxicity is a common pathogenic process in many neurological conditions including epilepsy. Prolonged seizures induce elevations in extracellular glutamate that contribute to excitotoxic damage, which in turn can trigger chronic neuroinflammatory reactions, leading to secondary damage to the brain. Blocking key inflammatory pathways could prevent such secondary brain injury following the initial excitotoxic insults. Prostaglandin E2 (PGE2) has emerged as an important mediator of neuroinflammation-associated injury, in large part via activating its EP2 receptor subtype. Herein, we investigated the effects of EP2 receptor inhibition on excitotoxicity-associated neuronal inflammation and injury in vivo. Utilizing a bioavailable and brain-permeant compound, TG6-10-1, we found that pharmacological inhibition of EP2 receptor after a one-hour episode of kainate-induced status epilepticus (SE) in mice reduced seizure-promoted functional deficits, cytokine induction, reactive gliosis, blood-brain barrier impairment, and hippocampal damage. Our preclinical findings endorse the feasibility of blocking PGE2/EP2 signaling as an adjunctive strategy to treat prolonged seizures. The promising benefits from EP2 receptor inhibition should also be relevant to other neurological conditions in which excitotoxicity-associated secondary damage to the brain represents a pathogenic event.

Peripherally Restricted, Highly Potent, Selective, Aqueous-Soluble EP2 Antagonist with Anti-Inflammatory Properties.

The prostaglandin E2 receptor, EP2, plays an important role in physiology and in a variety of pathological conditions. Studies indicate that EP2 is pro-inflammatory in chronic peripheral and central nervous system disease and cancer models. Thus, targeting the EP2 receptor with small molecules could be a therapeutic strategy for treating inflammatory diseases and cancer. We recently reported a novel class of competitive antagonists of the EP2 receptor. However, earlier leads displayed low selectivity against the DP1 prostanoid receptor, moderate plasma half-life, and low aqueous solubility, which renders them suboptimal for testing in animal models of disease. We now report a novel compound TG8-69, which has suitable drug-like properties. We present synthesis, lead-optimization studies, pharmacological characterization, and anti-inflammatory properties of this compound that support its use in chronic peripheral inflammatory diseases, including rheumatoid arthritis, endometriosis, and cancer, in which EP2 appears to play a pathogenic role.

Why Is It so Hard to Do Good Science?

"Good science" means answering important questions convincingly, a challenging endeavor under the best of circumstances. Our inability to replicate many biomedical studies has been the subject of numerous commentaries both in the scientific and lay press. In response, statistics has re-emerged as a necessary tool to improve the objectivity of study conclusions. However, psychological aspects of decision making introduce preconceived preferences into scientific judgment that cannot be eliminated by any statistical method. The psychology of decision making, expounded by Kahneman, Tversky, and Thaler, is well known in the field of economics, but the underlying concepts of cognitive psychology are also relevant to scientific judgments. I repeated experiments carried out on undergraduates by Kahneman and colleagues four to five decades ago, but with scientists, and obtained essentially the same results. The experiments were in the form of written reactions to scenarios, and participants were scientists at all career stages. The findings reinforce the roles that two inherent intuitions play in scientific decision making: our drive to create a coherent narrative from new data regardless of its quality or relevance and our inclination to seek patterns in data whether they exist or not. Moreover, we do not always consider how likely a result is regardless of its p value. Low statistical power and inattention to principles underpinning Bayesian statistics reduce experimental rigor, but mitigating skills can be learned. Overcoming our natural human tendency to make quick decisions and jump to conclusions is a deeper obstacle to doing good science; this too can be learned.

A New Approach for Epilepsy.

About one-third of the 65 million people worldwide affected by epilepsy are treatment-resistant, and the degree to which they suffer from seizures and convulsions can vary widely. Problems occur when nerve cells in the brain fail to communicate properly. A new study has found that inhibiting an enzyme that is critical in metabolic communication has an anti-seizure effect in epileptic mice. These findings, the authors believe, may very well initiate a shift to new therapeutic approaches.

The First 50 Years of Molecular Pharmacology.

In this Perspective, former and current editors of Molecular Pharmacology, together with the guest editors for this 50th Anniversary Issue, provide a historical overview of the journal since its founding in 1965. The substantial impact that Molecular Pharmacology has had on the field of pharmacology as well as on biomedical science is discussed, as is the broad scope of the journal. The authors conclude that, true to the original goals for the journal, Molecular Pharmacology today remains an outstanding venue for work that provides a mechanistic understanding of drugs, molecular probes, and their biologic targets.

When and how do seizures kill neurons, and is cell death relevant to epileptogenesis?

The effect of seizures on neuronal death and the role of seizure-induced neuronal death in acquired epileptogenesis have been debated for decades. Isolated brief seizures probably do not kill neurons; however, severe and repetitive seizures (i.e., status epilepticus) certainly do. Because status epilepticus both kills neurons and also leads to chronic epilepsy, neuronal death has been proposed to be an integral part of acquired epileptogenesis. Several studies, particularly in the immature brain, have suggested that neuronal death is not necessary for acquired epileptogenesis; however, the lack of neuronal death is difficult if not impossible to prove, and more recent studies have challenged this concept. Novel mechanisms of cell death, beyond the traditional concepts of necrosis and apoptosis, include autophagy, phagoptosis, necroptosis, and pyroptosis. The traditional proposal for why neuronal death may be necessary for epileptogenesis is based on the recapitulation of development hypothesis, where a loss of synaptic input from the dying neurons is considered a critical signal to induce axonal sprouting and synaptic-circuit reorganization. We propose a second hypothesis - the neuronal death pathway hypothesis, which states that the biochemical pathways causing programmed neurodegeneration, rather than neuronal death per se, are responsible for or contribute to epileptogenesis. The reprogramming of neuronal death pathways - if true - is proposed to derive from necroptosis or pyroptosis. The proposed new hypothesis may inform on why neuronal death seems closely linked to epileptogenesis, but may not always be.

Development of second generation EP2 antagonists with high selectivity.

EP2 receptor has emerged as an important biological target for therapeutic intervention. In particular, it has been shown to exacerbate disease progression of a variety of CNS and peripheral diseases. Deletion of the EP2 receptor in mouse models recapitulates several features of the COX-2 inhibition, thus presenting a new avenue for anti-inflammatory therapy which could bypass some of the adverse side effects observed by the COX-2 inhibition therapy. We have recently reported a cinnamic amide class of EP2 antagonists with high potency, but these compounds exhibited a moderate selectivity against prostanoid receptor DP1. Moreover they possess acrylamide moiety in the structure, which may result in liver toxicity over longer period of use in a chronic disease model. Thus, we now developed a second generation compounds that devoid of the acrylamide functionality and possess high potency and improved (>1000-fold) selectivity to EP2 over other prostanoid receptors.

Lead optimization studies of cinnamic amide EP2 antagonists.

Prostanoid receptor EP2 can play a proinflammatory role, exacerbating disease pathology in a variety of central nervous system and peripheral diseases. A highly selective EP2 antagonist could be useful as a drug to mitigate the inflammatory consequences of EP2 activation. We recently identified a cinnamic amide class of EP2 antagonists. The lead compound in this class (5d) displays anti-inflammatory and neuroprotective actions. However, this compound exhibited moderate selectivity to EP2 over the DP1 prostanoid receptor (∼10-fold) and low aqueous solubility. We now report compounds that display up to 180-fold selectivity against DP1 and up to 9-fold higher aqueous solubility than our previous lead. The newly developed compounds also display higher selectivity against EP4 and IP receptors and a comparable plasma pharmacokinetics. Thus, these compounds are useful for proof of concept studies in a variety of models where EP2 activation is playing a deleterious role.