Search

Nonselective glutamate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists are efficacious in chronic pain but have significant tolerability issues, likely arising from the ubiquitous expression of AMPA receptors in the central nervous system (CNS). Recently, LY3130481 has been shown to selectively block AMPA receptors coassembled with the auxiliary protein, transmembrane AMPA receptor regulatory protein (TARP) γ8, which is highly expressed in the hippocampus but also in pain pathways, including anterior cingulate (ACC) and somatosensory cortices and the spinal cord, suggesting that selective blockade of γ8/AMPA receptors may suppress nociceptive signaling with fewer CNS side effects. The potency of LY3130481 on recombinant γ8-containing AMPA receptors was modulated by coexpression with other TARPs; γ2 subunits affected activity more than γ3 subunits. Consistent with these findings, LY3130481 had decreasing potency on receptors from rat hippocampal, cortical, spinal cord, and cerebellar neurons that was replicated in tissue from human brain. LY3130481 partially suppressed, whereas the nonselective AMPA antagonist GYKI53784 completely blocked, AMPA receptor-dependent excitatory postsynaptic potentials in ACC and spinal neurons in vitro. Similarly, LY3130481 attenuated short-term synaptic plasticity in spinal sensory neurons in vivo in response to stimulation of peripheral afferents. LY3130481 also significantly reduced nocifensive behaviors after intraplantar formalin that was correlated with occupancy of CNS γ8-containing AMPA receptors. In addition, LY3130481 dose-dependently attenuated established gait impairment after joint damage and tactile allodynia after spinal nerve ligation, all in the absence of motor side effects. Collectively, these data demonstrate that LY3130481 can suppress excitatory synaptic transmission and plasticity in pain pathways containing γ8/AMPA receptors and significantly reduce nocifensive behaviors, suggesting a novel, effective, and safer therapy for chronic pain conditions.

Background Movement-evoked pain and impaired joint mobility are common comorbidities in inflammatory diseases such as Rheumatoid Arthritis (RA) and Osteoarthritis. The Janus kinase (JAK) pathway has been implicated in both inflammation and chronic pain. Clinical data suggests that baricitinib, a selective JAK 1/2 inhibitor, can robustly and rapidly alleviate pain in RA.1Utilizing a rodent forced-ambulation model, we have previously shown attenuation of gait deficits with analgesics such as opioids, nonsteroidal anti-inflammatory drugs (NSAIDs), and an anti-NGF (Nerve Growth Factor) antibody.2 However, in this same model a fusion protein blocker of TNFα (tumor necrosis factor alpha) signaling failed to demonstrate efficacy though inflammation was decreased. The present work investigated the potential of baricitinib, recently approved for treatment of RA, to reduce inflammagen-induced joint pain and gait impairment in this model of joint inflammation mediated pain. Objectives To determine if JAK 1/2 pathway inhibition is effective in treating inflammagen-induced joint pain and gait impairment. Methods Unilateral joint injury was induced in female Sprague Dawley rats (Harlan, Indianapolis, IN, USA) by unilateral intra-articular injection of 20 μg Complete Freund’s Adjuvant (CFA). Using the GaitScan (CleverSys Inc., Reston, VA) treadmill system, a composite gait score comprising of range of motion, normalized stance distance, stance/swing ratio, and paw print size was evaluated over 3 days post-injection.2 Rats were treated with vehicle, positive control (40 mg/kg Tramadol), or clinically relevant (based on plasma levels) increasing doses of baricitinib (1, 3, or 10 mg/kg p.o., 2-hrs prior to each test, q.d.). Dorsal root ganglion (DRG) were harvested post-gait evaluation and Total STAT3 (Cell Signaling, #4904) and phospho-STAT3 (Y705) (Cell Signaling, #9131) protein levels were examined via immunoblotting. The p-values were derived from repeated measures ANOVAs. Results In rat DRG homogenates, baricitinib significantly decreased phospho-STAT3 (Y705) protein levels in a dose-dependent manner (p Conclusion These data indicate that treatment with baricitinib attenuates CFA-induced joint deficits, a surrogate measure of joint pain. This effect correlated with the pharmacodynamic inhibition of JAK-STAT signaling in DRGs. These data support a role for JAK-STAT signaling in pain signaling and provide an opportunity to investigate the potential mechanism of action of baricitinib in joint pain. References [1] Keystone. Ann Rheum Dis. 2017;76(11):1853-1861. [2] Adams. Osteoarthritis Cartilage. 2016;24(11):1928-1939. Disclosure of Interests Kelly Knopp Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Akihiko Kato Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Theron Wall Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Jeff S Mcdermott Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Eric S Nisenbaum Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Benjamin Adams Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Michael Johnson Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company

Nonselective glutamate

A novel set of azabicyclic aryl amides have been identified as potent and selective agonists of the alpha7 nAChR. A two-pronged approach was taken to improve the potential hERG liability of previously disclosed alpha7 nAChR agonist, PNU-282,987, while maintaining the compound's other desirable pharmacological properties. The first approach involved further exploration of the aryl carboxylic acid fragment of PNU-282,987, while the second approach focused on modification of the azabicyclic amine portion of PNU-282,987. The best compounds from each series are characterized by rapid brain penetration, good oral bioavailability in rat, and demonstrate in vivo efficacy in a rat P50 auditory sensory gating assay. At least one analog from each series (1h, 1o, 2a, 9a, and 18a) shows an improved hERG safety profile over PNU-282,987.

N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]furo[2,3-c]pyridine-5-carboxamide (14, PHA-543,613), a novel agonist of the alpha7 neuronal nicotinic acetylcholine receptor (alpha7 nAChR), has been identified as a potential treatment of cognitive deficits in schizophrenia. Compound 14 is a potent and selective alpha7 nAChR agonist with an excellent in vitro profile. The compound is characterized by rapid brain penetration and high oral bioavailability in rat and demonstrates in vivo efficacy in auditory sensory gating and, in an in vivo model to assess cognitive performance, novel object recognition.

A library of benzamides was tested for alpha7 nicotinic acetylcholine receptor (nAChR) agonist activity using a chimeric receptor in a functional, cell-based, high-throughput assay. From this library, quinuclidine benzamides were found to have alpha7 nAChR agonist activity. The SAR diverged from the activity of this compound class verses the 5-HT(3) receptor, a structural homologue of the alpha7 nAChR. PNU-282987, the most potent compound from this series, was also shown to open native alpha7 nAChRs in cultured rat neurons and to reverse an amphetamine-induced gating deficit in rats.

Schizophrenic patients are thought to have an impaired ability to process sensory information. This deficit leads to disrupted auditory gating measured electrophysiologically as a reduced suppression of the second of paired auditoryevoked responses (P50) and is proposed to be associated with decreased function and/or expression of the homomeric alpha7 nicotinic acetylcholine receptor (nAChR). Here, we provide evidence that N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987), a novel selective agonist of the alpha7 nAChR, evoked whole-cell currents from cultured rat hippocampal neurons that were sensitive to the selective alpha7 nAChR antagonist methyllycaconitine (MLA) and enhanced GABAergic synaptic activity when applied to hippocampal slices. Amphetamine-induced sensory gating deficit, determined by auditory-evoked potentials in hippocampal CA3 region, was restored by systemic administration of PNU-282987 in chloral hydrate-anesthetized rats. Auditory gating of rat reticular thalamic neurons was also disrupted by amphetamine; however, PNU-282987 normalized gating deficit only in a subset of tested neurons (6 of 11). Furthermore, PNU-282987 improved the inherent hippocampal gating deficit occurring in a subpopulation of anesthetized rats, and enhanced amphetamine-induced hippocampal oscillation. We propose that the alpha7 nAChR agonist PNU-282987, via modulating/enhancing hippocampal GABAergic neurotransmission, improves auditory gating and enhances hippocampal oscillatory activity. These results provide further support for the concept that drugs that selectively activate alpha7 nAChRs may offer a novel, potential pharmacotherapy in treatment of schizophrenia.

Objective: A blocking monoclonal antibody to intercellular adhesion molecule-1 (ICAM-1), CL18/6, previously has been demonstrated to inhibit neutrophil attachment to isolated vascular endothelium and cardiomyocytes. Due to the well known participation of ICAM-1 in the inflammatory responses associated with myocardial ischemia/reperfusion injury, we investigated if CL18/6 could attenuate myocardial ischemia/reperfusion injury in vivo. Methods: Saline (3–5 ml, i.v., n = 6), non-blocking control MAb CL18/1D8 or CL18/6 (both 0.5 mg kg−1, i.v., n = 4) were administered prior to coronary occlusion (1 h) and subsequent reperfusion (5 h) produced by inflation of a coronary balloon angioplasty catheter in isoflurane-anesthetized, closed-chest dogs. Heart rate and arterial pressure were measured, and regional myocardial blood flow (rMBF), and myeloperoxidase activity (MPO) to index local neutrophil sequestration, were determined. Myocardial infarct size (IS) was evaluated using the tetrazolium staining technique and expressed as a percent of area at risk (AR). Results: Changes in heart rate and arterial pressure were insignificant throughout the experiment. rMBF (mean ± s.e.m.) in the ischemic subendocardium for each treatment group was: Saline (0.07 ± 0.02 ml min−1 g−1); CL18/1D8 (0.04 ± 0.02); CL18/6 (0.06 ± 0.02). IS/AR% was: saline (37 ± 3%); CL18/1D8 (39 ± 9%); CL18/6 (15 ± 4% *); * = significantly different from CL18/1D8 and saline, P < 0.05. MPO assayed from AR immediately adjacent to the infarct was significantly reduced below infarct MPO only in the CL18/6 treated group − 36%). Conclusions: The results indicate that CL18/6 antagonism of ICAM-1 provided cardioprotection associated with reduced neutrophil activity in vulnerable myocardium, and suggest that ICAM-1 mediated neutrophil sequestration in endangered cardiac tissue is an important mechanism of myocardial ischemia/reperfusion injury.

We wished to determine, using a novel specific antagonist of BK2, HOE 140, (a) if the angiotensin-converting enzyme (ACE) inhibitor, ramiprilat, reduces myocardial infarct size in a well-established animal model of ischemia/reperfusion with minimal coronary collateralization, and (b) if the reduction in myocardial infarct size occurred through a bradykinin-dependent mechanism Saline vehicle, ramiprilat, HOE 140, or ramiprilat plus HOE 140 (n = 6 each group), was administered intravenously (i.v.) in intact animal preparations of experimentally induced acute myocardial ischemia. Anesthetized, open-chest rabbits were instrumented for measurement of systemic hemodynamics and left ventricular pressure (LVP), from which LV + dP/dtmax was derived. Animals were subjected to 30-min left main coronary artery occlusion (marginal branch) followed by 2-h reperfusion. Ramiprilat (50 micrograms/kg) or saline was administered before reperfusion, and rabbits receiving HOE 140 were pretreated before occlusion (1 microgram/kg). In separate duration of action experiments (n = 6 each group), the above doses of ramiprilat or HOE 140 had significant vascular antagonism of sufficient duration against serial challenge with angiotensin I (AI) or bradykinin, respectively. After reperfusion, myocardial infarct size (IS) was determined by tetrazolium staining and expressed as a percentage of area at risk (AR). IS/AR% was significantly reduced in rabbits that received ramiprilat (20 +/- 6%, p < 0.05) as compared with those that received saline (41 +/- 6%), ramiprilat plus HOE 140 (47 +/- 2%), or HOE 140 alone (43 +/- 4%, mean +/- SEM). AR as a percentage of total LV mass was not different between any of the four treatment groups. Tachycardia was observed during early reperfusion in each group treated with ramiprilat.(ABSTRACT TRUNCATED AT 250 WORDS)

The angiotensin-converting enzyme inhibitor ramiprilat, the angiotensin II receptor antagonist losartan, angiotensin II, ramiprilat plus angiotensin II, or saline (N = 6 each group), were administered i.v. in anesthetized, open-chest rabbit preparations of acute myocardial ischemia. Animals were instrumented for measurement of systemic hemodynamics and left ventricular +dP/dtmax, then subjected to 30 min of left anterior descending coronary artery occlusion (marginal branch) followed by 2 h of reperfusion. Ramiprilat (50 μg/kg), losartan (10 mg/kg), or saline were administered prior to reperfusion, and angiotensin II (2.5 ng/kg per min) was infused 15 min prior to occlusion and throughout the remainder of the experiment. Losartan was supplemented (10 mg/kg) after 1 h of reperfusion. These non-hypotensive doses of ramiprilat and losartan were demonstrated to significantly antagonize the systemic pressor effects of i.v. challenge with angiotensin I (15% of control, maximum) and II (5% of control, maximum), respectively, for the duration of the experiment. Systemic hemodynamic and +dP/dtmax changes due to occlusion/reperfusion or drug administration were similar between treatment groups. Infarct size was measured post-experimentally using tetrazolium staining and is reported as a percent of area at risk. Infarcj size/area at risk (%) was significantly lower in rabbits administered ramiprilat only (20 ± 6%∗) or ramiprilat plus angiotensin II (26 ± 5%∗), compared to those receiving saline (41 ± 6%), angiotensin II (51 ± 4%), or losartan (52 ± 4%, mean ± S.E.M., P < 0.05). These data indicate that direct angiotensin II receptor stimulation or receptor antagonism does not alter the degree of myocardial necrosis. The results suggest that ramiprilat protects ischemic/reperfused myocardium independent of angiotensin II synthesis inhibition.

Several lines of evidence suggest a link between the alpha7 neuronal nicotinic acetylcholine receptor (nAChR) and brain disorders including schizophrenia, Alzheimer's disease, and traumatic brain injury. The present work describes a novel molecule, 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596), which acts as a powerful positive allosteric modulator of the alpha7 nAChR. Discovered in a high-throughput screen, PNU-120596 increased agonist-evoked calcium flux mediated by an engineered variant of the human alpha7 nAChR. Electrophysiology studies confirmed that PNU-120596 increased peak agonist-evoked currents mediated by wild-type receptors and also demonstrated a pronounced prolongation of the evoked response in the continued presence of agonist. In contrast, PNU-120596 produced no detectable change in currents mediated by alpha4beta2, alpha3beta4, and alpha9alpha10 nAChRs. PNU-120596 increased the channel mean open time of alpha7 nAChRs but had no effect on ion selectivity and relatively little, if any, effect on unitary conductance. When applied to acute hippocampal slices, PNU-120596 increased the frequency of ACh-evoked GABAergic postsynaptic currents measured in pyramidal neurons; this effect was suppressed by TTX, suggesting that PNU-120596 modulated the function of alpha7 nAChRs located on the somatodendritic membrane of hippocampal interneurons. Accordingly, PNU-120596 greatly enhanced the ACh-evoked inward currents in these interneurons. Systemic administration of PNU-120596 to rats improved the auditory gating deficit caused by amphetamine, a model proposed to reflect a circuit level disturbance associated with schizophrenia. Together, these results suggest that PNU-120596 represents a new class of molecule that enhances alpha7 nAChR function and thus has the potential to treat psychiatric and neurological disorders.