A Glutamate N-Methyl-d-Aspartate (NMDA) Receptor Subunit 2B-Selective Inhibitor of NMDA Receptor Function with Enhanced Potency at Acidic pH and Oral Bioavailability for Clinical Use

Abstract

We describe a clinical candidate molecule from a new series of glutamate N-methyl-d-aspartate receptor subunit 2B-selective inhibitors that shows enhanced inhibition at extracellular acidic pH values relative to physiologic pH. This property should render these compounds more effective inhibitors of N-methyl-d-aspartate receptors at synapses responding to a high frequency of action potentials, since glutamate-containing vesicles are acidic within their lumen. In addition, acidification of penumbral regions around ischemic tissue should also enhance selective drug action for improved neuroprotection. The aryl piperazine we describe here shows strong neuroprotective actions with minimal side effects in preclinical studies. The clinical candidate molecule NP10679 has high oral bioavailability with good brain penetration and is suitable for both intravenous and oral dosing for therapeutic use in humans. SIGNIFICANCE STATEMENT: This study identifies a new series of glutamate N-methyl-d-aspartate (NMDA) receptor subunit 2B-selective negative allosteric modulators with properties appropriate for clinical advancement. The compounds are more potent at acidic pH, associated with ischemic tissue, and this property should increase the therapeutic safety of this class by improving efficacy in affected tissue while sparing NMDA receptor block in healthy brain.

Fig. 1.

Pharmacology of NP10679 at NMDA receptors in oocytes. (A) NP10679 concentration-response curves for inhibition of GluN2B NMDA receptors expressed in Xenopus oocytes at pH 6.9 (blue symbols) and pH 7.6 (black symbols). Each fitted line and associated symbols represent the result from one experiment (i.e., frog injection, five total). The symbols are means from 12–22 oocytes from each experiment. The NP10679 effect on GluN2A (circles), GluN2C (squares), and GluN2D (triangles) NMDA receptors is shown at the indicated concentrations; mean ± S.E.M., n = 4 for GluN2A, 2C, and 2D results. The inset shows a representative current trace for NP10679 inhibition of GluN2B receptors at 0.1 μM at the two indicated pH values. Max G/G is the receptor current obtained in 100 μM glutamate and 30 μM glycine. (B) The pH potency boost for NP10679 (blue bar, n = 5 experiments) is compared with the pH boost for known preclinical and clinical GluN2B-selective inhibitors (n = 3 experiments each). The pH Boost is the ratio of IC₅₀ at pH 7.6/IC₅₀ at pH 6.9.

Fig. 2.

Neuroprotection by NP10679 reduces infarct volume in the MCAo model of transient ischemia in mice. (A) NP10679 reduces infarct volumes measured 24 hours post-MCAo with an ED₅₀ of 1 mg/kg, i.p. NP10679 was administered 15 minutes prior to initiation of transient MCAo for 60 minutes. In all experiments, the drug doses were randomized and the investigators were blinded from surgical procedure through analysis of stained sections to determine infarct volume. The plot is pooled data across three independent experiments. Data are means ± S.E.M. for n = 9 (0.2 mg/kg), 13 (0.5 mg/kg), 21 (1 mg/kg), 12 (2 mg/kg), 12 (5 mg/kg), 24 (10 mg/kg), and 34 (Veh) mice. **P < 0.01 from vehicle (ANOVA, Dunnett’s). (B and C) Representative images of TTC-stained sections of vehicle (B) and drug-treated brain. (C). A representative image is shown for each dose, with each brain cut into four sections (top to bottom), and both sides of the section are shown (left and right images). The red portion of the image is TTC stain of undamaged tissue. Vehicle (Veh) is the formulation alone as described in Methods.

Fig. 3.

Pharmacokinetics of NP10679 in mouse. (A) Total plasma levels of NP10679 after a 10 mg/kg oral dose (black symbols) and a 3 mg/kg i.v. dose (open symbols) in mice are plotted. (B) Free plasma levels of NP10679 after a 2 mg/kg (open symbols) and a 5 mg/kg i.p. (black symbols) dose in mice are given. For (A) and (B), data are means ± S.E.M. (n = 3 per data point). The gray dashed line indicates the IC₅₀ of NP10679 for pH 6.9 inhibition of GluN2B receptors, the green dashed line is the functional IC₅₀ at H₁ histamine receptors, and the blue dashed line is the functional IC₅₀ at hERG potassium channels.

Fig. 4.

Side effects measured in Rotarod challenge and locomotor activity. (A) Rotarod. Mice were trained on the Rotarod on 2 consecutive days (days 1 and 2), with four trials per day and an intertrial interval of 25 minutes. On day 3, mice were randomized to groups and administered vehicle (open circle), ifenprodil (open triangle, 30 mg/kg), or NP10679 at 2 mg/kg (light gray circle), 5 mg/kg (medium gray circle), or 10 mg/kg (black circle). The mean ± S.E.M. (n = 8) latency to fall for each trial was calculated for each group. *P < 0.01 from vehicle, ANOVA, Dunnett’s on day 3 within trials was determined. (B) Locomotor activity. Mice were habituated for 1 hour in a closed locomotor activity box and then removed and administered either vehicle (Veh, n = 6), MK-801 (MK at 0.3 mg/kg, n = 4) or NP10679 (20 mg/kg, n = 6 mice) by intraperitoneal injection and then placed back in the boxes, and the horizontal locomotor activity was counted for 2 hours. *P < 0.01, from vehicle (ANOVA, Dunnett’s). Total number of light beam breaks during the sample time are reported on the abscissa, which is representative of horizontal movement. Vehicle (Veh) is the formulation alone as described in Methods.