Assessment of glutamatergic synaptic transmission and plasticity in brain slices: relevance to bioelectronic approaches.

BACKGROUND: Glutamatergic neurons represent the largest neuronal class in the brain and are responsible for the bulk of excitatory synaptic transmission and plasticity. Abnormalities in glutamatergic neurons are linked to several brain disorders and their modulation represents a potential opportunity for emerging bioelectronic medicine (BEM) approaches. Here, we have used a set of electrophysiological assays to identify the effect of the pyrimidine nucleoside uridine on glutamatergic systems in ex vivo brain slices. An improved understanding of glutamatergic synaptic transmission and plasticity, through this type of examination, is critical to the development of potential neuromodulation strategies. METHODS: Ex vivo hippocampal slices (400 µm thick) were prepared from mouse brain. We recorded field excitatory postsynaptic potentials (fEPSP) in the CA1's stratum radiatum by stimulation of the CA3 Schaeffer collateral/commissural axons. Uridine was applied at concentrations (3, 30, 300 µM) representing the physiological range present in brain tissue. Synaptic function was studied with input-output (I-O) functions, as well as paired-pulse facilitation (PPF). Synaptic plasticity was studied by applying tetanic stimulation to induce post-tetanic potentiation (PTP), short-term potentiation (STP) and long-term potentiation (LTP). Additionally, we determined whether uridine affected synaptic responses carried solely by n-methyl-d-aspartate receptors (NMDARs), particularly during the oxygen-glucose deprivation (OGD) paradigm. RESULTS: The presence of uridine altered glutamatergic synaptic transmission and plasticity. We found that uridine affected STP and LTP in a concentration-dependent manner. Low-dose uridine (3 µM) had no effect, but higher doses (30 and 300 µM) impaired STP and LTP. Moreover, uridine (300 µM) decreased NMDAR-mediated synaptic responses. Conversely, uridine (at all concentrations tested) had a negligible effect on PPF and basal synaptic transmission, which is mediated primarily by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). In addition, uridine (100 µM) exerted a protective effect when the hippocampal slices were challenged with OGD, a widely used model of cerebral ischemia. CONCLUSIONS: Using a wide set of electrophysiological assays, we identify that uridine interacts with glutamatergic neurons to alter NMDAR-mediated responses, impair synaptic STP and LTP in a dose-dependent manner, and has a protective effect against OGD insult. This work outlines a strategy to identify deficits in glutamatergic mechanisms for signaling and plasticity that may be critical for targeting these same systems with BEM device-based approaches. To improve the efficacy of potential neuromodulation approaches for treating brain dysfunction, we need to improve our understanding of glutamatergic systems in the brain, including the effects of modulators such as uridine.

Ocular hypotensive activity of BOL-303259-X, a nitric oxide donating prostaglandin F2α agonist, in preclinical models.

The aim of the study was to investigate the ocular hypotensive activity of a nitric oxide (NO)-donating latanoprost, BOL-303259-X, following topical administration. The effect of BOL-303259-X (also known as NCX 116 and PF-3187207) on intraocular pressure (IOP) was investigated in monkeys with laser-induced ocular hypertension, dogs with naturally-occurring glaucoma and rabbits with saline-induced ocular hypertension. Latanoprost was used as reference drug. NO, downstream effector cGMP, and latanoprost acid were determined in ocular tissues following BOL-303259-X administration as an index of prostaglandin and NO-mediated activities. In primates, a maximum decrease in IOP of 31% and 35% relative to baseline was achieved with BOL-303259-X at doses of 0.036% (9 µg) and 0.12% (36 µg), respectively. In comparison, latanoprost elicited a greater response than vehicle only at 0.1% (30 µg) with a peak effect of 26%. In glaucomatous dogs, IOP decreased from baseline by 44% and 10% following BOL-303259-X (0.036%) and vehicle, respectively. Latanoprost (0.030%) lowered IOP by 27% and vehicle by 9%. Intravitreal injection of hypertonic saline in rabbits increased IOP transiently. Latanoprost did not modulate this response, whereas BOL-303259-X (0.036%) significantly blunted the hypertensive phase. Following BOL-303259-X treatment, latanoprost acid was significantly elevated in rabbit and primate cornea, iris/ciliary body and aqueous humor as was cGMP in aqueous humor. BOL-303259-X lowered IOP more effectively than latanoprost presumably as a consequence of a contribution by NO in addition to its prostaglandin activity. The compound is now in clinical development for the treatment of glaucoma and ocular hypertension.

Uridine ameliorates the pathological phenotype in transgenic G93A-ALS mice.

There is strong evidence from studies in humans and animal models to suggest the involvement of energy metabolism defects in neurodegenerative diseases. Uridine, a pyrimidine nucleoside, has been suggested to be neuroprotective in neurological disorders by improving bioenergetic effects, increasing ATP levels and enhancing glycolytic energy production. We assessed whether uridine treatment extended survival and improved the behavioral and neuropathological phenotype observed in G93A-ALS mice. In vitro and in vivo pharmacokinetic analyses in mutant SOD models provided optimal dose and assurance that uridine entered the brain. A dose-ranging efficacy trial in G93A mice was performed using survival, body weight, open-field analysis, and neuropathology as outcome measures. Urinary levels of 8-hydroxy-2'-deoxyguanosine, identifying DNA oxidative damage, were measured and used as a pharmacodynamic biomarker. Uridine administration significantly extended survival in a dose-dependent manner in G93A mice, while improving the behavioral and neuropathological phenotype. Uridine increased survival by 17.4%, ameliorated body weight loss, enhanced motor performance, reduced gross lumbar and ventral horn atrophy, attenuated lumbar ventral horn neuronal cell death, and decreased reactive astrogliosis. Consistent with a therapeutic effect, uridine significantly reduced urinary 8-hydroxy-2'-deoxyguanosine in G93A mice. These data suggest that uridine may be a therapeutic candidate in ALS patients.

A novel nitric oxide releasing prostaglandin analog, NCX 125, reduces intraocular pressure in rabbit, dog, and primate models of glaucoma.

PURPOSE: Nitric oxide (NO) is involved in a variety of physiological processes including ocular aqueous humor dynamics by targeting mechanisms that are complementary to those of prostaglandins. Here, we have characterized a newly synthesized compound, NCX 125, comprising latanoprost acid and NO-donating moieties. METHODS: NCX 125 was synthesized and tested in vitro for its ability to release functionally active NO and then compared with core latanoprost for its intraocular pressure (IOP)-lowering effects in rabbit, dog, and nonhuman primate models of glaucoma. RESULTS: NCX 125 elicited cGMP formation (EC(50) = 3.8 + or - 1.0 microM) in PC12 cells and exerted NO-dependent iNOS inhibition (IC(50) = 55 + or - 11 microM) in RAW 264.7 macrophages. NCX 125 lowered IOP to a greater extent compared with equimolar latanoprost in: (a) rabbit model of transient ocular hypertension (0.030% latanoprost, not effective; 0.039% NCX 125, Delta(max) = -10.6 + or - 2.3 mm Hg), (b) ocular hypertensive glaucomatous dogs (0.030% latanoprost, Delta(max)= -6.7 + or - 1.2 mm Hg; 0.039% NCX 125, Delta(max) = -9.1 + or - 3.1 mm Hg), and (c) laser-induced ocular hypertensive non-human primates (0.10% latanoprost, Delta(max) = -11.9 + or - 3.7 mm Hg, 0.13% NCX 125, Delta(max) = -16.7 + or - 2.2 mm Hg). In pharmacokinetic studies, NCX 125 and latanoprost resulted in similar latanoprost-free acid exposure in anterior segment ocular tissues. CONCLUSIONS: NCX 125, a compound targeting 2 different mechanisms, is endowed with potent ocular hypotensive effects. This may lead to potential new perspectives in the treatment of patients at risk of glaucoma.

L-Carnosine: multifunctional dipeptide buffer for sustained-duration topical ophthalmic formulations.

OBJECTIVES: The use of l-carnosine as an excipient in topical ophthalmic formulations containing gellan gum, a carbohydrate polymer with in-situ gelling properties upon mixing with mammalian tear fluid, was developed as a novel platform to extend precorneal duration. Specific utilisation of l-carnosine as a buffer in gellan gum carrying vehicles was characterised. METHODS: Buffer capacity was evaluated using 7.5, 13.3, and 44.2 mm l-carnosine in a pH range of 5.5-7.5. Accelerated chemical stability was determined by HPLC at l-carnosine concentrations of 5-100 mm. Combinations of 7.5 mm l-carnosine with 0.06-0.6% (w/v) gellan gum were characterised rheologically. l-Carnosine-buffered solutions of gellan gum were tested for acute topical ocular tolerance in vivo in pigmented rabbits. A unique formulation combining timolol (which lowers intraocular pressure) in l-carnosine-buffered gellan gum was compared with Timoptic-XE in normotensive dogs. KEY FINDINGS: l-Carnosine exhibited optimal pharmaceutical characteristics for use as a buffer in chronically administered topical ocular formulations. Enhancement trends were observed in solution-to-gel transition of l-carnosine-buffered vehicles containing gellan gum vs comparators. Topical tolerability of l-carnosine-buffered gellan gum formulations and lowering of intraocular pressure were equivalent with timolol and Timoptic-XE. CONCLUSIONS: Functional synergy between excipients in gellan gum formulations buffered with l-carnosine has potential for topical ocular dosage forms with sustained precorneal residence.