Main Research Findings

1) Administration of Neboglamine was found to act as a potential treatment for schizophrenia by inhibition of hyperlocomotion and frequency of rearing behavior, indicating activation of the frontostriatal system.

2) There was found to be a positive cooperative interaction between Neboglamine and glycine, indicating that the nootropic acts as an allosteric binding site to increase NMDA receptor functioning.

 

Selected Data

1) This study conducted by researchers Chiusaroli et al used a combination of immunohistochemistry, ex vivo tissue punch neurotransmitter-release assays, and behavioural pharmacology in adult male Wistar rats to characterize the neuroanatomical and functional consequences of Neboglamine (CR2249), compare its activity with the endogenous NMDA glycine-site agonist d‑serine and with prototypical antipsychotics clozapine and haloperidol, and test its ability to counteract phencyclidine (PCP)-induced NMDA hypofunction and hyperlocomotion. All animals weighing 200–250 g were housed under controlled lighting, temperature and humidity with ad libitum food and water. For immunohistochemistry single subcutaneous 20 mg/kg doses of Neboglamine, 50 mg/kg of d‑serine, 20 mg/kg of clozapine, and 1 mg/kg of haloperidol, were administered and animals were euthanized 2 h later under deep pentobarbital anesthesia [1].

Transcardial perfusion was performed first with saline and then with formalin to ensure fixation; brains were post‑fixed overnight, cryoprotected in 40% sucrose, cryosectioned at 28 µm, mounted and processed for Fos-like immunoreactivity (FLI) using a rabbit anti‑c‑Fos antibody and a Vector Elite rabbit immunostaining kit. Immunostaining was performed in PBS with 5% normal goat serum, 0.5% BSA and 0.3% Triton X‑100; positive nuclei were counted by light microscopy at 100× across six sections per area/hemisphere with groups of 3–5 animals per treatment. Quantitative comparisons used one‑way ANOVA with Holm–Sidak post hoc testing. To examine effects on NMDA‑mediated neurotransmission and PCP antagonism, frontal cortex tissue punches were prepared from 400 µm coronal slices cut with a McIlwain tissue chopper and punches were incubated with 0.2 µM 3,4‑[ring‑2,5,6‑3H]‑dopamine in the presence of blockers to avoid false labelling (6‑nitroquipazine 0.1 µM to block serotonin uptake; desipramine 0.1 µM to block noradrenergic uptake) and antioxidants/pargyline to reduce oxidation and MAO breakdown. Individual punches were washed and superfused in parallel chambers with Mg2+‑free artificial cerebrospinal fluid (aCSF) including 125 NaCl, 3 KCl, 1.2 CaCl2, 1.2 MgSO4, 1 NaH2PO4, 22 NaHCO3, and 10 glucose at 0.6 ml/min [1].

After a 45‑min equilibration, seven 5‑min fractions were collected; 300 µM NMDA was applied during a single 5‑min stimulation fraction, while PCP and Neboglamine (added 25 min before stimulation) were tested for their ability to inhibit or restore NMDA‑evoked [3H]dopamine overflow. Fractional release (FR) was calculated as sample radioactivity divided by total punch radioactivity and drug effects were expressed as overflow, NMDA‑stimulated FR minus first‑fraction FR. Release data were analyzed by one‑way ANOVA with Mann–Whitney U for pairwise comparisons where appropriate. For behavioural testing of PCP‑induced hyperlocomotion and rearing, animals were handled and habituated to the test room prior to experiments; drug solutions were prepared by dissolving test compounds in saline with a few drops of 2% lactic acid to aid solubility and pH adjustment for subcutaneous administration. In the primary experiment, rats received subcutaneous pre‑treatments of either saline, Neboglamine 20 mg/kg, d‑serine 50 mg/kg, clozapine 20 mg/kg, or haloperidol 1 mg/kg, administer 15 min before placement in an open‑field arena for a 30‑min habituation run.

After habituation rats were dosed with 2.5 mg/kg PCP while locomotor activity and rearing were recorded automatically for an additional 30 min using an overhead camera and tracking software. A second experiment tested oral Neboglamine at 0.3, 3 and 30 mg/kg or a vehicle of bi‑distilled water, to evaluate clinically relevant administration and dose–response. Animals were randomized and sessions balanced across day, time and arena position to minimize systematic bias. Behavioural data were analyzed as longitudinal time‑series of six 5‑min intervals per 30‑min block, using two‑way ANOVA with Holm–Sidak post hoc comparisons [1].

All ex vivo and behavioural assays incorporated appropriate vehicle controls run in parallel, and the release assays included controls for basal versus NMDA‑stimulated overflow and concentration–response tests of PCP. Sample sizes per assay and the inclusion of established comparator drugs clozapine, haloperidol, and d‑serine allowed both mechanistic inference about glycine‑site modulation and direct comparison with known antipsychotic pharmacological fingerprints [1].

2) This study completed by the research team of Lanze et al utilized male Wistar and Sprague-Dawley rats to examine the pharmacological actions of a novel glutamic acid derivative, Zeboglamine (CR 2249), focusing on its enantiomers (S)-CR 2249 and (R)-CR 2249. Animals were maintained under standard housing conditions with free access to food and water. Two complementary experimental approaches were employed: in vitro receptor binding assays using rat brain membranes and functional neurotransmitter release assays using rat hippocampal slices. For binding studies, separate preparations were made from cerebral cortex (for [3H]MK-801 assays) and cortex/hippocampus (for [3H]-5,7-dichlorokynurenic acid ([3H]-5,7-DCKA) assays) [2].

Cortices were homogenized in 0.32 M sucrose and subjected to sequential centrifugations (1000 g, 18 000 g, 50 00 g/40 000 g) with repeated wash steps before final resuspension in HEPES-KOH buffer at a concentration of 20 mM and storage at −80°C until use. Membrane aliquots were used in duplicate incubations with radioligand and test compounds; protein per assay was optimized at ~300 µg for MK-801 assays and ~25–75 µg for [3H]-5,7-DCKA, and non-specific binding was determined with excess MK-801 or D-serine. For [3H]MK-801 non-equilibrium binding, membranes were incubated with 2 nM [3H]MK-801 and 10 concentrations of test agonists, glutamate, glycine, and spermine, to define concentration–response relationships; the effects of (S)- and (R)-CR 2249 were examined at three concentrations, 10, 100, 1000 µM on modulation of these agonist concentration–response curves. Saturation and displacement protocols were used for [3H]-5,7-DCKA binding with a radioligand range of 3–300 nM, with triplicate determinations and D-serine to define non-specific binding; (S)-CR 2249 and D-serine were applied over broad concentration ranges to estimate IC50 and percent inhibition [2].

For functional release studies, coronal hippocampal slices measuring 0.40 mm thick were prepared using a McIlwain tissue chopper, labeled with 0.01 µM [3H]noradrenaline for 20 min at 37°C. They were then transferred to superfusion chambers for perfusion at 1 ml/min with Mg2+-free medium, with 0.1 µM 6-nitroquipazine included to block serotonin uptake and prevent false labeling of serotonergic terminals. After 48 min equilibration, nine 5-min superfusate fractions were collected; the first two served as basal release baseline. Drugs were applied during a single 5-min stimulation fraction (NMDA ± glycine ± CR 2249), with kynurenic acid and strychnine present where specified with kynurenate being added throughout the experiment starting 10 min before NMDA. Fractional release (FR) was calculated as the radioactivity in each superfusate divided by total slice radioactivity at that time; drug activity was expressed as percentage ratio of NMDA-stimulated FR to the first fraction’s FR. Experiments typically used 100 µM NMDA to evoke release. The design included testing (S)- and (R)-CR 2249 alone (up to 1000 µM) and their ability to modulate glycine-mediated reversal of kynurenate inhibition of NMDA-evoked [3H]noradrenaline release.

Statistical analyses employed curve-fitting to obtain EC50/IC50 values and maximal responses, with group comparisons by one-way ANOVA and post hoc Duncan tests where appropriate; polynomial analyses were used to break down variance in release experiments and to test linear trends. Data are reported as means ± SEM and significance was set at P ≤ 0.05. Reagents and radioligands were purchased from standard suppliers; CR 2249 enantiomers were synthesized in-house and prepared in minimal 0.1 N NaOH then adjusted to pH 7.0–7.4 with saline. Multiple controls were integrated: non-specific binding controls, vehicle controls, and both enantiomer comparisons to address stereoselectivity. Overall, the materials and methods combined rigorous membrane binding pharmacology with a physiologically relevant hippocampal slice release paradigm to probe both molecular and functional interactions of CR 2249 at NMDA receptor-associated sites [2].

 

Discussion

1) The study performed by Chiusaroli et al on Neboglamine, a functional modulator of the N-methyl-D-aspartate (NMDA) receptor’s glycine site, yielded several significant results concerning its potential antipsychotic-like effects, evaluated through immunohistochemical and behavioral paradigms in rats. The findings were systematically categorized into patterns of Fos-like immunoreactivity (FLI), effects on phencyclidine (PCP)-induced inhibition of NMDA-mediated transmission, and impacts on PCP-induced hyperlocomotion and rearing behavior [1].

First, the investigation into FLI patterns, a marker of neuronal activation, demonstrated distinct regional responses to Neboglamine and other antipsychotics. Both haloperidol (a first-generation antipsychotic, FGA) and clozapine (a second-generation antipsychotic, SGA) significantly increased FLI in the prefrontal cortex (PFCX), nucleus accumbens (NAc), and lateral septal nucleus (LSN). However, a crucial differentiation emerged in the dorsolateral striatum (DL-STR): haloperidol induced a profound increase in FLI in this region, while clozapine had minimal to no effect. Neboglamine’s effect on FLI across these brain regions closely mirrored that of clozapine, significantly increasing FLI in the PFCX, NAc, and LSN without affecting the DL-STR. Specifically, Neboglamine led to 3.2-, 4.8-, and 4.5-fold increases in FLI over control in PFCX, NAc, and LSN, respectively, with only a 1.3-fold increase in DL-STR.

This pattern is clinically important as high DL-STR activation is often associated with extrapyramidal symptoms, a common side effect of FGAs like haloperidol. D-serine, an endogenous agonist at the NMDA glycine site, produced a qualitatively similar FLI pattern to Neboglamine in the PFCX, NAc, and LSN, though the magnitude of increase was generally lower with 2.0-, 2.1-, and 3.0-fold increases in PFCX, NAc, and LSN, respectively. This suggests a shared mechanism of action or similar neurobiological footprint. Furthermore, Neboglamine increased overall neuronal activity without altering the balance of FLI across regions, unlike haloperidol and clozapine, which significantly changed the relative activity across different brain areas. Clozapine shifted the area with the highest FLI from the PFCX (as seen in saline-treated rats) to the LSN, while haloperidol induced an extreme response in the DL-STR, drastically altering the balance of neuronal activity [1].

Secondly, the study examined neboglamine’s impact on PCP-induced inhibition of NMDA-mediated transmission in rat frontal cortex punches. PCP is known to inhibit NMDA-stimulated [3H]dopamine release. Under experimental conditions where 300 μM NMDA evoked [3H]dopamine overflow, PCP at 100 nM significantly inhibited this overflow by 51.6 ± 4.2%. Neboglamine, when present in concentrations ranging from 0.3 to 30 μM, concentration-dependently prevented this inhibitory effect of PCP. At 30 μM, Neboglamine fully restored the efficiency of NMDA-mediated stimulation, effectively counteracting PCP’s disruptive action. Notably, in the absence of PCP, Neboglamine itself did not influence either the basal release of [3H]dopamine or the NMDA-evoked [3H]dopamine release, suggesting its action is specific to conditions of NMDA receptor hypofunction, such as those induced by PCP [1].

Lastly, the effects of Neboglamine on PCP-induced hyperlocomotion and rearing behavior were assessed. In the habituation session, before PCP administration, 20 mg/kg of Neboglamine and 50 mg/kg of D-serine did not significantly affect spontaneous locomotor activity. In contrast, both 1 mg/kg of haloperidol and 20 mg/kg of clozapine significantly reduced spontaneous activity, indicating a general motor impairment. Upon PCP administration at a dose of 2.5 mg/kg, which typically induces hyperlocomotion, all tested compounds significantly inhibited this PCP-induced hyperactivity.

Neboglamine and D-serine significantly inhibited PCP-induced hyperlocomotion without affecting basal activity, aligning with a more selective antipsychotic profile. Haloperidol and clozapine, as expected, completely antagonized the hyperlocomotion. A subsequent experiment with oral Neboglamine further confirmed its efficacy. Oral Neboglamine dose-dependently reduced both PCP-induced hyperlocomotion and the increased frequency of rearing behavior. Importantly, oral Neboglamine did not affect basal levels of locomotor or rearing activity, similar to the subcutaneous administration. The study found that Neboglamine inhibited stereotyped behavior (rearing) at a lower dose of 0.3 mg/kg than the 3 mg/kg dose required to inhibit locomotor activity, suggesting a differential sensitivity of these behavioral components to Neboglamine [1].

In conclusion, the study provides compelling molecular and behavioral evidence that Neboglamine, an NMDA glycine site modulator, exhibits a desirable antipsychotic-like profile. Its ability to normalize neuronal activation patterns similar to clozapine, without excessively activating the DL-STR, and its efficacy in reversing PCP-induced NMDA transmission deficits and hyperlocomotion, all without causing general motor impairment, strongly support its clinical evaluation as a potential treatment for schizophrenia [2].

2) The results of the study performed by Lanza et al revealed stereoselective and site-selective effects of CR 2249: both enantiomers showed negligible direct affinity for the glutamate recognition site, AMPA, or kainate receptors ([3H]CGS 19175 IC50 >1000 µM), but (S)-CR 2249 produced concentration-dependent increases in [3H]MK-801 binding in cortical membranes while (R)-CR 2249 was inactive up to 3000 µM. Classical NMDA agonists including glutamate, glycine, and spermine, each increased [3H]MK-801 binding with expected potencies and efficacies; glycine and (S)-CR 2249 had similar maximal efficacies (~100% enhancement) but markedly different potencies [2].

Importantly, (S)-CR 2249 did not change glycine affinity for its site but significantly increased glycine’s maximal effect on [3H]MK-801 binding in a concentration-dependent manner, with post-hoc analyses showing significant increases at 100 and 1000 µM. By contrast, (S)-CR 2249 had minimal or no effect on glutamate-stimulated MK-801 binding, only at a non-significant augmentation at high concentration for low glutamate, and only weak, non-significant modulation of spermine responses. These patterns indicate (S)-CR 2249 acts allosterically to enhance glycine-mediated potentiation of NMDA channel blockade by MK-801, plausibly via a separate modulatory site that positively cooperates with glycine action.

Further characterization using [3H]-5,7-DCKA, a ligand for the strychnine-insensitive glycine site, showed that D-serine fully displaced radioligand in a concentration-dependent manner, whereas (S)-CR 2249 partially reduced [3H]-5,7-DCKA binding with an IC50 ≈ 12.0 µM and a maximal inhibition only ~62% of D-serine’s effect. Hill coefficients suggested non-competitive interaction (Hill ~0.36 vs 1.03 for D-serine), supporting that (S)-CR 2249 modulates the glycine site indirectly or at an allosteric locus rather than acting as a direct competitive agonist/antagonist. Tests for interactions with the ifenprodil site and a battery of other receptor subtypes such as adrenergic, serotonergic, GABAergic, CCK, and muscarinic, showed no significant binding affinity for CR 2249 enantiomers, arguing for a NMDA-complex–related, stereoselective action of (S)-CR 2249 [2].

 

Figure 1: Changes in the effects of (S)-CR 2249 on [3H]-5,7-DCKA binding when enhanced through the addition of A) glycine, B) glutamate, or C) Spermine.

Functional hippocampal slice release experiments provided complementary physiological evidence. Neither (S)- nor (R)-CR 2249 altered basal [3H]noradrenaline release up to 1000 µM, nor did either enantiomer directly potentiate NMDA-evoked release when applied alone. However, when the glycine co-agonist effect was unmasked by partial blockade with 300 µM kynurenic acid, glycine produced a concentration-dependent reversal of kynurenate inhibition of NMDA-evoked [3H]noradrenaline release. In this setting, (S)-CR 2249 at doses ranging from 3–30 µM, produced a significant, concentration-related enhancement of glycine’s maximal effect without altering glycine EC50 values. Notably, in slice experiments the combination of 30 µM (S)-CR 2249 with >100 µM glycine achieved complete reversal of 300 µM kynurenate inhibition, an effect glycine alone could not fully produce under these conditions. (R)-CR 2249 was completely ineffective in modulating glycine’s action at the concentrations tested, reinforcing stereoselectivity [2].

Taken together, the results indicate (S)-CR 2249 selectively and stereoselectively enhances NMDA receptor functionality by increasing the efficacy of glycine at strychnine-resistant glycine-associated sites coupled to the NMDA receptor complex, acting allosterically rather than by increasing glycine affinity. The compound has the ability to potentiate glycine-dependent NMDA responses in a physiologically relevant hippocampal model of noradrenaline release. This supports its candidacy as a putative cognition enhancer, consistent with prior behavioral findings, while (R)-CR 2249 lacks these modulatory effects [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citation

[1] Chiusaroli R, Garofalo P, Espinoza S, Neri E, Caselli G, Lanza M. Antipsychotic-like effects of the N-methyl-D-aspartate receptor modulator neboglamine: an immunohistochemical and behavioural study in the rat. Pharmacol Res. 2010;61(5):430-436. doi:10.1016/j.phrs.2009.12.010

[2] Lanza M, Bonnafous C, Colombo S, Revel L, Makovec F. Characterization of a novel putative cognition enhancer mediating facilitation of glycine effect on strychnine-resistant sites coupled to NMDA receptor complex. Neuropharmacology. 1997;36(8):1057-1064. doi:10.1016/s0028-3908(97)00092-0