Main Research Findings

1) Neboglamine was found to increase noradrenaline release in the hippocampus, indicating the compound may have the potential to treat memory disorders by mediating direct and indirect actions on noradrenergic transmission.

2) 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.

 

Selected Data

1) The study performed by Garofalo et al investigated Neboglamine (CR 2249), a novel glutamic acid derivative, for its potential memory-enhancing effects using various behavioral and biochemical paradigms in rats and mice. For the animal models, male Wistar rats weighing 175-225g were utilized for passive avoidance, active avoidance, and open-field tests, while a separate group of male Wistar rats weighing 225-250g, was used for microdialysis and plasma level determination. Male CD-1 mice weighing 30-40g were employed for step-down passive avoidance tests. All animals, sourced from Charles River, were housed under controlled conditions with free access to food and water, and underwent a 30-60 minute acclimatization period before experiments. The investigational compound, CR 2249, along with other drugs such as noradrenaline, 3,4-dihydroxyphenylglycol, scopolamine hydrochloride, tacrine, and [3H]noradrenaline, were carefully prepared. Specifically, CR 2249 was dissolved in a minimal quantity of 1 M NaOH, brought to volume with saline, and its pH was adjusted to approximately 7.0 with 0.1 M HCl [1].

The determination of CR 2249 levels in plasma and cerebrospinal fluid (CSF) involved anaesthetizing rats with light ether, collecting blood from the abdominal aorta, and CSF from the fourth ventricle via the occipital bone. Samples were processed and analyzed using high-performance liquid chromatography (HPLC) coupled with fluorimetric detection, following pre-column derivatization with o-phthaldialdehyde. The chromatographic separation utilized a C18 reversed-phase column with a specific three-solvent discontinuous gradient program and a flow rate of 0.9 mL/min, achieving a retention time for CR 2249 of 18.7 min.

For in-vitro receptor binding studies, experiments were conducted according to established protocols to assess CR 2249’s affinity for various neurotransmitter receptors. Release studies from hippocampal slices involved decapitation of animals, preparing 0.40 mm thick coronal slices, and labeling them with 0.01 µM [3H]noradrenaline in a specific incubation medium containing 6-nitroquipazine to prevent false labeling. Labeled slices were then transferred to parallel superfusion chambers and superfused with a modified medium lacking Mg2+ ions. Fractions were collected every 10 minutes over a 60-min equilibration period, and CR 2249 was added throughout the experiments starting 20 minutes after the equilibration. Radioactivity was measured using liquid scintillation counting [1].

Behavioral assessments included passive and active avoidance tests. The step-through type passive-avoidance in rats used a two-compartment light/dark apparatus with a guillotine door and stainless-steel rod floor for scrambled foot-shock. Acquisition training involved placing rats in the illuminated compartment and recording the latency to enter the dark compartment, with a 60s cutoff. Rats received an inescapable foot-shock upon entry, and retention tests were performed 72 hours later without shock, recording step-through latency with a 120s maximum. Different foot-shock intensities ranging from 0-0.12 mA, were explored, with 0.09 mA chosen for subsequent CR 2249 testing. CR 2249 was administered intraperitoneally 45 min before training, immediately after training, or 45 min before retention to evaluate its effects on acquisition, consolidation, and retrieval processes, respectively.

The step-down passive-type avoidance in mice utilized a similar apparatus with a wooden platform and a scrambled foot-shock grid. Training involved placing mice on the platform and delivering a 0.2 mA shock until they returned to the platform. Mice with specific step-down and escape latencies between 3-30s and 3-60s were selected for retention testing. Immediately after training, mice received an electroconvulsive shock measuring at 20 mA, 50 Hz for 0.4s via ear electrodes. The retention test, performed 24 hours later, measured step-down latency without shock, with a 120s cutoff. CR 2249 and tacrine were administered orally 60 min before training [1].

Step-through active-avoidance conditioning in rats was conducted in a shuttle box apparatus with two compartments and a gate, featuring stainless-steel bars for a 0.4 mA scrambled foot-shock as an unconditioned stimulus and an overhead lamp/tone as a conditioned stimulus. Forty rats were subjected to five daily 40-trial sessions with 24-hour intervals. Animals received daily intraperitoneal injections of saline or CR 2249, 45 min before each session. Conditioned avoidance, uncoordinated escape, and spontaneous inter-trial responses were automatically recorded.

Motor activity was assessed by placing individual animals in a 60x60x30 cm box, with movements tracked by an automated system. Animals, randomly assigned to treatment groups receiving either CR 2249 or saline, were administered drugs intraperitoneally 45 min before the 10-min test, and total distances covered were recorded.

Microdialysis experiments involved anaesthetizing rats and implanting concentric dialysis probes into the hippocampus with location coordinates as follows: AP: 5.80 mm; ML: 4.80 mm; DV: 6.80 mm relative to bregma. After a 16-hour recovery period, modified Ringer’s solution was continuously perfused through the probe at 5.0 µL/min via a fluid swivel. Dialysis samples were collected at 20-min intervals and analyzed for noradrenaline and 3,4-dihydroxyphenylglycol content using HPLC with electrochemical detection. The mobile phase for HPLC consisted of a 0.1 M acetate buffer, 0.1 mM EDTA, and 0.34 mM octyl sulphonic acid, filtered and degassed. In-vitro experiments were also performed to determine the recovery rates of noradrenaline and 3,4-dihydroxyphenylglycol through the dialysis probes [1].

Statistical analysis for passive avoidance latencies involved survival analysis with a log-rank test. Active avoidance data were analyzed as mean percentages using ANOVA split-plot procedures on transformed differences from baseline. Inter-trial response data were presented as mean ± s.e.m. Hippocampal noradrenaline and 3,4-dihydroxyphenylglycol levels were expressed as mean percentage of baseline and compared using Student’s t-test after reciprocal transformation for homogeneous variances. Pharmacokinetic parameters like AUC, Tmax, lag-time, and half-life were calculated using specialized software [1].

2) 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 [2].

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 [2].

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 [2].

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 [2].

Discussion

1) The results of the study conducted by researchers Garofalo et al consistently demonstrated that CR 2249 possesses memory-enhancing properties across various behavioral models, often without affecting general behavior or motor activity. Pharmacokinetic analysis revealed that CR 2249 is rapidly absorbed into plasma following both oral and intraperitoneal administration, with a lag-time of approximately 0.12 hours and 0.10 hours, respectively. The maximum plasma concentration (Tmax) was reached around 60 minutes for both routes. Importantly, CR 2249 rapidly crossed the CSF barrier, with lag-times of 0.1 and 0.23 hours for oral and intraperitoneal administration, respectively, and maximum CSF concentrations were observed between 45-60 minutes. The CSF/plasma AUC ratios were significant at 47% after intraperitoneal and 60% after oral administration, suggesting substantial brain penetration and supporting the chosen 45-60 minute pre-treatment interval for behavioral tests [1].

In-vitro receptor binding studies indicated a selective interaction of CR 2249 with the NMDA receptor complex. While CR 2249 showed only weak affinity for the recognition site of the NMDA receptor and no significant binding affinity to AMPA or kainic receptors, it exhibited moderate affinity for the strychnine-insensitive glycine receptor. Furthermore, CR 2249 significantly increased the binding of [3H]MK-801, a non-competitive NMDA receptor antagonist, in rat cerebral cortex membranes. These results suggest that CR 2249 acts as an agonist for either the strychnine-insensitive glycine site or the polyamine site within the NMDA receptor complex. Crucially, CR 2249 showed no significant binding affinity to other major neurotransmitter receptors, including α1 and α2 adrenergic, 5-HT1a and 5-HT2a-ergic, cholecystokinin (CCKB), or muscarinic (M1) receptors, reinforcing its selective interaction with the NMDA receptor complex [1].

Complementary release studies from hippocampal slices further supported the NMDA receptor interaction. CR 2249 produced a dose-dependent increase in [3H]noradrenaline release from rat hippocampal slices, with a maximum 120% increase at 1000 µM concentration and an EC50 of 104.5 ± 17.8 µM. This finding is consistent with NMDA receptor activation leading to enhanced neurotransmitter release in the hippocampus.

In behavioral tests, CR 2249 demonstrated significant memory-enhancing effects. In the step-through type passive-avoidance paradigm in rats, pre-training administration of CR 2249 at intraperitoneal doses of 10 and 30 mg/kg significantly increased retention latencies compared to saline-treated rats. However, CR 2249 did not show an effect when administered immediately after training or 45 minutes before the retention test, suggesting its primary role in acquisition rather than consolidation or retrieval. This observation aligns with the pharmacokinetic data, as CR 2249 concentrations might not have been optimal during the immediate post-training consolidation period [1].

CR 2249 also effectively ameliorated scopolamine-induced memory impairment in the rat passive avoidance model. Scopolamine significantly shortened retention latencies, indicating amnesia. 1 mg/kg and 10 mg/kg of CR 2249 was found to dose-dependently increase these latencies, with the 10 mg/kg dose showing a statistically significant reversal of the deficit. Tacrine, a known memory enhancer, also reversed scopolamine’s effect at 10 mg/kg.

In the step-down passive-avoidance model in mice, where electroconvulsive shock (ECS) was used to induce amnesia, CR 2249 again proved efficacious. ECS significantly reduced step-down latencies, reflecting memory impairment. Oral administration of CR 2249 (3, 10, and 30 mg/kg) dose-dependently increased step-down latencies, with significant effects observed at 10 and 30 mg/kg, indicating a reversal of the ECS-induced amnesia. Notably, tacrine at comparable oral doses of 1, 3, and 10 mg/kg did not show any effect in this specific model [1].

Furthermore, CR 2249 improved performance in the step-through active avoidance conditioning task in rats. Intraperitoneal administration of CR 2249 in the dose range of 1-10 mg/kg significantly enhanced the percentage of conditioned avoidance responses over 5 consecutive days, with statistically significant dose-effect and treatment-by-days interactions observed at 3 and 10 mg/kg. This improvement in learning and memory was specific, as CR 2249 did not affect uncoordinated escape responses or spontaneous inter-trial responses, suggesting a direct effect on cognitive function rather than general motor changes. Supporting this specificity, subsequent motor activity tests in rats showed that intraperitoneal CR 2249 at 10 and 30 mg/kg had no significant effect on the total distance covered in the open field, confirming that the observed cognitive benefits were not attributable to altered spontaneous motility [1].

 

Figure 1: Change in the effects of CR 2249 on conditioned avoidance responses, uncoordinated avoidance responses, and inter-trial responses

Finally, in microdialysis experiments with freely moving rats, local application of 100 µM CR 2249 to the hippocampus significantly increased noradrenaline release to approximately 225% of baseline levels after 90 minutes. Concurrently, CR 2249 reduced the efflux of 3,4-dihydroxyphenylglycol, a noradrenaline metabolite, to 69% of baseline. These results indicate that CR 2249 modulates noradrenergic transmission in the hippocampus, a mechanism that could underlie its memory-enhancing properties. Overall, the behavioral and biochemical data strongly suggest CR 2249 as a promising memory enhancer, potentially mediated through noradrenergic pathways and interactions with the NMDA receptor complex [1].

2) 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 [2].

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 [2].

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.

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 [2].

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.

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].

 

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).

 

Citations

[1] Garofalo P, Colombo S, Lanza M, Revel L, Makovec F. CR 2249: a new putative memory enhancer. Behavioural studies on learning and memory in rats and mice. J Pharm Pharmacol. 1996;48(12):1290-1297. doi:10.1111/j.2042-7158.1996.tb03938.x

[2] 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