Antidepressant-like effect of CP-101,606: Evidence of mTOR pathway activation.

BACKGROUND: As a non-competitive N-methyl d-aspartate receptor antagonist, ketamine exerts rapid-onset and long-lasting antidepressant effects on depression, but some side effects limit its use. To identify a safer compound that may provide similar antidepressant effects, here we investigated whether CP-101,606, a selective NR2B receptor inhibitor, provides similar antidepressant effects and explored its underlying mechanisms. METHODS: To mimic depressive-like behavior, mice were subjected to chronic unpredictable mild stress (CUMS) for 21 days. Mice were treated with CP-101,606 at 10, 20, and 40 mg/kg doses for 7, 14, and 21 days, respectively, followed by a sucrose preference test (SPT), tail suspension test (TST), and forced swimming test (FST). Western blot analysis was performed on several targets (mTOR, p-mTOR, p70S6K, p-p70S6K, PSD-95, and GluA1), along with immunohistochemistry (GluA1) and immunofluorescence (p-mTOR) assays, using hippocampal tissue. RESULTS: CP-101,606 at 20 and 40 mg/kg doses for 7 and 14 days and fluoxetine 10 mg/kg and CP-101606 20 mg/kg for 21 days ameliorated depression-like behaviors in the SPT, TST, and FST. The effects of CP-101,606 were associated with a reversal of the CUMS-induced decrease in mTOR (Ser2448) and p70S6K (Thr389) phosphorylation and increasing PSD95 and GluA1 synthesis in the hippocampus. CONCLUSIONS: Our results demonstrate that CP-101,606 produces antidepressant effects in CUMS mice, which may be mediated by mTOR signaling cascade upregulation. Our findings suggest the possible utility of CP-101,606 as a treatment for depression.

Molecular Mechanisms of the Regulation of Liver Cytochrome P450 by Brain NMDA Receptors and via the Neuroendocrine Pathway-A Significance for New Psychotropic Therapies.

Recent investigations have highlighted the potential utility of the selective antagonist of the NMDA receptor GluN2B subunit for addressing major depressive disorders. Our previous study showed that the systemic administration of the antagonist of the GluN2B subunit of the NMDA receptor, the compound CP-101,606, affected liver cytochrome P450 expression and activity. To discern between the central and peripheral mechanisms of enzyme regulation, our current study aimed to explore whether the intracerebral administration of CP-101,606 could impact cytochrome P450. The injection of CP-101,606 to brain lateral ventricles (6, 15, or 30 µg/brain) exerted dose-dependent effects on liver cytochrome P450 enzymes and hypothalamic or pituitary hormones. The lowest dose led to an increase in the activity, protein, and mRNA level of CYP2C11 compared to the control. The activities of CYP2A, CYP2B, CYP2C11, CYP2C6, CYP2D, and protein levels of CYP2B, CYP2C11 were enhanced compared to the highest dose. Moreover, CP-101,606 increased the CYP1A protein level coupled with elevated CYP1A1 and CYP1A2 mRNA levels, but not activity. The antagonist decreased the pituitary somatostatin level and increased the serum growth hormone concentration after the lowest dose, while independently decreasing the serum corticosterone concentration of the dose. The findings presented here unveil a novel physiological regulatory mechanism whereby the brain glutamatergic system, via the NMDA receptor, influences liver cytochrome P450. This regulatory process appears to involve the endocrine system. These results may have practical applications in predicting alterations in cytochrome P450 activity and endogenous metabolism, and potential metabolic drug-drug interactions elicited by drugs that cross the blood-brain barrier and affect NMDA receptors.

The Effect of the Selective N-methyl-D-aspartate (NMDA) Receptor GluN2B Subunit Antagonist CP-101,606 on Cytochrome P450 2D (CYP2D) Expression and Activity in the Rat Liver and Brain.

The CYP2D enzymes of the cytochrome P450 superfamily play an important role in psychopharmacology, since they are engaged in the metabolism of psychotropic drugs and endogenous neuroactive substrates, which mediate brain neurotransmission and the therapeutic action of those drugs. The aim of this work was to study the effect of short- and long-term treatment with the selective antagonist of the GluN2B subunit of the NMDA receptor, the compound CP-101,606, which possesses antidepressant properties, on CYP2D expression and activity in the liver and brain of male rats. The presented work shows time-, organ- and brain-structure-dependent effects of 5-day and 3-week treatment with CP-101,606 on CYP2D. Five-day treatment with CP-101,606 increased the activity and protein level of CYP2D in the hippocampus. That effect was maintained after the 3-week treatment and was accompanied by enhancement in the CYP2D activity/protein level in the cortex and cerebellum. In contrast, a 3-week treatment with CP-101,606 diminished the CYP2D activity/protein level in the hypothalamus and striatum. In the liver, CP-101,606 decreased CYP2D activity, but not the protein or mRNA level, after 5-day or 3-week treatment. When added in vitro to liver microsomes, CP-101,606 diminished the CYP2D activity during prolonged incubation. While in the brain, the observed decrease in the CYP2D activity after short- and long-term treatment with CP-101,606 seems to be a consequence of the drug effect on enzyme regulation. In the liver, the direct inhibitory effect of reactive metabolites formed from CP-101,606 on the CYP2D activity may be considered. Since CYP2Ds are engaged in the metabolism of endogenous neuroactive substances, it can be assumed that apart from antagonizing the NMDA receptor, CP-101,606 may modify its own pharmacological effect by affecting brain cytochrome P450. On the other hand, an inhibition of the activity of liver CYP2D may slow down the metabolism of co-administered substrates and lead to pharmacokinetic drug-drug interactions.

The Selective NMDA Receptor GluN2B Subunit Antagonist CP-101,606 with Antidepressant Properties Modulates Cytochrome P450 Expression in the Liver.

Recent research indicates that selective NMDA receptor GluN2B subunit antagonists may become useful for the treatment of major depressive disorders. We aimed to examine in parallel the effect of the selective NMDA receptor GluN2B subunit antagonist CP-101,606 on the pituitary/serum hormone levels and on the regulation of cytochrome P450 in rat liver. CP-101,606 (20 mg/kg ip. for 5 days) decreased the activity of CYP1A, CYP2A, CYP2B, CYP2C11 and CYP3A, but not that of CYP2C6. The alterations in enzymatic activity were accompanied by changes in the CYP protein and mRNA levels. In parallel, a decrease in the pituitary growth hormone-releasing hormone, and in serum growth hormone and corticosterone (but not T3 and T4) concentration was observed. After a 3-week administration period of CP-101,606 less changes were found. A decrease in the CYP3A enzyme activity and protein level was still maintained, though no change in the mRNA level was found. A slight decrease in the serum concentration of corticosterone was also maintained, while GH level returned to the control value. The obtained results imply engagement of the glutamatergic system in the neuroendocrine regulation of cytochrome P450 and potential involvement of drugs acting on NMDA receptors in metabolic drug-drug interactions.

Enhancement of the GluN2B subunit of glutamatergic NMDA receptors in rat brain areas after cocaine abstinence.

BACKGROUND: Cocaine use disorder is associated with compulsive drug-seeking and drug-taking, whereas relapse may be induced by several factors, including stress, drug-related places, people, and cues. Recent observations strongly support the involvement of the N-methyl-D-aspartate (NMDA) receptors in cocaine use disorders and abstinence, whereas withdrawal in different environments may affect the intensification of relapse. METHODS: The aim of this study was to examine the GluN2B subunit expression and its association with the postsynaptic density protein 95 (PSD95) in several brain structures in rats with a history of cocaine self-administration and housed either in an enriched environment or in an isolated condition. Furthermore, a selective antagonist of the GluN2B subunit-CP 101,606 (10 and 20 mg/kg) administered during exposure to cocaine or a drug-associated conditional stimulus (a cue) was used to evaluate seeking behavior in rats. RESULTS: In rats previously self-administering cocaine, we observed an increase in the GluN2B expression in the total homogenate from the dorsal hippocampus under both enriched environment and isolation. Cocaine abstinence under isolation conditions increased the GluN2B and GluN2B/PSD95 complex levels in the PSD fraction of the prelimbic cortex in rats previously self-administering cocaine. Administration of CP 101,606 attenuated cue-induced cocaine-seeking behavior only in isolation-housed rats. CONCLUSION: In summary, in this study we showed region-specific changes in both the expression of GluN2B subunit and NMDA receptor trafficking during cocaine abstinence under different housing conditions. Furthermore, we showed that the pharmacological blockade of the GluN2B subunit may be useful in attenuating cocaine-seeking behavior.

Cocaine abstinence modulates NMDA receptor subunit expression: An analysis of the GluN2B subunit in cocaine-seeking behavior.

Cocaine use disorder develops in part due to the strong associations formed between drugs and the stimuli associated with drug use. Recently, treatment strategies including manipulations of drug-associated memories have been investigated, and the possibility of interfering with N-methyl-d-aspartate (NMDA)-mediated neurotransmission may represent an important option. The aim of this study was to examine the significance of the NMDA receptor subunit GluN2B at the molecular level (the expression of the GluN2B subunit, the Grin2B gene and the association of GluN2B with postsynaptic density protein 95 (PSD95)) in the brain structures of rats with a history of cocaine self-administration after i) cocaine abstinence with extinction training or ii) cocaine abstinence without instrumental tasks, as well as at the pharmacological level (peripheral or intracranial administration of CP 101,606, a GluN2B subunit antagonist during the cocaine- or cue-induced reinstatement). The GluN2B subunit levels and the GluN2B/PSD95 complex levels were either increased in the ventral hippocampus (vHIP) with higher levels of Grin2B gene expression in the HIP or decreased in the dorsal striatum (dSTR) after cocaine abstinence with extinction training. Moreover, CP 101,606, a GluN2B subunit antagonist, administered peripherally, attenuated the reinstatement of active lever presses induced by a priming dose of cocaine or by drug-associated conditioned stimuli, while injection into the vHIP reduced the cocaine- or cue with the subthreshold dose of cocaine-induced reinstatement. In cocaine abstinence without instrumental tasks, an increase in the GluN2B subunit levels and the level of the GluN2B/PSD95 complex in the dSTR was observed in rats that had previously self-administered cocaine. In conclusion, cocaine abstinence with extinction training seems to be associated with the up-regulation of the hippocampal GluN2B subunits, which seems to control cocaine-seeking behavior.

Identification and Preclinical Evaluation of a Radiofluorinated Benzazepine Derivative for Imaging the GluN2B Subunit of the Ionotropic NMDA Receptor.

The previously reported carbon-11 labeled GluN2B PET radioligand 11C-Me-NB1 served as a starting point for derivatization and led to the successful development of a radiofluorinated analogue designated (R)-18F-OF-Me-NB1. Given the short physical half-life of 20.3 min for carbon-11, (R)-18F-OF-Me-NB1 with a physical half-life of 109.8 min would allow satellite distribution to nuclear medicine facilities without an on-site cyclotron. Methods: Two fluorinated Me-NB1 derivatives, OF-Me-NB1 and PF-Me-NB1, were synthesized. Upon chiral resolution, the respective enantiomers were radiolabeled with carbon-11 and assessed in a proof-of-concept study by applying in vitro autoradiography on rodent brain sections. Based on the autoradiograms, (R)-OF-Me-NB1 was selected for radiofluorination and preclinical evaluation by ex vivo autoradiography, PET imaging, biodistribution and metabolite studies in Wistar rats. To rule out off-target binding to the σ1 receptor, the brain uptake of (R)-18F-OF-Me-NB1 in wild-type mice was compared with σ1 receptor knock-out mice. Results: Autoradiographic assessment revealed that both enantiomers of 11C-PF-Me-NB1 distributed homogenously across all brain regions on rodent brain sections. In contrast, the two enantiomers of 11C-OF-Me-NB1 exhibited an entirely different behaviour. While (S)-11C-OF-Me-NB1 bound virtually to all brain regions with considerable σ1 receptor binding, (R)-11C-OF-Me-NB1 exhibited high selectivity and specificity for the GluN2B-rich rat forebrain. These findings were confirmed for the radiofluorinated analogue (R)-11C-OF-Me-NB1, which was obtained via copper-mediated radiofluorination in radiochemical yields of 13-25% and molar activities ranging from 61-168 GBq/µmol. PET imaging and biodistribution studies in Wistar rats indicated appropriate pharmacokinetic profile and high in vivo specific binding of (R)-18F-OF-Me-NB1 as revealed by blocking studies with GluN2B-antagonist CP101,606. Off-target binding to the σ1 receptor was excluded by PET imaging with σ1 receptor knock-out mice. Receptor occupancy experiments with CP101,606 revealed a D50-value of 8.3 µmol/kg (intravenous). Conclusion: (R)-18F-OF-Me-NB1 is a promising radiofluorinated probe that exhibits specificity and selectivity for the GluN2B-containing N-methyl-D-aspartate (NMDA) complex and enables in vivo target occupancy studies in rodents.

Dissociable effects of NR2A and NR2B NMDA receptor antagonism on cognitive flexibility but not pattern separation.

RATIONALE: N-methyl-D-aspartate (NMDA) receptors play crucial roles in learning and memory, but the role of each NMDA receptor subtype in a specific cognitive process is unclear. Non-selective blockers of NMDA receptor are used to model the cognitive impairment in schizophrenia and Alzheimer's disease. Counter-intuitively selective NR2A and 2B NMDA receptor antagonists are thought to have pro-cognitive properties. These seemingly contrasting findings might in part be the result of different compounds and behavioral measures used across studies. OBJECTIVE: We compared the effect of NVP-AAM077 (NR2A antagonist), CP 101-606 (NR2B antagonist), and MK-801 (non-selective antagonist) in a series of touch screen tasks that can be used to measure spatial cognition and cognitive flexibility. METHODS: NVP-AAM077, CP 101-606, and MK-801 were administered prior to testing, in adult male Lister-hooded rats trained in tasks of location discrimination, paired associate learning (PAL), and trial unique non-match to location (TUNL). RESULTS: Results showed that MK-801 impaired performance on all the tasks. In contrast, CP 101-606 only impaired reversal learning in location discrimination and had minimal effect on working memory in TUNL and caused a modest improvement in accuracy in PAL and acquisition of a spatial discrimination. NVP-AAM077 had little effect on performance across tasks, although these data allude to a potential enhancement of acquisition of a spatial location and impairments in spatial reversal learning in a separation-dependent manner. CONCLUSIONS: These data demonstrated that non-selective NMDA antagonism will disrupt numerous aspects of cognitive function. However, selective antagonism is capable of impairing or enhancing cognitive function in a task-dependent fashion.

NR2B Antagonist CP-101,606 Abolishes Pitch-Mediated Deviance Detection in Awake Rats.

Schizophrenia patients exhibit a decreased ability to detect change in their auditory environment as measured by auditory event-related potentials (ERP) such as mismatch negativity. This deficit has been linked to abnormal NMDA neurotransmission since, among other observations, non-selective channel blockers of NMDA reliably diminish automatic deviance detection in human subjects as well as in animal models. Recent molecular and functional evidence links NR2B receptor subtype to aberrant NMDA transmission in schizophrenia. However, it is unknown if NR2B receptors participate in pre-attentive deviance detection. We recorded ERP from the vertex of freely behaving rats in response to frequency mismatch protocols. We saw a robust increase in N1 response to deviants compared to standard as well as control stimuli indicating true deviance detection. Moreover, the increased negativity was highly sensitive to deviant probability. Next, we tested the effect of a non-selective NMDA channel blocker (ketamine, 30 mg/kg) and a highly selective NR2B antagonist, CP-101,606 (10 or 30 mg/kg) on deviance detection. Ketamine attenuated deviance mainly by increasing the amplitude of the standard ERP. Amplitude and/or latency of several ERP components were also markedly affected. In contrast, CP-101,606 robustly and dose-dependently inhibited the deviant's N1 amplitude, and as a consequence, completely abolished deviance detection. No other ERPs or components were affected. Thus, we report first evidence that NR2B receptors robustly participate in processes of automatic deviance detection in a rodent model. Lastly, our model demonstrates a path forward to test specific pharmacological hypotheses using translational endpoints relevant to aberrant sensory processing in schizophrenia.