NMDA Receptor

Ro 25-6981 is a selective and activity-dependent blocker of NMDA receptors containing the NR2B subunit. It is structurally related to ifenprodil and has no affinity for the known binding sites of non-competitive antagonists such as phencyclidine or MK-801 Fischer et al., 1997, Lynch et al., 2001, Mutel et al., 1998. In Xenopus oocytes transfected with cDNA mixtures coding for NR1C and NR2B subunits, Ro 25-6981 acts as a potent antagonist. In contrast, its potency to antagonize NMDA responses in oocytes transfected with NR2A subunits is almost four orders of magnitude lower (Fischer et al., 1997). [1]

Rats lesioned with 6-hydroxydopamine (6-OHDA) exhibit counterrotation when administered intraperitoneally with Ro 25-6981 (0.39-12.5 mg/kg); nevertheless, normal rats do not exhibit any stimulation of locomotion [1]. In early postnatal development in rats, Ro 25-6981 (1.3 mg/kg; i.p.) shows age- and activation-dependent anticonvulsant effects [2]. Intrathecal injection of Ro 25-6981 (800 µg) significantly lowers postoperative hyperalgesia and has a considerable analgesic impact on incision pain in rats [3].

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N-methyl-D-aspartate (NMDA) receptor antagonists have antiakinetic and antidyskinetic effects in animals models of Parkinson's disease (PD). However, non-selective inhibition of NMDA receptors throughout the central nervous system may result in undesired effects such as ataxia and psychosis. We therefore studied Ro 25-6981, an activity-dependent antagonist of NMDA receptors containing the NR2B subunit which are predominantly expressed in the striatum. Ro 25-6981 induced contraversive rotations in 6-hydroxydopamine (6-OHDA)-lesioned rats without stimulating locomotion in normal rats and reversed parkinsonian symptoms in 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP)-treated common marmosets. Due to the small number of marmosets, there were no significant differences between Ro 25-6981 and vehicle though there was a significant trend toward differences, as shown by the Page test. Furthermore, Ro 25-6981 potentiated the action of levodopa in both species and attenuated the maximal levodopa response in 6-OHDA-lesioned rats chronically treated with levodopa without reducing the overall response. Ro 25-6981 also potentiated the action of the dopamine receptor agonists apomorphine, A68930 and quinpirole in 6-OHDA-lesioned rats. The present observations suggest a therapeutic potential of NR2B-selective NMDA receptor antagonists in the management of PD. [1]

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Ro 25-6981 maleate is a highly selective and activity-dependent antagonist of NMDA ionotropic glutamate receptors containing NR2B subunit (NR2B/NMDARs). The aim of our study was to investigate the influence of Ro 25-6981 administration in developing rats on physiological (single and paired pulse cortical interhemispheric evoked potentials) and epileptic brain activity (cortical afterdischarges (ADs)). Electrophysiological experiments were performed in animals with epidurally implanted electrodes at postnatal days (P) P12, P18, and P25. The drug was injected intraperitoneally at a dose of 1 or 3mg/kg. Control animals were injected with saline (1ml/kg). Single interhemispheric responses were evoked with 0.5-ms biphasic pulses with intensities increasing from 0.4 to 5mA, paired-pulse responses were elicited by twofold threshold intensity. The ADs were elicited by series of 15-s of 1-ms pulses at 8-Hz frequency. Firstly, six stimulations with stable suprathreshold intensity repeated at 30-min intervals were used to determine the time course of Ro 25-6981 effects against ADs in P12 animals. Secondly, similar experiment was performed in all age groups of animals but with 20-min intervals as well as a further experiment using stimulations with stepwise intensities increasing at 10-min intervals from 0.2 to 15 mA. Pretreatment with the 3-mg/kg (but not the lower) dose of Ro 25-9681 decreased significantly the amplitude of single responses evoked with higher stimulation intensities in P12 and P18 animals. Both doses affected responses in P25 animals, only the 1-mg/kg dose was more efficacious than the 3-mg/kg one. Paired pulse responses were not affected by either dose of Ro 25-6981 in any age group. Ro 25-9681 clearly influenced the duration of ADs only in P12 animals. The 1-mg/kg dose did not change the duration of ADs whereas the 3-mg/kg dose suppressed progressive prolongation of ADs with repeated stimulations. This effect was seen even 110-min after the drug injection. The modification of ADs, i.e. stimulations with stepwise increasing intensities (10 min intervals) was used to demonstrate possible dependence on activity. The Ro 25-6981 was administered immediately after the 4-mA stimulation (i.e. when rats experienced six ADs on the average). The 3-mg/kg dose resulted in shorter ADs after high stimulation intensities in P12. There were no significant effects in older animals, only a tendency to ADs shortening was observed in P25 rats. In conclusion, our results indicate that Ro 25-6981 as a selective antagonist of NR2B/NMDARs exhibit age- and activation-dependent anticonvulsant action at early postnatal development. In contrast, the influence of Ro 25-6981 on physiological excitability induced by single pulse stimulation of sensorimotor cortex does not depend on age. This compound may thus represent a useful antiepileptic agent in immature brain since its action against ADs prolongation can be observed even 110 min after the single administration of the drug. [2]

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Background: NR2B subunits (NMDA receptor 2B subunit) play an important role in generation of pain and forming central sensitization of pain. Ro 25-6981, a highly selective NR2B antagonist, gained much attention in recent years. In this study, we used a rat model of incisional pain to investigate effects of postoperative analgesia and changes of postoperative hyperalgesia induced by remifentanil through the pretreatment of intrathecal administration with Ro 25-6981.

Methods: The behavioral changes of rats have been evaluated by the paw withdrawal mechanical threshold and paw withdrawal thermal latency after intrathecal injection of Ro 25-6981. The expression of NR2B with tyrosine phosphorylation in the spinal dorsal horn was analyzed by Western blotting.

Results: Intrathecal injection of Ro 25-6981 significantly enhanced the paw withdrawal mechanical threshold and paw withdrawal thermal latency after the operation. Significant change has been observed after intrathecal injection of 800.0 μg of Ro 25-6981 and at 2h after operation in the oblique pull test degree and BBB rating score. Pretreatment of Ro 25-6981 decreased the high level expression of NR2B with tyrosine phosphorylation in spinal dorsal horn of the rat model after the operation.

Conclusions: Intrathecal injection of Ro 25-6981 had significant analgesic effects on incision pain in rats and effectively attenuated postoperative hyperalgesia induced by remifentanil. [3]

Animal/Disease Models: 6-OHDA injured rat [1]
Doses: 0.39-12.5 mg/kg
Route of Administration: intraperitoneal (ip) injection
Experimental Results: Dose-dependent induction of opposite tight nasal-caudal rotation and weak co-directional rotation response, indicating Effect of mildly non-specific stimulating compounds.

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Animal/Disease Models: Male albino rats of Wistar strain [2]
Doses: 1, 3 mg/kg
Route of Administration: Ip
Experimental Results: N1-P2 amplitude was Dramatically diminished at the higher stimulation intensity of 3 mg/kg, and demonstrated Age- and activation-dependent anticonvulsant effects in early postnatal development.\n

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\n\nTwenty-three months after exposure to MPTP, a group of four animals was treated with either vehicle or one of the three doses of Ro 25-6981 or levodopa in combination with one of two doses of Ro 25-6981. In these experiments, we used a Latin square design for the allocation of treatments. A 1-week recovery period was allowed between experiments. The animals had previously been treated with a selective D1 agonist for 25 days, and with a combination of a monoamine blocker in combination with levodopa/carbidopa for 30 days. Drug-free intervals between the experimental treatments were 9 and 12 months, respectively. These treatment did not lead to the development of dyskinesias. [1]

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\n\nEach age group was formed by control animals treated with saline and two groups treated with different doses of Ro 25-6981. Every age and dose group consisted of eight animals. \n \nRo 25-6981 maleate ((αR, βS)-α-(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidinepropanol maleate) was freshly dissolved in saline (1 mg/ml) before beginning of each experiment. The drug was administered intraperitoneally in doses of 1 or 3 mg/kg. [2]

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\n\nSingle pulse evoked potentials [2]
\nSingle 1-ms pulses with intensities increasing from 0.4 to 5.0 mA (0.4, 0.6, 0.8, 1.0, 1.4, 1.8, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 mA) were applied. First cycle of stimulations was a control one, then Ro 25-6981 or saline were injected and 20 min later the second stimulation series started. The software automatically averaged five subsequent responses (at each of 13 different current intensities used) and the amplitude was measured between peaks of N1 (first negative) and P2 (second positive) waves (Fig. 1a). First positive wave could not be used because it was often distorted by stimulation artifact.\n

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\n\nPaired pulse evoked potentials [2]
\nThe threshold stimulation intensity was found for each animal and double times this intensity was used to elicit paired responses with interpulse intervals from 50 to 1000 ms. Two cycles of stimulations were again performed: first before administration of the drug, and the second 20 min after Ro 25-6981 or saline administration. Amplitude of the first (A1) and second (A2) response was again measured between peaks of N1 (first negative) and P2 (second positive) waves (Fig. 1b). The A2/A1 ratio was calculated for each interval.\n

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\n\nCortical afterdischarges (ADs) [2]
\nSeries of 1-ms biphasic rectangular pulses were applied at 8-Hz frequency for 15 s (see an example of ADs recording in Fig. 2). Stimulation with suprathreshold current intensity was repeated six times. Intensity of 3.0 mA was reliably suprathreshold in P18 and P25 rats, higher stimulation intensity (up to 5.0 mA) was necessary in P12 animals due to immaturity of neuronal circuits (Mares et al., 2002). Intervals between two stimulation series were 20 min. The Ro 25-6981 or saline were always injected 10 min after the first AD (i.e. after the predrug control stimulation). Additional series with 30-min intervals were used in 12-day-old rats to determine the duration of effects of Ro 25-6981. \n\nRo 25-6981 was dissolved in DMSO (dimethyl sulfoxide, 5%) to a volume of 25 μl. Remifentanil (0.04 mg/kg) was dissolved in saline (NaCl 0.9%) to a volume of 0.4 ml. Intrathecal injection of Ro 25-6981 was performed at 30 min prior plantar incision. Remifentanil (0.04 mg/kg, 0.4 ml) was infused subcutaneously over a period of 30 min using an apparatus pump. The infusion rate was 0.8 ml/h. Control animals received the same volume of saline under identical conditions.\n

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\n\n Drug administration and experimental grouping [3]
\nAll rats were anesthetized with sevoflurane by a nose mask. Ro 25-6981 was dissolved in 5% DMSO. Remifentanil hydrochloride (0.04 mg/kg) was dissolved in saline (NaCl 0.9%) to a volume of 0.4 ml.\nAccording to the different doses administered, 54 SD rats were randomly divided into 9 groups (n = 6) (Table 1). Within two weeks before the experiment, the rats were placed in the test room for 2 h every day to accustom various apparatuses. The drug Ro 25-6981 was injected intrathecally before surgical incision. The detailed information of the dose was shown in Table 1. Intrathecal injections (i.t.) were made through the intervertebral space in all rats between the L4 and L5 of the spinal cord, as described by Hylden and Wilcox (1980). Ro 25-6981 (dissolved in 5% DMSO) at the dose of 25 μl was administrated i.t. with a 28-gauge 1/2-inch stainless steel needle connected to a 50 μl Hamilton microsyringe, the animal being lightly restrained to maintain the position of the needle. Puncture of the dura was indicated behaviorally by a slight flick of the tail. Because intrathecal injection of 5% DMSO solvent had no effect on the rat behavior (Qu et al., 2009), in order to maintain consistency, all the rats received intrathecal injection with 5% DMSO solvent. Rat models of incisional pain in the right back paw were prepared after intrathecal injection in all groups except group C. In group M, (R + M)1, (R + M)2 and (R + M)3, remifentanil (0.04 mg/kg, 0.4 ml) was infused subcutaneously during surgical incision with a pump for 30 min, and in group C, I, R1, R2 and R3, 0.9% saline (0.4 ml) was infused subcutaneously in identical conditions for 30 min. For behavioral studies, paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) of the rats were tested. The changes of rat behavior were measured at 24 h before intrathecal injection and at 2 h, 6 h, 24 h, and 48 h after operation (n = 6). And the motor function indexes (inclined pull test and BASSO, BEATTIE and BRESNAHAN (BBB) rating) were also examined at the same time points. According to the changes in behavioral indicators of pain, the specimens of all groups were collected at 2 h, 6 h, and 48 h after operation (n = 4) for Western blot analysis.

[1]. Antiparkinsonian activity of Ro 25-6981, a NR2B subunit specific NMDA receptor antagonist, in animal models of Parkinson's disease. Exp Neurol. 2004 May;187(1):86-93.

[2]. Different action of a specific NR2B/NMDA antagonist Ro 25-6981 on cortical evoked potentials and epileptic afterdischarges in immature rats. Brain Res Bull. 2015 Feb;111:1-8.

[3]. Antinociception and prevention of hyperalgesia by intrathecal administration of Ro 25-6981, a highly selective antagonist of the 2B subunit of N-methyl-D-aspartate receptor. Pharmacol Biochem Behav. 2013 Nov;112:56-63.

Ro 25-6981 is a piperidine compound with the structure 4-benzylpiperidine substituted at the 1-position with 3-hydroxy-3-(4-hydroxyphenyl)-2-methylpropyl (1R,2S-stereoisomer). It is a potent antagonist of the N-methyl-D-aspartate (NMDA) receptor GluN2B subunit. It exhibits various pharmacological effects, including NMDA receptor antagonism, anticonvulsant, antidepressant, and neuroprotective agent. It belongs to the piperidine, phenol, secondary alcohol, tertiary amine, and benzene classes. Ro 25-6981(1+) is a conjugate base. Ro 25-6981 is an activity-dependent NMDA receptor antagonist whose receptor contains the NR2B subunit, showing anti-Parkinson's disease activity in two related animal models of Parkinson's disease. Ro 25-6981 induces contralateral rotation in 6-OHDA-damaged rats but does not stimulate motor activity in normal rats. Furthermore, Ro 25-6981 increased motor activity and reduced disability in MPTP-treated marmosets. More importantly, Ro 25-6981 enhanced the effects of levodopa in both models and improved the efficacy of apomorphine and D1 and D2 selective dopamine receptor agonists in 6-OHDA-injured rats. Additionally, Ro 25-6981 attenuated the peak levodopa response in 6-OHDA-injured rats treated with long-term levodopa, but did not reduce the overall response. Ro 25-6981 did not cause any significant motor impairment. Therefore, the anti-Parkinson's disease effect of Ro 25-6981 is superior to any conventional competitive and non-competitive NMDA receptor antagonist because it combines intrinsic anti-Parkinson's disease activity with the ability to synergize with levodopa and D1 and D2 receptor agonists. Conversely, conventional NMDA receptor antagonists do not exhibit anti-Parkinson's disease activity in the absence of dopaminergic stimulation and selectively interact with subtype-specific agonists (Klockgether and Turski, 1990; Löschmann et al., 1997; Morelli et al., 1992). There are several reasons to speculate that the striatum mediates the anti-Parkinson's disease activity of Ro 25-6981. In situ hybridization studies have shown that the expression level of the NR2B subunit in the striatum is higher than that in other basal ganglia nuclei (Kosinski et al., 1998). Correspondingly, [3H]Ro 25-6981 binds more readily in the rat striatum than to the lateral globus pallidus (Fischer et al., 1997). Furthermore, intrastriatal injection of NMDA induces Parkinson's disease in rats, while intrastriatal injection of isifenprodil, another antagonist preferentially targeting the NR2B subunit NMDA receptor, reverses 6-OHDA-induced Parkinson's disease in marmosets (Klockgether and Turski, 1993; Mitchell et al., 1995). Current models of Parkinson's disease pathogenesis suggest that striatal neurons projecting to the lateral part of the globus pallidus (indirect pathway) become overactive after dopamine depletion, while the activity of striatal neurons projecting directly to the basal ganglia output nucleus (direct pathway) decreases (Albin et al., 1989). Since NR2B receptors are located on striatal projection neurons (Landwehrmeyer et al., 1995; Standaert et al., 1999), the effect of Ro 25-6981 is likely due to its blocking of NR2B receptors on striatal neurons projecting to the lateral part of the globus pallidus. This hypothesis implies that Ro 25-6981 selectively inhibits indirect pathways while having little effect on (inactive) direct pathways. This selectivity is best explained by the activity-dependent nature of Ro 25-6981's action. Ro 25-6981 binds with high affinity only to activated receptors, while having little effect on inactive receptors (Fischer et al., 1997). Indeed, studies have shown that after dopamine depletion, the ability of isifenprodil (a structural analogue of Ro 25-6981) to inhibit the binding of the NMDA channel blocker MK-801 in the striatum is enhanced fourfold (Nash et al., 1999). Ro 25-6981 enhances the anti-Parkinson's disease effects of the D1 receptor agonist A68930 and the D2 receptor agonist quipyrrol. Conversely, MK-801 enhanced the rotational behavior induced by the D1 receptor agonist SKF 38393, but weakened the rotational behavior induced by quipyrrol (Morelli et al., 1992). On the other hand, conditional position preference (CPP) enhanced the rotational behavior induced by quipyrrol, but had no effect on the rotational behavior induced by A68930 (Löschmann et al., 1997). We propose that the enhancing effect of Ro 25-6981 on quipyrrol-induced rotation is due to the synergistic inhibitory effect of the two compounds on the indirect pathway. Conversely, the synergistic effect of the D1 receptor agonist A68930 and Ro 25-6981 is best explained by the combined effect of A68930 activating the direct pathway and Ro 25-6981 inhibiting the indirect pathway. The use of traditional NMDA receptor antagonists as anti-Parkinson's disease drugs is limited because they readily induce ataxia and dyskinesia due to their action on NMDA receptors in the cerebellum (Löscher and Honack, 1991). Since the NR2B subunit is expressed at low or absent levels in the cerebellum (Standaert et al., 1994), NR2B-selective NMDA receptor antagonists are not expected to produce ataxia as a side effect as traditional NMDA antagonists. Indeed, in mice, intraperitoneal injection of Ro 25-6981 at doses up to 100 mg/kg did not induce dyskinesia (Boyce et al., 1999). This dose is about an order of magnitude higher than the dose required to produce an anti-Parkinson's effect. Therefore, the lack of ataxia and dyskinesia induced by Ro 25-6981 in rodents and primates may be one reason why it maintains its anti-Parkinson's activity even at high doses. Other antagonists that preferentially target NMDA receptors containing the NR2B subunit have also shown similarly favorable effects in animal models of Parkinson's disease. Ifenprodil stimulated motor activity in reserpine-treated rats, marmosets with bilateral 6-hydroxydopamine injury, and marmosets treated with MPTP (Mitchell et al., 1995; Nash et al., 1999; Nash et al., 2000). However, a small clinical trial in patients with Parkinson's disease yielded negative results (Montastruc et al., 1992). Another NR2B antagonist, CP 101,106, alleviated rigidity and akinesia in MPTP-treated nonhuman primates and haloperidol-treated rats (Steece-Collier et al., 2000). Several structurally unrelated antagonists were observed to have affinity for NMDA receptors containing the NR2B subunit and all exhibited potent anti-Parkinson's disease activity, making it unlikely that the effect of Ro 25-6981 is due to its action on other receptors, particularly dopamine receptors. In fact, combined experiments showed that Ro 25-6981 has extremely low affinity for dopamine receptors (Mutel et al., 1998). Given that Ro 25-6981 can induce contralateral rotation, its dopamine release effect is unlikely. Although the ability of compounds to induce contralateral rotation in 6-OHDA-damaged rats is generally considered to reflect their anti-Parkinson's disease activity (Kaakkola and Teravainen, 1990), numerous observations have shown that repeated and intermittent administration of levodopa leads to behavioral sensitization, manifested as enhanced response and shortened reaction time. This could serve as a model for motor dysfunction and decreased efficacy in Parkinson's disease patients after long-term levodopa treatment (Papa et al., 1994). Although these rats did not exhibit obvious dyskinesia, the changes in response following long-term levodopa treatment were similar to the response fluctuations observed in patients with advanced Parkinson's disease. The effectiveness of this model is supported by its ability to predict the anti-dyskinesia effects of the NMDA receptor antagonist amantadine (Papa et al., 1995; Verhagen et al., 1998). Given the causal relationship between the plasticity changes in the levodopa response in 6-OHDA-damaged rats and aberrant phosphorylation of the striatal NR2B subunit, we were interested in investigating the role of Ro 25-6981 in this model. As demonstrated in this series of experiments, chronic levodopa treatment led to an increase in the peak value of the rotational response, but not a shortening of its duration. This result differs from that published by Papa et al. (1994), possibly because we did not use levodopa methyl ester. Furthermore, we employed a more lenient selection procedure, using higher doses of apomorphine (0.1 mg/kg vs. 0.05 mg/kg), which may have resulted in the inclusion of some 6-OHDA-damaged rats (Papa et al., 1994). Ro 25-6981 reduced the peak rotational response in 6-OHDA-damaged rats treated with long-term levodopa, suggesting that Ro 25-6981 has a beneficial effect on levodopa-induced dyskinesia and response fluctuations. Based on the current results, selective antagonism of NMDA receptors containing NR2B subunits is sufficient to induce anti-Parkinson's disease effects in two relevant animal models of Parkinson's disease. NR2B antagonists, such as Ro 25-6981, may be beneficial for patients with Parkinson's disease and may be used alone or in combination with standard dopaminergic drugs for the treatment of Parkinson's disease. [1] It appears possible that, in P25 animals, the function of presynaptic NR2B/NMDAR autoreceptors can be restored with increasing stimulus intensity, as observed in adult patients with epilepsy (Yang et al., 2006). Therefore, GABA release activity regulated by NR2B/NMDAR (which may be restored by high-intensity stimulation) may not be completely blocked by a lower dose (1 mg/kg) of Ro 25-6981; thus, it may lead to a decrease in the amplitude of single evoked potentials. Furthermore, since the NR2B subunit is primarily expressed in the forebrain in adult animals (Loftis and Janowsky, 2003), Ro 25-6981 likely affects purely cortical activity in rats at the third and fourth weeks after birth. Unfortunately, we cannot currently explain why Ro 25-6981 has no effect whatsoever on cortical enhancement or inhibition induced by double-pulse stimulation. This may be due to the relatively low stimulation intensity (twice the threshold); in contrast, the effect on single-pulse responses is mainly observed at high stimulation intensities. In summary, our results indicate that the selective NR2B/NMDAR antagonist Ro 25-6981 exhibits a significant activation-dependent anticonvulsant effect on post-epileptic discharges (a myoclonic epilepsy model) only in rats at the second week of life. Therefore, we can infer that in older animals, receptors containing the NR2B subunit are not involved in the development of seizures in this model. Thus, Ro 25-6981 or other NR2B antagonists may be effective drugs for treating epileptic activity in immature brains. This drug also significantly reduces physiological excitability induced by single-pulse stimulation of the sensorimotor cortex, and this effect is age-independent and does not affect cortical excitability induced by double-pulse stimulation. Currently, we cannot fully explain this in vivo mechanism of action of Ro 25-6981, and further experiments are needed. However, the activation level and localization of NMDARs containing the NR2B subunit may play a role in the effect of Ro 25-6981 on cortical excitability. [2] The NR2B subunit plays an important role in pain and central sensitization (LoGrasso and McKelvy, 2003). The NR2B subunit has seven tyrosine phosphorylation sites at its C-terminus, with Tyr-1472 being the major site. Its phosphorylation has a significant effect on alterations in synaptic plasticity and the occurrence of long-term potentiation (LTP). LTP and opioid-induced hyperalgesia (OIH) share common pharmacological and signal transduction pathways (Drdla et al., 2009; Nakazawa et al., 2001). Many studies have shown that tyrosine phosphorylation of NR2B at Tyr-1472 in the dorsal horn of the spinal cord promotes hyperalgesia in neuropathic pain models (Abe et al., 2005) and inflammatory pain models (Guo et al., 2002). Therefore, we not only observed behavioral changes in rats, but also found that Ro 25-6981 has an analgesic effect in rat models of incision pain and hyperalgesia. Western blot analysis showed that intrathecal injection of Ro 25-6981 reduced tyrosine phosphorylation levels of NR2B in the dorsal horn of the spinal cord and alleviated hyperalgesia. Ro 25-6981, as a specific antagonist of the NR2B subunit, may alleviate incision pain and remifentanil-induced hyperalgesia through the NR2B pathway. In summary, behavioral tests and Western blot analysis indicated that intrathecal injection of Ro 25-6981 significantly reduced incision pain in rats and effectively prevented remifentanil-induced postoperative hyperalgesia. These results may be related to the inhibition of tyrosine phosphorylation of NR2B in the superficial layer of the rat spinal cord. The effect of Ro 25-6981 on rat motor function varied with dosage and drug metabolism. Although Ro 25-6981 is currently under investigation in animal studies, it has not yet been clinically applied. The mechanisms of action of these drugs may provide insights into finding more effective clinical treatments for incision pain and hyperalgesia. In conclusion, our study explored the analgesic effect of targeting NR2B in a rat model of incision pain. Our data suggest that targeting NR2B in the spinal cord may be a novel strategy for treating clinical pain. [3]

C22H29NO2

339.479

339.219

C, 77.84; H, 8.61; N, 4.13; O, 9.43

169274-78-6

Ro 25-6981 Maleate;1312991-76-6;Ro 25-6981 hydrochloride;919289-58-0

6604887

White to off-white solid powder

703.7ºC at 760mmHg

379.4ºC

2.52E-11mmHg at 25°C

1.587

3.666

2

3

6

25

366

2

C[C@@H](CN1CCC(CC1)CC2=CC=CC=C2)[C@H](C3=CC=C(C=C3)O)O

WVZSEUPGUDIELE-HTAPYJJXSA-N

InChI=1S/C22H29NO2/c1-17(22(25)20-7-9-21(24)10-8-20)16-23-13-11-19(12-14-23)15-18-5-3-2-4-6-18/h2-10,17,19,22,24-25H,11-16H2,1H3/t17-,22+/m0/s1

4-((1R,2S)-3-(4-benzylpiperidin-1-yl)-1-hydroxy-2-methylpropyl)phenol

Ro 25-6981; Ro25-6981; Ro-25-6981; Ro-256981; 169274-78-6; Ro 25-6981; Ro-25-6981; ro25-6981; Ro 25-6981 free base; CHEBI:92897; (alphar,betas)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidinepropanol; 4-[(1R,2S)-1-hydroxy-2-methyl-3-[4-(phenylmethyl)-1-piperidinyl]propyl]phenol; Ro 256981; Ro256981; Ro 25-6981 free base

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)