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].

---

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]

---

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]

---

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.

---

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.

---

Twenty-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]

---

Each 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. Ro 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]

---

---

Single pulse evoked potentials [2]
Single 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.

---

Paired pulse evoked potentials [2]
The 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.

---

Cortical afterdischarges (ADs) [2]
Series 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. Ro 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.

---

Drug administration and experimental grouping [3]
All 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. According 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 member of the class of piperidines that is 4-benzylpiperidine substituted by a 3-hydroxy-3-(4-hydroxyphenyl)-2-methylpropyl group at position 1 (the 1R,2S-stereoisomer). It is a potent antagonist of the GluN2B subunit of the N-methyl-D-aspartate (NMDA) receptor. It has a role as a NMDA receptor antagonist, an anticonvulsant, an antidepressant and a neuroprotective agent. It is a member of piperidines, a member of phenols, a secondary alcohol, a tertiary amino compound and a member of benzenes. It is a conjugate base of a Ro 25-6981(1+).

---

Ro 25-6981, an activity-dependent antagonist of NMDA receptors containing the NR2B subunit, had antiparkinsonian activity in two relevant animal models of PD. Ro 25-6981-induced contraversive rotations in 6-OHDA-lesioned rats without stimulating locomotor activity in normal rats. In addition, Ro 25-6981 increased locomotor activity and reduced disability in MPTP-treated marmosets. More importantly, Ro 25-6981 potentiated the action of levodopa in both models and increased the efficacy of apomorphine and a D1- and D2-selective dopamine receptor agonist in 6-OHDA-lesioned rats. Furthermore, Ro 25-6981 attenuated the peak levodopa response in 6-OHDA-lesioned rats chronically treated with levodopa without reducing the overall response. Ro 25-6981 did not lead to any obvious motor impairment. Thus, the antiparkinsonian action of Ro 25-6981 is superior to that of any conventional competitive and non-competitive NMDA receptor antagonist in that it combines intrinsic antiparkinsonian activity with the potency to synergistically interact with levodopa and both D1 and D2 receptor agonists. In contrast, conventional NMDA receptor antagonists do not have antiparkinsonian actions 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 numerous reasons to assume that the striatum mediates the antiparkinsonian action of Ro 25-6981. In situ hybridisation studies show that NR2B subunits are expressed in the striatum at higher levels than in other basal ganglia nuclei (Kosinski et al., 1998). Accordingly, binding of [3H]Ro 25-6981 was found to be high in the rat striatum compared to the external pallidum (Fischer et al., 1997). Furthermore, local intrastriatal injection of NMDA produces parkinsonism in rats, while intrastriatal injection of ifenprodil, another antagonist with preference for NR2B-containing NMDA receptors, reversed parkinsonism in 6-OHDA-lesioned marmosets Klockgether and Turski, 1993, Mitchell et al., 1995.
The current model of the pathogenesis of PD proposes that striatal neurons projecting to the external pallidum (indirect pathway) become overactive following dopamine depletion, while the activity of striatal neurons projecting directly to the basal ganglia output nuclei (direct pathway) is reduced (Albin et al., 1989). Since NR2B receptors are located on striatal projection neurons Landwehrmeyer et al., 1995, Standaert et al., 1999, it seems likely that the effects of Ro 25-6981 are the result of blockade of NR2B-containing NMDA receptors on striatal neurons projecting to the external pallidum. This assumption implies a selective suppressive action of Ro 25-6981 on the indirect pathway without major actions on the (underactive) direct pathway. This selectivity is best explained by the activity-dependance of the actions of Ro 25-6981. Ro 25-6981 only binds with high affinity to activated receptors, while it has negligible effects on non-activated receptors (Fischer et al., 1997). Indeed, the ability of ifenprodil, a structural analogue of Ro 25-6981, to inhibit binding of the NMDA channel blocker MK-801 in the striatum was shown to increase by a factor of four following dopamine depletion (Nash et al., 1999).
Ro 25-6981 potentiated the antiparkinsonian action of both, the D1 receptor agonist A68930 and the D2 receptor agonist quinpirole. In contrast, MK-801 potentiated rotational behaviour induced by the D1 receptor agonist SKF 38393, but reduced quinpirole-induced rotational behaviour (Morelli et al., 1992). CPP, on the other hand, potentiated quinpirole-induced rotational behaviour, but not A68930-induced rotations (Löschmann et al., 1997). We propose that the potentiating action of Ro 25-6981 on quinpirole-induced rotations is due to a synergistic suppressive action of both compounds on the indirect pathway. In contrast, the synergistic interaction of the D1 agonist A68930 and Ro 25-6981 is best explained by a combination of an activation of the direct pathway by A68930 and suppression of the indirect pathway by Ro 25-6981.
The usefulness of conventional NMDA antagonists as antiparkinsonian drugs is limited by their liability to produce incoordination and ataxia which is due to an action of these compounds on NMDA receptors in the cerebellum (Löscher and Honack, 1991). Since expression of NR2B subunits is low or absent in the cerebellum (Standaert et al., 1994), NR2B-selective NMDA receptor antagonists are predicted to be free of the ataxia-inducing side effects of conventional NMDA antagonists. Indeed, in doses up to 100 mg/kg i.p. Ro 25-6981 did not produce motor impairment in mice (Boyce et al., 1999). This dose is approximately one order of magnitude higher than the dose required to produce an antiparkinsonian action. The failure of Ro 25-6981 to induce ataxia and motor impairments in rodents and primates may therefore contribute to persistent antiparkinsonian activity of Ro 25-6981 even at high doses.
Other antagonists with preference for NR2B-containing NMDA receptors had similarly promising actions in animal models of PD. Ifenprodil stimulated locomotor activity in reserpine-treated rats, bilaterally 6-OHDA-lesioned marmosets and MPTP-treated marmosets Mitchell et al., 1995, Nash et al., 1999, Nash et al., 2000. However, a small clinical trial in PD patients was negative (Montastruc et al., 1992). Another NR2B antagonist, CP 101,106, reduced rigidity and akinesia in MPTP-treated non-human primates and haloperidol-treated rats (Steece-Collier et al., 2000). The observation that several structurally unrelated antagonists that share a preference for NMDA receptors containing the NR2B subunit have potent antiparkinsonian actions makes it unlikely that the action of Ro 25-6981 is due to actions at other receptors, in particular dopamine receptors. Indeed, binding experiments revealed only negligible affinity to dopamine receptors (Mutel et al., 1998). A dopamine releasing action of Ro 25-6981 is unlikely in view of the contraversive rotations induced by this compound.
While the ability of compounds to elicit contraversive rotations of 6-OHDA-lesioned rats is generally considered to reflect an antiparkinsonian activity (Kaakkola and Teravainen, 1990), numerous observations show that repeated and intermittent administration of levodopa results in a behavioural sensitization with an increased and shortened response that serves as a model of dyskinesias and the wearing-off phenomenon occurring in PD patients after long-term levodopa treatment (Papa et al., 1994). Although these rats do not develop overt dyskinesias, the response alterations occurring after chronic levodopa treatment are reminiscent of response fluctuations in patients with advanced PD. The validity of this model is supported by its ability to predict the antidyskinetic effect of the NMDA antagonist amantadine Papa et al., 1995, Verhagen et al., 1998. Since the plastic changes of the levodopa response in 6-OHDA-lesioned rats are causally associated with an abnormal phosphorylation of striatal NR2B subunits, we were interested to investigate the action of Ro 25-6981 in this model. Chronic levodopa treatment, as performed in this series of experiments, led to an increased peak, but not to a shortened rotational response. This discrepancy to the results published by Papa et al. (1994) may be because of the fact that we did not use the methylester of levodopa. In addition, we used a more liberal selection procedure with a higher dose of apomorphine (0.1 vs. 0.05 mg/kg) that may have resulted in inclusion of rats with only partial 6-OHDA lesions (Papa et al., 1994). Ro 25-6981 reduced the peak rotational response in chronically levodopa-treated 6-OHDA-lesioned rats suggesting a beneficial action of Ro 25-6981 on levodopa-induced dyskinesias and response fluctuations.
On the basis of the present results, it appears that selective antagonism at NR2B subunit containing NMDA receptors is sufficient to induce antiparkinsonian effects in two relevant animal models of PD. NR2B antagonists such as Ro 25-6981 could have favourable effects in PD patients and may be used alone or in combination with standard dopaminergic drugs in the management of PD. [1]

---

It seems possible, that after stimulation with increasing intensities the autoreceptor function of presynaptic NR2B/NMDARs in P25 animals can be restored, as it was observed in epileptic adults (Yang et al., 2006). Hence, modulated by the NR2B/NMDARs, GABA release activity, which probably was brought back by high intensity stimulation, might not be blocked completely by the lower (1-mg/kg) dose of Ro 25-6981; therefore, it is possible that it caused the decrease in amplitude of single evoked potentials. Moreover, as in adult animals NR2B subunit is primarily expressed in the forebrain (Loftis and Janowsky, 2003), it is also likely that purely cortical activity can be affected by Ro 25-6981 even in the third and fourth postnatal week in rats.
Unfortunately, at the moment we have no explanation on the complete lack of effects of Ro 25-6981 on cortical potentiation or depression induced by paired pulse stimulation. The reason might be in relatively moderate intensity of stimulation (twofold threshold); in contrast, the effects on single-pulse responses were observed mostly at high stimulation intensities.
In conclusion, our results show that Ro 25-6981, a selective antagonist of NR2B/NMDARs, exhibits clear activation-dependent anticonvulsant action against epileptic afterdischarges (model of myoclonic seizures) only during the second postnatal week in rats. Consequently, we can imply that in older animals, receptors containing NR2B subunit do not contribute to generation of seizures in this model. Thus, Ro 25-6981 or other NR2B antagonists may represent a useful tool in pharmacotherapy of epileptic activity in immature brain.
This drug also significantly reduces the level of physiological excitability induced by single pulse stimulation of sensorimotor cortex in an age-independent manner and did not affect cortical excitability evoked by paired pulse stimulation. At the moment we cannot fully explain this in vivo action of Ro 25-6981 and further tests will be necessary. However, the level of activation of NMDARs containing NR2B subunit and their localization may play a role in the effects of Ro 25-6981 on cortical excitability. [2]

---

NR2B subunit in the generation of pain and central sensitization plays an important role (LoGrasso and McKelvy, 2003). There are seven tyrosine phosphorylation sites in the cytoplasmic C-terminal of NR2B subunit and Tyr-1472 is the main site. Its phosphorylation has significant effect for changes of synaptic plasticity and occurrence of long-term potentiation (LTP). LTP and OIH have the common pharmacological and signal transduction pathway (Drdla et al., 2009, Nakazawa et al., 2001). Many studies have shown that tyrosine phosphorylation of the NR2B at Tyr-1472 in the spinal dorsal horn contributed to the development of hyperalgesia in neuropathic pain model (Abe et al., 2005) and inflammatory pain model (Guo et al., 2002). Therefore, we not only observed the behavioral changes in rats but also Ro 25-6981 induced antinociception in the rat model of incisional pain and hyperalgesia. Western blot analysis showed that pre-intrathecal injection of Ro 25-6981 decreased the higher level expression of tyrosine phosphorylation of NR2B in spinal dorsal horn and hyperalgesia. Ro 25-6981 may relieve incisional pain and remifentanil-induced hyperalgesia through the pathway of NR2B as the specific NR2B subunit antagonist.
In summary, behavioral test and Western blot analysis demonstrated pre-intrathecal injection of Ro 25-6981 had significant analgesic effects on incision pain in rats and effectively prevented postoperative hyperalgesia induced by remifentanil. These results might have some relation with inhibiting tyrosine phosphorylation of NR2B in superficial spinal cord of rats. The effects of Ro 25-6981 on locomotor functions of rats changed with the different doses and drug metabolism. Although Ro 25-6981 is currently studied for animal experiment, clinical application has not been carried out. The mechanism of these drugs may provide ideas to find out more value methods for the clinical treatment of incisional pain and hyperalgesia. Conclusively, our study investigated the analgesic effects through targeting NR2B in a rat incision pain model. And our data suggest that targeting NR2B in the spinal cord might be a new strategy for the treatment of 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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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