NMDA/N-methyl-D-aspartate receptor

Nicardipine, nifedipine and flunarizine showed anticonvulsive activity (reflected by significant elevations of the seizure threshold for tonic hindlimb extension) in doses of 20, 20 and 15 mg/kg, respectively. In combination studies, CGP 40116 [D-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid] or its methyl ester derivative (CGP 43487) was administered in a constant dose of 0.25 and 3.5 mg/kg, respectively. At these doses both competitive NMDA receptor antagonists were able to elevate significantly the convulsive threshold. Nicardipine, nifedipine, and flunarizine were administered at maximal doses (or lower) not affecting the convulsive threshold (15, 15 and 10 mg/kg, respectively). The protective activity of CGP 40116 and CGP 43487 was dose dependently potentiated by all three Ca2+ channel inhibitors. The combined treatment caused motor impairments (evaluated in the chimney test) and long-term memory deficits (measured in the passive avoidance task) similar to these produced by CGP 40116 or CGP 43487 alone. Our results indicate that nicardipine, nifedipine and flunarizine significantly potentiate the protective activity, but not the adverse effects, of CGP 40116 and CGP 43487 in mice.[1]

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The administration to rats of different doses of the non competitive NMDA receptor blocker MK-801 (0.03-1 mg/kg IP) induced stimulation or reduction of locomotor activity, depending on the dose, whereas the competitive NMDA antagonists CGP 43487 (0.188-6 mg/kg IP) and APV (2.5-20 micrograms/rat ICV) inhibited locomotion at the highest doses. Unlike MK-801 and APV treatment, the administration of CGP 43487 did not induce impairment of rota-rod test performance. Both competitive and non-competitive NMDA antagonists, at doses devoid of any behavioral effect per se, potentiated the responses elicited by apomorphine (0.25 mg/kg SC). In particular, the occurrence of episodes of licking was weakly affected by MK-801 administration, but significantly increased by CGP 43487 and APV treatment; the presence of gnawing was augmented by all the pretreatments; sniffing, locomotion, grooming and rearing occurrence were not affected by the administration of NMDA antagonists. The results suggest that the competitive antagonists which facilitated dopaminergic function without causing motor impairment could be useful supplements in the treatment of Parkinson's disease [2].

Locomotor activity [2]
Different groups of naive rats (n = 8 12 per group) were treated with various doses of MK-801 (0.03, 0.0625, 0.125, 0.25 and 1 mg/kg IP), CGP 43487 (0.188, 0,375, 0.75, 3 and 6 mg/kg IP), or APV (2,5, 5, 10 and 20 ~tg/rat, ICV) and 30 min, 180 or 5 min later, respectively, they were placed in actometric cages (Dall'Olio et al. 1988) where their motility was recorded for 60 min.

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Stereotyped behavior [2]
Stereotyped behavior induced by subcutaneous administration of apomorphine (0.25 mg/kg) was observed in different groups of rats (n=8-16 per group) pretreated with MK-801 (0.03 and 0.0625mg/kg IP, 30 rain before), CGP 43487 (0.375 and 0.75 mg/kg IP, 3 h before) or APV (2.5 and 5 pg/rat, ICV, 5 min before). The animals were placed in actometric cages and habituated to the experimental environment for 2 h before the dopaminomimetic injection. Starting 10 min after apomorphine administration, animals were assessed using a behavioral checklist technique (Fray et al. 1980; Molloy and Waddington 1984, 1987). Each rat was observed individually for 5-s periods at l-rain intervals over 5 consecutive minutes to determine the presence of the following individual behaviors (occurring alone or in combination): sniffing (Sn); licking (Lk); gnawing (Gn); grooming (Gr, of any form); stillness (St; motionless, with no behavior evident); locomotion (L); rearing (R). After assessment using the behavioral checklist, the animals were observed for the stereotypies (STR) using a conventional 0-6 point rating scale: 0 = asleep or inactive; 1 = episodes of normal activities; 2 = discontinuous activity with bursts of sniffing or rearing; 3 = continuous stereotyped activity such as sniffing or rearing along a fixed path; 4 = stereotyped sniffing or rearing fixated in one location; 5 = stereotyped behavior with bursts of licking or gnawing; 6 = continuous licking or gnawing. This cycle of observations was repeated three times at 10-rain intervals by observers unaware of the treatments. Rats were used only once (from 0900 hours to 1500 hours).

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Rota-rod test [2]
A rota-rod treadmill for rats was used. Rats were trained to the apparatus on four occasions at 30 min intervals. Only the animals that were able to maintain equilibrium on the rotating rod (speed 16 rpm) for at least 10 s in all the training trials were used for assaying the drug effects. Twenty-four hours after training, rats (n = 12 per group) were observed for their rota-rod performance before receiving MK-801 (0.5 and 1 mg/kg IP), CGP 43487 (3 mg/kg IP) or APV (10 pg/rat, ICV); after 30, 180 or 5 rain, respectively, the time spent by the animals on the rota-rod was evaluated again.
CGP 43487 was dissolved in saline and intraperitoneally injected.

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Chimney test [2]
The chimney test of Boissier et al. (1960) was used to evaluate the influence of CGP 40116 or CGP 43487 alone or in combination with the Ca 2+ channel inhibitors on motor performance. Motor impairment in this test was indicated by the inability of the animals to climb backwards up the tube (3 cm inner diameter, 25 cm length) within 60 s. The animals were pretrained 24 h before treatment and those unable to perform the test were rejected from the experimental groups. On the following day, mice were treated with the compounds either alone or in combination. Results were calculated as a percentage of animals failing to perform the test.

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Passive avoidance acquisition and retention testing [2]
According to Venault et al. (1986), the step-through passive avoidance task may be used as a measure of long-term memory. We used this test to compare the influence of CGP 40116, CGP 43487, nicardipine, nifedipine, flunarizine alone or in combination (an NMDA receptor antagonist+ a Ca 2- channel inhibitor) on passive avoidance acquisition in mice. Procedural details have been published elsewhere (Borowicz et al., 1995). Shortly, mice avoiding the dark compartment for over 60 s (on the day after their entry of this compartment had been punished by an electric footshock of 0.8 mA for 2 s) showed no long-term memory impairment and were regarded as remembering the task. Retention was expressed as the percentage of mice with no memory impairment.
Sterile saline was used to bring CGP 40116 and CGP 43487 into solution.

[1]. Ca2+ channel blockade and the antielectroshock activity of NMDA receptor antagonists, CGP 40116 and CGP 43487, in mice. Eur J Pharmacol. 1996 Sep 19;312(1):27-33.
[2]. The competitive NMDA antagonists CGP 43487 and APV potentiate dopaminergic function. Psychopharmacology (Berl). 1995 Apr;118(3):310-5.

Studies have shown that the Ca²⁺ channel agonist BAY k-8644 inhibits the anticonvulsant effects of competitive NMDA receptor antagonists (CGP 37849 and o-CPP-ene) only, while having no effect on the anticonvulsant effects of non-competitive NMDA receptor antagonists (MK-801) and non-NMDA receptor antagonists (NBQX and GYKI 52466) in a mouse electroconvulsive model (Czuczwar et al., 1994). These findings suggest that the enhanced anticonvulsant efficacy of CGP 40116 and CGP 43487 may be due to the centrally mediated effects of Ca²⁺ channel inhibitors. Similarly, centrally active Ca²⁺ channel inhibitors can enhance the anticonvulsant efficacy of conventional antiepileptic drugs, but verapamil cannot (Czuczwar et al., 1990a,b) because verapamil has limited brain penetration (Hamann et al., 1983). Epileptiform activity is a series of events that can be triggered by excitatory amino acids (Hayashi, 1954; Bradford and Peterson, 1987; Meldrum, 1991). First, L-glutamate activates the NMDA receptor/channel complex, leading to initial depolarization followed by Ca²⁺ ion influx into the cell through receptor-activated channels (Mayer and Miller, 1990). Furthermore, NMDA-activated depolarization diffuses to voltage-gated Ca²⁺ channels, ultimately leading to their activation and subsequent massive Ca²⁺ influx (Courtney et al., 1990; Mayer and Miller, 1990). Therefore, NMDA receptor antagonists may reduce Ca²⁺ influx directly through NMDA receptors or indirectly through voltage-gated Ca²⁺ channels. Similarly, Ca²⁺ channel inhibitors may also exhibit similar “crosstalk.” This explanation might account for the anticonvulsant activity of Ca²⁺ channel inhibitors themselves, as well as when used in combination with CGP 40116 or CGP 43487. There is also evidence that some Ca²⁺ channel inhibitors bind directly to the NMDA receptor complex and specifically block its function (Hashim et al., 1988; Skeen et al., 1993). However, with a few exceptions, this hypothesis lacks experimental support. Furthermore, the inhibitory effect of calcium channel inhibitors on the release of presynaptic excitatory amino acids cannot be completely ruled out (De Sarro et al., 1988; Janis and Triggle, 1991). Nicardipine, nifedipine, and flunarizine enhance the anticonvulsant effects of CGP 40116 and CGP 43487 in a similar manner, which may indicate that calcium channel blockade is the cause of this effect. Flunarizine, in addition to this mechanism of action, may also block sodium channels (Ashton and Wauquier, 1986), which may also be involved. Unfortunately, the potential antiepileptic application of NMDA receptor antagonists is limited by their side effects (Schmidt, 1994; Witkin, 1995). In fact, while Ca²⁺ channel inhibitors significantly enhance the protective effects of CGP 40116 or CGP 43487, they are accompanied by behavioral disorders. However, this novel strategy for treating epilepsy (Ca²⁺ channel inhibitor + NMDA receptor antagonist) should not be abandoned immediately, as more specific and less toxic compounds may soon become available. Whether this multipharmacological approach can be used to treat other neurological disorders remains to be further investigated. [1] According to other studies (Liljequist 1991; Ornstein et al. 1987; Starr and Starr 1994a,b), although low doses of MK-801 induce significant hyperactivity, neither CGP 43487 nor APV increases activity in rats. High doses of both non-competitive and competitive NMDA receptor antagonists reduced spontaneous movement in animals and resulted in different behavioral characteristics. MK-801 caused flattening of the posture and hind limb abduction, while non-competitive receptor antagonists only caused more pronounced sedation. In fact, rotarod tests showed no significant difference in motor coordination before and after CGP 43487 treatment, suggesting that competitive receptor antagonists have a smaller effect on spontaneous behavior than non-competitive receptor antagonists, and that CGP 43487 may have a significant therapeutic advantage among the antagonists studied. The most interesting result of this study relates to the effect of NMDA receptor antagonists on dopaminergic stimulation-induced behavioral effects. Both competitive and non-competitive NMDA receptor antagonists enhanced the response to the mixed D1/D2 receptor agonist apomorphine, even though these drugs themselves do not affect spontaneous behavior. However, unlike pretreatment with two competitive NMDA receptor antagonists, administration of MK-801 did not significantly alter the frequency of licking responses. According to Fray et al. (1980), licking behavior was not prominent under high doses of apomorphine; instead, biting behavior occurred. This suggests that overstimulation of dopamine receptors preferentially leads to biting behavior. However, in this study, MK-801 could not enhance the apomorphine response to counteract licking behavior, as even lower doses of non-competitive NMDA receptor antagonists did not increase the frequency of this behavior (data not shown). Furthermore, higher doses of competitive NMDA receptor antagonists enhanced the frequency of both licking and biting behaviors. Observations revealed that apomorphine-induced olfaction and motor responses were similar across all pretreatment groups, while rats treated with NMDA antagonists exhibited an increased biting frequency, which may suggest that the dopaminergic enhancement primarily occurs in the striatum, the main brain region where apomorphine-induced biting behavior is predominant (Ernst 1967; Ljungberg and Ungerstedt 1977). This is consistent with previous findings that have shown antagonism between the central glutamatergic and dopaminergic systems occurs in the striatum (Carlsson and Carlsson 1989; Carlsson and Svensson 1990). However, our data are not entirely consistent with other findings that have shown NMDA receptor blockade promotes D1-mediated motor responses in monoamine-depleted mice but does not affect D1/D2 dopamine receptor stimulation-induced behavior (Verma and Kulkarni, 1992; Starr and Starr, 1993). Administration of NMDA antagonists to 314 monoamine-depleted animals may have revealed the activation potential of other neurotransmitter systems (Carlsson and Carlsson, 1989; Carlsson and Svensson, 1990). Different experimental conditions (untreated animals versus monoamine-depleted animals) and species may explain our observed enhanced D1/D2 stimulation-induced responses by NMDA antagonists. In summary, the current findings are consistent with other data, suggesting that drugs that block the NMDA receptor complex have a stimulating effect on dopamine-mediated behavior, while competitive NMDA antagonists appear unlikely to cause motor disturbances and mental excitement. [2]

C8H16NO5P

237.19

237.077

C, 40.51; H, 6.80; N, 5.91; O, 33.73; P, 13.06

146388-56-9

6438792

Typically exists as solid at room temperature

1.312g/cm3

434.1ºC at 760 mmHg

216.3ºC

9.46E-09mmHg at 25°C

1.512

0.701

3

6

6

15

295

1

CCOC([C@@H](/C=C(/CP(=O)(O)O)\C)N)=O

OKDOWCKDTWNRCB-PTYLAXBQSA-N

InChI=1S/C8H16NO5P/c1-3-14-8(10)7(9)4-6(2)5-15(11,12)13/h4,7H,3,5,9H2,1-2H3,(H2,11,12,13)/b6-4+/t7-/m1/s1

[(E,4R)-4-amino-5-ethoxy-2-methyl-5-oxopent-2-enyl]phosphonic acid

Cgp-43487; Cgp43487; Cgp 43487; 146388-56-9; Cgp-43,487; 3-Pentenoic acid, 2-amino-4-methyl-5-phosphono-, 1-ethyl ester, (R-(E))-; [(E,4R)-4-amino-5-ethoxy-2-methyl-5-oxopent-2-enyl]phosphonic acid; (R,E)-(4-amino-5-ethoxy-2-methyl-5-oxopent-2-en-1-yl)phosphonic acid; CGP-39,551; Cgp 43487

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.)