(2R)-2-(methylamino)butanedioic acid

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The effect on the TCP (1-[1-(2-thienyl)-cyclohexyl]piperidine) binding was measured in rat brain synaptic membranes (SPM) prepared as previously described [J. B. Monahan & J. Michel; J. Neurochem. 48:1699-1708 (1987)]. Prior to their use in the binding assay, frozen SPM were thawed, diluted twenty fold with 50 mM Tris/acetate (pH 7.4 containing 0.04% (v/v) TritOn X-100), incubated for 30 min. at 37° C. and centrifuged at 95,000×g for 15 min. The Triton X-100 treated SPM were washed with 5 mM Tris/HCl, pH 7.4 and centrifuged a total of six times. The compound of Example #7 was incubated at different concentrations with SPM (0.2-0.4 mg protein) and 2 nM tritiated TCP, in a total volume of 0.5 ml of 5 mM Tris/HCl buffer pH 7.4 at 25° C. for 60 min. The samples were filtered through glass fiber filters (Schleicher & Schuell π32) which have been pretreated with 0.05% (v/v) polyethylenimine, washed 4 times with 2 ml of ice-cold 5 mM Tris/HCl buffer, and then counted on a Beckman LS 5800 liquid scintillation counter with automatic corrections for quenching and counting efficiency. Inhibition of TCP binding was measured as a decrease in the binding in the presence of 0.05 mM L-glutamate. Non-specific binding was defined as the residual binding in the presence of 60 mM phencyclidine. Results are shown in Table II. prepared Prior to their use in the binding assay, frozen SPM centrifuged at 95,000×g for 15 min The Triton X-100 treated SPM were washed with 5 mM Tris/HCl, pH 7.4 The compound of Example #7 was incubated at different concentrations with SPM (0.2-0.4 mg protein) and 2 nM tritiated TCP, in a total volume of 0.5 ml of 5 mM Tris/HCl buffer pH 7.4 at 25° C. for 60 min The samples were filtered through glass fiber filters (Schleicher & Schuell π32) which washed 4 times with 2 ml of ice-cold 5 mM Tris/HCl buffer for quenching Results

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Synaptic plasma membranes (SPM) were prepared as previously described Monahan, J. B. and Michel, J., "Identification and Characterization of an N-methyl-D-aspartate-specific L-[3H]-glutamate Recognition Site in Synaptic Plasma Membranes, J. Neurochem., 48, 1699-1708 (1987)]. The SPM were stored at a concentration of 10-15 mg/ml in 0.32M sucrose, 0.5 mM EDTA, 1 mM MgSO4, 5 mM Tris/sulfate, pH 7.4, under liquid nitrogen. The identity and purity of the subcellular fractions were confirmed by both electron microscopy and marker enzymes. Protein concentrations were determined by using a modification of the method of Lowry [Ohnishi, S. T. and Barr, J. K., "A Simplified Method of Quantitating Proteins Using the Biuret and Phenol Reagents", Anal. Biochem., 86, 193-197 (1978)]. The SPM were treated identically for the [3H]AMPA (QUIS), [3H]kainate and sodium-dependent L-[3H]-glumatate binding assays. The SPM were thawed at room temperature, diluted twenty-fold with 50 mM Tris/acetate, pH 7.4, incubated at 37° C. for 30 minutes, and centrifuged at 100,000 g for 15 minutes. The dilution, incubation and centrifugation were repeated a total of three times. Prior to use in the NMDA-specific L-[3H]-glutamate binding assay, the SPM were thawed, diluted twenty fold with 50 mM Tris/acetate (pH 7.4 containing 0.04% (v/v) Triton X-100), incubated for 30 minutes at 37° C. and centrifuged as described above. The Triton X-100 treated membranes were washed with 50 mM Tris/acetate (pH 7.4) and centrifuged at 100,000 g for 15 minutes a total of four times. The basic procedure for the receptor subclass binding assays was similar. This general method involved adding the radioligand (12.5 nM L-[3H]-glutamate; 0.5 nM [3H]kainate or 10nM [3H]AMPA) to the appropriate concentration of the test compound and initiating the assay by the addition of ice cold synaptic plasma membranes (0.2-0.45 mg). The binding assays were performed in 1.5 mL centrifuge tubes with the total volume adjusted to 1.0 mL. Additions of test compounds were made in 50 mM Tris/acetate (pH 7.4) and incubations were carried out at 0°-4° C. The incubation time for each of the NMDA and the AMPA binding assays was 10 minutes, for the kainate binding assay 60 minutes and for the sodium-dependent glutamate binding assay 15 minutes. The AMPA binding assay contained 100 mM KSCN and the sodium-dependent glutamate binding assay contained 150 mM sodium acetate in addition to the previously described reagents. To terminate the incubation, the samples were centrifuged for 15 minutes at 12,000 g and 4° C. in a Beckman Microfuge 12. The supernatant was aspirated and the pelleted membranes dissolved in Beckman BTS-450 tissue solubilizer for a minimum of 6 hours at room temperature. Beckman MP scintillation cocktail containing 7 mL/L acetic acid was then added and the samples counted on a Beckman LS 5800 or 3801 liquid scintillation counter with automatic corrections for quenching and counting efficiency. Nonspecific binding was defined as the residual binding in the presence of either excess L-glutamate (0.1-0.4 mM), kainate (0.01 mM), or NMDA (0.5 mM), and was 15-25% of the total binding in the NMDA binding assay, 19-27% in the AMPA binding assay, 20-30% in the kainate binding assay and 10-15% in the sodium-dependent binding assay. Radioligand binding to the synaptic plasma membranes was analyzed using Scatchard and Hill transformations and the Ki values of the compounds determined using logit-log transformations. Calculations and regression analysis were performed using templates developed for Lotus 1, 2, 3 as previously described [Pullan, L. M. "Automated Radioligand Receptor Binding Analysis with Templates for Lotus", Computer Appln. Biosci., 3 131 (1987)]. Binding results are reported in Table II for example compounds of the invention. Included in Table II are binding data for D,L-AP7[D,L-2-amino-7-phosphonoheptanoic acid]. Synaptic plasma membranes (SPM) were prepared as previously described Monahan, J Protein concentrations The SPM were treated identically for the [3H]AMPA (QUIS), [3H]kainate and sodium-dependent L-[3H]-glumatate binding assays were thawed at room temperature diluted twenty-fold with 50 mM Tris/acetate, pH 7.4 centrifuged at 100,000 g for 15 minutes diluted twenty fold with 50 mM Tris/acetate (pH 7.4 containing 0.04% (v/v) Triton X-100) incubated for 30 minutes at 37° C. The Triton X-100 treated membranes were washed with 50 mM Tris/acetate (pH 7.4) centrifuged at 100,000 g for 15 minutes This general method involved adding the radioligand (12.5 nM L-[3H]-glutamate; 0.5 nM [3H]kainate or 10nM [3H]AMPA) to the appropriate concentration of the test compound by the addition of ice cold synaptic plasma membranes (0.2-0.45 mg) were carried out at 0°-4° C was 10 minutes, for the kainate binding assay 60 minutes and for the sodium-dependent glutamate binding assay 15 minutes in addition to the previously described reagents To terminate the incubation the samples were centrifuged for 15 minutes at 12,000 g and 4° C. in a Beckman Microfuge 12 the pelleted membranes dissolved in Beckman BTS-450 tissue solubilizer for a minimum of 6 hours at room temperature Beckman MP scintillation cocktail containing 7 mL/L acetic acid was then added for quenching Binding results

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: FIG. 19 contains two graphs of data indicating pregnenolone sulfate (PS) differentially modulate NR1011/2A and NR1111/2A splice variants at pH 8.5. Data points are averaged normalized peak NMDA-induced current responses obtained from oocytes injected with (A) NR1011/2A or (B) NR1111/2A mRNAs. Concentration-response data for NMDA (circles) and for NMDA+100 μM PS (squares) were obtained in the presence of 10 μM glycine. Fitted parameters are (A) (control ◯, n=11), EC50=94 μM, Emax=1.30, nH=1.62; (+PS, open squares, n=10), EC50=58 μM, Emax=1.62, nH=1.44; (B) (control, closed circles, n=10), EC50=110 μM, Emax=1.35, nH=1.93; (+PS, closed squares, n=23), EC50=94 μM, Emax=1.98, nH=1.82. The data were normalized relative to the current induced by co-application of 200 μM NMDA and 10 μM glycine to the same oocyte. Error bars represent S.E.M. obtained from oocytes Concentration-response data for NMDA (circles)