IMIPRAMINE HYDROCHLORIDE IMIPRAMINE PAMOATE CHEMBL11 Imipramin BDBM50010859
Imipramine Presamine [3-(10,11-Dihydro-dibenzo[b,f]azepin-5-yl)-propyl]-dimethyl-amine; hydrochloride BDBM50028282 Janimine Pramine IMIPRAMINE HYDROCHLORIDE CHEMBL1692 Tofranil
- Martin, AR; Paradkar, VM; Peng, GW; Speth, RC; Yamamura, HI; Horn, AS Conformationally restricted tricyclic antidepressants. 1. Octahydrodibenzazepinonaphthyridines as rigid imipramine analogues. J Med Chem 23: 865-73 (1980)
- Eberlein, WG; Trummlitz, G; Engel, WW; Schmidt, G; Pelzer, H; Mayer, N Tricyclic compounds as selective antimuscarinics. 1. Structural requirements for selectivity toward the muscarinic acetylcholine receptor in a series of pirenzepine and imipramine analogues. J Med Chem 30: 1378-82 (1987)
- ChEBML_88911 Inhibition of binding of [3H]imipramine to imipramine receptor in rat brain
- ChEMBL_88911 (CHEMBL699813) Inhibition of binding of [3H]imipramine to imipramine receptor in rat brain
- ChEMBL_762027 (CHEMBL1816019) Displacement of [3H]imipramine from human 5HT transporter
- ChEMBL_1468619 (CHEMBL3413565) Displacement of [3H]imipramine from human SERT expressed in CHO cells
- ChEMBL_647228 (CHEMBL1217369) Displacement of [3H]imipramine from human SERT expressed in HEK293 cells
- ChEMBL_1361025 (CHEMBL3295403) Displacement of [3H]imipramine from human recombinant SERT expressed in CHO cells
- ChEMBL_643648 (CHEMBL1212512) Displacement of [3H]imipramine from human recombinant 5HT transporter expressed in CHO cells
- ChEMBL_675983 (CHEMBL1272921) Displacement of [3H]imipramine from human recombinant SERT receptor expressed in HEK293 cells
- ChEBML_201359 Equilibrium dissociation constant (KD) for Competitive binding between [3H]- imipramine and the compound at human transporter -hSERT
- ChEMBL_1559992 (CHEMBL3777270) Displacement of [3H]imipramine from human recombinant 5-HT transporter expressed in CHO cells
- ChEMBL_881850 (CHEMBL2211559) Displacement of [3H]imipramine from human SERT expressed in CHO cells by competition binding assay
- ChEMBL_201359 (CHEMBL804694) Equilibrium dissociation constant (KD) for Competitive binding between [3H]- imipramine and the compound at human transporter -hSERT
- ChEMBL_580516 (CHEMBL1053230) Displacement of [3H]imipramine from human serotonin transporter expressed in CHO cell membrane by scintillation counting
- ChEMBL_1864055 (CHEMBL4365030) Displacement of [3H]imipramine from recombinant human serotonin transporter after 120 mins by scintillation counting analysis
- ChEMBL_1978281 (CHEMBL4611416) Displacement of [3H]imipramine from SERT receptor (unknown origin) by cell based radioligand competitive binding analysis
- ChEBML_1711467 Displacement of [3H]-imipramine from recombinant human 5-HT transporter after 60 mins by scintillation counting relative to control
- ChEMBL_2145510 (CHEMBL5029790) Displacement of [3H]-imipramine from recombinant human 5-HT transporter after 60 mins by scintillation counting analysis
- ChEMBL_617082 (CHEMBL1100588) Displacement of [3H]imipramine from human SERT expressed in HEK293 cells after 30 mins by rapid filtration assay
- ChEBML_1690522 Displacement of [3H]-imipramine from human serotonin transporter expressed in HEK-293 cell membranes after 30 mins by scintillation counting
- ChEMBL_1767582 (CHEMBL4219694) Binding affinity to human SERT expressed in HEK293 cells after 60 mins in presence of [3H]imipramine by liquid scintillation counting
- ChEMBL_1767583 (CHEMBL4219695) Binding affinity to human NET expressed in HEK293 cells after 60 mins in presence of [3H]imipramine by liquid scintillation counting
- ChEMBL_1767584 (CHEMBL4219696) Binding affinity to human DAT expressed in HEK293 cells after 60 mins in presence of [3H]imipramine by liquid scintillation counting
- ChEMBL_1772964 (CHEMBL4229956) Displacement of [3H]imipramine from recombinant human SERT expressed in CHO cells after 60 mins by scintillation counting method
- ChEMBL_1848698 (CHEMBL4349239) Displacement of [3H]Imipramine from human 5-HT transporter expressed in HEK-293 cell membranes after 30 mins
- ChEMBL_1894262 (CHEMBL4396183) Displacement of [3H] imipramine from human recombinant 5-HT transporter measured after 60 mins by scintillation counter method
- ChEMBL_1515630 (CHEMBL3616095) Displacement of [3H]-imipramine from human serotonin transporter expressed in HEK293 cells after 30 mins by liquid scintillation counting analysis
- ChEMBL_1633618 (CHEMBL3876410) Displacement of [3H]imipramine from human recombinant 5HT transporter expressed in CHO cells measured after 60 mins by scintillation counting method
- ChEMBL_1814789 (CHEMBL4314363) Displacement of [3H]-imipramine from human serotonin transporter expressed in HEK293 cells membranes incubated for 30 mins by microbeta scintillation counting analysis
- ChEMBL_2070180 (CHEMBL4725714) Displacement of [3H]imipramine from recombinant human 5'-HT transporter expressed in CHO cells incubated for 60 mins by Scintillation counting method
- Binding Assay 5-HT transporter binding assay for evaluating binding of the compound to the serotonin transporter was carried out using human recombinant serotonin transporter membrane (PerkinElmer Life and Analytical Sciences, USA) expressed in HEK293 cells and radioisotope [3H]Imipramine (PerkinElmer).That is, the test drug, 2 nM [3H]Imipramine, serotonin transporter membrane (9 ug/well), 120 mM NaCl and 50 mM Tris-HCl buffer (pH 7.4) containing 5 mM KCl were added to obtain a reaction mixture with a final volume of 0.25 ml. After incubation for 30 minutes at 27° C., the mixture was quickly passed through a Filtermat A glass fiber filter (PerkinElmer) pre-soaked with 0.5% (w/v) PEI (polyethyleneimine) using Inotech Harvester (Inotech) to terminate the reaction. After washing with cold washing buffer (50 mM Tris-HCl, pH 7.4, 154 mM NaCl) solution, the filter was covered with MeltiLex and sealed in a sample bag. After drying in an oven, radioactivity was counted using MicroBeta Plus (Wallac).
- Binding Assay For serotonin transporter binding assays, a reaction mixture with a final volume of 0.25 ml was prepared by mixing a test compound, human serotonin transporter membrane expressed in HEK-293 cells (PerkinElmer, 5 ug/well), [3H]Imipramine (PerkinElmer, 2 nM) and 50 mM Tris-HCl (pH 7.4) buffer containing 120 mM NaCl and 5 mM KCl. The reaction mixture was incubated for 30 min at 27° C., and harvested through filtermate A glass fiber filter presoaked in 0.5% PEI with ice cold 50 mM Tris-HCl buffer (pH 7.4) containing 0.9% NaCl.
- In vitro Binding Affinity Assay The assay was performed in accordance with the method described by Owens et al. with slight modifications. Rat cerebral cortex was homogenized in 30 volumes of ice-cold 50mM Tris-HCl containing 150mM NaCl and 5mM KCl, pH 7.7, at 25°C and centrifuged at 20,000 x g for 20 min. Pellet was resuspended in 30 volumes of buffer and centrifuged again for 2 more times. The mixture of 240uL of the tissue suspension, 30uL of 1µM imipramine, 30uL of 1nM [3H]-citalopram and 100µL of the analyzed compound (10^-10 to 10^-4M) were incubated at 22°C for 1 h. Incubations were terminated by vacuum filtration and washed twice with 100µL of ice-cold buffer. The radioactivity was measured using a WALLAC 1409 DSA liquid scintillation counter.
- Radioligand Binding Assay (Ki) and Inhibition of Substrate Uptake (EC50/IC50) Binding affinity of each compound was measured by assessing the potency of inhibition of binding of radiolabeled RTI-55. Membranes were preincubated with compound before the addition of [125I]RTI-55. The reaction was terminated by filtration through Whatman GF/C filters using a 96-well Tomtech cell harvester (Tomtech, Orange, CT). Scintillation fluid was added to each filter spot and radioactivity remaining on the filter was determined using a Wallace beta-plate reader. IC50 values were converted to Ki values using the Cheng-Prusoff equation. Specific binding was defined as the difference in binding observed in the presence and absence of 5 uM mazindol (HEK-hDAT and -NET) or 5 uM imipramine (HEK-hSERT). EC50/IC50 values were obtained from inhibition of the reuptake of [3H] dopamine for DAT, [3H] serotonin for SERT, or [3H] norepinephrine for NET.
- Functional Uptake Assay (hNET) Inhibition of human norepinephrine reuptake transporter was assayed using the recombinant human norepinephrine transporter expressed in either HEK293 or MDCK cells using a published method (Galli A et al., J. Exp. Biol. 198: 2197-2212, 1995). The cells were plated before the assay. Test compound and/or vehicle was preincubated with cells in modified HEPES buffer pH 7.1 or pH 7.4 for 20 minutes at 18 to 25° C. Following the preincubation, 25 nM [3H]norepinephrine was added for an additional timed incubation period (10 to 20 minutes). After the cells were washed to remove [3H]norepinephrine not internalized, the cells were lysed, and the amount of tritium in the cell lysate was measured using a liquid scintillation counter to determine [3H]norepinephrine uptake. Non-specific binding of tritium was measured in a control reaction containing 10 uM imipramine (or 10 uM nisoxetine), and was subtracted from the counts for assays to correct for non-specific binding of tritium.
- Inhibition Assay Master solutions were prepared containing human liver microsomes (Gibco, 0.2 mg/mL) and MgCl2 (5 mM) in potassium phosphate buffer (10 mM). To aliquots (169 μL) of the microsome solution was added test compound in acetonitrile (1 μL) and DMSO (1 μL) to provide final test compound concentrations of 0, 0.005, 0.05, 0.25, 1, 5, 10, and 25 μM.NADPH (10 mM) in ultra-pure water (20 μL) was added, and this mixture was incubated at 37° C. for 30 minutes. The enzyme reaction then was initiated by the addition of enzyme substrate (dextromethorphan) dissolved in 1 μL of acetonitrile and 9 μL of ultra-pure water. The final substrate concentration was 10 μM.After 20 minutes, the incubation mixture was diluted with 3 volumes of cold methanol containing imipramine (200 nM), labetalol (200 nM), and ketoprofen (2 μM) as internal standards. Samples were centrifuged at 16,000 g for 10 minutes, then an aliquot of the supernatant (200 μL) was removed and a
- Rat Synaptosome Binding Assay The synaptosomes (150 μg) prepared from a rat brain were incubated at 37° C. for 15 minutes with 0.1 μCi [3H]5-HT in the absence or presence of the test compound or the reference compound in a buffer solution containing 106.2 mM NaCl, 4.5 mM KCl, 2.25 mM MgSO4, 1.08 mM NaH2PO4, 22.5 mM NaHCO3, 9.9 mM glucose, 9 μM EGTA and 45 μM ascorbic acid (pH 7.4).The basal control activity was determined by incubating the same mixture at 4° C. for 15 minutes in the presence of 10 μM imipramine to block the uptake of 5-HT, which was taken as the standard reference compound and tested in each experiment at several concentrations to obtain an inhibition curve.Following the incubation, the samples were filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) and rinsed twice with an ice-cold incubation buffer using a 96-sample cell harvester (Unifilter, Packard) to eliminate free [3H]5-HT. The filters were dried and the retained radioactivity was measured in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard). The experimental results were expressed as a percent inhibition of the control uptake of [3H]5-HT.Data AnalysisInhibition of serotonin transporter in rat synaptosome was measured by the concentrations of [3H]5-HT. The test compounds were required to be tested at least twice in the case of the concentration thereof being greater than 6 log, and the obtained data were subjected to a nonlinear regression analysis via a curve of Hill equation, to obtain IC50 value.
- hSERT and rVMAT2 Fluorometric Screening Assay For both hSERT and rVMAT2 screening experiments, respective singly transfected cells were seeded at a density of 0.09×106 cells/well in poly-D-Lysine (Alamanda Polymers, Inc.) coated white solid-bottom 96-well plates (Costar). Growth was permitted for approximately 44 hours in said aqueous media and at an incubation environment of 37° C. and 5% Carbon Dioxide. At the beginning of the experiment, the cellular growth solution was aspirated, and individual cells were rinsed with 150 μL of 1×Dulbecco's Phosphate Buffered Saline (PBS; HyClone). 63 μL of Experimental Media (consisting of the following contents: DMEM without phenol red but with 4.5 g/L of D-Glucose (Gibco), 1% (v/v) FBS (Atlanta Biologicals), 100 U/mL Penicillin (Gibco), and 10 μg/mL Streptomycin (Gibco)) with 2×tiered concentrations of inhibitor (or DMSO, the vehicle of these experiments) were added to the respective wells. Control inhibitors used in these studies include Imipramine for hSERT experiments, and Reserpine for rVMAT2 experiments (Eiden, L. E. and Weihe, E. 2011; Sette, M. et al. 1983). At the conclusion of the pre-incubation period (60 minutes for hSERT experiments and 30 minutes for rVMAT2 experiments), 63 μL of Experimental Media containing 2×various concentrations of tested inhibitor (or vehicle) along with a specified amount of fluorescent substrate, APP+ (Karpowicz, R. J. et al 2013) (final concentration: 1.1 μM for hSERT experiments) or FFN206 (Hu, G. et al. 2013) (final concentration: 0.75 μM for rVMAT2 experiments) were added to the present solution contained within the wells. After a required incubation period (30 minutes for hSERT experiments and 60 minutes for rVMAT2 experiments) for proper fluorescent probe uptake, the contents of each well were aspirated and consequently, rinsed twice with 120 μL of PBS. A final solution of 120 μL of PBS is finally added to all corresponding wells for cell maintenance before undergoing fluorescence uptake reading by a BioTek H1MF plate reader.