The metabolic stability was evaluated by incubation of compounds with human liver microsomes (Sigma-Aldrich, St. Louis, MO, USA) in 10 mM Tris–HCl buffer (pH 7.4) at 37 °C for 120 min in the presence of NADPH Regeneration System (Promega, Madison, WI, USA). The percentage of the remaining substrate and the most probable metabolic pathways were estimated by UPLC-MS analyses of the reaction mixtures.
Nadph regeneration system
Manufactured by Promega
Sourced in United States
The NADPH Regeneration System is a laboratory tool designed to maintain the availability of NADPH, an essential cofactor for various enzymatic reactions. This system provides a constant supply of NADPH, enabling researchers to conduct experiments that require sustained NADPH levels.
Automatically generated - may contain errors
Lab products found in correlation
Acquity uplc, by Waters Corporation (2 mentions)
Tqd mass spectrometer, by Waters Corporation (2 mentions)
Tq detector, by Waters Corporation (2 mentions)
P450 glo assay, by Promega (2 mentions)
Lumistar optima, by BMG Labtech (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Human liver microsomes, by Merck Group (1 mentions)
Nunc microwell 96 well microplate, by Thermo Fisher Scientific (1 mentions)
Cyp2d6 p450 glo, by Promega (1 mentions)
P450 glo cyp3a4, by Promega (1 mentions)
Luciferin detection reagent, by Promega (1 mentions)
Enspire, by PerkinElmer (1 mentions)
Tsq vantage triple stage quadrupole mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Glomax multi detection system, by Promega (1 mentions)
Quinidine, by Merck Group (1 mentions)
Potassium chloride (kcl), by Carl Roth (1 mentions)
Udpga, by Merck Group (1 mentions)
Glycerol, by Carl Roth (1 mentions)
Tris buffer, by Carl Roth (1 mentions)
19 protocols using nadph regeneration system
1
Metabolic Stability Evaluation of Compounds
2
CYP3A4 and CYP2D6 Enzymatic Assay
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The experiments were provided using commercially available luminescent CYP3A4 P450-Glo™ and CYP2D6 P450-Glo™ tests purchased from Promega (Madison, WI, USA). The enzymatic reactions were conducted in polystyrene, flat-bottom Nunc™ MicroWell™ 96-well microplates (Thermo Scientific, Waltham, MA, USA). The assays were carried out according to the procedures provided by the manufacturer, as described previously [38 (link),41 (link),42 (link)]. Compounds were tested in triplicate at the final concentrations in a range from 0.01 to 25 µM for both isoforms of CYP450. The references of CYP3A4 and CYP2D6 inhibitors (ketoconazole and quinidine, respectively) were tested in a range from 0.001 to 10 µM. Tested compounds were incubated in 100 mM Tris-HCl buffer separately with CYP3A4 and CYP2D6 membranes and the NADPH Regeneration System for 30 min at room temperature in triplicate. The bioluminescent signal was measured after the addition of the Luciferin Detection Reagent by using a microplate reader EnSpire PerkinElmer (Waltham, MA, USA). Both reagents (NADPH Regeneration System and Luciferin Detection Reagent) were purchased from Promega (Madison, WI, USA).
3
In Vitro Metabolic Profiling of Compounds
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Each compound (final 1 μM, duplicated) was added to each 0.1 M PBS solution (pH 7.4) containing liver microsome of three different species (human, dog, mouse). After a brief (5 m) incubation at 37 °C, the NADPH regeneration system (Promega Corp., Madison, WI, USA) was added, then the mixture was incubated at 37 °C for 30 m. The reaction was terminated by adding acetonitrile containing an internal standard (chlorpropamide), then the mixture was centrifuged at 15,000 rpm, 4 °C for 5 m. The supernatant solution was injected to LC/MS/MS system (TSQ Vantage Triple-Stage Quadrupole Mass Spectrometer, ThermoFisher Scientific, Waltham, MA, USA). The remaining substrates were analyzed with Xcalibur 4.4 s/w using an MRM (multiple reaction monitoring) quantitation mode.
4
BVR Activity Assay Protocol
5
Liver CYP3A Enzymatic Activity Assay
6
Metabolic Stability Evaluation of Compounds
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The in
vitro evaluation of the metabolic stability of the selected compound
was performed using human liver microsomes (HLMs) (Sigma-Aldrich,
St. Louis, MO, USA) as described previously.25 (link) The reaction mixture contained 50 μM of tested molecules HLMs
(1 mg/mL) in 10 mM tris–HCl buffer. Following 5 min of preincubation,
50 μL of the NADPH Regeneration System (Promega, Madison, WI,
USA) was added to induce the reaction. Next, the resulting reaction
mixture was incubated at 37 °C for 120 min. The reaction was
quenched with the addition of 200 μL of cold extra pure methanol
and then centrifuged at 14,000 rpm for 15 min. The resulting supernatant
was analyzed using an LC/MS Waters ACQUITY TQD system (Waters, Milford,
USA).
vitro evaluation of the metabolic stability of the selected compound
was performed using human liver microsomes (HLMs) (Sigma-Aldrich,
St. Louis, MO, USA) as described previously.25 (link) The reaction mixture contained 50 μM of tested molecules HLMs
(1 mg/mL) in 10 mM tris–HCl buffer. Following 5 min of preincubation,
50 μL of the NADPH Regeneration System (Promega, Madison, WI,
USA) was added to induce the reaction. Next, the resulting reaction
mixture was incubated at 37 °C for 120 min. The reaction was
quenched with the addition of 200 μL of cold extra pure methanol
and then centrifuged at 14,000 rpm for 15 min. The resulting supernatant
was analyzed using an LC/MS Waters ACQUITY TQD system (Waters, Milford,
USA).
7
Hepatic Biotransformation of H3R Ligands
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Commercial, pooled, human (adult male and female) liver microsomes (HLMs) were purchased from Sigma-Aldrich. The biotransformations were conducted using 1 mg/mL of HLMs in 200 µL of reaction buffer containing 0.1 M Tris–HCl (pH 7.4) and the respective test compound (H3R ligand) with a final volume of 50 µM. The reaction mixture was preincubated at 37°C for 5 min, and then, the reaction was commenced by adding 50 µL of NADPH Regeneration System (Promega, Madison, WI, USA). The reaction was ended after 120 min by the addition of cold methanol (200 µL). The mixture was next centrifuged at 14,000 rpm for 15 min, and the ultra-performance liquid chromatography–mass spectrometry (UPLC/MS) analysis of the supernatant was performed. Mass spectra were recorded on UPLC/MS system that consisted of a Waters Acquity UPLC (Waters, Milford, CT, USA), joined to a Waters TQD mass spectrometer (electrospray ionization mode ESI-tandem quadrupole). The in silico investigation was achieved by MetaSite 4.1.1 provided by Molecular Discovery Ltd, and the probability sites with the highest metabolism were analyzed during this study by liver computational model.
8
Rat Liver Microsome Biotransformation Assay
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Commercial, pooled, rat liver microsomes (RLMs) were purchased from Sigma-Aldrich (St. Louis, USA). The biotransformations were carried out using 1 mg/mL of RLMs in 200 μL of reaction buffer containing 0.1 M Tris-HCl (pH 7.4) and EMD386088 with final volume 50 μM. The reaction mixture was preincubated at 37 °C for 5 min and then, the reaction was initiated by adding 50 μL of NADPH Regeneration System (Promega, Madison, WI, USA). The reaction was terminated after 30 or 120 min by the addition of 200 μL of cold methanol. The mixture was next centrifuged at 14000 rpm for 15 min and the UPLC/MS analysis of the supernatant was performed. Mass spectra was recorded on UPLC/MS system consisted of a Waters Acquity UPLC (Waters, Milford, USA), coupled to a Waters TQD mass spectrometer (electrospray ionization mode ESI-tandem quadrupole).
9
In Vitro Inhibition of CYP2D6 Enzyme
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Chemicals and reagents. Quinidine (25 ) was purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA) as European Pharmacopoeia (EP) Reference Standard. The compounds that were selected for in vitro testing were kindly provided from the Department of Pharmacognosy, University of Innsbruck, Austria, i.e. 1 –3 , and the Griffith Institute for Drug Discovery, Griffith University, Australia, i.e. 4 –7 and 21 –23 . Compounds 8 –20 and 24 were purchased from SPECS (Zoetermeer, NL). The purity of the compounds that were purchased from SPECS was > 95% for 8 compounds i.e. 9 , 10 , 12 , 13 , 17 , 19, 20 and 24 , 95% for compound 8 and 90% for 5 compounds i.e. 11 , 14 , 15 , 16 and 18 . More information on the compounds from the university sources can be found in the Supplementary data section. ME-luciferin-EGE, luciferin-EGE, NADPH-regeneration system and the luciferin detection reagent with esterase were purchased from Promega Corporation (Madison, WI, USA). Recombinant human CYP2D6 BACULOSOMES Plus Reagent came from Life Technologies (Carlsbad, CA, USA). All other reagents were of the highest purity commercially available or at least HPLC grade.
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Chemicals and reagents. Quinidine (
10
Propranolol Metabolite Synthesis and Analysis
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Tris buffer, potassium chloride, glycerol and Triton X-100 were sourced from Carl Roth GmbH & Co. KG (Karlsruhe, Germany); UDPGA, (±)-propranolol, (R)-propranolol, and (±)-4-hydroxypropranolol were obtained from Sigma-Aldrich (Taufkirchen, Germany); (±)-5-hydroxypropranolol was purchased from Cayman Chemical Company (Ann Arbor, MI, USA); the NADPH regeneration system was sourced from Promega Corporation (Madison, WI, USA); and 4-methoxypropranolol was synthesized in-house as described previously [30 (link),31 (link),32 (link)]. Ammonium hydrogen carbonate was purchased from VWR International GmbH (Darmstadt, Germany); and phosphate buffered saline (PBS, pH 7.4) was purchased from VWR Chemicals LLC (Solon, OH, USA).
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