Human CYP3A4 expressed in Supersomes TM (CYP3A4-Supersomes TM ), microsomes isolated from insect cells transfected with a baculovirus construct containing cDNA of human CYP3A4, NADPH:cytochrome P450 oxidoreductase (POR) and cytochrome b5 (molar ratio of CYP3A4 to cytochrome b5 of 1 to 5), were purchased from Corning (Tewksbury, MA, USA). The enzymatic activity of the experimental CYP3A4-Supersomes TM system used was verified by studying its efficiency to catalyze 6--testosterone hydroxylation, a marker reaction for CYP3A4 (see below).
Supersome
Manufactured by Corning
Sourced in United States
Supersomes are a specialized type of microsomal preparation derived from insect cells that have been engineered to express specific human drug-metabolizing enzymes. They provide a standardized, reproducible system for evaluating the metabolism of drugs and other xenobiotics by individual human cytochrome P450 enzymes.
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Lab products found in correlation
Methanol, by Avantor (2 mentions)
Mes hydrate, by Merck Group (2 mentions)
Atp magnesium salt, by Merck Group (2 mentions)
Ammonium acetate, by Merck Group (2 mentions)
Sulfasalazine, by Merck Group (2 mentions)
Sodium orthovanadate, by Merck Group (2 mentions)
Itaq supermix, by Bio-Rad (2 mentions)
Nanodrop 2000 uv vis spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Qiazol, by Qiagen (2 mentions)
Indinavir, by Merck Group (1 mentions)
Nelfinavir, by Merck Group (1 mentions)
Ritonavir, by Merck Group (1 mentions)
Sulfasalazine, by Avantor (1 mentions)
Amprenavir, by Merck Group (1 mentions)
Saquinavir mesylate, by Merck Group (1 mentions)
Sodium orthovanadate, by Avantor (1 mentions)
Amp disodium salt, by Merck Group (1 mentions)
Trizma base, by Merck Group (1 mentions)
14 protocols using supersome
1
Characterization of Recombinant Human CYP3A4
2
Cytochrome P450 Enzyme Inhibition Assay
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Potassium phosphate monobasic, potassium phosphate dibasic, albendazole, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Acetonitrile, Optima™ LC/MS grade was purchased from Thermo Fisher (Waltham, MA, United States). Supersome™ containing human CYP3A4 (1 nmol/ml), oxidoreductase, and cytochrome b5 (product # 456202; lots 5070002, 6161001, and 9037004), Supersome™ containing human CYP3A7 (1 nmol/ml), oxidoreductase, and cytochrome b5 (product # 456237; lots 732001, 8045003, 8107003, 8249002, and 9218002), NADPH Regenerating System, Solution A (product # 451220), and Solution B (product # 451200), and low profile Axygen® single well reagent reservoir with 384-bottom troughs were purchased from Corning Inc. (Corning, NY, United States). MCA 96 nested 200 μl (product # 30038619) and 50 μl (product # 30038609) disposable tips were purchased from Tecan (Männedorf, Switzerland). 384-Well polypropylene sample collection plate of 250 µl (product # 186002632), and 100 µl (product # 186002631) were purchased from Waters (Milford, MA, United States).
3
Baculovirus-Expressed BCRP Transporter Assay
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The baculovirus-infected insect cell microsomes (Supersomes) containing human complementary DNA-expressed BCRP (Arg482, 5 mg protein/mL) and wildtype Supersomes without hBCRP (control membrane, 5 mg protein/mL) used as negative control were obtained from Corning (Amsterdam, The Netherlands). After delivery, Supersomes were thawed at 37 °C, aliquoted, snap-frozen in liquid nitrogen, and stored at -80 °C until use.
AMP disodium salt, ADP sodium salt, ATP magnesium salt, guanosine 5'diphospate (GDP) sodium salt, uridine 5'-phosphate (UDP) sodium salt hydrate, sulfasalazine, sodium orthovanadate, amprenavir, indinavir, nelfinavir, ritonavir, saquinavir mesylate, ammonium acetate, MES hydrate, and Trizma base were obtained from Sigma-Aldrich (Taufkirchen, Germany), formic acid (MS grade) from Fluka (Neu-Ulm, Germany), acetonitrile, methanol (both LC-MS grade), and all other chemicals from VWR (Darmstadt, Germany).
Stock solutions were prepared in bidistilled water for sodium orthovanadate (10 mM), AMP, ADP, ATP, GDP, and UDP (20 mM, respectively) or in methanol for sulfasalazine (0.5 mg/mL), amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir (1 mg/mL, respectively). Stock solutions were aliquoted and stored at -20 °C until use.
AMP disodium salt, ADP sodium salt, ATP magnesium salt, guanosine 5'diphospate (GDP) sodium salt, uridine 5'-phosphate (UDP) sodium salt hydrate, sulfasalazine, sodium orthovanadate, amprenavir, indinavir, nelfinavir, ritonavir, saquinavir mesylate, ammonium acetate, MES hydrate, and Trizma base were obtained from Sigma-Aldrich (Taufkirchen, Germany), formic acid (MS grade) from Fluka (Neu-Ulm, Germany), acetonitrile, methanol (both LC-MS grade), and all other chemicals from VWR (Darmstadt, Germany).
Stock solutions were prepared in bidistilled water for sodium orthovanadate (10 mM), AMP, ADP, ATP, GDP, and UDP (20 mM, respectively) or in methanol for sulfasalazine (0.5 mg/mL), amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir (1 mg/mL, respectively). Stock solutions were aliquoted and stored at -20 °C until use.
4
In Vitro CYP Inhibition Assay
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Inhibitory effects of the test drugs at 10 µM a g a i n s t 8 h u m a n P 4 5 0 s ( C Y P 1 A 1 , C Y P 1 A 2 , CYP1B1, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) were determined using the P450-Glo CYP1A1 Assay, P450-Glo CYP1A2 Induction/Inhibition assay, P450-Glo CYP1B1 Assay, P450-Glo CYP2B6 Assay, P450-Glo CYP2C9 Assay, P450-Glo CYP2C19 Assay, P450-Glo CYP2D6 Assay, and P450-Glo CYP3A4 Assay with Luciferin-IPA (Promega, Madison, WI, USA), as described previously (Watanabe et al., 2020) , with minor modifications. As enzyme sources, supersomes (Corning, Corning, NY, USA) were used and a brief summary of the reactions is presented in Table S2. The final DMSO concentration in the reaction mixture was < 0.1%, and equivalent amounts of DMSO were added to the vehicle control reactions. Typical P450 inhibitors (Table S2) were used to confirm assay conditions (Watanabe et al., 2020) . The test drugs were used at a constant concentration to compare their inhibitory activity under the same conditions, and 10 µM was selected because it was the highest concentration to be prepared without influence of DMSO on the enzymatic activity.
5
In Vitro Cytochrome P450 Assays
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Most microsomal preparations containing individual P450 enzymes were Supersomes®, the products of BD Gentest, formerly a part of Corning Life Sciences (Tewksbury, MA, USA). In the present study, we used preparations containing CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2E1, CYP3A4, and CYP3A5. All those preparations contained human CPR and cytochrome b5 co-expressed. The preparation of insect cell microsomes containing human CYP2D6 along with human CPR and cytochrome b5 (Baculosomes®) was the product of Thermo Fisher Scientific (Waltham, MA, USA).
6
In Vitro Metabolism of Telmisartan and Paclitaxel
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Chemicals Telmisartan and reduced nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Paclitaxel and fluvastatin were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Irbesartan, which was used as an internal standard (IS) for the determination of the fluvastatin metabolites, and candesartan were obtained from Astatech (Bristol, PA, U.S.A.). Docetaxel trihydrate, which was used as an IS for the determination of 6α-hydroxyPaclitaxel, and 6α-hydroxyPaclitaxel were purchased from Toronto Research Chemicals (Toronto, Canada) and Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.), respectively. Supersomes™ from insect cells that expressed recombinant human CYP2C9 (rCYP2C9)*1 (lot: 4141001), rCYP2C9*2 (lot: 5056008), rCYP2C9*3 (lot: 4203003), and control microsome (lot: 5217001), along with the 150-donor pooled human liver microsomes (pooled HLMs, lot: 38291) and monoclonal antibody for human CYP2C8 (MAB-2C8) derived from mouse were all obtained from Corning (Woburn, MA, U.S.A.). Metabolites of fluvastatin (M-2, M-3, and M-5) were kindly provided by Novartis Pharma (Tokyo, Japan). Other reagents and solvents were of either HPLC or special grades.
7
Quantification of Hepatic Cytochrome P450 Enzymes
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Liver tissue cells (25mg) were lysed using Qiazol (Qiagen), sonicated until completely homogenized, and then centrifuged at 10,000 × g for 12 minutes. Phase separation and binding of the mRNA was completed using a Qiagen RNEasy Kit. RNA purity and concentration was measured on a NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Scientific; Wilmington, DE) and RNA was diluted to 500 ng/μL for each sample. To ensure sample purity, only samples with an A260/A280 ratio between 1.9–2.1 were evaluated for gene expression. The isolated RNA was mixed with iScript™ cDNA Synthesis Kit (Biorad) and incubated at 25°C × 5 min, 42°C × 30 min, and then 85°C × 5 min to synthesize cDNA. The cDNA was mixed with iTaq™ Supermix (BioRad) and TaqMan Gene Expression Assay for CYP3A4, 3A5, 3A7, 2C9, 2C19, FMO1, FMO3 and 18s (Applied Bio-systems). Expression for each enzyme gene was measured (quantitative rt-PCR) and normalized to 18s rRNA. For protein quantitation of CYP3A and CYP2C families (signature peptide sequences shown in Supplemental Table S2 ), HLM were subjected to LC-MRM-based targeted quantitative proteomic analysis using recombinant proteins (Supersomes™; Corning Gentest, Woburn, MA) of known concentration as calibration standards (14 (link), 16 (link), 18 (link)). A more detailed description of the targeted quantitative proteomic methods was previously published (16 (link), 19 ).
8
Quantification of Hepatic Cytochrome P450 Enzymes
9
CES2-Mediated TAG and DAG Lipase Activity
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Supersomes transduced with human CES2 (Corning; #453322) or negative control Supersomes (Corning; #456200) were utilized in TAG lipase activity assays as previously described (Ahmadian et al., 2011 (link)). DAG lipase activity assay was assessed with cold and 14C-labeled diolein. Reactions were terminated, and lipids were extracted and separated by thin-layer chromatography as previously described (Massart et al., 2014 (link)).
10
Baculovirus-Insect Cell Microsomes for BCRP
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Baculovirus-infected insect cell microsomes (Supersomes) containing complementary DNA-expressed hBCRP (Arg482, 5 mg protein/mL) and wild-type Supersomes without hBCRP (control membrane, 5 mg protein/mL) used as negative control were obtained from Corning (Amsterdam, The Netherlands). After delivery, Supersomes were thawed at 37 °C, aliquoted, snap-frozen in liquid nitrogen, and stored at -80 °C until use. ADP sodium salt, ATP magnesium salt, sulfasalazine, rosuvastatin, sodium orthovanadate, ammonium acetate, MES hydrate, and TRIS base were obtained from Sigma-Aldrich (Taufkirchen, Germany), formic acid (MS grade) from Fluka (Neu-Ulm, Germany), acetonitrile, methanol (both LC-MS grade), and all other chemicals from VWR (Darmstadt, Germany). -ethyl-8-methoxy-1,2,3,4,6,7,12,12boctahydroindolo[2,3-a] quinolizin-2-yl]-3-methoxyprop-2-enoate (mitragynine) by the Department of Forensic Medicine, Johannes Gutenberg University (Mainz, Germany), where it was isolated from kratom leaves obtained from head&nature (Regensburg, Germany) (Philipp et al. 2009) . N-Allyl-N- [2-(5-methoxy-1H-indol-3-yl) ethyl]-2-propen-1-amine (5-MeO-DALT) was synthesized by use of established methods (Brandt et al. 2008 ) and provided by the School of Pharmacy and Biomolecular Sciences, John Moores University (Liverpool, UK).
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