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Easycyp bactosomes

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EasyCYP Bactosomes are a type of in vitro system used for the evaluation of drug metabolism and drug-drug interactions. They contain recombinant human cytochrome P450 enzymes expressed in bacterial cells.

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Lab products found in correlation

Formic acid, by Merck Group (3 mentions) Nadph, by Merck Group (3 mentions) Nootkatone, by Cayman Chemical (3 mentions) Corn oil, by Merck Group (2 mentions) β nicotinamide adenine dinucleotide 2 phosphate reduced tetrasodium salt hydrate, by Merck Group (2 mentions) Ketoconazole, by Cayman Chemical (2 mentions) Ammonium formate, by Thermo Fisher Scientific (1 mentions) Formic acid, by Thermo Fisher Scientific (1 mentions) Deuterated nmr solvents, by Cambridge Isotopes (1 mentions) Optima lc ms grade acetonitrile, by Thermo Fisher Scientific (1 mentions) Ay9944, by Merck Group (1 mentions) α naphthoflavone, by Merck Group (1 mentions) Ketoconazole, by Merck Group (1 mentions) Fluoxetine hydrochloride, by Merck Group (1 mentions) N acetylcysteine, by Merck Group (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Cysteine, by Merck Group (1 mentions) Semicarbazide, by Merck Group (1 mentions) Human liver microsomes, by Xenotech (1 mentions) Perhexiline maleate salt, by Merck Group (1 mentions)

8 protocols using easycyp bactosomes

1

Profiling Cytochrome P450 Metabolism

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Optima LC-MS grade acetonitrile, water, formic acid, and ammonium
formate used in liquid chromatography were purchased from Fisher Scientific
(Santa Clara, CA). BACs of various alkyl chain lengths (C10-,
C12-, C14-, and C16-BAC), the DHCR7
inhibitor AY9944, and all synthetic reagents and starting materials were from
Sigma-Aldrich (St. Louis, MO). Deuterated NMR solvents were from Cambridge
Isotopes Laboratories, Inc. (Andover, MA). Human liver tissues were obtained
from the School of Pharmacy Human Liver Bank at the University of Washington
(Seattle, WA). Human recombinant CYP enzymes individually-expressed in
Escherichia coli (EasyCYP Bactosomes) co-expressing human
cytochrome P450 reductase and, where indicated as +b5, supplemented
with purified human cytochrome b5 (CYPs 1A2, 2A6+b5, 2A13,
2B6+b5, 2C8+b5, 2C9+b5, 2C19+b5,
2D6, 2E1+b5, 2J2, 3A4+b5, 3A5+b5,
4A11+b5, 4F2+b5, and 4F3B+b5) and membrane
protein from E. coli with empty expression plasmid (EasyCYP
Control Bactosomes) were from XenoTech, LLC (Lenexa, KS). Human recombinant
CYP4F12 containing human cytochrome P450 reductase and cytochrome b5in insect microsomes isolated from baculovirus-transfected insect cells (CYP4F12
Supersomes) and negative control insect cell microsomes (Control Supersomes)
were from Corning (Corning, NY). NADPH co-factor was from Oriental Yeast (Tokyo,
Japan).
Seguin R.P., Herron J.M., Lopez V., Dempsey J.L, & Xu L. (2019). Metabolism of Benzalkonium Chlorides by Human Hepatic Cytochromes P450. Chemical research in toxicology, 32(12), 2466-2478.
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2

CYP Metabolism of STO-609 Compound

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Commercial STO-609 (STO-609C) (7-oxo-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinoline-3-carboxylic acid acetate) was purchased either from Tocris Bioscience (Bristol, U.K.) or Sigma-Aldrich (St. Louis, MO). α-Naphthoflavone, formic acid, and NADPH were obtained from Sigma-Aldrich (St. Louis, MO). Human liver microsomes (HLM), mouse liver microsomes (MLM) and the recombinant human CYP450s (EasyCYP Bactosomes) were purchased from XenoTech (Lenexa, KS). All the solvents for liquid chromatography-mass spectrometry (LC-MS) were of the highest grade from Honeywell - Burdick & Jackson (Muskegon, MI).
York B., Li F., Lin F., Marcelo K.L., Mao J., Dean A., Gonzales N., Gooden D., Maity S., Coarfa C., Putluri N, & Means A.R. (2017). Pharmacological inhibition of CaMKK2 with the selective antagonist STO-609 regresses NAFLD. Scientific Reports, 7, 11793.
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3

Cytochrome P450 Inhibitor Screening

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JQ1, [tert-butyl (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate], ketoconazole (KCZ), NADPH (β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate) and formic acid were obtained from Sigma-Aldrich (St. Louis, MO). Nootkatone was purchased form Cayman Chemical Company (Ann Arbor, MI). Human and mouse liver microsomes and recombinant human cytochrome P450s (EasyCYP Bactosomes) were purchased from XenoTech (Lenexa, KS). All solvents for liquid chromatography and mass spectrometry were of the highest grade commercially available.
Li F., MacKenzie K.R., Jain P., Santini C., Young D.W, & Matzuk M.M. (2020). Metabolism of JQ1, an inhibitor of bromodomain and extra terminal bromodomain proteins, in human and mouse liver microsomes. Biology of Reproduction, 103(2), 427-436.
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4

Quinazoline Derivative Metabolism

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ILB ((S)-2-(1-((9H-purin-6-yl)amino)propyl)-5-fluoro-3-phenylquinazolin-4(3H)-one) was purchased from Ark Pharm (Arlington Heights, IL). M1 was purchased from TLC Pharmaceutical Standards (Aurora, Ontario, Canada). β-nicotinamide adenine dinucleotide phosphate (NADPH), GSH, ketoconazole (KCZ), fluoxetine hydrochloride, dimethyl sulfoxide (DMSO), corn oil, cysteine, cysteine-glycine and N-acetyl cysteine were purchased from Sigma-Aldrich (St. Louis, MO). HLM and the recombinant human CYPs (EasyCYP Bactosomes) were purchased from XenoTech (Lenexa, KS). All solvents for ultra-performance liquid chromatography and quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS) analysis were of the highest grade commercially available.
Zhu J., Wang P., Shehu A.I., Lu J., Bi H, & Ma X. (2018). Identification of novel pathways in idelalisib metabolism and bioactivation. Chemical research in toxicology, 31(7), 548-555.
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5

Metabolic Profiling of Xenobiotic Compounds

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ATX, [(R)-N-Methyl-3-phenyl-3-(o-tolyloxy)propan-1-amine)], quercetin (QT), alpha-naphthoflavone (NF) and nootkatone (NT) were purchased from Cayman Chemical (Ann Arbor, Michigan). Quinidine (Qui), ticlopidine (TCP), ketoconazole (KCZ), methoxyamine hydrochloride, reduced glutathione (GSH), formic acid, titanium(III) chloride (TiCl3, ca. 12 wt. % solution in 12 wt. % hydrochloric acid), and NADPH (β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate) were obtained from Sigma-Aldrich (St. Louis, MO). HLM, MLM, RLM and the recombinant human CYPs (EasyCYP Bactosomes) were purchased from XenoTech (Lenexa, KS). All solvents for liquid chromatography and mass spectrometry were of the highest grade commercially available.
MacKenzie K.R., Zhao M., Barzi M., Wang J., Bissig K.D., Maletic-Savatic M., Jung S.Y, & Li F. (2020). Metabolic profiling of norepinephrine reuptake inhibitor atomoxetine. European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences, 153, 105488.
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6

Quantification of CYP-Mediated Metabolic Pathways

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DTG, methoxyamine (NH2OMe), isoniazid (INH), dimethyl sulfoxide (DMSO), corn oil and semicarbazide were purchased from Sigma-Aldrich (St. Louis, MO). 2,4- difluorobenzaldehyde was purchased from TCI America (Portland, OR). β- nicotinamide adenine dinucleotide phosphate (NADPH) and 2,3’,4,5’- tetramethoxystilbene (TMS) were purchased from Alfa Aesar (Tewksbury, MA) and Cayman (Ann Arbor, MI), respectively. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was purchased from Toronto Research Chemical (Toronto, Ontario). Human liver microsomes (HLM) and the recombinant human CYPs (EasyCYP Bactosomes) were purchased from XenoTech (Lenexa, KS). All solvents for ultra-performance liquid chromatography and quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS) analysis were of the highest grade commercially available.
Zhu J., Wang P., Li F., Lu J., Shehu A.I., Xie W., McMahon D, & Ma X. (2018). CYP1A1 and 1B1-mediated metabolic pathways of dolutegravir, an HIV-1 integrase inhibitor. Biochemical pharmacology, 158, 174-184.
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7

In Vitro Inhibition Profiling

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DLX, [(3S)-N-methyl-3-(1-naphthyloxy)-3-(2-thienyl)propan-1-amine)], alpha-naphthoflavone (α-NF), nootkatone (NK), quercetin (QT), ticlopidine (TCP), ketoconazole, (KCZ), and sulfaphenazole (SPA) were purchased from Cayman Chemical (Ann Arbor, MI). Quinidine (Quin), formic acid, and NADPH were obtained from Sigma-Aldrich (St. Louis, MO). HLM (XTreme 200 Mixed Gender Human Liver Microsomes), MLM (Mouse Liver Microsomes), and the recombinant human CYP450s (EasyCYP Bactosomes) were purchased from XenoTech (Lenexa, KS). All solvents for liquid chromatography and mass spectrometry (LC-MS) were of the highest grade commercially available.
Qin X., Hakenjos J.M., MacKenzie K.R., Barzi M., Chavan H., Nyshadham P., Wang J., Jung S.Y., Guner J.Z., Chen S., Guo L., Krishnamurthy P., Bissig K.D., Palmer S., Matzuk M.M, & Li F. (2022). Metabolism of a Selective Serotonin and Norepinephrine Reuptake Inhibitor Duloxetine in Liver Microsomes and Mice. Drug Metabolism and Disposition, 50(2), 128-139.
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8

CYP Inhibition Assay Protocol

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Williams’ Medium E, perhexiline maleate salt (racemic mixture), quinidine, β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH), formic acid, and dimethysulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). Cis-OH-Phx and trans-OH-Phx were purchased from Toronto Research Chemicals (Toronto, Canada), and no information is available on the enantiomeric and diastereomeric composition of these standards. α-Naphthoflavone, nootkatone, and ketoconazole were obtained from Cayman Chemical (Ann Arbor, MI). Xtreme 200-donor Mixed Gender Human Liver Microsomes and EasyCYP bactosomes (recombinant human CYPs) were purchased from XenoTech (Lenexa, KS). Antibiotic–antimycotic was obtained from Life Technologies (Carlsbad, CA). PureCol® Type I Collagen Solution was from Advanced BioMatrix (San Diego, CA). Fetal Bovine Serum (FBS) was purchased from Atlanta Biologicals (Lawrenceville, GA). All solvents for liquid chromatography and mass spectrometry were of the highest grade commercially available.
Nimura K., Takahashi H, & Yoshikawa H. (2001). Characterization of the dnaK Multigene Family in the Cyanobacterium Synechococcus sp. Strain PCC7942. Journal of Bacteriology, 183(4), 1320-1328.
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