All reagents and solvents unless noted were obtained from Sigma-Aldrich/Fluka (St. Louis, MO). Midazolam (MDZ) and 1′-hydroxy-midazolam (1′-OH-MDZ) were purchased from Tocris Bioscience (Bristol, UK) and Cayman Chemical (Ann Arbor, MI, USA), respectively. Podophyllotoxin (PPT) was purchased from Toronto Research Chemicals, Canada. 1-methyl-2-pyrrolidinone (NMP) was obtained from Avantor (Center Valley, PA, USA). CYP3A4-containing Baculosomes® (1 nmol/ml protein concentration) were purchased from Life Technologies (Carlsbad, CA, USA). Human liver microsomes pooled from 150 donors were purchased from Corning® Life Sciences (Tewksbury, MA, USA). Sprague-Dawley® rat liver microsomes from male donors were purchased from Invitrogen Co. (Carlsbad, CA, USA). Single jugular vein cannulated male Sprague Dawley rats were obtained from Charles River Laboratories (Malvern, PA, USA).
Human liver microsomes
Manufactured by Corning
Sourced in United States
Human liver microsomes are a preparation of subcellular fractions derived from human liver tissue. They contain metabolically active enzymes, primarily cytochrome P450 enzymes, that are involved in the biotransformation of various substances.
Automatically generated - may contain errors
Lab products found in correlation
Magnesium chloride salt, by Thermo Fisher Scientific (2 mentions)
Glucose 6 phosphate (g6p), by Merck Group (2 mentions)
Nadp disodium salt, by Merck Group (2 mentions)
Glucose 6 phosphate dehydrogenase, by Merck Group (2 mentions)
Ammonium formate, by Thermo Fisher Scientific (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
Formic acid, by Thermo Fisher Scientific (1 mentions)
Rat blood, by BioIVT (1 mentions)
Recombinant human ache, by Merck Group (1 mentions)
Intermedine, by Phytolab (1 mentions)
Jacobine, by Phytolab (1 mentions)
Senkirkine, by Phytolab (1 mentions)
Heliosupine, by Phytolab (1 mentions)
Europine n oxide, by Phytolab (1 mentions)
Senecionine n oxide, by Phytolab (1 mentions)
Intermedine n oxide, by Phytolab (1 mentions)
Jacobine n oxide, by Phytolab (1 mentions)
Heliosupine n oxide, by Phytolab (1 mentions)
Retrorsine n oxide, by Phytolab (1 mentions)
Echimidine n oxide, by Phytolab (1 mentions)
9 protocols using human liver microsomes
1
CYP3A4-Mediated Midazolam Metabolism
2
Optimized Metabolic Profiling Workflow
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PF74 (purity > 99%) was obtained from MedChemExpress (Shanghai, China), and the information for 11L (purity > 99%) is provided in Supplementary Materials . In addition, 7-EC (purity > 97%) was purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Acetonitrile, formic acid, water, and ammonium formate were purchased from Fisher Scientific (Fair Lawn, NJ, USA). All of the solvents used in the incubation and chromatographic system were UPLC-grade. Human liver microsomes (batch number: 38295) were obtained from Corning Incorporated (Corning, New York, NY, USA). Nicotinamide adenine dinucleotide phosphate (NADPH), phosphate buffer saline (PBS), and MgCl2 were obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).
3
Microsomal and Plasma Samples Acquisition
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Human liver microsomes (pooled from 20 donors, mixed gender) and rat liver microsomes (Sprague–Dawley, male) were purchased from Corning (Amsterdam, The Netherlands). Human plasma (pooled from 6 donors, mixed gender) were purchased from Zen-Bio, Inc (NC, USA). Rat plasma (Sprague–Dawley) was purchased from Innovative Research Inc. (MI, USA). For the rat samples further information on number of animals used to create the samples (liver microsomes and plasma) or on gender (plasma) was not provided by the provider. Rat blood was purchased from BioIVT (West Sussex, UK). Recombinant human AChE was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA), and the enzyme was stabilized with 1 mg/ml BSA.
4
Characterization of 5F-APINACA Metabolism
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All chemical solvents, salts and buffers were purchased from Thermo Fisher Scientific (Waltham, MA, USA). NADPH-regenerating system components NADP disodium salt, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate were purchased from Millipore-Sigma (St Louis, MO, USA), while magnesium chloride salt was purchased from Thermo Fisher Scientific. The substrate 5F-APINACA was provided by Dr. William Fantegrossi. Human liver microsomes pooled from 150 individuals were purchased from Corning (Corning, NY, USA). Dansylamide, used as the internal standard, was purchased from Millipore-Aldrich (St. Louis, MO, USA). ACD/ChemSketch 2017.2.1 software (Toronto, ON, Canada) was used for rendering structures of molecules.
5
Analytical Characterization of Pyrrolizidine Alkaloids
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Lasiocarpine (Las) and lasiocarpine N-oxide were ordered from Cfm Oskar Tropitzsch (Marktredwitz, Germany). Lycopsamine (Lyc), Lycopsamine N-oxide, europine (Eur), europine N-oxide, senecionine (Sen), senecionine N-oxide, echimidine (Ech), echimidine N-oxide, intermedine, intermedine N-oxide, jacobine, jacobine N-oxide, heliosupine, heliosupine N-oxide, retrorsine N-oxide, retronecine, and senkirkine were obtained from Phytolab (Vestenbergsgreuth, Germany). Retrorsine (Ret) was delivered by AppliChem (Darmstadt, Germany). Methanol (MeOH, LC–MS grade) and water (H2O, LC–MS grade) were purchased from Merck KGA (Darmstadt, Germany). 7,9-Diglutathionyl-6,7-dihydro-1-hydroymethyl-5H-pyrrolizidine was obtained from ASCA (Berlin, Germany). Potassium cyanide (KCN), isotopically labeled potassium cyanide (KCX, K13C15N), glutathione and isotopically labeled glutathione (GSX, 13C215N-GSH), and all other chemicals and co-factors were purchased from Sigma-Aldrich (Steinheim, Germany). All chemicals were obtained in analytical grade, if available. Human liver microsomes (mixed gender from 50 donors; protein concentration 20 mg/mL; cytochrome P450 content 310 pmol/mg protein) and rat liver microsomes (male Sprague–Dawley rats; protein concentration 20 mg/mL; cytochrome P450 content 700 pmol/mg protein) were obtained from Corning (Corning, USA).
6
Compound Characterization for Biological Evaluation
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Hydroxysafflor yellow A (X1 ), paeoniflorin (X2 ), oxypaeoniflorin (X3 ), albiflorin (X4 ), senkyunolides I (X5 ) and G (X7 ), tanshinol (X8 ), salvianolic acid B (X10 ), protocatechuic acid (X11 ), ferulic acid (X12 ) and 45 antibiotics (A1 –A45 ) were obtained commercially. Senkyunolide I-7-O-β-glucuronide (X6 ) and 3-O-methyltanshinol (X9 ) were prepared in-house using the method described previously35 (link), 36 (link). Purity of the compounds was ≥98%.
cDNA-expressed human P450 enzymes, uridine 5′-diphosphoglucuronosyltransferases (UGT), pooled human liver microsomes and pooled human liver cytosols were obtained from Corning Gentest (Woburn, MA, USA). Cryopreserved primary human hepatocytes from four donors XSM, HVN, DQB and OMA (Supporting Information Table S3 ) were obtained from BioreclamationIVT (Baltimore, MD, USA) and human embryonic kidney 293 (HEK-293) cells were obtained from American Type Culture Collection (Manassas, VA, USA). Human solute carrier (SLC) transporter expression plasmids were constructed commercially. Inside-out membrane vesicles [prepared from insect cells expressing human ATP-binding cassette (ABC) transporters] were obtained commercially. Probe substrates and positive inhibitors of test enzymes and transporters and positive inducers of P450 enzymes were also obtained commercially.
cDNA-expressed human P450 enzymes, uridine 5′-diphosphoglucuronosyltransferases (UGT), pooled human liver microsomes and pooled human liver cytosols were obtained from Corning Gentest (Woburn, MA, USA). Cryopreserved primary human hepatocytes from four donors XSM, HVN, DQB and OMA (
7
In vitro Metabolism of Novel Cannabinoids
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All chemical solvents, salts, and buffers were purchased from Thermo Scientific (Waltham, MA, USA). CYP isozyme inhibitors α-naphthoflavone, (+)-N-3-benzylnirvanol, ketoconazole, montelukast sodium, quinidine, sulfaphenazole, ticlopidine hydrochloride, and tranylcypromine sulfate were purchased from Millipore-Sigma. The substrate (5OH-APINACA) and CYP isozyme inhibitors (1-aminobenzotriazole and 4-methylpyrazole hydrochloride) were purchased from Cayman Chemical (Ann Arbor, MI, USA). NADPH-regenerating system components NADP disodium salt, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate were purchased from Millipore-Sigma (St. Louis, MO, USA), while magnesium chloride salt was purchased from Thermo Fisher Scientific. The substrate 5F-APINACA was provided by Dr. William Fantegrossi. Human liver microsomes pooled from 150 individuals, lot#38296, and P450 contribution of 0.380 nmol/mg, were purchased from Corning (Corning, NY, USA). CD-1 male mouse liver microsomes pooled from 6424 mice, lot#2010017, and P450 contribution of 0.671 nmol/mg, were purchased from Sekisui Xenotech (Kansas City, KS, USA). Internal standard acenocoumarol was purchased from Toronto Research Chemicals (Toronto, ON, Canada). ACD/ChemSketch 2017.2.1 software (Toronto, ON, Canada) was used for rendering structures of molecules.
8
Metabolic Stability Evaluation
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For the evaluation
of phase I metabolic stability, the compound (1 μM) was incubated
with 0.5 mg/mL pooled C57BL/6 mouse, Wistar rat liver microsomes (Xenotech,
Kansas City, USA), or human liver microsomes (Corning, New York, USA),
2 mM NADPH, and 10 mM MgCl2 at 37 °C for 120 min on
a microplate shaker (Eppendorf, Hamburg, Germany). The metabolic stability
of testosterone, verapamil, and ketoconazole was determined in parallel
to confirm the enzymatic activity of mouse/rat liver microsomes; for
human liver microsomes, testosterone, diclofenac, and propranolol
were used. Incubation was stopped after defined time points by precipitation
of aliquots of enzymes with 2 volumes of cold acetonitrile containing
an internal standard (15 nM diphenhydramine). Samples were stored
on ice until the end of the incubation, and precipitated protein was
removed by centrifugation (15 min, 4 °C, and 4000g). The concentration of the remaining test compound at the different
time points was analyzed by HPLC-MS/MS (TSQ Altis Plus, Thermo Fisher,
Dreieich, Germany) and used to determine the half-life (t1/2).
of phase I metabolic stability, the compound (1 μM) was incubated
with 0.5 mg/mL pooled C57BL/6 mouse, Wistar rat liver microsomes (Xenotech,
Kansas City, USA), or human liver microsomes (Corning, New York, USA),
2 mM NADPH, and 10 mM MgCl2 at 37 °C for 120 min on
a microplate shaker (Eppendorf, Hamburg, Germany). The metabolic stability
of testosterone, verapamil, and ketoconazole was determined in parallel
to confirm the enzymatic activity of mouse/rat liver microsomes; for
human liver microsomes, testosterone, diclofenac, and propranolol
were used. Incubation was stopped after defined time points by precipitation
of aliquots of enzymes with 2 volumes of cold acetonitrile containing
an internal standard (15 nM diphenhydramine). Samples were stored
on ice until the end of the incubation, and precipitated protein was
removed by centrifugation (15 min, 4 °C, and 4000g). The concentration of the remaining test compound at the different
time points was analyzed by HPLC-MS/MS (TSQ Altis Plus, Thermo Fisher,
Dreieich, Germany) and used to determine the half-life (t1/2).
9
In Vitro Cytochrome P450 Inhibition Assay
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Isoform-specific assay conditions
were employed. Test compound (0.1, 0.25, 1, 2.5, 10, 25 μM)
was incubated with pooled human liver microsomes (0.25 mg/mL; Corning
Life Sciences), an appropriate isoform-specific probe substrate, and
NADPH (1 mM) at 37 °C for an isoform-specific incubation time.
The reactions were terminated by the addition of an aliquot of the
incubation into methanol. The samples were centrifuged at 2500 rpm
for 30 min at 4 °C, and aliquots of the supernatant were diluted
for LC–MS/MS analysis. The formation of isoform-specific metabolites
acetaminophen (CYP 1A2), hydroxybupropion (CYP 2B6), 6α-hydroxypaclitaxel
(CYP 2C8), 4 hydroxydiclofenac (CYP 2C9), 4-hydroxymephenytoin (CYP
2C19), dextrorphan (CYP 2D6), 1-hydroxymidazolam, and 6β-hydroxytestosterone
(CYP 3A4) were monitored. A decrease in the amount of metabolite formed
compared with vehicle control was used to calculate an IC50 value for each experimental condition.
were employed. Test compound (0.1, 0.25, 1, 2.5, 10, 25 μM)
was incubated with pooled human liver microsomes (0.25 mg/mL; Corning
Life Sciences), an appropriate isoform-specific probe substrate, and
NADPH (1 mM) at 37 °C for an isoform-specific incubation time.
The reactions were terminated by the addition of an aliquot of the
incubation into methanol. The samples were centrifuged at 2500 rpm
for 30 min at 4 °C, and aliquots of the supernatant were diluted
for LC–MS/MS analysis. The formation of isoform-specific metabolites
acetaminophen (CYP 1A2), hydroxybupropion (CYP 2B6), 6α-hydroxypaclitaxel
(CYP 2C8), 4 hydroxydiclofenac (CYP 2C9), 4-hydroxymephenytoin (CYP
2C19), dextrorphan (CYP 2D6), 1-hydroxymidazolam, and 6β-hydroxytestosterone
(CYP 3A4) were monitored. A decrease in the amount of metabolite formed
compared with vehicle control was used to calculate an IC50 value for each experimental condition.
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