Determination of mycophenolic acid glucuronide in microsomal incubations using high performance liquid chromatography–tandem mass spectrometry

Mohamed, Mohamed‐Eslam F.; Harvey, Stephen S.; Frye, Reginald F.

doi:10.1016/j.jchromb.2008.06.020

Cited by 10 publications

(7 citation statements)

References 35 publications

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“…We have developed a sensitive LC–MS/MS assay for the simultaneous quantification of MPA, MPAG and AcMPAG in HepaRG cell culture medium, using MPA‐d 3 and MPAG‐d 3 as internal standards. LC–MS/MS assays for MPA and/or metabolites have been described in human plasma (Delavenne, Juthier, Pons, Mariat, & Basset, ; Figurski, Korecka, Fields, Waligorska, & Shaw, ; Kawanishi et al, ; Zegarska et al, ; Zhang, Chow, & Renbarger, ), urine (Benoit‐Biancamano et al, ; Klepacki et al, ), dried blood spots (Iboshi et al, ) and human liver microsomes (Mohamed, Harvey, & Frye, ). In contrast to other biological fluids (i.e.…”

Section: Resultsmentioning

confidence: 99%

Development and validation of a sensitive liquid‐chromatography tandem mass spectrometry assay for mycophenolic acid and metabolites in HepaRG cell culture: Characterization of metabolism interactions between p‐cresol and mycophenolic acid

Rong

Kiang

2019

Biomedical Chromatography

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Mycophenolic acid (MPA), a frequently used immunosuppressant, exhibits large interpatient pharmacokinetic variability. This study (a) developed and validated a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for MPA and metabolites [MPA glucuronide (MPAG) and acyl-glucuronide (AcMPAG)] in the culture medium of HepaRG cells; and (b) characterized the metabolism interaction between MPA and p-cresol (a common uremic toxin) in this in vitro model as a potential mechanism of pharmacokinetic variability. Chromatographic separation was achieved with a C 18 column (4.6 × 250 mm,5 μm) using a gradient elution with water and methanol (with 0.1% formic acid and 2 mM ammonium acetate). A dual ion source ionization mode with positive multiple reaction monitoring was utilized. Multiple reaction monitoring mass transitions (m/z) were: MPA (320.95 → 207.05), MPAG (514.10 → 303.20) and AcMPAG (514.10 → 207.05). MPA-d 3 (323.95 → 210.15) and MPAG-d 3 (517.00 → 306.10) were utilized as internal standards. The calibration curves were linear from 0.00467 to 3.2 μg/mL for MPA/MPAG and from 0.00467 to 0.1 μg/mL for AcMPAG. The assay was validated based on industry standards. p-Cresol inhibited MPA glucuronidation (IC 50 ≈ 55 μM) and increased MPA concentration (up to >2-fold) at physiologically relevant substrate-inhibitor concentrations (n = 3). Our findings suggested that fluctuations in p-cresol concentrations might be in part responsible for the large pharmacokinetic variability observed for MPA in the clinic.

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Section: Resultsmentioning

confidence: 99%

Development and validation of a sensitive liquid‐chromatography tandem mass spectrometry assay for mycophenolic acid and metabolites in HepaRG cell culture: Characterization of metabolism interactions between p‐cresol and mycophenolic acid

Rong

Kiang

2019

Biomedical Chromatography

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“…Incubations with MPA were performed as described previously with modifications (Mohamed et al, 2008). In brief, the incubation mixture (100 l) contained HLM (protein concentration, 0.16 mg/ml), alamethicin (100 g/mg protein), MgCl 2 (5 mM), 2% BSA, and 100 mM phosphate buffer, pH 7.4.…”

Section: Effect Of Herbal Extracts On Ugt1a4 1a6 and 1a9 Activitiesmentioning

confidence: 99%

“…MPAG was determined by liquid chromatography-tandem mass spectrometry on a Thermo Finnigan Surveyor series HPLC system connected to a TSQ Quantum triple quadrupole mass spectrometer (Thermo Fisher Scientific) using electrospray ionization, as described previously (Mohamed et al, 2008). In brief, 5 l of each sample was injected on a reverse-phase Synergi Fusion-RP18 column (100 ϫ 2 mm, 4 m; Phenomenex).…”

Section: Effect Of Herbal Extracts On Ugt1a4 1a6 and 1a9 Activitiesmentioning

confidence: 99%

Inhibitory Effects of Commonly Used Herbal Extracts on UDP-Glucuronosyltransferase 1A4, 1A6, and 1A9 Enzyme Activities

Mohamed

Frye²

2011

Drug Metabolism and Disposition

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ABSTRACT:The aim of this study was to investigate the effect of commonly used botanicals on UDP-glucuronosyltransferase (UGT) 1A4, UGT1A6, and UGT1A9 activities in human liver microsomes. The extracts screened were black cohosh, cranberry, echinacea, garlic, ginkgo, ginseng, milk thistle, saw palmetto, and valerian in addition to the green tea catechin epigallocatechin gallate (EGCG). Formation of trifluoperazine glucuronide, serotonin glucuronide, and mycophenolic acid phenolic glucuronide was used as an index reaction for UGT1A4, UGT1A6, and UGT1A9 activities, respectively, in human liver microsomes. Inhibition potency was expressed as the concentration of the inhibitor at 50% activity (IC 50 ) and the volume in which the dose could be diluted to generate an IC 50 -equivalent concentration [volume/dose index (VDI)]. Potential inhibitors were EGCG for UGT1A4, milk thistle for both UGT1A6 and UGT1A9, saw palmetto for UGT1A6, and cranberry for UGT1A9. EGCG inhibited UGT1A4 with an IC 50 value of (mean ؎ S.E.) 33.8 ؎ 3.1 g/ml. Milk thistle inhibited both UGT1A6 and UGT1A9 with IC 50 values of 59.5 ؎ 3.6 and 33.6 ؎ 3.1 g/ml, respectively. Saw palmetto and cranberry weakly inhibited UGT1A6 and UGT1A9, respectively, with IC 50 values >100 g/ml. For each inhibition, VDI was calculated to determine the potential of achieving IC 50 -equivalent concentrations in vivo. VDI values for inhibitors indicate a potential for inhibition of first-pass glucuronidation of UGT1A4, UGT1A6, and UGT1A9 substrates. These results highlight the possibility of herb-drug interactions through modulation of UGT enzyme activities. Further clinical studies are warranted to investigate the in vivo extent of the observed interactions.

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“…In vitro microsomal and recombinant human UGT incubation systems were performed as previously described elsewhere (Maul et al, 2015;Song et al, 2015). MPA (1 mM) was used as the probe substrate of UGT1A7 to evaluate the activity of microsomes and recombinant UGT1A7 enzyme (Mohamed et al, 2008).…”

Section: Methodsmentioning

confidence: 99%

Inhibitory Effects of Endogenous Linoleic Acid and Glutaric Acid on the Renal Glucuronidation of Berberrubine in Mice and on Recombinant Human UGT1A7, 1A8, and 1A9

Yang¹,

Li²,

Yan³

et al. 2018

Molecular Pharmacology

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Berberrubine (BRB) has a strong lipid-lowering effect and can be extensively metabolized into berberrubine-9---d-glucuronide (BRBG) in vivo. Recently, pharmacokinetics studies showed that the production of BRBG was significantly decreased in the urine of mice fed with a high-fat diet (HFD), indicating a decreased glucuronidation capacity. Based on the UDP-glucuronosyltransferase (UGT) isoform identification, hepatic and renal microsomal incubation, glucuronidation was examined to suggest the metabolism of BRB in liver and kidneys. The results showed that the renal UGT activity for metabolizing BRB markedly decreased, which may be highly related to the decreased expression and activity of renal Ugt1a7c. Surprisingly, in vitro studies revealed neither BRB nor BRBG inhibited the renal UGT activity. By employing an integrated strategy of metabolomics and pharmacokinetics, we identified and confirmed for the first time the inhibitory effect of some potential endogenous molecules on the renal glucuronidation of C57BL/6J mice, such as glutaric acid (GA) and linoleic acid (LA). By employing recombinant human UGTs, we found that GA and LA efficiently affect the activity of recombinant human UGT1A7, 1A9, and 1A8 at their normal or abnormal physiologic levels in vivo. GA (2 mM) markedly inhibited the activity of UGT1A7 by 89.4% and UGT1A9 by 32.8%. The inhibition rates reached 99.3% for UGT1A9, 48.3% for UGT1A7, and 46.8% for UGT1A8 with LA at 200 M. It has been suggested that the endogenous molecules have the potential to affect the efficiency of glucuronidation, which might be a key factor contributing to individual differences in drug metabolism.

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Determination of mycophenolic acid glucuronide in microsomal incubations using high performance liquid chromatography–tandem mass spectrometry

Cited by 10 publications

References 35 publications

Development and validation of a sensitive liquid‐chromatography tandem mass spectrometry assay for mycophenolic acid and metabolites in HepaRG cell culture: Characterization of metabolism interactions between p‐cresol and mycophenolic acid

Development and validation of a sensitive liquid‐chromatography tandem mass spectrometry assay for mycophenolic acid and metabolites in HepaRG cell culture: Characterization of metabolism interactions between p‐cresol and mycophenolic acid

Inhibitory Effects of Commonly Used Herbal Extracts on UDP-Glucuronosyltransferase 1A4, 1A6, and 1A9 Enzyme Activities

Inhibitory Effects of Endogenous Linoleic Acid and Glutaric Acid on the Renal Glucuronidation of Berberrubine in Mice and on Recombinant Human UGT1A7, 1A8, and 1A9

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