Simultaneous Measurement of In Vivo P‐glycoprotein and Cytochrome P450 3A Activities

Kirby, Brian J.; Kharasch, Evan D.; Thummel, Kenneth T.; Narang, Vishal S.; Hoffer, Christine; Unadkat, Jashvant D.

doi:10.1177/0091270006292625

Cited by 34 publications

(38 citation statements)

References 44 publications

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“…Using historical data for MDZ in healthy volunteers (Wang et al, 2001;Kirby et al, 2006), we conducted an a priori power analysis using the AUC of MDZ as the primary outcome measure. Assuming equal variance between control and treatment groups, our analysis indicated that n ϭ 7 would provide 80% power (␣ Ͻ 0.05) to discern 54 and 41% changes in oral and intravenous MDZ AUC, respectively.…”

Section: Methodsmentioning

confidence: 99%

Complex Drug Interactions of HIV Protease Inhibitors 1: Inactivation, Induction, and Inhibition of Cytochrome P450 3A by Ritonavir or Nelfinavir

Kirby¹,

Collier²,

Kharasch³

et al. 2011

Drug Metab Dispos

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ABSTRACT:Conflicting drug-drug interaction (DDI) studies with the HIV protease inhibitors (PIs) suggest net induction or inhibition of intestinal or hepatic CYP3A. As part of a larger DDI study in healthy volunteers, we determined the effect of extended administration of two PIs, ritonavir (RTV) or nelfinavir (NFV), or the induction-positive control rifampin on intestinal and hepatic CYP3A activity as measured by midazolam (MDZ) disposition after a 14-day treatment with the PI in either staggered (MDZ ϳ12 h after PI) or simultaneous (MDZ and PI coadministered) manner. Oral and intravenous MDZ areas under the plasma concentration-time curves were significantly increased by RTV or NFV and were decreased by rifampin. Irrespective of method of administration, RTV decreased net intestinal and hepatic CYP3A activity, whereas NFV decreased hepatic but not intestinal CYP3A activity. The magnitude of these DDIs was more accurately predicted using PI CYP3A inactivation parameters generated in sandwich-cultured human hepatocytes rather than human liver microsomes.

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Section: Methodsmentioning

confidence: 99%

Complex Drug Interactions of HIV Protease Inhibitors 1: Inactivation, Induction, and Inhibition of Cytochrome P450 3A by Ritonavir or Nelfinavir

Kirby¹,

Collier²,

Kharasch³

et al. 2011

Drug Metab Dispos

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101

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“…The samples were dried down and reconstituted in acetonitrile/water (9/1), and the concentration of 19-hydoxymidazolam was determined by liquid chromatography-mass spectrometry [Waters Alliance 2695 HPLC (Waters, Milford, MA) interfaced with Waters Micromass Quattro Micro] using an electrospray ion source. The mobile phase consisted of 0.1% (v/v) acetic acid in water and 0.1% (v/v) acetic acid in acetonitrile, as described previously (Kirby et al, 2006). The separation was performed with a Zorbax Eclipse SB-C18 column (2.1 Â 150 mm, 5 mm; Agilent Technologies, Santa Clara, CA) at a flow rate of 0.25 ml min…”

Section: Cyp3a Activity Measurementmentioning

confidence: 99%

Induction of Hepatic CYP3A Enzymes by Pregnancy-Related Hormones: Studies in Human Hepatocytes and Hepatic Cell Lines

2012

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CYP3A activity is induced by approximately 2-fold during the third trimester of human pregnancy. Placental growth hormone (PGH), estrogens (primarily 17b-estradiol), cortisol, and progesterone have the potential to modulate CYP3A activity. Therefore, we determined whether the elevated plasma concentrations of these hormones during pregnancy induce hepatic CYP3A expression. We incubated sandwich-cultured human hepatocytes (SCHH) from premenopausal female donors (n = 2) with the physiologic (unbound, 13 total) and the 103 total third trimester hormone plasma concentrations (individually and in combination) and determined their effect on CYP3A activity and the transcripts of CYP3A4, CYP3A5, and the respective hormone receptors (growth hormone receptor, glucocorticoid receptor, and estrogen receptor alpha). Of all the hormones, cortisol was the most potent inducer of CYP3A activity and CYP3A4, CYP3A5 mRNA expression. The combination of PGH/growth hormone and cortisol induced CYP3A activity and expression significantly more than did cortisol alone. When incubated with the unbound or total plasma concentration of all the hormones, CYP3A activity in SCHH was induced to an extent comparable to that observed in vivo during the third trimester. These hormones had only a modest effect on the mRNA expression of the hormone receptors. The pattern of induction observed in SCHH was reproduced in HepaRG cells but not in HuH7/ HepG2 cells. SCHH or HepaRG cells could be used to determine the mechanistic basis of CYP3A induction during pregnancy and to predict the magnitude of induction likely to be observed during the first and second trimesters, when phenotyping studies to measure in vivo CYP3A activity are logistically difficult to perform.

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“…A calibration curve ranging from 2.5 to 250 ng/ml of 1Ј-OH-MDZ was prepared in blank cell media. The samples were subjected to liquid-liquid extraction and the amount of 1Ј-OH-MDZ was measured by liquid chromatography-mass spectrometry as described previously (Kirby et al, 2006b) with the following minor modifications. To a 0.5-ml aliquot of cell lysate, a stable labeled internal standard (d 5 -1Ј-OH-midazolam, m/z 346.9, 25 ng) was added.…”

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confidence: 99%

Intestinal Human Colon Adenocarcinoma Cell Line LS180 Is an Excellent Model to Study Pregnane X Receptor, but Not Constitutive Androstane Receptor, Mediated CYP3A4 and Multidrug Resistance Transporter 1 Induction: Studies with Anti-Human Immunodeficiency Virus Protease Inhibitors

Gupta¹,

Mugundu²,

Desai³

et al. 2008

Drug Metab Dispos

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101

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ABSTRACT:Lack of an established cell line model to study induction of cytochromes P450 (P450s) and drug transporters poses a challenge in predicting in vivo drug-drug interactions. Although not well characterized, LS180 cells could be an excellent cell line to study induction of P450s and transporters because they express pregnane X receptor (PXR). Therefore, as part of a larger study of in vitro to in vivo prediction of inductive drug interactions, we determined induction of various P450s and drug transporters by the anti-human deficiency virus protease inhibitors (PIs) and the prototypic inducer, rifampin, in LS180 cells. Among these proteins, the various PIs significantly induced (n ‫؍‬ 3-5) only CYP3A4 and multidrug resistance transporter 1 (MDR1) transcripts (2-to 50-fold). CYP3A4 activity (1-hydroxymidazolam formation) was increased (2-fold) by rifampin (10 M) but was reduced by the PIs (1.5-to 7-fold). Surprisingly, constitutive androstane receptor 1 (CAR1) was not found to be expressed in these cells. Additionally, using a reporter assay, we found that PIs did not activate CAR3 (the natural splice variant of CAR1) but significantly activated PXR (2-to 24-fold), which correlated well with induction of CYP3A4 and MDR1 transcripts (ϳr ‫؍‬ 0.9). Furthermore, in a PXR-knockdown stable LS180 cell line, induction of CYP3A4 and MDR1 mRNA after treatment with PIs and rifampin was significantly reduced (1.4-to 5-fold) compared with that in PXR nonsilenced cells. Based on these data, we conclude that LS180 cells could be used as a readily available, high-throughput cell line to screen for PXR-mediated induction of CYP3A4 and MDR1 transcripts. These data also indicate that the majority of the PIs are likely to produce intestinal drug-drug interactions by inactivating or inhibiting CYP3A enzymes even though they induce CYP3A4 and MDR1 transcripts via PXR.To date, much of the focus on clinically significant drug interactions has been on metabolic-based inhibitory drug interactions. However, there is increasing appreciation that inductive drug interactions are more common than previously thought, and such interactions can also extend to transporters. For this reason, the U.S. Food and Drug Administration has recently issued draft guidance on models to study inductive drug interactions. This draft guidance states that "At this time, the most reliable method to study a drug's induction potential is to quantify the enzyme activity of primary hepatocyte cultures following treatments including the potential inducer drug, a positive control inducer drug [rifampin for CYP3A4], and vehicle-treated hepatocytes (negative control), respectively"

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Simultaneous Measurement of In Vivo P‐glycoprotein and Cytochrome P450 3A Activities

Cited by 34 publications

References 44 publications

Complex Drug Interactions of HIV Protease Inhibitors 1: Inactivation, Induction, and Inhibition of Cytochrome P450 3A by Ritonavir or Nelfinavir

Complex Drug Interactions of HIV Protease Inhibitors 1: Inactivation, Induction, and Inhibition of Cytochrome P450 3A by Ritonavir or Nelfinavir

Induction of Hepatic CYP3A Enzymes by Pregnancy-Related Hormones: Studies in Human Hepatocytes and Hepatic Cell Lines

Intestinal Human Colon Adenocarcinoma Cell Line LS180 Is an Excellent Model to Study Pregnane X Receptor, but Not Constitutive Androstane Receptor, Mediated CYP3A4 and Multidrug Resistance Transporter 1 Induction: Studies with Anti-Human Immunodeficiency Virus Protease Inhibitors

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