J. Christopher Gorski

The contribution of intestinal and hepatic CYP3A to the interaction between midazolam and clarithromycin

To assess the relative contribution of intestinal and hepatic CYP3A inhibition to the interaction between the prototypic CYP3A substrates midazolam and clarithromycin.

The effects of St John's wort (Hypericum perforatum) on human cytochrome P450 activity

St Johns wort(Hypericum perforatum) is a popular over‐the‐counter dietary supplement and herbal remedy that has been implicated in drug interactions with substrates of several cytochrome P450 (CYP) isozymes. The effect of St Johns wort on CYP activity in vivo was examined with a probe drug cocktail.

The Effect of Echinacea (Echinacea purpurea Root) on Cytochrome P450 Activity in Vivo

Echinacea is a widely available over‐the‐counter herbal remedy. Tinctures of echinacea have been shown to inhibit cytochrome P450 (CYP) in vitro. The effect of echinacea (Echinacea purpurea root) on CYP activity in vivo was assessed by use of the CYP probe drugs caffeine (CYP1A2), tolbutamide (CYP2C9), dextromethorphan (CYP2D6), and midazolam (hepatic and intestinal CYP3A).

The interaction between St John's wort and an oral contraceptive

The popular herbal remedy St Johns wort is an inducer of cytochrome P450 (CYP) 3A enzymes and may reduce the efficacy of oral contraceptives. Therefore we evaluated the effect of St Johns wort on thedisposition and efficacy of Ortho‐Novum 1/35 (Ortho‐McNeil Pharmaceutical, Inc, Raritan, NJ), a popular combination oral contraceptive pill containing ethinyl estradiol (INN, ethinylestradiol) and norethindrone (INN, norethisterone).

Semiphysiologically Based Pharmacokinetic Models for the Inhibition of Midazolam Clearance by Diltiazem and Its Major Metabolite

Prediction of the extent and time course of drug-drug interactions (DDIs) between the mechanism-based inhibitor diltiazem (DTZ) and the CYP3A4 substrate midazolam (MDZ) is confounded by time- and concentration-dependent clearance of the inhibitor. Semiphysiologically based pharmacokinetic (PBPK) models were developed for DTZ and MDZ with the major metabolite of DTZ, N-desmethyldiltiazem (nd-DTZ), incorporated in the DTZ model. Enzyme kinetic parameters (kinact and KI) for DTZ and nd-DTZ were estimated in vitro and used to model the time course of changes in the amount of CYP3A4 in the liver and gut wall, which in turn, determined the nonlinear elimination of MDZ and DTZ, and the corresponding DDI. The robustness of the model prediction was assessed by comparing the results of the prediction to published DTZ pharmacokinetic and DTZ/MDZ interaction data. A clinical study was conducted to further validate the predicted increase of MDZ exposure after DTZ treatment. The model predicted the nonlinear disposition of DTZ after single and multiple oral doses. The clinical study showed that DTZ treatment resulted in 4.1- and 1.6-fold increases in MDZ exposure after oral and intravenous MDZ administration, respectively, suggesting that the DDI in the gut wall plays an important role in the DTZ/MDZ interaction. The semi-PBPK model incorporating the DDI at the gut wall, and the effect of nd-DTZ successfully predicted the nonlinear disposition of DTZ and its interaction with MDZ. Moreover, model simulation suggested that both DTZ and nd-DTZ contributed to the overall inhibitory effect after DTZ administration, and the values of the in vitro estimated inhibition parameters and CYP3A4 turnover rate are critical for the prediction.

STOCHASTIC PREDICTION OF CYP3A-MEDIATED INHIBITION OF MIDAZOLAM CLEARANCE BY KETOCONAZOLE

Conventional methods to forecast CYP3A-mediated drug-drug interactions have not employed stochastic approaches that integrate pharmacokinetic (PK) variability and relevant covariates to predict inhibition in terms of probability and uncertainty. Empirical approaches to predict the extent of inhibition may not account for nonlinear or non-steady-state conditions, such as first-pass effects or accumulation of inhibitor concentration with multiple dosing. A physiologically based PK model was developed to predict the inhibition of CYP3A by ketoconazole (KTZ), using midazolam (MDZ) as the substrate. The model integrated PK models of MDZ and KTZ, in vitro inhibition kinetics of KTZ, and the variability and uncertainty associated with these parameters. This model predicted the time- and dose-dependent inhibitory effect of KTZ on MDZ oral clearance. The predictive performance of the model was validated using the results of five published KTZ-MDZ studies. The model improves the accuracy of predicting the inhibitory effect of increasing KTZ dosing on MDZ PK by incorporating a saturable KTZ efflux from the site of enzyme inhibition in the liver. The results of simulations using the model supported the KTZ dose of 400 mg once daily as the optimal regimen to achieve maximum inhibition by KTZ. Sensitivity analyses revealed that the most influential variable on the prediction of inhibition was the fractional clearance of MDZ mediated by CYP3A. The model may be used prospectively to improve the quantitative prediction of CYP3A inhibition and aid the optimization of study designs for CYP3A-mediated drug-drug interaction studies in drug development.

In vivo effects of interleukin‐10 on human cytochrome P450 activity

Injection of lipopolysaccharide into human volunteers leads to an increase in serum interleukin‐1β, interleukin‐6, and tumor necrosis factor‐α and a significant decrease in cytochrome P450 (CYP)–mediated drug metabolism. The in vivo effects of the noninflammatory cytokine interleukin‐10 (IL‐10) on CYP–mediated drug metabolism was examined.

Effects of HIV Protease Inhibitor Ritonavir on Akt-Regulated Cell Proliferation in Breast Cancer

Purpose: These studies were designed to determine whether ritonavir inhibits breast cancer in vitro and in vivo and, if so, how. Experimental Design: Ritonavir effects on breast cancer cell growth were studied in the estrogen receptor (ER)–positive lines MCF7 and T47D and in the ER-negative lines MDA-MB-436 and MDA-MB-231. Effects of ritonavir on Rb-regulated and Akt-mediated cell proliferation were studied. Ritonavir was tested for inhibition of a mammary carcinoma xenograft. Results: ER-positive estradiol-dependent lines (IC50, 12-24 μmol/L) and ER-negative (IC50, 45 μmol/L) lines exhibit ritonavir sensitivity. Ritonavir depletes ER-α levels notably in ER-positive lines. Ritonavir causes G1 arrest, depletes cyclin-dependent kinases 2, 4, and 6 and cyclin D1 but not cyclin E, and depletes phosphorylated Rb and Ser473 Akt. Ritonavir induces apoptosis independent of G1 arrest, inhibiting growth of cells that have passed the G1 checkpoint. Myristoyl-Akt, but not activated K-Ras, rescues ritonavir inhibition. Ritonavir inhibited a MDA-MB-231 xenograft and intratumoral Akt activity at a clinically attainable serum Cmax of 22 ± 8 μmol/L. Because heat shock protein 90 (Hsp90) substrates are depleted by ritonavir, ritonavir effects on Hsp90 were tested. Ritonavir binds Hsp90 (KD, 7.8 μmol/L) and partially inhibits its chaperone function. Ritonavir blocks association of Hsp90 with Akt and, with sustained exposure, notably depletes Hsp90. Stably expressed Hsp90α short hairpin RNA also depletes Hsp90, inhibiting proliferation and sensitizing breast cancer cells to low ritonavir concentrations. Conclusions: Ritonavir inhibits breast cancer growth in part by inhibiting Hsp90 substrates, including Akt. Ritonavir may be of interest for breast cancer therapeutics and its efficacy may be increased by sustained exposure or Hsp90 RNA interference.

Determination of cytochrome P450 3A4/5 activity in vivo with dextromethorphan N-demethylation

Dextromethorphan is used widely in vivo to phenotype the polymorphically expressed cytochrome P450 (CYP) 2D6. Dextromethorphan is N‐demethylated in vitro to 3‐methoxymorphinan by human CYP3A4/5. We examined whether the dextromethorphan/3‐methoxymorphinan urinary metabolic ratio (MR) could be used as an in vivo probe of CYP3A. Urinary excretion of 3‐methoxymorphinan was excretion rate‐limited in extensive metabolizers of CYP2D6, which necessitated a longer urine collection, 0 to 72 hours, to obtain true MR values for CYP3A. The urine excretion of dextromethorphan and 3‐methoxymorphinan was delayed in poor metabolizers of CYP2D6 but appeared to be formation rate‐limited. The delayed excretion in poor metabolizers necessitated longer urine collection intervals, 0 to 11 days, to estimate the true CYP3A MR and 0 to 8 days to estimate the true CYP2D6 MR. However, a 72‐hour collection in poor metabolizers was used as an index of the true dextromethorphan/3‐methoxymorphinan MR. Rifampin (300 mg b.i.d. for 7 days) significantly reduced the 0‐ to 72‐hour dextromethorphan/3‐methoxymorphinan MR consistent with an 830% (±1808%) induction of CYP3A activity (n = 8), whereas erythromycin (250 mg q.i.d. for 7 days) significantly increased the dextromethorphan/3‐methoxymorphinan MR, corresponding to a 34% ± 44% inhibition of activity (n = 7) in extensive metabolizers and poor metabolizers. The changes in CYP3A activity were independent of CYP2D6 phenotype and were also observed after 24‐ and 48‐hour urine collections in extensive metabolizers and poor metabolizers. In addition, MRs reflecting CYP2D6 and CYP3A were not significantly correlated. We conclude that the commonly used antitussive dextromethorphan can be used as an in vivo marker of CYP3A and CYP2D6 activity.

The effect of short- and long-term administration of verapamil on the disposition of cytochrome P450 3A and P-glycoprotein substrates

Verapamil has the capability to inhibit and induce cytochrome P450 (CYP) 3A and P‐glycoprotein (P‐gp), but the relative extent and time course of these events in vivo are unclear. The effect of verapamil on CYP3A and P‐gp activity was determined by examining its effect on its own disposition and on the disposition of fexofenadine, respectively.