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DURATION OF PLECONARIL EFFECT ON CYTOCHROME P450 3A ACTIVITY IN HEALTHY ADULTS USING THE ORAL BIOMARKER MIDAZOLAM

Amgen, Inc., Thousand Oaks, California (J.D.M.); Ordway Research Institute, Inc., Albany, New York (A.N.N., J.S.B.); Gilead, South San Francisco, California (G.R.); and Teva Neurosciences, Horsham, Pennsylvania (S.L.)

(Received October 12, 2005; Accepted February 1, 2006)

	Abstract

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The objective of this study was to evaluate the duration of oral pleconaril (a picornavirus inhibitor) effect on intestinal and hepatic cytochrome P450 (P450) 3A activity as assessed by oral midazolam. Healthy adults received oral midazolam (0.075 mg/kg) on days 1 (baseline), 7, 9, 13, 20, 27, and 34. Oral pleconaril (400 mg) three times daily for 15 doses was administered on days 2 through 7. Blood samples were collected during each day of midazolam dosing to determine plasma midazolam concentrations. On days 5, 6, and 7, blood samples were collected to determine plasma pleconaril concentrations. Midazolam pharmacokinetics were determined by noncompartmental analyses, with bioequivalence assessed by least-squares geometric mean ratios (LS-GMR) and 90% confidence intervals (90% CI). Eighteen subjects completed the study. Midazolam C_max (LS-GMR; 90% CI) decreased 24% on day 7 (0.76; 0.66–0.87). Midazolam oral clearance increased 53% on day 7 (1.53; 1.38–1.69). Midazolam oral clearance remained different on days 9 (1.38; 1.25–1.52) and 13 (1.19; 1.07–1.31) versus day 1. Midazolam volume of distribution (1.82; 1.57–2.11) and elimination half-life (1.19; 1.03–1.38) were also different on day 7 in comparison with day 1. Oral pleconaril increased intestinal and hepatic CYP3A activity. The duration of increased CYP3A activity by pleconaril was at least 6 days (but no longer than 13 days) after pleconaril discontinuation.

In vitro and animal studies showed a lack of cytochrome P450 (P450) 3A inhibition and induction by pleconaril (data on file, ViroPharma, Inc., Exton, PA; Rhodes et al., 2001). In healthy adults, oral pleconaril increases hepatic CYP3A activity after intravenous midazolam administration (Ma et al., 2006). Because of the prolonged half-life (180 h) of pleconaril (Rhodes and Liu, 2001a,b; Florea et al., 2003), the duration of increased CYP3A activity may be an important parameter in evaluating potential drug interactions with pleconaril. The purpose of this study was to evaluate the duration and extent of increased hepatic and intestinal CYP3A activity by pleconaril in healthy adults as assessed by oral midazolam.

	Materials and Methods

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Subjects. This study was approved by the Institutional Review Board of Bassett Healthcare, Cooperstown, NY, with written informed consent obtained from each subject. All subjects underwent a medical history, physical examination, electrocardiogram, and blood and urine laboratory tests. Subjects were 18 to 55 years of age, healthy, nonsmokers (for at least 6 months), and not on any medications known to affect CYP3A activity. Subjects refrained from consuming grapefruit and grapefruit juice for 7 days and caffeine-containing foods for 2 days before study start until study completion. Subjects with clinically significant findings by medical history, physical examination, laboratory safety testing, history of alcohol or drug abuse, history of medication or skin allergy, or intolerance to benzodiazepines were excluded.

Study Design. An open-label, single-sequence study design was used because of the prolonged elimination half-life of pleconaril (Florea et al., 2003). After an overnight fast, subjects were administered oral midazolam (0.075 mg/kg) on the morning of day 1 (baseline). Oral pleconaril (400 mg) (Picovir, 200-mg tablet; ViroPharma, Inc.) was administered three times daily for 15 doses. This dosing regimen was based on the expected clinical use for treatment of viral infections. The first pleconaril dose began in the afternoon of day 2. The last pleconaril dose was concurrently administered with midazolam on the morning of day 7. All pleconaril doses, except for the morning day 7 dose, were administered within 5 min after completion of a meal or snack because pleconaril bioavailability is increased with food (Abdel-Rahman and Kearns, 1998). Midazolam dosing was repeated on days 7, 9, 13, 20, 27, and 34. Venous blood samples were collected at predose and 5, 30, 60, 90, 120, 180, 240, 300, 360, and 480 min after each midazolam dose to determine plasma midazolam concentrations and on days 5, 6, and 7 before the morning pleconaril dose to determine plasma pleconaril concentrations.

Analytical Procedures. Plasma midazolam concentrations were analyzed by a liquid chromatography/mass spectrometry/mass spectrometry assay developed by Prevalere Life Sciences, Whitesboro, NY. Details of this procedure are described elsewhere (Kashuba et al., 1998). The linear range of the assay was 25 to 10,000 pg/ml. Intra- and interday percent coefficients of variation for midazolam were 9.4% at quality control samples of 75, 750, and 7500 pg/ml. For midazolam detection with mass spectrometry, the transition m/z 326291 was selected. Plasma pleconaril was analyzed by a liquid chromatography/mass spectrometry/mass spectrometry assay developed by MDS Pharma Services, Montreal, QC, Canada. Plasma pleconaril (0.1 ml) was isolated by a liquid-liquid extraction with the addition of an internal standard (VP 64027; 750 ng/ml) and 5 ml of hexanes. After mixing, samples were evaporated to dryness and reconstituted in 100 ml of mobile phase (85:15 acetonitrile/water). A BDZ-Hypersil C₁₈ column was coupled to a Sciex API 3000 or API III MS system (Perkin-Elmer Sciex, Rochester, NH) and an Alliance 2690 autosampler (Waters Corporation, Milford, MA). The linear range of the assay was 5 to 500 ng/ml, with intra- and interday percent coefficients of variation 10% at quality control samples of 5, 15, 150, and 350 ng/ml. For pleconaril detection with mass spectrometry, the transition m/z 382298 was selected.

View larger version (25K): [in this window] [in a new window]   FIG. 1. Mean plasma midazolam concentration versus time in healthy adults after oral administration of 0.075 mg/kg midazolam on days 1 (baseline), 7, 9, 13, 20, 27, and 34. Data on days 1, 7, 9, and 13 represent 19 subjects. Data on days 20, 27, and 34 represent 18 subjects.

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Statistical Analyses. Based on previous data (Ma et al., 2006) using = 0.05 and ß = 0.20 (power of 80%), an estimated sample size of 20 would detect a 25% difference in midazolam AUC_0-. Bioequivalence testing was done, and results are presented as least-squares geometric mean ratios (LS-GMR) with 90% confidence intervals (90% CI). A lack of bioequivalence (i.e., a drug interaction) was determined if the 90% CI were outside of the 0.80-to-1.25 interval (Food and Drug Administration, 2001). Statistical analyses were performed using SAS version 6.12 (SAS Institute, Cary, NC).

	Results

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Nineteen subjects were enrolled, and 18 subjects (nine males) completed the study. Median age was 31 years (range: 21–49 years). Weight (mean ± S.D.) was 76.5 ± 13.1 kg. One subject developed an upper respiratory infection and Bell's palsy, which was determined to be unrelated to pleconaril or midazolam, and was removed from the study on day 13. Data for this subject were used only for evaluation of plasma midazolam concentrations (Fig. 1) on days 1, 7, 9, and 13 and plasma pleconaril concentrations on days 5, 6, and 7. Mild sedation was noted with midazolam in all subjects. No significant adverse events were reported.

Plasma midazolam concentration versus time profiles are presented in Fig. 1. Pleconaril decreased midazolam C_max 24% on day 7 compared with day 1, and this decrease was maintained on day 9 (Table 1). Midazolam C_max values returned to baseline by day 13 and remained steady until day 34 when it increased slightly. Midazolam AUC_0- decreased 16 to 35% over days 7 to 13 and returned to baseline by day 20. Midazolam CL/F increased 19 to 53% over days 7 to 13 and returned to baseline by day 20. The observed results in midazolam CL/F and V/F were similar after weight normalization (data not shown). Midazolam t_1/2 increased on day 7 but decreased on days 13 and 20 (Table 1). Plasma pleconaril concentrations (mean ± S.D.) increased on days 5, 6, and 7 (697.5 ± 339.2, 787.7 ± 302.9, and 968.2 ± 366.8 ng/ml).

	Discussion

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This study evaluated the duration of increased intestinal and hepatic CYP3A activity by pleconaril in healthy adults. The increase in midazolam CL/F continued to day 13 and returned to baseline by day 20. These results are surprising because we expected the duration of increased CYP3A activity to be sustained until complete elimination of pleconaril (23–30 days).

Induction and heterotropic cooperativity are two possible explanations for increased CYP3A activity by pleconaril. The molecular mechanisms underlying P450 induction are not completely understood but generally result in a quantitative increase in CYP3A enzyme (Lin and Lu, 1998). The duration of CYP3A induction may be exponential and reflective of the degradation rate of CYP3A (Thummel and Wilkinson, 1998). Based on an irreversible enzyme inhibition model, the half-life of CYP3A is reported to be 8 h (Takanaga et al., 1999). However, there are limited and contrasting in vivo CYP3A data to support this hypothesis (Ohnhaus et al., 1989; Lee et al., 1993). The other explanation may be a qualitative change in existing CYP3A enzyme, termed heterotropic cooperativity. This has been reported primarily from in vitro experiments (Ekins et al., 1998; Tang and Stearns, 2001). Distinct binding sites in the active site of CYP3A or changes in CYP3A enzyme conformation during pleconaril and midazolam coadministration are possible but remain theoretical. In addition, a change of the kinetic characteristics of pleconaril (e.g., increase in V_max, decrease in K_m, or a change in both) may also be involved. Additional evidence to suggest CYP3A cooperativity comes from two short-term pleconaril administration studies (data on file, ViroPharma, Inc.). In one study, a pleconaril 800-mg loading dose followed by 400 mg every 6 h for three doses significantly decreased oral midazolam AUC_0- by 36%. In another, the AUC_0- of ethinyl estradiol (a CYP3A substrate) was significantly decreased by 17% after administration of a pleconaril 800-mg loading dose followed by 400 mg every 4 h for four doses. Heterotropic cooperativity may explain the discordance between pleconaril elimination half-life and the duration of increased CYP3A activity.

Midazolam V/F increased on day 7. This may be due to decreased midazolam oral bioavailability as a result of increased intestinal CYP3A activity by pleconaril. Midazolam t_1/2 also increased on day 7. Because t_1/2 is a dependent variable, changes in either the V/F or oral clearance may affect elimination half-life. The increase in V/F, to a greater extent than oral clearance, may account for the transient increase in the midazolam t_1/2 on day 7.

Pleconaril elimination half-life has been reported to be 6.7 to 34 h (Abdel-Rahman and Kearns, 1998, 1999), which is in contrast to the half-life of 180 h (Rhodes and Liu, 2001a,b; Florea et al., 2003). However, the shorter half-lives reported in the literature were determined from a truncated sample collection period (24–36 h). This did not allow for an accurate estimation of the terminal elimination half-life of the drug (Gilbaldi and Weintraub, 1971). The reported half-life of 180 h was based on a sample collection period that extended to 11 days after pleconaril administration and used analytical methods with increased sensitivity (Rhodes and Liu, 2001a,b).

The increases in intestinal and hepatic CYP3A activity are consistent with findings from another study of healthy adults, where oral pleconaril (400 mg three times daily for 16 doses) increased hepatic CYP3A activity after intravenous midazolam administration (Ma et al., 2006). Geometric mean day 1 and day 7 midazolam AUC_0- (ng min/ml) was 3093.6 and 2222.4, respectively. Midazolam AUC_0- decreased modestly [28% (LS-GMR; 90% CI: 0.718; 0.674–0.765)]. In comparison, rifampin (a potent CYP3A inducer) has been reported to decrease midazolam AUC_0- by 96% (Backman et al., 1996).

In summary, oral pleconaril increases intestinal and hepatic CYP3A activity. The duration was at least 6 days (but no longer than 13 days) after pleconaril discontinuation. The effect of pleconaril on CYP3A activity may be CYP3A induction, heterotropic cooperativity, or both. Duration and extent of increased CYP3A activity needs to be considered when evaluating the clinical significance and potential for drug interactions upon coadministration of pleconaril and other drugs primarily metabolized by CYP3A.

	Footnotes

 
Financial support was provided by ViroPharma, Incorporated.

This work was presented in part at the 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy, September 14–17, 2003, Chicago, IL.

J.S.B. has consulted for Viropharma, Inc., Ortho-McNeil, GlaxoSmithKline, and Merck. A.N.N. has consulted for Merck & Co. and Merck/Schering-Plough.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.105.007831.

ABBREVIATIONS: P450, cytochrome P450; AUC_0-last, area under the concentration-time curve extrapolated to the last measurable concentration; AUC_0-, area under the concentration-time curve extrapolated to infinity; CL, clearance; LS-GMR, least squares geometric mean ratios; 90% CI, 90% confidence interval(s).

Address correspondence to: Dr. Joseph S. Bertino, Jr., ORI Drug Development Center, Ordway Research Institute, Inc. 150 New Scotland Ave., Albany, NY 12208. E-mail: sbertino{at}ix.netcom.com

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