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Research ArticleArticle

Examination of the Human Cytochrome P4503A4 Induction Potential of PF-06282999, an Irreversible Myeloperoxidase Inactivator: Integration of Preclinical, In Silico, and Biomarker Methodologies in the Prediction of the Clinical Outcome

Jennifer Q. Dong, James R. Gosset, Odette A. Fahmi, Zhiwu Lin, Jeffrey R. Chabot, Steven G. Terra, Vu Le, Kristin Chidsey, Parya Nouri, Albert Kim, Leonard Buckbinder and Amit S. Kalgutkar
Drug Metabolism and Disposition May 2017, 45 (5) 501-511; DOI: https://doi.org/10.1124/dmd.116.074476

Abstract

The propensity for CYP3A4 induction by 2-(6-(5-chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide (PF-06282999), an irreversible inactivator of myeloperoxidase, was examined in the present study. Studies using human hepatocytes revealed moderate increases in CYP3A4 mRNA and midazolam-1′-hydroxylase activity in a PF-06282999 dose-dependent fashion. At the highest tested concentration of 300 μM, PF-06282999 caused maximal induction in CYP3A4 mRNA and enzyme activity ranging from 56% to 86% and 47% t0 72%, respectively, of rifampicin response across the three hepatocyte donor pools. In a clinical drug-drug interaction (DDI) study, the mean midazolam Cmax and area under the curve (AUC) values following 14-day treatment with PF-06282999 decreased in a dose-dependent fashion with a maximum decrease in midazolam AUC0–inf and Cmax of ∼57.2% and 41.1% observed at the 500 mg twice daily dose. The moderate impact on midazolam pharmacokinetics at the 500 mg twice daily dose of PF-06282999 was also reflected in statistically significant changes in plasma 4β-hydroxycholesterol/cholesterol and urinary 6β-hydroxycortisol/cortisol ratios. Changes in plasma and urinary CYP3A4 biomarkers did not reach statistical significance at the 125 mg three times daily dose of PF-06282999, despite a modest decrease in midazolam systemic exposure. Predicted DDI magnitude based on the in vitro induction parameters and simulated pharmacokinetics of perpetrator (PF-06282999) and victim (midazolam) using the Simcyp (Simcyp Ltd., Sheffield, United Kingdom) population-based simulator were in reasonable agreement with the observed clinical data. Since the magnitude of the 4β-hydroxycholesterol or 6β-hydroxycortisol ratio change was generally smaller than the magnitude of the midazolam AUC change with PF-06282999, a pharmacokinetic interaction study with midazolam ultimately proved important for assessment of DDI via CYP3A4 induction.

Introduction

The important role of cytochrome P450 (P450) enzymes in drug metabolism is highlighted by the fact that the CYP1, CYP2, and CYP3 families combined are responsible for the biotransformation of most xenobiotics including 70%–80% of all medicinal products in clinical use (Nelson, 2006; Guengerich, 2008; Zanger et al., 2008). Within the human P450 superfamily of drug-metabolizing enzymes, constitutively expressed CYP3A4 has the highest liver expression and plays a major role in the metabolism of ∼55% of drugs from almost all therapeutic categories (Wienkers and Heath, 2005; Bu, 2006; Liu et al., 2007). Drug-drug interactions (DDIs) caused by inhibition and/or induction of drug-metabolizing enzymes, particularly CYP3A4, can impact the efficacy and safety of coadministered drugs (Lin and Lu, 1998; Isoherranen et al., 2012). In vitro characterization of P450 inhibitory/induction potential of new investigational drug substances and interpretation of the generated results are critical steps in the drug development process, particularly in the design of relevant clinical DDI studies, and is also required for regulatory filing (http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm292362.pdf; http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2012/07/WC500129606.pdf).

2-(6-(5-Chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide (PF-06282999) (Fig. 1) is an irreversible inactivator of the human myeloperoxidase (MPO) enzyme (Ruggeri et al., 2015) and is a clinical candidate for the treatment of acute coronary syndrome and related cardiovascular disorders. PF-06282999 has been evaluated in first-in-human clinical studies for assessment of safety and tolerability (Dong et al., 2016). PF-06282999 is virtually resistant to metabolic turnover in human liver microsomes and human hepatocytes, and is principally eliminated in unchanged form via renal excretion in healthy adult volunteers (Dong et al., 2016). Although PF-06282999 was devoid of inhibitory (reversible and/or time-dependent) effects against major human P450 enzymes including CYP3A4, subsequent studies in cryopreserved human hepatocytes revealed a propensity for CYP3A4 induction upon pretreatment with PF-06282999. Because the degree of MPO inhibition that leads to clinical benefit in cardiovascular disease remains uncharacterized at the present time, there is much uncertainty around the clinically efficacious dose (and relevant systemic exposure) of PF-06282999 in relation to DDI potential with victim drugs (e.g., calcium channel blockers, lipid-lowering agents, etc.) that are subject to metabolism by CYP3A4 in a cardiovascular disease patient population (Niemi et al., 2003; Neuvonen et al., 2006).

Fig. 1.
Fig. 1.

Fold induction in mRNA expression (A) and enzyme activity (B) of CYP3A4 (A) in cultured human hepatocyte cultures treated with PF-06282999 (0.3, 2, 5, 10, 20, 30, 40, 50, 75, 100, 150, 200, and 300 µM) from three human donors (left-side bars, HH1089; middle bars, HC7-4; right-side bars, FOS). For purposes of comparison, increases in CYP3A mRNA and enzyme activity with rifampicin (10 μM) are also depicted. The chemical structure of PF-06282999 is also shown.

Assessment of clinical DDIs arising from inhibition and/or induction of CYP3A4 by investigational drug substances usually relies on the use of midazolam as a victim drug and an exogenous marker of CYP3A4 metabolic activity (Sinz et al., 2008; Fahmi et al., 2009; Kirwan et al., 2010; Jones et al., 2017). Apart from pharmacokinetics (PK) interaction studies with probe CYP3A4 substrates, changes in plasma and urinary ratios of 4β-hydroxycholesterol to cholesterol and 6β-hydroxycortisol to cortisol, respectively, can be used as endogenous biomarkers of CYP3A4 activity (Bodin et al., 2001; Nowak et al., 2002; Galteau and Shamsa, 2003; Konishi et al., 2004; Diczfalusy et al., 2011; Mårde Arrhén et al., 2013). The selective CYP3A4-mediated metabolic conversion of cholesterol to 4β-hydroxycholesterol and/or cortisol to 6β-hydroxycortisol, respectively, is used increasingly in clinical studies as noninvasive phenotypic markers of CYP3A4 induction, and are often implemented in phase 1 multiple ascending dose (MAD) clinical studies (Björkhem-Bergman et al., 2013; Dutreix et al., 2014). In our present work, a MAD study in healthy volunteers was conducted with PF-06282999 to assess the safety, tolerability, and PK upon repeated oral dosing, with a particular emphasis on the measurement of the endogenous plasma (4β-hydroxycholesterol/cholesterol) and urine (6β-hydroxycortisol/cortisol) biomarkers as surrogates for CYP3A4 induction. A DDI protocol was then integrated into the study design, which included the coadministration of PF-06282999 with midazolam to estimate the magnitude of the area under the curve (AUC) plasma concentration-time change for midazolam as a result of CYP3A4 induction by PF-06282999. Finally, in vitro CYP3A4 induction data in human hepatocyte cultures and the available clinical PK data were used to rationalize the clinical observations via dynamic physiologically based PK modeling using the Simcyp version 15.1 (Simcyp Ltd., Sheffield, United Kingdom) population-based simulator. The collective findings are summarized, herein.

Materials and Methods

Materials.

PF-06282999 (chemical purity >99% by high-performance liquid chromatography and nuclear magnetic resonance) was synthesized at Pfizer Worldwide Research and Development (Groton, CT). Hepatocyte thawing, plating, and incubation media as well as the Torpedo antibiotic mix for the media were purchased from Bioreclamation IVT Celsis (Baltimore, MD) and CellzDirect (Pittsboro, NC). Rifampin, midazolam, 1′-hydroxymidazolam, Dulbecco’s phosphate-buffered saline, and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). Cryopreserved human hepatocytes (Caucasian female, 57 years), HC7-4 (Caucasian male, 7 years), and FOS (Arabic male, 34 years) were purchased from In Vitro ADMET Laboratories (Columbia, MD), XenoTech (Lenexa, KS), and Bioreclamation IVT Celsis, respectively.

CYP3A4 Induction in Human Hepatocytes.

Cryopreserved human hepatocytes were used to examine CYP3A4 induction potential of PF-06282999 using established protocols (Fahmi et al., 2008b). Briefly, primary cultures of human hepatocytes from three donors (HH1089, HC7-4, and FOS) were seeded in collagen I-precoated 24-well plates, in which each well had a cell density of ∼2 × 105 viable cells. Hepatocyte cultures were treated for three consecutive days with medium containing solvent (0.1% DMSO) control, PF-0628999 (0.3, 2, 5, 10, 20, 30, 40, 50, 75, 100, 150, 200, and 300 µM), or prototypical CYP3A4 inducer rifampicin (0.01, 0.1, 0.5, 1, 5, and 10 µM). Following the incubation phase of the experiment, the medium containing test compounds or vehicle was removed, the cells were washed with phosphate-buffered saline, and then fresh medium was added to all wells for a 30-minute wash. Following removal of the wash media, midazolam (30 μM) in 0.1% DMSO was added to each well and the plate was returned to the incubator for 30 minutes. After incubation, 200 μl of the medium from each well was removed and analyzed for 1-hydroxymidazolam formation using established liquid chromatography–tandem mass spectrometry (LC-MS/MS) protocols (Walsky and Obach, 2004). Quantification of CYP3A4 mRNA was performed using the TaqMan method as previously described (Fahmi et al., 2008b).

In Vitro Data Analysis.

Data for mRNA and activity (normalized to the DMSO vehicle control) were plotted versus the concentration of PF-06282999 or rifampin. Individual fitting was carried out on each hepatocyte donor using GraphPad Prism (version 6; GraphPad Software, Inc; La Jolla, CA), and was fit to either a four-parameter sigmoidal model [eq. 1, fitting the maximal effect (Emax), Hill slope γ, and EC50, with the baseline fixed at 1] or a three-parameter sigmoidal model (eq. 2, fitting Emax and EC50, with the Hill slope and baseline each fixed at 1). The four-parameter model was preferred if adding the extra parameter produced a fit with P < 0.05 compared with the three-parameter model by the built-in GraphPad extra sum-of-squares F test.

Embedded Image(1)Embedded Image(2)

Clinical DDI Study Design.

The DDI potential of PF-06282999 was characterized in a clinical study (clinical trial identifier NCT01707082) comprised of healthy adult volunteers. This study was conducted according to the ethical principles of the Declaration of Helsinki and all protocols and informed consent documents were approved by the Ethics Committee for the study center. All subjects signed an approved written informed consent form before any study-related activities. There were two parts to this clinical study. Part A was a blinded, randomized placebo-controlled parallel group escalating multiple oral dose study in healthy overweight adult subjects in order to assess the safety, tolerability, and PK of PF-06282999 following 14 days of oral administration. A total of 48 male subjects and two female subjects (86% Caucasian, 4% African American, and 10% other) aged 22–55 years old, with a body weight range of 62.2–116.5 kg and a body mass index range of 27.0–34.8 kg/m2, were enrolled in five cohorts (10 per cohort; eight active and two placebo). The doses tested were 10, 30, 100, and 250 mg three times a day and 500 mg twice a day, all administered with food. PF-06282999 was administered as an immediate-release tablet during both periods of the study. The safety and tolerability of PF 06282999 was assessed by adverse events, electrocardiograms, vital signs, and safety laboratory measurements. In addition to the collection of safety and PK data, the concentrations of plasma (4β-hydroxycholesterol/cholesterol) and urinary (6β-hydroxycortisol/cortisol) biomarkers associated with CYP3A4 induction were also assessed in the various PF-06282999 dose groups.

Part B of the study was an open-label fixed-sequence DDI study in healthy adult volunteers evaluating the impact of PF-06282999 on the single-dose PK of oral midazolam. A total of 18 male subjects (94% Caucasian and 6% other) aged 20–48 years old, with a body weight range of 57.3–86.0 kg and a body mass index range of 19–27.3 kg/m2, were enrolled in two cohorts (nine per cohort) to evaluate the effect of two dose levels of PF-06282999 on the PK of oral midazolam. The two dose levels of PF-06282999 (125 mg three times a day and 500 mg twice a day) were selected based on review of emerging data from Part A of the study, including the review of the available urinary and plasma biomarker data. This DDI part of the study was conducted in two periods. On day 1 of period 1 after an overnight fast, subjects received a single 7.5 mg tablet oral dose of midazolam followed by collection of blood samples for up to 16 hours postdose for evaluation of midazolam PK. On the next day, period 2/day 1 commenced when subjects received their initial dose of PF-06282999. All doses were provided at approximately the same time of day throughout the study for 13 days. All doses of PF-06282999 were administered following completion of a standard meal. On day 14 of period 2, after an overnight fast, subjects received another single 7.5 mg oral dose of midazolam. Two hours later, PF-06282999 was administered following completion of a standard meal (the only dose to be administered on day 14). Midazolam was administered at a similar time on day 1 of period 1 and day 14 of period 2.

Evaluation of PF-06282999 PK and Endogenous Biomarkers of CYP3A4 Induction.

For part A, serial blood samples were collected prior to dosing and up to 48 hours after the first and last dose for quantitation of circulating levels of PF-06282999. Sparse sampling was also performed on subsequent dosing days to confirm the steady-state status of systemic exposure. For part B, only predose blood samples were collected on selected days to confirm adequate exposures to PF-06282999. Blood samples were centrifuged to generate plasma and concentrations of PF-06282999 were determined using a validated LC-MS/MS method (Dong et al., 2016). The PK parameters were calculated using noncompartmental analysis (eNCA, a Pfizer internally validated system of noncompartmental analysis). The AUC concentration-time curve over dosing interval (AUC0–τ) steady-state plasma PK parameters, peak plasma concentration (Cmax), time-to-peak concentration, minimum or trough concentration, average concentration over a dosing interval half-life (t1/2), and the observed accumulation ratio for AUC0–τ and Cmax were summarized descriptively by dose.

Measurement of plasma (cholesterol and 4β-hydroxycholesterol) and urine (cortisol and 6β-hydroxycortisol) biomarkers was performed at SGS Cephac Europe (Saint Benoît Cedex, France). Blood samples were also collected in both part A and B segments of the study for analysis of plasma cholesterol and 4β-hydroxycholesterol concentrations. Cholesterol levels were measured following chemical derivatization of plasma cholesterol to the corresponding dansylated derivative (Tang and Guengerich, 2010). Plasma samples were diluted (100-fold) with a solution of bovine serum albumin in phosphate-buffered saline (40 g/l) containing 1% butyl-4-hydroxytoluene (22.69 mM)), followed by the addition of internal standard [2H6]-cholesterol (50 μg/ml) to ∼100 μl portions of the diluted plasma samples. The samples were then treated with ethanolic sodium hydroxide solution (9 M) in water and incubated for 5–15 minutes at 37°C followed by the addition of 200 µl of derivatization solution [dansyl chloride (150 mg) and dimethylaminopyridine (150 mg) in 15 ml of dichloromethane] in the presence of 2% diiosopropylethylamine solution and heated to approximately 65°C for 1 hour as previously described (Tang and Guengerich, 2010). The samples were centrifuged, the organic phase was evaporated to dryness, and the residue was reconstituted with 200 μl of mobile phase (5 mM ammonium acetate in acetonitrile) prior to LC-MS/MS analysis. Samples (5–10 μl) were analyzed on an API-4000 triple-quadrupole mass spectrometer (AB Sciex, Framingham, MA) with a Phenomenex Kinetex C18 column (2.6 μm, 150 × 2.1 mm, 100 Å) at 40°C–55°C (Phenomenex, Torrance, CA). For separation of dansyl cholesterol from endogenous plasma components, liquid chromatography was performed under isocratic conditions using a mobile phase comprised of 5 mM ammonium acetate in acetonitrile at a flow rate of 0.5 ml/min. Under these conditions, the retention time for dansyl cholesterol (and its stable-labeled internal standard) was 1.8 minutes. Dansylated derivatives of cholesterol and the internal standard [2H6]-cholesterol were detected using electrospray ionization (positive ion mode) in the multiple-reactions monitoring mode, monitoring for mass-to-charge transitions 620 → 252 (cholesterol) and 626 → 252 ([2H6]-cholesterol). The lower limit of quantification was 1.0 µg/ml with an overall assay precision of ≤8.0%, and % relative error from −8.26% to 2.07%.

For measurement of 4β-hydroxycholesterol plasma levels, plasma samples (200 μl) were diluted with methanol (20 μl) containing the internal standard ([2H7]-4β-hydroxycholesterol (1250 ng/ml). The samples were centrifuged (3000g) for 10 minutes and the supernatant was dried under a steady stream of nitrogen. The residue was reconstituted with hexane containing 0.5% isopropanol, loaded onto solid-phase extraction cartridges (Biotage ISOLUTE Si 100 mg, 3cc; Biotage, Charlotte, NC), and eluted with 3 ml of hexane/isopropanol (70/30; v/v). The eluate was evaporated and reconstituted with the high-performance liquid chromatography mobile phase prior to analysis. The liquid chromatography method consisted of a 6-minute gradient with first 2 minutes at 20% solvent A [5 mM ammonium acetate in methanol/water (80/20; v/v)] and 80% solvent B (5 mM ammonium acetate in methanol) followed by gradual increase (1.5 minutes) to 100% solvent B. The flow rate was 0.3 ml/min and the retention time for 4β-hydroxycholesterol and its stable-labeled internal standard were 3.0 and 2.9 minutes, respectively. 4β-Hydroxycholesterol and [2H7]-4β-hydroxycholesterol were detected using electrospray ionization (positive ion mode) in the multiple-reactions monitoring mode, monitoring for mass-to-charge transitions 402 → 385 (4β-hydroxycholesterol) and 409 → 392 ([2H7]- 4β-hydroxycholesterol). The lower limit of quantification was 3.0 ng/ml with overall assay precision of <5.0%, and % relative error from −5.20% to 4.59%.

Twenty-four hour urine collections both before and after the 14-day oral administration of PF-06282999 were analyzed for the amount of urinary 6β-hydroxycortisol and cortisol. Urine samples (500 μl) diluted with 500 μl phosphate buffer (pH 8.0) were treated with 25 μl of the internal standard (30000/1000 ng/ml ([2H3]-6β-hydroxycortisol/[2H4]-cortisol in 50/50 methanol/water; v:v) and loaded onto preconditioned (with 0.5 ml methanol and 0.5 ml water) Oasis HLB cartridges and eluted with 300 μl of methanol. The samples were centrifuged (3500g, 1 minute at 5°C), evaporated to dryness under a steady stream of nitrogen, and the residue was reconstituted with 0.3 ml of a methanol/water (50/50) mixture containing 0.1% formic acid (Oasis, Milford, MA). The samples (20 µl) were analyzed on an API-4000 triple-quadrupole mass spectrometer (AB Sciex) with a Merck Chromolith RP-18e column (100 × 4.6 mm) at 30°C (Merck, Billerica, MA). Analytes were eluted using a binary gradient comprised of water/acetonitrile (90/10; v/v) with 0.5% formic acid (mobile phase A) and water/acetonitrile (80/20; v/v) with 0.5% formic acid (mobile phase B). Initial conditions (100% A) were held at 4 minutes at a flow rate of 2.0 ml/min, and then changed to 100% B over 7.5 minutes (flow rate = 3.0 ml/min) and held for 2 minutes. The flow rate was split into the mass spectrometer at a flow rate of 0.5 ml/min. The retention times for 6β-hydroxycortisol and cortisol were at 3.0 minutes. The retention times of the internal standards were at 9.8 minutes. The mass spectrometer was operated in positive electrospray mode and the mass-to-charge transitions used were 363 → 121 (cortisol), 379 → 343 (6β-hydroxycortisol), 367 → 121 ([2H4]-cortisol), and 382 → 346 ([2H3]-6β-hydroxycortisol). The lower limit of quantification of cortisol was 1.00 ng/ml with assay precision of ≤6.0% and % relative error from −4.40% to 0.33%. The lower limit of quantification of 6β-hydroxycortisol was 10.0 ng/ml with assay precision of ≤5.0% and % relative error from −0.89% to 6.56%.

The 4β-hydroxycholesterol/cholesterol and 6β-hydroxycortisol/cortisol ratios were calculated for individual subjects at pre- and post-treatment. For part A, the fold induction was derived as the ratio of post-treatment/pretreatment 4β-hydroxycholesterol ratio or 6β-hydroxycortisol ratio for each of the PF-06282999 dose groups or the placebo. Pairwise comparison between each PF-06282999 dose group versus placebo was performed using the t test to examine the dose-dependent changes in the biomarkers of CYP3A4 induction. For part B, the 4β-hydroxycholesterol and 6β-hydroxycortisol ratio data were log-transformed and then back-transformed to derive the fold induction with a 90% confidence interval (CI). The P values were from the t test and a 90% CI excluding 1 would indicate a statistically significant change from the baseline in the markers of CYP3A4 induction.

Assessment of Clinical DDI with Midazolam.

In the DDI part of the study, serial blood samples were collected for midazolam PK analysis (16-hour PK profile) on day 1 of period 1 for midazolam given alone and on day 14 of period 2 for midazolam and PF-06282999 coadministration. Plasma samples were analyzed for midazolam concentrations at Covance Bioanalytical Services (Shanghai, China) using a validated, sensitive, and specific LC-MS/MS method (Covance data on file). Calibration standard responses were linear over the range of 0.05–50 ng/ml, using a weighted (1/concentration2) linear least-squares regression. The lower limit of quantification for midazolam was 0.05 ng/ml. The overall assay precision was ≤7.9%, with % relative error from −5.6% to 0.0%. The PK parameters for midazolam were calculated using noncompartmental analysis (eNCA). Plasma midazolam PK parameters Cmax, AUC0–16 hours, and AUC0–inf were summarized descriptively by dose and period. The interactive effect on Cmax, AUC0–16 hours, and AUC0–inf of midazolam were determined by construction 90% CIs around the estimated difference between the test and reference treatments using a mixed-effects model based on the log-transformed data. The mixed-effects models, with the treatment as fixed effects and the subject as a random effect, was implemented using SAS PROC MIXED (SAS Institute, Inc., Cary, NC), with the restricted maximum likelihood estimation method and Kenward-Roger degrees of freedom algorithm. Estimates of the adjusted mean differences (test/reference) and corresponding 90% CIs for the differences were exponentiated to provide estimates of the ratio of adjusted geometric means (test/reference) and 90% CIs for the ratios. Midazolam alone was the reference treatment, while the midazolam coadministered with PF-06282999 was the test treatment.

Simcyp Model.

Physiologically based PK modeling was used to simulate the effect of PF-06282999 on midazolam metabolism. Simulations were carried out using the Simcyp population-based absorption, distribution, metabolism, and elimination simulator (Simcyp Ltd.). The PF-06282999 Simcyp perpetrator file was built using the clinical PK information to ensure the models reasonably recapitulated the day 14 plasma-concentration time profiles reported in part A. The input parameters for PF-06282999 are shown in Table 1; data for absorption and renal clearance parameters of PF-06282999 at the dose used in the clinical study were obtained from previous clinical studies (Dong et al., 2016). For midazolam, the Simcyp default midazolam model was used. Individual in vitro CYP3A4 mRNA induction data (EC50, Emax, and the Hill coefficient, γ) for PF-06282999 and rifampin from the three hepatocyte donors (Tables 2 and 3) were integrated into the Simcyp induction calibrator to allow for correction of the PF-06282999 induction parameters in reference to the known inducer rifampin (Table 4). The Emax and EC50 values were directly inputted as the maximum fold induction and the concentration that yields half of the Emax; media binding was calculated to be negligible for PF-06282999 (Kalvass and Maurer, 2002).

View this table:
TABLE 1

PF-06282999 Simcyp model input parameters

View this table:
TABLE 2

Values of EC50, Emax, and γ (Hill slope) (± S.E.) for induction of CYP3A4 mRNA and midazolam-1′-hydroxylase activity by PF-06282999

The S.E. values reflect the fit parameter uncertainty; n = number of replicates.

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TABLE 3

Values of EC50 and Emax (± S.E.) for induction of CYP3A4 mRNA and midazolam-1′-hydroxylase activity by rifampin

The S.E. values are determined by the variability in the fit parameters obtained for each independent run; n = number of independent runs.

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TABLE 4

Simcyp calibrated in vitro CYP3A4 induction parameters for PF-06282999

Simulations were performed for 100 healthy subjects of North American Caucasian origin (i.e., 10 different subjects were chosen randomly for each of the 10 trial simulations); simulated subjects had an average body weight of 74 kg, were aged between 20 and 50 years, and included both genders. The AUCT of the test (coadministration of midazolam and PF-06282999) and that of the reference (midazolam alone) treatment were determined by Simcyp algorithms. Dose, dose interval, and duration of administration of PF-06282999 and midazolam were set according to the clinical protocol, such that PF-06282999 was administered at 125 mg three times a day or 500 mg twice a day for 15 days with coadministration of midazolam (7.5 mg single dose), with the first dose of PF-06282999 on day 14.

Results

In Vitro Induction Assessment.

The potential for induction of CYP3A4 mRNA and enzyme activity was examined in cryopreserved human hepatocytes treated with PF-06282999 at several concentrations (0.3–300 μM). PF-06282999 demonstrated moderate induction of CYP3A4 mRNA levels and midazolam-1′-hydroxylase activity in a dose-dependent fashion in all three donor hepatocytes (Fig. 1). In contrast, treatment with positive control rifampicin led to a marked induction of CYP3A4 as measured from increases in mRNA and midazolam-1′-hydroxylase activity in cryopreserved human hepatocytes from the three donors. The estimated EC50 and Emax values for fold increases in CYP3A4 mRNA levels and enzyme activity in the three lots of human hepatocytes for PF-06282999 and rifampicin are shown in Tables 2 and 3. A cytotoxicity assay using MTS Cell Proliferation reagent (Promega, Madison, WI) and visual inspection showed no apparent reduction in hepatocyte viability after treatment with PF-06282999 at the tested concentrations.

Safety and Clinical PK.

PF-06282999 was safe and well tolerated after multiple oral doses up to 500 mg twice a day for 14 days in healthy adult volunteers. The corresponding Cmax value of PF 06282999 was achieved with a median time-to-peak concentration of 2.5–3.5 hours on day 1 and 2.0–3.0 hours on day 14 (Table 5). Steady-state exposures were attained by day 2 with minimal to modest accumulation (<2-fold) of PF 06282999 in plasma observed after dosing twice and three times a day, which was consistent with the t1/2 values in the range of 4.4–7.3 hours in this study as well as a previous single ascending dose study in healthy volunteers (Dong et al., 2016). Both Cmax and AUC0–τ increased proportionally with increasing dose from 10 to 250 mg three times a day and 500 mg twice a day, with maximum Cmax and AUC0–τ values of 5.14 µg/ml and 35.6 h/µg/ml, respectively, achieved at 500 mg twice a day (Table 5). For the midazolam DDI part of the study (part B), the mean minimum or trough PF-06282999 concentrations were 1.57 µg/ml on day 7 and 1.90 µg/ml on day 14 for 500 mg twice a day and 1.11 µg/ml on day 7 and 1.09 µg/ml on day 14 for 125 mg three times a day, respectively. These minimum or trough concentration values were comparable to those achieved in part A at similar doses (Table 5).

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TABLE 5

Summary of PF-06282999 pharmacokinetic parameters following daily PF-06282999 administrations for 14 days

The dosing intervals for τ are 12 hours for twice a day and 8 hours for three times a day.

Effects on Biomarkers of CYP3A4 Induction.

Because PF-06282999 induced CYP3A4 activity in human hepatocytes, endogenous clinical biomarkers of CYP3A4 activity were monitored in the MAD study with PF-06282999. Dose-dependent increases in both the plasma 4β-hydroxycholesterol and 24-hour urinary 6β–hydroxycortisol ratios were observed following daily administrations of PF-06282999 for 14 days, with an apparent induction effect starting at the 100 mg dose level three times a day (part A of study) (Fig. 2). Comparison between placebo and PF-06282999 dose groups was performed using the t test. For the 4β-hydroxycholesterol ratio, significant differences from placebo were detected in the 250 mg three times a day and 500 mg twice a day dose groups (P = 0.0044 and 0.0003, respectively). For the 6β-hydroxycortisol ratio, significant differences from placebo were found in the 100 and 250 mg three times daily and the 500 mg twice a day dose groups (P = 0.0206, 0.0029, and 0.0018, respectively).

Fig. 2.
Fig. 2.

Dose-dependent fold induction of urinary and plasma biomarkers of CYP3A4 induction, 4β-hydroxycholesterol/cholesterol ratio (4β-OH cholesterol ratio) and 6β-hydroxycortisol/cortisol ratio (6β-OH cortisol ratio), following daily administrations of placebo or PF-06282999 for 14 days. The numbers at the top designate the median fold induction in each treatment group. Pairwise comparison was performed between each treatment group versus placebo using the t test.

Midazolam DDI Study.

The observed changes in the biomarkers of CYP3A4 induction during part A of the study formed the basis of dose selection for the DDI portion of the study (part B), i.e., to confirm the extent of CYP3A4 induction following 125 mg three times daily and 500 mg twice daily PF-06282999 administrations for 15 days using oral midazolam as a probe. The median midazolam plasma concentrations over the 16-hour sampling interval were lower in the presence of multiple PF-06282999 administrations than when midazolam was administered alone. Midazolam was rapidly absorbed following a single 7.5 mg oral dose administered either alone or with multiple-dose PF 06282999 (500 mg twice a day or 125 mg three times a day), with similar median time-to-peak concentrations of 0.5–1 hours. The mean terminal t1/2 value for midazolam was similar when administered alone and with multiple-dose PF 06282999 ranging from 3.3 to 4.6 hours. Following the coadministration of midazolam and PF-06282999, the midazolam AUC and Cmax values were lower than those when midazolam was administered alone (Table 6). The results of the statistical comparison indicate that multiple-dose administration of PF-06282999 significantly decreased both peak and total midazolam exposures in a dose-dependent manner (Table 6). Following coadministration of 500 mg twice a day of PF-06282999, the mean (90% CI) midazolam AUC0–inf and Cmax values were decreased by 57.2% (52.1%, 61.7%) and 41.1% (24.6%, 54.06%), respectively, compared with when midazolam was administered alone. The exposures were decreased by 34.0% (26.2%, 41.0%) for AUC0–inf and 30.8% (11.4%, 46.0%) for Cmax, respectively, for midazolam with PF-06282999 125 mg three times a day.

View this table:
TABLE 6

Statistical summary of the pharmacokinetic parameters after an oral dose of 7.5 mg midazolam with and without coadministration of PF-06282999

For each PF-06282999 dose, n = 9 subjects; the ratios (and 90% Cis) are expressed as percentages.

Comparison of CYP3A4 Induction Biomarkers.

In the midazolam DDI part of the study, the CYP3A4 induction after 2 weeks of daily 125 mg three times a day or 500 mg twice a day of PF-06282999 treatment was also assessed by measuring the plasma 4β-hydroxycholesterol/cholesterol and urinary 6β-hydroxycortisol/cortisol ratios. There appeared to be comparable levels of baseline CYP3A4 enzyme activities (based on the plasma 4β-hydroxycholesterol/cholesterol ratio, urinary 6β-hydroxycortisol/cortisol ratio, and midazolam PK) before treatment with PF-06282999 within the two dose cohorts (Fig. 3). Statistical analysis comparing the values after PF-06282999 treatment with the baseline values revealed significant changes in the 4β-hydroxycholesterol/cholesterol and 6β-hydroxycortisol/cortisol ratios following the 500 mg PF-06282999 twice a day dosing regimen (Table 7). This was associated with moderate impact on midazolam PK (ratio to baseline of 0.44 for midazolam AUC0–16 hours and 0.59 for midazolam Cmax, respectively) (Table 7). Dosing of PF-06282999 at 125 mg three times a day did not demonstrate significant changes in the 4β-hydroxycholesterol/cholesterol ratio or the 6β-hydroxycortisol/cortisol ratio, albeit a modest impact on midazolam exposure was observed (Table 7). The magnitude of fold induction was the largest for the midazolam AUC changes, followed by the 6β-hydroxycortisol/cortisol ratio, with the plasma 4β-hydroxycholesterol/cholesterol ratio showing minimal change (Table 7). While both the 4β-hydroxycholesterol/cholesterol and 6β-hydroxycortisol/cortisol ratios performed similarly in detecting CYP3A4 induction, the 6β-hydroxycortisol/cortisol ratio in urine showed more interindividual variability (Fig. 3).

Fig. 3.
Fig. 3.

Comparison of three different markers for CYP3A4 activities: 4β-hydroxycholesterol/cholesterol ratio (4β-OH cholesterol ratio), 6β-hydroxycortisol/cortisol ratio (4β-OH cortisol ratio), midazolam AUC0–16h, and midazolam Cmax before and after 14 days of PF-06282999 administrations with graphical illustration of the treatment effect.

View this table:
TABLE 7

Comparison of three different markers for CYP3A4 activities: 4β-hydroxycholesterol/cholesterol ratio (4β-OH cholesterol ratio), 6β-hydroxycortisol/cortisol ratio (6β-OH cortisol ratio), midazolam AUC0–16 hours, and midazolam Cmax before and after 14 days of PF-06282999 administrations with statistical analysis of the treatment effect

For each PF-06282999 dose, n = 9 subjects. The ratio-to-baseline values have been back-transformed from the log scale and the t test was used to obtain the P values.

Simcyp Simulation.

Simcyp was used to simulate the effect of coadministration of PF-06282999 on midazolam PK. Model simulations for PF-06282999 successfully captured the day 14 plasma concentration-time profiles from part A (Fig. 4). Incorporation of the most sensitive estimate of rifampin-calibrated mRNA CYP3A4 induction parameters for PF-06282999 (donor FOS) reasonably characterized the magnitude of CYP3A4 enzyme induction on the PK of midazolam (Fig. 5). The Simcyp model predicted an AUC ratio (AUCR) of 0.62 (90% CI: 0.59–0.65) for midazolam with 500 mg twice a day of PF-06282999 compared with the observed interaction (AUCR = 0.43) in part B (Table 8). At the lower dose of 125 mg PF-06282999 three times a day the model predicted an AUCR of 0.91 (90% CI: 0.90–0.92) for midazolam compared with the observed interaction (AUCR = 0.66) (Table 8).

Fig. 4.
Fig. 4.

Simcyp-simulated versus observed plasma profile of PF-06282999 following oral administration of 500 mg twice a day for 14 days.

Fig. 5.
Fig. 5.

Simcyp-simulated versus observed plasma profiles following a single oral dose of 7.5 mg midazolam without and with 14 days coadministration of PF-06282999 500 mg twice a day (upper panel) and 125 mg three times a day (lower panel).

View this table:
TABLE 8

Summary of predicted versus observed DDI using Simcyp

Discussion

PF-06282999 is devoid of reversible and/or time-dependent inhibitory effects against major human P450 enzymes including CYP3A4, which largely mitigates the likelihood of clinical DDIs arising from P450 inhibition by PF-06282999 (Dong et al., 2016). However, concentration-dependent increases in CYP3A4 mRNA and midazolam-1′-hydroxylase activity were noted in human hepatocytes treated with PF-06282999, indicating that PF-06282999 is a CYP3A4 inducer in vitro. At the highest tested concentration of 300 μM, PF-06282999 caused maximal induction in CYP3A4 mRNA and enzyme activity ranging from 56% to 86% and 47% to 72%, respectively, of the positive control rifampicin response in the three individual hepatocyte lots. Consistent with previous reports (Fahmi et al., 2008a,b), significant variability in the EC50 (43–105 μM) and Emax (12.7–24.5) values for induction of CYP3A4 mRNA was noted among the three different lots of human hepatocytes. Resistance of PF-06282999 toward metabolic turnover in human hepatocytes (Ruggeri et al., 2015; Dong et al., 2016) suggests that CYP3A4 induction is solely mediated by the parent compound with little to no contribution of metabolites. Clinical PK and safety studies in healthy adult volunteers revealed that PF-06282999 was safe and well tolerated after administration of multiple oral doses (10 mg three times a day; 500 mg twice a day) for 14 days. Systemic exposures as reflected by the Cmax and AUC0–τ values increased in a dose-proportional manner; maximum Cmax and AUC0–τ values of 5.14 µg/ml and 35.6 h/µg/ml, respectively, were achieved at the top 500 mg twice a day dose group. The corresponding unbound Cmax (5.94 μM, PF-06282999, molecular weight = 325, and human plasma unbound fraction = 0.376) and average (1.71 μM) concentrations of PF-06282999 at the 500 mg twice a day dose were approximately 2.2- to 7.8-fold in excess over the IC50 value (0.76 μM) for inhibition of MPO activity in human whole blood (Ruggeri et al., 2015), and this dose was potentially considered to be a therapeutic dose to test proof-of-pharmacology in phase 2 efficacy studies.

Because PF-06282999 induced CYP3A4 activity in human hepatocytes, measurement of plasma 4β-hydroxycholesterol/cholesterol and urinary 6β-hydroxycortisol/cortisol ratios was incorporated in the MAD study. Daily administration of PF-06282999 for 14 days led to dose-dependent increases in both the plasma 4β-hydroxycholesterol/cholesterol and 24-hour urinary 6β-hydroxycortisol/cortisol ratios. The urinary biomarker 6β-hydroxycortisol/cortisol ratio was more variable than the plasma 4β-hydroxycholesterol/cholesterol ratio, although in the present scenario the 6β-hydroxycortisol/cortisol ratio appeared to be more sensitive in detecting mean fold induction upon repeated dosing with PF-06282999. High inter- and intrasubject variabilities in the urinary 6β-hydroxycortisol/cortisol ratio, previously noted across several clinical studies in healthy volunteers, have been attributed to interindividual variability in CYP3A4 expression in human liver and/or variability in cortisol secretion into the circulation that is influenced by several factors such as stress, infections, and circadian rhythm (Forrester et al., 1992; Galteau and Shamsa, 2003; Dutreix et al., 2014). Consistent with the dose-dependent increases in endogenous CYP3A4 biomarker ratios in the MAD study, the mean midazolam Cmax and AUC values following 14-day treatment with PF-06282999 decreased in a dose-dependent fashion with maximum decreases in the midazolam AUC0–inf and Cmax values of 57.2% and 41.1%, respectively, observed in the 500 mg twice a day PF-06282999 dose group. The moderate impact on midazolam PK at the 500 mg twice a day dose of PF-06282999 was also reflected in statistically significant changes in the plasma 4β-hydroxycholesterol/cholesterol and urinary 6β-hydroxycortisol/cortisol ratios. Interestingly, changes in plasma and urinary CYP3A4 biomarkers did not reach statistical significance at the 125 mg dose of PF-06282999 three times a day, despite a modest impact on midazolam systemic exposure.

In retrospect, both plasma and urinary biomarkers performed relatively well in the MAD study as early indicators of the CYP3A4 induction potential of PF-06282999. However, the magnitude of CYP3A4 induction determined by oral midazolam systemic exposure was larger than the fold induction ascertained from the plasma and urinary 4β-hydroxycholesterol/cholesterol and 6β-hydroxycortisol/cortisol ratios, respectively. Similar findings have also been recently reported in a clinical 14-day rifampin/midazolam DDI study (Björkhem-Bergman et al., 2013). Across several rifampin dose groups (10, 20, and 100 mg), the median fold induction from baseline was 2.0, 2.6, and 4.0 for the estimated midazolam clearance; 1.7, 2.9, and 3.1 for the 6β-hydroxycortisol/cortisol ratio; and 1.3, 1.6, and 2.5 for the 4β-cholesterol/cholesterol ratio. It is possible that the plasma 4β-hydroxycholesterol/cholesterol ratio was not at steady state in the 2-week MAD/DDI studies because of the slow turnover of 4β-hydroxycholesterol (t1/2 = 17 days), and therefore does not fully reflect the induced CYP3A4 levels (Wide et al., 2008; Diczfalusy et al., 2009). In contrast, the 6β-hydroxycortisol/cortisol ratio reaches steady state relatively faster because of the shorter t1/2 (∼1 hour) of cortisol and its 6β-hydroxyl metabolite, with little delay or lag time for changes in CYP3A4 activity in vivo (Weitzman et al., 1971; Dutreix et al., 2014). In addition, oral midazolam PK takes into account intestinal and hepatic metabolism by CYP3A4. Therefore, the greater fold induction of midazolam AUC changes may be due to induction of both intestinal and hepatic CYP3A4 activity by the perpetrator agents (e.g., PF-06282999 and/or rifampin). As such, Simcyp simulations of the steady-state PK of PF-06282999 in the 125 mg three times a day and 500 mg twice a day dose groups using the calibrated induction data predicted approximately similar increases in active CYP3A4 enzyme of ∼1.1- and 1.5-fold, respectively, further strengthening the hypothesis around DDI arising from induction of both intestinal and liver CYP3A4 activity. However, in accordance with previous literature reports (Dresser et al., 2003; Imai et al., 2008; Davis et al., 2012), lack of significant changes in the terminal t1/2 value for midazolam in the 14-day DDI study with both dose groups of PF-06282999 suggests that PF-06282999 mainly induces intestinal CYP3A4. Additional clinical studies between PF-06282999 and midazolam (e.g., measurement of intravenous and oral AUC and/or clearance of midazolam in the presence of PF-06282999) will be required to delineate DDIs arising from CYP3A4 induction in the gut wall or the liver or both. Overall, the clinical studies conducted to investigate the potential of PF-06282999 as a DDI perpetrator confirmed that this compound was a moderate inducer of CYP3A4 according to the U.S. Food and Drug Administration classification system (http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm072101.pdf).

Simcyp, a population-based absorption, distribution, metabolism, and elimination simulator, has been successfully used by various laboratories to quantitatively predict metabolism-based DDIs (Fahmi et al., 2009; Xu et al., 2011; Almond et al., 2016). Relying on the in vitro CYP3A4 induction data, the Simcyp simulator can predict the extent of in vivo induction in a virtual population using a concentration-dependent dynamic induction model, as was recently demonstrated with rifampin, carbamazepine, and phenobarbital (Xu et al., 2011). Consequently, a retrospective analysis of the clinical PF-06282999/midazolam DDI data was performed using Simcyp. The predicted steady-state PK of PF-06282999 (125 mg three times a day and 500 mg twice a day) and the PK (Cmax and AUC) of midazolam after a single 7.5 mg dose correlated reasonably well with the data obtained from our clinical study. The induction DDIs were simulated for CYP3A4 mRNA increase using PF-06282999 and midazolam kinetics and the in vitro induction parameters (EC50 and Emax for the three individual hepatocyte lots). The predicted magnitudes of DDIs were shown to be in reasonable agreement, particularly at the high PF-06282999 dose (500 mg twice a day). PF-06282999 exhibited dose- and dosing duration–dependent effects on the magnitude of midazolam victim DDI. Consistent with the conclusions noted in previous studies (Almond et al., 2016), Simcyp could be a useful tool for prospectively predicting clinical DDI risks for drug candidates that induce CYP3A4 enzyme levels.

Induction of expression of drug metabolizing enzymes (and hepatobiliary transport proteins) by small molecule xenobiotics, including clinical drugs, often occurs through activation of nuclear receptors, resulting in transcriptional upregulation of the receptor’s target genes. Nuclear receptors commonly involved in regulation of P450 enzymes include the aryl hydrocarbon receptor, pregnane X receptor (PXR), and constitutive androstane receptor (CAR). Activation of the aryl hydrocarbon receptor results in upregulation of the CYP1A genes, while activation of PXR and/or CAR results in upregulation of CYP3A, CYP2B, and CYP2C genes (Raucy et al., 2002; Raucy, 2003; Tirona and Kim, 2005; Hewitt et al., 2007; Sahi et al., 2009). Induction of CYP3A4 expression by xenobiotics (including drugs) primarily occurs via PXR activation, although there is overlap with CAR both in terms of ligand activators and target genes (Moore et al., 2000; Willson and Kliewer, 2002; Wang et al., 2003, 2004; Faucette et al., 2006, 2007). Induction of CYP3A4 expression by PF-06282999, which ultimately manifested in a clinical DDI with midazolam, has been clearly demonstrated in this study; however, the propensity for DDIs resulting from the potential induction of CYP2B6, CYP2C9, and/or CYP2C19 enzymes by PF-06282999 remains to be examined. As such, PF-06282999 (2–300 μM) does not induce CYP1A2-mediated ethoxyresorufin-O-deethylase activity in human hepatocytes, suggesting that PF-06282999 does not activate the aryl hydrocarbon receptor (Pfizer data on file). In vitro studies using PXR and CAR reporter assays (Luo et al., 2002) are currently in progress to ascertain whether the biochemical basis of CYP3A4 induction by PF-06282999 arises through activation of PXR and/or CAR. The knowledge gained from these ongoing studies can be potentially used to design the next generation of selective MPO inactivators that are devoid of the CYP3A4 induction liability in the clinic. Likewise, the clinical development plan for PF-06282999 has been modified to include additional in vitro studies in human hepatocytes to examine induction of CYP2C9 and/or CYP2C19 mRNA and activity levels, information that can be potentially used in designing appropriate clinical DDI studies with relevant isozyme-selective substrates of the CYP2C family.

Our studies clearly demonstrate the informative value of endogenous biomarker measurement in first-in-human studies to examine CYP3A4 induction in vivo; However, since the magnitude of the 4β-hydroxycholesterol or 6β-hydroxycortisol ratio change was generally smaller than the magnitude of an oral midazolam AUC change with PF-06282999, an oral midazolam DDI study is ultimately needed to gauge DDI via CYP3A4 induction, which is likely to be the case for weak to moderate CYP3A4 inducers or inducers that have a significant intestinal CYP3A4 induction component.

Authorship Contributions

Participated in research design: Dong, Fahmi, Terra, Le, Chidsey, Kim, Buckbinder, Kalgutkar.

Conducted experiments: Lin, Nouri.

Contributed new reagents or analytic tools: Nouri.

Performed data analysis: Dong, Gosset, Fahmi, Lin, Chabot, Le.

Wrote or contributed to the writing of the manuscript: Dong, Gosset, Fahmi, Chabot, Terra, Kalgutkar.

Footnotes

Abbreviations

AUC
area under the curve
AUCR
area under the curve ratio
CAR
constitutive androstane receptor
CI
confidence interval
DDI
drug-drug interaction
DMSO
dimethylsulfoxide
Emax
maximal effect
LC-MS/MS
liquid chromatography–tandem mass spectrometry
MAD
multiple ascending dose
MPO
myeloperoxidase
P450
cytochrome P450
PF-06282999
2-(6-(5-chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide
PK
pharmacokinetics
PXR
pregnane X receptor
t1/2
half-life

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CYP3A4 Induction Potential of PF-06282999

Jennifer Q. Dong, James R. Gosset, Odette A. Fahmi, Zhiwu Lin, Jeffrey R. Chabot, Steven G. Terra, Vu Le, Kristin Chidsey, Parya Nouri, Albert Kim, Leonard Buckbinder and Amit S. Kalgutkar
Drug Metabolism and Disposition May 1, 2017, 45 (5) 501-511; DOI: https://doi.org/10.1124/dmd.116.074476
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