Computational protocol: Physiologically Based Pharmacokinetic Modeling Framework for Quantitative Prediction ofan Herb–Drug Interaction

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[…] PBPK model development. The base model structure was adapted from the literature (), incorporating physiologic parameters obtained from the International Commission on Radiological Protection. Warfarin partition coefficients (Kps) and binding parameters were obtained from the literature (); absorption rate constants (kas) and clearance parameters were obtained by fitting the PBPK model to previously reported plasma concentration–time profiles. The reversible inhibition constant (Ki) of (R)-warfarin toward CYP2C9 activity was obtained from the literature. Midazolam Kps and ka were obtained from the literature,; intestinal and hepatic clearance parameters were extrapolated from in vitro data as described, (). Silybin A and silybin B Kps were predicted from physicochemical properties using GastroPlus (version 8.0; Simulations Plus, Lancaster, CA). Silibinin binding parameters were obtained from the literature; clearance parameters were generated by fitting the PBPK model to plasma concentration–time data from hepatitis C patients receiving silymarin (). Silybin A and silybin B mechanism-based (KI, kinact) and reversible inhibition kinetic parameters were obtained from the literature., Mechanism-based inhibition of CYP2C9 was not considered based on a previous publication showing no IC50 shift using (S)-warfarin as the probe substrate.PBPK interaction model simulations. PBPK models were developed for midazolam, (R)-warfarin, (S)-warfarin, silybin A, and silybin B using Berkeley Madonna (version 8.3; University of California at Berkeley, Berkeley, CA) with code compiled in MEGen (version 0.5; UK Health & Safety Laboratory, Buxton, UK) (Supplementary Materials and Methods). The PBPK model for perpetrator (silybin A and silybin B) and victim (warfarin or midazolam) compounds were linked through the reversible or mechanism-based inhibition of victim probe substrate. Initial simulations used doses of probe substrates and milk thistle products reported in previous studies. Simulations were considered accurate if the predicted primary pharmacokinetic outcomes (AUC and Cmax for (S)-warfarin and midazolam) were within 30% of observed outcomes. Following initial model evaluation, simulations were conducted with a higher dose of silibinin (1,650 mg/day) to determine whether a clinically important interaction is possible. Pharmacokinetic outcomes from the simulated profiles were recovered via noncompartmental analysis using Phoenix WinNonlin (version 6.3; Pharsight, Cary, NC).Analysis of silibinin product. Siliphos capsules (n = 28) (Thorne Research, Dover, ID) were analyzed using a modification of previously described methods, to ensure purity and content. Briefly, the contents of each capsule were weighed and extracted twice with 2 ml acetone. The extract was vortex mixed and centrifuged (13,000g for 2 minutes); the supernatant was transferred to a clean vial. Milk thistle constituents were quantified using an Acquity UPLC system with an HSS-T3 1.8 µm (2.1 × 100 mm) Acquity column and Empower 3 software (Waters, Milford, MA). Standards and Siliphos capsule extracts were analyzed using a gradient from 30:70 to 55:45 methanol:water (0.1% formic acid) over 5.0 minutes at a flow rate of 0.6 ml/minute at 50 °C; peaks were detected at 288 nm.Proof-of-concept clinical study. Healthy volunteers (six men and six nonpregnant women) were enrolled in an open-label, fixed sequence crossover study conducted at the UNC CTRC. The study protocol was approved by the UNC Office of Human Research Ethics Biomedical Institutional Review Board and the CTRC Advisory Committee. Eligibility to participate was based on screening evaluation and inclusion/exclusion criteria (Supplementary Table S1). Written informed consent was obtained from each subject prior to enrollment.The first (control) phase consisted of administration of 10 mg warfarin (Coumadin; Bristol Meyers Squibb, Princeton, NJ), 10 mg vitamin K (Mephyton; Aton Pharma, Lawrenceville, NJ), and 5 mg midazolam syrup (Ranbaxy; Jacksonville, FL). A negative pregnancy result was required before drug administration to women of childbearing potential. Vital signs (blood pressure, temperature, respiratory rate, pulse, and oxygen saturation) were obtained at baseline and every 15 minutes for the first 2 hours. All subjects underwent an international normalized ratio with prothrombin time. Blood (7 ml) was collected through an intravenous line before and from 0.25–12 hours following drug administration. Subjects continued to fast until after the 4-hour blood collection, when meals and snacks, devoid of fruit juices and caffeinated beverages, were provided. Subjects returned to the CTRC 24 and 48 hours post-drug administration for blood collection. Optimal study design simulations of previously reported clinical data demonstrated that a 0–48-hour collection was an accurate surrogate of total systemic exposure (AUC0–inf) for warfarin. Plasma was collected and stored at −80 °C pending analysis by high-performance liquid chromatography tandem mass spectrometry (HPLC/MS-MS).Following at least a 14-day washout, subjects received 480 mg silibinin (based on labeled content) to self-administer three times daily for 7 days. Each subject received his/her silibinin in a blister pack and was asked to complete a pill diary documenting the time of administration. Subjects were contacted at least twice during the week of silibinin self-administration to monitor compliance and adverse events, which were graded using a validated Adverse Events Scale. Subjects returned to the CTRC on day 7 for concomitant administration of silibinin, warfarin, vitamin K, and midazolam. Plasma was collected and stored as described for the first phase.Analysis of plasma for warfarin enantiomers, midazolam, silybin A, and silybin B. Concentrations of all analytes were quantified using a Sciex (Framingham, MA) API4000 Qtrap HPLC-MS/MS triple quadrupole mass spectrometer fitted with a Turbo ionspray interface operated in the positive ion mode. Plasma was treated with acetonitrile (6 volumes) containing the internal standard, warfarin-d5 (Toronto Research Chemicals; Toronto, Canada) or 1′-hydroxymidazolam-d4 (Cerilliant, Round Rock, TX), and centrifuged (3,000g). The supernatant was injected into the HPLC-MS/MS system. Warfarin enantiomers were separated on a Supelco Astec Chirobiotic V 15 cm × 2.1 mm 5 micron chiral column (Sigma Aldrich; St Louis, MO) and eluted with an isocratic mixture consisting of 75% 5 mmol/l ammonium acetate containing 0.01% (v/v) formic acid and 25% acetonitrile (flow rate, 0.4 ml/minute). Midazolam was eluted with a binary gradient mixture consisting of 10 mmol/l ammonium formate containing 1% (v/v) isopropyl alcohol and 0.1% (v/v) formic acid and methanol on a Varian Polaris C18-A 20 cm × 2.0 mm 5 micron column (Agilent, Santa Clara, CA) (flow rate, 0.65 ml/minute). Silybin A and silybin B were eluted with an isocratic mixture consisting of 44% water, 56% methanol, and 0.1% (v/v) formic acid on an Agilent Zorbax XDB C18 15 cm × 3.0 mm 3.5 micron column (Agilent) (flow rate, 0.7 ml/minute). Analyte concentrations were determined by interpolation from a linear standard curve with an assay dynamic range of 0.5–10,000 nmol/l (warfarin enantiomers) or 0.5–5,000 nmol/l (midazolam, silybin A, silybin B). Analytical methods were validated according to US Food and Drug Administration guidelines. Inter- and intraday variability for all analytes was less than 10%.Pharmacokinetic analysis. Pharmacokinetic outcomes were recovered by noncompartmental analysis using Phoenix WinNonlin. Concentrations below the limit of quantification were excluded. The terminal elimination rate constant (λz) was estimated by linear regression of the terminal portion of the log-transformed concentration–time profile using at least three data points. The terminal half-life (t1/2) was calculated as ln2/λz. The maximum observed concentration (Cmax), time to reach Cmax (tmax), and last measured concentration (Clast) were obtained directly from the concentration–time profile. AUC from time zero to Clast (AUC0–last) was determined using the trapezoidal method with linear up/log down interpolation. The AUC from time zero to infinity (AUC0–inf) was calculated as the sum of AUC0–last and the ratio of Clast to λz.Genotyping for common CYP2C9 variants. CYP2C9*2 and *3 polymorphisms were determined using a previously published polymerase chain reaction restriction fragment length polymorphism assay.Statistical analysis. All statistical analyses were conducted using SAS (version 9.2; SAS Institute, Cary, NC). The sample size for the proof-of-concept study (n = 12 evaluable subjects) was calculated based on 80% power to detect a 25% change in the primary endpoints with a type I error of 0.05; the primary endpoints were the treatment/control ratios of log-transformed AUC0–48 ((S)-warfarin) or AUC0–inf (midazolam) and Cmax ((S)-warfarin and midazolam), and the predefined no effect range was 0.75–1.33., Intraindividual variability in midazolam and warfarin AUC and Cmax were assumed to be ~20%.,, Secondary outcomes, t1/2 and tmax, were evaluated using a paired two-tailed Student's t-test on log-transformed data or Wilcoxon signed-rank test as appropriate, with 90% confidence intervals and ranges reported for t1/2 and tmax, respectively. A P value <0.05 was considered statistically significant. […]

Pipeline specifications

Software tools GastroPlus, Phoenix WinNonlin
Application Drug design
Organisms Silybum marianum
Chemicals Midazolam, Warfarin