day 13 and therefore were only included in the day 1 PK/PD analysis. Two other volunteers did not partici- pate in the repeated single-dose administration on day 34 and therefore were included in only the day 1 and day 13 PK/PD analyses. The baseline ITMN-191 demographics of the study participants are summarized in Table I. Effect of Concomitant Administration of Ketoconazole on Single-Dose Ruxolitinib Pharmacokinetics and Pharmacodynamics The plasma concentrations of ruxolitinib significantly increased with concomitant use of ketoconazole, as displayed in Figure 2A. Coadministration of keto- conazole 200 mg q12h significantly decreased the geometric mean oral clearance (CL/F) of ruxolitinib 4 J Clin Pharmacol   in geometric mean C max and a 91% increase (1.72-2.12) in the geometric mean AUC 0of ruxolitinib (Table II).

An observed 2.1-hour (60%) increase in geo- metric mean terminal-phase half-life with ketoconazole coadministration further supports that ruxolitinib metabolism was inhibited by ketoconazole. Concomitant treatment with ketoconazole caused a solitinib (+  LY450139 ketoconazole, n=16) * Day 1 Day 2-5 * Day 5 10 mg ruxolitinib (alone, n=15) 500 mg bid erythromycin 10 mg ruxolitinib (+ erythromycin, n=14) * # Day 1: 50 mg ruxolitinib (alone, n=12) Day 3-13: 600 mg qd rifampin * Day 13: 50 mg ruxolitinib (+ rifampin, n=10) Day 14-33: Drug washout * Day 34: 50 mg ruxolitinib (alone, n=8) Figure 1. An overview of the study design. *The study days in which pharmacokinetic/pharmacodynamic (PK/PD) samples were collected. # The PD samples collected on day 1 were partially lost in shipping, and the study was amended to redose ruxolitinib in the 8 study par- ticipants available on day 34 following a drug washout period of 20 days. ion ([MH + ]) to the m/z 186.2 product ion for ruxolitinib and the transition of the m/z 311.3 precursor ion to the m/z 190.2 product ion for the internal standard.

Using 50  plasma, this assay produced linear results over a plasma concentration range of 1.0 to 1000 nM for ruxolitinib ( R 2 > 0.993). Under these assay condi- tions, intra-assay Sorafenib VEGFR-PDGFR inhibitor precision and accuracy for quality control samples ranged from 1.8% to 6.0% and 90.9% to 108%, respectively, whereas interassay precision and accuracy ranged from 4.7% to 7.1% and 96.3% to 100%, respectively. A validated, GLP, LC/MS/MS analytical method was developed for the quantitation of 8 metabolites of ruxolitinib using synthetic standards, and the con- centrations of the metabolites were determined for the plasma samples collected in study B. Details of the assay will be published separately, and a brief description is provided here. Following the extraction of plasma samples using the identical procedure described above for the parent compound, the analytes (parent plus metabolites) were baseline separated by HPLC using gradient elution. Subsequently, the con- centrations of the analytes were determined by MS/ MS analysis on a Sciex API-4000 mass spectrometer (Applied Biosystems) operating in positive ion MRM mode. Using 100  plasma, this assay produced linear results over a plasma concentration range of 1.0 to 1000 nM for each of the ruxolitinib metabolites.

The intra- and interassay precision and accuracy for quality control samples for the buy Sorafenib ruxolitinib metabolites were comparable to those reported above for the parent com- pound and met the strict GLP requirement. Pharmacodynamic blood samples (300 ) were stimulated with 100 ng/mL of interleukin-6 (IL-6; R&D Systems, Minneapolis, Minnesota) to activate the JAK/ STAT pathway, the blood cells lysed, and the total cell extracts analyzed for levels of phosphorylated STAT3 (pSTAT3) using a specific enzyme-linked immuno- sorbent assay (ELISA) (Invitrogen/BioSource, Carlsbad, California). This Islamic physicians assay was performed under a specific 3 Downloaded from jcp.sagepub at Bobst Library, New York University on March 7, 2012 4  SHI ET AL standard operating procedure (SOP).