Phoenix winnonlin version 8

Blood samples for PK analysis of PXL770 were collected from each subject predose on Days 14 (fasting conditions) and 26 (fed conditions – standardized breakfast) at 30 min, 45 min, and 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 16, and 24 h postdose, and on Day 28 during the DNL assessment at the same time points as employed in the DNL procedure. Plasma concentrations of PXL770 were analyzed using validated protein precipitation and high-performance liquid chromatography tandem mass spectroscopy (HPLC-MS/MS) methods. Pharmacokinetic samples collected from subjects who received placebo were not analyzed for PXL770. The analytical phase was carried out in accordance with GCP guidelines. The lower limit of quantification for PXL770 plasma concentrations was 10 ng/mL. Pharmacokinetic data were analyzed using validated PK analysis software (Phoenix® WinNonlin® version 8.1, Certara, Princeton, NJ). All PK analyses were performed, saved, and audit trailed in a 21 Code of Federal Regulations Part 11 compliant Oracle database (Phoenix® Knowledgebase Server Online). All plasma PK parameters were calculated using actual time points relative to the time of study drug administration. If actual time was missing, nominal time was used.

Previous studies have demonstrated non-linearity with respect to clearance of opioids in horses [18 (link), 19 ]. Therefore, non-compartmental analysis was performed on plasma meperidine concentrations using a commercially available software (Phoenix WinNonlin Version 8.1, Certara, Princeton, NJ, USA) to assess whether similar behavior was seen with respect to meperidine clearance. Lambda z (λ_z) was used to calculate the terminal half-life (HL λ_z) using the Eq. 0.693/ λ. The area under curve (AUC) from time 0 to infinity (AUC_0→∞) was obtained by using the linear up log down trapezoidal rule. Clearance (Cl) and the apparent volume of distribution at steady state (V_ss) were determined using the following formulas:
$$V_{\mathit{ss}}={\mathit{MRT}}_{\mathit{inf}}x\mathit{Cl}$$
where MRT is the mean residence time.
Non-compartmental analysis, as described above was also used for determination of pharmacokinetic parameters for normeperidine.

For absolute lymphocyte count (ALC) analysis, blood samples were collected at the following time points: day −1: 0, 2, 4, 6, 8, and 12 h; day 1: 0 (predose), 2, 4, 6, 8, and 12 h (postdose); day 2–day 20 (predose); day 21: 0 (predose), 2, 4, 6, 8, 12, 24, 36, 48, 60, 72, 96, 120, 144, 168, 216, 264, 312, 360, 408, 456, and 504 h (postdose).
The maximum effect (E_max), the area under the ALC–time curve from time zero to the last measurable point (AUEC_last) and change of those parameters from the baseline (ΔE_max and ΔAUEC_last), and the time to maximum effect (TE_max) and maximum change of ALC from the baseline (CFB_max) were calculated using the noncompartmental method of Phoenix WinNonlin^® version 8.1 (Certara, Princeton, NJ, United States).
The baseline ALC of each subject was defined using the following formula: $\text{Baseline ALC}=\frac{{\text{AUEC}}_{0-24} of Day-1}{24}$
For heart rate analysis, 24-h Holter monitoring was performed the day before the first dosing (day 1), on the first dosing (day 1), and last dosing (day 21). The hourly average heart rate (HR) and the area under the hourly HR–time curve (AUEC_HR) were calculated for each individual subject.

For PK evaluation, serial blood samplings were performed at the following time points: day 1: 0 (predose), 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, and 24 h (postdose); day 4, 6, 8, 10, 12, 14, 16, 18, 19, and 20 (predose); and day 21: 0 (predose), 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 24, 36, 48, 72, 96, 120, 144, 168, 216, 264, 312, 360, 408, 456, and 504 h (postdose). Urine samples were collected at the following time points and intervals: day 1: 0 (predose), 0–24 h (postdose); day 21: 0 (predose), 0 to 24, 24 to 48, 48 to 72, 72 to 96, 96 to 120, 120 to 144, and 144–168 h postdose.
PK parameters were analyzed using a noncompartmental method using Phoenix WinNonlin^® version 8.1 (Certara, Princeton, NJ, United States). The maximum steady-state plasma concentration of LC51-0255 (C_max,ss), the time to reach maximum plasma concentration following administration of LC51-0255 at the steady state (T_max,ss), the area under the plasma concentration–time curve during a dosing interval at steady state (AUC_τ,ss), the terminal half-life (t_1/2), the accumulation ratio calculated from AUC_τ,ss and AUC_τ after single dosing (R_ac), the peak trough fluctuation over one dosing interval at steady state, apparent clearance at steady state (CL_ss/F), apparent volume of distribution during the terminal phase (V_z/F), the fraction of drug excreted into urine (f_e), and renal clearance (CL_R) were calculated.