Phoenix suite version 1

Blood samples were drawn from the retro-orbital sinus under light isoflurane anaesthesia for determination of plasma SET-M33 levels. The samples were taken on days 1 and 28 of treatment at the following times: control group: pre-dose and 30 min; 5, 9 and 15 mg/kg/day-group: pre-dose, 5, 15, 30, 60 min and 24 h after administration. Blood samples were collected into polypropylene test tubes containing lithium heparin anticoagulant and kept at room temperature for no longer than 60 min until centrifuging (1600 g for 10 min at 2–8 °C). The plasma obtained from each sample was transferred to a fresh polypropylene test tubes, immediately frozen in dry ice and stored at − 20 °C ± 5. Plasma concentrations of SET-M33 were measured by a previously validated LC–MS/MS method. Toxicokinetic parameters were determined for mean plasma concentrations at each dose level and time point, according to the validated method and with WinNonlin software, version 6.3, in the Phoenix Suite version 1.3 (Pharsight Corporation, Mountain View, CA, USA).

PK parameters were determined for each matrix by noncompartmental analysis using mean concentration values (right eyes, left eyes, and all eyes) for each matrix. Concentration and parameter values were rounded to two decimals, and coefficient of variation was rounded to one decimal.
PK analyses included determination of the maximum observed concentration (C_max), the time to C_max (t_max), and the area under the concentration–time curve (AUC_t) from time 0 to the last measurable time point. PK analysis was performed using validated WinNonlin^® Professional software, version 6.3, integrated in the Phoenix^® Suite, version 1.3 (Pharsight Corporation, Mountain View, CA, USA).
Arithmetic mean concentrations were calculated only when n ≥ 3 and at least two numerical values were above the lower limit of quantification (LLOQ). Concentration values below the LLOQ or no peak were treated as zero.