Besides directly observed maximum concentration (Cmax) and AUC0?72h, other derived PK parameters including area under the concentration curve till infinity (AUCinf), clearance (CL), volume at steady state (Vss), and half-life (T1/2) were reported. Evaluation of the Kidney and Liver Function Kidney function was evaluated by blood urea nitrogen (urea) and creatinine, measured by a colorimetric analysis using Konelab Clinical Chemistry Analyzers in the Renal Function Exploration platform of the Cordeliers Research Center. Kidney injury was evaluated by urea and creatinine in plasma and renal NGAL and HO-1 gene expression were measured. The pharmacokinetic properties of hemopexin (mass Pacritinib (SB1518) spectrometry) in the hemolytic mice were affected by the target-mediated drug disposition phenomenon due to the high affinity of binding of hemopexin to heme. Hemolysis induced complement overactivation and signs of mild renal dysfunction at 6 h, which were prevented by hemopexin, except for the NGAL upregulation. The heme-degrading capacity of the kidney, measured by the HO-1 expression, was not affected by the treatment. These results encourage further studies of hemopexin as a therapeutic agent in models of diseases with heme overload. Hpx exposure in presence and absence of induced hemolysis. (B) Mean SD plasma concentration vs. time plotted for human hemopexin administered Pacritinib (SB1518) to mice (100 mg/kg i.v.; = 3/timepoint). In presence of PHZ (0.125 mg/g weight, blue circles) or control (PBS, gray circles). Pharmacokinetic parameter estimates are shown in Table 1. (C) Mean SD plasma concentration vs. time plotted for human hemopexin administered to mice (500 mg/kg; = 3/timepoint). In presence of PHZ (0.125 mg/g weight, blue circles) or control (PBS, gray circles). Pharmacokinetic parameter estimates are shown in Table 1. A second experiment aimed to evaluate the complement inhibition capacity of Hpx and its impact on complement activation and renal function. Three groups of 8-week-old C56BL/6 male mice (= 10) were pretreated with human Hpx (CSL Behring) in i.v. with 100 or 500 mg/kg or equivalent volume of PBS (0 mg/kg) immediately before i.p. PHZ injection (0.125 mg/g body weight) (Number 2A). A control group of 8 mice received two injections of PBS, related to the volume of Hpx and PHZ. All mice were sacrificed by cervical dislocation, 6 or 72 h after Hpx administration. Whole blood was collected 3 days before experimentation and at day time 1 into microtubes filled with 2 L of heparin (Heparine Choay? 5000 ui/ L Sanofi) by venipuncture in the cheek. Pacritinib (SB1518) Microtubes were centrifuged at 604 g for 10 min at space temperature to separate plasma. Kidneys were harvested for immunofluorescence (IF) and gene manifestation analyses. Plasma and organs were directly freezing in liquid nitrogen and stored at ?80C. Open in a separate window Number 2 Heme scavenging upon PHZ induced hemolysis by hemopexin. (A) Mean total heme plasma concentration at 6 h, (B) imply cell free hemoglobin at 6 h and (C) hemopexin:heme complexes at 6 h, and (D) 72 h demonstrated for each group [(1) PBS; = 7, (2) PHZ, (3) PHZ + 100 mg/kg, and (4) PHZ + 500 mg/kg; = 10]. Package and whiskers plots represent means Min to Maximum. **** 0.0001, Ly6c ** 0.01, and * 0.05 comparisons to PBS treated, Two-way ANOVA Kruskal Wallis test, ns, not significant. Quantification of Human being Hpx in Animal Plasma by LC/MS Ten microliters of plasma sample were placed into a clean Eppendorf tube followed by the addition of 80 L MeOH to precipitate the protein. The methanol was eliminated after centrifugation and the pellet was air-dried and later on re-suspended in 50 mM NH4HCO3/0.16% ProteaseMAX containing a heavy-isotope labeled peptide, which is specific for human Hpx and is used as internal standard. After incubation at 56C/550 rpm for 45 min the samples were reduced by adding 0.5 M DTT (56C/550 rpm for 20 min). The samples were then alkylated by addition of 0. 5 M IAA and incubation for 20 min at RT safeguarded from light. Tryptic digestion was carried out at 37C/550 rpm and halted after 3 h by addition of formic acid. After centrifugation the samples were separated immediately on a C18 column (AdvanceBio Peptide Mapping, 2.1 150 mm). The measurements were carried out using an Agilent 1290 Infinity II C 6550 iFunnel QTOF LC-MS system. Data was analyzed by calculating the peak area of the analyte and the internal standard using Agilent MassHunter Quant software. A standard curve was created in Agilent MassHunter Quant by plotting the average response percentage of analyte to internal standard against concentration for each standard sample. The analyte concentration in the plasma samples Pacritinib (SB1518) was backcalculated using the standard curve equation. Preparation of Heme Hemin (Frontier Scientific) was dissolved.