K2edta coated vacutainers

Blood was drawn from a voluntary 30 y/o female donor (according to permit from Ethical Review Board in Lund: Dnr. 2015/801) in K₂EDTA-coated vacutainers (BD, Franklin Lakes, NJ, USA). The blood was transferred to a Falcon tube and centrifuged (800×g, 10 min) for blood fractionation. The RBCs were washed five times with PBS (pH 7.4) and afterwards diluted in PBS to 1% suspension (v/v). Thereafter, RBCs were incubated in 1.5 ml Eppendorf tubes for spontaneous hemolysis with increasing concentrations of HS6 (3.5, 14, 56, 225 or 1000 μg/ml) with or without recombinant A1M (220 μg/ml) or PBS in control samples. After 5 h of incubation in room temperature (end-over-end rotation, 8 rpm) the tubes were centrifuged (500×g, 5 min) and the supernatants were used for analysis of lactate dehydrogenase (LDH). The LDH release was measured with the CytoTox 96® Non-Radio Cytotoxicity Assay (Promega, Madison, WI, USA) according to instructions from the manufacturer. The absorbance was measured at 490 nm (VICTOR 1420 multilabel reader, PerkinElmer). Results are presented as a protection ratio where addition of only A1M is set to 1. Statistical comparison of differences was performed with a Kruskal-Wallis test with Dunn's multiple comparisons test using GraphPad Prism 8.3.0 for MacOS (GraphPad, Bethesda, MD, USA).

Venous blood samples were collected into K₂EDTA-coated vacutainers (BD Biosciences, Franklin Lakes, NJ, USA) for analyses of DNA methylation and mRNA expression. Serum samples were isolated from venous blood samples collected in silica-coated vacutainers (BD Biosciences, Franklin Lakes, NJ, USA) for the determination of protein markers of muscle damage and inflammatory cytokines at the Pre-ex, Post-ex, Post-ex + 1 h, Post-ex + 3 h and Post-ex + 48 h timepoints (Figure 1B). Blood cell counts were also performed at each time point using a Yumizen H500 system (Horiba Medical, Kyoto, Japan). Skeletal muscle biopsies were obtained for the determination of DNA methylation and mRNA expression from 6 of the 8 participants (2 participants opted out of biopsies but completed the remaining parts of the study). Following collection, the skeletal muscle tissue was blotted dry and any visible fat or connective tissue was removed, snap-frozen in liquid nitrogen and stored at −80 °C prior to the analysis.

The dosing and sampling involved a three-treatment (R = reference drug, T1 = Test 1 and T2 = Test 2 drugs are first and second treatment, respectively), three-period, and six-sequence schedule with a 2-week washout period between treatments. Before starting any drug administration, the statistical department of Ipharma S.A. randomized drug allocation (R, T1 or T2, file code, and subject code) with a balanced design using the Mersenne Twister algorithm and R statistical software. The participants were blinded to treatment. About 18 h before the first dose administration, the subjects were admitted to the clinical site and served a standard dinner (<800 kcal). The next morning, after an overnight fast, ATV was administered orally as a single 80-mg dose tablet (Pfizer, New York, NY, USA) with 240 mL of water. A standardized lunch was served 4 h after dosing and standardized dinner at 12 h after dosing. Peripheral blood (4-mL samples) was collected in K₂EDTA-coated Vacutainers (BD Diagnostics, Franklin Lakes, NJ, USA), a pre-dose sample was taken (time 0), while other samples were taken at 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, 24, 36 and 48 h after drug administration. Cells and plasma were separated (10 min at 1600 × g at 4 °C). Plasma was stored at −65 ± 15 °C until use while DNA was extracted from cells.