Production of HbV, phase 1 study design, adverse event and change of systemic blood pressure. (A) Outline of production scheme of HbV: carbonylhemoglobin (HbCO) solution was purified from nucleic acid amplification test (NAT)-inspected human red blood cell concentrate. The lipids for Hb encapsulation in liposomes comprised 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), cholesterol, 1,5-O-dihexadecyl-N-succinyl- l-glutamate (DHSG), and 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-poly(ethylene glycol) (DSPE-PEG). The HbCO encapsulation and particle size control were performed using the kneading method.¹¹ The entire procedure was performed under Good Manufacturing Practice (GMP) controls (supplemental Methods). (B) Phase 1 study design: (a) Twelve healthy volunteers were scheduled to receive single doses of HbV with no premedication in 2 consecutive dosing cohorts 1 and 2 (10 mL and 50 mL). In each cohort (n = 4), HbV suspension was administered to 1 subject in week 1 and to 3 subjects in week 2. (b) For a subsequent cohort 3 (n = 4), administration was made of a single dose of 100 mL of HbV suspension to 1 subject per week with premedication. After completion of administration, subjects were housed for 4 days and were monitored as ambulatory on days 5, 8, and 15. In some cases, an additional follow-up study was conducted to confirm recovery from the deviated laboratory variable. ∗Review of safety and tolerability by the Data and Safety Monitoring Committee. (C) Summary of adverse events associated with the infusion of HbV: these adverse events were resolved without medication. Premedication: dexamethasone 6.6 mg (IV), famotidine 20 mg (PO), and acetaminophen 500 mg (PO) were premedicated 1 hour before infusion of the HbV suspension. ∗Same subject. (D) Change of systemic systolic blood pressure: the systemic systolic pressure was measured sequentially. No significant change was found among blood pressure measurements of the respective cohorts. P values (2-tailed) of cohorts 1, 2, and 3 were, respectively, 0.996, 0.866, and 0.073; post, immediately after the start of infusion (in cohorts 2 and 3, only saline was being infused at this time point); closed small circle, blood pressure of each subject; open large circle, mean blood pressure.