This was an investigator-initiated, unblinded, single-center prospective cohort study. A total of three cohorts (n = 8) with different dosing schedules were planned. The study protocol was approved by the ethical committee of the Amsterdam Medical Center (NL 54915, EUDRACT number 2015–003902-17) and registered at: NCT02836340. The study was conducted in adherence to (inter) national standards of good clinical practice and was monitored by the Clinical Research Centre of the Amsterdam University Medical Centers.

After the completion of each cohort, inclusion was temporarily stopped. The results were analyzed and discussed with the Data and Safety Monitoring Safety Board (DSMB). If necessary, the study protocol was adapted and submitted to the ethical committee for review and approval.

Consecutive adult patients admitted to the ICU after OHCA were screened for eligibility by the physician on call. If the patients fulfilled the inclusion criteria, one of the investigators (MMA and JH) came to the hospital for inclusion and informed consent. Inclusion criteria for cohort A were as follows: (1) OHCA due to a cardiac cause, (2) coma on ICU admission, (3) treatment with TTM at 36°C, and (4) possibility of administration of study medication within 6 h after OHCA via a central venous catheter. After analyzing the results of cohort A, two criteria were added for cohort B and C: (1) duration of resuscitation not longer than 30 min and (2) shockable rhythm as presenting rhythm. Administration via a central venous catheter was not required anymore. Instead, using a peripheral intravenous catheter was allowed based on additional safety data from other trials (7, 8). Exclusion criteria in all cohorts were as follows: (1) female patient aged < 50 years, (2) known co-morbidity with a life expectancy of less than 6 months prior to OHCA, (3) known severe cognitive impairment prior to OHCA, and (4) no informed consent.

As all patients were unconscious on ICU admission, a legal representative was informed about the study and asked for informed consent before the start of study medication. If the patient recovered, informed consent for the use of collected data and follow-up was asked.

All patients received intravenously administered 2-IB (Neurophyxia BV, ‘s Hertogenbosch, Netherlands). Preferably, a central venous catheter was used for administration, but if this was not available, administration using a peripheral intravenous catheter was allowed. Each patient received a total of six dosages of study medication in 24 h (every 4 h), infused in 15 min.

A total of three cohorts of patients were included using a dose-escalation design: In cohort A, cohort B, and cohort C, an exposure was targeted at an AUC_0-24h of 600–1,200 ng*h/mL, 2,100–3,300 ng*h/mL, and 7,200–8,400 ng*h/Ml, respectively. Based on preclinical data and human neonatal pharmacokinetics (PK) data, a dose of 0.055 mg/kg/dose was administered in cohort A (8). After completion of cohort A, exposure was found to be higher than expected and renal function turned out to be an important co-variate in the determination of this exposure. Therefore, in cohort B and C, dosing was based on renal function, which was determined using the estimated glomerular filtration rate (eGFR) on hospital admission (see Supplementary Table S1 for the dosing schedule). The eGFR was calculated using the Modification of Diet in Renal Disease (MDRD) equation: 186 × (Creat/88.4)^–1.154 × (Age)^–0.203 × (0.742 if female) × (1.210 if the patient is black) (9). The MDRD is the clinical standard in Netherlands to calculate eGFR.

Standard ICU care for OHCA patients consisted of TTM with a target of 36°C for 24 h during which patients were sedated using propofol and, if needed, remifentanil. Mechanical ventilation, hemodynamic monitoring, and, if necessary, vasopressive and inotropic support, were applied in all patients. After completion of TTM, sedative medication was stopped, and the neurological situation was evaluated. If the patient remained comatose, the neurologist was consulted for advice on prognostication. The neurologist was unaware of the participation in the current study and national guidelines for prognostication were used.

The primary objective was to explore the short-term safety, tolerability, and PK of 2-IB. The main study parameters used for evaluating the short-term safety and tolerability were (1) vital signs, i.e., heart rate, blood pressure, ST segment changes as sign of cardiac ischemia, assessed every 5 min before and until 15 min after 2-IB administration, (2) biochemistry and hematology measured as part of clinical care, (3) the occurrence of serious adverse events (SAEs) up to 7 days in the ICU or until ICU discharge, whichever occurred first, and (4) adverse events (AE) up to 30 days.

For outcome assessment, the Cerebral Performance Category (CPC) scale was used (10). The best CPC score in 30 days after OHCA was registered. Poor outcome was defined as a CPC score of 3–5 [severely disabled, vegetative state, and (brain) death].

Brain injury was assessed using the neurobiomarker neuron specific enolase (NSE), S100ß and neurofilament light chain (NfL) at 24 h and 48 h (± 1 h) after the first dose of study medication (11–13).

Other data collected include demographic and baseline characteristics, OHCA details, cardiac arrest hospital prognosis (CAHP) score, hospital data (laboratory results, neurological examination after clearance of sedative drugs, and length of stay (LOS) in the ICU and in the hospital) (14).

For evaluation of PK of 2-IB, nine 3.5 mL plasma samples were taken from an arterial catheter at 1 min after the end of the first administration, 45 min and 1 h 15 min after the start of the first administration and just before the start of the second administration, 1 min before the 4th, 5th, or 6th administration, 1 min after the end of this administration and 45 min, and 1 h 15 min and 4 h after the start of the chosen administration. Except for the first blood sample, in all these time points, a margin of ± 5 min was used. Blood samples were centrifuged, and the supernatant was kept frozen at − 80°C until determination. The following PK parameters were determined: observed maximum plasma concentration (C_max), area under the plasma concentration–time curve from time 0–24 h after administration (AUC_0–24h), time at maximum plasma concentration (T_max), terminal elimination half-life (t_1/2), clearance (CL), and volume of distribution (Vd).

Pharmacokinetics analysis was performed using nonlinear mixed effects modeling (NONMEM; version 7.4; ICON Development Solutions, Ellicott City, MD, United States), with the main aim to obtain individual exposure metrics for 2-IB (15). With the data from cohort A, an initial population PK model was built. The estimation method was the first-order conditional estimation with an interaction option in NONMEM. Simulation studies were performed to identify the best dose for use in cohort B with dose adjustment for renal function at admission. Using the eGFR on admission, a dosing schedule for cohort B was defined. In order to reach the target of cohort C, a three times higher dose compared to cohort B was given in cohort C based on eGFR. The population PK model was reevaluated with data from cohort A and B after cohort B was completed, and again with all data after completion of cohort C.

Baseline characteristics, distributions on the CPC, and outcome measures are presented in a descriptive way as mean ± SEM or as percentages when appropriate. Not normally distributed data were reported as median [with interquartile range (IQR)].

Baseline characteristics, OHCA details, and outcome of the included patients are presented in Table 1. The duration of resuscitation was longer, and CAHP scores were higher in cohort A compared with cohort B and C, which was caused by a change in inclusion criteria. Study medication was started at a median of 5.3 h (IQR 4.8–5.6) after OHCA. In 20 patients, all six dosages were administered as scheduled. One patient (patient 18) died due to refractory cardiogenic shock with multiorgan failure before the last dosage was administered.

In all patients, the study medication was well tolerated, and no safety concerns were encountered during the 24 h of administration of 2-IB. No statistically significant or clinically relevant changes in heart rate or blood pressure were observed up to 15 min after 2-IB administration compared to before administration in either cohort (see Figures 2A–C). No signs of cardiac ischemia (ST segment changes) were seen during the 24 h administration period of the study medication in either cohort. The laboratory results of standard blood tests did not show any unexpected deviations for this patient population.

In total, eight patients died (four in cohort A, one in cohort B, and three in cohort C), which was reported as an SAE to the DSMB and regulation authorities. In all cases, the SAE was judged as not related to the study medication. One patient in cohort A suffered another cardiopulmonary arrest, consisting of bradycardia with loss of circulation. This patient was still in the ICU, and CPR was performed immediately and successfully. This SAE occurred more than 24 h after the last administration of the study drug. In cohort B, one patient woke up very agitated after sedative drugs were stopped. It was decided to extubate the patient to see if this affected the agitation. As no improvement occurred, sedative medication had to be restarted which necessitated reintubation and mechanical ventilation for another 24 h. Finally, in cohort C, one patient was readmitted to the hospital after discharge because of shortness of breath and cystitis with urinary retention. This occurred within 30 days after inclusion in the study and was scored as an SAE. After treatment with diuretics and antibiotics, the patient recovered rapidly. These three SAEs were also judged as not related to the study medication.

A total of eight patients died, seven due to brain injury leading to postanoxic coma and a decision to withdraw life-sustaining therapy (Table 1). One patient died due to cardiogenic shock. Brain injury markers are presented in Table 2 and Figure 3.

In all samples of cohort A, B, and C, 2-IB concentrations could be measured; no concentrations were below the lower limit of quantification. The median value of clearance was 12.3 L/h, volume of distribution was 10 L, and half-life was 1.5 h. Individual values can be found in Supplementary Table S2.