CERE‐LYSE‐1 2012.

Study characteristics
Methods	Study design: prospective, randomised, placebo‐controlled, double‐blind trial Study grouping: parallel group Losses to follow‐up: 19 (16%) Trial protocol registration: retrospective (4 years difference between study start date (2005) and the date registration record posted (2009), when the trial was already completed in 2008)
Participants	Baseline characteristics: Cerebrolysin Participants: 60 Mean age: 65.5 years (SD 11.30) Smokers: 15 (25%) Men: 40 (66.7%) Mean time from first symptoms to rtPA infusion: 142.4 minutes (SD 27.39) Mean NIHSS score: 12.3 (SD 5.39) Medical history: Hypertension: 46 (76.7%) Hyperlipidaemia: 20 (33.3%) Arrhythmia: 17 (28.3%) Coronary heart disease: 15 (25%) Obesity: 12 (20%) Diabetes of old age: 10 (16.7%) Earlier TIA: 6 (10.0%) Mean time from first symptoms to hospital admission: 82.6 minutes (SD 38.91) Mean time from first symptoms to rtPA infusion: 142.4 minutes (SD 27.39) Mean time from hospital admission to rtPA infusion: 59.9 minutes (SD 36.59) Placebo Participants: 59 Mean age: 67.0 years (SD 10.56) Smokers: 12 (20.7%) Men: 37 (62.7%) Mean NIHSS score: 11.0 (SD 5.44) Medical history: Hypertension: 41 (69.5%) Hyperlipidaemia: 16 (27.1%) Arrhythmia: 17 (28.8%) Coronary heart disease: 12 (20.3%) Obesity: 9 (15.3%) Diabetes of old age: 7 (11.9%) Earlier TIA: 6 (10.2%) Mean time from first symptoms to hospital admission: 72.5 minutes (SD 30.86) Mean time from first symptoms to rtPA infusion: 133.4 minutes (SD 34.37) Mean time from hospital admission to rtPA infusion: 60.9 minutes (SD 29.04) Inclusion criteria: men and women, 18 to 80 years, who had a clinical diagnosis of acute ischaemic hemispheric stroke that had commenced within 3 hours prior to initiation of administration of rtPA, and had stroke symptoms being present for at least 30 minutes with no significant improvement before treatment, were eligible (further inclusion and exclusion criteria, see Table 1). All participants had to meet the admission standards of the European Medicines Agency (EMA) consensus criteria for the application of thrombolytic therapy with alteplase (rtPA): (1) clinical diagnosis of ischaemic stroke causing a measurable neurological deficit defined as impairment of language, motor function, cognition and/or gaze, vision or neglect. Ischaemic stroke is defined as an event characterised by the sudden onset of an acute focal neurologic deficit presumed to be due to cerebral ischaemia after CT scan excluded haemorrhage, (2) informed consent Exclusion criteria: evidence of intracranial haemorrhage on the CT scan; participation in another therapeutic clinical trial 3 months before baseline; people with any history of prior stroke and concomitant diabetes; prior stroke within the last 3 months; platelet count below 100 to 103/mm3; blood glucose < 50 or > 400 mg/dL (< 2.77 or > 22.15 mmol/L); known haemorrhagic diathesis; manifest or recent severe or dangerous bleeding; known bacterial endocarditis, pericarditis; acute pancreatitis; documented ulcerative gastrointestinal disease during the last 3 months, oesophageal varices, arterial‐aneurysm, arterial/venous malformation; neoplasm with increased bleeding risk; severe liver disease, including hepatic failure, cirrhosis, portal hypertension, oesophageal varices, and active hepatitis; major surgery or significant trauma in past 3 months; multiple serious drug allergies; hypersensitivity or allergy to 1 of the components of the drug; severe renal impairment; systolic blood pressure > 185 mmHg or diastolic blood pressure > 110 mmHg, or aggressive management (intravenous medication repeatedly) needed to reduce blood pressure to these limits; recent (less than 10 days) traumatic external heart massage, obstetrical delivery, recent puncture of a non‐compressible blood vessel (e.g. subclavian or jugular vein puncture); chronic intoxication or chronic substance use disorder with pharmaceuticals, drugs, alcohol, or industrial poisons; symptoms of ischaemic attack began more than 3 hours prior to start of thrombolytic therapy or if time of symptom onset is unknown; minor neurological deficit or symptoms rapidly improving before start of infusion; severe stroke as assessed clinically (e.g. NIHSS > 25) and/or by appropriate imaging techniques; epilepsy; symptoms suggestive of subarachnoid haemorrhage, even if the CT scan is normal; known history of or suspected intracranial haemorrhage; suspected subarachnoid haemorrhage or condition after subarachnoid haemorrhage from aneurysm; any history of CNS damage (i.e. neoplasm, aneurysm, intracranial, or spinal surgery); haemorrhagic retinopathy, e.g. in diabetes (vision disturbances may indicate haemorrhagic retinopathy); administration of heparin within the previous 48 h and a thromboplastin time exceeding the upper limit of normal for laboratory; people receiving oral anticoagulants, e.g. warfarin, sodium; people receiving nifedipine for acute treatment Pretreatment: the 2 groups were well balanced with respect to baseline prognostic variables, and no significant differences between treatment groups were observed
Interventions	Cerebrolysin Frequency of dosage: once daily for 10 consecutive days: intravenous infusion of 30 mL of Cerebrolysin diluted with 70 mL of 0.9% physiological saline to a total volume of 100 mL. Cerebrolysin starting immediately 1 hour after thrombolytic treatment Standard treatment: the thrombolytic therapy with rtPA was administered as intravenous infusion over 60 minutes. Immediately thereafter, the first intravenous infusion of the study medication (Cerebrolysin/placebo) was administered over a time period of 30 minutes. Placebo Frequency of dosage: once daily for 10 consecutive days: an identical amount of physiological saline (100 mL) was used as placebo Standard treatment: the thrombolytic therapy with rtPA was administered as intravenous infusion over 60 minutes. Immediately thereafter, the first intravenous infusion of the study medication (Cerebrolysin/placebo) was administered over a time period of 30 minutes.
Outcomes	Poor functional outcome defined as death or dependence at the end of the follow‐up period (dichotomous outcome) Early death (dichotomous outcome) All‐cause death (dichotomous outcome) Serious adverse events (dichotomous outcome) Adverse effects specifically associated with Cerebrolysin (dichotomous outcome) Total number of participants with adverse events (dichotomous outcome)
Identification	Sponsorship source: not mentioned. Only the Conflict of Interest statement: "Wilfried Lang has served as consultant for Bayer, Boehringer Ingelheim, EVER, MSD, Sanofi‐Aventis and Pfizer and has received speaking honoraria from these companies. Christian Stadler has received speaker honoraria from EVER. Zdavka Poljakovic received Principal Investigator fee for the clinical study. David Fleet is a freelance consultant statistician undertaking statistical contracts on behalf of pharmaceutical/biotechnology organizations and as such was contracted by EVER. All authors have no other financial interest in the company or its products." Country: 5 countries: Austria, Croatia, the Czech Republic, Slovakia, Slovenia Setting: inpatient (hospital) Author: Wilfried Lang Institution: Department of Neurology, Hospital St John, Austria Email: wilfried.lang@bbwien.at
Notes	No results posted on trial registration platform.
Risk of bias
Bias	Authors' judgement	Support for judgement
Allocation concealment	Low risk	Quote: "The vials containing the study drug and the placebo were visually identical." Comment: though the quote refers to potential blinding and not allocation concealment, we judged this as low risk
Sequence Generation	Unclear risk	Quote: "according to a pre‐compiled 1:1 randomization schedule, stratified by centre." Comment: there was not only "insufficient information to permit judgement of low risk or high risk" as the basis for a judgement of unclear risk as per the Cochrane Handbook. The described procedure does not fit with any of the criteria for an assessment of low risk of bias, i.e. referring to a random number table; using a computer random number generator; coin tossing; shuffling cards or envelopes; throwing dice; drawing of lots; or minimisation. There is no information about the process of generation of the randomisation sequence. In addition to retrospective protocol registration and statistician contracted by Cerebrolysin manufacture EVER Neuro Pharma, we judged this as unclear risk.
Incomplete outcome data All outcomes	High risk	Quote: "Two patients received the incorrect study medication assignment." Quote: "Based on statistical information from the third interim analysis, it was decided to terminate the study, as no significant result for the main outcome criteria was expected to be reached." Quote: "All patients were included in the ITT population with 60 patients being assigned to Cerebrolysin and 59 assigned to placebo. In the PP population, 100 patients were included with 49 receiving Cerebrolysin and 51 receiving placebo (Fig. 1)." Comment: 19 participants of 119 (16%) were lost to follow‐up. Attrition bias. Information not available by outcome. Furthermore, the study authors used the 'last observation carried forward' (LOCF) method to fill in the missing data points. There is not a single peer‐reviewed statistical publication that describes general conditions under which LOCF provides a statistically unbiased result.
Blinding of outcome assessors All outcomes	Low risk	Quote: "All study personnel and participants were blinded to treatment assignment for the duration of the study." Comment: however, it was retrospective protocol registration, and the statistician was contracted by Cerebrolysin manufacturer EVER Neuro Pharma
Selective outcome reporting	Unclear risk	Quote: "There were no obvious differences between either treatment arms. In each treatment group, four patients died, but in none of the cases was any relationship to the study medication seen. The number of patients with serious adverse events was slightly higher in the Cerebrolysin group compared to the placebo group (12 vs. 7, respectively). In total, 19 (16%) patients experienced at least one serious adverse event (Table 5). " Comment: the study was stopped because of no significant result for the main outcome criteria. According to the study authors, there was no causal relationship to the study drug for any of the deaths observed. Neither reasons for nor timing of deaths is presented. Timing of adverse events, serious adverse events not presented. Study protocol was registered retrospectively. We judged this to be unclear risk of bias.
Blinding of participants and personnel All outcomes	Low risk	Quote: "All study personnel and participants were blinded to treatment assignment for the duration of the study."
Other sources of bias	High risk	Quote: "Ljubljana, Ljubljana/Slovenia) ClinicalTrials.gov identifier: NCT00840671 Conflicts of interest: Wilfried Lang has served as consultant for Bayer, Boehringer Ingelheim, EVER, MSD, Sanofi‐Aventis and Pfizer and has received speaking honoraria from these companies. Christian Stadler has received speaker honoraria from EVER. Zdavka Poljakovic received Principal Investigator fee for the clinical study. David Fleet is a freelance consultant statistician undertaking statistical contracts on behalf of pharmaceutical/ biotechnology organizations and as such was contracted by EVER. All authors have no other financial interest in the company or its products." Comment: no information on funding sources for the trial. Statistician was contracted by EVER, the manufacturer of Cerebrolysin. There is no information about the provider of Cerebrolysin. Retrospective protocol registration. Early stopping of the trial after an interim analysis