manufacturer bial refused the regulator's request to release the investigator's brochure and the product dossier (investigational medicinal product dossier), citing french law on trade secrets. trial details the study was entitled "a double-blind, randomised, placebo-controlled, combined single and multiple ascending dose study including food interaction, to investigate the safety, tolerability, pharmacokinetic and pharmacodynamic profile of bia 10-2474, in healthy volunteers". the trial commenced on 9 july 2015 at a single centre in the city of rennes, and commenced recruitment of 128 healthy volunteers, both men and women aged 18 to 55. participants of the study were to receive €1,900 and, in turn, asked to stay at biotrial's facility for two weeks during which time they would take the drug for ten days and undergo tests. the study drug was presented as capsules in three different strengths (0.25, 2.5 and 10 mg). the protocol describes four distinct parts to the study: a single dose ascending part a cross-over part to evaluate fed vs. fasting conditions (could be either single or multiple dose) a multiple dose ascending part a pharmacodynamics part to evaluate the effect of bia 10-2474 vs placebo with different challenge agents the protocol specifies the first three parts would be double-blind but the pharmacodynamics part would be open label. according to the protocol, dose levels and numbers of groups could be increased, or were not yet defined, and would depend on what was observed with initial dosing, (an approach known as adaptive trial design). thus many details of the key multi-dose part are not included in the trial protocol. the absence of these details was criticised by scientists and in the media, before the ansm published further details of the dosing. the royal statistical society was particularly critical, stating that it had "clear statistical reservations about the trial's study design", and that the protocol lacked features such as a risk assessment, recommended in the wake of the tgn1412 incident to prevent severe adverse events occurring in these circumstances. starting dose and subsequent doses for the single dose part of the study, the protocol describes eight groups of eight volunteers (3:1 randomised) who were to receive single doses of bia 10–2474 at 0.25, 1.25, 2.5, 5.0, 10, 20, 40 and 100 mg, with the possibility of additional groups to be added if no maximum tolerated dose was reached. in describing the rationale for the starting dose, the authors of the protocol conclude that: no target organ was identified during toxicology studies and few adverse clinical findings were observed at the highest dose tested. for the single ascending dose part , a starting dose of 0.25 mg was judged to be safe for a first-in-human administration. the protocol provides for the first two subjects of the first single dose cohort to receive the first dose as a sentinel dose, that is, either 0.25 mg bia 10-2474 or a placebo on the first day, then wait for 24h before treating the other 5:1 subjects. in the event no safety concerns had emerged in the foregoing, all other single- and multi-dose groups were to be dosed with a 10-minute interval between recipients. for the multiple ascending dose part of the study, the protocol planned four groups of eight volunteers (3:1 randomised) who were to receive a single dose orally, once daily for 10 days at different dose levels. however, the protocol defines no doses for these groups, stating that this will be based on the outcome of the single dose portion of the trial. further dose groups up to a maximum of eight groups could be added depending on whether adverse events were observed. the authors note that nonetheless, the starting dose will not exceed 33% of the maximum tolerated dose (mtd) identified in the single dose groups (or 33% of the maximum administered dose if the mtd is not reached). further details of the study as it was actually conducted have been published by the french agency (ansm). the dose groups for the single dose part were as described in the protocol, with no additional groups undertaken. in the crossover part, a single 40 mg dose was given to a group of 12 subjects. in the multiple dose ascending part, the doses were 2.5, 5.0, 10, 20 and 50 mg bia 10–2474, each to be given once per day for 10 days to groups of 8 volunteers (3:1 randomised). the severe adverse events were observed in the 50 mg dose group. the ansm expert committee reported that complete faah inhibition should have been achieved by a dose of 1.25 mg remarking that: it appears unjustified to plan to test a dose (100 mg) 80 times higher than that presumed to induce complete and prolonged faah inhibition. further, the ansm committee remarked that the gap between the 20 mg and 50 mg dose cohorts, in effect, skipped a dose based on extrapolation from the single dose part of the study, and that the progression to 50 mg was too large a jump. on reviewing the subject data from the trial itself, the committee noted that bia 10-2474 showed non-linear pharmacokinetics at doses between 40 and 100 mg (that is, the molecule appeared to be accumulating at higher doses) and that most likely the elimination mechanism had become saturated. thus dosing at 50 mg daily was - each day - 40 times more than required to achieve complete inhibition, and in practice this dose level resulted in accumulation. trial status at the time of serious adverse events according to bial and the rennes university hospital, by the time the serious adverse reactions emerged, 116 subjects had been recruited and 84 other volunteers had received the drug during the trial, with no serious adverse events being reported. the single dose part (up to 100 mg bia 10–2474), the fed vs. fasting part, and the first four dose groups of the multi-dose part of the study had each been completed in 2015. notably, the administration of 20 mg bia 10-2474 once daily for 10 consecutive days elicited no serious adverse events in the six volunteers who received it. dosing of eight volunteers in the highest multi-dose group in the bia 10-2474 trial commenced on 6 january 2016. six of the participants received 50 mg per day of the drug while two received placebo. the first subject became ill on the evening of the 5th day of dosing (10 january). on the following day, the other subjects received a 6th dose at 8:00am before the trial was suspended later that day (11 january). death and serious adverse events the fifth dose level (50 mg per day for 10 days) of the multi-dose part of study had been underway for five days when the first volunteer became ill and was hospitalized at the rennes university hospital on the evening of 10 january 2016 with symptoms similar to a stroke. the following day, the man lapsed into a coma and was shortly thereafter declared brain dead. according to the hospital, the man died at midday on 17 january 2016. four of the other five men in the same dosage group were also hospitalized between 10 and 13 january suffering injuries similar to the man who died, including deep haemorrhagic and necrotic lesions seen on brain mri. all the mri findings, though widely varying in severity, were of the same form and seen in the hippocampus and pons of the affected individuals. biotrial stopped the study on 11 january and both the ansm and regional ethics committee were notified on 14 january. the men who were hospitalised were all from the group which received the highest dose of the multiple ascending part of the trial. a neurologist at the university of rennes hospital center, professor pierre-gilles edan, stated in a press conference with the french minister for health, that 3 of the 4 men who were displaying neurological symptoms "already have a severe enough clinical picture to fear that even in the best situation there will be an irreversible handicap" and were being given corticosteroids to control the inflammation. the sixth man from the group was not showing adverse effects but was hospitalized on 15 january 2016 for observation. other study volunteers who had received doses with no ill effects were asked to return for further testing. the man who died was later named by local news media as guillaume molinet, 49, an artist and father of four from guilliers, a town in the breton department of morbihan. according to the man's brother laurent molinet, mr molinet was recruited as a stand-by and went to rennes not expecting to be dosed, only being given bia 10-2474 because another volunteer dropped out. molinet's family said they were originally told he had had a stroke that was unrelated to the clinical trial, though this quickly turned out not to be the case. molinet's family objected that key information was hidden by bial/biotrial and is reported to have launched a manslaughter lawsuit. reaction and investigations the events in rennes were made public on 15 january 2016 and were reported widely in the media in france, internationally in mainstream news and scientific media. all these reports drew comparisons between this incident and the tgn1412 trial at northwick park, london in which six volunteers developed life-threatening drug reactions during a phase i study in 2006. the journal nature quoted bial spokeswoman susana vasconcelos as saying the trial had been conducted "in accordance with all the good international practices guidelines, with the completion of tests and preclinical trials" and that "the company is committed to determine thoroughly and exhaustively the causes which are at the origin of this situation". bial also denounced the unauthorised release of the trial protocol and was critical of the wide-ranging speculation by scientists and the media about the possible cause of the incident. in july 2016, bial's executive director antónio portela confirmed his company's decision to permanently abandon development of the molecule. the journal also sought comment from jean-marc gandon, the president and chief executive of the cro biotrial, who said "he cannot immediately respond to queries from nature, that he is focused on trying to save the patients and that the company will respond later". biotrial stated its position that "the trial was conducted in full compliance with the international regulations and biotrial's procedures, in particular the emergency procedures". as of march 2016 it remained unclear whether bial disclosed the adverse animal findings to biotrial, including the deaths of monkeys and dogs in several studies. françois peaucelle, the director general of biotrial, is reported to have told le figaro. "we received a 15-page summary of the tests that was based on data that would have filled a lorry. from that data, there was nothing worrying in view of the dosage we were administering to humans." french authorities the agence nationale de sécurité du médicament (ansm) announced an investigation, and that an inspection of the trial site was already underway. and formation of a specialist committee of pharmacologists, toxicologists and neurologists to review all existing data on faah inhibitor drugs. the french health minister marisol touraine, who visited the trial site in rennes and spoke with the victims' families, called the events "an accident of exceptional gravity" and promised to investigate the matter via the inspector general for social affairs (igas), with a final report due end of march. a judicial investigation was also initiated after the office of the chief prosecutor for paris announced it would look into potential charges of involuntary injury through its public health enforcement section, seeking assistance from the gendarmerie in rennes and the ministry of justice's office for the environment and public health. the oniam (office national d'indemnisation des accidents médicaux), responsible for medical injury compensation, stated only 10 accidents had occurred during clinical trials over the past 15 years per its records, and that those cases had "consequences infinitely less serious" than the incident in rennes. initial reports from the authorities suggested that the causes of the brain injuries was likely to be due to the mechanism of bia 10-2474 and the doses employed in the trial. ansm director general dominique martin said "it is clearly the molecule that is the cause", following the publication of the ansm expert committee's initial findings on 7 march 2016. in may 2016, the french health