BIA 10-2474 is an experimental fatty-acid amide hydrolase (FAAH) inhibitor developed by the Portuguese pharmaceutical company Bial-Portela & Ca. SA. It interacts with the human endocannabinoid system. The drug was in development for the treatment of a range of different medical conditions from anxiety disorder to Parkinson's disease, also for the treatment of chronic pain of multiple sclerosis, cancer, hypertension, or the treatment of obesity. A clinical trial with this drug was underway in Rennes, France, in January 2016, in which serious adverse events occurred affecting five participants, including the death of one man. The underlying mechanism that caused the acute neurotoxicity of this molecule remains unknown.
Structure and action The chemical name of BIA-10-2474 is 3-(1-(cyclohexyl(methyl)carbamoyl)-1H-imidazol-4-yl)pyridine 1-oxide. BIA-10-2474 is a long-acting inhibitor of fatty acid amide hydrolase (FAAH) that increases levels of the neurotransmitter anandamide in the central nervous system and in peripheral tissues (that is, the rest of the body other than the brain and spinal cord).
In normal tissues, the enzyme FAAH degrades anandamide and other endocannabinoid neurotransmitters, which relieve pain and can affect eating and sleep patterns. FAAH inhibitors have been proposed for a range of nervous system disorders including anxiety disorders, alcoholism, pain and nausea.
The Portuguese pharmaceutical company Bial holds several patents on FAAH enzyme inhibitors. The structure and synthesis of BIA 10-2474 is disclosed in a Bial patent as 'compound 362', part of a Bial patent family dating from December 2008.
The patent discloses limited details about BIA 10–2474, mainly the screening assay results for each of the several hundred candidate compounds to evaluate the effect on FAAH activity. For compound 362 (that is, BIA 10–2474), an in vitro assay in rat brain showed only modest FAAH inhibition, however, mice given compound 362 at 3 mg/kg orally had less than 2% the normal level of FAAH activity in both brain and liver tissues after 8 hours. Inhibition of other enzymes affected by cannabinoids (monoacylglycerol lipase and liver carboxylesterase) was performed as a screen for biological selectivity for a small number of compounds, but compound 362 was not included. These results appear to be high-throughput screening data only, and no more definitive data are included (such as inhibitory concentration (IC50) or inhibition constant (Ki) values that characterise the potency of the molecule's inhibition of, or binding with, the target).
The Agence Nationale de Sécurité du Médicament et des Produits de Santé (ANSM) reported that the compound has a rat IC50 of 1.1-1.7 micromolar, and that this is 200 times the concentration required for inhibition with a different FAAH inhibitor developed by Pfizer (that is, much weaker). As such, the ANSM described the molecule as "a compound with a relatively poor specificity for the endocannabinoid FAAH." In the same report, the ANSM also noted that the inhibitor is irreversible, not reversible as claimed by the manufacturer Bial.
Publication of the chemical structure created considerable interest among chemists, with some sharing online their assessment of likely binding interactions between BIA 10-2474 and in vivo targets. At least one analysis using standard software modelling packages found that although FAAH emerged as the primary target for BIA 10–2474, a number of other proteins rated highly as well. These other targets included histone deacetylases, macrophage-stimulating protein receptor and hormone-sensitive lipase.
Although the exact mechanism of action leading to BIA 10-2474 toxicity remains unknown, the final report by the ANSM Committee concluded it was likely one of two possible mechanisms: "inhibition of other serine hydrolases, or harmful effects from the imidazole-pyridine leaving group." The report also theorised that this leaving group "may produce an isocyanate to which many brain proteins are likely to bind." A 2017 research article suggested the off-target activity of BIA 10-2474 may affect lipid metabolism in neurones.
Preclinical studies According to a company statement, a project to develop FAAH inhibitors was initiated by Bial in 2005, and studies with this compound began in 2009 with pre-clinical in vitro and in vivo pharmacological and toxicological evaluation. The French medicines regulator (ANSM) released a version of the clinical trial protocol, after the newspaper Le Figaro leaked a (more recent) version. The protocol presents a summary of what appears to be a full package of pharmacodynamic, pharmacokinetic and toxicological studies that might be expected to support a first-in-man study. The manufacturer Bial refused the regulator's request to release the Investigator's brochure and the product dossier (the Investigational Medicinal Product Dossier; IMPD), citing French law on trade secrets.
An expert committee, established by the French medicines regulator after the trial, requested clarification from Bial of a range of preclinical issues.
Pharmacodynamics and pharmacokinetics In terms of animal pharmacodynamics, the trial protocol reports that the biological activity of BIA 10-2474 was tested in models of predictive efficacy in treating pain. "BIA 10-2474 produced analgesic/anti-inflammatory activity in the mouse Formalin-Paw and Tail-Flick tests in a time-and dose-dependent manner. BIA 10-2474 also markedly potentiated the antinociceptive effects of exogenous anandamide in the mouse Formalin-Paw and Tail-Flick tests". In other words, BIA 10-2474 worked as a pain-killer in mice using two different tests (the "analgesic/anti-inflammatory" effect); and, in mice that had been given a dose of the neurotransmitter anandamide, BIA 10-2474 also improved its effects in numbing pain ("antinociceptive" effect). The expert committee of the ANSM took the view that this was insufficient basis for commencing human trials, and that further evidence of BIA 10-2474 as an analgesic was warranted. The committee's final report noted that in fact the original formalin paw study report presented additional data for the gabapentin comparator which was omitted in the Investigators Brochure. The final ANSM report stated that "doses used in these tests differ greatly (from 0.3 to 10 mg/Kg), without it being possible to trace a dose-effect curve or to estimate an effective dose 50 (which is a surprising shortcoming)".
In terms of animal pharmacokinetics, in rats and dogs given a radio-labelled dose of BIA 10–2474, the drug was detectable in the blood for up to a day afterwards (oral or i.v.). Oral bioavailability was not reported. Terminal half-life (persistence in the blood) of the BIA 10–2474 in rats was 45 hours (oral) or 4 hours (i.v.) and in dogs it was 104 hours (oral) or 52 hours (i.v.). The authors made no prediction of a likely half-life in humans. Around two-thirds of the total dose was eliminated in the urine, about one-fifth in the faeces and the remainder was heavily metabolized in all species studied (rat, mouse, dog, monkey). Metabolism of BIA 10-2474 was essentially complete by 72 hours. Main metabolites were not described. The studies used total detectable radioactivity to calculate half-life and did not assess what proportion of this was due to metabolites.
The preliminary report by the French Inspector General for Social Affairs (IGAS), for the French Ministry of Health, remarked on the difference between the rat oral vs. I.V. half life, and, that in light of the adverse events seen in humans on repeated dosing, this may indicate "a mechanism of accumulation". Separately, accumulation of BIA 10-2474 given orally in humans was supported by pharmacokinetic data from the trial itself, reviewed by the ANSM expert committee. The molecule showed non-linear pharmacokinetics at doses between 40 and 100 mg, suggesting elimination pathways had become saturated, leading to accumulation.
The final report by the ANSM Committee noted the drug had a very steep dose-effect curve in humans "going from absence of to almost complete inhibition" over a narrow concentration range. The report also found, based on clinical data from the Rennes trial, that complete inhibition by BIA 10-2474 was very long-acting, and persisted well beyond the point where it was no longer detectable in blood of the trial subjects.
Safety pharmacology and toxicology The protocol presents a summary of safety pharmacology studies in two species (rat, dog) and repeated dose toxicity studies in four species (13-week sub-chronic studies in mouse, dog and monkey; a 26-week chronic study in the rat). Of note, few adverse events were observed in any of the studies, with the oral No Observed Adverse Effect Level (NOAEL) varying between 10 mg/kg/day in rats to 75 mg/kg/day in monkeys. The authors suggest that these were the maximum doses tested in these studies, though it is not clear. The authors also report no effects of significance in the animal models used for the CNS safety pharmacology studies, which studied a dose of up to 300 mg/kg/day. The protocol nominates a human NOAEL of 100 mg as a human equivalent dose to the 26-week rat NOAEL, though with no description how this was calculated. The summary presented however includes no assessment of the relevance of the animal species selected for study (that is, in terms of physiological and genetic similarities with humans and the mechanism of action of the study drug). Early reports that the molecule had been studied in chimpanzees turned out to be incorrect.
At a hearing convened by the ANSM in March 2016, Bial clarified that the apparently extensive animal toxicology studies (and number of species) was due to a delay in commencement of clinical development, thus some studies had already been completed that would not have been required for a Phase I study. The ANSM Committee found no evidence that the studies had been performed because the company had doubts as to the tolerance of the molecule.
Notably absent from the protocol were calculations of receptor occupancy; predictions of in vivo ligand binding saturation levels; measures of target affinity or assessment of non-target binding interactions as suggested by the European guidance for Phase I studies (depending on whether BIA 10-2474 could be considered to require 'special consideration' as outlined in the guideline). On these issues, the French regulator's expert committee pointed out, based on the company's IC50 data, that complete FAAH inhibition should have been achieved with a dose of 1.25 mg in humans. In fact, the trial tested doses up to 80 times more (100 mg BIA 10–2474) than should have been required. The ANSM committee also requested data on non-target enzyme affinity from the manufacturer Bial.
Although the protocol summary reports no animal deaths during the studies, the ANSM expert committee reported that in fact several monkeys died or had to be euthanised during the dose escalation studies and that an explanation from Bial was pending. Also, two animals had to be euthanised in the 13-week dog study due to lung lesions - both from the top dose group. None of these animal deaths were described in the trial protocol.
It is not clear whether Bial disclosed these adverse animal findings either to Biotrial or to the ANSM in their application for clinical trial authorisation.