Inhibition of fatty acid amide hydrolase by BIA 10-2474 in rat brain

Abstract

In a recent clinical trial, the drug BIA 10-2474, a putative fatty acid amide hydrolase(FAAH) inhibitor, was responsible for severe adverse events (SAEs), including one death. To date, there has been little reliable information divulged about the potency of BIA 10-2474 at FAAH in the central nervous system. We synthesised BIA 10-2474 and determined its ability to inhibit FAAH ex vivo in rat brain using a FAAH selective radiotracer. BIA 10-2474 proved to be a potent FAAH inhibitor with IC₅₀s of 50–70 µg/kg (i.p.) in various brain regions. This information may be useful for determining the cause of the SAEs.

Introduction

In January 2016, severe adverse events (SAE) occurred in the Phase I clinical trial using the drug BIA 10-2474 including one death. The Agence Nationale de Sécurité du Médicament et des Produits de Santé (ANSM) set up a Temporary Specialist Scientific Committee (TSSC) to investigate the tragedy. ¹ BIA 10-2474 is a putative inhibitor of Fatty Acid Amide Hydrolase (FAAH) and thus the events have had an impact on on-going research and trials which engage FAAH (ANSM). Nowadays, the rationale for testing a drug in humans is typically to induce a pharmacological effect by specific target engagement. To the best of our knowledge, the BIA 10-2474 Phase 1 trial or pre-trial development did not collect information on target engagement in the central nervous system (CNS) and crucially, the SAEs were CNS derived.

We have developed several radiotracers for positron emission tomography (PET) imaging of FAAH. One, [¹¹C]-CURB, has been used in human PET studies extensively^2–4 while another, [¹⁸F]-DOPP, has been shown to bind to FAAH with exquisitely high signal-to-noise and specificity in rat and non-human primate.^5,6 We took advantage of the selective ex vivo binding properties of [¹⁸F]-DOPP to FAAH to assess the in vivo potency of BIA 10-2474 to inhibit FAAH in rat brain regions.

Methods

All animal experiments were carried out under humane conditions, with approval from the Animal Care Committee at the Centre for Addiction and Mental Health (CAMH), in accordance and with the guidelines set forth by the Canadian Council on Animal Care. Rats (male, Sprague-Dawley, 350–380 g) were kept on a reversed 12-h light/12-h dark cycle and allowed food and water ad libitum. [¹⁸F]-3-(4,5-Dihydrooxazol-2-yl)phenyl (5-fluoropentyl)carbamate ([¹⁸F]-DOPP) was synthesised as previously described ⁶ with specific activities greater than 100 GBq/µmole and radiochemical purities greater than 98%. Results are reported in compliance with the Animal Research: Reporting In Vivo Experiments (ARRIVE guidelines). Unless stated, otherwise all radioactivity measurements were normalised for radioactive decay.

N-Cyclohexyl-N-methyl-4-(pyridin-3-yl)-1H-imidazole-1-carboxamide

Under N₂, an ice-cold mixture of triphosgene (50 mg, 16.85 µmol) and K₂CO₃ (100 mg, 72.4 µmol) in dry CH₃CN (2 mL) was well-stirred and treated with a solution of freshly distilled N-methylcyclohexylamine (67 µL, 51.4 µmol) in dry CH₃CN (0.5 mL). The mixture was stirred for 2 h at ambient temperature, and then 3-(3H-imidazol-4-yl)pyridine hydrochloride (110.2 mg, 50.5 µmol) and pyridine (150 µL) were added. The mixture was heated to reflux for 30 h, cooled to ambient temperature, and then water (20 mL) was added. The mixture was stirred at 0℃ for 2 h and the white precipitate was collected by vacuum filtration, and then washed with cold 10% aq. MeOH, water, and heptane, and dried in vacuo to give 79 mg (55%) of a white solid; mp 159℃–162℃.

3-(1-(Cyclohexyl(methyl)carbamoyl)-1H-imidazol-4-yl)pyridine 1-oxide (BIA 10-2474)

A slurry of N-cyclohexyl-N-methyl-4-(pyridin-3-yl)-1H-imidazole-1-carboxamide (70 mg, 24.6 µmol) and 3-chloroperoxybenzoic acid (100 mg, <72%) in dichloromethane (7 mL) was stirred at ambient temperature for 24 h. The residue obtained upon removing the dichloromethane was stirred with aq. NaHCO₃ (1 M, 70 mL) for 1 h and the resultant solid was collected by vacuum filtration, and then washed with water and heptane. Recrystallisation from 2-propanol gave a white solid (53 mg, 74%); mp 215℃–218℃ (decomposed).

Inhibition of FAAH by BIA 10-2474 in rat brain

Groups of rats (n = 4) were pre-treated with solutions of the challenge drug (2 mL/kg in 5% Tween 80 in saline i.p.) or with vehicle 40 min prior to radiotracer injection. Rats, in a restraining box, received 3–4 MBq of high-specific activity [¹⁸F]-DOPP (<0.03 nmol/kg) in 0.3 mL of 10% ethanol in citrate buffer (pH 6) via the tail vein which had been vasodilated in a warm water bath. They were sacrificed by decapitation at 40 min after radiotracer administration, the brain surgically removed from the skull and stored on ice. Brain regions were excised, blotted, and weighed while blood was collected (from the trunk). Radioactivity in tissues was assayed in an automated gamma counter, back corrected to time of injection, using diluted aliquots of the initial injected dose as standards. Values are reported as standard uptake values (SUVs, mean ± standard deviation) defined as percentage of injected dose/g of tissue divided by rat weight in kilogram. Brain tissue uptake was corrected for a 4% vascular component. Percentage FAAH inhibition was defined as; 100 × (SUV_control – SUV_drug)/SUV_control.

Measurement of BIA 10-2474 levels in plasma

Blood collected from trunk after decapitation of each rat was centrifuged and 2 mL of plasma treated with 1 mL of 25% acetic acid. The acidified plasma was passed through a pre-conditioned Waters Oasis HLB cartridge (3 mL, 30 mg) and washed with 5 mL of 5% methanol in water. BIA 10-2474 was eluted with 0.4 mL of ethanol followed by 1.6 mL of 1% aq. formic acid. The concentration of BIA 10-2474 was determined by high-performance liquid chromatography analysis by comparison with prepared standard solutions of BIA 10-2474 using an ACT ACE C18 column, 5 µ, 250 × 4.6 mm, with CH₃CN/H₂O + 0.1 N ammonium formate (pH 4) at 2 mL/min and ultraviolet detection at 260 nm.

Results

BIA 10-2474 was synthesised in two steps by coupling 3-(3H-imidazol-4-yl)pyridine with N-methylcyclohexylamine using triphosgene followed by oxidation with 3-chloroperbenzoic acid (Scheme SI 1) in an overall yield of 41%. BIA 10-2474 was characterised by ¹H and ¹³C NMR and high resolution mass spectrometry (See supporting information). Purity was >99.5% assessed by high performance liquid chromatography (Figure SI 1).

In control experiments iv, administration of [¹⁸F]-DOPP to rats resulted in high brain uptake of radioactivity, with a distribution consistent with the known distribution of FAAH and in good agreement with previous studies in rats using [¹⁸F]-DOPP. ⁶ Pre-treatment with the potent, selective, and well-characterised FAAH inhibitor, PF-04457845 ⁷ served as a positive control and abolished greater than 98% of [¹⁸F]-DOPP in brain regions (Figure 1).

Figure 1.

Inhibition of [¹⁸F]-DOPP binding to FAAH in rat brain regions by pre-treatment with PF-04457845 (0.1 mg/kg) or BIA 10-2474. Drugs were given i.p. 40 min before radiotracer injection. Mean and SD values given for groups of four animals.

To study the effects of BIA 10-2474 on [¹⁸F]-DOPP binding, we initially treated animals with either 0.5 or 5 mg/kg drug (i.p.), 40 min before radiotracer injection. Both doses completely abolished [¹⁸F]-DOPP brain uptake in all regions, i.e. both doses completely inhibited FAAH activity in the rat brain, showing that BIA 10-2474 was more potent a FAAH inhibitor than would have been inferred from the limited available information in the patent issued to BIAL. ⁸ Lower doses of BIA 10-2474, ranging from 1 to 500 µg/kg, were then used to more fully characterise its in vivo potency (Figure 1).

The IC₅₀ of BIA 10-2474 was calculated from a sigmoidal dose–response relationship using GraphPad Prism software (V4.0c) allowing a variable Hill slope (Figure 2). IC₅₀ values for brain regions were 52 (cerebellum), 67 (rest of brain), 68 (cortex), and 71 µg/kg (hypothalamus) with corresponding Hill slope numbers of 0.88, 1.1, 1.1, and 1.7, respectively. IC₅₀ values were not significantly different between regions (F (DFn, DFd): 1.675 (6, 88)).

Figure 2.

Brain regional inhibition of FAAH by doses of BIA 10-2474 as measured by inhibition of [¹⁸F]-DOPP.

We measured levels of BIA 10-2474 in rat plasma for rats receiving the three highest doses and were 25 ± 3, 116 ± 22, and 1380 ± 71 ng/ mL for rats dosed with 100, 500, and 5000 µg/kg, respectively, at time of sacrifice (corresponding to 83, 386, and 4595 nM). Other groups, receiving lower doses of BIA 10-2474, had plasma levels of BIA-10-2474 below detection limits of our assay (<15 ng/mL).

Discussion

The TSSC reported that BIA 10-2474 has an IC₅₀ in rats of 1.1 to 1.7 µM and is thus a compound with relatively low affinity and poor specificity for FAAH. ¹ Presumably this information was provided to the TSSC by BIAL as their patent only reports 22% inhibition of FAAH in rat brain homogenates at 100 nM concentration of BIA 10-2474 and 98% inhibition of FAAH ex vivo in mice treated with 3 mg/kg BIA 10-2474 (compound #362, Table 1 in patent). ⁸

Our results demonstrate that BIA 10-2474 is indeed a potent inhibitor of FAAH in rat brain as measured by inhibition of FAAH binding by the radiotracer [¹⁸F]-DOPP. Its IC₅₀ of 52–71 µg/kg is comparable to other established FAAH inhibitors such as URB597 (0.15 mg/kg), ⁹ URB694 (40 µg/kg), ⁴ and PF-04457835 (>30 but<100 µg/kg). ⁷ The IC₅₀ values reported here for BIA 10-2474 may err on the high side as we used SUV reduction as a surrogate for % inhibition rather than an input function. ^a

While allometric extrapolation from rodents to human is fraught with error, it is highly likely that very high levels of FAAH inhibition were achieved in the CNS in subjects in the Phase 1 trial. Indeed an oral dose of 1 mg of PF-04457845 inhibits >95% FAAH activity in human brain. ¹⁰ In light of the availability of specific FAAH radiotracers for both animal studies and human PET imaging, it is unfortunate that target engagement in the CNS by BIA 10-2474 was not assessed by these or similar techniques prior to dose escalation in the clinical trial. Dissection or small animal PET imaging studies using FAAH specific radiotracers would have provided a rational basis for dose selection in planned clinical trials. Target engagement in the human brain can only be directly measured by human PET scans or similar imaging modalities. A dosing regime based on effective FAAH inhibition in the CNS rather than maximum tolerated dose might have helped to prevent the tragic occurrence of the SAEs in the BIA 10-2474 clinical trial.