PET imaging of fatty acid amide hydrolase in the brain: synthesis and biological evaluation of an 11C-labelled URB597 analogue

https://doi.org/10.1016/j.nucmedbio.2010.03.009Get rights and content

Abstract

Introduction

Fatty acid amide hydrolase (FAAH) is part of the endocannabinoid system (ECS) and has been linked to the aetiology of several neurological and neuropsychiatric disorders. So far no useful PET or SPECT tracer for in vivo visualisation of FAAH has been reported. We synthesized and evaluated a carbon-11-labeled URB597 analogue, biphenyl-3-yl [11C]-4-methoxyphenylcarbamate or [11C]-1, as potential FAAH imaging agent.

Methods

The inhibitory activity of 1 was determined in vitro using recombinant FAAH. Radiosynthesis of [11C]-1 was performed by methylation using [11C]-CH3I, followed by HPLC purification. Biological evaluation was done by biodistribution studies in wild-type and FAAH knock-out mice, and by ex vivo and in vivo metabolite analysis. The influence of URB597 pretreatment on the metabolisation profile was assessed.

Results

[11C]-1 was obtained in good yields and high radiochemical purity. Biodistribution studies revealed high brain uptake in wild-type and FAAH knock-out mice, but no retention of radioactivity could be demonstrated. Metabolite analysis and URB597 pretreatment confirmed the non-FAAH-mediated metabolisation of [11C]-1. The inhibition mechanism was determined to be reversible. In addition, the inhibition of URB597 appeared slowly reversible.

Conclusions

Although [11C]-1 inhibits FAAH in vitro and displays high brain uptake, the inhibition mechanism seems to deviate from the proposed carbamylation mechanism. Consequently, it does not covalently bind to FAAH and will not be useful for mapping the enzyme in vivo. However, it represents a potential starting point for the development of in vivo FAAH imaging tools.

Introduction

The endocannabinoid system (ECS) is a neuromodulatory system in the brain that comprises cannabinoid receptors CB1 and CB2, endogenous ligands termed endocannabinoids like anandamide (AEA), transporters and several synthesizing and degrading enzymes [1], [2]. Fatty acid amide hydrolase (FAAH), an integral membrane-bound enzyme, is one of the main enzymes of the ECS. A key role of FAAH in hydrolyzing AEA was first reported in 1993 where it was called an ‘anandamide amidase’ enzyme [3]. Studies in FAAH knock-out (FAAH−/−) mice confirmed the primary role of FAAH in controlling endogenous AEA levels in the brain and, consequently, its importance as a regulatory enzyme for key physiological functions [4]. It is thus conceivable that disease-associated changes in tissue concentration of endocannabinoids in part reflect corresponding changes in their inactivation and therefore an up- or down-regulation of FAAH. Research on the role of FAAH in pathological conditions is rising, and accumulating data suggest that the symptoms of several neurological and neuropsychiatric disorders could be caused by changes in the endocannabinoid biosynthesis and degradation. Indeed, a naturally occurring single nucleotide polymorphism (SNP) in the human FAAH gene C385A (cytosine 385→adenosine) was found to be strongly associated with street drug use and problem drug/alcohol use [5]. A direct role for FAAH in the regulation of alcohol consumption was also demonstrated in a study on FAAH knock-out mice [6], [7], [8], [9]. Furthermore, FAAH knock-out mice have reduced anxiety-like behaviour, as revealed by two different behavioural models [10]. Also, pharmacological inhibition of FAAH elicits anxiolytic-like and antidepressant-like effects in rodents [11], [12], [13], [14], indicating a critical role played by the ECS in the pathophysiology of depression and anxiety, as well as the potential usefulness of inhibiting the enzyme. Nevertheless, the therapeutic potential of the ECS and more specifically FAAH has yet to be fully determined, and the number of disorders that may be treated will likely continue to grow.

The availability of a positron emission tomography (PET) or single photon emission computed tomography (SPECT) tracer for in vivo evaluation of FAAH in the brain would be of great interest to elucidate the specific role of the enzyme in those neurological and neuropsychiatric disorders and would greatly improve the knowledge on their aetiology. Mapping FAAH in the brain by means of a PET or SPECT tracer would stimulate the research for novel therapeutic strategies and would be useful for the evaluation of efficacy of potential FAAH inhibitors applicable in the treatment of FAAH-related disorders like anxiety and depression. In general, two approaches are being applied for characterisation of enzymes using PET or SPECT. A first approach is based on the design of radioactive substrate analogues and on the principle of metabolic trapping. This principle implies trapping of the radiolabelled hydrolysis products of the radiotracer in the tissue where the enzyme is present. The distribution of radioactivity in the brain thus reflects the distribution of the enzyme as well as its functionality [15]. A second approach is the use of a radiolabelled inhibitor for the enzyme of interest. The selective binding of the radiolabelled inhibitor to the enzyme will reflect the regional distribution of the enzyme within the brain [16]. Several radioligands based on either of the two approaches have been developed for in vivo evaluation of enzymes like acetylcholinesterase [17], [18], [19], [20], [21], [22] and the monoamine oxidases MAO-A [23], [24] and MAO-B [25], [26], [27] in the human brain by PET or SPECT. To our knowledge, so far no radioligands for PET or SPECT exist for the in vivo visualisation of FAAH in the brain [28], [29].

The high lipophilicity associated with the fatty acid chain present in all up-to-now known endogenous and synthetic FAAH substrates increases the risk of nonspecific binding and rapid metabolic turnover of the tracers [30], [31] and thus limits the use of the principle of metabolic trapping in the development of FAAH tracers. Therefore, we chose to focus on the development of a radiolabelled FAAH inhibitor for in vivo mapping of the enzyme in the brain. Since FAAH is a member of the class of serine hydrolases, it is susceptible to inhibition by most classical serine hydrolase-directed inhibitors, including fluorophosphonates, trifluoromethyl ketones, α-ketoheterocycles and carbamates [32]. The last class includes the potent and selective O-arylcarbamate URB597 (cyclohexylcarbamic acid 3′-carbamoylbiphenyl-3-yl ester) (Fig. 1), which has a nanomolar affinity for FAAH (44 nM in our hands, using recombinant human FAAH) and is devoid of CB1 cannabinoid receptor affinity [33]. URB597 is reported to inhibit FAAH by an irreversible, substrate-like inhibition mechanism proposed to involve carbamylation of the catalytic nucleophile Ser241, with the O-biaryl group serving as the leaving group [34], [35]. Based on the structure of URB597, we synthesized analogues while focusing on the prospect of possible labelling with carbon-11 or fluorine-18. In the present study, we describe the synthesis, in vitro and in vivo evaluation of biphenyl-3-yl [11C]-4-methoxyphenylcarbamate ([11C]-1) as PET tracer for in vivo visualisation of FAAH in the brain. The proposed mechanism for visualisation of FAAH using [11C]-1 is based on carbamylation of the catalytic nucleophile of FAAH by the inhibitor, similar to the proposed inhibitory mechanism of URB597. This carbamylation of the Ser241 would leave the 11C-methoxyanilino group bound to the enzyme and thus provide an opportunity for mapping the enzyme in distinct brain regions using PET (Fig. 2).

Section snippets

Synthesis of compounds

All chemical reagents were obtained from commercial sources (Sigma-Aldrich Fluka, Acros Organics, Belgium) and used without further purification. Solvents used were of high-performance liquid chromatography (HPLC) grade and were purchased from ChemLab (Belgium). Nuclear magnetic resonance (1H NMR, 13C NMR) spectra were recorded on a Bruker Avance 400-MHz Ultrashield. Chemical shifts (δ) are reported relative to the tetramethylsilane peak set at 0 ppm. In the case of multiplets, the signals are

FAAH Inhibition assay

The FAAH inhibitory action of compound 1 was assessed by its ability to prevent the enzyme from hydrolyzing anandamide tritiated in the ethanolamine part ([3H]-AEA). Experiments were performed using 10 concentrations of test compound 1 in the range of 1 mM–1 nM. With this assay, 1 was determined to inhibit the hydrolysis of [3H]-AEA by hFAAH with an IC50 value of 436 nM. An IC50 value of 40 nM was obtained for URB597 when tested in the same assay.

Radiosynthesis of [11C]-1, quality control and partition coefficient determination

[11C]-1 was synthesized by carbon-11 methylation

Discussion and conclusions

Increasing evidence suggests a pivotal role of FAAH in several neurological and neuropsychiatric disorders. Studies in FAAH knock-out mice and the finding of a SNP in the human FAAH gene linked FAAH with addiction. Also, the observation that genetic or pharmacological inactivation of FAAH results in anxiolytic [13] and antidepressant [11], [41] phenotypes in rodents suggests that FAAH may play an important therapeutic target for these and possibly many other CNS disorders. The exact picture of

Acknowledgment

The authors sincerely wish to thank Maarten Dhaenens for the protein content determination. The authors especially thank Johan Sambre for his excellent technical assistance. We are grateful to Dr. Benjamin F. Cravatt for providing the FAAH knock-out mice.

References (52)

  • wyffelsL. et al.

    Radiosynthesis, in vitro and in vivo evaluation of I-123-labeled anandamide analogues for mapping brain FAAH

    Bioorg Med Chem

    (2009)
  • WaterhouseR.

    Determination of lipophilicity and its use as a predictor of blood–brain barrier penetration of molecular imaging agents

    Mol Imaging Biol

    (2003)
  • PajouheshH. et al.

    Medicinal chemical properties of successful central nervous system drugs

    J Am Soc Exp Therap

    (2005)
  • AlexanderJ.P. et al.

    Mechanism of carbamate inactivation of FAAH: implications for the design of covalent inhibitors and in vivo functional probes for enzymes

    Chem Biol

    (2005)
  • WilsonA. et al.

    An admonition when measuring the lipophilicity of radiotracers using counting techniques

    Appl Rad Isotopes

    (2001)
  • DesarnaudF. et al.

    Anandamide amidohydrolase activity in rat-brain microsomes — Identification and partial characterization

    J Biol Chem

    (1995)
  • NunezE. et al.

    Glial expression of cannabinoid CB2 receptors and fatty acid amide hydrolase are beta amyloid-linked events in Down's syndrome

    Neurosci

    (2008)
  • ReichC.G. et al.

    Differential effects of chronic unpredictable stress on hippocampal CB1 receptors in male and female rats

    Behav Brain Res

    (2009)
  • AhnK. et al.

    Discovery and characterization of a highly selective FAAH inhibitor that reduces inflammatory pain

    Chem Biol

    (2009)
  • ZhangD. et al.

    Fatty acid amide hydrolase inhibitors display broad selectivity and inhibit multiple carboxylesterases as off targets

    Neuropharmacology

    (2007)
  • De PetrocellisL. et al.

    The endocannabinoid system: a general view and latest additions

    Br J Pharm

    (2004)
  • LambertD.M. et al.

    The endocannabinoid system: drug targets, lead compounds, and potential therapeutic applications

    J Med Chem

    (2005)
  • DeutschD.G. et al.

    Enzymatic-synthesis and degradation of anandamide, a cannabinoid receptor agonist

    Biochem Pharmacol

    (1993)
  • CravattB.F. et al.

    Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase

    Proc Natl Acad Sci U S A

    (2001)
  • SipeJ.C. et al.

    A missense mutation in human fatty acid amide hydrolase associated with problem drug use

    Proc Natl Acad Sci U S A

    (2002)
  • BlednovY.A. et al.

    Role of endocannabinoids in alcohol consumption and intoxication: studies of mice lacking fatty acid amide hydrolase

    Neuropsychopharmacology

    (2007)
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