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Christophe Mallet,1 Jules Desmeules,2 Rassa Pegahi,3 Alain Eschalier1 1Université Clermont Auvergne, INSERM, NEURO-DOL Basics & Clinical Pharmacology of Pain, Clermont-Ferrand, France; 2Faculty of Medicine and The School of Pharmaceutical Sciences, Faculty of Sciences, Geneva University, Geneva, Switzerland; 3Medical Affairs Department, UPSA, Paris, FranceCorrespondence: Alain Eschalier, Faculté de Médecine, UMR Neuro-Dol, 49 Bd François Mitterrand, Clermont-Ferrand, 63000, France, Email alain.eschalier@uca.frAbstract: Paracetamol remains the recommended first-line option for mild-to-moderate acute pain in general population and particularly in vulnerable populations. Despite its wide use, debate exists regarding the analgesic mechanism of action (MoA) of paracetamol. A growing body of evidence challenged the notion that paracetamol exerts its analgesic effect through cyclooxygenase (COX)-dependent inhibitory effect. It is now more evident that paracetamol analgesia has multiple pathways and is mediated by the formation of the bioactive AM404 metabolite in the central nervous system (CNS). AM404 is a potent activator of TRPV1, a major contributor to neuronal response to pain in the brain and dorsal horn. In the periaqueductal grey, the bioactive metabolite AM404 activated the TRPV1 channel‐mGlu5 receptor‐PLC‐DAGL‐CB1 receptor signaling cascade. The present article provides a comprehensive literature review of the centrally located, COX-independent, analgesic MoA of paracetamol and relates how the current experimental evidence can be translated into clinical practice. The evidence discussed in this review established paracetamol as a central, COX-independent, antinociceptive medication that has a distinct MoA from non-steroidal anti-inflammatory drugs (NSAIDs) and a more tolerable safety profile. With the establishment of the central MoA of paracetamol, we believe that paracetamol remains the preferred first-line option for mild-to-moderate acute pain for healthy adults, children, and patients with health concerns. However, safety concerns remain with the high dose of paracetamol due to the NAPQI-mediated liver necrosis. Centrally acting paracetamol/p-aminophenol derivatives could potentiate the analgesic effect of paracetamol without increasing the risk of hepatoxicity. Moreover, the specific central MoA of paracetamol allows its combination with other analgesics, including NSAIDs, with a different MoA. Future experiments to better explain the central actions of paracetamol could pave the way for discovering new central analgesics with a better benefit-to-risk ratio.Keywords: paracetamol, acetaminophen, AM404, analgesia, molecular targets

[Display omitted] • AM404 infused into hippocampus CA1 area blocks consolidation in a GC-dependent way. • AM404 blocks the induction of LTP in the CA1 area of the hippocampus. • AM404 amnestic effect is mediated by CB1 and M4, but possibly not TRPV1 receptors. • Optimal level of extracellular Anandamide is required in CA1 for memory consolidation. The endocannabinoid system is involved in the fine-tuning of local synaptic plasticity in the hippocampus during the initial steps of memory formation/transformation. In spite of extensive studies, endocannabinoid modulation of these processes is still poorly understood. Here we studied the effects of intra-CA1 infused AM404, an anandamide (AEA) transport/metabolism inhibitor, upon an aversive memory consolidation with or without prior systemic administration of metyrapone, as well the concomitant intra-CA1 administration of AM404 plus AM251 (CB1 receptor inverse-agonist), capsazepine (TRPV1 receptor antagonist) or tropicamide (M4 receptor antagonist). We also investigated the effect of AM404 on memory retrieval and Long-Term Potentiation induction. Adult male Wistar rats were trained in the Contextual Fear Conditioning task and tested 48 h later. AM404 disrupted both memory consolidation and retrieval, and abolished LTP induction. The post-training effect, however, was reverted by metyrapone – which was amnestic by itself – corroborating the known co-dependency between glucocorticoids and endocannabinoids, and suggesting that some level of aversiveness is necessary for an adequate consolidation. In the coadministration experiments, while AM251 and tropicamide were able to revert the AM404 amnestic effect, capsazepine had no effect. This confirms that CB1 actually mediate the amnestic effect caused by the augmented AEA pool, but TRPV1 does not. The tropicamide result suggests an interesting comodulatory interaction between the endocannabinoid and the cholinergic systems. We propose a steady-state model centered in the idea of an optimal, stable extracellular concentration of anandamide as a necessary condition to ensure the consolidation of a stable memory trace in the CA1 area. [ABSTRACT FROM AUTHOR]

Study Question: Do paracetamol (N-acetyl-para-aminophenol (APAP) or acetaminophen) and/or its metabolites affect human sperm Ca2+-signalling and function?Summary Answer: While APAP itself does not interact with Ca2+-signalling in human sperm, its metabolite N-arachidonoyl phenolamine (AM404), produced via fatty acid amide hydrolase (FAAH), interferes with human sperm Ca2+-signalling and function through a suggested CatSper channel-dependent action.What Is Known Already: Studies have shown that adult men with high urinary levels of over-the-counter mild analgesic APAP have impaired sperm motility and increased time-to-pregnancy.Study Design, Size, Duration: This study consists of (i) an in vivo human pharmaceutical APAP exposure experiment to understand to what degree APAP reaches the sperm cells in the seminal fluid; (ii) in vitro calcium imaging and functional experiments in freshly donated human sperm cells to investigate CatSper channel-dependent activation by APAP and its metabolites; and (iii) experiments to understand the in situ capabilities of human sperm cells to form APAP metabolite AM404.Participants/materials, Setting, Methods: Three healthy young males participated in the in vivo human exposure experiment after prior consent. Human semen samples were provided by healthy young volunteer donors after prior consent on the day of the in vitro experiments.Main Results and the Role Of Chance: Pharmaceutical APAP exposure reaches the seminal plasma in high micromolar concentrations and accumulates in the seminal plasma between 3 and 5 days of exposure (P-value 0.023). APAP and its primary metabolite 4-aminophenol (4AP) do not interact with human sperm Ca2+-signalling. Instead, the APAP metabolite AM404 produced via FAAH interferes with human sperm Ca2+-signalling through a CatSper-dependent action. Also, AM404 significantly increases sperm cell penetration into viscous mucous (P-value of 0.003). FAAH is functionally expressed in human sperm cells in the neck/midpiece region, as evidenced by immunohistochemical staining and the ability of human sperm cells to hydrolyse the fluorogenic FAAH substrate arachidonyl 7-amino, 4-methyl coumarin amide in an FAAH-dependent manner. Importantly, human sperm cells have the capacity to form AM404 in situ after exposure to 4AP (P-value 0.0402 compared to vehicle-treated sperm cells).Limitations, Reasons For Caution: The experiments were conducted largely in vitro. Future studies are needed to test whether APAP can disrupt human sperm function in vivo through the action of AM404.Wider Implications Of the Findings: We hypothesize that these observations could, at least in part, be responsible for the negative association between male urinary APAP concentrations, sperm motility and time-to-pregnancy.Study Funding/competing Interest(s): D.M.K. is funded by the Lundbeck Foundation, grant number R324-2019-1881, and the Svend Andersen Foundation. A.R. is funded by a BRIDGE-Translational Excellence Programme grant funded by the Novo Nordisk Foundation, grant agreement number: NNF18SA0034956. All authors declare no competing interests.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

The mechanism by which acetaminophen produces its analgesic effects is not fully understood. One possible mechanism is the activation of the spinal 5-hydroxytryptamine (5-HT) receptor, although direct evidence of spinal 5-HT release has not yet been reported. N-arachidonoylphenolamine (AM404), a metabolite of acetaminophen, is believed to be the key substance that contributes to the analgesic effects of acetaminophen. In this study, we examined whether acetaminophen and AM404 induce spinal 5-HT release and the mechanism through which spinal 5-HT receptor activation exerts analgesic effects in a rat formalin test in an inflammatory pain model. Spinal 5-HT release was examined by intrathecal microdialysis in conscious and freely moving rats. Acetaminophen was administered orally, and AM404 was administered intracerebroventricularly. In rat formalin tests, oral acetaminophen and intracerebroventricular AM404 induced significant spinal 5-HT release and produced analgesic effects. The analgesic effect of oral acetaminophen was partially antagonized by intrathecal administration of WAY100135 (a 5-HT1A receptor antagonist) and SB269970 (a 5-HT7 receptor antagonist). In contrast, the analgesic effect of intracerebroventricular AM404 was completely antagonized by WAY100135, while SB269970 had no effect. Our data suggest that while oral acetaminophen and intracerebroventricular AM404 activate the spinal 5-HT system, the role of the spinal 5-HT system activated by oral acetaminophen differs from that activated by intracerebroventricular AM404.

An emerging number of studies address the involvement of neuroinflammation and oxidative stress in the pathophysiology of central nervous system (CNS) disorders such as depression, schizophrenia, anxiety, and neurodegenerative diseases. Different cytokines and molecules, such as prostaglandin (PG) E2, are associated with neuroinflammatory processes. The active acetaminophen metabolite AM404 has been shown to prevent inflammation and neuroinflammation in primary microglia and organotypic hippocampal slice cultures. However, its effects on pathophysiological conditions in the CNS and especially on neurons are still poorly understood. In this study, we therefore evaluated the effects of AM404 and acetaminophen on the arachidonic acid cascade and oxidative stress induced by interleukin (IL)-1β in human SK-N-SH neuronal cells. We observed that AM404 and acetaminophen significantly and concentration-dependent inhibited IL-1β-induced release of PGE2, independent of cyclooxygenases (COX)-1 and COX-2 enzymatic activity as well as COX-2 mRNA and protein levels in SK-N-SH-cells. The reduction of IL-1β-induced PGE2-release by AM404 and acetaminophen treatment might be mediated by the 8-iso-PGF2α pathway since IL-1β-induced synthesis of this free radical marker is dose-dependently reduced by both compounds, respectively. Therefore, understanding of the potential therapeutic properties of AM404 in neuroinflammation and oxidative stress might lead to future treatment options of different neurological disorders. [ABSTRACT FROM AUTHOR]

An emerging number of studies address the involvement of neuroinflammation and oxidative stress in the pathophysiology of central nervous system (CNS) disorders such as depression, schizophrenia, anxiety, and neurodegenerative diseases. Different cytokines and molecules, such as prostaglandin (PG) E2, are associated with neuroinflammatory processes. The active acetaminophen metabolite AM404 has been shown to prevent inflammation and neuroinflammation in primary microglia and organotypic hippocampal slice cultures. However, its effects on pathophysiological conditions in the CNS and especially on neurons are still poorly understood. In this study, we therefore evaluated the effects of AM404 and acetaminophen on the arachidonic acid cascade and oxidative stress induced by interleukin (IL)-1β in human SK-N-SH neuronal cells. We observed that AM404 and acetaminophen significantly and concentration-dependent inhibited IL-1β-induced release of PGE2, independent of cyclooxygenases (COX)-1 and COX-2 enzymatic activity as well as COX-2 mRNA and protein levels in SK-N-SH-cells. The reduction of IL-1β-induced PGE2-release by AM404 and acetaminophen treatment might be mediated by the 8-iso-PGF2α pathway since IL-1β-induced synthesis of this free radical marker is dose-dependently reduced by both compounds, respectively. Therefore, understanding of the potential therapeutic properties of AM404 in neuroinflammation and oxidative stress might lead to future treatment options of different neurological disorders.

More effective treatments to reduce pathological alcohol drinking are needed. The glutamatergic system and the NMDA receptor (NMDAR), in particular, are implicated in behavioral and molecular consequences of chronic alcohol use, making the NMDAR a promising target for novel pharmacotherapeutics. Ethanol exposure upregulates Fyn, a protein tyrosine kinase that indirectly modulates NMDAR signaling by phosphorylating the NR2B subunit. The Src/Fyn kinase inhibitor saracatinib (AZD0530) reduces ethanol self-administration and enhances extinction of goal-directed ethanol-seeking in mice. However, less is known regarding how saracatinib affects habitual ethanol-seeking. Moreover, no prior studies have assessed the effects of Src/Fyn kinase inhibitors on alcohol-seeking or consumption in human participants. Here, we tested the effects of saracatinib on alcohol consumption and craving/seeking in two species, including the first trial of an Src/Fyn kinase inhibitor to reduce drinking in humans. Eighteen male C57BL/6NCrl mice underwent operant conditioning on a variable interval schedule to induce habitual responding for 10% ethanol/0.1% saccharin. Next, mice received 5 mg/kg saracatinib or vehicle 2 h or 30 min prior to contingency degradation to measure habitual responding. In the human study, 50 non-treatment seeking human participants who drank heavily and met DSM-IV criteria for alcohol abuse or dependence were randomized to receive 125 mg/day saracatinib (n = 33) or placebo (n = 17). Alcohol Drinking Paradigms (ADP) were completed in a controlled research setting: before and after 7–8 days of treatment. Each ADP involved consumption of a priming drink of alcohol (0.03 mg%) followed by ad libitum access (3 h) to 12 additional drinks (0.015 g%); the number of drinks consumed and craving (Alcohol Urge Questionnaire) were recorded. In mice, saracatinib did not affect habitual ethanol seeking or consumption at either time point. In human participants, no significant effects of saracatinib on alcohol craving or consumption were identified. These results in mice and humans suggest that Fyn kinase inhibition using saracatinib, at the doses tested here, may not reduce alcohol consumption or craving/seeking among those habitually consuming alcohol, in contrast to reports of positive effects of saracatinib in individuals that seek ethanol in a goal-directed manner. Nevertheless, future studies should confirm these negative findings using additional doses and schedules of saracatinib administration. [ABSTRACT FROM AUTHOR]

Different studies have demonstrated that inflammation and alterations in glutamate neurotransmission are two events contributing to the pathophysiology of neurodegenerative or neurological disorders. There are evidences that N-arachidonoylphenolamine (AM404), a cannabinoid system modulator and paracetamol metabolite, modulates inflammation and exerts neuroprotective effects on Huntington’s (HD) and Parkinson’s diseases (PD), and ischemia. However, the effects of AM404 on the production of inflammatory mediators and excitotoxicity in brain tissue stimulated with N-methyl-D-aspartic acid (NMDA) are not elucidated. In this present study, we investigated the effects of AM404 on the production of inflammatory mediators and neuronal cell death induced by NMDA in organotypic hippocampal slices cultures (OHSC) using qPCR, western blot (WB), and immunohistochemistry. Moreover, to comprehend the mechanism of excitotoxicity, we evaluated the effects of AM404 on glutamate release in hippocampal synaptosomes and the NMDA-induced calcium responses in acute hippocampal slices. Our results showed that AM404 led to a significant decrease in cell death induced by NMDA, through a mechanism possibly involving the reduction of glutamate release and the calcium ions responses. Furthermore, it decreased the expression of the interleukin (IL)-1β. This study provides new significant insights about the anti-inflammatory and neuroprotection effects of AM404 on NMDA-induced excitotoxicity. To understand the effects of AM404 in these processes might contribute to the therapeutic potential of AM404 in diseases with involvement of neuroinflammation and neurodegeneration and might lead to a possible future treatment of neurodegenerative diseases.

Abstract Background N-arachidonoylphenolamine (AM404), a paracetamol metabolite, is a potent agonist of the transient receptor potential vanilloid type 1 (TRPV1) and low-affinity ligand of the cannabinoid receptor type 1 (CB1). There is evidence that AM404 exerts its pharmacological effects in immune cells. However, the effect of AM404 on the production of inflammatory mediators of the arachidonic acid pathway in activated microglia is still not fully elucidated. Method In the present study, we investigated the effects of AM404 on the eicosanoid production induced by lipopolysaccharide (LPS) in organotypic hippocampal slices culture (OHSC) and primary microglia cultures using Western blot, immunohistochemistry, and ELISA. Results Our results show that AM404 inhibited LPS-mediated prostaglandin E2 (PGE2) production in OHSC, and LPS-stimulated PGE2 release was totally abolished in OHSC if microglial cells were removed. In primary microglia cultures, AM404 led to a significant dose-dependent decrease in the release of PGE2, independent of TRPV1 or CB1 receptors. Moreover, AM404 also inhibited the production of PGD2 and the formation of reactive oxygen species (8-iso-PGF2 alpha) with a reversible reduction of COX-1- and COX-2 activity. Also, it slightly decreased the levels of LPS-induced COX-2 protein, although no effect was observed on LPS-induced mPGES-1 protein synthesis. Conclusions This study provides new significant insights about the potential anti-inflammatory role of AM404 and new mechanisms of action of paracetamol on the modulation of prostaglandin production by activated microglia.

Different studies have demonstrated that inflammation and alterations in glutamate neurotransmission are two events contributing to the pathophysiology of neurodegenerative or neurological disorders. There are evidences that N-arachidonoylphenolamine (AM404), a cannabinoid system modulator and paracetamol metabolite, modulates inflammation and exerts neuroprotective effects on Huntington's (HD) and Parkinson's diseases (PD), and ischemia. However, the effects of AM404 on the production of inflammatory mediators and excitotoxicity in brain tissue stimulated with N-methyl-D-aspartic acid (NMDA) are not elucidated. In this present study, we investigated the effects of AM404 on the production of inflammatory mediators and neuronal cell death induced by NMDA in organotypic hippocampal slices cultures (OHSC) using qPCR, western blot (WB), and immunohistochemistry. Moreover, to comprehend the mechanism of excitotoxicity, we evaluated the effects of AM404 on glutamate release in hippocampal synaptosomes and the NMDA-induced calcium responses in acute hippocampal slices. Our results showed that AM404 led to a significant decrease in cell death induced by NMDA, through a mechanism possibly involving the reduction of glutamate release and the calcium ions responses. Furthermore, it decreased the expression of the interleukin (IL)-1β. This study provides new significant insights about the anti-inflammatory and neuroprotection effects of AM404 on NMDA-induced excitotoxicity. To understand the effects of AM404 in these processes might contribute to the therapeutic potential of AM404 in diseases with involvement of neuroinflammation and neurodegeneration and might lead to a possible future treatment of neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Neuropathic pain consequent to peripheral nerve injury has been associated with local inflammation. Following noxious stimulation afferent fibres release substance P (SP) and calcitonin-gene related peptide (CGRP), which are closely related to oedema formation and plasma leakage. The effect of the anandamide transport blocker AM404 has been studied on plasma extravasation after chronic constriction injury (CCI) which consists in a unilateral loose ligation of the rat sciatic nerve (Bennett and Xie, 1988). AM404 (1-3-10 mg kg(-1)) reduced plasma extravasation in the legated paw, measured as mug of Evans Blue per gram of fresh tissue. A strong effect on vascular permeability was also produced by the synthetic cannabinoid agonist WIN 55,212-2 (0.1-0.3-1 mg kg(-1)). Using specific antagonists or enzyme inhibitors, we demonstrate that cannabinoids act at several levels: data on the 3rd day suggest a strong involvement of substance P (SP) and calcitonin gene-related peptide (CGRP) in the control of vascular tone, whereas at the 7th and 14th days the major role seems to be played by prostaglandins (PGs) and nitric oxide (NO). Capsaicin injection in ligated paws of AM404- or WIN 55,212-2-treated rats resulted in an increase of Evans Blue extravasation, suggesting the involvement of the cannabinergic system in the protective effect of C fibres of ligated paws. Taken together, these data demonstrate the efficacy of cannabinoids in controlling pain behaviour through the modulation of several pain mediators and markers of vascular reactivity, such as SP, CGRP, PGs and NO.

Necroptosis and apoptosis contribute to the pathogenesis of myocardial ischaemia/reperfusion (I/R) injury and subsequent heart failure. N-arachidonoylphenolamine (AM404) is a paracetamol lipid metabolite that has pleiotropic activity to modulate the endocannabinoid system. However, the protective role of AM404 in modulating I/R-mediated myocardial damage and the underlying mechanism remain largely unknown. A murine I/R model was generated by occlusion of the left anterior descending artery. AM404 (20 mg/kg) was injected intraperitoneally into mice at 2 and 24 h before the I/R operation. Our data revealed that AM404 administration to mice greatly ameliorated I/R-triggered impairment of myocardial performance and reduced infarct area, myocyte apoptosis, oxidative stress and inflammatory response accompanied by the reduction of receptor interacting protein kinase (RIPK)1/3- mixed lineage kinase domain-like (MLKL)-mediated necroptosis and upregulation of the immunosubunits (β2i and β5i). In contrast, administration of epoxomicin (a proteasome inhibitor) dramatically abolished AM404-dependent protection against myocardial I/R damage. Mechanistically, AM404 treatment increases β5i expression, which interacts with Pellino-1 (Peli1), an E3 ligase, to form a complex with RIPK1/3, thereby promoting their degradation, which leads to inhibition of cardiomyocyte necroptosis in the I/R heart. In conclusion, these findings demonstrate that AM404 could prevent cardiac I/R damage and may be a promising drug for the treatment of ischaemic heart disease. [Display omitted] [ABSTRACT FROM AUTHOR]

The endocannabinoid system mediates in the pharmacological actions of ethanol and genetic studies link endocannabinoid signaling to alcoholism. Drugs activating cannabinoid CB1 receptors have been found to promote alcohol consumption but their effects on self-administration of alcohol are less clear because of the interference with motor performance. To avoid this problem, a novel pharmacological approach to the study of the contribution of the cannabinoid system in alcoholism may be to use drugs that locally amplify the effects of alcohol on endogenous cannabinoids. In the present study we addressed this model by studying the effects of the anandamide transport inhibitor N-(4-hydroxyphenyl) arachidonoyl-ethanolamide (AM404) on both ethanol self-administration and reinstatement of alcohol-seeking behavior in rats. The results show that AM404 significantly reduced ethanol self-administration in a dose-dependent manner but failed to modify reinstatement for lever pressing induced by the stimulus associated with alcohol. This effect was not due to a motor depressant effect and was not related to a decrease in general motivational state, as it was not effective in other reward paradigms such as lever pressing for a saccharin solution. The mechanism of action of AM404 does not involve cannabinoid CB1 receptors as the CB1-selective antagonist SR141716A did not block the reduction of ethanol self-administration induced by the anandamide uptake blocker. Moreover, 3-(1,1-dimethylheptyl)-(-)-11-hydroxy-delta 8-tetrahydrocannabinol (HU-210), a classical cannabinoid receptor agonist, did not affect ethanol self-administration. The effects of AM404 are not mediated by either vanilloid VR1 receptors or cannabinoid CB2 receptors because it is not antagonized by either the VR1 receptor antagonist capsazepine or the CB2 antagonist AM630. These results indicate that AM404 may be considered as an innovative approach to reduce alcohol consumption.

The endocannabinoids anandamide and 2-arachidonoyglycerol (2-AG) may contribute to the regulation of mood and emotion. In this study, we investigated the impact of the endocannabinoid transport inhibitor AM404 on three rat models of anxiety: elevated plus maze, defensive withdrawal and separation-induced ultrasonic vocalizations. AM404 (1-5 mg kg(-1), intraperitoneal (i.p.)) exerted dose-dependent anxiolytic-like effects in the three models. These behavioral effects were associated with increased levels of anandamide, but not 2-AG, in the prefrontal cortex and were prevented by the CB(1) cannabinoid antagonist rimonabant (SR141716A), suggesting that they were dependent on anandamide-mediated activation of CB(1) cannabinoid receptors. We also evaluated whether AM404 might influence motivation (in the conditioned place preference (CPP) test), sensory reactivity (acoustic startle reflex) and sensorimotor gating (prepulse inhibition (PPI) of the startle reflex). In the CPP test, AM404 (1.25-10 mg kg(-1), i.p.) elicited rewarding effects in rats housed under enriched conditions, but not in rats kept in standard cages. Moreover, AM404 did not alter reactivity to sensory stimuli or cause overt perceptual distortion, as suggested by its lack of effect on startle or PPI of startle. These results support a role of anandamide in the regulation of emotion and point to the anandamide transport system as a potential target for anxiolytic drugs.

The endogenous cannabinoid, anandamide, has been shown to attenuate naloxone-precipitated opiate withdrawal in rodents. Here we show that the spontaneous, but not the naloxone-precipitated withdrawal syndrome in morphine-dependent mice is attenuated by the inhibitor of carrier-mediated anandamide transport N-(4-hydroxyphenyl) arachidonylethanolamide (AM404) (2 and 10 mg/kg, i.p.). These results suggest that spontaneous but not opioid antagonist-precipitated withdrawal is associated with dynamic changes in endogenous cannabinoid signaling.

Chhaya V Sharma,1 Jamie H Long,2 Seema Shah,1 Junia Rahman,1 David Perrett,3 Samir S Ayoub,4 Vivek Mehta1 1Pain & Anaesthesia Research Centre, St Bartholomew’s and The Royal London Hospitals, Barts Health NHS Trust, London, UK; 2Barts & The London School of Medicine, Queen Mary University of London, London, UK; 3BioAnalytical Science, Barts & The London School of Medicine, Queen Mary University of London, London, UK; 4School of Health, Sport and Bioscience, Medicines Research Group, University of East London, London, UK Abstract: Paracetamol is arguably the most commonly used analgesic and antipyretic drug worldwide, however its mechanism of action is still not fully established. It has been shown to exert effects through multiple pathways, some actions suggested to be mediated via N-arachidonoylphenolamine (AM404). AM404, formed through conjugation of paracetamol-derived p-aminophenol with arachidonic acid in the brain, is an activator of the capsaicin receptor, TRPV1, and inhibits the reuptake of the endocannabinoid, anandamide, into postsynaptic ­neurons, as well as inhibiting synthesis of PGE2 by COX-2. However, the presence of AM404 in the central nervous system following administration of paracetamol has not yet been demonstrated in humans. Cerebrospinal fluid (CSF) and blood were collected from 26 adult male patients between 10 and 211 minutes following intravenous administration of 1 g of paracetamol. Paracetamol was measured by high-performance liquid chromatography with UV detection. AM404 was measured by liquid chromatography-tandem mass spectrometry. AM404 was detected in 17 of the 26 evaluable CSF samples at 5–40 nmol⋅L–1. Paracetamol was measurable in CSF within 10 minutes, with a maximum measured concentration of 60 μmol⋅L–1 at 206 minutes. This study is the first to report on the presence of AM404 in human CSF following paracetamol administration. This may represent an important finding in our understanding of paracetamol’s mechanism of action, although measured concentrations were far below the previously documented IC50 for this metabolite. Keywords: acetaminophen, paracetamol, paracetamol: mechanism of action, AM404, N-­arachidonoylphenolamine, pharmacology - theories of analgesic action

We characterized the pharmacological properties of the anandamide transport inhibitor N-(4-hydroxyphenyl)-arachidonamide (AM404) in rats and investigated the effects of this drug on behavioral responses associated with activation of dopamine D(2) family receptors. Rat brain slices accumulated [(3)H]anandamide via a high-affinity transport mechanism that was blocked by AM404. When administered alone in vivo, AM404 caused a mild and slow-developing hypokinesia that was significant 60 min after intracerebroventricular injection of the drug and was reversed by the CB1 cannabinoid receptor antagonist SR141716A. AM404 produced no significant catalepsy or analgesia, two typical effects of direct-acting cannabinoid agonists. However, AM404 prevented the stereotypic yawning produced by systemic administration of a low dose of apomorphine, an effect that was dose-dependent and blocked by SR141716A. Furthermore, AM404 reduced the stimulation of motor behaviors elicited by the selective D(2) family receptor agonist quinpirole. Finally, AM404 reduced hyperactivity in juvenile spontaneously hypertensive rats, a putative model of attention deficit hyperactivity disorder. The results support a primary role of the endocannabinoid system in the regulation of psychomotor activity and point to anandamide transport as a potential target for neuropsychiatric medicines.

Acetaminophen is a popular antipyretic analgesic medicine that has weaker anti-inflammatory properties and lower incidence of side effects than nonsteroidal anti-inflammatory drugs (NSAIDs). However, acetaminophen causes hepatotoxicity due to the reactive metabolite N -acetyl- p -benzoquinone imine (NAPQI). We have obtained acetaminophen analogues in 57–99% yields by using aniline derivatives with protected α-amino acids and fatty acids via the corresponding mixed carbonic carboxylic anhydrides in aqueous MeCN. We have also succeeded in synthesizing AM404 analogues in 76–97% yields, which are expected to be promising candidates for reducing hepatotoxicity. [ABSTRACT FROM AUTHOR]

The compulsive, habitual behaviors that have been observed in individuals diagnosed with substance use disorders may be due to disruptions in the neural circuits that mediate goal‐directed actions. The endocannabinoid system has been shown to play a critical role in habit learning, but the role of this neuromodulatory system in habit expression is unclear. Here, we investigated the role of the endocannabinoid system in established habitual actions using contingency degradation in male C57BL/6 mice. We found that administration of the endocannabinoid transport inhibitor AM404 reduced habitual responding for food and that antagonism of cannabinoid receptor type 1 (CB1), but not transient receptor potential cation subfamily V (TRPV1), receptors produced a similar reduction in habitual responding. Moreover, pharmacological stimulation of CB1 receptors increased habitual responding for food. Co‐administration of an enzyme inhibitor that selectively increases the endocannabinoid 2‐arachidonoyl glycerol (2‐AG) with AM404 partially restored habitual responding for food. Together, these findings demonstrate an important role for the endocannabinoid system in the expression of habits and provide novel insights into potential pharmacological strategies for reducing habitual behaviors in mental disorders. [ABSTRACT FROM AUTHOR]

Rationale: Hyperglycemia accelerates the progression of Alzheimer's disease (AD), and GSK3β plays a potential link between AD and hyperglycemia. Therefore, a direct or indirect GSK3β inhibition may have potential to delay the progression of AD. Our previous biochemical assay identified AM404 as a GSK3β inhibitor at high dose (IC50 = 5.353 μM); however, other study suggests that AM404 impaired synaptic plasticity of hippocampus at high dose (10 mg/kg; i.p.). Therefore, the dose and duration of treatment are crucial for the effects of AM404.Objective: The effects and molecular mechanisms of AM404 at low dose were evaluated from in vitro to in vivo models.Methods: AM404 (0.1-0.5 μM) was tested on tau hyperphosphorylated mouse hippocampal primary cultures treated with Wortmannin (WT) and GF109203X (GFX). Hyperglycemic triple transgenic AD (3×Tg-AD) mice at 6 months old were intraperitoneally injected with AM404 (0.25 mg/kg) for 4 weeks. The spatial learning and memory of mice were measured using the Morris water maze. Mouse brain and serum samples were collected for pathological analyses.Results: AM404 (0.5 μM) exhibited significantly augmented neuroprotection toward tau hyperphosphorylation in primary cultures. The chronic systemic administration of AM404 (0.25 mg/kg) attenuated cognitive deficits in hyperglycemic 3×Tg-AD mice. Moreover, chronic low dose of AM404 significantly attenuated Aβ production, tau protein phosphorylation, and inflammation associated with an increase of pS473Akt and pS9-GSK3β. Therefore, AM404 at low dose, not only increased neuroprotection, but also ameliorated cognitive deficit, could be partly by regulating the Akt/GSK3β signaling, which may contribute to downregulation of Aβ, tau hyperphosphorylation, and inflammation in hyperglycemic 3×Tg-AD mice.Conclusions: These results highlight that chronic administration of AM404 at low dose may be through the Akt/GSK3β pathway to ameliorate the impairment in hyperglycemic 3×Tg-AD mice. [ABSTRACT FROM AUTHOR]

Several classes of biologically occurring fatty acid amides have been reported from mammalian and plant sources. Many amides conjugated with fatty acids of mammalian origin exhibit specific activation of individual receptors. Their potential as pharmacological tools or as lead compounds towards the development of novel therapeutics is of great interest. Hence, access to such amides by a practical, high-yielding and scalable protocol without affecting the geometry or position of sensitive functionalities is needed. A protocol that meets all these requirements involves activation of the corresponding acid with carbonyl diimidazole (CDI) followed by reaction with the desired amine or its hydrochloride. More than fifty compounds have been prepared in generally high yields. [ABSTRACT FROM AUTHOR]

Aims The anandamide reuptake inhibitor N -arachidonoylaminophenol (AM404) and the reactive substance N -acetyl- p -benzoquinone imine (NAPQI) are both metabolites of acetaminophen and may contribute to acetaminophen-induced analgesia by acting at TRPV1 expressed in the peripheral or central nervous system. While NAPQI slowly sensitizes and activates TRPV1 by interacting with distinct intracellular cysteine residues, detailed properties of AM404 as an agonist of TRPV1 have not yet been reported on. We explored the effects of AM404 on recombinant human TRPV1 and in rodent dorsal root ganglion (DRG) neurons. Materials and methods HEK 293 cells expressing different isoforms of recombinant TRPV1 and rodent DRG neurons were employed for patch clamp and calcium imaging experiments. Cytotoxicity was assessed by propidium iodide and Annexin V staining on TRPV1-HEK 293 cells and with trypan blue staining on DRG neurons. Key findings AM404 activates hTRPV1 at concentrations > 1 μM and in a concentration-dependent manner. AM404 also potentiates TRPV1-mediated currents evoked by heat and anandamide. Moreover, AM404-evoked currents are potentiated by NAPQI. While the partly capsaicin-insensitive rabbit (o) TRPV1 fails to respond to AM404, AM404-sensitivity is restored by insertion of the capsaicin binding-domain of rat TRPV1 into oTRPV1. In DRG neurons, AM404-evoked calcium influx as well as cell death is mediated by TRPV1. Significance AM404 gates TRPV1 by interacting with the vanilloid-binding site, and TRPV1 is the main receptor for AM404 in DRG neurons. While direct activation of TRPV1 requires high concentrations of AM404, it is possible that synergistic effects of AM404 with further TRPV1-agonists may occur at clinically relevant concentrations. [ABSTRACT FROM AUTHOR]

Background: N-arachidonoylphenolamine (AM404), a paracetamol metabolite, is a potent agonist of the transient receptor potential vanilloid type 1 (TRPV1) and low-affinity ligand of the cannabinoid receptor type 1 (CB1). There is evidence that AM404 exerts its pharmacological effects in immune cells. However, the effect of AM404 on the production of inflammatory mediators of the arachidonic acid pathway in activated microglia is still not fully elucidated.Method: In the present study, we investigated the effects of AM404 on the eicosanoid production induced by lipopolysaccharide (LPS) in organotypic hippocampal slices culture (OHSC) and primary microglia cultures using Western blot, immunohistochemistry, and ELISA.Results: Our results show that AM404 inhibited LPS-mediated prostaglandin E2 (PGE2) production in OHSC, and LPS-stimulated PGE2 release was totally abolished in OHSC if microglial cells were removed. In primary microglia cultures, AM404 led to a significant dose-dependent decrease in the release of PGE2, independent of TRPV1 or CB1 receptors. Moreover, AM404 also inhibited the production of PGD2 and the formation of reactive oxygen species (8-iso-PGF2 alpha) with a reversible reduction of COX-1- and COX-2 activity. Also, it slightly decreased the levels of LPS-induced COX-2 protein, although no effect was observed on LPS-induced mPGES-1 protein synthesis.Conclusions: This study provides new significant insights about the potential anti-inflammatory role of AM404 and new mechanisms of action of paracetamol on the modulation of prostaglandin production by activated microglia. [ABSTRACT FROM AUTHOR]

Paracetamol is arguably the most commonly used analgesic and antipyretic drug worldwide, however its mechanism of action is still not fully established. It has been shown to exert effects through multiple pathways, some actions suggested to be mediated via N-arachidonoylphenolamine (AM404). AM404, formed through conjugation of paracetamol-derived p-aminophenol with arachidonic acid in the brain, is an activator of the capsaicin receptor, TRPV1 and inhibits the reuptake of the endocannabinoid, anandamide, into postsynaptic neurons, as well as inhibiting synthesis of PGE2 by COX-2. However, the presence of AM404 in the central nervous system following administration of paracetamol has not yet been demonstrated in humans. Cerebrospinal fluid (CSF) and blood were collected from 26 adult male patients between 10 and 211 minutes following intravenous administration of 1 g of paracetamol. Paracetamol was measured by high-performance liquid chromatography with UV detection. AM404 was measured by liquid chromatography-tandem mass spectrometry. AM404 was detected in 17 of the 26 evaluable CSF samples at 5-40 nmol·L-1. Paracetamol was measurable in CSF within 10 minutes, with a maximum measured concentration of 60 μmol·L-1 at 206 minutes. This study is the first to report on the presence of AM404 in human CSF following paracetamol administration. This may represent an important finding in our understanding of paracetamol's mechanism of action, although measured concentrations were far below the previously documented IC50 for this metabolite. [ABSTRACT FROM AUTHOR]

The development of exaggerated avoidance behavior is largely responsible for the decreased quality of life in patients suffering from anxiety disorders. Studies using animal models have contributed to the understanding of the neural mechanisms underlying the acquisition of avoidance responses. However, much less is known about its extinction. Here we provide evidence in mice that learning about the safety of an environment (i.e., safety learning) rather than repeated execution of the avoided response in absence of negative consequences (i.e., response extinction) allowed the animals to overcome their avoidance behavior in a step-down avoidance task. This process was context-dependent and could be blocked by pharmacological (3 mg/kg, s.c.; SR141716) or genetic (lack of cannabinoid CB1 receptors in neurons expressing dopamine D1 receptors) inactivation of CB1 receptors. In turn, the endocannabinoid reuptake inhibitor AM404 (3 mg/kg, i.p.) facilitated safety learning in a CB1-dependent manner and attenuated the relapse of avoidance behavior 28 days after conditioning. Safety learning crucially depended on endocannabinoid signaling at level of the hippocampus, since intrahippocampal SR141716 treatment impaired, whereas AM404 facilitated safety learning. Other than AM404, treatment with diazepam (1 mg/kg, i.p.) impaired safety learning. Drug effects on behavior were directly mirrored by drug effects on evoked activity propagation through the hippocampal trisynaptic circuit in brain slices: As revealed by voltage-sensitive dye imaging, diazepam impaired whereas AM404 facilitated activity propagation to CA1 in a CB1-dependent manner. In line with this, systemic AM404 enhanced safety learning-induced expression of Egr1 at level of CA1. Together, our data render it likely that AM404 promotes safety learning by enhancing information flow through the trisynaptic circuit to CA1. [ABSTRACT FROM AUTHOR]

Changes in rhythmic activity can serve as early biomarkers of pathological alterations, but it remains unclear how different types of rhythmic activity are altered during neurodegenerative processes. Glutamatergic neurotoxicity, evoked by kainic acid (KA), causes hyperexcitation and acute seizures that result in delayed brain damage. We employed wide frequency range (0.1–300 Hz) local field potential recordings in guinea pigs to study the oscillatory activity of the hippocampus, entorhinal cortex, medial septum, and amygdala in healthy animals for three months after KA introduction. To clarify whether the activation of endocannabinoid (eCB) system can influence toxic KA action, AM404, an eCB reuptake inhibitor, and URB597, an inhibitor of fatty acid amide hydrolase, were applied. The cannabinoid CB1 receptor antagonist AM251 was also tested. Coadministration of AM404 or URB597 with KA reduced acute behavioral seizures, but electrographic seizures were still registered. During the three months following KA injection, various trends in the oscillatory activities were observed, including an increase in activity power at all frequency bands in the hippocampus and a progressive long-term decrease in the medial septum. In the KA- and KA/AM251-treated animals, disturbances of the oscillatory activities were accompanied by cell loss in the dorsal hippocampus and mossy fiber sprouting in the dentate gyrus. Injections of AM404 or URB597 softened alterations in electrical activity of the brain and prevented hippocampal neuron loss and synaptic reorganization. Our results demonstrate the protective potential of the eCB system during excitotoxic influences. [ABSTRACT FROM AUTHOR]

Rationale: N-(4-hydroxyphenyl)-arachidonamide (AM404) is an anandamide transport inhibitor shown to reduce rewarding and relapse-inducing effects of nicotine in several animal models of tobacco dependence. However, the reinforcing/rewarding effects of AM404 are not clear. Objectives: We investigated whether AM404 maintains self-administration behavior or reinstates extinguished drug seeking in squirrel monkeys. Methods and results: In monkeys with a history of anandamide or cocaine self-administration, we substituted injections of AM404 (1-100 μg/kg/injection). Using a 10-response, fixed-ratio schedule, self-administration behavior was maintained by AM404. Dose-response curves had inverted U shapes, with peak response rates occurring at a dose of 10 μg/kg/injection. In anandamide-experienced monkeys, we also demonstrated self-administration of another anandamide transport inhibitor VDM11. In addition to supporting self-administration, priming injections of AM404 (0.03-0.3 mg/kg) reinstated drug-seeking behavior previously reinforced by cannabinoids (∆-tetrahydrocannabinol (THC) or anandamide) or cocaine. Both AM404 self-administration behavior and reinstatement of drug seeking by AM404 were reduced by treatment with the cannabinoid CB1 receptor antagonist/inverse agonist rimonabant (0.3 mg/kg). Moreover, the reinforcing effects of AM404 were potentiated by the treatment with the fatty acid amide hydrolase (FAAH) inhibitor URB597 (0.3 mg/kg) suggesting a major role of anandamide in these effects. Finally, AM404 (0.3 mg/kg) potentiated the reinforcing effects of anandamide but not those of cocaine. Conclusions: In non-human primates, AM404 effectively reinforced self-administration behavior and induced reinstatement of drug-seeking behavior in abstinent monkeys. These effects appeared to be mediated by cannabinoid CB1 receptors. Therefore, compounds that promote actions of endocannabinoids throughout the brain by inhibiting their membrane transport may have a potential for abuse. [ABSTRACT FROM AUTHOR]

Rationale: The contribution of two major endocannabinoids, 2-arachidonoylglycerol (2-AG) and anandamide (AEA), in the regulation of fear expression is still unknown. Objectives: We analyzed the role of different players of the endocannabinoid system on the expression of a strong auditory-cued fear memory in male mice by pharmacological means. Results: The cannabinoid receptor type 1 (CB1) antagonist SR141716 (3 mg/kg) caused an increase in conditioned freezing upon repeated tone presentation on three consecutive days. The cannabinoid receptor type 2 (CB2) antagonist AM630 (3 mg/kg), in contrast, had opposite effects during the first tone presentation, with no effects of the transient receptor potential vanilloid receptor type 1 (TRPV1) antagonist SB366791 (1 and 3 mg/kg). Administration of the CB2 agonist JWH133 (3 mg/kg) failed to affect the acute freezing response, whereas the CB1 agonist CP55,940 (50 μg/kg) augmented it. The endocannabinoid uptake inhibitor AM404 (3 mg/kg), but not VDM11 (3 mg/kg), reduced the acute freezing response. Its co-administration with SR141716 or SB366791 confirmed an involvement of CB1 and TRPV1. AEA degradation inhibition by URB597 (1 mg/kg) decreased, while 2-AG degradation inhibition by JZL184 (4 and 8 mg/kg) increased freezing response. As revealed in conditional CB1-deficient mutants, CB1 on cortical glutamatergic neurons alleviates whereas CB1 on GABAergic neurons slightly enhances fear expression. Moreover, 2-AG fear-promoting effects depended on CB1 signaling in GABAergic neurons, while an involvement of glutamatergic neurons remained inconclusive due to the high freezing shown by vehicle-treated Glu-CB1-KO. Conclusions: Our findings suggest that increased AEA levels mediate acute fear relief, whereas increased 2-AG levels promote the expression of conditioned fear primarily via CB1 on GABAergic neurons. [ABSTRACT FROM AUTHOR]

Summary Status epilepticus (SE) is a medical emergency associated with a high rate of mortality if not treated promptly. Exogenous and endogenous cannabinoids have been shown to possess anticonvulsant properties both in vivo and in vitro. Here we study the influence of endocannabinoid metabolism on the development of kainic acid-induced SE in guinea pigs. For this purpose, the inhibitors of endocannabinoid transport, AM404, and enzymatic (fatty acid amide hydrolase) degradation, URB597, were applied. Cannabinoid CB1 receptor antagonist, AM251, was also tested. Animal behavior as well as local electric field potentials in four structures: medial septum, hippocampus, entorhinal cortex and amygdala were analyzed when AM404 (120 nmol), URB597 (4.8 nmol) or AM251 (20 nmol) were administrated alone or together with 0.4 μg of kainic acid. All substances were injected i.c.v. AM404, URB597 or AM251 administered alone did not alter markedly local field potentials of all four studied structures in the long-term compared with their basal activity. AM404 and URB597 significantly alleviated kainic acid-induced SE, decreasing behavioral manifestations, duration of seizure events and SE in general without changing the amplitude of local field potentials. AM251 did not produce distinct effects on SE in terms of our experimental paradigm. There was no apparent change of the seizure initiation pattern when kainic acid was coadministrated with AM404, URB597 or AM251. The present study provides electrophysiologic and behavioral evidences that inhibition of endocannabinoid metabolism plays a protective role against kainic acid-induced SE and may be employed for therapeutic purposes. Further investigations of the influences of cannabinoid-related compounds on SE genesis and especially epileptogenesis are required. [ABSTRACT FROM AUTHOR]

Itch and pain are two irritating sensations sharing a lot in common. Considering the antinociceptive effects of blockade of endocannabinoid degrading enzymes in pain states, we attempted to reduce scratching behavior by endocannabinoid modulation, i.e. by inhibiting fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL), or cellular uptake of endocannabinoids. Scratching behavior was induced by intradermal injection of serotonin to Balb/c mice. URB597 (10 mg/kg, i.p.), a FAAH inhibitor, JZL184 (16 mg/kg, i.p.), a MAGL inhibitor, and AM404 (10 mg/kg, i.p.), an endocannabinoid transport inhibitor, were given to evaluate the effects of endocannabinoid modulation on scratching responses. Then, the CB1 receptor antagonist, AM251 (1 mg/kg, i.p.), and the CB2 receptor antagonist, SR144528 (1 mg/kg, i.p.), were administered to determine whether cannabinoid receptors mediate these effects. URB597 and JZL184, but not AM404, attenuated serotonin-induced scratches. The inhibitory effect of URB597 was reversed by SR144528, but cannabinoid receptor antagonists had no other effects on modulation by the inhibitors. We propose that augmenting the endocannabinoid tonus by inhibition of degradative enzymes, FAAH and MAGL, but not cellular uptake, may be a novel target for the development of antipruritic agents. [ABSTRACT FROM AUTHOR]

N -arachidonoylphenolamine (AM404), a paracetamol lipid metabolite, is a modulator of the endocannabinoid system endowed with pleiotropic activities. AM404 is a dual agonist of the Transient Receptor Potential Vanilloid type 1 (TRPV1) and the Cannabinoid Receptor type 1 (CB 1 ) and inhibits anandamide (AEA) transport and degradation. In addition, it has been shown that AM404 also exerts biological activities through TRPV1- and CB 1 -independent pathways. In the present study we have investigated the effect of AM404 in the NFAT and NF-κB signaling pathways in SK-N-SH neuroblastoma cells. AM404 inhibited NFAT transcriptional activity through a CB 1 - and TRPV1-independent mechanism. Moreover, AM404 inhibited both the expression of COX-2 at transcriptional and post-transcriptional levels and the synthesis of PGE 2 . AM404 also inhibited NF-κB activation induced by PMA/Ionomycin in SK-N-SH cells by targeting IKKβ phosphorylation and activation. We found that Cot/Tlp-2 induced NFAT and COX-2 transcriptional activities were inhibited by AM404. NFAT inhibition paralleled with the ability of AM404 to inhibit MMP-1, -3 and -7 expression, cell migration and invasion in a cell-type specific dependent manner. Taken together, these data reveal that paracetamol, the precursor of AM404, can be explored not only as an antipyretic and painkiller drug but also as a co-adjuvant therapy in inflammatory and cancer diseases. [ABSTRACT FROM AUTHOR]

The endocannabinoid (eCB) system, consisting of eCB ligands and the type 1 cannabinoid receptor (CB1R), subserves retrograde, activity-dependent synaptic plasticity in the brain. eCB signaling occurs 'on-demand,' thus the processes regulating synthesis, mobilization and degradation of eCBs are also primary mechanisms for the regulation of CB1R activity. The eCBs, N-arachidonylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), are poorly soluble in water. We hypothesize that their aqueous solubility, and, therefore, their intracellular and transcellular distribution, are facilitated by protein binding. Using in silico docking studies, we have identified the nonspecific lipid binding protein, sterol carrier protein 2 (SCP-2), as a potential AEA binding protein. The docking studies predict that AEA and AM404 associate with SCP-2 at a putative cholesterol binding pocket with ∆G values of −3.6 and −4.6 kcal/mol, respectively. These values are considerably higher than cholesterol (−6.62 kcal/mol) but consistent with a favorable binding interaction. In support of the docking studies, SCP-2-mediated transfer of cholesterol in vitro is inhibited by micromolar concentrations of AEA; and heterologous expression of SCP-2 in HEK 293 cells increases time-related accumulation of AEA in a temperature-dependent fashion. These results suggest that SCP-2 facilitates cellular uptake of AEA. However, there is no effect of SCP-2 transfection on the cellular accumulation of AEA determined at equilibrium or the IC50 values for AEA, AM404 or 2-AG to inhibit steady state accumulation of radiolabelled AEA. We conclude that SCP-2 is a low affinity binding protein for AEA that can facilitate its cellular uptake but does not contribute significantly to intracellular sequestration of AEA. [ABSTRACT FROM AUTHOR]

Multiple studies suggest a pivotal role of the endocannabinoid system in the regulation of the reinforcing effects of various substances of abuse. Different approaches have been used to modulate endocannabinoid neurotransmission including the use of endogenous cannabinoid anandamide reuptake inhibitors. Previously, the effects of one of them, N-(4-hydroxyphenyl)-arachidonamide (AM404), have been explored in rodents trained to self-administer ethanol and heroin, producing some promising results. Moreover, AM404 attenuated the development and reinstatement of nicotine-induced conditioned place preference (CPP). In this study, we used the nicotine intravenous self-administration procedure to assess the effects of intraperitoneal administration of 0, 1, 3 and 10 mg/kg AM404 on nicotine-taking and food-taking behaviors under fixed-ratio and progressive-ratio schedules of reinforcement, as well as on reinstatement of nicotine-seeking induced by nicotine priming and by presentation of nicotine-associated cues. The ability of AM404 to produce place preference was also evaluated. AM404 did not produce CPP and did not modify nicotine-taking and food-taking behaviors. In contrast, AM404 dose-dependently attenuated reinstatement of nicotine-seeking behavior induced by both nicotine-associated cues and nicotine priming. Our results indicate that AM404 could be a potential promising therapeutic option for the prevention of relapse to nicotine-seeking in abstinent smokers. [ABSTRACT FROM PUBLISHER]

Abstract: Diabetes is often associated with painful neuropathy. The current treatments are symptomatic and ineffective. Cannabinoids have been proposed as promising drugs for chronic pain treatment and its antinociceptive effect has already been related in nerve injury models of neuropathic pain, but little has been investigated in painful diabetic neuropathy models. Thus, the current study aims to investigate the potential antinociceptive effect of drugs that alter endocannabinoid system when injected subcutaneously into the dorsal surface of the ipsilateral hind paw in chemical hyperalgesia induced by formalin in both normoglycemic (Ngl) and streptozotocin-diabetic (Dbt) rats. Diabetic rats exhibited exaggerated flinching behaviors during first and second phases of the formalin test, indicating the presence of hyperalgesia. AM404, an anandamide (AEA) re-uptake inhibitor, AEA (an agonist of CB1/CB2 receptors) or ACEA (a selective CB1 receptor agonist) induced antinociception in both phases of formalin test in Ngl and Dbt rats. In both groups, the antinociceptive effect of ACEA was prevented by AM251, a CB1 inverse agonist while the antinociceptive effect of AEA was prevented by AM251 or AM630, a CB2 receptor antagonist. In Ngl rats, the antinociceptive effect of AM404 was prevented by AM251 or capsazepine only during first phase of the formalin test while in Dbt rats, this effect was blocked by pretreatment with AM251 (both phases) or AM630 (second phase). Taken together, these results demonstrated broad-spectrum antinociceptive properties of cannabinoids in a model of painful diabetic neuropathy. Peripheral activation of both cannabinoid receptors seems to mediate the antinociceptive effect of exogenous or endogenous anandamide. [Copyright &y& Elsevier]

Summary: Anandamide, an endogenous agonist of CB1 receptors, also activates TRPV1 but at a higher concentration. Studies demonstrate the anticonvulsant activity of anandamide via CB1 receptors, while its action through TRPV1 is still ambiguous. Thus, the present study investigated the influence of anandamide on pentylenetetrazole-induced seizures in mice pretreated with TRPV1 or CB1 receptor antagonists. Acute intracerebroventricular administration of low doses of anandamide (10, 20, or 40μg/mouse) produced anticonvulsant effect, while the pro-convulsant effect was evident at high doses (80 or 100μg/mouse). Interestingly, AM251 (2μg/mouse), a CB1 antagonist pretreatment blocked the anticonvulsant effect, but augmented the pro-convulsant effect. Conversely, in the presence of inactive dose of capsazepine (1μg/mouse), a TRPV1 antagonist, anandamide exhibited significant anticonvulsant effect even at high doses with no change in its anticonvulsant effect. Moreover, mice treated with capsaicin, a TRPV1 agonist (10, or 100μg/mouse) exhibited pro-convulsant activity that was blocked by capsazepine pretreatment. However, capsazepine, per se at doses 10 or 100μg/mouse exhibited anticonvulsant effect. Like anandamide, the agents (AM404 and URB597), which increase its synaptic concentrations produced similar biphasic effects. Thus, these results indicate that anandamide exhibits both pro- and anticonvulsant activities by activating TRPV1 and CB1 receptor respectively. [ABSTRACT FROM AUTHOR]

Abstract: Activation of cannabinoid CB1 receptor is shown to inhibit marble-burying behavior (MBB), a behavioral model for assessing obsessive-compulsive disorder (OCD). Anandamide, an endogenous agonist at CB1 receptor also activates the transient receptor potential vanilloid type 1 (TRPV1) channels but at a higher concentration. Furthermore, anandamide-mediated TRPV1 effects are opposite to that of the CB1 receptor. Therefore, the present study was carried out to investigate the influence of low and high doses of anandamide on MBB in CB1 and TRPV1 antagonist pre-treated mice. The results revealed that i.c.v. administration of lower doses of anandamide (1–10μg/mouse) or its analogues (AM404 or URB597; 1–5μg/mouse) inhibited MBB indicating the anticompulsive activity. Conversely, at higher doses (40 or 20μg/mouse) these compounds increased MBB similar to capsaicin (TRPV1 agonist, 100μg/mouse) exhibiting a pro-compulsive effect. Pretreatment with AM251 (CB1 antagonist, 1μg/mouse) antagonized the anticompulsive effect of these compounds, while their pro-compulsive effect at higher doses was attenuated by inactive dose of capsazepine (TRPV1 antagonist, 10μg/mouse). However, capsazepine per se at a higher dose (100μg/mouse) inhibited MBB. When given daily for 14 days, the anticompulsive effect of anandamide and its analogues gradually disappeared, whereas capsazepine either alone or with URB597 produced consistent inhibition of MBB comparable to fluoxetine. Thus, the study indicates the biphasic influence of anandamide on MBB, and chronic administration of capsazepine either alone or with URB597 might be an effective tool in the treatment of OCD. [Copyright &y& Elsevier]

Abstract: Endocannabinoid analogues exhibit antidepressant and anti-compulsive like effects similar to that of serotonin selective reuptake inhibitors (SSRIs) indicating a parallelism between the effects of serotonin and endocannabinoids. Therefore, the present study was designed to investigate the role of endocannabinoids in the antidepressant and anti-compulsive like effect of fluoxetine using mice model of forced swim test (FST) and marble-burying behavior (MBB). The results revealed that intracerebroventricular injections of endocannabinoid analogues, anandamide, a CB1 agonist (AEA: 1–20μg/mouse); AM404, an anandamide transport inhibitor (0.1–10μg/mouse); and URB597, a fatty acid amide hydrolase inhibitor (0.05–10μg/mouse) produced antidepressant-like effect dose-dependently, whereas influenced the MBB in a biphasic manner (produced a U-shaped dose–response curve). Fluoxetine (2.5–20mg/kg, i.p.) dose dependently decreased the immobility time as well as burying behavior. Co-administration of sub-effective dose of fluoxetine (2.5mg/kg, i.p.) potentiated the effect of sub-effective dose of AEA (0.5μg/mouse, i.c.v.), AM404 (0.05μg/mouse, i.c.v) or URB597 (0.01μg/mouse, i.c.v) in both the paradigms. Interestingly, pretreatment with AM251, a CB1 antagonist, blocked the effect of fluoxetine in FST and MBB at a dose (1μg/mouse, i.c.v) that per se had no effect on either parameter. Similar effects were obtained with endocannabinoid analogues in AM251 pretreated mice. However, AM251 increased the burying behavior in MBB at a highest dose tested (5μg/mouse). None of the treatments had any influence on locomotor activity. Thus, the study indicates an interaction between endocannabinoid and serotonergic system in regulation of depressive and compulsive-like behavior. [Copyright &y& Elsevier]