Journal of Pain Research Dovepress open access to scientific and medical research Open Access Full Text Article ORIGINAL RESEARCH First evidence of the conversion of paracetamol WR$0LQKXPDQFHUHEURVSLQDOÁXLG This article was published in the following Dove Press journal: Journal of Pain Research Chhaya V Sharma,1 Jamie H Abstract: Paracetamol is arguably the most commonly used analgesic and antipyretic drug Long,2 Seema Shah,1 Junia worldwide, however its mechanism of action is still not fully established. It has been shown to Rahman,1 David Perrett,3 exert effects through multiple pathways, some actions suggested to be mediated via N-arachi- Samir S Ayoub,4 Vivek donoylphenolamine (AM404). AM404, formed through conjugation of paracetamol-derived Mehta1 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, 1Pain & Anaesthesia Research Centre, as well as inhibiting synthesis of PGE by COX-2. However, the presence of AM404 in the cen- St Bartholomew’s and The Royal 2 London Hospitals, Barts Health NHS tral nervous system following administration of paracetamol has not yet been demonstrated in Trust, London, UK; 2Barts & The humans. Cerebrospinal fluid (CSF) and blood were collected from 26 adult male patients between London School of Medicine, Queen Mary University of London, London, 10 and 211 minutes following intravenous administration of 1 g of paracetamol. Paracetamol UK; 3BioAnalytical Science, Barts & was measured by high-performance liquid chromatography with UV detection. AM404 was The London School of Medicine, measured by liquid chromatography-tandem mass spectrometry. AM404 was detected in 17 of Queen Mary University of London, – London, UK; 4School of Health, Sport the 26 evaluable CSF samples at 5–40 nmol⋅L 1. Paracetamol was measurable in CSF within 10 and Bioscience, Medicines Research minutes, with a maximum measured concentration of 60 μmol⋅L–1 at 206 minutes. This study is Group, University of East London, London, UK the first to report on the presence of AM404 in human CSF following paracetamol administra- tion. 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 Video abstract for this metabolite. Keywords: acetaminophen, paracetamol, paracetamol: mechanism of action, AM404, N- arachidonoylphenolamine, pharmacology - theories of analgesic action Introduction Despite its discovery over a century ago and ubiquitous clinical use, the mechanism of action of paracetamol remains undetermined, although involvement of multiple central pathways has been demonstrated. These include effects on prostaglandin production,1,2 on serotonergic pathways,3,4 and more recently, there has been increasing evidence for the roles of the endocannabinoid system and TRPV1 receptor in paracetamol’s Point your SmartPhone at the code above. If you have a antinociceptive (though not its hypothermic) effects.5–8 QR code reader the video abstract will appear. Or use: http://youtu.be/0umrSpQgoeU Arachidonoyl ethanolamine (AEA, anandamide) is an endogenous cannabinoid present in the human nervous system, with demonstrated antinociceptive actions. Its actions are terminated by a two-step inactivation process beginning with its reup- Correspondence: Chhaya V Sharma take from the synaptic cleft into the postsynaptic neuron by a recently characterized Department of Anaesthesia, 4th Floor, FAAH-like anandamide transporter (FLAT).9 Inside the neuron, AEA is hydrolyzed North Tower, Royal London Hospital, by the enzyme FAAH. Paracetamol has been suggested to exert its analgesic actions Whitechapel, E1 1BB, London, UK Email [email protected] by interfering with the reuptake of AEA by using FAAH to form a novel metabolite submit your manuscript | www.dovepress.com Journal of Pain Research 2017:10 2703–2709 2703 Dovepress © 2017 Sharma et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. http://dx.doi.org/10.2147/JPR.S143500 php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Sharma et al Dovepress N-arachidonoylphenolamine (AM404) through conjugation Sample collection and storage in brain tissue of paracetamol-derived para-aminophenol with CSF and blood sample were collected into EDTA tubes, arachidonic acid.10 The consequence of this is increased AEA stored on ice, centrifuged at 1,400× g at 4°C for 15 minutes, within the synaptic cleft, producing increased cannabinoid and then frozen at -20°C until analysis. 10,11 receptor (CB1) activation. AM404 is also an activator of TRPV1, otherwise known as the capsaicin receptor.7,10 Materials Although the potential central antinociceptive role of Paracetamol reference samples were from Sigma-Aldrich Co. AM404 has been investigated in animal models, its role (St Louis, MO, USA); AM404 and palmitoylethanolamide- in humans is not clear. AM404 has been found only as a d4 (PEA-d4) were from Cayman Chemicals (Ann Arbor, metabolite of paracetamol and is not an endogenous com- MI, USA); ammonium acetate, potassium phosphate, and ponent of human or animal nervous systems.7,12 However, its EDTA were from VWR (Lutterworth, UK). Analytical grade presence in cerebrospinal fluid (CSF) following paracetamol methanol, acetonitrile, H2O, and formic acid were from Fisher administration in humans has not been reported. The purpose Scientific UK Ltd (Loughborough, UK). of this pilot study was to investigate whether AM404 is present in human CSF following a single intravenous dose Measurement of paracetamol in CSF and of paracetamol. plasma High-performance liquid chromatography (HPLC) analysis Methods was performed on a Jasco system. CSF (100 μL) was diluted Study design and patient randomization with 100 μL 50 mM ammonium acetate, pH 5.5, containing Following approval by the East London Research Ethics 183 mmol.mL–1 3-aminophenol (internal standard). One Committee (ref: 2010-019488-12), EudraCT registration (ref: hundred microliters of plasma was precipitated with 300 μL 2010-019488-12), and receipt of written informed consent acetonitrile containing 183 mmol.mL–1 3-aminophenol. from all participants, 30 American Society of Anesthesiology Extracted plasma was centrifuged at 13,000× g for 10 min- classification of perioperative risk of anesthesia I–III adult utes and 100 μL supernatant was diluted with 100 μL 50 mM male and female patients, aged between 18 and 80 years, potassium phosphate, pH 6.5. Extracts were injected (20 μL were recruited to the study. These patients were scheduled for CSF, 10 μL for plasma) and separated on a Thermo Sci- to undergo elective urological surgery under spinal anesthe- entific C18 column (100 × 4.6 mm, 5 μm). For CSF, elution sia. Exclusion criteria were any contraindication to spinal was carried out with 50 mM ammonium acetate (pH 5.5) con- anesthesia, known hypersensitivity to paracetamol or severe taining 1 mM EDTA and 5% methanol. For plasma, 50 mM hepatocellular insufficiency, patients taking paracetamol potassium phosphate (pH 6.5) containing 1 mM EDTA and in the preceding 12 hours, or pregnancy or lactation in 5% methanol was used. The column was eluted at 1 mL.min–1 women. Patients were given a single intravenous dose of 1 using a Jasco PU-980 pump. Detection was at 242 nm using g paracetamol (100 mL solution, Perfalgan; Bristol-Myers a Jasco UV-975 detector. The retention time for paracetamol Squibb Pharmaceutical Limited, Middlesex, UK) as an was 7.05 minutes and that of the internal standard was infusion over 10 minutes, and were randomized for sample 3.5 minutes. The HPLC for paracetamol was calibrated to a collection at 15, 30, or 120 minutes post-infusion, prior to six-point curve of 0–1.65 mmol⋅L–1. The matrix was PBS for their spinal anesthetic (administration to sampling interval). CSF and paracetamol-free human plasma for plasma samples. At the time of the lumbar puncture but before injection of The limit of detection for paracetamol in CSF and plasma intrathecal local anesthetic agents, CSF (5 mL) and blood were 54 nmol⋅L–1 and 200 nmol⋅L–1, respectively (S/N=3). (10 mL) samples were collected. The limits of quantitation for paracetamol in CSF and plasma Randomization was achieved using sealed envelopes were 180 nmol⋅L–1 and 600 nmol⋅L–1, respectively (S/N=10). containing the study group allocation (for sampling at 15, 30, or 120 minutes post-paracetamol administration). The sealed Measurement of AM404 in CSF and envelope was selected by a member of the research team not plasma involved in data collection or analysis.13 As outcomes of the Liquid chromatography-tandem mass spectrometry (LC- study were measured values of paracetamol and its metabo- MS/MS) analysis was performed on a Thermo Scientific lite, AM404, rather than subjective patient experience, clini- Accela system attached to a TSQ Vantage mass spectrom- cal bias was not a concern and masking was not carried out. eter. One hundred microliters of CSF was diluted with 2704 submit your manuscript | www.dovepress.com Journal of Pain Research 2017:10 Dovepress Dovepress Paracetamol metabolism to AM404 in human CSF 100 μL H2O. One hundred microliters of plasma was pre- set as follows: collision gas was argon, spray voltage was cipitated with 300 μL acetonitrile containing 329 nmol/L 3,500 V, vaporizer temperature was 300°C, sheath gas pres- internal standard PEA-d4. Extracts (10 μL) were injected sure was 60 psi, capillary temperature was 270°C, collision onto a Waters Acquity BEH phenyl column (2.1 × 50 mm, cell pressure was 1.5 mTorr, and no declustering voltage 1.7 μm), maintained at 50°C, and eluted at 0.5 mL⋅min–1.