Conversion of Acetaminophen to the Bioactive N-Acylphenolamine AM404 via Fatty Acid Amide Hydrolase-dependent Arachidonic Acid Conjugation in the Nervous System

Acetaminophen (paracetamol) is a popular domestic analgesic and antipyretic agent with a weak anti-inflammatory action and a low incidence of adverse effects as compared with aspirin and other non-steroidal anti-inflammatory drugs. Here we show that acetaminophen, following deacetylation to its primary amine, is conjugated with arachidonic acid in the brain and the spinal cord to form the potent TRPV1 agonist N-arachidonoylphenolamine (AM404). This conjugation is absent in mice lacking the enzyme fatty acid amide hydrolase. AM404 also inhibits purified cyclooxygenase (COX)-1 and COX-2 and prostaglandin synthesis in lipopolysaccharide-stimulated RAW264.7 macrophages. This novel metabolite of acetaminophen also acts on the endogenous cannabinoid system, which, together with TRPV1 and COX, is present in the pain and thermoregulatory pathways. These findings identify fatty acid conjugation as a novel pathway for drug metabolism and provide a molecular mechanism for the occurrence of the analgesic N-acylphenolamine AM404 in the nervous system following treatment with acetaminophen. Acetaminophen (paracetamol) is a popular domestic analgesic and antipyretic agent with a weak anti-inflammatory action and a low incidence of adverse effects as compared with aspirin and other non-steroidal anti-inflammatory drugs. Here we show that acetaminophen, following deacetylation to its primary amine, is conjugated with arachidonic acid in the brain and the spinal cord to form the potent TRPV1 agonist N-arachidonoylphenolamine (AM404). This conjugation is absent in mice lacking the enzyme fatty acid amide hydrolase. AM404 also inhibits purified cyclooxygenase (COX)-1 and COX-2 and prostaglandin synthesis in lipopolysaccharide-stimulated RAW264.7 macrophages. This novel metabolite of acetaminophen also acts on the endogenous cannabinoid system, which, together with TRPV1 and COX, is present in the pain and thermoregulatory pathways. These findings identify fatty acid conjugation as a novel pathway for drug metabolism and provide a molecular mechanism for the occurrence of the analgesic N-acylphenolamine AM404 in the nervous system following treatment with acetaminophen. Acetaminophen was introduced into clinical medicine more than a century ago, but its mechanism of action is still a matter of debate. The analgesic, antipyretic, and anti-inflammatory effects of non-steroidal anti-inflammatory drugs are believed to depend on their ability to inhibit COX 1The abbreviations used are: COX, cyclooxygenase; FAAH, fatty acid amide hydrolase; LPS, lipopolysaccharide; PMSF, phenylmethylsulfonyl fluoride.1The abbreviations used are: COX, cyclooxygenase; FAAH, fatty acid amide hydrolase; LPS, lipopolysaccharide; PMSF, phenylmethylsulfonyl fluoride. 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Bobrov M. Gretskaya N.M. Bezuglov V.V. De Petrocellis L. Di Marzo V. Biochem. J. 2000; 351: 817-824Crossref PubMed Scopus (316) Google Scholar, 26Guhring H. Hamza M. Sergejeva M. Ates M. Kotalla C.E. Ledent C. Brune K. Eur. J. Pharmacol. 2002; 454: 153-163Crossref PubMed Scopus (109) Google Scholar). In line with these studies, AM404 produces analgesia in the mouse formalin and hot plate tests (26Guhring H. Hamza M. Sergejeva M. Ates M. Kotalla C.E. Ledent C. Brune K. Eur. J. Pharmacol. 2002; 454: 153-163Crossref PubMed Scopus (109) Google Scholar, 27Burstein S.H. Rossetti R.G. Yagen B. Zurier R.B. Prostaglandins Other Lipid Mediat. 2000; 61: 29-41Crossref PubMed Scopus (130) Google Scholar) and potentiates the analgesic effect of anandamide in the mouse hot plate test (13Beltramo M. Stella N. Calignano A. Lin S.Y. Makriyannis A. Piomelli D. Science. 1997; 277: 1094-1097Crossref PubMed Scopus (717) Google Scholar). Furthermore, AM404 decreases the expression of c-fos in the spinal cord of neuropathic rats, an effect inhibited by both TRPV1 and cannabinoid receptor antagonists (28Rodella L.F. Borsani E. Rezzani R. Ricci F. Buffoli B. Bianchi R. Eur. J. Pharmacol. 2005; 508: 139-146Crossref PubMed Scopus (33) Google Scholar).MATERIALS AND METHODSIn Vivo ExperimentsAcetaminophen (30–300 mg/kg), p-aminophenol (10–100 mg/kg), or vehicle (saline) at volumes of 2–3 ml (rat) or 0.25–0.3 ml (mice) were given to female Wistar-Hannover rats (female; 200–300 g) or fatty acid amide hydrolase (FAAH) gene knock-out mice (29Cravatt B.F. Demarest K. Patricelli M.P. Bracey M.H. Giang D.K. Martin B.R. Lichtman A.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9371-9376Crossref PubMed Scopus (1097) Google Scholar) and their homozygous controls (female and male; 25–30 g) by intraperitoneal injections. After 20 min, the animals were killed to collect brain, liver, spinal cord, and arterial blood. The tissues were homogenized in Tris buffer (10 mm, pH 7.6) containing EDTA (1 mm). Phenylmethylsulfonyl fluoride (PMSF; 0.1 mm) and ascorbic acid (0.3 mm) were also present in the Tris buffer and added to the blood samples to prevent degradation of fatty acid amides and p-aminophenol, respectively. Aliquots (200 μl) of blood and homogenates were precipitated with 1 ml of ice-cold acetone containing 1 μm2H8-labeled anandamide as internal standard. The samples were kept on ice until the acetone phase was vacuum evaporated.Tissue Homogenate ExperimentsThe brain, liver, spinal cord, and dorsal root ganglia from Wistar-Hannover rats (female; 200–300 g) or FAAH gene knock-out mice and their homozygous controls (female and male; 25–30 g) were homogenized in a Tris buffer (10 mm, pH 7.6) containing EDTA (1 mm) at volumes of 5–10 ml/g tissue. We carried out experiments in aliquots of 200 μl homogenate at 37 °C as further explained in the text. The reactions were stopped by adding 1 ml of ice-cold acetone containing 1 μm [2H8]anandamide. The samples were kept on ice until the acetone phase was vacuum evaporated.Rat Purified FAAH Enzyme AssayThe enzyme assay was performed in triplicate with rat purified FAAH (>95% purity), containing an N-terminal His6 tag and a truncation of amino acid residues 1–29, which constitute a predicted transmembrane domain (30Patricelli M.P. Lashuel H.A. Giang D.K. Kelly J.W. Cravatt B.F. Biochemistry. 1998; 37: 15177-15187Crossref PubMed Scopus (144) Google Scholar). The amount of FAAH protein in the assay was 100 nm. Reactions were conducted in 10 mm Tris buffer, pH 7.6, with 5 mm p-aminophenol and 100 μm arachidonic acid. Both substrate stocks were prepared in ethanol, which was not >5% of the total volume of 300 μl. The reaction was initiated by addition of FAAH or Tris buffer for controls. At each time point (0, 5, 10, 15, 20, 30, 45, and 60 min), 50 μl of the reaction was removed and quenched by addition to 300 μl of 0.1 m hydrochloric acid. It was extracted in 300 μl of ethyl acetate, which was removed and concentrated to dryness under a stream of nitrogen. The initial rate of formation of AM404 was obtained from a linear fit of time (0–20 min) versus AM404 produced.Quantitative AnalysesFor in vivo and tissue homogenate experiments, the extraction residues were reconstituted in 100 μl of methanol except for p-aminophenol, for which 100 μl of 0.5% acetic acid was used. The quantitative analyses were performed using a PerkinElmer Life Sciences 200 liquid chromatography system with autosampler (Applied Biosystems) coupled to an API 3000 tandem mass spectrometer (Applied Biosystems/MDS-SCIEX). All mobile phases were water-methanol gradients containing 0.5% acetic acid, and the flow rate was 200 μl/min except for arachidonic acid, where it was 400 μl/min. In experiments with rat purified FAAH, the residue was reconstituted in 50 μl of ethanol. The quantitative analyses were performed on a Hewlett-Packard series 1100 LC with a mass spectrometer detector.AM404 and Anandamide—Sample aliquots of 5 μl were injected on a Genesis C8 column (20 × 2.1 mm; Jones Chromatography). Initially, the mobile flow was 25% water for 5.5 min. Then a linear gradient to 100% methanol was applied in 0.2 min, and the mobile phase was kept at 100% methanol for 2.3 min, after which the column was reconditioned in 25% water for 2 min. The electrospray interface was operating in the positive ion mode at 370 °C, the ion spray voltage was 4500 volts, and the declustering potential was 40 volts. m/z 396.1/109.8 with a collision energy of 27 volts was used for the determination of AM404, whereas m/z 348.2/61.6 with a collision energy of 35 volts was used for the determination of anandamide. m/z 356.4/62.2 with a collision energy of 35 volts was used for the internal standard, 2H8-labeled anandamide. In experiments with rat purified FAAH, sample aliquots of 40 μl were injected on a Haisil C4 column (150 × 4.6 mm; Higgins Analytical, Inc.). A linear gradient elution was applied from 100% buffer A (95:5 water/methanol with 0.1% formic acid) to 100% buffer B (60:35:5 isopropanol/methanol/water with 0.1% formic acid) in 30 min. The MH+ (396.6) and MNa+ (418.6) mass peaks were extracted and integrated for quantification of AM404.p-Aminophenol—Sample aliquots of 2 μl were injected on a Genesis phenyl column (150 × 2.1 mm; Jones Chromatography). The mobile flow was initially 97% water for 2 min. Then a linear gradient to 100% methanol was applied in 1 min and the mobile phase was kept at 100% methanol for 2 min, after which the column was reconditioned in 97% water for 3 min. The electrospray ion source was set at 450 °C and used in the positive ion mode. The ion spray voltage and declustering potential were set to 4500 volts and 55 volts, respectively. m/z 109.9/64.6 with a collision energy of 31 volts was used for the quantitative determinations of p-aminophenol.Acetaminophen—Sample aliquots of 5 μl were injected on a Genesis C18 column (50 × 2.1 mm; Jones Chromatography). The mobile flow was initially 90%, and a linear gradient to 80% methanol was immediately applied in 5 min. The mobile phase was kept at 80% methanol for 1 min, after which the column was reconditioned in 90% water for 3 min. The electrospray ion source was set at 370 °C and used in the positive ion mode. The ion spray voltage and declustering potential were set to 4500 volts and 55 volts, respectively. m/z 151.9/109.8 with a collision energy of 25 volts was used for the quantitative determinations of acetaminophen.[2H8]Arachidonic Acid—Sample aliquots of 5 μl were injected on a Genesis C18 column (50 × 2.1 mm; Jones Chromatography). High performance liquid chromatography was operated isocratically at 20% water and 80% methanol. The electrospray ion source was operating in the negative ion mode at 370 °C, the ion spray voltage was –3000 volts, and the declustering potential was –120 volts. m/z 310.8/267.0 with a collision energy of –22 volts was used for the quantitative determinations.COX-1 and COX-2 AssaysCOX-1 activity and COX-2 activity were determined in the presence of 10 μm arachidonic acid using a COX (ovine) inhibitor screening assay (Cayman). Drugs were incubated with the enzyme preparation 8 min before the application of arachidonic acid. Prostaglandin formation was used as a measure of COX activity and quantified via enzyme immunoassay.COX-2 Activity in RAW264.7 MacrophagesRAW264.7 cells, obtained from American Type Culture Collection, were cultured in Dulbecco's modified Eagle's medium containing 1 mm sodium pyruvate, 1 mm non-essential amino acids, 2 mm glutamine, penicillin (100 units/ml), streptomycin (100 μg/ml), and 10% fetal calf serum (all from Sigma). Experiments were performed on confluent cells grown in 6-well Falcon plates (passages 3–6) and then exposed to lipopolysaccharide (LPS) (1 μg/ml) for 18 h (37 °C, 5% CO2) to induce COX-2. The medium was replaced with fresh medium, and test substances or vehicles (0.1% Me2SO for AM404 and NS-398; distilled water for indomethacin) were added 1 h before the cells were further exposed to arachidonic acid (1 μm) or its vehicle (0.1% Me2SO) for 1 h. Aliquots of the medium were removed, and the prostaglandin E2 content was determined using a monoclonal enzyme immunoassay kit (Cayman).Recording of TensionExperiments were performed on mesenteric arteries from female Wistar-Hannover rats (250 g) as described (31Zygmunt P.M. Petersson J. Andersson D.A. Chuang H. Sorgard M. Di Marzo V. Julius D. Högestätt E.D. Nature. 1999; 400: 452-457Crossref PubMed Scopus (1845) Google Scholar). Briefly, the arteries were cut into ring segments and mounted in tissue baths containing aerated physiological salt solution (5% CO2 and 95% O2 at 37 °C, pH 7.4). Experiments were carried out in the presence of NG-nitro-l-arginine (0.3 mm) and indomethacin (10 μm) to eliminate any contribution of nitric oxide and cyclooxygenase products, respectively. We studied relaxant responses in preparations contracted with phenylephrine. When stable contractions were obtained, substances were added cumulatively to determine concentration-response relationships.Calculations and StatisticsData are presented as means ± S.E. (vertical lines in Figs. 2, 3, 4, 5, 6), and n indicates the number of animals unless stated otherwise. GraphPad Prism 3.0 software was used for curve fitting (non-linear regressions) and calculations of pEC50 values. A Mann-Whitney U test or Student's t test on log-transformed values was used for statistical analysis. Statistical significance was accepted when p < 0.05.Fig. 3The formation of AM404 is dependent on the enzyme fatty acid amide hydrolase (FAAH). A and B, rat brain homogenates were incubated for 1 h with p-aminophenol plus arachidonic acid (each 100 μm) to generate AM404 (n = 4) (A) or [2H8]-anandamide (10 μm) to study its hydrolysis (B). Prior incubation for 1 h with the FAAH inhibitor PMSF (100 μm) prevents the production of AM404 (n = 4) and the formation of [2H8]arachidonic acid ([2H8]AA), the breakdown product of anandamide (n = 4). C, time-dependent formation of AM404 when p-aminophenol (5 mm) and arachidonic acid (100 μm) are incubated with rat purified FAAH (filled circles) but not with its vehicle (open circles; n = 3–6). D, in brain homogenates from FAAH–/– mice or their control littermates (FAAH+/+) incubated with p-aminophenol (100 μm) for 1 h (n = 5), AM404 could only be detected in FAAH+/+ mice. E, treatment of rats with PMSF (10 mg/kg subcutaneously) but not vehicle (control) for 20 min before exposure to acetaminophen (300 mg/kg intraperitoneally) for 20 min abolishes the AM404 production as measured in brain (n = 5). F, no AM404 could be detected in brains from FAAH–/– mice exposed to acetaminophen (300 mg/kg intraperitoneally) for 20 min (n = 5). G, the formation of p-aminophenol from acetaminophen (300 mg/kg intraperitoneally; 20 min) is partially inhibited in brain of rats pretreated with PMSF (10 mg/kg subcutaneously) for 20 min (n = 5). H, p-aminophenol levels were determined in liver homogenates from FAAH+/+ and FAAH–/– mice after incubation with acetaminophen (1 mm) for 1 h (n = 5). *, p < 0.05 compared with control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Acetaminophen and p-aminophenol are metabolized to AM404 by fatty acid amide hydrolase (FAAH) in the liver. A, time-dependent formation of AM404 in rat liver homogenates incubated with acetaminophen (100 μm; triangles; n = 4) or p-aminophenol (10 μm; circles; n = 8) but not in homogenates incubated with vehicle (n = 4). B and C, in rat liver homogenates, the formation of AM404 from acetaminophen (100 μm) (B) and p-aminophenol (10 μm) (C) after 1 h is inhibited by1hof pre-treatment with PMSF (100 μm)(n = 4). D and E, the formation of AM404 in mouse liver homogenates after 1 h exposure to acetaminophen (1 mm) (D) and p-aminophenol (100 μm) (E) is dependent on FAAH (n = 5).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5AM404 inhibits the production of prostaglandins. A, AM404 causes concentration-dependent inhibition of COX-1 (open circles) and COX-2 (filled circles) activity in isolated enzyme preparations (n = 4–5). Indomethacin (10 μm) and the COX-2 selective inhibitor NS-398 (10 μm) also abolished COX-1 (6 ± 0.4%, n = 4) and COX-2 (11 ± 2%, n = 6) activity, respectively (not shown). COX activity was measured as prostaglandin (PG) formation in the presence of 10 μm arachidonic acid. B, AM404 reduces the formation of prostaglandin E2 (PGE2) in RAW264.7 macrophages. Cells were exposed to LPS for 18 h to induce COX-2 activity. The medium was replaced with fresh medium, and cells were incubated with different concentrations of AM404, the selective COX-2 inhibitor NS-398, or the non-selective COX-inhibitor indomethacin (Indo) for 1 h. Arachidonic acid (AA) was then added to the medium for another 1 h, after which the prostaglandin E2 content was determined. The data are obtained from six to eight independent experiments performed in duplicate or triplicate and expressed as a percentage of the maximal prostaglandin E2 formation in LPS-stimulated cells. *, p < 0.001 compared with maximal prostaglandin E2 production.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6AM404 but neither acetaminophen nor p-aminophenol acts on native TRPV1 receptors in rat-isolated mesenteric arteries. A, AM404 is a potent vasodilator (open circles) of arterial segments contracted with phenylephrine (n = 11). The action of AM404 is inhibited by the competitive TRPV1 receptor antagonist capsazepine (3 μm; filled circles; n = 5) and the non-competitive TRPV1 receptor antagonist ruthenium red (1 μm; diamonds; n = 4). AM404 was unable to relax arteries pre-treated with capsaicin (1 μm) for 30 min (n = 4; not shown), causing desensitization and/or depletion of sensory neuropeptides (31Zygmunt P.M. Petersson J. Andersson D.A. Chuang H. Sorgard M. Di Marzo V. Julius D. Högestätt E.D. Nature. 1999; 400: 452-457Crossref PubMed Scopus (1845) Google Scholar). The broken line with triangles shows the relaxant effect of “endogenous” AM404 from rat homogenates incubated with p-aminophenol (mean of four arterial segments from the same rat). Endogenous AM404 was purified using liquid chromatography and quantified by liquid chromatography along with tandem mass spectrometry as described. Tension traces show relaxant responses to increasing concentrations of exogenous (upper trace) and endogenous (lower trace) AM404. B, concentration-response curves for capsaicin in arterial segments contracted with phenylephrine after treatment with 1 mm acetaminophen (triangles), 100 μm p-aminophenol (squares), or vehicle (circles) for 30 min (n = 5). The experiments with p-aminophenol were performed in the presence of ascorbic acid to prevent its decomposition. Representative traces showing no response to acetaminophen (AcAP) or p-aminophenol (p-AP) in arterial segments contracted with phenylephrine (n = 5). Capsaicin (CAP) always relaxed these arteries. Dashed line indicates the basal tension level before the addition of drugs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DrugsAcetaminophen, p-aminophenol, NG-nitro-l-arginine, ascorbic acid, phenylephrine, PMSF, and ruthenium red were from Sigma. [2H4]Acetaminophen (CDN Isotopes) and indomethacin (Confortid, Dumex) were dissolved in and diluted with distilled water. AM404, capsaicin, and capsazepine were from Tocris. [2H8]Anandamide, [2H8]arachidonic acid, and NS-398 were from Cayman. Anandamide (Biomol) and arachidonic acid (Sigma) were all dissolved in and diluted with ethanol. Me2SO was substituted for ethanol as a solvent in studies with RAW264.7 macrophages. The batch of acetaminophen contained either no or <0.001% (w/w) of p-aminophenol, as determined by liquid chromatography coupled with tandem mass spectrometry.RESULTSDeuterium-labeled AM404 and p-aminophenol were detected in rat brain 20 min after an intraperitoneal injection of deuterium-labeled acetaminophen (Fig. 1). We could not, however, detect the corresponding non-deuterium-labeled compounds in these animals (data not shown). The formation of AM404 and p-aminophenol from acetaminophen was dose-dependent (Table I). After the injection of 300 mg/kg acetaminophen, AM404 could be detected in the spinal cord in two of five animals but was absent in liver and blood (Fig. 2A). p-Aminophenol was present in all tissues, of which the liver contained the highest level (Fig. 2A). AM404 and p-aminophenol could not be detected in vehicle-treated animals (n = 4).Fig. 1Acetaminophen is metabolized to the primary amine p-aminophenol, which is further conjugated with arachidonic acid to form the bioactive fatty acid amide N-arachidonoyl phenolamine (AM404). Representative chromatograms of samples obtained from rat brain showing the presence of [2H4]AM404 and [2H4]p-aminophenol in animals treated with 300 mg/kg [2H4]acetaminophen for 20 min (n = 3). The tandem mass spectrometer was operated to select the protonated molecular ion of AM404 at m/z 400.1 and p-aminophenol at m/z 113.8 in the first quadrupole mass separator, whereas the mass fragments at m/z 113.8 and m/z 68.6 after fragmentation of AM404 and p-aminophenol, respectively, in the collision cell were selected by the second quadrupole.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IDose-dependent formation of AM404 and p-aminophenol in rat brain following administration (intraperitoneally) of acetaminophen or p-aminophenolnAM404aThe levels of AM404 and p-aminophenol were determined 20 min after administration of acetaminophen or p-aminophenol. AM404 and p-aminophenol were not detected in vehicle-treated animals (n = 4).np-AminophenolaThe levels of AM404 and p-aminophenol were determined 20 min after administration of acetaminophen or p-aminophenol. AM404 and p-aminophenol were not detected in vehicle-treated animals (n = 4).pmol/gnmol/gAcetaminophen30 mg/kg90.14 ± 0.0770.30 ± 0.16100 mg/kg81.6 ± 0.161.8 ± 0.2300 mg/kg1010.3 ± 1.9106.7 ± 0.9p-Aminophenol10 mg/kg53.2 ± 1.352.1 ± 0.730 mg/kg544 ± 13536 ± 7100 mg/kg5667 ± 1145531 ± 82a The levels of AM404 and p-aminophenol were determined 20 min after administration of acetaminophen or p-aminophenol. AM404 and p-aminophenol were not detected in vehicle-treated animals (n = 4). Open table in a new tab Intraperitoneal injection of p-aminophenol also led to a dose-dependent formation of AM404 in rat brain (Table I). At a dose of 100 mg/kg p-aminophenol, both AM404 (165 ± 39 pmol/g) and p-aminophenol (60 ± 8 nmol/g) were detected in the spinal cord (n = 5).Incubation with p-aminophenol (1