European Journal of Medicinal Chemistry 213 (2021) 113042
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper Paracetamol analogues conjugated by FAAH induce TRPV1-mediated antinociception without causing acute liver toxicity
Johan L. Å.Nilsson a, Christophe Mallet b, c, Kiseko Shionoya d, Anders Blomgren a, Anders P. Sundin e, Lars Grundemar a, Ludivine Boudieu b, c, Anders Blomqvist d, * Alain Eschalier b, c, Ulf J. Nilsson e, Peter M. Zygmunt f, a Division of Clinical Chemistry and Pharmacology, Department of Laboratory Medicine, Lund University, Box 117, SE-221 00, Lund, Sweden b Universit e Clermont Auvergne, INSERM, NEURO-DOL Basics & Clinical Pharmacology of Pain, F-63000, Clermont-Ferrand, France c ANALGESIA Institute, Faculty of Medicine, F-63000, Clermont-Ferrand, France d Division of Neurobiology, Department of Biomedical and Clinical Sciences, Linkoping€ University, SE-581 85, Linkoping,€ Sweden e Centre for Analysis and Synthesis, Department of Chemistry, Lund University, 221 00, Lund, Sweden f Department of Clinical Sciences Malmo,€ Lund University, SE-214 28, Malmo,€ Sweden article info abstract
Article history: Paracetamol, one of the most widely used pain-relieving drugs, is deacetylated to 4-aminophenol (4-AP) Received 17 September 2020 that undergoes fatty acid amide hydrolase (FAAH)-dependent biotransformation into N-arach- Received in revised form idonoylphenolamine (AM404), which mediates TRPV1-dependent antinociception in the brain of ro- 3 November 2020 dents. However, paracetamol is also converted to the liver-toxic metabolite N-acetyl-p-benzoquinone Accepted 16 November 2020 imine already at therapeutic doses, urging for safer paracetamol analogues. Primary amine analogues Available online 21 November 2020 with chemical structures similar to paracetamol were evaluated for their propensity to undergo FAAH- dependent N-arachidonoyl conjugation into TRPV1 activators both in vitro and in vivo in rodents. The Keywords: Paracetamol antinociceptive and antipyretic activity of paracetamol and primary amine analogues was examined with Acetaminophen regard to FAAH and TRPV1 as well as if these analogues produced acute liver toxicity. 5-Amino-2- TRP channel methoxyphenol (2) and 5-aminoindazole (3) displayed efficient target protein interactions with a TRPV1 dose-dependent antinociceptive effect in the mice formalin test, which in the second phase was FAAH dependent on FAAH and TRPV1. No hepatotoxicity of the FAAH substrates transformed into TRPV1 ac- Analgesic tivators was observed. While paracetamol attenuates pyrexia via inhibition of brain cyclooxygenase, its Pain antinociceptive FAAH substrate 4-AP was not antipyretic, suggesting separate mechanisms for the Fever antipyretic and antinociceptive effect of paracetamol. Furthermore, compound 3 reduced fever without a brain cyclooxygenase inhibitory action. The data support our view that analgesics and antipyretics without liver toxicity can be derived from paracetamol. Thus, research into the molecular actions of paracetamol could pave the way for the discovery of analgesics and antipyretics with a better benefit-to- risk ratio. © 2020 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
1. Introduction crisis urging for the discovery of new analgesics. Paracetamol (acetaminophen) is one of the most used fever and pain-relieving The pharmacological options for analgesic therapy are limited drugs worldwide and its consumption increases [1,2]. It is with only a few distinct drug classes on the market. For many currently recommended as first-line pharmacological therapy for chronic pain conditions the analgesic treatments do not offer many pain conditions, although some observations show that adequate pain relief and are often associated with adverse effects paracetamol has a limited efficacy, particularly in several chronic that limit a long-term use. Today, this is highlighted by the opioid painful conditions including osteoarthritis [3e6] and low back pain [3,7]. Variation in human genotype may also contribute to the variable effectiveness of paracetamol as an analgesic [8]. Further- more, paracetamol is converted into the liver-toxic metabolite N- * Corresponding author. acetyl-p-benzoquinone imine (NAPQI) already at therapeutic doses, E-mail address: [email protected] (P.M. Zygmunt). https://doi.org/10.1016/j.ejmech.2020.113042 0223-5234/© 2020 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). J.L. Å.Nilsson, C. Mallet, K. Shionoya et al. European Journal of Medicinal Chemistry 213 (2021) 113042
Abbreviations
4-AP 4-aminophenol ALT alanine aminotransferase AM404 N-arachidonoylphenolamine AST aspartate aminotransferase COX cyclooxygenase FAAH fatty acid amide hydrolase HMBA 4-(aminomethyl-2-methoxyphenol) LPS lipopolysaccharide NAPQI N-acetyl-p-benzoquinone imine PG prostaglandin TRP transient receptor potential TRPV1 transient receptor potential vanilloid 1 TX thromboxane
causing hepatotoxicity, a well-known adverse effect of the drug [9e11] making paracetamol the principal cause of acute liver failure in the Western world [12e15]. In a series of publications, we have developed a conceptual framework for the antinociceptive action of paracetamol, involving fatty acid amide hydrolase (FAAH) and TRPV1 [16e20]. Accordingly, paracetamol as a prodrug undergoes hepatic deacetylation into 4- aminophenol (4-AP) followed by conjugation of 4-AP with arach- idonic acid through a FAAH-dependent mechanism in the brain yielding N-arachidonoylphenolamine (AM404). While the endog- enous role of FAAH mainly is to hydrolyse anandamide and related N-acylethanolamines [21], it can also synthesize N-arachidonoyl conjugates, including AM404 and arvanil [16e18]. Activation of TRPV1 in the brain by AM404 and arvanil induces antinociception, and previous reports have demonstrated that modulation of supraspinal FAAH and TRPV1 activity affects nociceptive signaling as well as that FAAH and TRPV1 are critical for the antinociceptive efficacy of paracetamol in mice [17e20,22e26]. It has also been shown that AM404 inhibits cyclooxygenase (COX)-2 [16,27], which could explain the antipyretic effect of paracetamol. Noteworthy, AM404 has not only been identified in the brain of rodents, but also in human cerebrospinal fluid after paracetamol administration [28]. Furthermore, there is genetic evidence that TRPV1 is involved in the analgesic action of paracetamol in humans [8]. The objective of this study was to understand the structure- activity relationship of paracetamol and primary amine analogues that undergo a FAAH-dependent biotransformation into N-arach- idonoyl conjugates (Fig. 1) mediating a TRPV1-induced anti- nociception, and to examine if these analogues could produce antinociception without causing acute liver toxicity. The antipyretic Fig. 1. The chemical structures of the primary amines 4-aminophenol (4-AP), 4- effect of paracetamol and analogues was also examined. (aminomethyl)-2-methoxyphenol (HMBA), 4-amino-2-methoxyphenol (1), 5-amino- 2-methoxyphenol (2), 5-aminoindazole (3), 5-aminomethyl indazole (4) and their 2. Methods corresponding N-arachidonoyl conjugates. Shown is also the structure of the proto- typic TRPV1 activator capsaicin.
2.1. Drugs
4-Aminophenol, 4-(aminomethyl)-2-methoxyphenol (HMBA), methyl arachidonyl fluorophosphonate (MAFP) and 30-carbamoyl- 4-amino-2-methoxyphenol (1), 5-amino-2-methoxyphenol (2), 5- 6-hydroxy-[1,10-biphenyl]-3-ylcyclohexylcarbamate (URB937) aminoindazole (3), 5-aminomethyl indazole (4), paracetamol, were purchased from Cayman Chemicals (Ann Arbor, MI, USA). N- u capsazepine, ibuprofen and N -nitro-L-arginine and phenylephrine (4-hydroxy-3-methoxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide were purchased from Sigma-Aldrich (St Louis, MO, USA). Indo- (1aa; conjugate of 4-amino-2-methoxyphenol) and N-(3-hydroxy- ® methacin (Confortide ) was obtained from Dumex (Copenhagen, 4-methoxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (2aa; conju- Denmark). N-arachidonoyl conjugates: N-(4-hydroxyphenyl)- gate of 5-amino-2-methoxyphenol) were purchased from Bioduro 5Z,8Z,11Z,14Z-eicosatetraenamide (AM404), N-(4-hydroxyphenyl)- (San Diego, CA, USA). N-(1H-indazol-5-yl) 5Z,8Z,11Z,14Z-eicosate- 5Z,8Z,11Z,14Z-eicosatetraenamide-d4 (AM404-d4), N-(4-hydroxy- traenamide (3aa; conjugate of 5-aminoindazole) and N-(1H- 3-methoxybenzyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (arvanil),
2 J.L. Å.Nilsson, C. Mallet, K. Shionoya et al. European Journal of Medicinal Chemistry 213 (2021) 113042 indazol-5-ylmethyl) 5Z,8Z,11Z,14Z-eicosatetraenamide (4aa; con- were dissected and mounted in organ baths between two force jugate of 5-aminomethyl indazole) were synthesised by Red Glead sensitive wires at a tension corresponding to a blood pressure of Discovery AB (Lund, Sweden). All N-arachidonoyl conjugates were 90 mm Hg. The surrounding buffer was composed of a physiological dissolved in ethanol. Primary amines were dissolved in physiolog- salt solution (NaCl 119, KCl 4.6, CaCl2 1.5, MgCl2 1.2, NaHCO3 15, ical saline, occasionally supplemented with 5% DMSO. Intraperito- NaH2PO4 1.2 and D-glucose 6 (mM); pH 7.4) which was 1 neal injections were given in volumes of 5e20 ml kg . temperature-controlled (37 C) and aerated with 95% O2 and 5% Capsazepine and URB937 were dissolved in DMSO/saline (1:9) for CO2. To create an environment in which phenylephrine (dissolved in vivo use. Drug solutions were prepared immediately before use. in saline) induces stable and long-lasting contractions all experi- ments were performed in the presence of indomethacin (10 mM) 2.2. Computational studies and Nu-nitro-L-arginine (100 mM), both dissolved in saline, to diminish the production of COX- and nitric oxide synthase-derived All molecular modelling was performed using the Schrodinger€ vasodilators, respectively. After submaximal vasoconstriction 2018 software suite (Schrodinger€ Release 2017-3, Schrodinger,€ LLC, mediated by phenylephrine (3 mM), the arteries were exposed to N- New York, NY, 2017). The model of FAAH was constructed based on arachidonoyl conjugates (dissolved in ethanol) to estimate their electron diffraction data (X-ray, PDB: 3PR0, 2.2 Å), whereas the sensory-nerve mediated vasodilator properties in the absence and model of TRPV1 was constructed from a cryo-EM structure (PDB: presence of the TRPV1 antagonist capsazepine (3 mM) dissolved in 5IRZ, 3.28 Å) [29,30]. Protein structures for docking simulations ethanol. The final concentration of ethanol reached 1% and does not were prepared using Maestro’s Protein Preparation Wizard. The affect blood vessel contractile properties [16,18,34]. Changes in the corresponding N-arachidonoyl conjugates of 4-AP (i.e. AM404), and tension of vessels were registered by the force-displacement of the paracetamol analogues 5-amino-2-methoxyphenol (com- transducer model FT03C (Grass Instruments; Rhode Island, USA). pound 2) and 5-aminoindazole (compound 3) were manually placed in the capsaicin-binding pocket of TRPV1, and FAAH models 2.5. Evaluation of FAAH-mediated conjugation in vitro of bound N-arachidonoyl conjugates were constructed by super- þ þ imposing each conjugate with anandamide covalently bound to Brains from FAAH /- and FAAH / mice were removed and FAAH. These procedures were followed by an energy minimization frozen in liquid nitrogen and homogenized in 4 ml ice-cold 10 mM with the OPLS-3 force field and the GB/SA solvation method for Tris buffer supplemented with 1 mM EDTA and 0.3 mM ascorbic water. acid. The homogenate was divided into aliquots of 200 ml and preheated to 37 C prior to addition of primary amines (0.1 mM). 2.3. Animals The samples were incubated at 37 C under stirring for 20 min before the reaction was stopped by adding 1 ml of ice-cold acetone All experiments were approved by the Linkoping€ and Lund containing an internal standard (AM404-d4). The conjugate con- Animal Ethic Committees (ethical permission No. 44/13 (ID1854), tent of samples was determined using the LC-MS/MS technique M48e13, M 49e134818/2017, 44-13; ID 1854) as well as the local (see below). For details on methodology, see Refs. [16e18]. French ethical committees (CEMEA Auvergne, France, ethical permission No. 02175.02). Experiments were performed according 2.6. Evaluation of FAAH-mediated conjugation in vivo to European legislation (Directive 2010/63/EU) on the protection of þ þ animals used for scientific purposes, and complied with the rec- FAAH /- and FAAH / mice were injected intraperitoneally (i.p.) ommendations of the International Association for the Study of with the primary amines (100 or 300 ml kg 1) dissolved in saline Pain [31]. The design, analysis and reporting of the research com- (occasionally supplemented with 5% DMSO). The animals were plied with the ARRIVE guidelines [32]. All efforts were made to anesthetized with isoflurane and decapitated 20 min after the in- minimize discomfort and use as few animals as possible. Male jection. The brains were removed and promptly frozen in liquid Wistar rats (200e250 g) were purchased from Charles River Lab- nitrogen, and stored at 80 C. They were homogenized in 2 ml ice- oratories (RRID:RGD_10395233, Sulzfeld, Germany). FAAH /- and cold 10 mM Tris buffer, which had been supplemented with 1 mM þ þ FAAH / mice, originally generated by Benjamin Cravatt [33] and EDTA and 0.3 mM ascorbic acid and 10 mM of the FAAH inhibitor back-crossed with C57BL/6 mice for at least ten generations in our MAFP to prevent N-arachidonoyl formation and degradation post laboratory, were obtained from our own homozygous breeding for mortem. To determine N-arachidonoyl conjugate levels, aliquots of 2e3 generations. To minimize the number of animals used for the 200 ml homogenate were precipitated with 1 ml ice-cold acetone in vitro and in vivo FAAH metabolic studies, both male (25e35 g) containing 0.3 mM ascorbic acid, 5 nM AM404-d4 (Cayman and female (18e25 g) mice were used as previous studies have not Chemicals) and 10 nM PGE1-d4 (Cayman Chemicals), as internal revealed any difference between sex with regard to such studies standards, and analyzed by the LC-MS/MS technique (see below). [16e18]. For behavioural and temperature recording studies, eight- For details on methodology, see Refs. [16e18]. week-old male C57BL/6 mice were obtained from Janvier Labora- tories and (RRID:MGI:5657942, Le Genest-Saint-Isle, France). All 2.7. Lipopolysaccharide-induced fever and prostanoid levels in vivo mice were kept 4e6 per cage at specific pathogen free facilities and fed ad libitum under controlled conditions with a 12 h light-dark Lipopolysaccharide (LPS), 100 mgkg 1 i.p. from Escherichia coli cycle. At the end of the experiment animals were euthanized by (O111:B4; Sigma-Aldrich, St. Louis, MO), or saline was administered 1 CO2 inhalation. For liver toxicity studies, eight-week old male 3 h prior to i.p. injection of paracetamol (100 and 150 mg kg ), 4- C57BL/6 mice were obtained from Charles River Laboratories AP (30 mg kg 1), 3 (25 mg kg 1), or their vehicle [Ringer’s acetate þ (RRID:RGD_10395233, Sulzfeld, Germany). solution, Fresenius Kabi AB, Uppsala, Sweden (Na 131 mM, Cl þ þ þ 112 mM, acetate 30 mM, K 4 mM, Ca2 2 mM, Mg2 1 mM)]. Forty 2.4. TRPV1-mediated vasorelaxation minutes after the injection, the animals were anesthetized with isoflurane and decapitated. Blood was collected from carotid ar- The TRPV1 potency of N-arachidonoyl conjugates was assessed teries into heparin tubes containing sodium citrate and the COX- using mesenteric arteries from rats by recording the nerve- inhibitor ibuprofen. The brain was removed and the dienceph- mediated vasorelaxation [16,18,34]. In short, arterial segments alon, including the hypothalamus (considered to be the site of the
3 J.L. Å.Nilsson, C. Mallet, K. Shionoya et al. European Journal of Medicinal Chemistry 213 (2021) 113042