On the mechanism of paracetamol’s (acetaminophen's) analgesic activity and a note on NSAIDs mechanisms.
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On the mechanism of paracetamol’s (acetaminophen's) analgesic activity and a note on NSAIDs mechanisms.



A novel hypothesis is presented in this manuscript that paracetamol (or acetaminophen in the US) acts by covalently modifying several pain signaling proteins via the reactive metabolite ...

A novel hypothesis is presented in this manuscript that paracetamol (or acetaminophen in the US) acts by covalently modifying several pain signaling proteins via the reactive metabolite N-acetyl-para-benzoquinoneimine (NAPQI). Cysteine proteases (caspases and some cathepsins) are suggested to be key targets since many of these are involved in biochemical processes where pain signalling molecules, such as certain cytokins (IL-1b, IL-6, and more) are generated, An extended hypothesis states that analgesic and anti-inflammatory effects of some NSAIDs that can form reactive metabolites might receive additional (over COX inhibition) analgesic activity via analogous mechanisms.
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    On the mechanism of paracetamol’s (acetaminophen's) analgesic activity and a note on NSAIDs mechanisms. On the mechanism of paracetamol’s (acetaminophen's) analgesic activity and a note on NSAIDs mechanisms. Document Transcript

    • Alf ClaessonAwametox Consulting, Lilldalsvägen 17 A, SE-14461 Rönninge, Sweden. E-mail: alf.cl@telia.comKey words: paracetamol, acetaminophen, NAPQI, aspirin, diclofenac, indometacin, reactive metabolite, hepatotoxicity, bioactivation,NSAID.This paper should be cited as follows: Author and title of paper. Manuscript published on SlideShare.net February 4, 2013.ABSTRACT: The analgesic mechanism of action of paracetamol (acetaminophen, also known as APAP) has been the object of considerablebewilderment but the hypotheses brought forward have not reached wide recognition. A novel hypothesis is presented here that APAP acts bycovalently modifying several pain signaling proteins via the reactive metabolite N-acetyl-para-benzoquinoneimine (NAPQI). The most likelyprotein targets, it is suggested here, are cysteine proteases that take part in generation of pain signaling molecules, for example caspase-1 thatacts on proIL-1beta. An extended hypothesis states that analgesic and anti-inflammatory effects of some NSAIDs that can form reactive me-tabolites might receive additional analgesic activity via analogous mechanisms. Testing the hypothesis in vivo is difficult since the proposedmechanism consists of a great number of concurrent targets. However, it should be possible to indicate some cysteine proteases as targets invivo using radiolabeling and protein separation. Implications of the hypothesis are that the concept of exploiting reactive metabolites is a non-plausible route for design of new analgesics. Paracetamol (N-acetyl-p-aminophenol or APAP), also hydroxyphenyl)arachidonylamide (AM404) which has CB1known as acetaminophen in the US, is a widely used non- receptor-modulating properties, inhibit the enzyme fatty acidprescription drug with antipyretic and analgesic actions but amide hydrolase-1, and also interferes with the uptake ofwith very weak anti-inflammatory activity (review, [1]). It is anandamide [2]. Some authors even declared “mystery re-an old drug, first synthesized in 1878, within the acetani- solved” [3]. There was also an observation that “the blockadelides, a group that includes phenacetin and acetanilide (Fig. of cannabinoid CB1 receptors has been shown to completely1). It has an exceptionally simple structure with a molecular prevent the analgesic activity of paracetamol in rats” [3]. Itweight of only 151 Da that is less than most anesthetic gases was also suggested that AM404 would activate TRPV1 in thehave. CNS thereby evoking an analgesic activity [4]. Other claims regarding the mechanism(s) of action of APAP involve inhibition of prostaglandin synthesis via COX inhibition, in particular COX-2 [5]. In 2002 the finding of a new cyclooxygenase (COX-3), which is synthesized from RNA splice variants in canines and mice was published [6]. It was suggested that a similar enzyme might be the thera- peutic target in humans since the canine COX-3 enzyme that was studied was particularly sensitive to APAP compared with standard NSAIDs. However, other researchers have argued that the COX-3 hypothesis lacks credibility because Figure 1. Structures of and relationships between the acetanilideanalgesics. All these structures can be metabolized to the reactive of the unlikely existence of this enzyme in humans [7-9].metabolite N-acetyl-para-benzoquinoneimine (NAPQI). The ion channel TRPA1 has also been suggested as the target via metabolites of APAP. In 2011 Andersson et al. There have been much bewilderment as to the mecha- published results that might indicate antinociceptive effectsnism(s) of action of this simple compound and a few sugges- in mice of APAP, acting via its reactive metabolites NAPQItions involving the following unique mechanisms have been (Fig. 1) and benzoquinone that would activate spinal TRPA1brought forward. [10]. APAP was administered to trpa1-/- mice locally at the spinal cord (intrathecal injection) and also systemically; A relatively modern hypothesis that gained wide publicity NAPQI was administered intrathecally. The report will beis based on the proven in vivo formation from APAP of N-(4-
    • discussed below. A few other mechanisms have been men- inhibit also the many (15+) human cysteine proteases bytioned in reviews on APAP [1, 11]. reacting with their active cysteine thiol, as demonstrated for So far, none of the mechanisms can be considered proven. NAPQI with papain [15]. Cysteine proteases are key playersThe main purpose of the current paper is to present a novel in a great number of cellular processes many of which in-hypothesis of mechanism of action that is not based on spe- volve formation of molecules that have been reported tocific receptors or enzymes but which fits with all the known take part in pain signalling [16-23] and in generation of neu-pharmacology of APAP and analogues. The previous hy- ropathies (possibly via initiation of apoptosis) [24]. In fact,potheses will be discussed in this context. such proteases have been explicitly referred to in the litera- ture as potential targets for new analgesics [22, 25]. The pro- teases that have been mentioned in connection with painA novel hypothesis of mechanism of action signalling include, for example caspase-1, caspase-3, cathep- The cited works present theories on narrow mechanisms of sin B, K, L and S. It is of some interest that both oral inhibi-action of APAP. The novel hypothesis brought forward in tors and an irreversible inhibitor of microglial cathepsin Sthe current paper is, like that of Andersson et al. [10], based were claimed to be active in neuropathic pain models [26,on the formation of NAPQI and other reactive metabolites 27]. The implications of the latter finding are limited by the(RM), e.g. benzoquinone. These RMs were previously sug- intrathecal route of administration.gested [10] to elicit analgesia in mice through, somewhatsurprising, specific activation of the noxious sensor TRPA1(limited to local action at the spinal cord). The current papersuggests instead that the analgesic activity of APAP originatesfrom reactions of RMs with a larger number of proteins withmany varied physiological roles in pain signaling. The goodand the bad actions balance in slight favor toward the posi-tive side. This idea puts the mechanism of action more in Figure 2. Aspirin is metabolized to the hydroquinone gentisic ac-line with the now well recognized complexity of pain signal- id which can readily form a reactive benzoquinone metabolite.ing than earlier hypotheses and does not limit the target ofthese RMs to a single protein. In the complex world of pain signaling, it would be pre- If there is some truth to this notion of “analgesic RMs” sumptuous to try to pinpoint one particular cysteine prote-the hypothesis might be expanded also to connect the phar- ase whose systemic inhibition should entail particularly posi-macology of APAP to older reports of RMs from NSAIDs. tive effects. However, one might just speculate that the pro-For example, gentisic acid (Fig. 2), formed in vivo from aspi- teases that take part in the processing of procytokines, suchrin, might be a pain relieving mediator via its easily formed as those generating IL-1beta and IL-6, could be key targets;benzoquinone-2-carboxylic acid. The hypotheses can be they have also been mentioned as drug targets, mostly forsummarized in the following statements that will be enlarged therapies involving control of apoptosis [28]. The enzymein the following text. caspase-1, also called interleukin-1beta converting enzyme  APAP acts by covalently modifying pain signaling (ICE) is one of these, which has also been targeted in new proteins via the reactive metabolite NAPQI. drug research [29]. Patent applications also claim irreversible  The target proteins are mainly cysteine proteases. caspase-1 inhibitors as pain relieving agents[30]. An extension of the hypothesis: It might be argued that NAPQI should not be available for enzyme inhibition throughout the human body since it is  Analogous mechanisms might contribute to the generated primarily in the liver by actions of cytochrome P- analgesic and anti-inflammatory effects of some 450 enzymes and is quickly made harmless by reaction with NSAIDs. glutathione. However, several other oxidizing enzymes are NAPQI is a well-known liver and kidney toxin, which like also able to generate NAPQI (and other RMs from APAP),similar quinoid RMs, is an indiscriminate electrophile and including monophenol monooxygenase (tyrosinase), peroxi-arylating species (and is also an oxidizing agent) that react dases and COX and there are reports of proteins in thepreferentially with nucleophilic thiols in proteins or peptides periphery that have formed conjugates with APAP in vivo[12], as happens in the irritant sensor TRPA1. Reactive me- [31-33]. See also comments at references [51-52].tabolites (RMs) of drugs are notorious for causing severe A fraction usually ranging from 5 to 15% of APAP is me-side-effects such as drug-induced liver injury [13], and are tabolized via the oxidation pathway. Since the bioavailabilitythought to lie behind the hepatotoxicity of a great number of APAP is high, 60-89%, a normal therapeutic dose of 700-of drugs, many of which have been withdrawn from the 1000 mg gives rise to high plasma concentrations, in themarket, for example lumiracoxib in 2007-8 and sitaxentan in range 33-132 µM, e.g. [34]. APAP is uniformly distributed2010. The largest group of RMs is the quinoids [14] to throughout most body fluids, freely crosses the placenta andwhich also NAPQI belongs. Since these electrophilic com- penetrates the blood-brain barrier. This means that a num-pounds react preferentially with thiols they should be able to ber of extrahepatic oxidative enzymes have the possibility© 2013 Alf Claesson 2
    • to generate RMs from APAP in the system, usually with neg- This might provide a mechanistic explanation for the analge-ative consequences (for example, proposed to occur in the sic/anti-inflammatory activity of salicylic acid which is a weaklungs [35]) but maybe, for a limited set of proteins, with a COX inhibitor but yet has analgesic activity comparable withslightly positive, pain relieving outcome. Also, APAP forms aspirin [41]. Other authors have also sought explanations inp-aminophenol by hydrolysis and this can also be oxidized to this direction to try to explain the discrepancy [42]. OtherRMs. NSAIDs than the ones mentioned also form para- hydroxylated drug metabolites, en route to quinoid species, when they are so predisposed.An extrapolation of the hypothesis to certain In principle, to verify this hypothesis it should be possibleNSAIDs to differentiate the common NSAIDs based on their analge- Here, it is relevant to point to the often made observations sic activity (in relation to free plasma concentration in hu-that NSAIDs have many effects that do not primarily involve mans) plotted against COX inhibition constants. This plotprostaglandins. The point and hypothesis, related to NAPQI might reveal whether there is any correlation with propensityas the presumed explanation for the analgesic activity of to form RMs (of the quinoid class).APAP, is that some NSAIDs might receive extra analgesicactivity from similar quinoid metabolites (Fig. 2 and Fig. 4) A further point is that every reactive metabolite generated,that react with a number of proteins, other than COX, rele- be it from APAP or an NSAID, has a unique structure andvant in pain signaling. therefore should have its own spectrum of pain-relevant tar- get proteins which might confer a certain pharmacology. It The time-dependent irreversible inhibition of “the fatty ac- might not be a coincidence that the the carboxylic acid gen-id oxygenase”, i.e. COX, by aspirin and indometacin was tisic acid (via its reactive metabolite, benzoquinone-2-first reported in 1971 [36]. Some structural requirements carboxylic acid, Fig. 2) inhibits COX, possibly preferentially.for time-dependent inhibition of prostaglandin biosynthesisby anti-inflammatory drugs were published in 1975 [37] andin 1994 [38]. It was noted that the compounds ibuprofen The hypothesis in relation to earlier onesand flurbiprofen (Fig. 3) which have Ki values of 3 and 1 Regarding the proposal that APAP works via an arachidon-µM, respectively, behave differently in that only flurbiprofen ic amide metabolite there is no doubt that APAP formsgives time-dependent inhibition. The inhibition of prosta- AM404 in vivo in rats but the hypothesis of a resolved mech-glandin cyclooxygenase by gentisic acid (metabolite of salicyl- anism of action cannot be considered strong; as the authorsic acid, Fig. 2) was further studied [39]. themselves state: “.. it can only be hypothesized that AM404 Since effective NSAIDs have been shown to form RMs it is is formed in man and contributes to the pharmacologicalnot far-fetched to think that certain compounds of this class, effects of acetaminophen.” [2]. Also, it should be noted thatfor example indometacin and diclofenac, might provide ad- the high dose of 300 mg/kg of APAP to rats only led to for-ditional analgesic activity from quinoid metabolites (Fig. 4), mation of tiny amounts (< 11 pg/g) of AM404 in the brains.similar to NAPQI, that react with a number of proteins rele- One might add as a side-note that the whole concept of can-vant in pain signalling, including COX [37, 38]. As stated, nabinoids as analgesics, although having been a topic forthis might be considered an extended hypothesis from that decades, so far has failed to mature beyond the limited ap-of APAP forming analgesic NAPQI. Obviously, this is a bold plications of some compounds as adjunct pain relievers [43].speculation but it is not unreasonable based on evidence of The most recent efforts in the field have focused on fattynon-prostaglandin effects of NSAIDs. For example, diclo- acid amide hydrolase-1 (FAAH) as a target; here, PF-fenac showed a dose-dependent anti-apoptotic effect that, it 04457845 failed to show analgesic activity in patients withwas hypothesized, went via a non-prostaglandin “suppression pain due to osteoarthritis of the knee [44].of the activation of caspases” [40]. Also, the first hydroxylated metabolite from analgesic sali-cylic acid is gentisic acid and this hydroquinone is only oneeasy step from the reactive benzoquinone-2-carboxylic acid(Fig. 2) which should quickly react with available proteinthiols. Figure 4. Reactive quinoid metabolites of diclofenac (left) and indometacin. There have been much interest in the COX enzymes as a target of APAP. Hinz [5] has pointed to COX-2 inhibition at Figure 3. Structures of flurbiprofen (left), which has an irreversi- therapeutic plasma concentrations but the evidence wasble component as inhibitor of COX-1, and ibuprofen which is a based on lowered PGE2 excretion from thrombocytes andpurely competitive inhibitor. not on direct enzyme inhibition. There is, however, clear indications that APAP, acting as a reducing agent, can influ-© 2013 Alf Claesson 3
    • ence the oxidation state of COX and thereby decrease the Conclusionsoutput of prostaglandins, especially from intact cells [45, 46]. The quest for the mechanism of action of APAP might The earlier mentioned hypothesis [10] that is closest to the have gone too far toward seeking narrow and elaborate ex-current one proposes to see antinociceptive effects in mice of planations. The hypothesis brought forward here, even ifAPAP given systemically and locally at the spinal cord (in- difficult to test and far from detailed, obeys the principle oftrathecal injection) to trpa1-/- mice; the reactive metabolites Occams razor since it provides the simplest imaginable an-NAPQI and benzoquinone were only administered intrathe- swer in agreement with all known data.cally. The authors relied on the hot-plate, cold-plate and At the same time as reactive metabolites might providewrithing tests to measure what they declare are antinoceptive some analgesic effects, according to the current hypothesis,effects in wildtype (trpa1+/+) mice. The trpa1-/- mice, on the their covalent binding to proteins is problematic. In mostother hand, did not show an antinociceptive response under cases these nonspecific reactions should cause negative ef-any of these conditions. The observations provided the basis fects, which means drug side-effects. The devastating harmfulfor an audacious conclusion: “Our study provides a molecu- effects of overdoses of APAP worldwide is a grim reminderlar mechanism for the antinociceptive effect of acetamino- of how we value pain relief in relation to drug safety [50].phen and discloses spinal TRPA1 activation as a potential Thinking about new drugs based on general reactivity orpharmacological strategy to alleviate pain.” The far-reaching affinity labeling, there is a delicate cost/benefit balance toimplication of the experiments is that the antinociceptive observe. The concept of exploiting reactive metabolites doeseffects of APAP seen in mice should be ascribed to activa- not seem a plausible route for design of new analgesics.tion of the noxious sensor TRPA1 in the spinal cord (byinhibition of pain signals thereby overriding the noxiouseffects of TRPA1 activation in the periphery). This would Competing interestsseem a rather specific action of a compound that generates None.indiscriminate reactive metabolites and the conclusionsshould be further scrutinized. As mentioned, the authors Referencesalso rely on rodent models of pain that are notoriously prob-lematic to translate into analgesia in man [47]. 1. Bertolini A, Ferrari A, Ottani A, Guerzoni S, Tacchi R, Leone S: Paracetamol: new vistas of an old drug. CNS Drug Rev 2006, 12:250-275.Testing the hypothesis The mechanism proposed consists of concurrent attacks 2. Hogestatt ED, Jonsson BA, Ermund A, Andersson DA, Bjorkon a great number of targets the sum of which balances in H, Alexander JP, Cravatt BF, Basbaum AI, Zygmunt PM: Con-favour of an overall (weak) analgesic activity. Testing of this version of acetaminophen to the bioactive N-acylphenolaminepolypharmacology in vivo is therefore difficult. However, it AM404 via fatty acid amide hydrolase-dependent arachidonic acid conjugation in the nervous system. J Biol Chem 2005,might be possible to test the effect of scavengers of NAPQI 280:31405-31412.(and of benzoquinone), such as the ones, e.g. N-acetylcysteine, used in APAP intoxications, to antagonize the 3. Ottani A, Leone S, Sandrini M, Ferrari A, Bertolini A: Theanalgesic effects of APAP in animal models or even in hu- analgesic activity of paracetamol is prevented by the blockademan. Obviously, it should be possible to study the NAPQI of cannabinoid CB1 receptors. Eur J Pharmacol 2006, 531:280-inhibition of a few selected caspases or cathepsins in vitro as 281.was already done with papain [15]. It should also be possibleto assess ex vivo whether a few selected cysteine proteases 4. Mallet C, Barriere DA, Ermund A, Jonsson BA, Eschalier A,become inhibited by conjugation with administered APAP. Zygmunt PM, Hogestatt ED: TRPV1 in brain is involved in acetaminophen-induced antinociception. PLoS One 2010, In contrast to the vast number of liver proteins that be- 5:e12748.come covalently bound to RMs from APAP [48] fewer stud-ies have dealt with the extent of binding to peripheral pro- 5. Hinz B, Cheremina O, Brune K: Acetaminophen (paraceta-teins [31-33]. Whole body autoradiography using radioactive- mol) is a selective cyclooxygenase-2 inhibitor in man. Thely labeled APAP does not seem to have been reported in a FASEB Journal} 2008, 22:383-390.way meaningful for the current discussion although APAPdistribution in the CNS has been studied [49]. These au- 6. Chandrasekharan NV, Dai H, Roos KL, Evanson NK, Tomsikthors used [3H]-APAP, which is not ideal, and they did not J, Elton TS, Simmons DL: COX-3, a cyclooxygenase-1 variantsee any specific binding. Using an immunochemical assay inhibited by acetaminophen and other analgesic/antipyreticWare et al. found protein adducts in rat enterocytes with drugs: cloning, structure, and expression. 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