1. Alf Claesson
Awametox Consulting, Lilldalsvägen 17 A, SE-14461 Rönninge, Sweden. E-mail: alfeaclaesson@gmail.com
Key words: paracetamol, acetaminophen, NAPQI, aspirin, diclofenac, indometacin, reactive metabolite, hepatotoxicity, liver injury, bioacti-
vation, NSAID.
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This paper might be cited as follows: Author and title of paper. Manuscript first 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 considerable
bewilderment but the hypotheses brought forward have not reached wide recognition. A novel hypothesis is presented here that APAP acts by
covalently modifying several pain signaling proteins via the reactive metabolite N-acetyl-para-benzoquinoneimine (NAPQI). The most likely
protein targets, it is suggested here, are cysteine proteases that take part in generation of pain signaling molecules, for example caspase-1 that
acts 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 proposed
mechanism consists of a great number of concurrent targets. However, it should be possible to indicate some cysteine proteases as targets in
vivo 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
known as acetaminophen in the US, is a widely used non-
prescription drug with antipyretic and analgesic actions but
with very weak anti-inflammatory activity (review, [1]). It is
an old drug, first synthesized in 1878, within the acetani-
lides, a group that includes phenacetin and acetanilide (Fig.
1). It has an exceptionally simple structure with a molecular
weight of only 151 Da that is less than most anesthetic gases
have.
Figure 1. Structures of and relationships between the acetanilide
analgesics. All these structures can be metabolized to the reactive
metabolite N-acetyl-para-benzoquinoneimine (NAPQI).
There have been much bewilderment as to the mecha-
nism(s) of action of this simple compound and a few sugges-
tions involving the following unique mechanisms have been
brought forward.
A relatively modern hypothesis that gained wide publicity
is based on the proven in vivo formation from APAP of N-(4-
hydroxyphenyl)arachidonylamide (AM404) which has CB1
receptor-modulating properties, inhibit the enzyme fatty acid
amide hydrolase-1, and also interferes with the uptake of
anandamide [2]. Some authors even declared “mystery re-
solved” [3]. There was also an observation that “the blockade
of cannabinoid CB1 receptors has been shown to completely
prevent the analgesic activity of paracetamol in rats” [3]. It
was also suggested that AM404 would activate TRPV1 in the
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 com-
pared with standard NSAIDs. However, other researchers
have argued that the COX-3 hypothesis lacks credibility be-

2. © 2013 Alf Claesson 2
cause of the unlikely existence of this enzyme in humans [7-
9].
The ion channel TRPA1 has also been suggested as the
target via metabolites of APAP. In 2011 Andersson et al.
published results that might indicate antinociceptive effects
in mice of APAP, acting via its reactive metabolites NAPQI
(Fig. 1) and benzoquinone that would activate spinal TRPA1
[10]. APAP was administered to trpa1-/-
mice locally at the
spinal cord (intrathecal injection) and also systemically;
NAPQI was administered intrathecally. The report will be
discussed below. A few other mechanisms have been men-
tioned in reviews on APAP [1, 11].
So far, none of the mechanisms can be considered proven.
The main purpose of the current paper is to present a novel
hypothesis of mechanism of action that is not based on spe-
cific receptors or enzymes but which fits with all the known
pharmacology of APAP and analogues. The previous hy-
potheses will be discussed in this context.
A novel hypothesis of mechanism of action
The cited works present theories on narrow mechanisms
of action of APAP. The novel hypothesis brought forward in
the current paper is, like that of Andersson et al. [10], based
on the formation of NAPQI and other reactive metabolites
(RM), e.g. benzoquinone. These RMs were previously sug-
gested [10] to elicit analgesia in mice through, somewhat
surprising, specific activation of the noxious sensor TRPA1
(limited to local action at the spinal cord). The current paper
suggests instead that the analgesic activity of APAP origi-
nates from reactions of RMs with a larger number of pro-
teins with many varied physiological roles in pain signaling.
The good and the bad actions balance in slight favor toward
the positive side. This idea puts the mechanism of action
more in line with the now well recognized complexity of
pain signaling than earlier hypotheses and does not limit the
target of these RMs to a single protein.
If there is some truth to this notion of “analgesic RMs”
the hypothesis might be expanded also to connect the
pharmacology of APAP to older reports of RMs from
NSAIDs. For example, gentisic acid (Fig. 2), formed in vivo
from aspirin, might be a pain relieving mediator via its easily
formed benzoquinone-2-carboxylic acid. The hypotheses can
be summarized in the following statements that will be en-
larged in the following text.
 APAP acts by covalently modifying pain signaling
proteins via the reactive metabolite NAPQI.
 The target proteins are mainly cysteine proteases.
An extension of the hypothesis:
 Analogous mechanisms might contribute to the
analgesic and anti-inflammatory effects of some
NSAIDs.
NAPQI is a well-known liver and kidney toxin, which like
similar quinoid RMs, is an indiscriminate electrophile and
arylating species (and is also an oxidizing agent) that react
preferentially with nucleophilic thiols in proteins or peptides
[12], as happens in the irritant sensor TRPA1. Reactive me-
tabolites (RMs) of drugs are notorious for causing severe
side-effects such as drug-induced liver injury [13], and are
thought to lie behind the hepatotoxicity of a great number
of drugs, many of which have been withdrawn from the
market, for example lumiracoxib in 2007-8 and sitaxentan in
2010. The largest group of RMs is the quinoids [14] to
which also NAPQI belongs. Since these electrophilic com-
pounds react preferentially with thiols they should be able to
inhibit also the many (15+) human cysteine proteases by
reacting with their active cysteine thiol, as demonstrated for
NAPQI with papain [15]. Cysteine proteases are key players
in a great number of cellular processes many of which in-
volve formation of molecules that have been reported to
take part in pain signalling [16-23] and in generation of neu-
ropathies (possibly via initiation of apoptosis) [24]. In fact,
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 pain
signalling include, for example caspase-1, caspase-3, cathep-
sin B, K, L and S. It is of some interest that both oral inhibi-
tors and an irreversible inhibitor of microglial cathepsin S
were claimed to be active in neuropathic pain models [26,
27]. The implications of the latter finding are limited by the
intrathecal route of administration.
Figure 2. Aspirin is metabolized to the hydroquinone gentisic ac-
id which can readily form a reactive benzoquinone metabolite.
In the complex world of pain signaling, it would be pre-
sumptuous to try to pinpoint one particular cysteine prote-
ase whose systemic inhibition should entail particularly posi-
tive effects. However, one might just speculate that the pro-
teases that take part in the processing of procytokines, such
as those generating IL-1beta and IL-6, could be key targets;
they have also been mentioned as drug targets, mostly for
therapies involving control of apoptosis [28]. The enzyme
caspase-1, also called interleukin-1beta converting enzyme
(ICE) is one of these, which has also been targeted in new

3. © 2013 Alf Claesson 3
drug research [29]. Patent applications also claim irreversible
caspase-1 inhibitors as pain relieving agents[30].
It might be argued that NAPQI should not be available for
enzyme inhibition throughout the human body since it is
generated primarily in the liver by actions of cytochrome P-
450 enzymes and is quickly made harmless by reaction with
glutathione. However, several other oxidizing enzymes are
also able to generate NAPQI (and other RMs from APAP),
including monophenol monooxygenase (tyrosinase), peroxi-
dases and COX and there are reports of proteins in the
periphery, including serum albumin [53], that form conju-
gates with APAP in vivo [31-33]. See also comments in list of
references at [51-52].
A fraction usually ranging from 5 to 15% of APAP is me-
tabolized via the oxidation pathway. Since the bioavailability
of APAP is high, 60-89%, a normal therapeutic dose of 700-
1000 mg gives rise to high plasma concentrations, in the
range 33-132 µM, e.g. [34]. APAP is uniformly distributed
throughout most body fluids, freely crosses the placenta and
penetrates the blood-brain barrier. This means that a num-
ber of extrahepatic oxidative enzymes have the possibility to
generate RMs from APAP in the system, usually with nega-
tive consequences (for example, proposed to occur in the
lungs [35]) but maybe, for a limited set of proteins, with a
slightly positive, pain relieving outcome. Also, APAP forms
p-aminophenol by hydrolysis and this can also be oxidized to
RMs.
An extrapolation of the hypothesis to certain NSAIDs
Here, it is relevant to point to the often made observations
that NSAIDs have many effects that do not primarily involve
prostaglandins. The point and hypothesis, related to NAPQI
as the presumed explanation for the analgesic activity of
APAP, is that some NSAIDs might receive extra analgesic
activity from similar quinoid metabolites (Fig. 2 and Fig. 4)
that react with a number of proteins, other than COX, rele-
vant in pain signaling.
The time-dependent irreversible inhibition of “the fatty ac-
id oxygenase”, i.e. COX, by aspirin and indometacin was
first reported in 1971 [36]. Some structural requirements
for time-dependent inhibition of prostaglandin biosynthesis
by anti-inflammatory drugs were published in 1975 [37] and
in 1994 [38]. It was noted that the compounds ibuprofen
and flurbiprofen (Fig. 3) which have Ki values of 3 and 1
µM, respectively, behave differently in that only flurbiprofen
gives time-dependent inhibition. The inhibition of prosta-
glandin cyclooxygenase by gentisic acid (metabolite of salicyl-
ic acid, Fig. 2) was further studied [39].
Since effective NSAIDs have been shown to form RMs it is
not far-fetched to think that certain compounds of this class,
for example indometacin and diclofenac, might provide
additional analgesic activity from quinoid metabolites (Fig.
4), similar to NAPQI, that react with a number of proteins
relevant in pain signalling, including COX [37, 38]. As stat-
ed, this might be considered an extended hypothesis from
that of APAP forming analgesic NAPQI. Obviously, this is a
bold speculation but it is not unreasonable based on evi-
dence of non-prostaglandin effects of NSAIDs. For example,
diclofenac showed a dose-dependent anti-apoptotic effect
that, it was hypothesized, went via a non-prostaglandin
“suppression of the activation of caspases” [40].
Also, the first hydroxylated metabolite from analgesic sali-
cylic acid is gentisic acid and this hydroquinone is only one
easy step from the reactive benzoquinone-2-carboxylic acid
(Fig. 2) which should quickly react with available protein
thiols.
Figure 3. Structures of flurbiprofen (left), which has an irreversi-
ble component as inhibitor of COX-1, and ibuprofen which is a
purely competitive inhibitor.
This might provide a mechanistic explanation for the anal-
gesic/anti-inflammatory activity of salicylic acid which is a
weak COX inhibitor but yet has analgesic activity compara-
ble with aspirin [41]. Other authors have also sought expla-
nations in this direction to try to explain the discrepancy
[42]. Other NSAIDs than the ones mentioned also form
para-hydroxylated drug metabolites, en route to quinoid
species, when they are so predisposed.
In principle, to verify this hypothesis it should be possible
to differentiate the common NSAIDs based on their analge-
sic activity (in relation to free plasma concentration in hu-
mans) plotted against COX inhibition constants. This plot
might reveal whether there is any correlation with propensi-
ty to form RMs (of the quinoid class).
A further point is that every reactive metabolite generated,
be it from APAP or an NSAID, has a unique structure and
therefore should have its own spectrum of pain-relevant tar-
get proteins which might confer a certain pharmacology. It
might not be a coincidence that the the carboxylic acid gen-
tisic acid (via its reactive metabolite, benzoquinone-2-
carboxylic acid, Fig. 2) inhibits COX, possibly preferentially.
The hypothesis in relation to earlier ones
Regarding the proposal that APAP works via an arachidon-
ic amide metabolite there is no doubt that APAP forms
AM404 in vivo in rats but the hypothesis of a resolved mech-
anism of action cannot be considered strong; as the authors

4. © 2013 Alf Claesson 4
themselves state: “.. it can only be hypothesized that AM404
is formed in man and contributes to the pharmacological
effects of acetaminophen.” [2]. Also, it should be noted that
the high dose of 300 mg/kg of APAP to rats only led to
formation of tiny amounts (< 11 pg/g) of AM404 in the
brains. One might add as a side-note that the whole concept
of cannabinoids as analgesics, although having been a topic
for decades, so far has failed to mature beyond the limited
applications of some compounds as adjunct pain relievers
[43]. The most recent efforts in the field have focused on
fatty acid amide hydrolase-1 (FAAH) as a target; here, PF-
04457845 failed to show analgesic activity in patients with
pain due to osteoarthritis of the knee [44].
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
therapeutic plasma concentrations but the evidence was
based on lowered PGE2 excretion from thrombocytes and
not on direct enzyme inhibition. There is, however, clear
indications that APAP, acting as a reducing agent, can influ-
ence the oxidation state of COX and thereby decrease the
output of prostaglandins, especially from intact cells [45, 46].
The earlier mentioned hypothesis [10] that is closest to the
current one proposes to see antinociceptive effects in mice
of APAP given systemically and locally at the spinal cord
(intrathecal injection) to trpa1-/-
mice; the reactive metabo-
lites NAPQI and benzoquinone were only administered
intrathecally. The authors relied on the hot-plate, cold-plate
and writhing tests to measure what they declare are antino-
ceptive effects in wildtype (trpa1+/+
) mice. The trpa1-/-
mice,
on the other hand, did not show an antinociceptive re-
sponse under any of these conditions. The observations pro-
vided the basis for an audacious conclusion: “Our study
provides a molecular mechanism for the antinociceptive
effect of acetaminophen and discloses spinal TRPA1 activa-
tion as a potential pharmacological strategy to alleviate
pain.” The far-reaching implication of the experiments is
that the antinociceptive effects of APAP seen in mice should
be ascribed to activation of the noxious sensor TRPA1 in
the spinal cord (by inhibition of pain signals thereby overrid-
ing the noxious effects of TRPA1 activation in the periph-
ery). This would seem a rather specific action of a com-
pound that generates indiscriminate reactive metabolites
and the conclusions should be further scrutinized. As men-
tioned, the authors also rely on rodent models of pain that
are notoriously problematic to translate into analgesia in
man [47].
Testing the hypothesis
The mechanism proposed consists of concurrent attacks
on a great number of targets the sum of which balances in
favour of an overall (weak) analgesic activity. Testing of this
polypharmacology in vivo is therefore difficult. However, it
might be possible to test the effect of scavengers of NAPQI
(and of benzoquinone), such as the ones, e.g. N-
acetylcysteine, used in APAP intoxications, to antagonize the
analgesic effects of APAP in animal models or even in hu-
man. Obviously, it should be possible to study the NAPQI
inhibition of a few selected caspases or cathepsins in vitro as
was already done with papain [15]. It should also be possi-
ble to assess ex vivo whether a few selected cysteine proteases
become inhibited by conjugation with administered APAP.
In contrast to the vast number of liver proteins that be-
come covalently bound to RMs from APAP [48] fewer stud-
ies have dealt with the extent of binding to peripheral pro-
teins [31-33]. Whole body autoradiography using radioac-
tively labeled APAP does not seem to have been reported in
a way meaningful for the current discussion although APAP
distribution in the CNS has been studied [49]. These au-
thors used [3
H]-APAP, which is not ideal, and they did not
see any specific binding. Using an immunochemical assay
Ware et al. found protein adducts in rat enterocytes with
APAP that had not passed the liver [33]. It might be of value
to run whole body autoradiography using stably labeled
[14
C]-APAP, possibly in combination with microautoradiog-
raphy, to find out just how diffuse or localized the covalent
binding to peripheral proteins is.
Conclusions
The quest for the mechanism of action of APAP might
have gone too far toward seeking narrow and elaborate ex-
planations. The hypothesis brought forward here, even if
difficult to test and far from detailed, obeys the principle of
Occam's razor since it provides the simplest imaginable an-
swer in agreement with all known data.
At the same time as reactive metabolites might provide
some analgesic effects, according to the current hypothesis,
their covalent binding to proteins is problematic. In most
cases these nonspecific reactions should cause negative ef-
fects, which means drug side-effects. The devastating harm-
ful effects of overdoses of APAP worldwide is a grim re-
minder of how we value pain relief in relation to drug safety
[50]. Thinking about new drugs based on general reactivity
or affinity labeling, there is a delicate cost/benefit balance to
observe. The concept of exploiting reactive metabolites does
not seem a plausible route for design of new analgesics.

5. © 2013 Alf Claesson 5
Competing interests
None.
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