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Key words: paracetamol, acetaminophen, NAPQI, aspirin, diclofenac, indometacin, reactive metabolite, hepatotoxicity, liver injury, bioacti-
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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, ). 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
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
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 . Some authors even declared “mystery re-
solved” . 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” . It
was also suggested that AM404 would activate TRPV1 in the
CNS thereby evoking an analgesic activity .
Other claims regarding the mechanism(s) of action of
APAP involve inhibition of prostaglandin synthesis via COX
inhibition, in particular COX-2 . In 2002 the finding of a
new cyclooxygenase (COX-3), which is synthesized from
RNA splice variants in canines and mice was published .
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-