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New insights into the mechanism of action

of acetaminophen: Its clinical


pharmacologic characteristics reflect its
inhibition of the two prostaglandin H2
synthases
David M. Aronoff, MD, John A. Oates, MD, and Olivier Boutaud, PhD Ann Arbor,
Mich, and Nashville, Tenn

Acetaminophen (INN, paracetamol) possesses highly pharmacologic inhibitors of these enzymes (discussed
selective analgesic and antipyretic effects that result later). However, in this commentary we use the term
from its inhibitory actions on the synthesis of prosta- PGHS to describe the enzymes and limit the use of
glandins (PGs). PGs are lipid mediators derived from COX to refer specifically to the PGHS cyclooxygenase
arachidonic acid that play central roles in the pathogen- active site and POX to refer to the PGHS peroxidase
esis of inflammation, fever, and pain.1 However, acet- active site (as discussed in the “Catalytic mechanisms
aminophen differs from the majority of nonsteroidal of PGHS” section).
anti-inflammatory drugs (NSAIDs) and selective inhib- Acetaminophen is an inhibitor of both PGHS iso-
itors of prostaglandin H2 synthase (PGHS) 2 because it forms in purified enzyme preparations.6 However, it
lacks significant antiinflammatory activity.2 Moreover, exhibits a highly variable capacity to inhibit PG syn-
as opposed to aspirin, acetaminophen is a poor inhibitor thesis by different cell and tissue types. For example,
of platelet function at doses that are antipyretic.3,4 the analgesic and antipyretic effects of acetaminophen
PGs are generated by the oxygenation of arachidonic follow its inhibition of prostaglandin E2 (PGE2) gener-
acid to the unstable intermediate prostaglandin H2 ation within the central nervous system (CNS),7,8
(PGH2) by PGHS, of which there are 2 major iso- whereas the failure of acetaminophen to inhibit platelet-
forms—the constitutive PGHS-1 and the (generally) derived thromboxane A2 synthesis9 and inflammatory
inducible PGHS-2 (discussed later).5 These enzymes PGE2 synthesis10 accords with its weak antiplatelet and
are also commonly referred to as cyclooxygenase anti-inflammatory effects. Recent investigations of the
(COX) 1 and 2, respectively, in reference to the specific mechanism of action of acetaminophen on the PGHSs
enzymatic active site that catalyzes arachidonic acid have elucidated the basis for this cellular selectivity.
oxygenation and provides the target for the majority of This commentary brings together past investigations
and recent insights regarding the nature of
From the Division of Infectious Diseases, Department of Internal acetaminophen-PGHS interactions and provides a
Medicine, University of Michigan Health System, Ann Arbor, and model that best explains the selective clinical behavior
Departments of Internal Medicine and Pharmacology, Division of
Clinical Pharmacology, Vanderbilt University, Nashville. of acetaminophen.
Financial support was provided by National Institutes of Health
grants HL007749 and GM15431, the Parker B. Francis Founda- MECHANISM OF ACTION OF
tion, and grants from Merck-Frosst and McNeil. ACETAMINOPHEN
Received for publication July 25, 2005; accepted Sept 15, 2005.
Reprint requests: Olivier Boutaud, PhD, Division of Clinical Phar- In the latter half of the 19th century, phenacetin and
macology, Department of Pharmacology, Vanderbilt University, acetaminophen, structurally related aniline dye deriva-
Nashville, TN 37232-6602. tives, were developed as antipyretic agents.11,12 The
E-mail: olivier.boutaud@vanderbilt.edu safety profile of phenacetin was mistakenly favored
Clin Pharmacol Ther 2006;79:9-19. over acetaminophen, leading to the popular clinical use
0009-9236/$32.00
Copyright © 2006 by the American Society for Clinical Pharmacology of the former drug for nearly 80 years.13 However, in
and Therapeutics. the late 1940s Brodie and Axelrod14 elegantly demon-
doi:10.1016/j.clpt.2005.09.009 strated that phenacetin’s therapeutic efficacy was a

9
CLINICAL PHARMACOLOGY & THERAPEUTICS
10 Aronoff, Oates, and Boutaud JANUARY 2006

Fig 1. Catalytic mechanism of prostaglandin H synthases as proposed by Karthein et al.32 The


different steps are described in the text. Fe(IV)⫽OPP●⫹, Ferryl protoporphyrin IX radical cation;
FE(III), resting enzyme; Fe(IV)⫽O, ferryl protoporphyrin IX; Tyr●, tyrosine 385 radical; AH,
reducing cosubstrate; AA, arachidonic acid; AA●, arachidonic acid radical; PGG2, prostaglandin G2;
PGG2●, prostaglandin G2 with peroxyl radical on carbon 15; ROOH, hydroperoxide substrate; ROH,
alcohol product; A●, oxidized cosubstrate; HPETE, hydroperoxide of fatty acids.

result of its major hepatic metabolite, acetaminophen. hol, PGH2, in the POX catalytic site. The reduction of
These seminal experiments published in the Journal of hydroperoxides in the POX site yields an oxidized
Pharmacology and Experimental Therapeutics were heme radical, which generates the tyrosine 385 radical
among the first to demonstrate the pharmacologic ac- in the COX site via intramolecular electron transfer.
tivity of a drug metabolite formed in vivo and resulted As stated previously, initiation of COX-catalyzed
in the rapid popularization of acetaminophen for clini- arachidonic acid oxygenation requires that the tyrosine
cal use. Within 2 decades, acetaminophen was shown to 385 residue within the active site must first be oxidized
reduce prostaglandin E production in vivo,15 inciting to a radical (Tyr●) (Fig 1). This priming event is made
researchers to investigate the molecular basis for such possible by the initial reduction of an available hy-
effects. droperoxide substrate by the POX site, generating a
ferryl protoporphyrin IX radical cation [Fe(IV)PP●⫹]
Biochemical differences within cells that affect (Fig 1, step 1). The POX site can reduce a broad range
acetaminophen-PGHS interactions of hydroperoxides with variable efficacy: hydroperox-
Catalytic mechanisms of PGHS. PGHSs catalyze ides of fatty acids are preferential substrates, whereas
the oxygenation of arachidonic acid to yield PGH2. hydrogen peroxide is a very weak substrate.16 The
This reaction occurs in 2 steps catalyzed by distinct Fe(IV)PP●⫹ radical can then be transferred to the
active sites within the same enzyme.16 The hydrogen neighboring Tyr 385 in the COX active site, yielding
atom on carbon 13 of arachidonic acid is abstracted by the requisite Tyr● and a partially reduced form of the
the tyrosine 385 radical present in the COX active site. heme [Fe(IV)] (Fig 1, step 2), which must be further
The resulting arachidonyl radical incorporates 2 mole- reduced by 1 electron to regenerate the resting enzyme
cules of oxygen to yield the hydroperoxyendoperoxide [Fe(III)] (Fig 1, step 6). This heme reduction is per-
intermediate prostaglandin G2 (PGG2). PGG2 then un- formed by any number of reducing cosubstrates within
dergoes a 2-electron reduction to the equivalent alco- cells that have not been characterized. Using phenol,
CLINICAL PHARMACOLOGY & THERAPEUTICS
2006;79(1):9-19 Mechanism of action of acetaminophen 11

Harvison et al17 were first able to demonstrate that PGHS-1– expressing cells of different lineage were
reducing cosubstrates was necessary for POX activity. found to exhibit variable susceptibilities to inhibition
Since then, many molecules have been characterized as by acetaminophen.29
POX cosubstrates with variable efficiencies.18 These The understanding that acetaminophen inhibits
molecules facilitate the regeneration of the resting en- PGHSs by reducing the higher oxidative state of the
zyme, a process that is necessary for starting a new enzymes derives from an accumulation of evidence.
POX cycle. Numerous in vitro studies have demonstrated that acet-
The Tyr● initiates the oxygenation of arachidonic aminophen can reduce the higher oxidative state of the
acid by abstracting a proton from C-13 of the molecule PGHS-POX (and other peroxidase enzymes) to the
(Fig 1, step 3), generating a radical species that is ferric [Fe(III)] or “resting” state.17,18,23 The capacity of
further oxidized by 2 molecules of oxygen (Fig 1, step acetaminophen to act as a reducing cosubstrate for
4). This process yields a PGG2 radical that is reduced to PGHS-POX is illustrated in Fig 2, A. It would be
PGG2, regenerating the Tyr● in the process (Fig 1, step predicted from this that reduction of the POX heme by
5; mechanism extensively reviewed in references 19 and acetaminophen would produce oxidized radicals of the
20
). However, some “leakage” of the PGG2 radical from drug, which has indeed been shown,22,24,25 and this
the COX active site can occur, thereby interrupting the reaction is dependent on generation of PGG2 by ara-
COX cycle that regenerates the Tyr●; this necessitates chidonic acid oxidation in the COX active site.26 By
reactivation of COX activity by heme-dependent hy- contrast, ibuprofen, indomethacin, and flurbiprofen, in-
droperoxide reduction. hibitors that block access of substrates to the PGHS-
The COX active site is the target of the majority of COX catalytic site, do not act as cosubstrates of
NSAIDs, as well as the COX-2–specific inhibitors, PGHS-POX.18
which essentially inhibit PGHS function by the same As illustrated in Fig 2, B, the reduction of the POX
mechanism, that is, noncovalently binding within the heme to its resting state by acetaminophen essentially
COX site to physically obstruct entry of arachidonic reverses the hydroperoxide-driven oxidation of the
acid (aspirin is unique among these inhibitors by virtue heme moiety. Accordingly, it was envisioned that acet-
of its ability to covalently modify the COX site). How- aminophen could most effectively keep the PGHS in a
ever, this mechanism is not shared by acetaminophen, resting state when the concentration of peroxide is low.
which was not found to inhibit PGHSs from within the Hanel and Lands30 hypothesized that peroxides, by
COX site. This was evidenced by its inability to protect oxidizing the enzyme to its catalytically active state,
the enzyme against the time-dependent COX inhibition would oppose the action of drugs that reduce the oxi-
by indomethacin (INN, indometacin), diclofenac, or dized form(s) of the enzyme back to the catalytically
aspirin.3,21 In fact, acetaminophen appears to inhibit inactive resting state. They provided evidence for this
PGHS activity through its capacity to serve as a reduc- by demonstrating that lowering the peroxide concen-
ing cosubstrate for the POX active site.17,18,22-26 In the tration with the enzyme glutathione peroxidase en-
next sections we illustrate this unique mechanism of hanced the inhibitory action of a number of reducing
inhibition and explain how ambient levels of hydroper- agents on PGHS-1, including acetaminophen; this find-
oxide molecules might prevent acetaminophen’s action. ing has recently been extended to PGHS-2.21 This
Acetaminophen-PGHS interactions. After the dis- effect of peroxide concentration is consistent with the
covery that both constitutive and inducible isoforms of catalytic mechanism of PGHSs, in which the Tyr● in
PGHS exist,27 it was realized that inhibitors might the PGHS-COX site is required for the COX activity
exhibit selectivity toward one isoform over the other.28 (Fig 2, B).31,32
However, the selective inhibition of either PGHS-1 or Increasing the concentration of the arachidonic acid
-2 would not explain acetaminophen’s poor inhibitory substrate attenuates the inhibitory action of acetamino-
activity against both platelets (which only express phen by elevating levels of PGG2. Increasing the con-
PGHS-1) and activated leukocytes (which rely on centration of arachidonic acid has been shown to reduce
PGHS-2 for eicosanoid production). In fact, in vitro the inhibitory action of acetaminophen on the purified
assays of purified PGHS-1 and -2 enzyme preparations PGHSs.6,21,33 This phenomenon would appear to be the
demonstrate either no isoform selectivity6 or a greater consequence of the elevated level of PGG2 formed by
tendency of acetaminophen to inhibit PGHS-1.21 It the increased concentration of substrate, rather than a
appears most likely that acetaminophen inhibits cellular result of a competition of acetaminophen with arachi-
PGHS-1 or -2 (or both), depending on the cell type and donic acid at the COX active site, because it has been
experimental conditions (discussed later). For example, reported that acetaminophen does not prevent the inhi-
CLINICAL PHARMACOLOGY & THERAPEUTICS
12 Aronoff, Oates, and Boutaud JANUARY 2006

Fig 3. Effect of concentration of enzyme on inhibition of


PGHS-2 by acetaminophen. Inhibition by 1-mmol/L acet-
aminophen of arachidonic acid oxygenation by different con-
centrations of purified PGHS-2 was monitored as described
by Boutaud et al.6 The percentage of PGHS-2 inhibition by
acetaminophen is represented. Each data point represents the
mean ⫾ SEM of 3 values. The significance between each
concentration of enzyme was determined by the 2-tailed
Student t test.
Fig 2. A, Effect of acetaminophen on prostaglandin H2
synthase (PGHS) 1 peroxidase function. Conversion of PPHP
to PPA by ovine PGHS-1 reconstituted with hematin was
monitored as a function of the concentration of acetamino- bition of the enzyme by either indomethacin or aspi-
phen, as described by Aronoff et al.64 The PGHS-peroxidase rin.3,21 Confirming this hypothesis, similar results were
activity is expressed as a percentage of the control to which obtained when exogenous PGG2 was added to the re-
no acetaminophen was added. Each data point represents the action vessel.6
mean ⫾ SEM of 6 values. (Adapted with permission from In studies with purified PGHSs, the concentration of
Aronoff DM, Boutaud O, Marnett LJ, Oates JA. Inhibition of PGG2 also can be increased by raising the concentra-
prostaglandin H2 synthases by salicylate is dependent on the tion of the enzyme. Thus we demonstrated that the
oxidative state of the enzymes. J Pharmacol Exp Ther 2003; inhibitory potency of acetaminophen could be signifi-
304:589-95.) B, Hypothetic mechanism of inhibition of cantly diminished by increasing the amount of PGHS
PGHS by acetaminophen. Acetaminophen reduces enzyme present (Fig 3). These results essentially rule
Fe(IV)⫽OPP●⫹ to Fe(IV)⫽O. This reaction competes with out a direct competition between arachidonic acid and
the electron transfer between the tyrosine 385 residue and the acetaminophen because their ratio does not change
heme radical, which yields the tyrosine radical in the cyclo- under these experimental conditions. The concept that
oxygenase active site. Acetaminophen also provides the sec- the inhibitory potency of acetaminophen is determined
ond electron necessary to the regeneration of Fe(III), a pro- by the concentration of enzyme becomes very impor-
cess that is prerequisite for starting a new peroxidase cycle. tant when assaying the inhibition of PGHSs by acet-
PPHP, 5-Phenyl-4-pentenyl hydroperoxide; PPA, 5-phenyl- aminophen. Thus comparison of the inhibitory potency
4-pentenyl alcohol; ApAP, acetaminophen; ApAP●, acet- of acetaminophen for different PGHS isoforms must be
aminophen radical. made with amounts of the isoforms that have the same
catalytic activity. As seen in Fig 4, the potency of
acetaminophen is markedly enhanced when low con-
CLINICAL PHARMACOLOGY & THERAPEUTICS
2006;79(1):9-19 Mechanism of action of acetaminophen 13

Fig 4. Effect of experimental conditions on inhibition by


acetaminophen of PGHS-2. Inhibition of PGHS-2 activity by
acetaminophen was monitored in different experimental con-
ditions. Carbon 14 arachidonic acid was incubated with the
enzyme at 37°C as described later. After 8 seconds, the
reaction was terminated and the oxidized products were mon- Fig 5. Comparative inhibition by acetaminophen of prosta-
itored by scanning thin-layer chromatography as described in noid synthesis by human platelets, human umbilical vein
reference 64. “Typical” oxygraph conditions consisted of endothelial cells (HUVECs), and RAW 264.7. Synthesis of
performing the assay in the presence of 200-nmol/L PGHS-2 6-keto–prostaglandin F1␣ in HUVECs, thromboxane B2 in
and 50-␮mol/L arachidonic acid. “Enzymologic” conditions washed human platelets, and prostaglandins E2 and D2 in
consisted of performing the assay in the presence of 10- RAW 264.7 was assayed in the presence of increasing con-
nmol/L PGHS-2 and 0.5-␮mol/L arachidonic acid. The arrow centrations of acetaminophen. Arachidonic acid was added at
represents the increase of PGG2 produced by PGHS-2 in the a final concentration of 2.0 ␮mol/L. After 15 minutes at 37°C,
different conditions. prostanoids were extracted on a SepPak (Waters Corp, Mil-
ford, Mass) cartridge and derivatized for analysis by gas
chromatography–negative ion chemical ionization–mass
centrations of both substrate and enzyme are used. With
spectrometry as described in reference 65. The concentration
the high substrate and enzyme concentrations used in
of prostanoids present in the medium is represented as a
oxygraph experiments, acetaminophen is unable to in-
percent of the control to which no acetaminophen was added.
hibit PGHS-2 at concentrations as high as 3 mmol/L,
Each data point represents the mean ⫾ SEM of at least 3
whereas the 50% inhibitory concentration (IC50) falls
values.
within the micromolar range when assayed in condi-
tions of low substrate and low enzyme concentrations
that favor low peroxide tone.
Influence of cellular peroxide tone on acetamino- aminophen (Fig 5). These investigations have been
phen function. The aforementioned discovery that ac- facilitated by our development of the pyrogen-
etaminophen’s inhibitory potency against PGHS could stimulated human umbilical vein endothelial cell
be antagonized by hydroperoxides (such as PGG2) sug- (HUVEC) model and the demonstration of potent inhi-
gests that intracellular hydroperoxide levels (the “per- bition of epoprostenol (previously known as prostacy-
oxide tone” of the cell) might best explain the ability of clin) biosynthesis by acetaminophen in these cells when
the drug to block prostanoid synthesis in a cell-specific low levels of exogenous arachidonic acid are provided
manner. For example, the potency of acetaminophen as substrates.6 In interleukin 1␣–stimulated endothelial
against platelet PGHS-1 when the concentration of cells (which predominantly express PGHS-2), the IC50
exogenous arachidonic acid was 0.5 ␮mol/L (IC50 ⫽ of acetaminophen in the presence of 20 ␮mol/L of
210 ␮mol/L) was much lower than that observed if the arachidonic acid (72 ␮mol/L) was lower if the concen-
arachidonic acid concentration was 20 ␮mol/L (IC50 tration of added arachidonic acid was reduced to 2
⬎3000 ␮mol/L).6 To explore this hypothesis, we have ␮mol/L (IC50 ⫽ 4.3 ␮mol/L).6 The HUVECs, similar
characterized the effect of acetaminophen on cells that to other endothelial cells,34 are cells that are sensitive to
are germane to the pharmacologic selectivity of acet- the PGHS-inhibiting action of acetaminophen at phar-
CLINICAL PHARMACOLOGY & THERAPEUTICS
14 Aronoff, Oates, and Boutaud JANUARY 2006

macologically relevant concentrations. An endothelial drug. Consistent with the lack of an anti-inflammatory
site of action of acetaminophen is particularly germane effect of acetaminophen, we have found that the PGHS
to its antipyretic action, in light of evidence that activity of the activated monocyte-macrophage cell line
PGHS-2 is induced in brain endothelial cells during RAW 264.7 was very resistant to inhibition by acet-
fever35-37 and is required for the pyretic response.38,39 aminophen. Low concentrations of acetaminophen that
That acetaminophen’s inhibitory strength in the en- block PGHS activity in HUVECs by more than 90%
dothelial cell is inversely proportional to the “peroxide actually stimulate PG biosynthesis in RAW 264.7 cells
tone” of the cell was also demonstrated by use of the (Fig 5); only when the acetaminophen concentration
cell-permeable organic hydroperoxide tert-butyl hy- reached 1 mmol/L did we observe inhibition of PGE2
droperoxide.6 Treatment of HUVECs with increasing synthesis by RAW cells stimulated with 2 ␮mol/L of
amounts of tert-butyl hydroperoxide completely abro- exogenous arachidonic acid. These results strongly sup-
gated the inhibitory effects of acetaminophen. These port the hypothesis that cellular “peroxide tone” deter-
results have been duplicated in a pulmonary epithelial mines the cell-selective inhibition of PGHSs by
A549 cell line.40 acetaminophen.
Compared with these cells, the platelet is relatively Thus the lack of appreciable antithrombotic3,46 and
resistant to the action of 0.6- to 1-g oral doses of anti-inflammatory10 effects of acetaminophen may be
acetaminophen, which exert no effects on ex vivo ag- ascribed to the resistance of platelets and activated
gregation and only moderate (40%-70%) inhibition of macrophages to PGHS inhibition in vivo, whereas the
thromboxane A2 biosynthesis in most human volun- antipyretic and analgesic effects of acetaminophen
teers studied; only infrequently is more substantial in- could be attributed to PG suppression in vascular en-
hibition of thromboxane A2 biosynthesis and inhibition dothelial cells6,34,47 and neurons.8,48 Future studies to
of aggregation found, perhaps related to an association assess the “peroxide tone” of cells and tissues in vivo
with the highest concentrations of acetaminophen in and assess the relationship of such levels to acetamin-
plasma.4 The minimal effect of acetaminophen on ophen’s clinical action are required.
platelets at doses that are antipyretic is consistent with
our finding of an IC50 of 120 ␮mol/L for inhibition of Acetaminophen and “COX-3”
thromboxane A2 biosynthesis in thrombin-activated The ability of acetaminophen to effectively inhibit
platelets by acetaminophen.6 This can be explained by CNS PGE2 production during fever15 and pain8 led to
the fact that the explosive activation of phospholipase the hypothesis that an exquisitely acetaminophen-
A2 in platelets by receptor-dependent stimuli results in sensitive variant of PGHS exists within the CNS49,50
a burst of PGG2 formation, which will cause the resis- or vascular endothelium.28 In 2002 Chandrasekharan
tance of the platelet to the inhibitory action of acet- et al51 reported to have found such an enzyme within
aminophen. Furthermore, on activation, a substantial the canine cerebral cortex, which they designated
amount of the lipid hydroperoxide 12-hyroperoxy- “COX-3.” This enzyme is the product of an alterna-
icosatetraenoic acid (12-HpETE) is formed via the tively spliced messenger ribonucleic acid (mRNA) of
platelet 12-lipoxygenase,41 which is a good PGHS- the COX-1 gene that is identical to COX-1 mRNA
POX substrate.42 We have found that 12-HpETE is a except that intron 1 is retained. Because it is a
potent antagonist of acetaminophen action on purified product of alternative splicing of PGHS-1 and not a
PGHS-1 and likely contributes to the poor antiplatelet genetically distinct entity, the name COX-3 has been
effect of acetaminophen in vivo.6 When stimulated rejected by many authors.52,53 Therefore we refer to
with the same concentration of exogenous arachidonic it as PGHS-1b in this commentary. The PGHS-1b
acid, platelets are resistant to acetaminophen at concen- transcript (approximately 2.6 kilobases) was present
trations that completely block PGHS activity in at about 5% of the level of COX-1 mRNA in the dog
HUVECs (Fig 5). cerebral cortex.51
Hydroperoxide-generating lipoxygenase enzymes The investigators cloned and expressed canine
(such as the 12/15 leukocyte type lipoxygenase and PGHS-1b, as well as murine PGHS-1 and PGHS-2
5-lipoxygenase) also are highly expressed in inflamma- enzymes in insect (Sf9) cells, and then compared
tory leukocytes,43-45 and their products, in addition to their catalytic activities by measuring PGE2 genera-
peroxynitrite and hydrogen peroxide generated by ac- tion in response to exogenous arachidonic acid.51
tivated macrophages, might attenuate the action of acet- The expressed enzymes demonstrated quite hetero-
aminophen in inflammatory settings, thereby contribut- geneous activities, with PGHS-2 displaying the
ing to the lack of an anti-inflammatory effect of the greatest catalytic activity. The PGHS-1 construct
CLINICAL PHARMACOLOGY & THERAPEUTICS
2006;79(1):9-19 Mechanism of action of acetaminophen 15

possessed only approximately 20% of the activity of


PGHS-2, and PGHS-1b was only approximately 4%
as active as PGHS-2.51 The reason for this variability
was not determined.
This experimental system was used to compare the
sensitivity of the 3 enzymes to inhibition by various
compounds, including acetaminophen. The results
were striking: Acetaminophen inhibited PGHS-1,
PGHS-2, and PGHS-1b with IC50 values of 133
␮mol/L, 5887 ␮mol/L, and 64 ␮mol/L, respectively
(in the presence of 5-␮mol/L arachidonic acid).51
Moreover, with 30-␮mol/L arachidonic acid, the po-
tency of acetaminophen for all 3 isoforms was re-
duced, with only PGHS-1b being inhibited with mi-
cromolar concentrations (460 ␮mol/L). This inverse
relationship between substrate (arachidonic acid)
concentration and inhibitory potency accords with
the hypothesis that higher amounts of PGG2 produc-
tion antagonize the ability of acetaminophen to in-
hibit PGHS activity (discussed previously).6,21 The Fig 6. Comparison of inhibition of PGHS in platelets by
authors suggested that “COX-3 in the CNS may be acetaminophen and p-aminophenol. The synthesis of thrombox-
an essential target of both analgesic/antipyretics and ane A2 in washed human platelets incubated with 20-␮mol/L
standard NSAIDs.”51 However, a closer analysis of arachidonic acid was assayed in the presence of increasing
their data (and subsequent data from other investi- concentrations of acetaminophen or p-aminophenol as described
gators) suggests that PGHS-1b is not a pharmaco- in reference 6. The amount of thromboxane B2 (TxB2) present in
logic target of acetaminophen’s actions. the medium was determined by gas chromatography–negative
These studies were conducted before the discovery ion chemical ionization–mass spectrometry as described in ref-
that the inhibitory potency of acetaminophen was an erence 65 and normalized to the value of the control (no inhibitor
inverse function of the concentration of the enzyme.6 was added). Each data point represents the mean ⫾ SEM of at
Thus an important limitation of the PGHS-1b study least 3 values.
resulted from the authors’ use of PGHS constructs
having disparate activity levels. This experimental de-
sign confounds an accurate determination of the rela-
tive inhibitory potency of acetaminophen against more potent on PGHS-1b than on PGHS-1 and
PGHS-1, -2, and -1b, for reasons expressed previously. PGHS-2.
Because the ability of acetaminophen to inhibit PGHS There is no evidence that a catalytically functional
enzymes is inversely proportional to enzyme concen- PGHS-1b protein is expressed in humans. Indeed,
tration/activity (Figs 3 and 6), it would be predicted a Chandrasekharan et al51 noted that intron 1 in humans
priori that acetaminophen would inhibit the PGHS-1b differs from that in the dog by 1 nucleotide and there-
construct with low catalytic activity more potently than fore its inclusion in a human PGHS-1b would shift the
cells transfected with either PGHS-1 or -2, which have remainder of the protein out of frame. Subsequently,
higher catalytic activity. Therefore a meaningful com- Dinchuk et al54 cloned 12 human genomic fragments
parison of the inhibitory potency of acetaminophen spanning this region and confirmed that intron 1 of
against different PGHS isoforms requires that the ex- human PGHS-1 is 94 nucleotides long and that inclu-
perimental conditions are designed to achieve compa- sion of this intron in a human PGHS-1 would shift the
rable degrees of enzymatic activity among the 3 con- remaining sequence of PGHS-1 out of frame. Further-
structs.6 Future studies using purified PGHS-1, -2, and more, the rat53 and mouse55 PGHS-1b transcripts are
-1b with equivalent catalytic activity are warranted shifted out of frame by the retention of intron 1 and
before sound conclusions can be drawn regarding the would not be expected to yield an active enzyme.
relative potency of acetaminophen against the 3 pro- Indeed, the rat PGHS-1b protein was recently cloned
teins. The experiments reported by Chandrasekharan et and does not have COX activity.53 Thus, in the absence
al51 do not provide evidence that acetaminophen is of evidence for an acetaminophen-sensitive human
CLINICAL PHARMACOLOGY & THERAPEUTICS
16 Aronoff, Oates, and Boutaud JANUARY 2006

PGHS-1b, there is no basis for implicating it in the (AM404), most likely via the enzymatic conjugation
pharmacology of acetaminophen in humans. of p-aminophenol to arachidonic acid by fatty acid
amide hydrolase. This is notable because AM404 is
Possible role for metabolites of acetaminophen itself an analgesic agent63 that activates both va-
Another hypothesis for cellular selectivity in the nilloid and cannabinoid receptors and inhibits the
response to acetaminophen is that the metabolic fate cellular reuptake of cannabinoid compounds. Hoges-
of the drug differs among cells in a way that could tatt et al62 further reported that AM404 could inhibit
modify its efficacy either through formation of an both PGHS-1 and -2 in vitro, which may be relevant
active metabolite or by accelerated inactivation of to the molecular basis of acetaminophen’s inhibition
drug. There is abundant information on the biotrans- of these enzymes. Additional studies are needed to
formation of acetaminophen, and some of this does prove that this metabolic pathway for acetaminophen
indeed indicate such selectivity. Metabolism of acet- is necessary for its analgesic behavior and that such
aminophen largely occurs in the liver, via glucu- pathways exist in humans.62
ronidation and sulfate conjugation (reviewed exten-
sively in reference 56). A minor pathway of CONCLUSIONS
metabolism consists in the deacetylation of acet- Acetaminophen is a commonly used antipyretic
aminophen to yield p-aminophenol, a potent nephro- and analgesic agent that distinguishes itself from
toxicant. In explorations of the mechanism of other NSAIDs and aspirin by its poor antiplatelet and
acetaminophen renal toxicity, deacetylation of acet- anti-inflammatory actions. The pharmacologic basis
aminophen to p-aminophenol was demonstrated in for this distinct clinical behavior likely results from
the mouse kidney.57 Subsequently, acetaminophen its unique ability to inhibit PGHS enzymes at the
has been shown to undergo deacetylation to level of the POX catalytic site. Such an inhibitory
p-aminophenol followed by reacetylation back to mechanism would be predicted to exhibit a sensitiv-
acetaminophen, a “futile deacetylation” that con- ity to ambient peroxide levels, and this appears to be
founds attempts to measure the extent of conversion the case with acetaminophen. Whereas its analgesic
of acetaminophen to p-aminophenol unless appropri- and antipyretic effects likely follow PGHS inhibition
ate steps are taken to account for the futile deacety- within vascular endothelial cells and neurons, higher
lation pathway.58 Thus it is of interest that acetamin- concentrations of lipid and nonlipid hydroperoxides
ophen is quite potent as an inhibitor of renal PGHS.59 within activated leukocytes and platelets prevent
Furthermore, inhibition of acetaminophen deacetyla- acetaminophen from substantially affecting such pro-
tion reduces the nephrotoxicity caused by acetamin- cesses as inflammation and platelet thrombosis. Such
ophen but not that resulting from p-aminophenol, a model explains acetaminophen’s unique pharmaco-
suggesting an important role of this metabolic path- logic profile in humans.
way in nephrotoxicity.60 On the basis of this and on
evidence that p-aminophenol is an inhibitor of PGHS ADDENDUM
in rat renal medulla,61 we have demonstrated that After this article was accepted for publication, Qin et
inhibition of biosynthesis of thromboxane A2 in al66 reported the low-level expression of a functional
washed platelets is far greater with p-aminophenol PGHS-1 splice variant in human tissues, which was not
than with acetaminophen (Fig 6). Although these more sensitive to inhibition by acetaminophen than the
findings provide a clear rationale for determining the wild-type enzyme.
extent of acetaminophen deacetylation as it relates to
Dr Oates has received grant support from Merck-Frosst and Mc-
cellular selectivity in the action of acetaminophen, Neil. Drs Aronoff and Boutaud have not disclosed any conflict of
any role that cell- or tissue-specific deacetylation of interest with respect to the manuscript.
acetaminophen may play in determining its clinical
behavior remains to be explored. References
A very recent study advances the hypothesis that a
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