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Home , Arachidonic acid, Prostaglandin E, Prostaglandin G2

New insights into the mechanism of action of acetaminophen: Its clinical pharmacologic characteristics reflect its inhibition of the two H2 synthases

David M. Aronoff, MD, John A. Oates, MD, and Olivier Boutaud, PhD Ann Arbor, Mich, and Nashville, Tenn

Acetaminophen (INN, ) possesses highly pharmacologic inhibitors of these (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 mediators derived from COX to refer specifically to the PGHS that play central roles in the pathogen- active site and POX to refer to the PGHS peroxidase esis of inflammation, , 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- 6 itors of synthase (PGHS) 2 because it forms in purified preparations. However, it lacks significant antiinflammatory activity.2 Moreover, exhibits a highly variable capacity to inhibit PG syn- as opposed to , acetaminophen is a poor inhibitor thesis by different and tissue types. For example, of 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 (PGE2) gener- 7,8 acid to the unstable intermediate prostaglandin H2 ation within the central nervous system (CNS), (PGH2) by PGHS, of which there are 2 major iso- whereas the failure of acetaminophen to inhibit platelet- 9 forms—the constitutive PGHS-1 and the (generally) derived A2 synthesis and inflammatory 5 10 inducible PGHS-2 (discussed later). These enzymes PGE2 synthesis 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, 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: [email protected] 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 13 Copyright © 2006 by the American Society for Clinical Pharmacology of the former drug for nearly 80 years. However, in 14 and Therapeutics. the late 1940s Brodie and Axelrod 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)ϭOPP●ϩ, Ferryl protoporphyrin IX cation; FE(III), resting enzyme; Fe(IV)ϭO, ferryl protoporphyrin IX; Tyr●, tyrosine 385 radical; AH, ● reducing cosubstrate; AA, arachidonic acid; AA , arachidonic acid radical; PGG2, ; ● PGG2 , prostaglandin G2 with peroxyl radical on carbon 15; ROOH, hydroperoxide substrate; ROH, 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 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●ϩ] 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 16 the oxygenation of arachidonic acid to yield PGH2. hydrogen is a very weak substrate. The This reaction occurs in 2 steps catalyzed by distinct Fe(IV)PP●ϩ 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 22,24,25 ). However, some “leakage” of the PGG2 radical from drug, which has indeed been shown, 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, , 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 , 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, ), , 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 (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 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 6 demonstrate either no isoform selectivity 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 Ϯ 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- 3,21 phen, as described by Aronoff et al.64 The PGHS-peroxidase rin. 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- 6 no acetaminophen was added. Each data point represents the action vessel. mean Ϯ 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)ϭOPP●ϩ to Fe(IV)ϭO. 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, in and 50-␮mol/L arachidonic acid. “Enzymologic” conditions washed human platelets, and 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. were extracted on a SepPak (Waters Corp, Mil- ford, Mass) cartridge and derivatized for analysis by gas centrations of both substrate and enzyme are used. With chromatography–negative ion chemical ionization–mass the high substrate and enzyme concentrations used in spectrometry as described in reference 65. The concentration oxygraph experiments, acetaminophen is unable to in- of prostanoids present in the medium is represented as a hibit PGHS-2 at concentrations as high as 3 mmol/L, percent of the control to which no acetaminophen was added. Each data point represents the mean Ϯ SEM of at least 3 whereas the 50% inhibitory concentration (IC50) falls within the micromolar range when assayed in condi- values. 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 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 ␮ ϭ ␮ 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 Ͼ ␮ 6 ␮ ϭ ␮ 6 3000 mol/L). To explore this hypothesis, we have mol/L (IC50 4.3 mol/L). 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 -macrophage cell line PGHS-2 is induced in 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

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- 6,34,47 8,48 hibition of thromboxane A2 biosynthesis and inhibition dothelial cells and neurons. 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 6 15 8 platelets by acetaminophen. This can be explained by CNS PGE2 production during fever and pain led to the fact that the explosive activation of the hypothesis that an exquisitely acetaminophen- 49,50 A2 in platelets by -dependent stimuli results in sensitive variant of PGHS exists within the CNS 28 a burst of PGG2 formation, which will cause the resis- or vascular endothelium. 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-,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 43-45 tory leukocytes, and their products, in addition to their catalytic activities by measuring PGE2 genera- peroxynitrite and 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- 6,21 hibit PGHS activity (discussed previously). 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 A in washed human platelets incubated with 20-␮mol/L 51 2 standard NSAIDs.” 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 6 inverse function of the concentration of the enzyme. was added). Each data point represents the mean Ϯ 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 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 receptors and inhibits the response to acetaminophen is that the metabolic fate cellular reuptake of cannabinoid compounds. Hoges- 62 of the drug differs among cells in a way that could tatt et al 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 62 aminophen largely occurs in the , via glucu- pathways exist in humans. 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 61 in rat renal medulla, we have demonstrated that After this article was accepted for publication, Qin et 66 inhibition of biosynthesis of thromboxane A2 in al 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 cellular selectivity in the action of acetaminophen, Dr Oates has received grant support from Merck-Frosst and Mc- 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 1. Funk CD. Prostaglandins and : advances in downstream metabolite of p-aminophenol partici- eicosanoid biology. Science 2001;294:1871-5. pates in the analgesic properties of acetaminophen.62 62 2. Aronoff DM, Neilson EG. Antipyretics: mechanisms of Hogestatt et al demonstrated that, after its admin- action and clinical use in fever suppression. Am J Med istration to the rat, acetaminophen is metabolized to 2001;111:304-15. the bioactive N-acylphenolamine compound N-(4- 3. Catella-Lawson F, Reilly MP, Kapoor SC, Cucchiara AJ, hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide DeMarco S, Tournier B, et al. Cyclooxygenase inhibitors CLINICAL PHARMACOLOGY & THERAPEUTICS 2006;79(1):9-19 Mechanism of action of acetaminophen 17

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