Drug Resistance and Pseudoresistance: An Unintended ...phdres.caregate.net/jclub-articles/Sbong.11513.pdfEnteric coating retards aspirin absorption markedly and renders this process

DOI: 10.1161/CIRCULATIONAHA.112.117283

Drug Resistance and Pseudoresistance:

An Unintended Consequence of Enteric Coating Aspirin

Running title: Grosser et al.; Aspirin Resistance and Pseudoresistance

Tilo Grosser, MD*1; Susanne Fries, MD*1; John A. Lawson, MS1; Shiv C. Kapoor, PhD1;

Gregory R. Grant, PhD1,2,3; Garret A. FitzGerald, MD1

* contributed equally

1Institute for Translational Medicine and Therapeutics, Perelman School of Medicine; 2Department of Genetics; 3Penn Center for Bioinformatics, University of Pennsylvania,

Philadelphia, PA

Address for Correspondence:

Garret A. FitzGerald, MD

Institute for Translational Medicine and Therapeutics, Translational Research Center

3400 Civic Center Boulevard, Building 421, Room 116

Perelman School of Medicine, University of Pennsylvania

Philadelphia, PA 19104-5158

Tel: 215-898-1185

Fax: 215-573-9135

E-mail: [email protected]

Journal Subject Codes: [118] Cardiovascular Pharmacology; [122] Secondary prevention; [71] Antiplatelets; [92] Platelets; [178] Aggregation

1

* contributed equally

111IInsttitititututute fofor r TrT anslational Medicine and TThhheerrapeutics, Pereeelml anan SSSchc ool of Medicine; 2Departmementnn oof f f GeGeG nenenetititicscscs;;; 333PePePennnnn CCCeenterrr ffofor BBBioooinfffoorrmamaatititicscs,, UnUnUniiviveere ssiityty ooff f PePeennnnnnsysysylvlvl anananiaiaia,,

Phillladddelpphphiaia, PAPAPA

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Abstract:

Background—Low dose aspirin reduces the secondary incidence of myocardial infarction and

stroke. Drug resistance to aspirin might result in treatment failure. Despite this concern, no clear

definition of “aspirin resistance” has emerged and estimates of its incidence have varied

remarkably. We aimed to determine the commonality of a mechanistically consistent, stable and

specific phenotype of true pharmacological resistance to aspirin – such as might be explained by

genetic causes.

Methods and Results—Healthy volunteers (n=400) were screened for their response to a single

oral dose of 325 mg immediate release or enteric coated aspirin. Response parameters reflected

the activity of aspirin’s molecular target, cyclooxygenase-1. Individuals who appeared “aspirin

resistant” on one occasion underwent repeat testing and if still “resistant” were exposed to low

dose enteric coated aspirin (81 mg) and clopidogrel (75 mg) for one week each. Variable

absorption caused a high frequency of apparent resistance to a single dose of 325 mg enteric

coated aspirin (up to 49%) but not to immediate release aspirin (0%). All individuals responded

to aspirin upon repeated exposure, extension of the post dosing interval or addition of aspirin to

their platelets ex vivo.

Conclusions—Pharmacological resistance to aspirin is rare; this study failed to identify a single

case of true drug resistance. Pseudoresistance, reflecting delayed and reduced drug absorption,

complicates enteric coated but not immediate release aspirin administration.

Clinical Trial Registration Information— clinicaltrials.gov. Identifier: NCT00948987.

Key words: antiplatelet drug resistance; antiplatelet therapy

2

oral dose of 325 mg immediate release or enteric coated aspirin. Response parammmeteteree ss rerereflflflececectetedd

he activity of aspirin’s molecular target, cyclooxygenase-1. Individuals who appeared “aspirin

eesisiistststananant”t”t” ooon n oone ee oococcasion underwent repeat testttininingg and if still “rereesis stanannt”t”t” were exposed to low

ddodoseese enteric coaoattet ddd asaspipiriririnn n (8(88111 mgmgg)) ) anandd cclopppidddogrrrelll (75755 mmggg) fforor onnene wweeeek k eaeaeacchch. VaVaVaririr ababableee

abbbsososorprprptitit onon cccaauauseseedd a a hihiighhh ffrer quququenenencycycy oof f f apapppapaparrer ntntnt rreeesiisistaaancncceee ttoto aaa ssiininglglgle e ett dddossese ooof f f 32323 5 5 mgmgm eeenttterericicc

coated aspirininn (((upupup tttooo 49494 %)%)% bbbutuu nnnototot tttooo immmmememedidid atata e e e rerereleleleasasa ee asasaspipipiririr n n n (0(0(0%)%)%). . AlAlA ll l ininndidivivividududualala s s s rer sponded


Introduction

The efficacy of low dose aspirin in the secondary prevention of important vascular events, such

as stroke and myocardial infarction, is well established while its relative benefit versus the risk of

gastrointestinal bleeding in primary prevention is still debated.1-4 The mechanism by which this

benefit is accrued is explained sufficiently by the irreversible acetylation of Ser530 in the enzyme

prostaglandin G/H synthase-1 (commonly termed cyclooxygenase [COX]-1) and the consequent

suppression of thromboxane (Tx) A2 formation. 3, 5 TxA2 is synthesized from arachidonic acid in

activated platelets and released as a local signal which amplifies activation and recruits

additional platelets to the site of clot formation. 6, 7 However, TxA2 is but one of several

endogenous agonists that result in platelet activation and vasoconstriction, both pertinent to

clinically important vascular occlusive events. 3, 7 Thus, inhibition of TxA2 signaling limits

substantially the aggregation response, but it does not prevent completely clot formation. Given

this capacity for redundancy, the magnitude of aspirin’s clinical impact is perhaps surprising; for

example, it reduces both death and myocardial infarction by 50% in placebo controlled trials of

patients with unstable angina. 8, 9 What is unsurprising is that treatment failures occur on aspirin,

as they do in patients taking other cardiovascular drugs. Many reasons for this have been

invoked, from patients failing to take their medication to aspirin insensitive mechanisms of

platelet activation to drug-drug interactions. 10-14 Despite this, the concept of “aspirin resistance”

has emerged,15 prompting the development of point-of-care diagnostic approaches to assessing

the inhibition of platelet aggregation in patients taking aspirin. 16 Estimates of the incidence of

such a “resistant” phenotype have varied widely 16-20 and the reliance on an assay of platelet

aggregability as a surrogate for enzyme acetylation and suppression of platelet TxA2 formation 21

and for clinical outcomes 15 has been criticized. Similarly, reliance on post hoc analysis of the

3

endogenous agonists that result in platelet activation and vasoconstriction, both ppepertrttininenent tt tototo

clinically important vascular occlusive events. 3, 7 Thus, inhibition of TxA2 signaling limits

uubsbsbstatatantntntiaiaialllllly yy tthe e agagagggregation response, but it doeseses nnnot prevent compmm leetetetelylyly clot formation. Given

hhhiss ccapacity fofor r reeeduundndn ananancycycy,, thththe e mamamagngnnitttuudee oof asppiirrin’’’s s clcliininiiccalal immpmpacact iiss pepeperhrhrhapapsss susuurprppriisisisingngg; for

exxamamamplplple,e,e, iitt t rerereduducceces s boboothhh ddeaeaththt aaandndnd mmmyoyoyocacacardrdrdiaaall l ininffafarrcr titiionnn bbbyy y 505050%%% ininin ppllalacceeboboo cconono trtrtroloolleddd trtrriaialslss ooof f

patients with h unununststababablele aaangnn ininina.a.a. 888, 999 WWWhahh t t t isisis uuunsnsnsurururprprprisissininng g isisis ttthahahat tt trtrtreaeaeatmmmenenent t t fafafailillururu esese oooccccccururur on aspirin,,,


major urinary Tx metabolites 22 as a biomarker of resistance in large scale trials 23, 24 has been

questioned. 15

True drug resistance to aspirin would result from (i) a failure to reach its molecular target,

(ii) a failure to acetylate COX-1 despite sufficient concentrations in the platelet or (iii) a failure

of COX-1 acetylation to suppress thromboxane formation. In the present study, we sought to

parse the variance in response to aspirin and to estimate the incidence of an internally consistent

phenotype of true drug resistance, reconciling biochemical and functional assays of aspirin

action that are stable over time and specific for the effect of aspirin on platelets. Healthy

volunteers (n=400) were screened for their response to a single oral dose of 325 mg regular

“immediate release” aspirin or enteric coated aspirin, which was designed to reduce local

damage of the gastric mucosa. Platelet aggregation induced by arachidonic acid, serum

thromboxane formation and urinary excretion of a Tx metabolite (TxM) – all reflecting the

activity of aspirin’s molecular target, COX-1 – were measured before and after dosing.

Individuals who appeared to be “aspirin resistant” underwent repeat testing. Individuals who

failed to respond to aspirin twice were exposed to low dose enteric coated aspirin (81 mg) and

clopidogrel (75 mg) for one week each in a cross over design.

While the distinct estimates of aspirin action were congruent and apparent “resistance”

based on platelet aggregation inhibition at a single time point was common, this reflected

variable drug exposure due to enteric coating of aspirin and was either inconstant over time or

could be overcome by addition of aspirin ex vivo. Thus, we failed to find a single case of true

drug resistance in this study of 400 healthy volunteers.

Methods

4

“immediate release” aspirin or enteric coated aspirin, which was ded signed to reduduuceee locococalalal

damage of the gastric mucosa. Platelet aggregation induced by arachidonic acid, serum

hhroroombmbmboxoxananane formrmrmata ion and urinary excretion offf aa TTTx metabolite (((TxT M)M)M) – all reflecting the fff

acctiivvity of aspipiriirin’n’s mmomoleleecucuc lalaar r r tatargrggetetet, COCOCOX-1 –– wweerree mmmeeaeasusuureeed d bbbeffoforere aaanndd aaafftfteerer ddoososinining.g.

nndididivivividudud alalsss whwhw o o appppepep aaareeded too bebebe ““asasaspippiririin n rereresisisistttananant”t”” uuundndderrrwewewentntt rrreeppepeatatat tteesestiiingng. InInndidivvividdudualalls whwhw ooo

failed to respsppononond d d tototo aaspspspiriri innn tttwiww cecece wwwerere e exexexpopoposesesed d d totoo lllowowow dddososose ee enenenteteterirr cc cococoatatatededed aaaspsppiriririninin (((818181 mg) and


Study Design

The study protocol was approved by the Institutional Review Board of the University of

Pennsylvania and conducted in University’s Clinical Translational Research Center (CTRC). 13

Healthy, non-smoking volunteers (18-55 years) who provided written informed consent were

enrolled and abstained from all medications and nutritional supplements for the duration of the

study.

In Phase 1 fasted healthy volunteers (n=400) received a single oral dose of 325 mg

aspirin and drug response parameters were assessed before and after dosing. Two distinct aspirin

formulations were administered with 250 ml water, followed by a light standardized meal 2

hours later. Drug responsiveness was assessed at 4 or 8 hours after administration to create three

groups (Figure 1). Group 1 (n=40) received regular, immediate release aspirin (Genuine Bayer

Aspirin, Bayer Health Care, Morristown, NJ) and their response was assessed 8 hours after

dosing. Group 2 (n=210) received enteric coated aspirin (Safety Coated Bayer Aspirin) and their

response was measured 8 hours after dosing. Group 3 (n=150) received enteric coated aspirin

and their response was assessed at 4 hours. Enteric coating retards aspirin absorption markedly

and renders this process much more variable. 25, 26 Thus, Groups 2 and 3 were characterized by

decreasingly stringent conditions for the identification of variation in the response to aspirin.

This would be expected to increase the likelihood of identifying host factors (e.g. genetics,

nutritional habits, the composition of the gut microbiome) which might influence reproducibly

the response to aspirin. Decisions regarding inclusion of subjects into the next study phases were

made based on a “point-of-care” assessment, the platelet aggregation response to arachidonic

acid, which was performed immediately following the blood draw. Individuals who showed less

than 60% inhibition of their maximal arachidonic acid induced platelet aggregation (comparing

5

hours later. Drug responsiveness was assessed at 4 or 8 hours after administrationonn tto oo ccrreaeaeatetete ttthrhhree

groups (Figure 1). Group 1 (n=40) received regular, immediate release aspirin (Genuine Bayer

AsAspipipiriririnn,n, BBBayayayeeer HHHeaeaealtl h Care, Morristown, NJ) andndnd thhheir response waww s asasssesesessed 8 hours after

ddodosisiingn . Group p 222 (nnn=2=2210100))) rererecceceiivivededd eeentntereriic cooaoatted asasppiririinn n (S(SSafaffetetyy CCooatatedede BBayayayeerer AsAsspipipiriririn)n)n aaa dndnd thhheir

eespspsponononsese wwwasasas mmeeeassusurereed 88 hohouuru ss afafafteteter r ddodosisisingngng. GrGrGroouupp p 3 3 (n(nn==11150)0)0) rrececeieieiveveed enenteteteririiccc cocooaatateddd aaspsps irirrinin

and their respspponononsesee wwwasass aaasssesese seses d d d atatat 444 hhououoursrsrs.. EnEnEnteteteririric c c cococ atttininingg g rereretatatardrdrds asasa pipip ririr nn n abababsosoorprprptitit ononon mmarkedly


post vs. pre dose aggregation) were classified as “non-responders”, who might potentially be

resistant to aspirin and entered Phase 2 of the study. Additionally, a subset of individuals who

showed an adequate inhibition of 60% or more was invited to participate in Phase 2 as

“responder” controls.

Phase 2 addressed the stability of the phenotype of aspirin resistance. Following a

washout period of at least 14 days, the two cohorts identified in Phase 1 (108 “non-responders”

and 149 “responders”) underwent repeat testing with a single dose of aspirin. Again, individuals

showing less than 60% inhibition of platelet aggregation using arachidonic acid as a stimulus

were classified as “non-responders”. Those who were “non-responders” in Phase 1 AND Phase

2 were invited to proceed to Phase 3. They were matched by gender, age (± 2.5 years) and race

with subjects who were “responders” in Phase 1 AND Phase 2.

Phase 3 determined whether resistance persisted when the dose of aspirin was altered and

multiple doses were administered. It also assessed whether the phenotype was specific for aspirin

or extended to another platelet inhibitor, clopidogrel. Two cohorts (27 “non-responders” and 25

“responders”, who completed the study) received 81 mg enteric coated aspirin and 75 mg

clopidogrel for 7 days, in a crossover design randomized by order and separated by a washout

period of at least 14 days (Figure 1). Fifteen of the 42 volunteers, who were classified “non-

responders” in both Phase 1 and Phase 2, were unavailable for re-testing in Phase 3 mostly due

to geographical mobility. However, the “non-responder’ cohort included all individuals who had

appeared “resistant” in Phase 2 even when aspirin was added to their platelets ex vivo. Outcome

measurements were obtained prior to treatment begin, 4 hours after the first dose, just prior to the

last dose and 4 hours after the last dose. Aspirin response status was assessed based on the last

inhibition detected in the last measurement relative to baseline. Those who were responsive to

6

2 were invited to proceed to Phase 3. They were matched by gender, age (± 2.5 yeyyeararars))) aandndnd rrracaca e e

with subjects who were “responders” in Phase 1 AND Phase 2.

PhPhPhasasa eee 3 dddeeteteermined whether resistance ppperere ssiisted when theee dosse ee oofof aspirin was altered and

mmulltltiple dosess wwwerrre adada mimiminininistststeerereded.. ItItIt aalslsoo assesesseddd wwwheetethheher r thhhe e phppheenenototyypypee wwawasss spspeececififificiic ffooror aaspspspirin

orr eeextxtxtenene dededd d tototo aanononoththererr ppplalatet lelelet inininhihihibibib totoor,r, ccclllopoopidididogogogreeel.l TTTwowowo ccohohohororrtsts (2(2(277 “nnnonon-r-r- esesespopopondndnderrrs”s” aandndd 22255

“responders”,, whwhw ooo cococ mpmpmpleeeteteted d d thhhe e e ststtuduu y)y)y) rrrecececeieiiveveveddd 888111 mgmgmg eeentntntererericicic cccoaaateteted d d asasaspipipiririn n n ananand d d 757575 mg d


clopidogrel but showed an impaired response only in the COX-1 pathway as determined by

arachidonic acid induced platelet aggregation and ex vivo whole blood TxB2 release in all three

study phases were considered pharmacologically “aspirin resistant”.

Efficacy Assessments

These included (i) platelet function tests ex vivo involving traditional optical platelet aggregation

testing of four pathways of platelet activation (500 μg/mL arachidonic acid, 20 μmol/L ADP, 2

μg/mL collagen, 100 μmol/L the protease receptor-1 activating peptide [PAR-1AP]). 13 The

sensitivity of platelets to aspirin added ex vivo was determined by addition of freshly prepared

aspirin (dissolved in ethanol) at final concentrations of 30 and 100 μmol/L to PRP, 15 minutes

before platelets were stimulated. (ii) The capacity of platelets to form Tx during clotting was

assessed as an index of COX-1 enzymatic activity ex vivo. Serum TxB2 was detected by enzyme

immunoassay (Cayman Chemicals, Ann Arbor, Michigan). 27 (iii) The urinary excretion of the

Tx metabolite, 11-dehydro TxB2 (TxM) – an index of platelet COX-1 activity in vivo – was

quantified as its methoxyamine derivative by liquid chromatography tandem mass spectrometry

in spot urine samples that were collected 30 minutes after voiding. 28

Statistical considerations

Empirical cumulative frequency distributions were graphed in R (cran.r-project.org). The effect

of aspirin exposure on the outcome variables (post vs. pre dose comparison) was assessed with

the Wilcoxon Matched-Pairs Signed Ranks Test as implemented in R. A p-value below 0.05 was

considered statistically significant.

Responder status to aspirin in the arachidonic acid induced aggregation and the serum

TxB2 assays was reported using thresholds that were derived from data sets generated previously

in our laboratory. 13, 29 Inhibition of aggregation and TxB2 formation relative to baseline (prior to

7

before platelets were stimulated. (ii) The capacity of platelets to form Tx duringg ccclooottttininnggg wawawas s sm

assessed as an index of COX-1 enzymatic activity ex vivo. Serum TxB2 was detected by enzyme

mmmumumunnonoasasssasas yyy (CCCayayaymman Chemicals, Ann Arbor, MMMicchigan). 27 (iii)i)) Thee uuurririnary excretion of the

TTx mmmetabolite,e 1111--deehyhyh drdrdrooo TxTxTxBBB22 (((TTxTxMM)M) – aann iindeeexx off pppllalateteeleeet t CCOOX-X-111 acactiivivivitytyy iin n vivivivovov –– wwwasass

ququanana tititififif eded aaas ss ititss memeththooxoxyayay mimiinenen ddderereriivivatattivvvee e bbyby llliqiqi uuuidd d chhhrooommamatooogrgrg aapaphyhyhy ttaanandeded mm m mmamasssss ssspepeectctrrorommemetrtrry yqq

n spot urinee sasasampmpmpleleles s thththata wwwererere cococollllecece teeed d d 303030 mmminininutututeseses aaftftterere vvvoioo dididingngn . 282828


aspirin administration) was calculated. Failure of aspirin to inhibit arachidonic acid induced

aggregation more than 60% or serum Tx formation more than 95% was classified as “non-

response”. These threshold values were chosen before the study was conducted.

“Responders”/”non-responders” did not exhibit any differences in baseline AA aggregation

measures before the administration of aspirin.

For all other outcome measures (maximal aggregation, maximal slope of aggregation,

absolute serum TxB2 concentration, urinary TxM excretion), a data driven classification

threshold for “responder” vs. “non-responder” status was identified using a modified Receiver

Operating Characteristics (ROC) analyses with the inhibition of serum TxB2 formation as the

gold standard. Thus, all values of a given outcome measure (e.g. maximal arachidonic acid

induced aggregation) were considered as hypothetical cutoffs to predict class membership. For

each hypothetical cutoff all instances of an outcome measurement were assigned a hypothetical

class label. However, instead of assigning the “true” class labels based on an arbitrary threshold

of serum TxB2 inhibition we allowed the best fitting random forest model 30 of serum TxB2

inhibition to define “true” class membership. The random forest classifier as implemented in the

R package “randomForest” was run in classification mode for each hypothetical threshold. Each

random forest model, consisting of 4000 trees, generated a confusion matrix with the classes

predicted by the hypothetical cutoff and the “true” classes derived from the random forest fit of

the serum TxB2 inhibition distribution. Graphs of “true” positive and “true” negative rates were

used to evaluate the performance of the hypothetical thresholds. The random forest algorithm

also generates an error estimate of the model fit, which is the error rate we report here together

with the classification accuracy.

8

gold standard. Thus, all values of a given outcome measure (e.g. maximal arachihiddodoninin cc acacacididid

nduced aggregation) were considered as hypothetical cutoffs to predict class membership. For

eaachchh hhhyypypototothehehetticaaall l ccucutoff all instances of an outcooommeme measurementntt werere ee aaassigned a hypothetical

cllasssss label. Howowevevverr, , ininstststeaeaeadd d oofof aasssss igigi nnninnng thhehe “truueue”” clllaassss llal bbebelsls ff bbaasesed dd oonn aaannn ararbibiitrtrt aarary y thhhrrereshshshooold

offf sssererrumumu TTTxBxBxB22 iinhnhnhibibitittioion n wewe aallllllowowowedeed ttthehee bbbeeestt fffititttitingngg rrrananndododom m fofoforrereststst mmmoododeelel 33030 ooof f f seseerururum TxTxxBB222

nhibition to dededefifif nenene “trtrrueueu ” clclc aaassss mmmememmbeeersrsrshihih p.p.p TTThehehe rrrananandodoom m m fofoforereeststst cclasasassisisifififiererer aaas s imimmplplplemememene ted in thee


Results

Parsing variability in the response to aspirin

We studied a population of relatively young and healthy individuals (Table 1) to assess the inter-

and intra-individual variability in the pharmacological response to aspirin without

“environmental” variability contributed by disease. High inter- and low intra individual

variability would suggest that host factors which predict the response to aspirin might be

identified even in a much more heterogeneous population with cardiovascular disease. All 40

subjects administered immediate release aspirin (Group 1) in Phase 1 responded with a reduction

of platelet aggregation induced by direct stimulation of the COX-1 pathway by arachidonic acid

by more than 60% comparing pre and post dose measurements (Table 2). By contrast, 17%

qualified as “non-responders” 8 hours after dosing with enteric coated aspirin (Group 2),

increasing to 49% 4 hours after the dose (Group 3).

If platelets are not inhibited by ingestion of aspirin, but can be inhibited by addition of

aspirin ex vivo, this points to a pharmacokinetic, rather than a pharmacodynamic explanation for

such a “non-responder” status. The addition of aspirin ex vivo dose dependently “corrected” non-

responder status in the two groups of volunteers administered enteric coated aspirin, falling from

17 % to 0% 8 hours (Group 2) and from 49% to 12% 4 hours post dosing (Group 3) (Table 2).

Assessment of the response to aspirin by quantitation of serum TxB2, which is considered

a reliable biochemical measure of platelet COX-1 function, but is not practical as a point-of-care

assay, 31 showed that all but one subject receiving immediate release aspirin (Group 1)

responded with inhibition of serum TxB2 formation by more than 95% (Table 3). 21 All subjects

responded by suppression of the absolute post dose serum TxB2 concentration to below 10

ng/mL. 29 There was a similar increase in apparent non-response after enteric coated aspirin

9

by more than 60% comparing pre and post dose measurements (Table 2). By coonnntraaastst,, 171717%%%

qualified as “non-responders” 8 hours after dosing with enteric coated aspirin (Group 2),

nncrcrreaeaeasisisingngg tttoo 4949% % % 44 hours after the dose (Grouppp 333).

If plateeleletst arere nototot iiinhnhnhibibibititededed bbyy innngessstioon oofof aspppirirrinin,, bubuut t cccannn bebe iinnhhibibbiititeeded bbbyy y adadaddidd tiiioonon ooff f

asspipipiririinn n exex vvvivivivo,o, ttthihiis s ppopoinnntsts tooo a ppphahaharrmrmaacacokokokinininettticicic, , rarattheerer ttthahahann aaa pphhaharmrmrmacaccododdynynnamamamicic eeexxpxplalaananaatitioonon ffforo

uch a “non-r-resesespopopondndndererr” statatatususus. ThThThe e adaa dididititiiononon ooof f asasspipipiriririn n exexex vvvivivvo o o dododoseee dddepepepenenendededentnttlylyly “cococorrrrr ected” nonnn


when measured 8 hours (Group 2; 24%) and 4 hours (Group 3; 59%) post dosing as observed

with platelet aggregation using the relative inhibition of serum TxB2 by more than 95%.

Phase 2 addressed the stability of the phenotype of “aspirin resistance”. Following a washout

period, two cohorts identified in Phase 1 (108 “non-responders” and 149 “responders”)

underwent repeat testing before and after a single dose of aspirin to assess the intraindividual

variability of the drug response within each of the three Groups. While “non-response” defined

either in terms of suppression of platelet aggregation or serum TxB2 was relatively common after

enteric coated aspirin in Phase 1 of the study, the persistence of the phenotype in the same

individuals in Phase 2 of the study was comparatively rare (Table 2). None of the volunteers

who received plain aspirin (Group 1) was a “non-responder” in both phases of the study. Only

6% (Group 2) and 20% (Group 3) by arachidonic acid induced platelet aggregation and 7%

(Group 2) and 26% (Group 3) by inhibition of serum TxB2 formation remained “non-

responders” in Phase 1 AND Phase 2 (Table 3).

Acute dose studies bias in favor of detection of “resistance”. Cumulative inhibition of

platelet COX-1 during chronic drug administration would be expected to minimize the incidence

attributable to insufficient drug exposure. Phase 3 determined whether resistance persisted

during repeated daily administration of a reduced dose of enteric coated aspirin to mimic a

typical, chronic low dose aspirin therapy. It also assessed whether the phenotype was specific for

aspirin or extended to another platelet inhibitor, clopidogrel, which targets an adenosine

diphosphate (ADP) receptor (the P2Y12). Twenty-seven individuals, who were “non-responders”

and 25 who were “responders” based on arachidonic induced platelet aggregation in both Phase

1 AND Phase 2 received 81 mg aspirin for 7 days and 75 mg clopidogrel for 7 days, in a

crossover design (Figure 1). Only one volunteer in the “non-responder” cohort failed to respond

10

who received plain aspirin (Group 1) was a “non-responder” in both phases of ththhee ststs ududdy.y.y. OOOnlnlnlyy

6% (Group 2) and 20% (Group 3) by arachidonic acid induced platelet aggregation and 7%

GrGrrouououpp p 222))) anana dd 26266%%% (Group 3) by inhibition of sserere uumm TxB2 formamaatit onn rrremememained “non-

eespppono ders” inn PPhP aasasee 11 AAANDNDND PhPPhasasasee e 22 (TTTabllle 3).

AcAcutututee e dodoseee sstutuudiiieses bbiaiaias ininin fffaavavooror oooff f dddeteteectctc iiiononn ooff “r“r“resesesisistatatancncce”e””.. CuCuummumulalaatitiivevev iiinhnhnhibbiiitiooon n ooof

platelet COXX-1-11 ddurururinining g g chcc roroonininic c drdrdrugugug aadmdmdminininisisistrtrtratatatioioon n n wowow ulululd d d bebebe eeexpxpxpeccctttrrr ededed ttto o o mimim niniimimimizezeze ttthehh incidenceee


to the weeklong treatment with aspirin in Phase 3 as assessed by arachidonic acid induced

platelet aggregation and serum TxB2 inhibition (Figure 2). However, platelets from this subject

responded to aspirin addition ex vivo. Furthermore, this apparent “resistance” was not specific as

this individual also failed to respond to clopidogrel (not shown). Interestingly, two individuals

who entered Phase 3 as “controls” with demonstrated ability of their platelets to respond to

aspirin administration in Phase 1 and 2 now failed to respond in Phase 3 (Figure 2). One of

them also failed to respond to clopidogrel (not shown), but both responded to addition of aspirin

ex vivo.

Diagnosis of an individual’s aspirin response status

The study sought individuals who showed an inadequate mechanistic response to the

administration of aspirin as assessed by platelet aggregation ex vivo, serum TxB formation 2 – an

index of the capacity of platelets to form COX-1 dependent TxA2 – and the urinary excretion of

the thromboxane metabolite, 11-dehydro TxB (TxM) 2 – an index of actual thromboxane

biosynthesis. All three measurements detected a significant difference between the pre and post

dose measurement in each treatment group (p<0·01, data not shown). Plotting their relative

cumulative frequency distributions illustrates the utility of the drug response markers to

determine response status at the individual level (Figure 3). The distribution curves for

arachidonic acid induced platelet aggregation and serum TxB2 concentration, but not for urinary

TxM excretion, allowed the data driven detection of a threshold value – a plateau in the

distribution curve – which distinguishes between “responders” and “non-responders”. A

threshold of 16% (95% confidence interval 12% to 62%) residual arachidonic acid induced

platelet aggregation resulted in a test accuracy of >90% and an error rate of 10%. Indeed, the

extended plateau in the frequency distribution renders arachidonic acid induced platelet

11

The study sought individuals who showed an inadequate mechanistic response totoo theheh

administration of aspirin as assessed by platelet aggregation ex vivo, serum TxB formation 2 – an–

nndedeexx x ofofof ttthehehe cccappacacacititi yy of platelets to form COX-111 dedeependent TxAA222 – aandndnd tthe urinary excretion of –

hhhe ththromboxananeee mmemetataabooolililitetete,, 11111-d-ddehehehydyddrooo TxxBxB (TTxTxM)M) 2 –– aann innndedexx ofof aaccctuuauall– thththrorombmbmboxoxoxananneee

biiosososynynynththt esesisisis. AlAlll l thhhrereeee mmemeasa uururememmenenentsts dddetetetecececttted dd a a a sssigngngniffficccananantt t dididifffff eeerenenencecec beetetweweeenenn tthehehe pppreee aandndn pppoosst t

dose measureremmmenenentt t ininin eeeacaca h h trtrt eaee tmtmmenenentt t grgrrouououp pp (p(p(p<0<0<0·0001,1,1 ddatattaa a nononott t shshshowowo n)n)n).. PlPlPlototottititingngn ttheheheirirr rrrelee ative


aggregation essentially a dichotomous outcome measurement (see, for example, the post dose

distribution of Group 3, the group with approximately 50% apparent “resistance”, in Figure 3C).

Stimuli of platelet aggregation which activate platelet pathways in which COX-1 dependent

events are downstream of the initial stimulus – collagen and ADP – were much less reliable

diagnostic tools for the assessment of the response status (Figure 4). They resulted in accuracies

of 80% and 75% and error rates of 21% and 36%. As expected, platelet activation with

proteinase activated receptor-1 activating peptide (PAR-1AP), which ligates a thrombin receptor,

did not associate with inhibition of serum Tx formation by aspirin.

Measurement of the absolute serum TxB2 concentration (rather than “serum TxB2

inhibition” based on the comparison of pre and post dose concentrations) is an assessment that

could be performed in patients without stopping aspirin therapy. Analysis of the frequency

distribution of the absolute serum TxB2 concentration also revealed a plateau that segregated the

population into apparent responders and apparent non-responders (Figure 3). A threshold of 29

ng/mL (95% confidence interval 26 ng/mL to 78 ng/mL) serum TxB2 resulted in an accuracy of

98 % and an error rate of 2%.

The congruence between arachidonic acid induced aggregation and serum TxB2

formation for the diagnosis of “non-response” was high. One-hundred five of 108 (97%) subjects

classified as “non-responsive” by arachidonic acid induced platelet aggregation in Phase 1 also

showed incomplete suppression of serum TxB2 formation. However, measurement of serum

TxB2 identified 32 of 400 (8 %) individuals as “non-responders” who had not been recognized

by arachidonic induced platelet aggregation. Most of these had serum TxB2 levels between 10

and 100 ng/ml, which represents a partially suppressed range when compared to median pre and

post dose concentrations of 4 ng/ml (IQR: 2 – 133 ng/ml) and 315 ng/ml (IQR: 193 – 450

12

nhibition” based on the comparison of pre and post dose concentrations) is an aassssesesssmmmennent tt thththatat

could be performed in patients without stopping aspirin therapy. Analysis of the frequency

diiststtririribububutititiononon oof thhheee ababsolute serum TxB2 concentttrarar tiiion also revealaledee a a plplplaatateau that segregated the

poppupulation intto o aaapppaparereentntt rreseespopopondndererersss aanndnd apppparrrentt nnoon---rereespsppononondederrrs ((FiFigguurrere 333)).). AA ttthrhrhresese hohh ldldd oof ff 2229

ngng/m/m/mLLL (9(9(95%5%5% ccononnfiidedenncncee e ininteervrvr alalal 222666 ngngg/m/mmLL L ttoto 77788 ngngng/m/mmL)L)L) ssseeerumumum TTxBxBxB22 rresessululteteted dd inin aaannn accccuuuraracyccy oof

98 % and an n erererroror r r r rararatetee ooof 2%2%2%...


ng/ml).

Analysis of the urinary TxM excretion data did not result in a threshold value that would

allow for distinction of “responders” from “non-responders” at the individual level with any

reasonable accuracy (Figure 3).

Discussion

Aspirin blocks just one pathway of platelet activation and treatment failures may result from the

dominant role of other platelet agonists; 11, 12, 32, 33 combination of aspirin with other antiplatelet

drugs, such as antagonists of the P2Y12 receptor for ADP, sometimes confers a measurable

incremental benefit. 34 Other reasons for treatment failure include failure to consume the

medication, 10, 35 accelerated platelet turn over, 36, 37 or interaction with reversible, competitive

active site inhibitors of the COX-1 enzyme, such as ibuprofen 13 or naproxen. 14

The term “aspirin resistance” is often used to imply treatment failure although clinical

outcomes have rarely been assessed – not unlike studies of the pharmacogenomics of

clopidogrel. 38 More commonly, a patient is classified as “resistant” to aspirin, if platelet

aggregation, measured on a single occasion, is not depressed to an arbitrary degree. Estimates of

the frequency of “aspirin resistance” have varied from 5% to 20% in most studies. 16-20 Here we

wished to determine the commonality of an internally consistent, specific phenotype of

pharmacological resistance to aspirin that was stable over time, such as might result from gene

variants in aspirin metabolizing enzymes or in the platelet COX-1-TxA2 synthesis/response

network. This might be more readily discernible in a younger population without confounding

disease variables and would be measurable by tests that reflected the mechanism of drug action.

The irreversible acetylation of platelet COX-1 is a sufficient mechanism to explain the

13

ncremental benefit. 34 Other reasons for treatment failure include failure to conssumumume e thththee

medication, 10, 35 accelerated platelet turn over, 36, 37 or interaction with reversible, competitive

acctitiiveveve sssitititeee inininhhhibiitototorsrs of the COX-1 enzyme, suchhh asss ibuprofen 13 ooor r naaprprproooxen. 14

The teermrm ““asaspipip riinn n rereresisisistststanancecece””” iiss oooftennn uuuseddd ttoo impmpmplylyy trrereatatmementnt fffaiailulureree aaaltlthohoouguggh hh clclinininiccicalal

ouutctctcomomomeses hhhavavave e rraarereelylyy bbeeeeenn asasssesessssssededed – nnnototot uuunnnlikikikee ssstuuudiieies s ofofof tthehehe pphahaarmrmrmaaacogogogenennomommiciccss s ooof

clopidogrel. 383838 MMMororore e e cococommmmmmonononlyyy,, a a a papapatienenent t t isiss ccclalalasssssififfieieed d asasas “rereresisisistststanana t”t tttooo asasaspipipiririr n,n,, iiif f f plplplatatatele et


cardioprotection afforded by aspirin, however direct assays 39 have yet to be deployed to

characterize resistance. The immediate consequence of drug action is suppression of formation

of platelet TxA2, quantitatively reflected by the analysis of its inactive hydrolysis product, TxB2

in serum generated under controlled conditions. 31 Measurement of serum TxB2 reflects dose

dependent suppression by aspirin, cumulative inhibition during repeated daily dosing and

delayed recovery of platelet thromboxane formation after cessation of aspirin, driven by platelet

turnover time. 40 Urinary Tx metabolites also reflect the pharmacodynamic impact of aspirin on

platelets41 and, indeed, derive largely (~ 80%) from platelets under physiological conditions.42

However, other non-platelet sources of Tx biosynthesis are also reflected by urinary metabolites

and their contribution may increase under conditions of disease or physiological perturbation. 43

Here, we found that large variability in urinary TxM excretion renders it impracticable to assess

even a healthy individual’s aspirin response status although the reduction in mean urinary TxM

by aspirin was clearly detectable in a group of treated individuals. Finally, platelet aggregation

responses induced by the COX enzyme substrate, arachidonic acid, depend on its transformation

to TxA2 and are suppressed by aspirin. Although other agonists aggregate platelets directly,

secondary release of TxA2 may amplify the response and result in partial inhibition of the

aggregation signal by aspirin. 7 A complication is that the relationship between suppression of

platelet thromboxane and aggregation is very nonlinear – the capacity of platelets to generate

thromboxane has to be suppressed by >95% before functional inhibition of the aggregation

response is attained. 29 This explains why diagnostic approaches which tested the COX pathway

directly had the most favorable test sensitivities and false classification rates for identification of

“non-responders”.

We found that under acute dosing conditions, the frequency of “resistance” based solely

14

and their contribution may increase under conditions of disease or physiologicall pppererrtuturbrbbatatatioioion.n.n 43

Here, we found that large variability in urinary TxM excretion renders it impracticable to assess

evvenenen aaa hhheaeaaltltlthyhyh indndndivivi idual’s aspirin response stattususu aaalthough the reeedud ctctioioionnn in mean urinary TxM

bbyy aaasps irin wass cclell aaarlyly ddeteetececectatatablbble e ininin aa gggroooup offf treeaatteed ininindidivvviddudualalls. FFininalaa llyy,, pplplaatateleletetet aaaggggrereggagatititionnn

eespspsponononsesess ininindudduceceed d bybyy tthhee CCOXOXOX eeenznznzyymymee e sususubsbsbstrrratatate,e,e, aarracchchidididoononiccc aacccid,d,d dddepepepeennd d ononon iitsts ttrraransssfooormrmaaatiioion

o TxA2 andd aaarerere ssupupupprprpresessseed d d bybyb aaaspspspiriririni ... AlAlAlthththouououghghgh oooththhere agagagonononisisistststs aaagggggrereregagagatetete plplp atatteleleletetets s s didid rectly,


on the failure to suppress the aggregation response to arachidonic acid was conditioned by the

formulation of aspirin and the timing of the measurement. “Resistance” was measurable after

enteric coated, but not immediate release formulations of aspirin and appeared more frequent

after the former when measurements were performed 4 hours rather than 8 hours after dosing.

This pattern of variance was reflected both by the aggregation response to arachidonic acid and

by serum TxB2. By contrast, aspirin had minor effects on aggregation induced by ADP and

collagen and no detectable effect on the activation of aggregation through the thrombin pathway

by PAR-1AP. The incidence of apparent “resistance” to enteric coated aspirin based on a single

measurement of aggregation was 49% at 4 hours (Group 3) dropping to 17% at 8 hours (Group

2) after dosing in Phase 1 of the study. Strikingly, there was marked discordance between the

individuals who seemed “resistant” on the two occasions; only in 20% at 4 hours and 6% at 8

hours was “resistance” stable over time. Consistent with the suggestion that apparent

“resistance” was largely reflective of variable drug exposure in the patients receiving enteric

coated aspirin, we corrected the phenotype in 101 of 108 cases by adding aspirin ex vivo in

Phase 1 or 2 and all of the remaining 7 cases responded to adding aspirin ex vivo in Phase 3.

In the Phase 3 of the study we compared cases who had remained “resistant” to inhibition

of platelet aggregation by ingestion of aspirin in the first two phases of the study and matched

them by gender and age with controls, then subjecting both groups to a weeklong administration

of aspirin and clopidogrel in a cross over design. Because 15 of the original 400 subjects who

fulfilled the inclusion criteria for Phase 3 were lost-to follow up, we may have underestimated

the incidence of true biological resistance. However, of the 27 cases enrolled in this phase only

one appeared now to be “resistant” to aspirin as assessed by arachidonic acid induced

aggregation and this individual had responsiveness restored by addition of aspirin ex vivo. While

15

2) after dosing in Phase 1 of the study. Strikingly, there was marked discordancee bbetete weweweenenen ttthehehe

ndividuals who seemed “resistant” on the two occasions; only in 20% at 4 hours and 6% at 8 t

hoourururs s wwawasss “r“r“reeesisstatatanncnce” stable over time. Consistetetentnt with the suggggesee tiononn ttthhat apparent

““rresssisi tance” wwasas laaargegeelyyy rrefefefleleecctctivivee e oofof vvvaarriabblle druug expppoososururre inin ttthehee ppatattieientnts s rereececeivivvininng g eenenteteteriiriccc r

cooatatatededed aaspspspiriririninin, , wwwe ccororrrerectctc eded ttthehee ppphehehenonootytytypepepe iiin 11101011 ooof f 11010888 ccacaseees ss bbyby aaaddddd iiingg g asasspipipiririr n n exexex viivvoo o inin n

Phase 1 or 22 aaandndnd aaallllll oof f f ththt ee rereremamamainininininng gg 7 7 7 cacacasesesess rererespspspononondeded d d d tototo aaaddddddinining g asasaspipipiriririn n n exexex vvvivivivo o o ininin PhPP ase 3.


this was consistent with “resistance” due to limited drug exposure in vivo, as might be caused by

genetic variants in aspirin metabolism, the effect was not specific to aspirin: the volunteer failed

also to exhibit clopidogrel induced inhibition of ADP induced platelet aggregation. The

unreliability of basing a diagnosis of “aspirin resistance” on a single measurement of platelet

aggregation was further emphasized by two individuals who appeared “resistant” amongst the

controls in Phase 3, having been responsive to aspirin in the two earlier phases of the study.

Here, again, responsiveness to aspirin was apparent by addition of the drug ex vivo and the

subject was also unresponsive to clopidogrel. Thus, such “resistance” may have reflected a

failure to ingest aspirin.

In summary, we performed studies in 400 healthy volunteers seeking to determine the

commonality of a mechanistically consistent, temporally stable and specific phenotype of true

aspirin resistance, such as might be attributable to genomic variation in aspirin metabolism or in

the platelet COX-1 / TxA2 synthesis response pathway. We failed to find a single person who

satisfied these criteria. By contrast, pseudoresistance, due to delayed and reduced drug

absorption was common after ingestion of enteric coated aspirin. 25, 26 These observations

question the value of seeking to diagnose aspirin resistance with single point-of-care diagnostic

approaches and support the finding of inconsistent platelet inhibition following enteric coated

preparations of aspirin. 44-46

Acknowledgements: G.A.F. is the Robert McNeill Professor of Translational Medicine and

Therapeutics.

Funding Sources: This work was supported by the National Heart Lung and Blood Institute (HL

54500), the National Center for Research Resources (UL1-RR-024134), the American Heart

Association (T.G.: Scientist Development Grant 0430148N), and Bayer Health Care

16

In summary, we performed studies in 400 healthy volunteers seeking to dddeteteree mimiminenene ttthehehe

commonality of a mechanistically consistent, temporally stable and specific phenotype of true

asspipipiriririnn n reresisisistststaaancecee, , susuch as might be attributable tototo gggenomic variaatitiiono iinn n aasaspirin metabolism or in

hhhe pplatelet COXOXX-111 // TTTxAxAA22 ssynynynththesesesisiss rresespponnssee patthhwwway.y.y. WWeee ffafailileeded tto o fififinddd aa ssininnglgleee pepeersrsononn wwhohoho

aatititisfsfsfieieiedd ththhesesesee crcrriiteereriaia. BByBy cononontrtrrasasast,t,t, ppseseeudududorororesesisisistatancncnce,e, ddueueue tttoo dededelalaayeyeed d d aanandd d reredududucecec d d d drdrdrugugg

absorption wwasasas cccomomommomomon nn afaffteteter r r innngegegessstitit onnn oooff f enenentetet ririr c cc cococoata ededd aaaspspspirirrininin... 252525,, 262626 TTThehehesesese ooobsbsbsererervavavatitions


(Morristown, NJ, Study No X05-215). The funding sources were not involved in the study

design, or in the collection, analysis, and interpretation of data.

Conflict of Interest Disclosures: T.G. received consultancy fees from PLx Pharma. S.F. has no

potential conflict of interest. J.A.L. has no potential conflict of interest. S.C.K has no potential

conflict of interest. G.R.G has no potential conflict of interest. G.A.F. received research funding

from Bayer Health Care to support partial funding of this study.

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27277. PPaP trignaanini P,, PPPanaararaa MMRRR, GGreecooo AA, FFFusccco O, NaNaN totolilili CCC, IaIaacobbebellli S,, CCCippolollolol nen FFF, GaGaanncci AAA, CrCremememinininonon CCC,, MaMaaclllououff f J,J, PPatatrororononoo CCC.. BiBiiococchehehemmimicacacall aanndnd ppphahaharmrmrmacacacolollogogogicicicalall chhhararaacacteteteririzazazattitionnn ooof f ththhecyclclooooxygegenan sese actctiviviti y ofof hhumumanan bbloodd pprrosostataglglandidinn enendodopeperoxixidede ssynththasa ese .. J J Phhara mamacocol Exp Ther. 199949494;2;2;2717171:1: 7070705-17171712121 . ..


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33. Folts JD, Schafer AI, Loscalzo J, Willerson JT, Muller JE. A perspective on the potential problems with aspirin as an antithrombotic agent: A comparison of studies in an animal model with clinical trials. J Am Coll Cardiol. 1999;33:295-303.

34. Mehta SR, Yusuf S, Peters RJ, Bertrand ME, Lewis BS, Natarajan MK, Malmberg K, Rupprecht H, Zhao F, Chrolavicius S, Copland I, Fox KA. Effects of pretreatment with clopidogrel and aspirin followed by long-term therapy in patients undergoing percutaneous coronary intervention: The pci-cure study. Lancet. 2001;358:527-533.

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36. Pascale S, Petrucci G, Dragani A, Habib A, Zaccardi F, Pagliaccia F, Pocaterra D, Ragazzoni E, Rolandi G, Rocca B, Patrono C. Aspirin-insensitive thromboxane biosynthesis in essential thrombocythemia is explained by accelerated renewal of the drug target. Blood. 2012;119:3595-3603.

37. Rocca B, Santilli F, Pitocco D, Mucci L, Petrucci G, Vitacolonna E, Lattanzio S, Mattoscio D, Zaccardi F, Liani R, Vazzana N, Del Ponte A, Ferrante E, Martini F, Cardillo C, Morosetti R, Mirabella M, Ghirlanda G, Davi G, Patrono C. The recovery of platelet cyclooxygenase activity explains interindividual variability in responsiveness to low-dose aspirin in patients with and without diabetes. J Thromb Haemost. 2012, 10:1220-1230.

38. Holmes MV, Perel P, Shah T, Hingorani AD, Casas JP. Cyp2C19 genotype, clopidogrel metabolism, platelet function, and cardiovascular events: A systematic review and meta-analysis. JAMA. 2011;306:2704-2714.

39. Li X, Fries S, Ricciotti E, Lawson JA, Blair AI, FitzGerald GA, Grosser T. Assessment of the pharmacological response to aspirin by quantitation of platelet cyclooxygenase acetylation. Circulation. 2010;122:A16834.

40. Pedersen AK, FitzGerald GA. Dose-related kinetics of aspirin. Presystemic acetylation of platelet cyclooxygenase. N Engl J Med. 1984;311:1206-1211.

41. Patrignani P, Filabozzi P, Patrono C. Selective cumulative inhibition of platelet thromboxane production by low-dose aspirin in healthy subjects. J Clin Invest. 1982;69:1366-1372.

42. FitzGerald GA, Oates JA, Hawiger J, Maas RL, Roberts LJ, 2nd, Lawson JA, Brash AR. Endogenous biosynthesis of prostacyclin and thromboxane and platelet function during chronic administration of aspirin in man. J Clin Invest. 1983;71:676-688.

20

36. Pascale S, Petrucci G, Dragani A, Habib A, Zaccardi F, Pagliaccia F, Pocaterrra a D,D,D RRagaggazazzozoniE, Rolandi G, Rocca B, Patrono C. Aspirin-insensitive thromboxane biosynthesisiss iin nn esssesesentntntiaiaialllhrombocythemia is explained by accelerated renewal of the drug target. Blood. 20200121212;1;11191919:3:3:35959595-5-5

3603.

377. . RoRoRocccccaaa B,BB, Sananntititilllli F, Pitocco D, Mucci L, Petttrurur ccci G, Vitacoloonnnn a E,E,E LLattanzio S, Mattoscio D,D,D, ZZZaccarddi FFF, LLiaiaianini RR,, VaVaVazzzzz anannaaa N,N,N DDelel PPonontete AA, FFeerrananntetete EEE,, MaMaMartrttininii F,F,F, CCarrdidillllllooo C,C, MMorororosossetetettiti R,MMirrarabella M, GGGhhih rrllanandadad GGG,, DaDaDavivi GGG,, , PaPaatrroonoo CCC. Thhhee recococoveverrry oof f plplatatelelleeet cccycycclololooxoxygyggenennasasse aacactititiviiity exexxplllaia ns intererini ddidivvviduuaal varriiaiabbbiliityt iiin nn rreressps onnnsiiivennnessss tto oo lololowww-dddossee aaspirrrinnn iinnn papaatitienttst wwiiithhh anddd wiwiithththouououtt didiabababeteeteses. JJ J ThThhroroombmb HHHaeaeemomomosstst. 202020121212,, , 1000:1:1: 2222200-12121 303030.

38. Holmes MMMV,V,V, PPPerererelell PPP, , ShShShahaha TTT,, HiHiHingnggorororananani i i ADADAD,,, CaCaCasas sss JPJPJP... CyCyCyp2p2p2C1C1C19 9 9 gegegenononotytyt pepepe,, , clclclopopopidi ogrelmemetatabobolilismsm plplatateleletet ffununctctioionn aandnd cacardrdioiovavascsculularar eeveventnts:s: AA ssysystetemamatiticc rereviviewew aandnd mmetetaa-ananalalysysisiss


43. Catella F, FitzGerald GA. Paired analysis of urinary thromboxane B2 metabolites in humans. Thromb Res. 1987;47:647-656.

44. Maree AO, Curtin RJ, Dooley M, Conroy RM, Crean P, Cox D, Fitzgerald DJ. Platelet response to low-dose enteric-coated aspirin in patients with stable cardiovascular disease. J Am Coll Cardiol. 2005;46:1258-1263.

45. Cox D, Maree AO, Dooley M, Conroy R, Byrne MF, Fitzgerald DJ. Effect of enteric coating on antiplatelet activity of low-dose aspirin in healthy volunteers. Stroke. 2006;37:2153-2158.

46. Peace A, McCall M, Tedesco T, Kenny D, Conroy RM, Foley D, Cox D. The role of weight and enteric coating on aspirin response in cardiovascular patients. J Thromb Haemost.2010;8:2323-2325.

Table 1. Demographics of the study population

Group 1 Group 2 Group 3 TotalGroup characteristics Aspirin formulation Plain EC ECAssessment time 8 hours 8 hours 4 hours Ethnicity / race (n)American Indian or Alaskan Native 1 1Asian 2 27 18 47Native Hawaiian or Pacific Islander 1 1Black (Non-Hispanic) 13 23 26 62Hispanic 1 10 8 19White (Non-Hispanic) 23 144 96 263Other or more than one race 1 5 1 7Totals 40 210 150 400Gender (%) Female 65 52 60 57Age (y) Median 28 25 27 261. Quartile 23 23 23 233. Quartile 34 30 35 31BMI (kg/m2)Median 24.0 23.4 24.0 23·51. Quartile 21.1 21.2 21.7 21·53. Quartile 28.8 26.0 26.2 26·3

21

Group 1 Group 2 Group 3 TotatallGroup characteristics Aspirin formulation Plain EC ECAssessment time 8 hours 8 hours 4 hoursEtEthnhnhnicicicititity y y / / / raraacee (((nn)n)AAmAmeererican Indnddiaiaannn ooro AAAlalalaskskskannan NNNatatativivive e e 111 111AAsA iaiaan 222 272727 18 4777NaNaatitit veveve HHawawwaiaiaiiaiannn ooror PPacacififi icic IIIslss ananandedederrr 111 111BlBlacackk (N(N( onon-H-Hisispapap ninic)c)) 1313 2323 2626 6262Hisppanic 11 11010 888 19191


Table 2. “Non-response” to administration of 325 mg aspirin in study Phases 1 and 2 as assessed by arachidonic acid induced platelet aggregation. Regular or enteric coated (EC) aspirin was administered and the response assessed either 8 or 4 hours post dosing. Platelet function was also measured in platelet rich plasma that was treated with 100 micromolar aspirin ex vivo for 15 minutes. “Non-responders” were arbitrarily defined as individuals who had an inhibition of platelet aggregation induced by arachidonic acid of less than 60% when comparing to pre dose assessments.

% (N)

Group 1 Plain aspirin,

8 hrs post drug N=40

Group 2 EC aspirin,

8 hrs post drugN=210

Group 3 EC aspirin,


Aspirin ex vivo - + - + - +

“Non-responders” in Phase 1

0(0/40)

0(0/40)

17(35/210)

0(0/210)

49(73/150)

12(18/150)

“Non-responders” in Phase 1 AND Phase 2

0(0/40)

0(0/40)

6(12/209)

0(0/210)

20(30/150)

4(6/150)

Table 3. “Non-response” to administration of 325 mg aspirin in study Phases 1 and 2 as assessed by serum TxB2 measurement. Regular or enteric coated aspirin was administered and the response assessed either 8 or 4 hours post dosing. “Non-responders” were arbitrarily defined as individuals who had less than 95% inhibition of serum TxB2, when comparing post and pre dosing assessments.

%(N)

Group 1 Plain aspirin,

8 hrs post drugN=40

Group 2 EC aspirin,


Group 3 EC aspirin,


Non-responders in Phase1

3(1/40)

24(35/210)

59(88/150)

Non-responders in Phase1 AND Phase 2

0(0/40)

7(14/199)

26(39/146)

22

“Non-responders” in Phase 1 AND Phase 2

0(0/40)

0(0/40)

6(12/209)

0(0/210)

202020(30/0/1515150)0)0)

444(6(6(6/1/1/1505050)))

TTaTabblble 3. “NNononon-r-r-resesespopoonsnsnse”e”e ttto o o adadadmimimininin stststrararatitit ononn ooof f 323255 mgmg aaaspspspiriririnini iiinnn ststtudududy y y PhPhP asseseses 111 aaandndn 222 aaas s asseeesss ed by seerurum m TxTxBB222 mmeaeaassusurer memementntt. RRReguullaar oorr eentererericic cccoatatededd aaaspiririnin wwaasas aadmdmmininisisi teteereed d aaanddd hhhe e rrerespponsee aaasseeesssed eieittherr 8 oror 4 hhhououursss pposst dossiinngg. “N“N“Nononn-rreesppponnndersss” weweereee aarbr iittraraaririlyly defffinined

asas iiindndndivivividididuauauallsls wwwhohoho hhhaadad lllesese s ththhanan 9995%55% iiinhnhnhibibibititi ioioion n n oofof ssserererumumum TTTxBxBB22,, whwhwhenenen ccomomompapapaririringngng pppososstt t anannd d d prprpre e edodosisingngg aassssesessmsmenentsts..


Figure Legends:

Figure 1. Study design (A) Phase 1 and 2; (B) Phase 3.

Figure 2. Response to 7 days of low dose aspirin (81mg, enteric coated) in Phase 3. Assessment

of platelet aggregation using arachidonic acid as the stimulus in the absence and presence of

aspirin (100 mol/L) added to isolated platelets ex vivo. (A) Individuals who qualified as “non-

responders” in Phase 1 AND Phase 2 (n=27). One subject showed uninhibited platelet

aggregation following one week of aspirin treatment, but responded to aspirin addition ex vivo.

(B) Individuals who were classified as responders on two occasions of single dose aspirin

administration in Phase 1 and Phase 2 (n= 25). Two subjects showed uninhibited platelet

aggregation but responded to aspirin addition ex vivo.

Figure 3. Relative cumulative frequency distributions of the arachidonic acid (325 mg) induced

platelet aggregation response (A-C), serum thromboxane formation (D-F) and urinary

thromboxane metabolite excretion (G-I) stratified by drug formulation and assessment time. The

density of the data points is visualized by color coding – green high density/frequency, red – low

density /frequency. Classification thresholds (“responders” vs. “non-responders”) as determined

iteratively using random forest classifiers are shown as blue arrows, which also depict the

fraction of responders in the study groups. Urinary TxM excretion failed to predict individual

response status although pre- and postdose distributions segregated in each group (p<0.05).

Figure 4. Consistency of the response to distinct stimuli of platelet aggregation. Four stimuli

23

B) Individuals who were classified as responders on two occasions of single doossese aaaspppirirrininin

administration in Phase 1 and Phase 2 (n= 25). Two subjects showed uninhibited platelet

agggrgrgregegegatatioioion nn bbbut rereresspsponded to aspirin addition exx vvvivvo.

FiFigugugureree 333.. RRReleelatativiviveee cucuumumumulalativeveve fffrererequququenencycycy dddisisistrribibibutututioononss ofoff ttthhhe aaarrarachchchidididononnicc aaciciid d d (3(3(3252525 mmgg)g) iiindnduuuceeded

platelet aggrereregagagatitit ononon rrresese popop nsnsnseee (A-A-A-C)C)C), seeerururum m m thththrororombmbmboxoxoxannne ee fofoformrmrmatatatioii nn n (D(D(D-F-F-F))) anana d d d urururinininararary yy


24

were used to assess the functional impact of aspirin treatment on platelet aggregation:

arachidonic acid (A), collagen (B), adenosine diphosphate (ADP, C) and protease activated

(thrombin) receptor 1 activating peptide (PAR-1 AP, D). Data are displayed as scattergrams of

slope vs. maximum aggregation. Data points are color coded by the response status as

determined by 95% inhibition of serum TxB2 concentration. Thus, “responders” are plotted in

green and “non-responders” in red. Shades of gray represent densities of the two-dimensional

distribution. Classification thresholds as determined iteratively using random forest classifiers

and 95% confidence intervals are shown in blue. (A) The test sensitivity of arachidonic acid

induced platelet aggregation was >90% with an error rate of 10% when volunteers with a

residual platelet function following aspirin administration of 16% maximal aggregation (95%

confidence interval 12% to 62%) or 11%/min slope (95% confidence interval 8%/min to

17%/min) were classified as “non-responders”. (B, C) Inducing platelet aggregation with

collagen or ADP resulted in sensitivities of 80% and 75% and error rates of 25% and 36% at

thresholds of 41%/min slope (95% confidence interval 39%/min to 42%/min) and 75% maximal

aggregation (95% confidence interval 72% to 76%), respectively. Maximal aggregation was not

a predictor of response status in collagen stimulated platelets; slope was not a predictor of

response status in ADP stimulated platelets. (D) As expected, the PAR-1AP response did not

associate with inhibition of serum Tx formation by aspirin.

esidual platelet function following aspirin administration of 16% maximal aggrregeegatatatioioon nn (9(9(95%5%5%

confidence interval 12% to 62%) or 11%/min slope (95% confidence interval 8%/min to

177%/%/%/mimimin)n)n) wwweeere clclclaasassified as “non-responders”. (((BB,B C) Inducing ppplall teleleett t aaaggregation with

coollllaga en or ADDP PP rreesusultl ededd iiinn n sesesensnsitittivivviti iieesss of 88000% aaanndd 7755%% % aanand d ererrrooor rarattet sss ofoff 2225%5% aaandndnd 3336%%% aaat t

hhrereeshshhololo dsds ooof ff 4141%/%/%/miminnn sslslopope e (9(9(95%5%5% cccononnfifiidededennnceee inini ttterrvrvaalal 3339%9%9%/mmmininn tooo 42424 %/%/%/mimiin)n)n aaandndd 7775%5%% mmmaxaxximimmala

aggregation (9(9(95%5%5% ccconononfififidddennncecece intntnterere vavaval 727272% %% tototo 77766%)%)%),,, rer spspspececectitiivevevelylyly. MaMaMaxixiximamamal l l agagggrgrgregegegatatatioioi n was not

Phase 1Test dose: 325 mg aspirin p.o.

“Responders”Point-of-care test: AA induced aggregation

Criterion: Inhibition of Aggregation ≥ 60%

Subjects: n=292

Post-hoc test: Serum TxB2, urinary TxM

“Non-Responders”Point-of-care test: AA induced aggregation

Criterion: Inhibition of Aggregation < 60%

Subjects: n=108


Formulation: EC aspirin

Assessment: pre dose,

4 hours post dose

Subjects: n=150

Group 3Formulation: EC aspirin


8 hours post dose

Subjects: n=210

Group 2Formulation: Plain aspirin


8 hours post dose

Subjects: n=40

Group 1

Phase 2Test dose: 325 mg aspirin p.o.



Subjects: n=292




Subjects: n=108


Formulation: EC aspirin


4 hours post dose

Subjects: n=120

Group 3Formulation: EC aspirin


8 hours post dose

Subjects: n=125

Group 2Formulation: Plain aspirin


8 hours post dose

Subjects: n=12

Group 1

7 “Non-responders”

discontinued study

133 “Responders”

completed study

“Responders”

in Phase 1 AND Phase 2

n=120

“Non-responders”


n=42

“Responders”

in Phase 1 OR Phase 2

n=95

9 “Responders”

discontinued study



n=42

“Responders”


n=120

45 Matched “Responders”

invited to participate in Phase 3

42 “Non-responders”

invited to participate in Phase 3

18 Discontinued study

1 Lost to follow up

2 Exclusion criteria

15 Matched case

discontinued

15 Discontinued study

11 Lost to follow up


2 Subject request

Period 1

Aspirin 81 mg for 7 days

or

Clopidogrel 75 mg for 7 days

Period 2

Aspirin 81 mg for 7 days

or

Clopidogrel 75 mg for 7 days

2 Discontinued study1 Lost to follow up


27 “Non-Responders”completed Phase 3

25 “Responders”

completed Phase 3

Cro

ss ove

r

Phase 3

A

B

matched by age, gender, race

Figure 1



Subjects: n=292




Subjects: n=108


“Respondponders”er

in Pn Pin Phasehase 11 ANDANDAND PhasPhasPh e 2e 2

n=1n==120

“Nonon-r-res-respondponders”ers”

in Phahashase 1 ANDANDAND PhaPhase 2se 2e 2

n==4242

“ResRe ponpondderrss”

ininin PhasPhasP e 1e OORR PPhhase 2

n=9955

“Non-respondeon rs”

inin PPhasehasehase 1 ANNDND PhPhahase 2sese 2

n=42

“Responders”

in Pin Pn Phasehasehas 1 ANDANDAND PhasPhasPhasee 22

n=120

B

Figure 2

Group 3Coated aspirin - 4 hrs

Group 2Coated aspirin - 8 hrs

Group 1Plain aspirin - 8 hrs

100

100

100

0 25 50 75 100

025

5075

Max. Aggregation (%)

Cum

ulat

ive F

requ

ency

(%)

predose

postdose

postdose

predose

postdose

postdose

100

025

5075

Serum TxB2 (ng/mL)

Cum

ulat

ive F

requ

ency

(%)

100

025

5075

Serum TxB2 (ng/mL)

Cum

ulat

ive F

requ

ency

(%)

100

025

5075

Serum TxB2 (ng/mL)

Cum

ulat

ive F

requ

ency

(%)

025

5075

TxM (ng/mg Creatinine)

Cum

ulat

ive F

requ

ency

(%

025

5075

TxM (ng/mg Creatinine)

Cum

ulat

ive F

requ

ency

(%

025

5075

TxM (ng/mg Creatinine))

Cum

ulat

ive F

requ

ency

(% 100

100

100

predose

postdose

predose

postdose

predose

postdose

0.1 1 10 100 1000 0.1 1 10 100 1000 0.1 1 10 100 1000

0.1 1 10 0.1 1 10 0.1 1 10

Agg

rega

tion

(AA

)Se

rum

TxB

2U

rinar

y Tx

M

A B C

D E F

G H I

0 25 50 75 100

025

5075


Cum

ulat

ive F

requ

ency

(%)

predose

postdose

predosepredose

0 25 50 75 100

025

5075


Cum

ulat

ive F

requ

ency

(%)

predose

postdose

“responders”

“responders”

“responders”

“responders”“responders”

“responders”

Figure 3

predose 0

Serumum TxB2 (ng/mL)

Cum

u

0

Serum TxB2 (ng/mL)

Cum

u

0

Serum TxB2 (ng/mL)

Cum

u

025

50775

TxMTxM (n(ng/mg/mg Cg Creareatintinineine))

Cum

Cul

aul

ative

Fere

qeque

nue

cy (%

025

5075

TxMTxM (n(ng/mg/mg Cg Creareatintinineine))

umC

ula

ulat

itivF

e Fr

eqenue

cy (%(%

025

5075

TxMTxM (n(ng/mg/mg Cg Creareatintinineine))))

umC

ula

ulat

ive F

requ

ency

(%% 100

100

100

10000

prepredp osee

postostdosedos

predprepredososese

posposstdoosse

predprededoseose

postpos dosedos

0.1 1 10 100 1000 0.1 1 10 100 1000 0.1 1 1010 100100 1000

0.10.1 1 1010 0.10.1 1 1010 0.10.1 1 10

GGG H I

predpredoosepredose

0 20 40 60 80 100

020

4060

80Max Aggregation (%)

Slo

pe (%

x m

in-1)

0 20 40 60 80 100

020

4060

80

Max Aggregation (%)

0 20 40 60 80 100

020

4060

80

Max Aggregation (%)

0 20 40 60 80 100

020

4060

80

Max Aggregation (%)

A

B

C

D

Arachidonic Acid

Collagen

ADP

PAR1-AP




Slo

pe (%

x m

in-1)

Slo

pe (%

x m

in-1)

Slo

pe (%

x m

in-1)

Figure 4

Max Aggregation (%)

0 202 40 660 800 1000

020

040

6080

MMax AgA gre tgatiion (%(%))

800

C

D

ADPAAAAAAAAAAAAAAAAAAAAAAAAAAAADADADADADADADADADDDDDDDDDDDDDDDDDDDDDDDDDDDDPPPPPPPPPPPPPPPPPPPPPPPPPPP

PAR1-APPPPPPPPPPPPPPPPPPAPAPAPAPAPAPAPAAAAAAAAAAAAAAAAAAAAAAAAARRRRRRRRRRRRRRRRRRRRRRRRR1R1R1R1R1R1R1R111111111----AAAAAAAAAAAAAAAAAAAAAAAAAPAPAPAPAPAPAPAPPPPPPPPPPPPPPPPP

“N“Non--“““““““““““NNNNNN“N“N“N“N“NNNNNNNNNNNNNNNNNNNNNNNoooooooooooooooooonononononononnnnnnnnnnnnnnn------resspoonderss”rrrrrrrrerererererereeeeeeeeesesesesesesessssssssssssssssssssssppppppppppppppppppoooooooooooooooooooooonononononononnnnnnnnnnnnnnndddddddddddddddddddedededededededdeeeeeeeeeeeeeerrrrrrrrsrsrsrsrsrsrssssssssssssss”ss”””””””S

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Drug Resistance and Pseudoresistance: An Unintended ...phdres.caregate.net/jclub-articles/Sbong.11513.pdfEnteric coating retards aspirin absorption markedly and renders this process