See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/328971785

The association between clopidogrel and 2-oxo-clopidogrel plasma levels and

the long-term clinical outcome after acute myocardial infarction

Article  in  International journal of clinical pharmacology and therapeutics · November 2018

DOI: 10.5414/CP203190

CITATIONS READS

0 41

7 authors, including:

Milan Pavlović Svetlana Apostolović

Klinički Centar Niš Cardiology clinic
103 PUBLICATIONS   185 CITATIONS    80 PUBLICATIONS   205 CITATIONS   

SEE PROFILE SEE PROFILE

Dragana Stokanovic
University of Niš
26 PUBLICATIONS   56 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Ev.No. 1991 View project

Herbal and synthetic bioactive products of the new generation View project

All content following this page was uploaded by Dragana Stokanovic on 11 December 2018.

The user has requested enhancement of the downloaded file.

The association between clopidogrel and 2-oxo-
clopidogrel plasma levels and the long- term clinical
outcome after acute myocardial infarction

Milan Pavlovic1,2, Svetlana Apostolovic1,2, Dragana Stokanovic3, Jelena Lilic4, Sandra S.

Konstantinovic5, Jelena B. Zvezdanovic5, and Valentina N. Nikolic3

1Department of Internal Medicine – Cardiology, Medical Faculty, University of Nis, 2Clinic for
Cardiovascular Diseases, Clinical Centre Nis, 3Department of Pharmacology and Toxicology,
Medical Faculty, 4Medical Faculty, University of Nis, Nis, and 5Department of Chemistry, University
of Nis, Faculty of Technology, Leskovac, Serbia
Correspondence to Valentina N. Nikolic
Department of Pharmacology and Toxicology, Medical Faculty, University of Nis, Bulevar dr Zorana Djindjica 81, Nis,Serbia
valentina@ medfak.ni.ac.rs

Key words clopidogrel – acute myocardial infarction –clinical outcome – coronary artery disease

Abstract. Background and objectives: A significant number of ischemic events occur after acute
myocardial infarction (MI), even when adhering to dual antiplatelet therapy including aspirin and
clopidogrel. The aim of our study was to investigate the association between the concentration of the
prodrug clopidogrel and its intermediary metabolite 2-oxo-clopidogrel plasma as well as demographic and
clinical factors, and the long-term clinical outcome in patients with their first acute MI, ST-elevation
myocardial infarction (STEMI) or non-ST-elevation myocardial infarction NSTEMI, treated with percutaneous
coronary intervention (PCI). Materials and methods: This study included 172 consecutive patients with their
first acute MI, 88 STEMI, and 84 NSTEMI, treated with PCI. On the third day of hospitalization, blood
samples were collected from each patient to measure the concentration of clopidogrel and its metabolite 2-
oxo-clopidogrel using the UHPLC-DAD-MS method. The following clinical outcomes were registered
during the 28-month follow-up: mortality from cardiovascular causes, nonfatal MI, nonfatal stroke, and
hospitalization for urgent myocardial revascularization or heart failure. Results: Lower dose-adjusted
clopidogrel concentrations (p < 0.05) were measured in NSTEMI patients with a com- posite of the hard
clinical endpoint events of cardiovascular mortality, non-fatal MI, or a nonfatal stroke. During the follow-up,
there was a 3.4 times higher risk of hard clinical endpoint events (p < 0.05) for each unit dec- rement of the
dose-adjusted clopidogrel plas- ma concentration. Lower dose-adjusted concentrations of clopidogrel in these
patients were associated with lower left ventricular ejection fraction (p < 0.001), and fentanyl (p < 0.001)
and pantoprazole administration (p < 0.01) during the acute phase of MI. Conclusion: In patients with acute MI
treated with PCI, lower dose-adjusted clopidogrel and dose-adjusted 2-oxo-clopidogrel plasma concentrations
were associated with an in- creased risk of ischemic events.

Introduction
Dual antiplatelet treatment with aspirin and clopidogrel is recommended for the secondary prevention of
ischemic events after acute coronary syndromes [1, 2]. However, a significant number of ischemic events
occur even when adhering strictly to this therapy [3, 4]. There is an interindividual as well as
intraindividual variability in response to clopidogrel. Various mechanisms leading to a poor response to
clopidogrel have not yet been fully clarified. Clopidogrel is a prodrug, and numerous studies have shown
that a response to clopidogrel might be caused by pharmacokinetic variables, such as intestinal absorption and
metabolic transformation in the liver, which are affected by genetic polymorphisms [5, 6, 7]. It has been
shown that clinical factors, such as obesity, insulin resistance, and the nature of a coronary event, may also
contribute to variability in the response to clopidogrel [8, 9, 10]. Numerous studies have shown that a poor
response to clopidogrel is associated with an unfavorable patient clinical outcome, especially after pecutaneous
coronary intervention (PCI) [11]. The objective of our study was to investigate the association between the
prodrug clopidogrel and concentrations of intermediary metabolite 2-oxo-clopidogrel determined on the
third day of hospitalization, and the long-term 28-month patient clinical outcome in patients with their first
acute myocardial infarction (MI), treated with PCI. We analyzed the association between the clopidogrel

•
and 2-oxo-clopidogrel concentrations and demographic, clinical, and angiographic factors as well as the
administered medicaments. The objective of our study was also to investigate the association between patients’
clinical characteristics and the occurrence of cardiovascular mortality, nonfatal MI, nonfatal stroke, or
hospitalization for urgent myocardial revascularization or heart failure after the 28-month follow-up.
Study population
This study included 172 consecutive patients with their first acute MI, treated with PCI. There were 88
patients wih ST-elevation myocardial infarction (STEMI) treated with primary PCI within 6 hours of the
chest- pain onset, and 84 patients with non-ST-elevation myocardial infarction (NSTEMI) treated within 72
hours after hospital admission. Patients were included in the study if they had symptoms consistent with acute
MI, ECG changes, and increased levels of serum markers of myocardial necrosis. No patients with previous
MI or patients with previous percutaneous or surgical myocardial revascularization were included in the study,
neither were any patients with surgical revascularization or patients with vascular events during initial
hospitalization enrolled.
All patients had medication, as well as PCI procedures, in accordance with the institution doctrine and
current European Society of Cardiology (ESC) and American College of Cardiology/American Heart
Association (ACC/AHA) guidelines. Dual antiplatelet treatment consisted of acetylsalicylic acid and
clopidogrel in all patients included in the study. The clopidogrel loading dose was 600 mg in 119 patients
(69.2%), and 300 mg in 53 patients (30.8%), and the aspirin loading dose was 300 mg in all patients. Both
STEMI and NSTEMI patients received an oral antiplatelet loading dose immediately after diagnosis was
established. The clopidogrel maintenance dose was 75 mg in 151 pa- tients (87.8%) and 150 mg in 21 (12.2%)
patients during the first week of treatment, and 75 mg after the first week. During follow-up visits, special
attention was paid to the patients’ adherence, and the necessity for strict adherence to the prescribed
treatment was emphasized. The research was performed following the principles highlighted in the 1975
Helsinki Declaration and was approved by the Ethics Committee of the Faculty of Medicine, University of
Nis, Nis, Serbia. Informed consent was obtained from all the patients before their inclusion in the study.

Materials and methods

On the third day of hospitalization, blood samples were collected from each patient 2 hours after
clopidogrel administration. A sample of plasma was used to measure the concentration of clopidogrel and its
metabolite 2-oxo-clopidogrel using the UHPLC- DAD-MS ((ultrahigh-performance liquid chromatography-
diode array detector-mass spectrometry) method, as described previously [12]. The following clinical
outcomes were registered: mortality from cardiovascular causes, nonfatal MI, nonfatal stroke, and
hospitalization for urgent myocardial revascularization and heart failure.

Statistical analysis
We used the Statistical Package for Social Sciences (SPSS 21.0; Chicago, IL, USA) for data analysis.
Continuous variables were pre- sented as the mean value with standard deviation (SD). The association
between continuous variables was tested using the Pearson or Spearman correlation test. The association
between two categorical variables was examined using the χ2 independence test with Yates’s correction
for continuity or based on the Fisher exact probability test. The method of standard multivariate linear
regression was used to create models predicting the dependent variable change based on multiple
independent variables. The method of Cox- regression was used to determine the significance of the
parameters for the occurrence of the clinical outcome events. The statistical significance was determined at p
< 0.05.

•
Table 1. Patient characteristics.

STEMI NSTEMI t or χ2 (p)

mean ± SD or N (%) mean ± SD or N (%)
Age (years) 59.86 ± 10.52 61.38 ± 11.92 0.886 (0.377)
Gender (male) 68 (77.3%) 55 (65.5%) 2.385 (0.122)
BMI (kg/m2) 27.13 ± 3.51 25.97 ± 3.95 1.491 (0.139)
DM 20 (22.7%) 28 (33.3%) 1.905 (0.168)
HTA 54 (61.4%) 54 (64.3%) 0.057 (0.811)
Smoking (current smokers) 39 (44.3%) 20 (23.8%) 7.137 (0.008)
CRP (mg/L) 11.57 ± 22.27 27.06 ± 40.52 2.971 (0.004)
Fibrinogen (g/L) 4.53 ± 1.64 4.81 ± 2.61 0.690 (0.492)
Creatinine (µmol/L) 94.68 ± 19.91 115.33 ± 61.63 2.926 (0.004)
Creatinine clearance (mL/min) 84.86 ± 22.96 75.75 ± 29.04 1.571 (0.120)
Urea (mmol/L) 6.03 ± 1.95 7.57 ± 4.23 3.048 (0.003)
Cholesterol (mmol/L) 5.65 ± 1.34 5.51 ± 1.39 0.663 (0.509)
LDL (mmol/L) 3.65 ± 1.11 3.62 ± 1.25 0.178 (0.859)
HDL (mmol/L) 1.19 ± 0.74 1.03 ± 0.31 1.808 (0.072)
Triglycerides (mmol/L) 2.22 ± 3.62 2.16 ± 3.49 0.104 (0.917)
AST (U/L) 194.79 ± 208.82 76.87 ± 82.00 4.793 (0.000)
ALT (U/L) 58.09 ± 64.13 31.52 ± 18.97 3.650 (0.000)
hs-TnI (ng/L) 25.52 ± 40.61 7.60 ± 20.43 2.421 (0.020)
Creatine kinase MB isoenzyme (U/L) 122.65 ± 131.18 72.66 ± 103.98 2.627 (0.010)
Platelet count (×109/L) 254.62 ± 73.78 251.65 ± 87.11 0.240 (0.811)
LVEF (%) 51.13 ± 11.56 50.61 ± 12.15 0.265 (0.791)
EDD (mm) 53.29 ± 5.63 51.24 ± 7.32 1.815 (0.072)
ESD (mm) 37.80 ± 7.24 36.64 ± 8.86 0.820 (0.414)
1-vessel disease 44 (50.0%) 38 (45.2%) 4.040 (0.133)
2-vessel disease 23 (26.1%) 17 (20.2%)
3-vessel disease 21 (23.9%) 29(34.5%)
LAD prox/med 53 (60.2%) 52 (61.9%) 0.000 (1.000)

Results
There were 172 patients with acute MI, 123 (71.5%) male and 49 (28.5%) female patients, with an average
age of 60.6 ± 11.2 years (from 30 to 89 years). We included 88 STEMI patients: 44 (50.0%) with 1-vessel, 23
(26.1%) with 2-vessel, and 21 (23.9%) with 3-vessel CAD, and 84 NSTEMI patients: 38 (45.2%) with 1-
vessel, 17 (20.2%) with 2-vessel, and 29 (34.5%) with 3-vessel CAD. 48 patients (27.9%) were previously
diagnosed with diabetes mellitus type 2, and 108 (62.8%) patients had arterial hypertension (Table 1). 59
patients (34.3%) were current smokers, while 39 (22.7%) were former smokers. The culprit coronary artery
lesion in STEMI patients was located on the LAD in 71 (41.3%) patients, on the LCx in 43 (25.0%), and on
the RCA in 58 (33.7%) patients.
The average follow-up period in patients with their first acute MI treated with PCI was 28.8 ± 18.2 months.
During this period, 24 (14.0%) patients died, in all cases due to cardiovascular causes. Nonfatal MI occurred
in 9 patients (5.2%), while 7 patients (4.1%) had a nonfatal stroke. In total, hard clinical outcome events were
observed in 32 patients (18.6%). 24 patients (14.0%) were hospitalized due to urgent revascularization during
the follow-up period and 71 patients (41.3%) due to heart failure. The composite outcome of mortality from
cardiovascular causes, nonfatal MI, nonfatal stroke, or hospitalization for urgent myocardial revascularization
or heart failure was registered in 76 patients (44.2%). There was no difference in the incidence of the events
registered between the STEMI and NSTEMI patients, except for heart failure hospitalization, which had a
higher incidence in NSTEMI patients (χ2 = 12.909, p < 0.001).

Clopidogrel and 2-oxo-clopidogrel plasma levels in association with

demographic and clinical factors

Considering that all patients were not on the same daily clopidogrel dose at the time of sample collection,
we calculated their dose-adjusted concentrations along with measurements of clopidogrel and 2-oxo-
clopidogrel concentrations. By performing both the univariate and multivariate linear regression modeling

•
method, we determined the factors predicting the dose-adjusted clopidogrel and 2-oxo-clopidogrel
concentrations (Table 2). The dose-adjusted concentration of clopidogrel may be predicted with the 3-
independent variable model, what accounts for 32.2% of its variance (F = 8.892, p < 0.001). Lower dose-
adjusted concentrations of clopidogrel were associated with a lower left ventricular ejection fraction (p <
0.001), higher cholesterol concentration (p < 0.001), and the presence of pantoprazole as comedication (p <
0.01). In the NSTEMI group, the most important predictors of a dose-adjusted clopidogrel concentration, up to
64.4% of this variable variance, may be explained by the model obtained by linear regression modeling (F =
19.556, p < 0.001). Three independent variables remained independently significant. Throughout the study
group, lower dose-adjusted concentrations are associated with a lower left ventricular ejection fraction (p
< 0.01), higher urea concentration (p < 0.05), and concomitant administration of pantoprazole (p < 0.001).
After a sequence of univariate linear regression analysis and subsequent multivariate regression modeling,
we obtained a model that explains 15.0% of the variance in the dose-adjusted 2-oxo-clopidogrel plasma
concentration (F = 8.230, p < 0.001) (Table 3). Two variables remained independently significant: lower 2-
oxo-clopidogrel con- centrations were measured in patients with a higher echocardiographically-determined
end-diastolic diameter (EDD) (p < 0.001), and concomitant administration of pantoprazole (p < 0.01). In the
NSTEMI group, where a dose-adjusted 2-oxo-clopidogrel concentration was taken as a dependent variable in
linear regression modeling, a statistically significant model (F = 12.707, p < 0.001) accounting for 31.0% of its
variance, was ob- tained. The dose-adjusted 2-oxo-clopidogrel concentration was lower in cotreatment with
pantoprazole (p < 0.001).

Table 2. Univariate and multivariate regression modeling of dose-adjusted clopidogrel concentration.

Dose-adjusted clopidogrel concentration in STEMI and NSTEMI patients

Univariate Multivariate
Adjusted R2 B p B SE Beta p
LVEF (%) 0.085 0.016 0.000 0.013 0.004 0.252 0.001
ESD (mm) 0.024 –0.014 0.043
Cholesterol (mmol/L) 0.052 –0.102 0.002 –0.109 0.033 –0.243 0.001
LDL (mmol/L) 0.037 –0.1.02 0.008
Platelet count (×109/L) 0.029 –0.001 0.016
Urea (mmol/L) 0.044 0.038 0.003
Pantoprazole 0.053 –0.466 0.001 –0.422 0.150 –0.208 0.006
ACE inhibitor/ARB 0.038 –0.254 0.006 –0.183 0.097 –0.142 0.061

Dose-adjusted clopidogrel concentration in NSTEMI group

Univariate Multivariate
Adjusted R2 B p B SE Beta p
LVEF (%) 0.116 0.021 0.002 0.015 0.006 0.251 0.010
Cholesterol (mmol/L) 0.076 –0.147 0.007 –0.088 0.050 –0.172 0.084
LDL (mmol/L) 0.050 –0.140 0.027
Platelet count (×109/L) 0.065 –0.002 0.012
Urea (mmol/L) 0.106 0.055 0.001 0.038 0.017 0.222 0.030
Pantoprazole 0.231 –1.139 0.000 –0.854 0.224 –0.367 0.000

•
 Table 3. Univariate and multivariate regression modeling of dose-adjusted 2-oxo-clopidogrel
concentration.

Dose-adjusted 2-oxo-clopidogrel concentration in STEMI and NSTEMI patients

Univariate Multivariate
Adjusted R2 B p B SE Beta p
LVEF (%) 0.050 0.166 0.003
EDD (mm) 0.063 –0.347 0.002 –0.381 0.117 –0.274 0.001
ESD (mm) 0.046 –0.250 0.008
Pantoprazole 0.036 –5.443 0.007 –7.034 2.610 –0.225 0.008
Amiodarone 0.055 6.722 0.001
Fentanyl 0.036 –3.419 0.010 –2.467 1.527 –0.136 0.109
Fentanyl dose (mL) 0.024 –1.592 0.030

Dose-adjusted 2-oxo-clopidogrel concentration in NSTEMI group

Univariate Multivariate
Adjusted R2 B p B SE Beta p
EDD (mm) 0.083 –0.485 0.010 0.122 0.180 0.150 0.501
ESD (mm) 0.053 –0.336 0.035 –0.183 0.144 –0.280 0.210
LDL (mmol/L) 0.045 –1.100 0.033 –0.587 0.518 –0.123 0.262
Pantoprazole 0.038 –7.929 0.041 –9.117 2.000 –0.501 0.000

Table 4. Clopidogrel and 2-oxo-clopidogrel concentrations in association with clinical outcome.

Dose-adjusted clopidogrel Dose-adjusted 2-oxo-clopido-

concentration (ng/mL/mg) grel concentration (ng/mL/mg)
Cardiovascular Yes
mortality mean ± SD 0.16 ± 0.23 1.52 ± 2.25
Md (IQR) 0.0 (0.0 – 0.3) 0.2 (0.0 – 3.4)
No
mean ± SD 0.21 ± 0.42 4.10 ± 9.98
Md (IQR) 0.0 (0.0 – 0.2) 1.1 (0.0 – 4.4)
Z (p) 0.155 (0.877) 1.278 (0.201)
Composite of Yes
cardiovascular mean ± SD 0.26 ± 0.42 1.63 ± 2.09
mortality, nonfatal Md (IQR) 0.1 (0.0 – 0.5) 0.3 (0.0 – 3.3)
myocardial infarction, No
or nonfatal stroke mean ± SD 0.19 ± 0.39 4.37 ± 10.21
occurrence Md (IQR) 0.0 (0.0 – 0.2) 1.3 (0.0 – 4.6)
Z (p) 1.085 (0.278) 1.116 (0.265)

Clopidogrel and 2-oxo-clopidogrel concentrations in association

with the clinical outcome
Using the Mann-Whitney U-test, there was no statistically significant difference in the clopidogrel and 2-
oxo-clopidogrel plasma concentrations when divided into two groups by cardiovascular mortality and the
composite of hard clinical endpoint events of cardiovascular mortality, nonfatal MI, or a nonfatal stroke, after
the 28-month follow-up(Table 4). Lower dose-adjusted clopidogrel concentrations (Z = 1.964, p < 0.05) were
measured on the third day of hospitalization in NSTEMI patients with the composite of hard clinical endpoint
events of cardiovas- cular mortality, nonfatal MI, or a nonfatal stroke. After the 28-month follow-up, there was
a 3.4 times higher risk of hard clinical endpoint events (p < 0.05) for each decre- ment of the dose-adjusted
clopidogrel plas- ma concentration. For each decrement of the dose-adjusted 2-oxo-clopidogrel concentra-
tion, there was a 1.04 times higher risk of heart failure development in the entire study group (p < 0.01), and in
the NSTEMI group (p < 0.05).

•
 Table 5. Cox-regression analysis of cardiovascular mortality.

Cardiovascular mortality in STEMI and NSTEMI patients

Univariate Multivariate
OR (95% CI for OR) p OR (95% CI for OR) p
Age (years) 1.086 (1.024 – 1.153) 0.006 1.091 (1.023 – 1.165) 0.009
Smoking 0.119 (0.016 – 0.915) 0.041
Creatinine (µmol/L) 1.007 (1.002 – 1.012) 0.010
Urea (mmol/L) 1.117 (1.043 – 1.196) 0.002
3-vessel CAD 3.989 (1.261 – 12.613) 0.019 3.884 (1.228 – 12.280) 0.021
Trimetazidine 4.610 (1.491 – 14.257) 0.008

Cardiovascular mortality in NSTEMI patients

Univariate Multivariate
OR (95% CI for OR) p OR (95% CI for OR) p
3-vessel CAD 5.895 (1.075 – 32.331) 0.041 5.406 (0.909 – 32.145) 0.064
Creatinine (µmol/L) 1.005 (1.000 – 1.011) 0.048
Urea (mmol/L) 1.091 (1.015 – 1.173) 0.018
Trimetazidine 7.539 (1.864 – 30.499) 0.005 10.988 (1.858 – 64.972) 0.008

Table 6. Cox-regression analysis of composite of cardiovascular mortality, nonfatal myocardial infarction, or nonfatal stroke
occurrence.

Composite of hard endpoint events in STEMI and NSTEMI patients

Univariate Multivariate
OR (95% CI for OR) p OR (95% CI for OR) p
Age (years) 1.079 (1.027 – 1.133) 0.002 1.076 (1.015 – 1.140) 0.014
CAD (N vessels) 1.974 (1.070 – 3.641) 0.029
3-vessel CAD 3.080 (1.155 – 8.217) 0.025
Cholesterol (mmol/L) 0.699 (0.490 – .996) 0.048
Creatinine (µmol/L) 1.006 (1.002 – 1.011) 0.009
Creatinine clearance (mL/min) 0.967 (0.942 – 0.993) 0.012
Urea (mmol/L) 1.106 (1.037 – 1.179) 0.002
DM type II 2.513 (1.021 – 6.190) 0.045 3.553 (1.230 – 10.262) 0.019
Amlodipine 2.836 (1.019 – 7.896) 0.046
Trimetazidine 3.958 (1.495 – 10.481) 0.006 3.515 (1.166 – 10.599) 0.026

Composite of hard endpoint events in NSTEMI patients

Univariate Multivariate
OR (95% CI for OR) p OR (95% CI for OR) p
Age (years) 1.078 (1.005 – 1.156) 0.036
CAD (N vessels) 3.312 (1.107 – 9.909) 0.032
3-vessel CAD 6.947 (1.344 – 35.917) 0.021 6.137 (1.098 – 34.284) 0.039
Dose-adjusted clopidogrel concentration 3.388 (1.056 – 10.865) 0.040
Creatinine (µmol/L) 1.006 (1.000 – 1.011) 0.036
Creatinine clearance (mL/min) 0.952 (0.912 – 0.993) 0.023
Urea (mmol/L) 1.096 (1.021 – 1.177) 0.012
Fibrinogen 2.135 (1.032 – 4.419) 0.041
Trimetazidine 8.627 (2.361 – 31.529) 0.001 12.679 (2.324 – 69.183) 0.003

•
 Table 7. Cox-regression analysis of the composite clinical outcome events of cardiovascular mortality,
nonfatal myocardial infarction, nonfatal stroke, or hospitalization for urgent myocardial revascularization
or heart failure occurrence.

Composite clinical outcome events in STEMI and NSTEMI patients

Univariate Multivariate
OR (95% CI for OR) p OR (95% CI for OR) p
CAD (N vessels) 1.544 (1.105 – 2.158) 0.011 1.145 (0.739 – 1.775) 0.545
1-vessel CAD 0.492 (0.267 – 0.906) 0.023
3-vessel CAD 1.297 (1.079 – 3.441) 0.027
LAD prox/med 2.307 (1.176 – 4.528) 0.015 1.527 (0.643 – 3.626) 0.337
Heart failure 9.653 (4.554 – 20.463) 0.000 8.649 (4.620 – 16.192) 0.000
DM type II 2.589 (1.518 – 4.414) 0.000 1.957 (1.044 – 3.671) 0.036
Long-lasting nitrates 1.952 (1.136 – 3.353) 0.015 1.352 (0.739 – 1.775) 0.545

Composite clinical outcome events in NSTEMI patients

Univariate Multivariate
OR (95% CI for OR) p OR (95% CI for OR) p
hs-TnI (ng/L) 1.016 (1.002 – 1.030) 0.022 1.013 (0.999 – 1.026) 0.060
Creatine kinase (U/L) 1.000 (1.000 – 1.001) 0.034 1.000 (1.000 – 1.001) 0.071
DM type II 3.299 (1.517 – 7.173) 0.003 3.687 (1.399 – 9.715) 0.008
ACE inhibitor/ARB 2.737 (1.031 – 7.270) 0.043

Other predictors of the clinical outcome

We tested various demographic and clinical data, including comorbidities, coronary angiography data, and
comedication as predictors of the clinical outcome. We obtained a model, using the Cox-regression method
(Table 5), showing that patients with 3-vessel CAD had a 3.9 times higher risk of cardiovascular mortality (p <
0.05). Also, elderly patients had a 1.1 times higher risk (p < 0.01). In the NSTEMI group, cardiovascular
mortality was predicted by a two-variable model, but only treatment with trimetazidine was independently
significant. Patients taking trimetazidine had an 11 times higher risk of mortality (p < 0.01).
The occurrence of a composite of cardiovascular mortality, nonfatal MI, or a nonfatal stroke was predicted
using a model that included three independent variables (Table 6). This risk increased 1.1 times with each year
of age (p < 0.05). Patients suffering from dia- betes mellitus type II had a 3.6 times higher risk (p < 0.05). The
use of trimetazidine in- creased the risk 3.5 times (p < 0.05). In the NSTEMI group, we obtained a model
showing 3-vessel CAD and trimetazidine pharmacotherapy as independently-significant predictors.
Patients with a 3-vessel disease had a 6.1 times higher risk of a composite of hard endpoint events (p <
0.05). In the case of trimetazidine, this risk was as high as 12.7 (p < 0.01).
The composite clinical outcome events of cardiovascular mortality, nonfatal MI, a nonfatal stroke, and
hospitalization for urgent myocardial revascularization or heart failure were recorded during the 28-month
follow-up. According to the results of the Cox-regression analysis, the composite clinical outcome events
(Table 7) were registered with a 1.1 times higher incidence in patients with more severe CAD with each new
vessel affected (p < 0.05). In the NSTEMI patients, the composite clinical outcome events oc- curred with
higher incidence in patients with diabetes mellitus type 2 (p < 0.01).
Although the univariate Cox-regression analysis identified two predictors of hospitalization for heart
failure after MI, after the multivariate analysis, only the biomarker for myocardial necrosis CKMB (p < 0.05)
(Table 8) was independently significant. In the NSTEMI group, the only independently-significant predictor of
heart failure development after an acute MI was the creatine kinase MB isoenzyme activity (OR = 1.007, p <
0.001), which was the case for the entire STEMI and NSTEMI group.

•
Table 8. Cox-regression analysis of the hospitalization for the heart failure.

Hospitalization for the heart failure in STEMI and NSTEMI patients

Univariate Multivariate
OR (95% CI for OR) p OR (95% CI for OR) p
Dose-adjusted 2-oxo-clopidogrel (mg/mL/mg) 1.041 (1.015 – 1.069) 0.002 1.044 (0.977 – 1.116) 0.199
Creatine kinase MB (U/L) 1.003 (1.000 – 1.005) 0.034 1.003 (1.000 – 1.006) 0.025

Hospitalization for the heart failure in NSTEMI patients

Univariate Multivariate
OR (95% CI for OR) p OR (95% CI for OR) p
Dose-adjusted 2-oxo-clopidogrel (ng/mL/mg) 1.036 (1.007 – 1.066) 0.014 1.062 (0.941 – 1.198) 0.329
Creatine kinase MB (U/L) 1.007 (1.003 – 1.011) 0.001 1.007 (1.003 – 1.011) 0.001

Discussion
Clopidogrel and 2-oxo-clopidogrel plasma levels in association with
demographic and clinical factors
In our study, a lower dose-adjusted concentration of clopidogrel, measured on the third day of
hospitalization of patients with their first acute MI 2 hours after clopidogrel administration, was associated
with a lower left ventricular ejection fraction and concomitant administration of pantoprazole. It is considered
that the variability of the pharmacodynamics of clopidogrel is to a high degree caused by its variable
bioavailability [13, 14]. The pharmacokinetics of clopidogrel is exceptionally important, particularly regarding
STEMI. A reduction in the maxi- mum clopidogrel concentration and the ac- tive metabolite concentration has
been reported [15]. Furthermore, increased residual platelet activity has been found in patients with STEMI.
Clopidogrel absorption may be reduced in patients due to impaired left ventricular function, an increased
sympathetic tone, and blood flow redistribution from the splanchnic vascular bed. Moreover, the release of
atrial natriuretic peptide caused by increased venous pressure reduces the intestinal motility and
permeability. Information available on the impaired bioavailability of clopidogrel in STEMI is very scarce,
and the study by Heestermans et al. [15] is the only study comparing the pharmacokinetics of a high
loading dose of clopidogrel in STEMI patients and healthy controls. To the best of our knowledge, there
have been no such studies for NSTEMI patients.
In our study, a lower dose-adjusted concentration of clopidogrel, measured on the third day of
hospitalization, was associated with cotreatment with pantoprazole – a proton pump inhibitor. In the STEMI
group, the most important predictor of a dose-adjusted clopidogrel concentration during the acute phase of MI
was fentanyl administration, which reduced the clopidogrel plasma concentration (unpublished results). In the
other cohort, in the NSTEMI group, as in the en- tire study group, the dose-adjusted clopidogrel concentration
was lower in patients after pantoprazole coadministration. Previous publications have suggested that opioid
administration has deleterious effects on the pharmacokinetics of clopidogrel [16]. This is not only due to its
delayed absorption, but also due to the impaired metabolic conversion of clopidogrel. Additionally, as a recent
study showed, opioid administration had a similar effect on the onset of action of the newer oral P2Y12
inhibitors, prasugrel and ticagrelol [17, 18, 19, 20]. Consistent with these results were findings by Kubica et al.
[21] who suggested that due to a delayed onset of the action of ticagrelor, the optimal level of platelet
inhibition was not achieved 2 to 4 hours after oral loading doses in STEMI patients. This observation was
less expected because ticagrelor does not require metabolic conversion to its active form. Such a
pharmacokinetic interaction is maybe the basis of the neutral effect of prehospital treatment with
ticagrelor in STEMI patients (ATLANTIC study). Moreover, the administration of morphine either alone
or in combination with nitroglycerin in NSTEMI patients was followed by increased mortality [22]. In the
actual European Society of Cardiology STEMI Guidelines from 2017 [20], the class of recommendation for
morphine administration in the acute phase of MI was downgraded from Class I to Class IIa. Evidence level
C indicates that additional studies regarding this issue are required. Along with anginal pain relief, an
additional reason for morphine ad- ministration was the treatment of acute heart failure, a complication of
MI. Our patients with heart failure in the acute phase of MI had lower clopidogrel and 2-oxo-clopidogrel
plasma concentrations compared with the patients without heart failure, but the difference did not reach
statistical significance.
In the whole study group, the only covariate examined that affected the dose-adjusted concentration of

•
clopidogrel was pantoprazole administration. Since clopidogrel is a prodrug that requires two-step
pharmacokinetic activation, mainly by cytochrome P450 2C19 and CYP3A4 enzymes, there is a possibility
that the concomitant administration of drugs, such as statins, proton-pump inhibitors, and calcium-channels
blockers that are metabolized by these isoenzymes, may alter the plasma concentration of clopidogrel.
Pantoprazole as a weak CYP2C19 inhibitor [23] has less effect on the antiplatelet activity of clopidogrel than
omeprazole [24]. The importance of clarifying the possibility of clopidogrel-pantoprazole interaction arose
after learning the fact that pantoprazole prescription has significantly increased in the past years instead of
known CYP2C19 inhibitors such as omeprazole [25]. However, even though it is the general opinion that
pantoprazole is the safest of all proton-pump inhibitors, and that there are no interactions with clopidogrel
[13], there are studies that cast doubt on this assertion. It is suggested that interaction with pantoprazole is
significant only in loss-of-function CYP2C19*2 allele carriers, through lowering the concentration of
clopidogrel active metabolite [26]. On the other hand, it has been shown that pantoprazole diminishes the
antiaggregation effect of clopidogrel, independently of CYP2C19 polymorphisms, both in the first days and
after a month of antiplatelet therapy and pantoprazole [27].
Statins are frequently coadministrated with clopidogrel in ACS patients. The pharmacokinetic interaction
between atorvastatin and clopidogrel has been proven in vitro and in vivo. In our study, it was not possible to
examine this interaction because the majority of the patients had statin therapy. Some data exist about the
negative clinical consequences of coadministration of clopidogrel and statins metabolized by CYP3A4
[28]. The effect of pharmacokinetic interaction between clopidogrel and atorvastatin may be overshadowed by
its numerous beneficial effects in patients with MI, thus improving the outcome [29].

Clopidogrel and 2-oxo-clopidogrel plasma concentrations in association with

clinical outcome

In our study, lower dose-adjusted clopidogrel plasma concentrations were mea- sured on the third day of
hospitalization in NSTEMI patients with the hard clinical endpoint events of cardiovascular mortality, nonfatal
MI, or a nonfatal stroke, after long- term follow-up. Lower dose-adjusted 2-oxo-clopidogrel plasma
concentrations were measured in patients hospitalized for heart failure after MI during the follow-up period. In
this study, blood samples were collected at equal time intervals after clopidogrel administration, considering
that both drug and metabolite concentrations may significantly vary depending on the time elapsed from drug
administration [13, 30, 31]. The pharmacokinetics of clopidogrel was assessed based on concentrations of
clopidogrel and 2-oxo-clopidogrel, and maintenance dose-adjusted concentrations. The active thiol metabolite
level was not measured due to this metabolite’s instability and the need for immediate processing of the
samples collected [13, 32, 33]. It was shown in an earlier study that there is a significant correlation
between the pharmacokinetic parameters of maximum concentration and the area below the concentration-
time curve for clopidogrel as well as clopidogrel’s intermediary metabolite 2-oxo-clopidogrel and the active
H4 thiol metabolite [13]. This makes it possible to predict the patient exposure to the active metabolite in
situations in which only the measurements of the parent compound and intermediate metabolite are
available. In the same study, the existence of a significant correlation between the maximum concentration of
the active clopidogrel metabolite and suppression level of platelet aggregation was found in the patients with
coronary disease.
Consistent with most other reports in the literature, we found that MI patients with 3-vessel CAD, as
well as elderly patients, demonstrated a higher risk of cardiovascular mortality in the long-term follow-up.
Advanced age, as well as diabetes mellitus type II and treatment with trimetazidine, were associated with a
higher risk of the composite of hard endpoint events of cardiovascular mortality, nonfatal MI, or a nonfatal
stroke. Trimetazidine was administered primarily to patients with diffuse coronary artery disease, at the
discretion of the attending physician. This drug was also given to the patients with disease in the branches of
their coronary arteries with small lumens, in which a complete myocardial revascularization could not be
performed. In general, trimetazidine was given to the patients with advanced coronary artery disease, and
more severe ischemic heart disease rather than drug administration was the cause of the more un-
favorable clinical outcome in such patients. Finally, it should be noted that patients who experience an
adverse clinical outcome are frequently older and have more comorbidities. In summary, the strongest
predictor of clinical outcome was the severity of coro- nary artery disease. Considering that the im- pact of
CYP2C19 polymorphisms on high-on- clopidogrel platelet reactivity is restricted to a few weeks [29], as
well as that it accounts for only 4.6% of the total variability [34], further study needs to be carried out in

•
order to solve the unex- plained variability of clopidogrel.

Limitations of the study

The most important limiting factor of this study is the number of patients included, and consequently the
number of events registered, despite a long follow-up of 28 months, on average. The relatively low
incidence of registered clinical events may be explained by the exclusion of all the patients with the
events during the period of hospitalization, the high percentage of 1-vessel CAD, and the average patient
age of 61 years. In addition to this, the clopidogrel and 2-oxo-clopidogrel concentrations were measured
only once, on the third day after the MI. There- fore, possible changes in the concentrations of
clopidogrel and its metabolite in the fol- lowing months were not considered. Finally, another limitation to
the present study is its single-center design with some residual confounding.

Conclusion
In conclusion, we showed that lower dose-adjusted clopidogrel plasma concentra- tions were associated
with an adverse clinical outcome.

Funding
This study was funded by Grants No. 44004 and No. III 41018, given by the Serbian Ministry of
Education, Science and Techno- logical Development.

Conflict of interest
The authors declare that they have no conflict of interest.

References
[1] Roffi M, Patrono C, Collet JP, Mueller C, Valgimigli M, Andreotti F, Bax JJ, Borger MA, Brotons C, Chew DP, Gencer B,
Hasenfuss G, Kjeldsen K, Lancellotti P, Landmesser U, Mehilli J, Mukherjee D, Storey RF, Windecker S; ESC Scientific Docu-
ment Group. 2015 ESC Guidelines for the man- agement of acute coronary syndromes in patients presenting without persistent
ST-segment eleva- tion: task force for the management of acute coro- nary syndromes in patients presenting without persistent
ST-segment elevation of the European Society of Cardiology (ESC). Eur Heart J. 2016; 37: 267-315.
[2] Levine GN, Bates ER, Bittl JA, Brindis RG, Fihn SD, Fleisher LA, Granger CB, Lange RA, Mack MJ, Mauri L, Mehran R,
Mukherjee D, Newby LK, O’Gara PT, Sabatine MS, Smith PK, Smith SC Jr. 2016 ACC/AHA guideline focused update on
duration of dual antiplatelet therapy in patients with coronary artery disease: A report of the Amer- ican college of
cardiology/American heart associa- tion task force on clinical practice guidelines. J Am Coll Cardiol. 2016; 68: 1082-1115.
Erratum: https://doi.org/10.1016/j.jacc.2016.07.715
[3] Chen ZM, Jiang LX, Chen YP, Xie JX, Pan HC, Peto R, Collins R, Liu LS; COMMIT (ClOpidogrel and Metoprolol in
Myocardial Infarction Trial) collaborative group. Addition of clopidogrel to aspirin in 45,852 patients with acute myocardial
infarction: randomised placebo-controlled trial. Lancet. 2005; 366: 1607-1621.
[4] Mega JL, Close SL, Wiviott SD, Shen L, Walker JR, Simon T, Antman EM, Braunwald E, Sabatine MS. Genetic variants in
ABCB1 and CYP2C19 and cardiovascular outcomes after treatment with clopidogrel and prasugrel in the TRITON-TIMI 38
trial: a pharmacogenetic analysis. Lancet. 2010; 376: 1312-1319.
[5] Brandt JT, Close SL, Iturria SJ, Payne CD, Farid NA, Ernest CS II, Lachno DR, Salazar D, Winters KJ. Common polymorphisms
of CYP2C19 and CYP2C9 affect the pharmacokinetic and pharma- codynamic response to clopidogrel but not prasu- grel. J
Thromb Haemost. 2007; 5: 2429-2436.
[6] Giusti B, Abbate R. Response to antiplatelet treat- ment: from genes to outcome. Lancet. 2010; 376: 1278-1281.
[7] Taubert D, von Beckerath N, Grimberg G, Lazar A, Jung N, Goeser T, Kastrati A, Schömig A, Schömig E. Impact of P-
glycoprotein on clopido- grel absorption. Clin Pharmacol Ther. 2006; 80: 486-501.
[8] Jakubowski JA, Angiolillo DJ, Zhou C, Small DS, Moser BA, Ten Berg JM, Brown PB, James S, Winters KJ, Erlinge D. The
influence of body size on the pharmacodynamic and pharmacokinetic response to clopidogrel and prasugrel: a retro- spective
analysis of the FEATHER study. Thromb Res. 2014; 134: 552-557.
[9] Angiolillo DJ, Jakubowski JA, Ferreiro JL, Tello- Montoliu A, Rollini F, Franchi F, Ueno M, Darling- ton A, Desai B, Moser BA,
Sugidachi A, Guzman LA, Bass TA. Impaired responsiveness to the platelet P2Y12 receptor antagonist clopidogrel in patients
with type 2 diabetes and coronary artery disease. J Am Coll Cardiol. 2014; 64: 1005-1014.
[10] James S, Angiolillo DJ, Cornel JH, Erlinge D, Husted S, Kontny F, Maya J, Nicolau JC, Spinar J, Storey RF, Stevens SR,
Wallentin L; PLATO Study Group. Ticagrelor vs. clopidogrel in pa- tients with acute coronary syndromes and diabe- tes: a
substudy from the PLATelet inhibition and patient Outcomes (PLATO) trial. Eur Heart J. 2010; 31: 3006-3016.
[11] Bonello L, Camoin-Jau L, Armero S, Com O, Arques S, Burignat-Bonello C, Giacomoni M-P, Bonello R, Collet F, Rossi P,
Barragan P, Dignat- George F, Paganelli F. Tailored clopidogrel loading dose according to platelet reactivity monitoring to

•
 prevent acute and subacute stent thrombosis. Am J Cardiol. 2009; 103: 5-10.
[12] Stokanovic D, Nikolic VN, Konstantinovic SS, Zvezdanovic JB, Lilic J, Apostolovic SR, Pavlovic M, Zivkovic VS, Jevtovic-
Stoimenov T, Jankovic SM. P-glycoprotein polymorphism C3435T is as- sociated with dose-adjusted clopidogrel and 2-oxo-
clopidogrel concentration. Pharmacology. 2016; 97: 101-106.
[13] Karaźniewicz-Łada M, Danielak D, Burchardt P, Kruszyna L, Komosa A, Lesiak M, Główka F. Clinical pharmacokinetics of
clopidogrel and its metabolites in patients with cardiovascular dis- eases. Clin Pharmacokinet. 2014; 53: 155-164.
[14] Cattaneo M. Response variability to clopidogrel: is tailored treatment, based on laboratory testing, the right solution? J Thromb
Haemost. 2012; 10: 327-336.
[15] Heestermans AACM, van Werkum JW, Taubert D, Seesing TH, von Beckerath N, Hackeng CM, Schömig E, Verheugt FWA, ten Berg
JM. Impaired bioavailability of clopidogrel in patients with a ST-segment elevation myocardial infarction. Thromb Res. 2008;
122: 776-781.
[16] Khan N, Cox AR, Cotton JM. Pharmacokinetics and pharmacodynamics of oral P2Y12 inhibitors during the acute phase of a
myocardial infarction: A systematic review. Thromb Res. 2016; 143: 141-148.
[17] Silvain J, Storey RF, Cayla G, Esteve J-B, Dillinger J-G, Rousseau H, Tsatsaris A, Baradat C, Salhi N, Hamm CW, Lapostolle F, Lassen
JF, Collet J-P, Ten Berg JM, Van’t Hof AW, Montalescot G. P2Y12 receptor inhibition and effect of morphine in pa- tients
undergoing primary PCI for ST-segment elevation myocardial infarction. The PRIVATE- ATLANTIC study. Thromb Haemost.
2016; 116:
369-378.
[18] Montalescot G, van ’t Hof AW, Lapostolle F, Silvain J, Lassen JF, Bolognese L, Cantor WJ, Cequier A, Chettibi M, Goodman SG,
Hammett CJ, Huber K, Janzon M, Merkely B, Storey RF, Zeymer U, Stibbe O, Ecollan P, Heutz WMJM, Swahn E, et al; ATLANTIC
Investigators. Prehospital ticagrelor in ST-segment elevation myocardial infarction. N Engl J Med. 2014; 371: 1016-1027.
[19] Thomas MR, Morton AC, Hossain R, Chen B, Luo L, Shahari NNBM, Hua P, Beniston RG, Judge HM, Storey RF. Morphine
delays the onset of ac- tion of prasugrel in patients with prior history of ST-elevation myocardial infarction. Thromb Hae- most.
2016; 116: 96-102.
[20] Ibanez B, James S, Agewall S, Antunes MJ, Bucciarelli-Ducci C, Bueno H, Caforio ALP, Crea F, Goudevenos JA, Halvorsen S,
Hindricks G, Kastrati A, Lenzen MJ, Prescott E, Roffi M, Valgimigli M, Varenhorst C, Vranckx P, Widimský
P. ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The
Task Force for the management of acute myocardial infarction in pa- tients presenting with ST-segment elevation of the European
Society of Cardiology (ESC). European Heart Journal 2017. ESC GUIDELINES. 2017;
00: 1-66.
[21] Kubica J, Adamski P, Ostrowska M, Sikora J, Kubica JM, Sroka WD, Stankowska K, Buszko K, Navarese EP, Jilma B, Siller-Matula
JM, Marszałł MP,Rość D, Koziński M. Morphine delays and attenu- ates ticagrelor exposure and action in patients with myocardial
infarction: the randomized, dou- ble-blind, placebo-controlled IMPRESSION trial. Eur Heart J. 2016; 37: 245-252.
[22] Meine TJ, Roe MT, Chen AY, Patel MR, Washam JB, Ohman EM, Peacock WF, Pollack CV Jr, Gibler WB, Peterson ED;
CRUSADE Investiga- tors. Association of intravenous morphine use and outcomes in acute coronary syndromes: results from
the CRUSADE Quality Improvement Initia- tive. Am Heart J. 2005; 149: 1043-1049.
[23] Ogilvie BW, Yerino P, Kazmi F, Buckley DB, Ros- tami-Hodjegan A, Paris BL, Toren P, Parkinson The proton pump inhibitor,
omeprazole, but not lansoprazole or pantoprazole, is a metabolism- dependent inhibitor of CYP2C19: implications for
coadministration with clopidogrel. Drug Metab Dispos. 2011; 39: 2020-2033.
[24] Siller-Matula JM, Spiel AO, Lang IM, Kreiner G, Christ G, Jilma B. Effects of pantoprazole and esomeprazole on platelet
inhibition by clopido- grel. Am Heart J. 2009; 157: 148.e1-148.e5.
[25] Guérin A, Mody R, Carter V, Ayas C, Patel H, Lasch K, Wu E. Changes in Practice Patterns of Clopidogrel in Combination
with Proton Pump Inhibitors after an FDA Safety Communication. PLoS One. 2016; 11: e0145504.
[26] Simon N, Finzi J, Cayla G, Montalescot G, Collet JP, Hulot JS. Omeprazole, pantoprazole, and CY- P2C19 effects on clopidogrel
pharmacokinetic- pharmacodynamic relationships in stable coro- nary artery disease patients. Eur J Clin Pharmacol. 2015; 71:
1059-1066.
[27] Parri MS, Gianetti J, Dushpanova A, Della Pina F, Saracini C, Marcucci R, Giusti B, Berti S. Pan- toprazole significantly
interferes with antiplatelet effect of clopidogrel: results of a pilot randomized trial. Int J Cardiol. 2013; 167: 2177-2181.
[28] Brophy JM, Babapulle MN, Costa V, Rinfret S. A pharmacoepidemiology study of the interaction between atorvastatin and
clopidogrel after percu- taneous coronary intervention. Am Heart J. 2006; 152: 263-269.
[29] Bates ER, Lau WC, Angiolillo DJ. Clopidogrel- drug interactions. J Am Coll Cardiol. 2011; 57: 1251-1263.
[30] Ernest CS II, Small DS, Rohatagi S, Salazar DE, Wallentin L, Winters KJ, Wrishko RE. Population pharmacokinetics and
pharmacodynamics of pra- sugrel and clopidogrel in aspirin-treated patients with stable coronary artery disease. J Pharmacoki-
net Pharmacodyn. 2008; 35: 593-618.
[31] Jung JA, Kim T-E, Kim J-R, Kim M-J, Huh W, Park K-M, Lee S-Y, Ko JW. The pharmacokinetics and safety of a fixed-dose
combination of acetyl- salicylic acid and clopidogrel compared with the concurrent administration of acetylsalicylic acid and
clopidogrel in healthy subjects: a randomized, open-label, 2-sequence, 2-period, single-dose crossover study. Clin Ther. 2013; 35:
985-994.
[32] Elsinghorst PW. Quantitative determination of clopidogrel and its metabolites in biological sam- ples: a mini-review. J Chromatogr
B Analyt Tech- nol Biomed Life Sci. 2013; 917-918: 48-52.
[33] Mullangi R, Srinivas NR. Clopidogrel: review of bioanalytical methods, pharmacokinetics/pharmacodynamics, and update on
recent trends in drug-drug interaction studies. Biomed Chro- matogr. 2009; 23: 26-41.
[34] Hochholzer W, Trenk D, Fromm MF, Valina CM, Stratz C, Bestehorn HP, Büttner HJ, Neumann FJ. Impact of cytochrome P450
2C19 loss-of-func- tion polymorphism and of major demographic characteristics on residual platelet function after loading and
maintenance treatment with clopido- grel in patients undergoing elective coronary stent placement. J Am Coll Cardiol. 2010; 55:
2427-2434.

View publication stats