Chronic inhibition of lipoprotein-associated phospholipase A2 does not improve coronary endothelial function: A prospective, randomized-controlled trial

International Journal of Cardiology 253 (2018) 7–13

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International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard

Chronic inhibition of lipoprotein-associated phospholipase A2 does not improve coronary endothelial function: A prospective, randomized-controlled trial☆ Megha Prasad a, Ryan Lennon b, Gregory W. Barsness a, Abhiram Prasad a, Rajiv Gulati a, Lilach O. Lerman a, Amir Lerman a,⁎ a b

Mayo Clinic, Department of Cardiovascular Diseases, Rochester, MN, United States Mayo Clinic, Department of Health Sciences Research, Rochester, MN, United States

a r t i c l e

i n f o

Article history: Received 19 July 2017 Received in revised form 7 September 2017 Accepted 18 September 2017 Keywords: Inflammation Lipoprotein-associated phospholipase A2 Endothelial function

a b s t r a c t Aims: Lipoprotein-associated phospholipase A2 (Lp-PLA2), a novel biomarker for vascular inflammation, is associated with coronary endothelial dysfunction (CED) and independently predicts cardiovascular events. The current study aimed to determine whether darapladib, an orally administered Lp-PLA2 inhibitor, improved CED. Methods and results: Fifty-four patients with CED were enrolled in a double-blinded randomized placebocontrolled trial, and were randomized to receive oral darapladib, 160 mg daily, or placebo. Coronary angiography and invasive coronary endothelial function assessment were performed at baseline and post-6 months of treatment. Primary endpoints were change in coronary artery diameter and coronary blood flow in response to acetylcholine. Additionally, Lp-PLA2 activity was measured at baseline and on follow-up to evaluate for adherence and drug effect. Fifty-four patients were randomized to placebo (n = 29) and darapladib (n = 25). Mean age in darapladib group was 55.2. ± 11.7 years vs. 54.0 ± 10.5 years (p = 0.11). On follow-up, there was no significant difference in the percent response to acetylcholine of coronary artery diameter in treatment vs. placebo group (+3 (IQR −9, 15) vs. +3 (−12, 19); p = 0.87) or coronary blood flow (−5 (IQR −24, 54) vs. 39 (IQR −26, 67); p = 0.41). There was significant reduction in Lp-PLA2 activity in the treatment arm vs. placebo (−76 (IQR −113, −52) vs. −7(−21, −7); p b 0.001). Discussion: Lp-PLA2 inhibition with darapladib did not improve coronary endothelial function, despite significantly reduced Lp-PLA2 activity with darapladib. This study suggests endogenous Lp-PLA2 may not play a primary role in coronary endothelial function in humans. Clinicaltrials.gov Identifier: NCT01067339 © 2017 Elsevier B.V. All rights reserved.

1. Background Inflammation may play a key role in coronary endothelial dysfunction, atherosclerosis, and cardiovascular morbidity and mortality (1–3). Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a novel biomarker of inflammation, and is independently associated with endothelial dysfunction, atherosclerosis, ischemic stroke, and

Abbreviations: Lp-PLA21, lipoprotein-associated phospholipase A2; hsCRP, highsensitivity C-reactive protein; PLACA, Lp-PLA2 activity; DSMB, data safety monitoring board; FDA, federal drug administration; IRB, institutional review board. ☆ Dr. Amir Lerman is a member of the advisory board of Itamar Medical, a company that produces EndoPAT, a device for noninvasive endothelial function detection. This device was not used in this study. Dr. Lilach O Lerman is his spouse. All other authors have no disclosures. ⁎ Corresponding author at: Mayo Clinic, 200 First St SW, Rochester, MN 55905, United States. E-mail address: [email protected] (A. Lerman).

https://doi.org/10.1016/j.ijcard.2017.09.171 0167-5273/© 2017 Elsevier B.V. All rights reserved.

cardiovascular risk. Lp-PLA2 is a proinflammatory, proatherogenic molecule, that when inhibited has been deemed to have therapeutic potential in reducing cardiovascular morbidity and mortality (4–9). Darapladib is a selective reversible inhibitor of Lp-PLA 2 . Initial pre-clinical and clinical studies showed that Lp-PLA 2 inhibition may prevent necrotic core expansion and reduce plaque vulnerability, with potential to reduce cardiovascular events (5,10,11). The independent association between Lp-PLA2 levels and cardiovascular risk may be explained by an indirect effect on coronary endothelial function (12,13). Preliminary studies showing that levels of secretory non-pancreatic type II phospholipase A2 were found to be elevated in patients with established coronary disease, and this was in turn associated with impaired endothelial-dependent forearm flow (14). Moreover, circulating Lp-PLA2 levels have been found to be an independent predictor of coronary endothelial function, even after adjustment for cardiac risk factors (12,13). These data support a potential role of Lp-PLA2 as a marker of endothelial dysfunction.

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Thus, vascular inflammation may link traditional risk factors, endothelial dysfunction and cardiovascular events (15,16). Previous studies have demonstrated that coronary endothelial dysfunction has been associated with an increase in Lp-PLA2 level and activity (12,13). However, there are no prospective studies investigating the mechanistic role of endogenous inflammation in patients with early coronary atherosclerosis and endothelial dysfunction (17,18). The current study was designed to test the hypothesis that the inhibition of the endogenous Lp-PLA2 pathway will improve coronary endothelial function in humans. 2. Methods This trial was a single-center, phase 3, randomized, double-blinded controlled trial that evaluated the safety and efficacy of darapladib (GlaxoSmithKline; Brentford, London), an Lp-PLA2 inhibitor, in improving coronary endothelial function. Patients enrolled underwent baseline laboratory testing and coronary angiography with invasive coronary endothelial function assessment (19–23). Patients were then randomized by a statistical program to darapladib 160 mg by mouth daily vs. placebo administrated by the pharmacy, and were evaluated after 6 months with repeat laboratory testing, and coronary angiography with invasive coronary endothelial function assessment. The study was performed between February 2010 and February 2015. The study protocol was approved by the Mayo Clinic Institutional Review board. This work was supported by the National Institutes of Health (NIH Grants HL-92954, AG-31750, DK20092, and DK102325), and GlaxoSmithKline provided the study drug darapladib. 3. Eligibility criteria, randomization and study medication 3.1. Patient enrollment and eligibility Study subjects were enrolled between February 2010 and February 2015 from the Chest Pain and Coronary Physiology Clinic as well as from outpatient and inpatient practices in the Cardiovascular Division at Mayo Clinic Rochester. Patients N18 years old age, b 85 years of age, with clinical indication for coronary angiography and nonobstructive coronary artery disease on index angiography were deemed eligible. Patients with acute ST-segment elevation myocardial infarction, acute non-ST segment elevation myocardial infarction, and unstable angina within last 3 months prior to cardiac catheterization, cardiac arrest or stroke in previous 6 months, acute or chronic heart failure with left ventricular ejection fraction b45%, increased QTc interval, chronic hepatic disorders, history of renal transplant or contrast nephropathy, uncontrolled diabetes or hypertension, were excluded. Pregnancy or concurrent treatment with CYP3A4 inhibitors were also exclusion factors. Those patients with significant coronary disease were also excluded. 3.2. Randomization and study drug A total of 70 patients were screened, and of these 5 patients withdrew prior to randomization. Sixty-five patients were randomized to either the placebo arm (n = 34) or the darapladib arm (n = 31). During the course of the study, 5 patients withdrew from the placebo arm, and 6 patients withdrew from the darapladib arm. These were not related to the study drug. Thus, 29 patients were analyzed in the placebo arm, and 25 patients were analyzed in the daradpladib arm (Fig. 1). All patients had been referred to the cardiac catheterization laboratory for further evaluation of coronary artery disease and were found to have nonobstructive coronary disease, and subsequently underwent comprehensive coronary physiology testing including assessment of endothelial function and non-endothelium-dependent coronary flow reserve. Patients found to have endothelial dysfunction on testing who provided informed consent were randomized to receive either standard

Fig. 1. Depicts a flow chart of the trial design used to complete this study.

medical therapy including a statin in addition to darapladib, a Lp-PLA2 inhibitor, or standard medical therapy including statin with placebo. A dose of 160 mg by mouth administered daily was used. Each patient was randomly assigned to either placebo or treatment groups. A computer-generated code with either placebo or darapladib was generated and patients were treated according to their randomized assignment. Participants, study staff, and investigators were blinded to treatment assignments. Only the pharmacist technician was aware of the assignment of a particular patient. Study and placebo tablets were distributed in identical bottles by the pharmacist technician. 3.3. Follow-up Patients were followed by phone call every month for the duration of the study by a blinded, trained nurse. Cardiac events and side effects were monitored during this time. After 6 months of follow-up, the patient returned for coronary angiography and coronary endothelial function assessment. This was performed by an independent investigator who was blinded to treatment allocation. On repeat angiography and invasive acetylcholine endothelial function testing, patients had repeat measurement of % change in coronary blood flow with intracoronary administration of graded doses of acetylcholine and % change in coronary artery diameter measured by quantitative coronary angiography. In addition, patients underwent additional laboratory testing. The same protocol was used by a blinded investigator to assess these parameters. The primary endpoints were percent change in coronary artery diameter and coronary blood flow in response to acetylcholine. 4. Outcome assessment 4.1. Laboratory testing Patients were followed for 6 months, and underwent basic blood testing, including assessment of Lp-PLA2 activity (PLACA) and CRP levels both at baseline and at follow-up using stored samples. High sensitivity C-reactive protein (hs-CRP) was measured using a latex particleenhanced immunoturbidimetric assay (Roche Diagnostics, Indianapolis,

M. Prasad et al. / International Journal of Cardiology 253 (2018) 7–13

IN) and Lp-PLA2 was measured using an enzymatic colorimetric activity assay (Diazyme Laboratories, Poway, CA) with a 5-point calibration curve (0–400 nmol/min/mL) on the Cobas 6000/c501 instrument (Roche Diagnostics, Indianapolis, IN). 4.2. Quantitative coronary angiography Digital images were analyzed to measure coronary artery diameter as previously described (24). The left anterior descending artery was divided into three segments; proximal, middle and distal. Measurements were performed in the region which showed the greatest change with administration of acetylcholine. The reference diameter for calculation of stenosis was found using an angiographically smooth segment of the proximal, middle and distal segments of the left anterior descending artery which had no branch vessels or overlap. Calibration of the images was performed so as to find the diameter of the guide catheter, and enddiastolic images were used. The diameter of each segment was calculated at baseline prior to acetylcholine infusion and after. The proximal segment of the left anterior descending artery was the internal control as there was no acetylcholine infused here. This assessment was performed at baseline and after 6 months of follow-up. 4.3. Assessment of coronary blood flow Endothelium-dependent coronary vasoreactivity was assessed according to standardized protocol as previously described (23,25,26). The Doppler guidewire was advanced within the coronary-infusion catheter and placed in the mid-left anterior descending coronary artery. Acetylcholine was administered in the left anterior descending artery for 3 min each with increasing concentrations. Doppler velocity, and hemodynamic tracings were measured during each infusion. According to previous studies, microvascular endothelial dysfunction was defined as a b 50% increase in coronary blood flow in response to maximal dose of acetylcholine when compared to baseline coronary blood flow or change in coronary artery diameter of b 0% in response to acetylcholine (24,27).

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measures, and so the correlation is at least (ρ), and the standard deviation of the differences will be σ x square root (2 × 1 −ρ). ρ N 0.2 was set as low correlation and σ ≤ 100 for percent change in coronary blood flow, and σ ≤ 30 was set for % change in coronary artery diameter, (12) meaning that the standard deviations of the differences between pre-and post-treatment measures no N 126% and 38% respectively. A two-sample two-sided t-test at the 0.025 significant level showed that the mean of the differences is not equal between the two treatment groups and will have 80% power with 35 patients in each arm to detect a mean difference of 95% in the microvascular function difference measure and 29% in the macrovascular function difference measure. 6. Results 6.1. Baseline characteristics Mean age in darapladib group was 55.2 ± 11.7 years vs. 54.0 ± 10.5 years (absolute standardized difference, ASD, 0.11) (Table 1). Hypertension was present in 58% of patients in darapladib arm and 52% of patients in the placebo arm. Subjects in the darapladib arm were less likely to have hyperlipidemia (44% versus 72%), a family history of CAD (56% versus 76%) and were more likely to be taking aspirin (80% versus 52%). Baseline hsCRP levels were slightly lower in the darapladip group (median [IQR]: 1 [1,4] versus 2 [1,5]) while Lp-PLA2 activity was slightly higher (135 [116, 149], 123 [106, 149]). 6.2. Lipids At baseline, prior to enrollment, 44% in the darapladib and 48% in the placebo group used lipid lowering drugs. At baseline, median total cholesterol level in the darapladib group was 171 (IQR 148, 226) mg/dL vs. 185(IQR 160, 212) mg/dL in the placebo group. Median triglycerides were 100 (IQR 79,115) mg/dL in the darapladib group and 98(IQR 83, 181) mg/dL in the placebo group (ASD = 0.37). Median HDL was 56 (IQR 44, 67) mg/dL in the darapladib group and 60 (IQR 43, 83) mg/dL in the placebo group. Post-treatment, there was no significant difference in change in total cholesterol, triglycerides, HDL, and LDL (p N 0.05).

4.4. Safety 6.3. Endothelial function Throughout the study, a data safety monitoring board (DSMB) consistently monitored patients' safety. This served to oversee the clinical study and reviewed all serious adverse events as well as side effects of the medication. The principal investigator reported to the DSMB on a quarterly basis with information regarding number of participants, as well as any potential side effects. All serious adverse events were reported to the IRB, FDA, and DSMB. 5. Statistical analyses Continuous variables are presented as mean (standard deviation) if normally distributed and median (25th, 75th percentile) if not normally distributed. Discrete variables are summarized as frequency (group percentage). Because subjects were randomized to either placebo or darapladib, baseline comparisons between groups are summarized using the absolute standardized difference. Post-treatment comparisons were made with Student's two-sample t-test. Due to 2 primary endpoints (%change in CAD and %change in CBF), an alpha level of 0.025 was used for hypothesis tests. Linear regression models were used to model the effect of treatment on follow-up endothelial function measure adjusting for the baseline measures. Statistical analyses were performed by an independent statistician. Pre-treatment and post-treatment difference in % change in coronary artery diameter in response to acetylcholine and % change in coronary blood flow in response to acetylcholine were primary endpoints. For a priori power calculations, the post-treatment measures were assumed to have the same standard deviation as the pre-treatment

Baseline maximal coronary flow reserve was similar in both groups. At baseline, percent change in coronary artery diameter with acetylcholine administration was median-10 (IQR − 27, − 4) in the darapladib group and − 14(IQR − 27, − 5) in the placebo group. At baseline, percent change in coronary blood flow was 16(IQR − 4, 66) in the darapladib group and 7 (IQR − 14, 41) in the placebo group. On follow-up endothelial function assessment after 6 months of darapladib therapy, maximum non-endothelium dependent coronary flow reserve was 3(IQR 3,3) in the darapladib arm and 3(IQR 3,3) in the placebo arm. The percent change in coronary artery diameter on follow-up was 3(IQR −9.15) in the darapladib arm and 3(IQR −12,19) in the placebo group. Overall, there was no significant difference in the improvement in percent change in coronary artery diameter on follow-up (p = 0.87). There was also no significant improvement in percent change in coronary blood flow on follow-up (p = 0.48) (Fig. 1). When using analysis of covariance to account for the baseline measure, the effect of darapladib on percent change in coronary artery diameter at 6 months was 4.4% (95% CI, − 4.4, 12.9; p = 0.32) (Fig. 2), and the percent change in coronary blood flow at 6 months was 6.0% (95% CI, − 33.9, 46.0; p = 0.77) (Fig. 3) Similar results were obtained when adjusting for additional covariates. 6.4. Hs-CRP and Lp-PLA2 activity Hs-CRP levels were assessed on baseline and on follow-up and there was no significant change in hs-CRP levels with darapladib use

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Table 1 Baseline characteristics. Baseline characteristics Variable

Darapladib (N = 25)

Placebo (N = 29)

Absolute std. diff.

Age, yrs. (mean (SD)) Men, n (%) Hypertension, n (%) Diabetes, n (%) Hyperlipidemia, n (%) Body Mass Index Family history, n (%) Post menopause, n (%) Smoking status, n (%) Never smoked Former smoker Current smoker Stress, n (%) Depression, n (%) (Baseline) Total Chol Mean, SD Median (Q1, Q3) (Baseline) Triglycerides Mean, SD Median (Q1, Q3) (Baseline) HDL Chol Mean, SD Median (Q1, Q3) (Baseline) LDL Chol Mean, SD Median (Q1, Q3) Antiarrhythmic use, n (%) Beta Blocker use, n (%) Aspirin use, n (%) Calcium Channel Blocker use, n (%) Lipid lowering drug use, n (%) Anticoagulant use, n (%) Antihypertensive use, n (%) (Baseline) hs-CRP, mg/L Mean, SD Median (Q1, Q3) (Baseline) PLACA, nmol/min/mL Mean, SD Median (Q1, Q3) (Baseline) Max CFR Mean, SD Median (Q1, Q3) (Baseline) %chg.CAD Mean, SD Median (Q1, Q3) (Baseline) %chg.CBF Mean, SD Median (Q1, Q3)

55.2 8 14 3 11 31.2 14 12

(11.7) (32%) (58%) (12%) (44%) (5.7) (56%) (50%)

54.0 3 15 2 21 31.0 22 11

(10.5) (10%) (52%) (7%) (72%) (7.0) (76%) (46%)

0.111 0.550 0.133 0.175 0.602 0.024 0.429 0.083

18 7 0 8 13

(72%) (28%) (0%) (38%) (52%)

20 8 1 7 9

(69%) (28%) (3%) (33%) (32%)

0.067 0.009 0.221 0.100 0.411 0.045

186.0 171

(47.7) (148, 226)

188.1 185

(44.1) (160, 212)

106.6 100

(53.4) (79, 115)

131.1 98

(76.4) (83, 181)

57.9 56

(15.5) (44, 67)

64.1 60

(22.7) (43, 83)

106.8 95 8

(42.1) (76, 126) (32%)

97.8 97 7

(35.5) (72, 115) (24%)

0.176

10 20 9

(40%) (80%) (36%)

6 15 11

(21%) (52%) (38%)

0.430 0.625 0.040

11

(44%)

14

(48%)

0.086

7 14

(28%) (56%)

3 17

(10%) (59%)

0.460 0.053

2.3 1

(2.3) (1, 4)

3.3 2

(3.4) (1, 5)

0.372

0.321

0.231

0.356

0.268 137.2 135

(32.8) (116, 149)

128.2 123

(34.5) (106, 149)

3.0 3

(0.8) (3, 3)

3.0 3

(0.7) (2, 4)

−13.8 −10

(16.4) (−27, −4)

−19.2 −14

(21.4) (−27, −5)

38.3 16

(71.8) (−4, 66)

13.5 7

(50.2) (−14, 41)

0.042

0.282

0.401

n = number of patients. SD: standard deviation. hs-CRP: high-sensitivity C-reactive protein. PLACA: Lp-PLA2 activity. %chg CAD: % change in coronary artery diameter in response to acetylcholine. %chg CBF: %change in coronary blood flow.

(p = 0.90). Darapladib use, was however, associated with significant reduction in Lp-PLA2 activity on follow-up vs. baseline (%change in Lp-PLA2 activity −76(−113, −52) vs. −7(−21,7); p b 0.001). 6.5. Adverse events There were a total of five serious adverse events reported. One patient was admitted to the hospital with chest pain which resolved after nitroglycerin and morphine. Another patient was admitted with recurrent angina likely secondary to an anomalous pulmonary vein

and concomitant coronary endothelial dysfunction, which responded to morphine. A third patient was hospitalized for tricuspid valve repair and anomalous pulmonary vein repair, and required preponement of surgery due to increasing chest pain. The last adverse event involved a patient admitted for chest pain the day of follow-up angiography. Work up was unrevealing and pain was thought to be secondary to endothelial dysfunction. These were all thought to be unrelated to treatment after review by the DSMB. 7. Discussion The current study demonstrates that in patients with early coronary atherosclerosis and endothelial dysfunction, endogenous Lp-PLA2 inhibition with darapladib for a duration of 6 months did not improve coronary endothelial function. The current study does not support a role for the endogenous Lp-PLA2 pathways in coronary endothelial function in humans. There is a large body of literature demonstrating that Lp-PLA2 is associated with cardiovascular events.(28–31) Several large studies have suggested that Lp-PLA2 and CRP may have a role in identifying individuals with increased cardiovascular risk, even after adjustment for traditional risk factors.(32) Gerber and colleagues found that Lp-PLA2 levels early post-myocardial infarction are independently associated with mortality and may be useful in predicting cardiovascular risk.(33) Thus, these studies suggest that Lp-PLA2 may be an independent predictor of adverse cardiovascular events. Despite these data, chronic Lp-PLA2 inhibition may not reduce cardiovascular morbidity/mortality as seen in two recent large randomized studies studying the use of darapladib on cardiovascular events. In the STABILITY trial, darapladib was not associated with an improvement in cardiovascular death, rate of myocardial infarction or stroke in patients with stable CAD.(34,35) In the SOLID-TIMI 52 trial, darapladib was also not associated with any reduction in cardiovascular death, rate of myocardial infarction or need for urgent coronary revascularization.(35) Thus, in spite of the evidence supporting Lp-PLA2 as a biomarker for cardiovascular events, inhibition of the Lp-PLA2 pathway did not result in a reduction in cardiovascular events. The findings of the current study are in accord with these two larger randomized studies showing no effect on cardiovascular morbidity and mortality. Previous studies have shown that therapies associated with an improvement in overall cardiovascular morbidity and mortality, may do so by improving endothelial function. Also, treatment of endothelial function has been associated with reduction in future cardiovascular morbidity/mortality.(36–38) Beta-blocker therapy, statins, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, and specific diabetes therapies have been shown to play an important role in improving both endothelial function as well as cardiovascular outcomes.(38–43) The lack of coronary endothelial function improvement in this study may at least partly explain the lack of benefit seen in large randomized studies with Lp-PLA2 inhibition on cardiovascular events. Moreover, drugs shown not to improve endothelial function have also been shown to not be associated with reduction in cardiovascular events.(38) Thus, the effect of a specific intervention on endothelial function may serve as a potential mechanism for a beneficial effect on cardiovascular events. The findings of the current study are not consistent with prior studies showing a significant association of Lp-PLA2 with plaque vulnerability, inflammation, endothelial dysfunction and cardiovascular events. Several studies have suggested that Lp-PLA2 inhibition is associated with reduced inflammatory burden, improved plaque stability and potentially improved cardiovascular outcomes.(9,44) Sustained dosedependent inhibition of plasma Lp-PLA2 has been linked to reduced inflammatory burden based on Il-6 and hs-CRP levels.(8) Reduced development of advanced coronary atherosclerosis in the diabetic and hypercholesterolemic swine with darapladib further supported this anti-inflammatory effect.(45) Significantly reduced necrotic core area

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Fig. 2. Panel A depicts % change in coronary artery diameter at baseline (Panel A) on follow-up after six months (Panel B), and the change from baseline to follow-up in %change in coronary artery diameter in response to acetylcholine (Panel C). CAD: coronary artery diameter.

and medial destruction with darapladib in this model was thought to translate to reduced plaque progression and increased plaque stability as well.(45). While mechanistically, Lp-PLA2 inhibition may be associated with reduced progression of advanced lesions, this does not appear to translate to improved endothelial function or cardiovascular outcomes. Even though Lp-PLA2 was associated with adverse events in patients with unstable plaque, the role in those with early atherosclerosis and endothelial dysfunction is less clear. Lp-PLA2 may not play a key role in the early stage of atherosclerosis, potentially explaining the lack of change in endothelial function with Lp-PLA2 inhibition. Further studies examining the role of LpPLA2 inhibition and subclinical atherosclerosis may further explain the potential use of darapladib for management of endothelial function. While Lp-PLA2 has been associated with endothelial dysfunction, another possibility is that this may simply be association and not causation due to underlying mechanistic differences, explaining why inhibition of LpPLA2 was not associated with improvement in endothelial function or cardiovascular outcomes. Lp-PLA2 may serve as a biomarker for disease activity, and not an ideal therapeutic target.

A major strength of our study is the data we provide on Lp-PLA2 activity and hsCRP levels after 6 months of follow-up. With darapladib use, patients have significant reduction in Lp-PLA2 activity, which suggests adherence with the medication. Interestingly, hsCRP levels do not decrease with darapladib use however. This may imply that the darapladib dose used is not sufficient to reduce hsCRP levels and may provide a mechanistic explanation for the negative findings described in both our study as well as others. Our study has several limitations. First, we have a small sample size of patients. However, previous studies from our group were able to demonstrate an effect on coronary endothelial function with a similar group size.(27,46) Secondly, patients were not enrolled based on Lp-PLA2 activity levels. Screening based on Lp-PLA2 activity levels may identify those patients who would benefit from darapladib inhibition. Genetic polymorphisms in the LpPLA2 gene were not assessed in this study as well, and may play a role on effect of darapladib as well.(47–49) Genetic variations of the Lp-PLA2 gene may have a role in degree of enzyme activity. There are several Lp-PLA2 associated single nucleotide polymorphisms that are associated with Lp-PLA2 mass and/or activity and the presence or absence of these may determine

Fig. 3. Panel A depicts % change in coronary blood flow at baseline (Panel A) on follow-up after six months (Panel B), and the change from baseline to follow-up in %change in coronary blood flow in response to acetylcholine (Panel C). CBF: coronary blood flow.

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the ultimate role Lp-PLA2 and Lp-PLA2 inhibition plays in the occurrence of cardiovascular events.(47). Dosage and duration of therapy is another variable that may affect the results. We used similar doses as those used in previous large studies.(34,35,50) It is certainly possible that this dosage or duration of therapy may be insufficient to see an improvement in endothelial function and/or cardiovascular outcomes. Although patients were followed closely with frequent phone follow-ups, an inherent limitation of both our study as well as the STABILITY and SOLID-TIMI 52 trials, is the inability to test for medication adherence and patient compliance. However, the reduction in the levels of LpPLA2 in the treatment arm suggests that there was a sufficient degree of adherence in this group. 8. Conclusions While Lp-PLA2 has been implicated in vascular inflammation and promotion of endothelial dysfunction and cardiovascular disease, studies have shown that chronic inhibition of Lp-PLA2 has not been associated with reduction in cardiovascular events and mortality. The current study is consistent with this body of literature, and shows that there is no significant improvement in coronary endothelial function with darapladib in humans despite reduction in LpPLA2 activity. These results further support the notion that therapies shown to improve cardiovascular risk and mortality have also been shown to improve endothelial function suggesting that endothelial function plays a key role in explaining the reduced risk seen with these patients. Conflict of interest The authors report no relationships that could be construed as a conflict of interest. Perspectives What is known? Lipoprotein phospholipase A2 (LpPLA2) has been associated with increased risk of cardiovascular events, however large randomized studies using darapladib, an Lp-PLA2 inhibitor, have not shown any reduction in cardiovascular morbidity or mortality. What is new? The current randomized-controlled trial shows that despite adequate reduction in Lp-PLA2 activity, darapladib does not improve coronary endothelial function. This provides a mechanistic explanation for previous findings that darapladib is not associated with improved cardiovascular morbidity or mortality. What is next? Further investigation is necessary to better understand therapies for coronary endothelial dysfunction. Darapladib at this dose is not associated with improved endothelial function. Funding and disclosures This work was supported by the National Institutes of Health (NIH Grants HL-92954, AG-31750, DK20092, and DK102325), and the Mayo Foundation. GlaxoSmithKline provided the study drug darapladib as well as the placebo.

References [1] P. Libby, Role of inflammation in atherosclerosis associated with rheumatoid arthritis, Am. J. Med. 121 (10 Suppl 1) (2008) S21–31. [2] P. Libby, P.M. Ridker, Inflammation and atherosclerosis: role of C-reactive protein in risk assessment, Am. J. Med. 116 (Suppl 6A) (2004) 9S–16S. [3] P. Libby, Inflammation in atherosclerosis, Nature 420 (6917) (2002) 868–874. [4] R.S. Fenning, M.E. Burgert, D. Hamamdzic, E.G. Peyster, E.R. Mohler, S. Kangovi, et al., Atherosclerotic plaque inflammation varies between vascular sites and correlates with response to inhibition of lipoprotein-associated phospholipase A2, J Am. Heart Assoc. 4 (2) (2015), https://doi.org/10.1161/JAHA.114.001477. [5] K.L. Carpenter, I.F. Dennis, I.R. Challis, D.P. Osborn, C.H. Macphee, D.S. Leake, et al., Inhibition of lipoprotein-associated phospholipase A2 diminishes the deathinducing effects of oxidised LDL on human monocyte-macrophages, FEBS Lett. 505 (3) (2001) 357–363. [6] S.M. Boekholdt, R.J. de Winter, J.J. Kastelein, Inhibition of lipoprotein-associated phospholipase activity by darapladib: shifting gears in cardiovascular drug development: are antiinflammatory drugs the next frontier? Circulation 118 (11) (2008) 1120–1122. [7] D. Divchev, C. Grothusen, M. Luchtefeld, M. Thoenes, F. Onono, R. Koch, et al., Impact of a combined treatment of angiotensin II type 1 receptor blockade and 3-hydroxy-3-methylglutaryl-CoA-reductase inhibition on secretory phospholipase A2-type IIA and low density lipoprotein oxidation in patients with coronary artery disease, Eur. Heart J. 29 (16) (2008) 1956–1965. [8] E.R. Mohler 3rd, C.M. Ballantyne, M.H. Davidson, M. Hanefeld, L.M. Ruilope, J.L. Johnson, et al., The effect of darapladib on plasma lipoprotein-associated phospholipase A2 activity and cardiovascular biomarkers in patients with stable coronary heart disease or coronary heart disease risk equivalent: the results of a multicenter, randomized, double-blind, placebo-controlled study, J. Am. Coll. Cardiol. 51 (17) (2008) 1632–1641. [9] P.W. Serruys, H.M. Garcia-Garcia, P. Buszman, P. Erne, S. Verheye, M. Aschermann, et al., Effects of the direct lipoprotein-associated phospholipase A(2) inhibitor darapladib on human coronary atherosclerotic plaque, Circulation 118 (11) (2008) 1172–1182. [10] K.E. Suckling, C.H. Macphee, Lipoprotein-associated phospholipase A2: a target directed at the atherosclerotic plaque, Expert Opin. Ther. Targets 6 (3) (2002) 309–314. [11] A. Zalewski, C. Macphee, Role of lipoprotein-associated phospholipase A2 in atherosclerosis: biology, epidemiology, and possible therapeutic target, Arterioscler. Thromb. Vasc. Biol. 25 (5) (2005) 923–931. [12] E.H. Yang, J.P. McConnell, R.J. Lennon, G.W. Barsness, G. Pumper, S.J. Hartman, et al., Lipoprotein-associated phospholipase A2 is an independent marker for coronary endothelial dysfunction in humans, Arterioscler. Thromb. Vasc. Biol. 26 (1) (2006) 106–111. [13] S. Lavi, R. Lavi, J.P. McConnell, L.O. Lerman, A. Lerman, Lipoprotein-associated phospholipase A(2): review of its role as a marker and a potential participant in coronary endothelial dysfunction, Mol Diagn Ther. 11 (4) (2007) 219–226. [14] S. Fichtlscherer, M. Kaszkin, S. Breuer, S. Dimmeler, A.M. Zeiher, Elevated secretory nonpancreatic type II phospholipase A2 serum activity is associated with impaired endothelial vasodilator function in patients with coronary artery disease, Clin Sci (Lond). 106 (5) (2004) 511–517. [15] A. Honda, N. Tahara, Y. Nitta, A. Tahara, S. Igata, M. Bekki, et al., Vascular inflammation evaluated by [18F]-fluorodeoxyglucose-positron emission tomography/computed tomography is associated with endothelial dysfunction, Arterioscler. Thromb. Vasc. Biol. 36 (9) (2016) 1980–1988. [16] A. Csiszar, Z. Ungvari, Endothelial dysfunction and vascular inflammation in type 2 diabetes: interaction of AGE/RAGE and TNF-alpha signaling, Am. J. Physiol. Heart Circ. Physiol. 295 (2) (2008) H475–6. [17] B.J. von Scholten, H. Reinhard, T.W. Hansen, C.G. Schalkwijk, C. Stehouwer, H.H. Parving, et al., Markers of inflammation and endothelial dysfunction are associated with incident cardiovascular disease, all-cause mortality, and progression of coronary calcification in type 2 diabetic patients with microalbuminuria, J. Diabetes Complicat. 30 (2) (2016) 248–255. [18] T. Matsubara, T. Ishibashi, T. Hori, K. Ozaki, T. Mezaki, A. Nasuno, et al., Coronary endothelial dysfunction and inflammation of coronary atherosclerosis, J. Mol. Cell. Cardiol. 33 (6) (2001) (A74-A). [19] B.J. Choi, Y. Matsuo, T. Aoki, T.G. Kwon, A. Prasad, R. Gulati, et al., Coronary endothelial dysfunction is associated with inflammation and vasa vasorum proliferation in patients with early atherosclerosis, Arterioscler. Thromb. Vasc. Biol. 34 (11) (2014) 2473–2477. [20] A.A. Elesber, M.M. Redfield, C.S. Rihal, A. Prasad, S. Lavi, R. Lennon, et al., Coronary endothelial dysfunction and hyperlipidemia are independently associated with diastolic dysfunction in humans, Am. Heart J. 153 (6) (2007) 1081–1087. [21] A.J. Flammer, T. Anderson, D.S. Celermajer, M.A. Creager, J. Deanfield, P. Ganz, et al., The assessment of endothelial function: from research into clinical practice, Circulation 126 (6) (2012) 753–767. [22] D. Hasdai, A. Lerman, The assessment of endothelial function in the cardiac catheterization laboratory in patients with risk factors for atherosclerotic coronary artery disease, Herz 24 (7) (1999) 544–547. [23] M. Prasad, M. Reriani, S. Khosla, M. Gossl, R. Lennon, R. Gulati, et al., Coronary microvascular endothelial dysfunction is an independent predictor of development of osteoporosis in postmenopausal women, Vasc. Health Risk Manag. 10 (2014) 533–538. [24] D. Hasdai, R.J. Gibbons, D.R. Holmes Jr., S.T. Higano, A. Lerman, Coronary endothelial dysfunction in humans is associated with myocardial perfusion defects, Circulation 96 (10) (1997) 3390–3395. [25] M. Reriani, A.J. Flammer, J. Li, M. Prasad, C. Rihal, A. Prasad, et al., Microvascular endothelial dysfunction predicts the development of erectile dysfunction in men with coronary atherosclerosis without critical stenoses, Coron. Artery Dis. 25 (7) (2014) 552–557. [26] R.J. Widmer, W.Y. Chung, J. Herrmann, K.L. Jordan, L.O. Lerman, A. Lerman, The association between circulating microRNA levels and coronary endothelial function, PLoS One 9 (10) (2014), e109650. [27] M. Reriani, E. Raichlin, A. Prasad, V. Mathew, G.M. Pumper, R.E. Nelson, et al., Long-term administration of endothelin receptor antagonist improves coronary endothelial function in patients with early atherosclerosis, Circulation 122 (10) (2010) 958–966.

M. Prasad et al. / International Journal of Cardiology 253 (2018) 7–13 [28] M.J. Caslake, C.J. Packard, Lipoprotein-associated phospholipase A2 (platelet-activating factor acetylhydrolase) and cardiovascular disease, Curr. Opin. Lipidol. 14 (4) (2003) 347–352. [29] C.A. Garza, V.M. Montori, J.P. McConnell, V.K. Somers, I.J. Kullo, F. Lopez-Jimenez, Association between lipoprotein-associated phospholipase A2 and cardiovascular disease: a systematic review, Mayo Clin. Proc. 82 (2) (2007) 159–165. [30] M.S. Sabatine, D.A. Morrow, M. O'Donoghue, K.A. Jablonksi, M.M. Rice, S. Solomon, et al., Prognostic utility of lipoprotein-associated phospholipase A2 for cardiovascular outcomes in patients with stable coronary artery disease, Arterioscler. Thromb. Vasc. Biol. 27 (11) (2007) 2463–2469. [31] C.J. Packard, D.S. O'Reilly, M.J. Caslake, A.D. McMahon, I. Ford, J. Cooney, et al., Lipoproteinassociated phospholipase A2 as an independent predictor of coronary heart disease. West of Scotland coronary prevention study group, N. Engl. J. Med. 343 (16) (2000) 1148–1155. [32] C.M. Ballantyne, R.C. Hoogeveen, H. Bang, J. Coresh, A.R. Folsom, G. Heiss, et al., Lipoprotein-associated phospholipase A2, high-sensitivity C-reactive protein, and risk for incident coronary heart disease in middle-aged men and women in the Atherosclerosis Risk in Communities (ARIC) study, Circulation 109 (7) (2004) 837–842. [33] Y. Gerber, J.P. McConnell, A.S. Jaffe, S.A. Weston, J.M. Killian, V.L. Roger, Lipoproteinassociated phospholipase A2 and prognosis after myocardial infarction in the community, Arterioscler. Thromb. Vasc. Biol. 26 (11) (2006) 2517–2522. [34] H.D. White, C. Held, R. Stewart, E. Tarka, R. Brown, R.Y. Davies, et al., Darapladib for preventing ischemic events in stable coronary heart disease, N. Engl. J. Med. 370 (18) (2014) 1702–1711. [35] M.L. O'Donoghue, E. Braunwald, H.D. White, M.A. Lukas, E. Tarka, P.G. Steg, et al., Effect of darapladib on major coronary events after an acute coronary syndrome: the SOLID-TIMI 52 randomized clinical trial, JAMA 312 (10) (2014) 1006–1015. [36] Y. Kitta, J.E. Obata, T. Nakamura, M. Hirano, Y. Kodama, D. Fujioka, et al., Persistent impairment of endothelial vasomotor function has a negative impact on outcome in patients with coronary artery disease, J. Am. Coll. Cardiol. 53 (4) (2009) 323–330. [37] M.G. Modena, L. Bonetti, F. Coppi, F. Bursi, R. Rossi, Prognostic role of reversible endothelial dysfunction in hypertensive postmenopausal women, J. Am. Coll. Cardiol. 40 (3) (2002) 505–510. [38] Y. Matsuzawa, R.R. Guddeti, T.G. Kwon, L.O. Lerman, A. Lerman, Treating coronary disease and the impact of endothelial dysfunction, Prog. Cardiovasc. Dis. 57 (5) (2015) 431–442. [39] Bank AJ, A.S. Kelly, A.M. Thelen, D.R. Kaiser, J.M. Gonzalez-Campoy, Effects of carvedilol versus metoprolol on endothelial function and oxidative stress in patients with type 2 diabetes mellitus, Am. J. Hypertens. 20 (7) (2007) 777–783.

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[40] M.A. Potenza, S. Gagliardi, C. Nacci, M.R. Carratu, M. Montagnani, Endothelial dysfunction in diabetes: from mechanisms to therapeutic targets, Curr. Med. Chem. 16 (1) (2009) 94–112. [41] V.L. Franklin, F. Khan, G. Kennedy, J.J. Belch, S.A. Greene, Intensive insulin therapy improves endothelial function and microvascular reactivity in young people with type 1 diabetes, Diabetologia 51 (2) (2008) 353–360. [42] T.F. Luscher, M. Pieper, M. Tendera, M. Vrolix, W. Rutsch, F. van den Branden, et al., A randomized placebo-controlled study on the effect of nifedipine on coronary endothelial function and plaque formation in patients with coronary artery disease: the ENCORE II study, Eur. Heart J. 30 (13) (2009) 1590–1597. [43] J.A. Vita, A.C. Yeung, M. Winniford, J.M. Hodgson, C.B. Treasure, J.L. Klein, et al., Effect of cholesterol-lowering therapy on coronary endothelial vasomotor function in patients with coronary artery disease, Circulation 102 (8) (2000) 846–851. [44] H.M. Garcia-Garcia, R.M. Oemrawsingh, S. Brugaletta, P. Vranckx, J. Shannon, R. Davies, et al., Darapladib effect on circulating high sensitive troponin in patients with acute coronary syndromes, Atherosclerosis 225 (1) (2012) 142–147. [45] R.L. Wilensky, Y. Shi, E.R. Mohler 3rd, D. Hamamdzic, M.E. Burgert, J. Li, et al., Inhibition of lipoprotein-associated phospholipase A2 reduces complex coronary atherosclerotic plaque development, Nat. Med. 14 (10) (2008) 1059–1066. [46] A. Lerman, J.A. Suwaidi, J.L. Velianou, L-Arginine: a novel therapy for coronary artery disease? Expert Opin. Investig. Drugs 8 (11) (1999) 1785–1793. [47] L.M. Xu, J.Q. Zhou, S. Huang, Y. Huang, Y.P. Le, D.J. Jiang, et al., An association study between genetic polymorphisms related to lipoprotein-associated phospholipase A(2) and coronary heart disease, Experimental and Therapeutic Medicine 5 (3) (2013) 742–750. [48] H. Grallert, J. Dupuis, J.C. Bis, A. Dehghan, M. Barbalic, J. Baumert, et al., Eight genetic loci associated with variation in lipoprotein-associated phospholipase A2 mass and activity and coronary heart disease: meta-analysis of genome-wide association studies from five community-based studies, Eur. Heart J. 33 (2) (2012) 238–251. [49] R. Schnabel, J. Dupuis, M.G. Larson, K.L. Lunetta, S.J. Robins, Y.Y. Zhu, et al., Clinical and genetic factors associated with lipoprotein-associated phospholipase A(2) in the Framingham Heart Study, Atherosclerosis 204 (2) (2009) 601–607. [50] H. White, C. Held, R. Stewart, D. Watson, R. Harrington, A. Budaj, et al., Study design and rationale for the clinical outcomes of the STABILITY Trial (STabilization of Atherosclerotic plaque By Initiation of darapLadIb TherapY) comparing darapladib versus placebo in patients with coronary heart disease, Am. Heart J. 160 (4) (2010) 655–661.