Inflammation and platelet reactivity during adjunctive colchicine versus aspirin in patients with acute coronary syndrome treated with potent P2Y12 inhibitor

Background In patients undergoing percutaneous coronary intervention (PCI), the use of anti-inflammatory therapy with colchicine is associated with a reduction of recurrent ischemic events. The mechanisms of such findings are not fully elucidated. Objectives To investigate the effects of colchicine versus aspirin on inflammation and platelet reactivity in patients with acute coronary syndrome (ACS) undergoing PCI. Methods This observational study compared laboratory measurements in ACS patients receiving single antiplatelet therapy with ticagrelor or prasugrel plus colchicine (MACT) (n = 185) versus conventional dual-antiplatelet therapy (DAPT) with aspirin plus ticagrelor or prasugrel (n = 497). The primary outcome was the frequency of high residual inflammation, defined as high-sensitivity C-reactive protein (hs-CRP) ≥2 mg/L at 1 month post-PCI. Multiple sensitivity analyses were performed for the primary outcome, including multivariable adjustment, propensity-score matching, and inverse-probability weighted methods. Results One month after PCI, patients treated with MACT had significantly lower levels of hs-CRP compared to those treated with DAPT (0.6 [0.4–1.2] vs. 0.9 [0.6–2.3] mg/L, p < 0.001). The frequency of high residual inflammation was also lower in the MACT group (10.8% vs. 27.2%, p < 0.001) (odds ratio [95% confidence interval] = 0.33 [0.20–0.54], p < 0.001). This effect was consistent across sensitivity analyses. There was no difference in platelet reactivity between MACT and DAPT (49.6 ± 49.0 vs. 51.5 ± 66.4 P2Y12 reaction unit [PRU] measured by VerifyNow, p = 0.776). Conclusion In ACS patients undergoing PCI, MACT was associated with a lower rate of high residual inflammation without increasing platelet reactivity compared to conventional DAPT. Clinical trial registration NCT04949516 for MACT pilot trial and NCT04650529 for Gyeongsang National University Hospital registry.


Introduction
Dual antiplatelet therapy (DAPT) with aspirin and a P2Y 12 inhibitor has represented the standard-of-care for the treatment and prevention of thrombotic events in patients with an acute coronary syndrome (ACS) (1) undergoing percutaneous coronary intervention (PCI) (2).In this setting, prasugrel or ticagrelor are the preferred oral P2Y 12 inhibitors, in the absence of contraindications, in light of their superior efficacy over clopidogrel (3,4).However, DAPT is associated with an increased risk of bleeding which is exacerbated with the prolongation of treatment (5,6).Given the adverse prognosis associated with bleeding, over the past years a number of novel DAPT regimens have been tested with the goal of mitigating the risk of bleeding while preserving efficacy (7).Among these, P2Y 12 inhibitor monotherapy after a brief period of DAPT has emerged as a promising bleeding reduction strategy (8)(9)(10).
Inflammation plays a crucial role in the pathophysiology of ACS.Persistent elevation of inflammatory markers in ACS patients has been linked to future atherothrombotic events (11)(12)(13).Early inhibition of inflammation may be associated with enhanced benefits, particularly in patients with heightened inflammatory levels such as those presenting with ACS (14).Colchicine's anti-inflammatory effects have been shown to improve cardiovascular outcomes (15)(16)(17), it was added to DAPT in ACS patients within the past month or chronic stable patients (18).However, the early effect of colchicine on residual inflammation has not been compared with aspirin, which is recommended after PCI.In addition to its anti-inflammatory effect, colchicine possesses antithrombotic properties which may contributed to its effects on cardiovascular outcomes (19,20).Recently, the Mono-Antiplatelet and Colchicine Therapy (MACT) pilot trial demonstrated the feasibility of omitting aspiring and maintaining single antiplatelet therapy with ticagrelor or prasugrel combined with colchicine in ACS patients undergoing PCI (21).However, larger studies are warranted to better define the benefits of this approach.
Our study aims to investigate the effects of MACT on inflammation and platelet reactivity in ACS patients, comparing it to conventional DAPT.

Study design and population
This observational comparative study utilized the data from the MACT pilot trial (NCT04949516) (21) and the Gyeongsang National University Hospital (GNUH) registry (NCT04650529) (22).The MACT pilot trial assessed the feasibility of ticagrelor or prasugrel P2Y 12 inhibitor monotherapy combined with low-dose colchicine (0.6 mg once daily) in ACS patients immediately after PCI with drug eluting stent (DES) (21).The GNUH registry, a two-center database of PCI-treated patients, examined various hemostatic, vascular, and physiological parameters from January 2010 to November 2018 (22).Figure 1 provides an overview of the study flow.Among a total of 200 patients from the MACT pilot trial, 13 patients did not undergo highsensitivity C-reactive protein (hs-CRP) examination at 1 month follow-up and 2 patients received aspirin.Thus, 185 ACS patients treated with MACT in the MACT pilot trial, who underwent serial hs-CRP examinations at both admission and 1 month follow-up, were included in the present study (MACT group).From the GNUH registry, 519 ACS patients who underwent DES implantation treated with conventional DAPT, including aspirin (100 mg once daily) plus ticagrelor or prasugrel, were assessed; among them, 22 patients without statin therapy were additionally excluded to minimize bias since all patients from MACT were treated with statins.Therefore, the final analysis included 497 patients (DAPT group).The patients from both groups were not exposed to other anti-inflammatory drugs.Both studies received approval from the institutional review boards, and written informed consent was obtained from all patients in the MACT pilot trial, while consent was waived for the GNUH registry.

Laboratory measurements
The hs-CRP levels were measured using the UniCel ® DxC 800 Synchron ® Clinical System (Beckman Coulter, Inc., Brea, CA, United States) with a commercially available enzyme-linked Abbreviations: ACS, acute coronary syndrome; CV, cardiovascular; DAPT, dual antiplatelet therapy; DES, drug-eluting stent; hs-CRP, high-sensitivity C-reactive protein; MACT, mono-antiplatelet and colchicine therapy; PCI, percutaneous coronary intervention; PRU, P2Y 12 reaction unit.) and low platelet reactivity (<85 PRU) were defined based on a consensus document (23).At 1 month, hs-CRP and VeirfyNow P2Y 12 assessments were performed using blood collected from the antecubital vein at 2 to 6 h after the last drug administration.

Study outcomes
The primary outcome was the frequency of high residual inflammation, defined as a hs-CRP level of 2 mg or more per liter at 1 month post-PCI (24).Secondary outcomes were the temporal change in inflammation between admission and one month and platelet reactivity at 1 month.In addition, routine laboratory tests, including complete blood count, creatinine, aspartate aminotransferase, alanine aminotransferase, total cholesterol, highdensity lipoprotein, and low-density lipoprotein were performed at 1 month.Patients were categorized into the four groups based on the temporal change in hs-CRP levels relative to the threshold of 2 mg/L: (1) persistently high inflammation (hs-CRP ≥ 2 mg/L at both admission and 1 month); (2) aggravated inflammation (hs-CRP < 2 mg/L at admission, but ≥2 mg/L at 1 month); (3) attenuated inflammation (hs-CRP ≥ 2 mg/L at admission, but <2 mg/L at 1 month); and (4) persistently low inflammation (hs-CRP < 2 mg/L at both admission and 1 month) (11).

Statistical analysis
Continuous variables were presented as either mean ± standard deviation or median (interquartile range) and compared using appropriate statistical tests: Student's t-test, Mann-Whitney U test, or Wilcoxon signed-rank test.Categorical variables were reported as numbers (percentages) and compared using the chi-square test.Logistic regression was employed to calculate the odds ratio (OR) and 95% confidence intervals (Cis) for comparing the primary outcome between the MACT and DAPT groups.The correlation between hs-CRP levels and PRU was determined using Spearman's rank test.
Multiple sensitivity analyses were performed to adjust for baseline differences, including multivariable regression, propensity-score matching, and inverse-probability weighted analysis.The multivariable model included covariates with p < 0.10 in univariate analysis, including dyslipidemia, white blood count, hs-CRP on admission, and colchicine.For propensity-score matching and inverse-probability weighted analysis, variables that could confound the relationship between treatment and outcome were included, such as age, male, body mass index, hypertension, diabetes, dyslipidemia, clinical

Baseline characteristics
Table 1 presents the baseline characteristics of the MACT and DAPT groups.Compared with the DAPT group, the MACT group had a higher proportion of male patients and a higher body mass index, but had lower proportions of dyslipidemia, chronic kidney disease and clinical presentation of acute myocardial infarction.The MACT group also had lower white blood cell counts, but higher hemoglobin levels.Patients in the MACT group showed a lower frequency of multi-vessel disease, and were less likely to receive ticagrelor and angiotensin blockade compared to the DAPT group.

Subgroup analysis
Figure 4 shows a forest plot indicating the risk of high residual inflammation between the MACT and DAPT groups according to subgroups.The lower risk of high residual inflammation observed in the MACT group remained consistent across different baseline characteristics and low-density lipoprotein-cholesterol level at 1 month, without any significant interaction.

Discussion
This analysis represents the first comparison between colchicine and aspirin, along with a potent P2Y 12 inhibitor on their early effects on residual inflammation and platelet reactivity in ACS patients undergoing PCI.The key findings of this analysis are as follows: (1) colchicine reduced the risk of high residual inflammation (hs-CRP ≥ 2 mg/L) in comparison to aspirin; (2) the frequency of persistently high inflammation (hs-CRP ≥ 2 mg/L at both admission and 1 month) was also lower in colchicine therapy; (3) the lower risk of high residual inflammation with colchicine was consistent across subgroups such as age, diabetes, clinical presentation, systolic heart   function, renal insufficiency, baseline inflammatory activity, and residual low-density lipoprotein cholesterol level; and (4) the addition of colchicine to a potent P2Y 12 inhibitor achieved a similar antiplatelet effect to standard DAPT, as assessed by ADP-induced platelet reactivity using the VerifyNow PRU assay.These findings suggest that colchicine may offer advantages over aspirin in terms of reducing The rationale for the MACT strategy is based on two key aspects.First, in presence of potent P2Y 12 inhibition with standard doses of prasugrel or ticagrelor, aspirin has shown to have limited adjunctive antithrombotic efficacy as shown in both pharmacodynamic and clinical studies; moreover, omitting aspirin markedly reduces bleeding (25)(26)(27)(28)(29). Second, the anti-inflammatory effects of colchicine which may be of enhanced benefit early after an ACS presentation when inflammation is heightened (14).By combining these concepts, MACT aims to reduce both ischemic and bleeding events compared to conventional DAPT.Colchicine has emerged as a potential candidate with more specific anti-inflammatory efficacy than aspirin.Its anti-inflammatory effects translate into improved ischemic outcomes (15,16).Although a previous MACT pilot trial demonstrated the feasibility of this strategy in ACS patients undergoing PCI, it did not compare the effects of MACT on residual inflammation and platelet reactivity, both of which are established predictors for future atherothrombotic events, with conventional DAPT (21).The present analysis reveals a significant decrease in high residual inflammation among patients treated with MACT, regardless of baseline inflammatory activity.Specifically, the statistical significance of baseline hs-CRP level was attenuated after adjusting colchicine therapy in the multivariable model.This finding is particularly important as persistently high inflammation has been associated with higher rates of mortality and myocardial infarction (11,12,30).Furthermore, the efficacy of colchicine therapy appears to be consistent across subgroups and unaffected by clinical factors.However, there remains controversy regarding the clinical benefit of low-dose colchicine according to hs-CRP levels in patients with acute myocardial infarction.Two randomized trials reported inconsistent results despite a meta-analysis suggesting a reduction in hs-CRP and interleukin-6 levels in patients with coronary artery disease (17,31,32).
Colchicine disrupts the cellular cytoskeleton by inhibiting mitosis and intracellular transport through the suppression of tubulin polymerization.This can explain why colchicine can inhibit platelet aggregation, degranulation, and the formation of platelet-derived extracellular vesicles.Colchicine also inhibits leucocyte recruitment and activity in inflamed vascular areas (33,34).Additionally, colchicine hinders the activation of the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domaincontaining protein 3 inflammasome, as well as the secretion of tumor necrosis factor-α (33,34).Although there is lack of data regarding plaque stabilization with colchicine therapy, an observational study conducted by Vaidya et al. found that low-dose colchicine therapy led to beneficial modifications in coronary plaque, manifested by a decrease in both low attenuation plaque volume and hs-CRP levels when compared to controls (35).In addition, colchicine has been shown to modulate low-grade inflammation of adipose tissue (34) and may also modify the inflammatory activity of epicardial adipose tissue, considering its pro-inflammatory effects and contribution to high-risk plaque progression (36).However, further research is necessary to comprehensively investigate the action of antiinflammatory therapy for the treatment of coronary artery disease and explore the potential role of colchicine in current DAPT for patients undergoing PCI.
The present study found no difference in ADP-mediated platelet reactivity, assessed by PRU at 1 month post-PCI, between colchicine and aspirin when combined with a potent P2Y 12 inhibitor.Colchicine has been reported to directly inhibit the release of platelet-derived microparticles and the expression of platelet activation markers (37, ).Additionally, reduction of residual inflammation by colchicine therapy may impact platelet reactivity, as elevated levels of inflammatory markers like hs-CRP and fibrinogen have been linked to increased platelet reactivity (39,40).Although the present study did not demonstrate a correlation between hs-CRP and PRU measures, a previous study demonstrated that higher hs-CRP levels were associated with greater platelet reactivity in PCI patients treated with clopidogrel (41).Therefore, the current findings suggest that ticagrelor or prasugrel might be less susceptible to the pro-aggregatory effects of inflammation compared to clopidogrel.

Limitations
The present analysis was performed using two cohorts with different characteristics.Confounding factors or unmeasured variables such as the presence of peripheral artery disease may have affected the present results despite multiple sensitivity analyses.Additionally, potential selection bias may have led to biased results due to the different nature of the studies being compared.The baseline PRU levels were not adjusted because they were not measured in the MACT trial.However, most patients included in this study were naïve to P2Y 12 inhibitors on admission.The MACT cohort was also smaller than the DAPT cohort.Only one platelet function assay using ADP agonist was used.Therefore, although the present study showed that colchicine has a limited effect on ADP-induced platelet reactivity, it cannot be excluded that other platelet signaling pathways may be affected, thus warranting more comprehensive studies to assess the effects of colchicine on platelet biology.

Conclusion
Compared to conventional DAPT, MACT is associated with a lower risk of high residual inflammation and similar ADP-induced platelet reactivity at 1 month after PCI in ACS patients.Larger studies are warranted to understand the clinical implications of these findings.

Figure 3
Figure 3 illustrates the temporal change in inflammation between admission and 1 month follow-up.At admission, hs-CRP levels were

FIGURE 3
FIGURE 3    Temporal change in inflammation between admission and 1 month follow-up.(A) hs-CRP levels; (B) Frequency of temporal inflammation criteria.DAPT, dual antiplatelet therapy; hs-CRP, high-sensitivity C-reactive protein; MACT, mono-antiplatelet and colchicine therapy.

FIGURE 5
FIGURE 5 Platelet reactivity at one month after PCI in ACS patients.(A) P2Y 12 reaction unit levels; (B) Frequency of platelet reactivity.DAPT, dual antiplatelet therapy; MACT, mono-antiplatelet and colchicine therapy.

TABLE 1
Baseline characteristics between MACT and DAPT.

TABLE 2
High residual inflammatory risk of MACT from multiple sensitivity analyses.