FormalPara Key Summary Points

Why carry out this study?

Cardiovascular disease is the leading cause of morbidity and mortality worldwide, and aspirin is recommended in patients with coronary artery disease to reduce the risk of major adverse cardiovascular events (MACE).

Aspirin resistance (AR) describes a phenomenon whereby patients receiving aspirin therapy respond in a suboptimal manner, evidenced by continued MACE and/or the lack of attenuated platelet reactivity. Studies demonstrate that approximately 25% of patients with vascular disease are resistant to aspirin therapy, conferring an almost fourfold increased risk of MACE.

What was learned from the study?

Participants on Bayer® low-dose enteric-coated aspirin 81 mg tablets (LD EC-ASA) (Bayer AG, Leverkusen, North Rhine-Westphalia, Germany) had a mean aspirin reaction unit (ARU) score of 426, with 19.1% having an ARU > 550; patients on Vazalore® low-dose phospholipid-aspirin liquid-filled 81 mg capsules (LD PL-ASA) (PLx Pharma Inc., Sparta, NJ, USA) derived a mean ARU score of 435, with 14.9% achieving an ARU > 550. There were no significant differences in AR (ARU > 550) according to the formulation (Bayer® LD EC-ASA vs. Vazalore® LD PL-ASA) used.

This dedicated pharmacodynamic study could potentially be informative and applicable for Trinidadian patients on dual antiplatelet therapy.

Introduction

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide, and aspirin is recommended in patients with coronary artery disease (CAD) to attenuate major adverse cardiovascular events (MACE) [1,2,3]. Several studies reveal discordant findings regarding the preferred dosage and formulation of aspirin in patients with CAD with varying risk–benefit profiles [4,5,6,7]. Absorption of enteric-coated aspirin (EC-ASA) tablets is suboptimal, which can result in decreased clinical effectiveness. Phospholipid-aspirin liquid-filled capsules (PL-ASA) are a novel immediate-release formulation, approved by the Federal Drug Administration (FDA) in 2022, that can mitigate adverse gastrointestinal effects [8].

High on-treatment platelet reactivity (HPR) to clopidogrel is significantly higher in South Asians (> 60% of patients), which has severe clinical repercussions considering the cardiovascular disease pandemic [9, 10]. South Asians are four times as likely to carry the CYP2C19*2 allele than Caribbean Black patients [9, 11]. The combination of aspirin resistance (AR) with HPR may be potentially catastrophic in these patients with CVD, alluding to a “double-hit” phenomenon.

We conducted this exploratory pilot study to assess the antiplatelet pharmacodynamic effect of Bayer® low-dose enteric-coated aspirin 81 mg tablets (LD EC-ASA) (Bayer AG, Leverkusen, North Rhine-Westphalia, Germany) compared to Vazalore® low-dose phospholipid-aspirin liquid-filled 81 mg capsules (LD PL-ASA) (PLx Pharma Inc., Sparta, NJ, USA) on platelet reactivity with respect to aspirin reaction units (ARU) and in a Trinidadian subpopulation comprising healthy participants.

Methods

Study Design and Patient Population

The study complied with the tenets of Declaration of Helsinki, International Conference on Harmonization, Good Clinical Practice (ICH-GCP), and was authorized by the Campus Research Ethics Committee (CREC) of the University of the West Indies, St. Augustine (UWI STA), Trinidad (CEC1153/06/19) [12, 13]. All healthy volunteers consented to participate in a prospective, open-label, single-arm crossover study that evaluated the effect of Bayer® LD EC-ASA (Bayer AG, Leverkusen, North Rhine-Westphalia, Germany) daily for 14 days, with a washout period of 28 days, followed by Vazalore® LD PL-ASA (PLx Pharma Inc., Sparta, NJ, USA) daily for 14 days, again followed by aspirin reaction unit (ARU) testing. Healthy volunteers were screened and recruited between January 2024 and March 2024 at the ambulatory outpatient center at our institution, Trinidad Institute of Medical Technology (TIMT), Trinidad and Tobago). They met selection criteria if they were above 18 years of age and healthy, with no pre-existing medical condition. Pre-specified exclusion included any aspirin reaction, allergy or hypersensitivity, declining participation, and any prior medical condition. Patients were instructed to self-administer aspirin during or shortly after meal ingestion. The patients were monitored for 28 days post-completion to evaluate for any serious adverse events.

VerifyNow™ Aspirin Reaction Unit (ARU) Testing

Aspirin was withheld on the day of testing, almost 18 h before baseline blood sampling, which ensured the determination of trough levels. Whole blood samples were acquired by antecubital puncture utilizing a 21-gauge needle (Greiner Bio-One North America, Monroe, NC, USA, #450097). They were placed into Vacuette (Greiner Bio-One North America, Monroe, NC, USA) citrated blood collecting vials (containing 3.8% trisodium citrate) (#454322) after discarding the first 5 ml of waste. Blinded clinical research associates processed samples. VerifyNow™ ARU assays (Werfen, Bedford, MA, USA) were utilized in accordance with standardized protocols [14, 15]. The assay reports the results as aspirin reaction units (ARU). The pre-specified level of aspirin resistance was ≥ 550 ARU. The enrolled patients were then treated with Bayer® aspirin 81 mg once daily for 14 days, with a 28-day washout period, followed by Vazalore® aspirin 81 mg once daily for 14 days, with pill accountability verified by the clinical research associate (Fig. 1).

Fig. 1
figure 1

Consolidated Standards of Reporting Trials (CONSORT) diagram

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Patient Interview and Case Report Form

The patients’ demographic data were transcribed on a case report form (CRF). They included the patient’s age, gender, ethnicity, anthropometric data, respective ARUs on Bayer® LD EC-ASA and Vazalore® LD PL-ASA, and adverse drug reactions such as gastrointestinal upset or severe bleeding.

Statistical Analysis

The calculated sample size was 47 patients based on a paired proportion sample, an alpha (α) value of 0.05, power of 80%, estimated baseline prevalence of 45% of ARU > 550, and an absolute delta of 22.5% (expected prevalence of 22.5% of ARU > 550) [16]. The accrued data were assessed using descriptive statistics as well as SPSS version 28.0 software. The descriptive data were presented using frequencies with percentages for categorical variables, means for continuous data, and medians for ordinal data. Independent comparisons were performed using Fisher’s exact test for categorical variables. Pairwise comparisons were made using McNemar’s test for paired proportions, and Wilcoxon signed-rank test for ordinal data. The Kruskal–Wallis test was used to compare medians across three categories. Spearman’s correlation was used to evaluate associations between non-normal continuous variables. A p value of < 0.05 was used to indicate statistical significance. All participants were 100% compliant with administration of both study drugs, verified with pill accountability, and completed this study without any protocol deviations.

Results

Of a total of 47 healthy participants, the average age was 34 (standard deviation (SD) ± 10.9) years, with 55% representative of the male gender. Caribbean South Asians comprised 68%, Caribbean Blacks 17%, and mixed and/or interracial 15% of the study population. The mean body mass index (BMI) was 25.9 (SD ± 6.11) kg/m2, and the mean body surface area (BSA) was 1.84 (SD ± 0.23) m2 (Table 1). There were no statistically significant differences in aspirin resistance (ARU > 550) according to the brand (Bayer® vs. Vazalore®) used (Fig. 2, Table 2). Aspirin resistance was independent of ethnicity regardless of the brand used (Table 3). In addition, there were no significant associations between body surface area (BSA) and Bayer® ARU value (p value 0.788) or Vazalore® ARU value (p value 0.477) (Table 3). No patients experienced any serious adverse events nor treatment-emergent adverse events during their active participation and follow-up period of 28 days.

Table 1 Patients’ demographic information
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Fig. 2
figure 2

Comparison of patients’ aspirin reaction units (ARU) on Bayer® low-dose enteric-coated aspirin 81 mg tablets (LD EC-ASA) (Bayer AG, Leverkusen, North Rhine-Westphalia, Germany) compared to Vazalore® low-dose phospholipid-aspirin liquid-filled 81 mg capsules (LD PL-ASA) (PLx Pharma Inc., Sparta, NJ, USA) followed by ARU testing

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Table 2 Comparison of patients’ aspirin reaction units (ARU) on Bayer® low-dose enteric-coated aspirin 81 mg tablets (LD EC-ASA) (Bayer AG, Leverkusen, North Rhine-Westphalia, Germany) compared to Vazalore® low-dose phospholipid-aspirin liquid-filled 81 mg capsules (LD PL-ASA) (PLx Pharma Inc., Sparta, NJ, USA)
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Table 3 Comparison of patients’ aspirin reaction units (ARU) on Bayer® low-dose enteric-coated aspirin 81 mg tablets (LD EC-ASA) (Bayer AG, Leverkusen, North Rhine-Westphalia, Germany) compared to Vazalore® low-dose phospholipid-aspirin liquid-filled 81 mg capsules (LD PL-ASA) (PLx Pharma Inc., Sparta, NJ, USA) with respect to gender, age, ethnicity, body mass index (BMI), and body surface area (BSA)
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Discussion

Acetylsalicylic acid irreversibly inhibits the activity of platelet cyclooxygenase-1 (COX-1), thereby inhibiting the platelet production of thromboxane A2 (TXA2) [17, 18]. By preventing the formation of TXA2, ASA reduces platelet activation and aggregation promoted by TXA2 [19, 20].

ASA is frequently prescribed with meals to alleviate dyspeptic symptoms. EC-ASA is erratically absorbed, and the bioavailability decreases with the ingestion of food. The novel lipid-aspirin complex (PL-ASA) liquid capsule formulation is an immediate-release aspirin with consistent bioavailability that has been specifically designed to attenuate gastrointestinal (GI) effects [6, 21]. There has been a recent paradigm shift in the pharmaceutical development and commercialization of phosphatidylcholine–nonsteroidal anti-inflammatory drugs (PC-NSAIDs), namely Vazalore®, that reduce GI injury, such as ulcers, without compromising therapeutic activity [22, 23]. The PC-NSAID complex (PLxGuard™ delivery platform, PLx Pharma Inc., Sparta, NJ, USA) remains intact in low pH environments, such as the stomach, limiting free aspirin contact with the gastric mucosa [24]. A recent prospective, randomized crossover study demonstrated that LD PL-ASA resulted in faster platelet inhibition compared with EC-ASA, which could not be attributed to ASA formulation [25]. Lipid excipients used in LP-ASA did not impair the pharmacodynamic effects or alter platelet function [26].

Aspirin resistance (AR) describes a phenomenon where patients receiving aspirin therapy respond in a suboptimal manner, evidenced by continued MACE and/or the lack of attenuated platelet reactivity. Studies demonstrate that approximately 25% of patients with vascular disease are resistant to aspirin therapy, conferring an almost fourfold increased risk of MACE [27]. Novel antithrombotic strategies incorporating clopidogrel, ticagrelor, and direct oral anticoagulants such as rivaroxaban improve outcomes in patients with vascular disease [27]. Many investigators postulate that AR is implicated in major adverse cardiovascular and cerebrovascular events (MACCE) [28,29,30,31]. AR is a heterogeneous entity and remains challenging to define clinically or biochemically [32]. Fundamentally, it is the inability of aspirin to attenuate TXA2 production with effects on platelet activation and aggregation. Contributory factors include inappropriate dosage dose, drug–drug interactions, polymorphisms of COX-1 genes regulating thromboxane biosynthesis, upregulation of other sources of thromboxane production, and accentuated platelet turnover [33, 34]. Conditions that influence AR include age, gender, acute coronary syndromes, heart failure, chronic kidney disease, anemia, diabetes mellitus, tobacco use, concomitant NSAID use, malabsorption, and genetic factors [35, 36]. Compliance is also a key factor in the suboptimal response to ASA [28]. Medication compliance is a critical aspect of this phenomenon and potentially contributes to its overreporting. Thus, counseling patients with respect to the benefits of antiplatelet therapy may enhance compliance [37]. Our study did not show any statistically significant association with gender, ethnicity, body mass index, or body surface area. Our participants’ compliance was verified by pill accountability by the consultant cardiologists.

The VerifyNow™ (VN) (Werfen, Bedford, MA, USA) is a bedside, point-of-care platelet function analyzer in which a blood sample is exposed to fibrinogen-coated beads and platelet agonists. Light transmission is accentuated with platelet aggregation, signifying the absence of an antiplatelet effect and suggesting aspirin resistance [33]. VerifyNow™ test results are expressed in aspirin reaction units (ARU), and the cutoff for determination of aspirin resistance was ≥ 550 ARU [14]. The prevalence of aspirin resistance is estimated to be between 5.5 and 60% in patients, contingent on the definition, assessment, and parameters utilized [16]. When the VN-ARU test is used, the prevalence ranges from 7 to 27% [38, 39]. However, using the “gold standard” methodology, optical aggregometry, the prevalence is about 0.4–9%. Our study displayed similar results, with a mean ARU score of 426 and 19.1% of participants having an ARU > 550 on LD EC-ASA. LD PL-ASA derived a mean ARU score of 435, with 14.9% of participants achieving an ARU > 550, of which neither parameter reached statistical significance. There is the caveat that our participants were all healthy volunteers without any pre-existing comorbidity.

Currently, the decision whether to initiate LD ASA use for the primary prevention of CVD in middle-aged adults who have a 10% or greater 10-year CVD risk should be tailored to each individualized, as the net clinical benefit is marginal. The United States Preventive Services Task Force (USPSTF) recommends against the use of LD ASA for the primary prevention of CVD in older adults [40]. However, it is crucial to note that these overarching recommendations are derived from a completely different ethnic profile from that encountered in the Caribbean.

Important information including prospective data demonstrating that AR is detrimental, evidence that alternative antiplatelet therapies and strategies can achieve a clinical effect, and support for a reliable test that can assess AR status and predict outcomes is pivotal in attenuating CVD burden and MACE [41,42,43].

Study Limitations

Despite being powered for pharmacodynamic ARU values, this study was not designed for clinical outcomes and, thus, is limited with respect to external validity for the efficacy and safety of aspirin formulations in patients with CAD.

This study was of a prospective, open-label, single-arm crossover design. The study was not randomized or blinded, as there are currently no pharmaceutical division resources for interaction response technology, such as interaction web randomization services in this region. As a result, selection bias during enrolment and inaccurate estimates of the treatment effects of the two aspirin regimens can occur. Currently, there are also no contract research organizations (CROs) that operate within Trinidad and Tobago. Therefore, the study investigators decided to pursue an open-label, pragmatic study for transparency and supervised all other aspects, as there were no third-party entities to specifically coordinate randomization and/or blinding, as would be expected in large-scale trials. Conducting clinical trials, even small pilot studies, in the Caribbean is very difficult as there are limited resources with respect to grant financing, logistical planning, and experienced personnel who are qualified and trained in clinical trial management.

The study showed a predilection for Caribbean South Asian patients (68%), which is strikingly similar to other exploratory pilot studies performed by this group, suggestive of an inherent selection bias [15, 44,45,46].

Unfortunately, the prevalence of AR is contingent on the assay utilized, which has relatively poor inter-test reliability [32]. Comprehensive platelet function testing biochemical and pharmacokinetic data may prove to be more informative; however, due to several logistical issues, these are not currently available in Trinidad.

Conclusions

There were no statistically significant differences in aspirin resistance between Bayer® LD EC-ASA and Vazalore® LD PL-ASA. This dedicated pharmacodynamic study could be potentially informative for Caribbean patients on dual antiplatelet therapy (DAPT). Further prospective studies are required to confirm these results.