Utility of VerifyNow to assess the immediate pharmacodynamic response of chewed and swallowed aspirin: comparison with aggregometry

Abstract

VerifyNow (VN) test is a less laborious method to assess pharmacodynamics (PD) compared to light transmittance aggregometry (LTA). VN assay has not been used to study the immediate PD effects of acetylsalicylic acid (ASA). Ten healthy volunteers were randomly assigned to a single 162 or 650 mg dose of chewed and swallowed ASA. Pharmacodynamic and pharmacokinetic measurements were performed at baseline and serially up to 60 min after ASA administration. Onset by VN was 20 ± 7 min with 162 mg and 13 ± 7 min with 650 mg ASA (p = .07). Onset by 1 mM AA-induced PA was 13 ± 12 min with 162 mg and 7 ± 3 min with 650 mg ASA (p=NS). VN correlated with AA-induced PA (r = 0.80, p < .001) and serum TxB₂ levels (r = 0.76, p < .001). 95% inhibition of serum TxB₂ was achieved at 38 ± 22 min and 22 ± 8 min with the 162 and 650 mg ASA, respectively (p = .08). The onset and extent of the antiplatelet effect of 650 mg ASA is numerically faster and greater than the 162 mg dose. VN identifies the onset, extent, and dose response to ASA therapy. The ease of using VN should facilitate multicenter PD investigations of ASA.

Plain Language Summary

Aspirin (acetylsalicylic acid) is an important drug widely used to prevent adverse ischemic events in patients with cardiovascular disease. Platelet aggregation and thromboxane B₂ levels in blood samples by complex laboratory methods are used to assess platelet response to aspirin. VerifyNow assay is a simple laboratory test that has not been used to assess the immediate effect of aspirin. In this study, conducted in 10 healthy volunteers, we compared the immediate platelet response to aspirin by serially assessing platelet aggregation by aggregometry and VerifyNow assay, and thromboxane B₂ levels. We also measured plasma levels of acetylsalicylic acid and salicylic acid. Our study demonstrated that the VerifyNow Aspirin test identifies the onset, extent, and dose-response to aspirin therapy. The ease of using the VerifyNow test should facilitate multicenter pharmacodynamic investigations of aspirin.

Keywords:

• Acetylsalicylic acid
• platelet aggregation
• thromboxane B₂
• VerifyNow aspirin test

Introduction

VerifyNow (VN) and light transmittance aggregometry (LTA) tests are widely used to measure the pharmacodynamic (PD) effects of antiplatelet agents and their relation to clinical outcomes in translational research studies.¹ The VN Aspirin test is a Clinical Laboratory Improvement Amendments (CLIA) waived point-of-care test that does not require an experienced technician or certified laboratory for processing. The United States Food and Drug Administration (FDA) mandates the measurement of serum thromboxane (Tx)B₂ to assess the PD effect of acetylsalicylic acid (ASA). A >95% inhibition has been used as the surrogate to indicate clinically effective inhibition.^2,3 However, there are controversies regarding the extent of TxB₂ inhibition required for thrombosis protection by ASA.⁴ There have been no PD studies employing serial measurements to assess the immediate effects of ASA administration using VN performed concomitantly with an assessment of pharmacokinetics (PK). The primary aim of the current study was to assess the immediate (within 1 h) PD effects of 162 and 650 mg doses of chewed and swallowed ASA in healthy subjects using the VN Aspirin Test in comparison with other PD assays.

Methods

This was an open-label, single dose, two treatment method comparison exploratory study conducted in 10 healthy ASA and NSAID naïve adult volunteers (5 males and 5 females) aged 18–55 years. The study was approved by the Internal Review Board (IRB) of the LifeBridge Health System. This study was performed in accordance with the principles stated in the Declaration of Helsinki.

After screening and obtaining written informed consent, subjects were randomly assigned to receive a single dose of 162 mg (n = 5) or 650 mg (n = 5) soluble ASA (Bayer Corporation, Whippany, NJ, USA). Venous blood samples were collected using an indwelling intravenous catheter prior to ASA administration (baseline, time 0) and at 2, 5, 10, 20, 30, 40 and 60 min after administration of ASA for PD and PK measurements. Arachidonic (AA) acid-induced platelet aggregation (PA) was measured using VN assay (Werfen, Bedford, MA, USA) and LTA (Chronolog Corp. Havertown, PA, USA) as previously described.^5,6 A predefined cutoff of <550 aspirin reaction units (ARU) by VN assay and ≤20% AA-induced PA by LTA has been used to define ASA therapeutic response and ASA onset.^5,6

For serum TxB₂ measurements, blood samples were collected in glass tubes (BD, Franklin Lakes, NJ, Ref 366430) without any anticoagulant and incubated immediately in a 37°C water bath for 60 min. Serum was separated after incubation and stored immediately at −80°C. For PK measurements of ASA and salicylic acid (SA), blood was collected in K₂EDTA tubes (BD, Franklin Lakes, NJ, Ref 367856), processed immediately and plasma samples were stored at −80°C. Both serum and plasma samples were shipped on dry ice (−80°C) to Syneos Health Laboratories (Québec, Canada) for further analysis. Serum TxB₂ and plasma concentrations of ASA and SA were also measured by HPLC/MS at Syneos Health Laboratories and calculated by model independent (compartment-free) methods using WinNonlin® software, version 4.1.a. (Pharsight Corporation, Mountain View, CA, USA).

Continuous variables were expressed as mean ± SD and categorical variables were expressed as number and percentages. The change in aggregation from baseline was assessed using a paired t-test; if the normality assumption was not met, a non-parametric (Wilcoxon signed-rank) method was used. The dose groups were compared at each timepoint using an independent t-test. We used polynomial regression analysis to assess the correlation between ARU by VN assay and serum TxB₂ and 1 mM AA-induced PA, since it provided a superior fit across the examined relationships. Receiver operating characteristic (ROC) curve analysis was performed to assess the relation between PD and PK parameters. p < .05 was considered as statistically significant. MedCalc® Statistical Software version 20.023 was used for statistical analysis (MedCalc Software Ltd, Ostend, Belgium).

Results

Among 11 healthy subjects enrolled in the study, one subject was unable to complete the study due to syncope upon venipuncture. The 10 remaining subjects with complete data were included in the per protocol population for analysis. There were 5 males and the overall mean age was 31 year (18–47) years. All subjects had vital signs and hematologic measurements within normal limits.

Pharmacodynamics

At pre-dose, all subjects were above the 550 ARU threshold indicating that all subjects were ASA naïve prior to dosing. Onset by VN was 20 ± 7 min with 162 mg and 13 ± 7 min with 650 mg ASA (p = .07) (Figure 1a). At pre-dose, mean 1 mM AA-induced maximum platelet aggregation was ~ 76% with both doses indicating that all subjects were ASA naïve prior to dosing. Onset was 13 ± 12 min with 162 mg and 7 ± 3 min with 650 mg ASA (p=NS) (Figure 1b). Numerically, the antiplatelet effect was earlier and greater with the 650 mg dose with both assays but a difference between the groups only reached statistical significance at 40 min with VN assay. Pre-dose TxB₂ levels were 261 ± 80 and 364 ± 157 ng/ml in the 162 mg and 650 mg ASA groups, respectively. There was a sequential decrease in serum TxB₂ after ASA administration. Mean TxB₂ levels were reduced to 20 ± 37 and 0.4 ± 0.2 ng/ml at 60 min with 162 and 650 mg ASA, respectively (p < .001). 95% inhibition of serum TxB₂ level (therapeutic ASA response) was achieved at 38 ± 22 min and 22 ± 8 min with the 162 and 650 mg ASA doses, respectively (p=NS). Numerically, the 650 mg dose was associated with faster and greater inhibition. However, differences between groups did not reach statistical significance at any timepoint (Figure 1c).

Figure 1. (a) VerifyNow aspirin test ARU = aspirin reaction units dotted red line indicates therapeutic ASA response. (b) 1 mM-Arachidonic acid-indued platelet aggregation. Dotted red line indicates therapeutic ASA response. (c) Serum thromboxane B₂. (d) Plasma acetyl salicylic acid. (e) Plasma salicylic acid.

Pharmacokinetics

There was a sequential increase in plasma exposure to ASA. Mean maximum level of ~2,000 ng/ml ASA was observed at 20 min with 162 mg and ~4,000 ng/ml ASA with 650 mg dose. Similarly, plasma exposure to SA reached mean maximum levels of ~7,500 and ~10,000 ng/ml at 20 min with 162 mg and 650 mg ASA, respectively. Plasma exposure to ASA and SA was numerically faster and greater with the 650 mg dose, but statistically significant differences between groups was observed at 60 min (the latest measured time point for PK and PD assessments) (Figure 1d,e).

Correlation between markers

A good correlation was observed between VN ARU and 1 mM AA-induced platelet aggregation (r = 0.80, p < .001) and serum TxB₂ levels (r = 0.76, p < .001) (Figure 2a,b). ROC curve analysis indicated that a cut point of ≤558 ARU by VN test was associated with >95% inhibition of serum TxB₂ (change from pre-dose) with an area under the curve (AUC) = 0.912 (specificity = 74.5, sensitivity = 95.7, p < .0001). ARU ≤ 550 was associated with ≤81 ng/ml serum TxB₂ with an AUC 0.940 (specificity = 89.7, sensitivity = 90.9 and p < .0001). In the ROC curve analysis, the 686 ng/mL ASA cut off point was associated with ≤ 585 ARU (AUC = 0.936, specificity = 90.48%, sensitivity = 92.68%, p < .001) and 4907 ng/mL SA cut off point was associated with ≤558 ARU (AUC = 0.955, specificity = 81.82%, sensitivity = 96.55%, p < .001).

Figure 2. (a) Correlation between VerifyNow aspirin test and AA-induced platelet aggregation. (b) Correlation between VerifyNow aspirin test and serum thromboxane B₂.

Discussion

The current method comparison study is the first study to assess immediate PD effects of 162 and 650 mg doses of chewed and swallowed soluble ASA using VN test and 1 mM AA-induced platelet aggregation by LTA in healthy subjects and to correlate with serum TxB₂. The important findings are: (1) the VN Aspirin test with a threshold level of < 550 ARU can be used to assess the immediate PD response to ASA, (2) the VN Aspirin test correlated with AA- induced platelet aggregation by LTA and Serum TxB₂ levels, and (3) ROC curve analysis demonstrated that a cut point of ≤558 ARU by VN Aspirin test is associated with >95% inhibition of serum TxB₂ (clinically relevant response) and ARU ≤550 was associated with ≤81 ng/ml serum TxB₂ in the presence of ASA.

The most widely used methods to assess the PD effects of ASA in clinical and translational investigations are AA-induced PA by LTA and inhibition of serum TxB₂.^1,2 Since these assays are time consuming, cumbersome, expensive and require experienced laboratory personnel, they are challenging to perform in assessing immediate effects of ASA with repeated PD measurements and in multicenter studies assessing antiplatelet effects of ASA in relation to clinical outcomes. Although, the VN aspirin test is available commercially, it has not been employed to assess PD effect immediately after ASA administration. In the current study, we have demonstrated that the VN Aspirin test can be used to assess the immediate antiplatelet effect of ASA as it is correlated with AA-induced PA and serum TxB₂ levels. A cutoff of 550 ARU can be used to indicate ASA response based on the conventional cut point of 95% inhibition of serum TxB₂. It appears that very early effects (<5 min) of ASA effect are better detected by serum TxB₂ and 1 mm AA-induced PA than the VN aspirin test. However, with an increased number of subjects, the VN test would have detected very early effects of ASA. The current study included a total of 560 PD and PK measurements in 10 subjects for comparing VN aspirin test with other PD assays that we believe is sufficient to reach our conclusions.^7–9 The current PD and PK measurements were comparable to earlier studies where these measurements were performed repeatedly within 1 h.^10,11 Limitations of this exploratory study include the number of subjects may be low for comparison between baseline and on-treatment time points and also to study heavier subjects where response to aspirin dose may be different. In addition, aspirin response may be influenced by comorbidities and co-medications in studies with patients and further studies in patients are required to explore these interactions.

A novel inhaled nanoparticle ASA preparation that can be administered with a dry powder inhaler (Vectura, Inc., New York, NY) is being developed to achieve earlier and greater platelet inhibition than occurs after chewing and swallowing soluble ASA. Immediate inhibition of platelet function is essential to prevent evolving arterial thrombus formation in the early stages of an acute coronary syndrome event. In a pilot study with this novel inhaled nanoparticle ASA, we have previously demonstrated faster and greater platelet inhibition using LTA and serum TxB₂ measurements with the inhaled ASA formulation compared to chewed and swallowed ASA.⁹ The VN Aspirin test is being used in 2 ongoing multicenter center studies to compare the immediate PD effects of inhaled aspirin with chewed and swallowed ASA in healthy volunteers (NCT05625334) and in adult subjects with obstructive or restrictive pulmonary function (NCT05625347). The point-of-care VN test will assist in analyzing the antiplatelet effect of a novel inhaled ASA formulation during multicenter drug development studies and potentially assist in monitoring ASA effects in the future. We feel that our study is novel and provides an important message to the translational researcher, particularly those who are involved in investigations where rapid (within minutes) information on aspirin response is critical- for example in the care of the acute stroke or myocardial infarction patient. In conclusion, the VN Aspirin test identifies the onset, extent, and dose response to ASA therapy. The ease of using VN Aspirin test may facilitate multicenter pharmacodynamic investigations of aspirin.

Disclosure statement

Dr. Gurbel has received consulting fees and/or honoraria from Bayer, Otitopic/Vectura, Janssen, UpToDate, Cleveland Clinic, Adeno, Wolters Kluwer Pharma, Web MD Medscape, Baron and Budd, North American Thrombosis Forum, Innovative Sciences; institutional research grants from the Haemonetics, Janssen, Bayer, Instrumentation Laboratories, Amgen, Idorsia, Otitopic, Hikari Dx, Novartis, and R-Pharma International; in addition, Dr. Gurbel has two patents, Detection of restenosis risk in patients issued and Assessment of cardiac health and thrombotic risk in a patient. Dr. Gurbel was an expert witness in a lawsuit associated with Plavix.

Additional information

Funding

The study was sponsored by Platelet and Thrombosis Research, LLC and funded by Vectura, Inc (120 Park Avenue, New York, NY, USA).Serum Thromboxane B₂, and plasma acetyl salicylic acid and salicylic acid measurements were done at Syneos Health (Syneos Health Clinique inc. 2500, rue Einstein, Québec, Canada).