Impact of cerebral small vessel disease burden and drug level at admission on direct oral anticoagulant associated intracerebral hemorrhage.

INTRODUCTION: Direct oral anticoagulant (DOAC)-associated intracerebral hemorrhage (ICH) is a catastrophic complication. The aim of this study was to investigate the association between computed tomography (CT)-based cerebrovascular small vessel disease (SVD) burden and DOAC-ICH as well as the DOAC concentration upon hospital admission and ICH outcome. PATIENTS AND METHODS: The study included two cohorts: (1) DOAC-ICH: patients who suffered from DOAC-ICH and underwent drug level measurements upon admission; (2) DOAC-non-ICH: stable DOAC users who underwent head CT without ICH during treatment. We categorized the DOAC levels of the DOAC-ICH patients as low (<50 ng/mL), medium (50-300 ng/mL), and high (>300 ng/mL). The CT-based SVD burden (including white matter lesions [WML], lacunes, and cerebral atrophy) was evaluated, and SVD scores (range, 0-3) were used to evaluate SVD severity. RESULTS: A total of 43 DOAC-ICH patients and 177 DOAC-non-ICH patients were enrolled. DOAC-ICH patients were more likely to have WML, lacunes, or cerebral atrophy compared to DOAC-non-ICH patients. After adjustment, the SVD burden was associated with DOAC-ICH, with a higher risk of more severe SVD (SVD score of 2; odds ratio [OR], 10.3 [3.17, 33.3]; score of 3; OR, 16.8 [4.50, 62.6]). The proportions of patients with high, medium, and low drug levels in the DOAC-ICH group were 16.3%, 55.8%, and 27.9%, respectively. Additionally, the high-level group displayed a larger hematoma size and had worse functional outcomes at 3 months than the other two groups. DISCUSSION AND CONCLUSION: The severity of SVD burden was associated with DOAC-ICH. Furthermore, high DOAC levels in ICH were associated with unfavorable clinical outcomes. To address the potential selection bias from these two cohorts, a prospective study to investigate the co-contribution of drug levels and SVD to DOAC-ICH is essential.

Impact of Direct Oral Anticoagulant Concentration on Clinical Outcomes in Asian Patients with Atrial Fibrillation.

A real-world association between direct oral anticoagulant (DOAC) concentration and clinical outcomes among Asian patients with atrial fibrillation (AF) is reported herein. Patients with AF aged ≥ 20 years who used DOAC for ≥ 3 days were enrolled. Trough and peak DOAC concentrations were measured and compared with the expected range reported in clinical trials. The Cox proportional hazard model was used to investigate the association between concentration and outcomes. From January 2016 to July 2022, a total of 859 patients were enrolled. Among them, 22.5%, 24.7%, 36.4%, and 16.4% were on dabigatran, rivaroxaban, apixaban, and edoxaban, respectively. Compared with clinical trials, the proportion of DOAC concentrations higher or lower than the expected range were 9.0% and 14.6% for trough, respectively, and 20.9% and 12.1% for peak, respectively. The average follow-up duration was 2.4 ± 1.6 years. The incidence of stroke and systemic thromboembolism (SSE) was 1.31 per 100-person years, and low trough concentration predicted SSE (hazard ratio (HR) = 2.78 (1.20, 6.46)). The incidence of major bleeding was 1.64 per 100-person years, and high trough was associated with major bleeding (HR = 2.63 (1.09, 6.39)). The association between peak concentration and SSE or major bleeding was nonsignificant. Off-label underdosing (odds ratio (OR) = 2.69 (1.70, 4.26)), once daily DOAC dosing (OR = 3.22 (2.07, 5.01)), and high creatinine clearance (OR = 1.02 (1.01, 1.03)) caused low trough concentration. Contrarily, congestive heart failure was significantly associated with high trough concentration (OR = 1.71 (1.01, 2.92)). In conclusion, trough DOAC concentration measurements should be considered among patients at risk of out-of-expected range DOAC concentrations.

Impact of amiodarone on plasma concentration of direct oral anticoagulant in patients with atrial fibrillation.

BACKGROUND/PURPOSE: Amiodarone increases exposure of direct oral anticoagulants (DOACs). We aimed to analyze the effects of concurrent amiodarone use on DOAC concentrations and clinical outcomes. METHODS: Patients who were ≥20 years of age, had atrial fibrillation, and took DOAC were enrolled to provide trough and peak samples for DOAC concentration measurements using ultra-high-performance liquid chromatography-tandem mass spectrometry. The results were compared with concentrations reported in clinical trials to define above, within, or under the expected range. The outcomes of interest were major bleeding and any gastrointestinal bleeding. Multivariate logistic regression and Cox proportional hazards model were used to determine the impact of amiodarone on above-range concentration and clinical outcomes, respectively. RESULTS: A total of 722 participants (420 men, 58.2%) were enrolled to provide 691 trough samples and 689 peak samples. Among them, 21.3% concurrently used amiodarone. The proportion of patients with above-range trough and peak concentrations was 16.4% and 30.2%, respectively, for amiodarone users, in contrast to 9.4% and 19.8% for amiodarone non-users. The use of amiodarone was associated with above-range trough and peak concentrations (odds ratio [OR] = 2.00 [1.16, 3.47] and 1.82 [1.19, 2.79], respectively). However, amiodarone was not a significant predictor of major bleeding or any gastrointestinal bleeding. CONCLUSION: Concurrent amiodarone use led to increased DOAC concentration but was not associated with a higher risk of major bleeding or any gastrointestinal bleeding. Therapeutic monitoring of DOAC users concurrently taking amiodarone may be recommended for patients with an additional risk of increased DOAC exposure.

Site-specific labeling of SBDS to monitor interactions with the 60S ribosomal subunit.

The Shwachman-Diamond syndrome (SDS) is a rare inherited ribosomopathy that is predominantly caused by mutations in the Shwachman-Bodian-Diamond Syndrome gene (SBDS). SBDS is a ribosomal maturation factor that is essential for the release of eukaryotic translation initiation factor 6 (eIF6) from 60S ribosomal subunits during the late stages of 60S maturation. Release of eIF6 is critical to permit inter-subunit interactions between the 60S and 40S subunits and to form translationally competent 80S monosomes. SBDS has three key domains that are highly flexible and adopt varied conformations in solution. To better understand the domain dynamics of SBDS upon binding to 60S and to assess the effects of SDS-disease specific mutations, we aimed to site-specifically label individual domains of SBDS. Here we detail the generation of a fluorescently labeled SBDS to monitor the dynamics of select domains upon binding to 60S. We describe the incorporation of 4-azido-l-phenylalanine (4AZP), a noncanonical amino acid in human SBDS. Site-specific labeling of SBDS using fluorophore and assessment of 60S binding activity are also described. Such labeling approaches to capture the interactions of individual domains of SBDS with 60S are also applicable to study the dynamics of other multi-domain proteins that interact with the ribosomal subunits.

Development of the dried blood spot preparation protocol for comprehensive evaluation of the hematocrit effect.

The application of dried blood spots (DBS) has gradually increased in different fields because of its several advantages. The hematocrit (Hct) effect is one major analytical challenge that may affect the quantification accuracy of DBS samples and should be investigated when developing a novel DBS method. However, previous studies usually overlooked the Hct-related distribution bias when evaluating the Hct effect. This study aimed to propose an effective DBS preparation protocol for the comprehensive evaluation of the Hct effect. We selected voriconazole and posaconazole as the demonstration drugs. Fifteen microliters of the blood samples were spotted on DBS cards followed by whole spot extraction. An LC-MS/MS method was first developed to quantify voriconazole and posaconazole in DBS samples. The quantitation accuracy for both azole drugs was within 93.5%-111.7%, except for the accuracies of posaconazole at the LLOQ, which were less than 119%. The intra- and interday precision were below 11%. The validated LC-MS/MS method was used to develop the DBS preparation protocol for evaluating the Hct effect. Three critical parameters that may affect the observed Hct effect were investigated. The results showed that using the solid-state of the target analytes, spiking the target analytes before preparing different Hct levels, and allowing enough equilibrium time after spiking target analytes can provide a more holistic Hct effect evaluation. The validity of the proposed new protocol was verified by conversion factors obtained from 71 paired DBS and plasma samples. Conversion factors calculated by clinical samples were consistent with the Hct effect evaluated by manually prepared DBS samples. This new DBS preparation protocol eliminated the common pitfalls in studying the Hct effect and offered a comprehensive strategy to assess the Hct effect for further DBS studies.

Impact of different renal function equations on direct oral anticoagulant concentrations.

The purpose of this study is to investigate the correlation between glomerular filtration rate (GFR) estimated by different renal function equations and non-vitamin K antagonist oral anticoagulant concentration. Atrial fibrillation patients who aged ≥ 20 years and used dabigatran, rivaroxaban, or apixaban for thromboembolism prevention were enrolled to collect blood samples and measure drug concentrations using ultra-high-performance liquid chromatography with tandem mass spectrometry. The GFR was estimated using the Cockroft-Gault formula (abbreviated as creatinine clearance, CrCL), Chronic Kidney Disease Epidemiology Collaboration formula (CKD-EPI) featuring both creatinine and cystatin C, and the Modification of Diet in Renal Disease Study equation (MDRD). Multivariate regression was used to investigate the associations of different renal function estimates with drug concentrations. A total of 511 participants were enrolled, including 146 dabigatran users, 164 rivaroxaban users and 201 apixaban users. Compared to clinical trials, 35.4% of dabigatran, 4.9% of rivaroxaban, and 5.5% of apixaban concentrations were higher than the expected range (p < 0.001). CKD-EPI and MDRD estimates classified fewer patients as having GFR < 50 mL/min than CrCL in all 3 groups. Both CrCL and CKD-EPI were associated with higher-than-expected ranges of dabigatran or rivaroxaban concentrations. Nevertheless, none of the renal function equations was associated with higher-than-expected apixaban concentrations. For participants aged ≥ 75 years, CKD-EPI may be associated with higher-than-expected trough concentration of dabigatran. In conclusion, CrCL and CKD-EPI both can be used to identify patients with high trough concentrations of dabigatran or rivaroxaban. Among elderly patients who used dabigatran, CKD-EPI may be associated with increased drug concentration.

Measurement of Dabigatran Concentration Using Finger Prick Dried Blood Spot Sample Collection.

Background and Purpose: Real-world laboratory monitoring of dabigatran activity is challenging. The purpose of the present study was to demonstrate the feasibility and accuracy of finger prick sampling with dried blood spot (fpDBS) cards in measuring the dabigatran concentration. Material and Methods: Patients >20 years of age with atrial fibrillation and receiving dabigatran therapy for more than 7 days were included in the study. Peak and trough dabigatran concentrations were collected by simultaneous finger prick and venous puncture. The dabigatran concentration was measured by ultra-high performance liquid chromatography with tandem mass spectrometry. Our previously developed post-column infused internal standard (PCI-IS) method was applied to estimate the blood spot volume on fpDBS and to calibrate the drug concentration. Deming regression was used to analyze the correlation between dabigatran concentration on fpDBS cards and in plasma samples, followed by Bland-Altman analysis to compare the bias between two sampling techniques. Results: A total of 33 patients were enrolled and contributed 66 plasma and 55 fpDBS dabigatran samples. The average patient age was 74.6 ± 7.9 years, mean creatinine clearance 58.1 ± 18.3 mL/min, and CHA2DS2-VASc score 3.5 ± 1.6 points. The dabigatran concentration ranged from 41.8-1421.7 ng/mL. The plasma and DBS dabigatran concentrations correlated well (r = 0.98), and the conversion factor for fpDBS to plasma dabigatran concentration was 1.28. The Bland-Altman analysis showed that 94.5% of the fpDBS-predicted concentration fell within 20% of bias. Conclusions: The study showed that fpDBS measurement of dabigatran concentration is reliable and can be applied in clinical scenarios.

Using the PCI-IS Method to Simultaneously Estimate Blood Volume and Quantify Nonvitamin K Antagonist Oral Anticoagulant Concentrations in Dried Blood Spots.

Nonvitamin K antagonist oral anticoagulants (NOACs) have emerged as the preferred choice for the treatment of atrial fibrillation (AF). The establishment of a therapeutic range to minimize bleeding and thrombosis is important for personalized treatment of NOACs. The importance of dried blood spots (DBSs) has increased in medical care. An efficient and effective DBS analytical method could facilitate the concentration management of NOACs. The postcolumn infused internal standard (PCI-IS) method was applied to estimate spot volume and quantify dabigatran, rivaroxaban, and apixaban concentrations on DBS cards. The extraction solvent contented 0.1% formic acid and 70% ACN with a successive extraction procedure. Paired DBS and plasma samples from patients undergoing NOAC therapy (n = 269) were used to calculate conversion factors. [13C6]-Rivaroxaban was selected as the PCI-IS. The quantification accuracy for the three NOACs was within 88.9-104.3%. The RSDs of the repeatability and intermediate precision were below 10%. The obtained conversion factors of DBS to plasma concentrations of dabigatran, apixaban, and rivaroxaban were 1.81, 1.59, and 1.31, respectively. Bland-Altman analysis showed that the % differences between predicted and measured plasma concentrations were within a bias of ±20%. The result showed that PCI-IS was an accurate and efficient LC-MS/MS method to simultaneously estimate blood volume and NOAC concentrations on DBS cards. The stability results revealed that the DBS sampling strategy could improve compound stability. The developed method offers a new strategy for the therapeutic drug monitoring of NOACs and may improve the safe use of these drugs.