Measurement of Anti-Factor Xa Activity in Patients on Apixaban for Non-Valvular Atrial Fibrillation

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1 2584 OSANAI H et al. Circulation Journal ORIGINAL ARTICLE Official Journal of the Japanese Circulation Society Arrhythmia/Electrophysiology Measurement of Anti-Factor Xa Activity in Patients on Apixaban for Non-Valvular Atrial Fibrillation Hiroyuki Osanai, MD; Masayoshi Ajioka, MD; Tomohiro Masutomi, MD; Tasuku Kuwayama, MD; Sota Ishihama, MD; Yusuke Sakamato, MD; Naoya Otaka, MD; Teruhiro Sakaguchi, MD; Yosuke Inoue, MD; Takahiro Kanbara, MD; Yoshihito Nakashima, MD; Hiroshi Asano, MD; Kazuyoshi Sakai, MD Background: Chromogenic anti-factor Xa activity (AXA) assay is reported to be the most appropriate method to measure the pharmacodynamics of apixaban, but the distribution of AXA in non-valvular atrial fibrillation (NVAF) patients on apixaban therapy has not been fully elucidated. Methods and Results: Steady-state trough and peak AXA were measured in 124 NVAF patients taking apixaban. In 25 patients, baseline, first peak, and trough AXA were also examined, and were 0.01±0.02 IU/ml, 0.83±0.43 IU/ml, and 0.34±0.17 IU/ml, respectively. First trough AXA was significantly lower than steady-state trough AXA, although it was significantly higher than baseline (P<0.0001). Similarly, first peak AXA was significantly lower than steady-state peak AXA (P<0.0001). In 124 patients, steady-state peak AXA was significantly higher in the 5-mg b.i.d. group than in the 2.5-mg b.i.d. group (2.05±0.73 IU/ml vs. 1.51±0.65 IU/ml, respectively; P<0.001), although there was no significant difference in trough AXA. Other than dose, age and serum creatinine were significantly related to both trough and peak AXA. Conclusions: The distribution of AXA in Japanese NVAF patients on apixaban therapy in daily clinical practice both in the acute and steady-state phase was measured. In patients taking apixaban, measurement of AXA clearly showed the pharmacodynamic profile of this drug. (Circ J 2015; 79: ) Key Words: Anti-factor Xa activity; Apixaban; Atrial fibrillation Apixaban is an oral, direct, and selective factor-xa (FXa) inhibitor approved worldwide for the prevention of stroke in patients with non-valvular atrial fibrillation (NVAF). 1 4 The usually recommended dosage of apixaban is a 5-mg tablet twice daily (b.i.d.), while a 2.5-mg tablet b.i.d. is recommended for patients with at least 2 of the following conditions: age 80 years, body weight 60 kg, or serum creatinine 1.5 mg/dl. 1 Similar to other non-vitamin K oral anticoagulants (NOAC), biological monitoring and dose adjustment following the results of monitoring are not usually recommended in patients on apixaban due to its proven efficacy and safety. 1,5 8 These findings were obtained in large-scale clinical trials and may be applicable only to patients who met the inclusion criteria for each trial. Thus, the lack of predictable drug level measurement may hinder the management of patients on anticoagulant therapy with NOAC in real clinical practice. That is to say, monitoring anticoagulant activity might be useful in dealing with patients undergoing emergency surgery or with hemorrhage, to determine the optimal dose in individual patients, excluding under or over dosage, and estimating adherence to therapy, especially in cases of unexpected thromboembolic events. 9 One study reported that prothrombin time (PT) measured using specific reagents can predict apixaban-associated anticoagulant activity, although there is significant interpatient variability. 10 In contrast, chromogenic anti-factor Xa activity (AXA) assay was found to be the most appropriate method to measure the pharmacodynamics of apixaban, and the broadly used coagulation assays such as PT or activated partial thromboplastin time (aptt) are not appropriate to effectively estimate plasma drug concentration due to several limitations and a lack of sensitivity. 8,11 13 Editorial p 2539 Therefore, the aims of the present study were to determine the distribution of trough and peak AXA in Japanese NVAF patients receiving apixaban and to assess the impact of AXA on clinical outcome. Received April 28, 2015; revised manuscript received August 25, 2015; accepted September 6, 2015; released online October 6, 2015 Time for primary review: 13 days Division of Cardiology, Tosei General Hospital, Seto, Japan Mailing address: Hiroyuki Osanai, MD, Division of Cardiology, Tosei General Hospital, 160 Nishi Oiwake-cho, Seto , Japan. osanai@tosei.or.jp ISSN doi: /circj.CJ All rights are reserved to the Japanese Circulation Society. For permissions, please cj@j-circ.or.jp

2 Anti-Factor Xa Activity on Apixaban 2585 Table 1. Patient Characteristics Total (n=124) 5 mg b.i.d. (n=76) 2.5 mg b.i.d. (n=48) P-value Age (years) 77.5± ± ±6.2 <0.01 Male sex 62 (50.0) 43 (56.6) 19 (39.6) 0.07 Body weight (kg) 56.8± ± ±10.0 <0.01 Paroxysmal AF 58 (46.8) 37 (48.7) 21 (43.8) 0.59 Under dose 9 (7.3) 9 (18.8) Serum creatinine (mg/dl) 1.02± ± ±0.43 <0.05 Mean creatinine clearance (ml/min) 51.0± ± ±15.7 <0.01 Mean CHADS2 score 2.1± ± ±1.1 < (8.1) 9 (11.8) 1 (2.1) (25.0) 24 (31.6) 7 (14.6) < (33.1) 24 (31.6) 17 (35.4) (33.9) 19 (25.0) 23 (47.9) <0.01 Congestive heart failure 49 (39.5) 18 (39.9) 31 (64.6) <0.01 Hypertension 84 (67.7) 53 (67.5) 31 (64.6) 0.55 Age 75 years 85 (70.2) 39 (61.1) 46 (95.8) <0.01 Diabetes mellitus 27 (21.8) 16 (20.2) 11 (22.9) 0.81 Baseline stroke/tia/systemic embolism 10 (8.1) 6 (7.9) 4 (8.3) 0.93 Previous anticoagulants None 74 (59.7) 50 (65.8) 24 (50.0) 0.08 Warfarin 41 (33.1) 20 (26.3) 21 (43.8) <0.05 Dabigatran 4 (3.2) 2 (2.6) 2 (4.2) 0.64 Rivaroxaban 5 (4.0) 4 (5.3) 1 (2.1) 0.38 Concurrent antiplatelet drugs Total 29 (23.0) 16 (21.1) 13 (27.1) 0.44 Aspirin 10 (7.9) 5 (6.6) 5 (10.4) 0.44 Clopidogrel 14 (11.1) 8 (10.5) 6 (12.5) 0.74 Cilostazol 2 (1.6) 2 (2.6) 0 (0) 0.26 Aspirin+clopidogrel 3 (2.4) 1 (1.3) 2 (4.2) 0.31 Data given as mean ± SD or n (%). AF, atrial fibrillation; TIA, transient ischemic attack. Methods Study Design and Participants This observational study of Japanese patients with NVAF on apixaban was conducted in routine clinical practice at Tosei General Hospital, Aichi, Japan. From 1 October 2013 to 31 October 2014, 203 patients were started on apixaban and were considered for study entry. Trough and peak AXA after repeated apixaban intake were measured in 124 of these patients in the outpatient clinic; in 25 of these patients, AXA was measured immediately before, 3 h after, and 12 h after the first dose of apixaban in the hospital. The correlations between AXA and routine coagulation markers (PT and aptt) were also examined in these 124 patients after repeated apixaban intake. Patients gave written, informed consent to participate, and this study was conducted in accordance with the ethics policies of Tosei General Hospital. The observation period lasted until 31 January Creatinine clearance was determined using the Cockcroft- Gault formula: men, [(140 age)/(serum creatinine)] (weight/72); women, 0.85 [(140 age)/(serum creatinine)] (weight/72)]. Measuring AXA and Routine Coagulation Markers The HemosIL Liquid Heparin Kit (Instrumentation Laboratory, Lexington, KY, USA) was used for the chromogenic AXA assay. This 1-stage assay is based on the reaction between FXa and a synthetic chromogenic substrate. For Liquid Heparin, 10 μl of plasma were mixed with 100 μl chromogenic substrate Figure 1. Baseline, first peak and trough and steady-state trough and peak anti-factor Xa activity (AXA) in 25 patients. The respective measurements were 0.01±0.02 IU/ml, 0.83±0.43 IU/ml, 0.34±0.17 IU/ml, 0.92±0.52 IU/ml, and 1.61±0.62 IU/ml. First trough AXA was significantly lower than steady-state trough AXA, although it was significantly higher than baseline. Similarly, first peak AXA was significantly lower than steady-state peak AXA.

3 2586 OSANAI H et al. Figure 2. Plot of steady-state trough and peak anti-factor Xa activity (AXA) according to the dosage and consistency with the recommendation. Steady-state peak AXA was significantly higher than trough AXA in (A) the 5-mg b.i.d. group (n=76, P<0.0001), (B) the 2.5-mg b.i.d. group (n=39, P<0.0001); and (C) the under-dosed 2.5-mg b.i.d. group (n=9, P<0.05). and incubated at 37 C. After 75 μl of bovine FXa were added, the chromogenic substrate was hydrolyzed by FXa releasing p-nitroaniline. The quantity of p-nitroaniline released was determined on photometry at 405 nm, and the measurement was performed with the ACL TOP Hemostasis Testing System (Instrumentation Laboratory). 11 The standard HemosIL Heparin Calibrators (Instrumentation Laboratory) were used, and AXA was expressed in heparin international units (IU)/ml. AXA at trough and peak times after repeated apixaban intake was measured >72 h after the start of treatment. The trough time was defined as immediately before the intake of apixaban, and the peak time was defined as 3 h after the intake of apixaban. To ensure measurement at the precise times corresponding to trough concentration, patients took the drug at the hospital after the first blood collection in the morning. Then, blood was collected again 3 h later to measure peak concentration accurately. These measurements were defined as steady-state trough AXA and peak AXA. In contrast, AXA at the beginning of apixaban therapy was measured immediately before, 3 h after, and 12 h after the first intake of apixaban on admission, and was defined as baseline AXA, first peak AXA, and first trough AXA, respectively. PT was measured using HemosIL RecombiPlasTin (Instrumentation Laboratory), and aptt was measured using HemosIL APTT-SP (Instrumentation Laboratory). Outcomes Outcome events included stroke or any other form of embolism affecting a major organ. In the safety evaluation, major and non-major clinically relevant bleeding events were included. A major bleeding event was defined, according to the International Society on Thrombosis and Haemostasis criteria, as clinically overt bleeding accompanied by a decrease in hemoglobin 20 g/l or transfusion 2 units of packed red cells occurring at a critical site or resulting in death. 1,14 A non-major clinically relevant bleeding event was defined as clinically overt bleeding that did not satisfy the criteria for major bleeding but that required medical intervention or resulted in the impairment of daily activities. Statistical Analysis Categorical variables are presented as numbers and percentages. Continuous variables are presented as mean ± SD. To measure the strength of the relationship between 2 variables, Spearman s

4 Anti-Factor Xa Activity on Apixaban 2587 Figure 3. Distribution of steady-state trough and peak anti-factor Xa activity (AXA) according to dosage and consistency with the recommendation. (A) Trough AXA did not differ significantly among the 3 groups (1.00±0.66 IU/ml, 0.84±0.47 IU/ml, and 0.59±0.38 IU/ml, respectively; P=0.146). (B) Peak AXA was significantly higher in the 5-mg b.i.d. group than in the 2.5-mg b.i.d. group (2.05±0.73 IU/ml vs. 1.51±0.65 IU/ml, respectively; P<0.001) and in the under-dosed 2.5-mg b.i.d. group (2.05±0.73 IU/ml vs. 1.23±0.46 IU/ml, respectively; P<0.01). Figure 4. Relationship between steady-state trough and peak anti-factor Xa activity (AXA) and prothrombin time (PT) in the overall patients. Significant and moderate positive correlation was observed between the AXA and PT both at (A) trough (r=0.60, P<0.001) and (B) peak (r=0.69, P<0.0001).

5 2588 OSANAI H et al. Figure 5. Relationship between steady-state trough and peak anti-factor Xa activity (AXA) and activated partial thromboplastin time (aptt) in the overall patients. Significant but weak positive correlation was observed between AXA and aptt both at (A) trough (r=0.28, P<0.01) and (B) peak (r=0.37, P<0.001). Table 2. Significant Indicators of Trough and Peak AXA Variable β P-value Factors that raised trough AXA Dose * Female sex Age * Body weight Serum creatinine * Factors that raised peak AXA Dose <0.0001* Female sex Age * Body weight Serum creatinine * *P<0.05. AXA, anti-factor Xa activity. correlation coefficient was used. To compare parameters between groups, Mann-Whitney U-test, Wilcoxon signed-rank test, chisquared test, or Kruskal-Wallis test was used. To evaluate the factors affecting trough and peak AXA, multiple regression analysis was performed. P<0.05 was considered significant. All statistical analysis was conducted using Ekuseru-Tokei (Ekuseru-Tokei 2010, Social Survey Research Information, Tokyo, Japan). Results Patient Characteristics The characteristics of the patients whose AXA levels were evaluated are listed in Table 1. Of the patients, 76 received apixaban 5 mg b.i.d. and 48 received 2.5 mg b.i.d., including 9 patients who received 2.5 mg b.i.d. based on the decision of the attending physician, although the recommended dose was 5 mg b.i.d. The mean age, weight, serum creatinine, and creatinine clearance of these 9 patients were 76.1±4.9 years, 56.6±12.4 kg, 1.33±0.61 mg/dl, and 39.8±15.0 ml/min, respectively. AXA In the 25 patients in whom baseline, first peak, and trough AXA were measured, the respective levels were 0.01±0.02 IU/ml, 0.83±0.43 IU/ml, and 0.34±0.17 IU/ml. First trough AXA was significantly lower than steady-state trough AXA (P<0.0001), although it was significantly higher than baseline (P<0.0001). Similarly, first peak AXA was significantly lower than steadystate peak AXA (P<0.0001; Figure 1). Steady-state AXA was compared between the 3 groups according to dosage and consistency with the recommendation, namely 5 mg b.i.d. consistent with the recommendation (5-mg b.i.d. group), 2.5 mg b.i.d. consistent with the recommendation (2.5-mg b.i.d. group), and 2.5 mg b.i.d. despite a recommendation of 5 mg b.i.d. (under-dosed 2.5-mg b.i.d. group). Peak AXA was significantly higher than trough AXA in all 3 groups (Figure 2). Trough AXA did not differ significantly between the 3 groups (1.00±0.66 IU/ml, 0.84±0.47 IU/ml,

6 Anti-Factor Xa Activity on Apixaban 2589 Table 3. Patient Characteristics vs. Presence of Bleeding With bleeding Without bleeding (n=7) (n=117) P-value Age (years) 76.0± ± Male sex 5 (71.4) 57 (48.7) 0.24 Body weight (kg) 67.9± ± mg b.i.d. 4 (57.1) 72 (61.5) 0.82 Mean creatinine clearance (ml/min) 44.1± ± Mean CHADS2 score 2.7± ± Concurrent antiplatelet drugs 2 (28.6) 27 (23.1) 0.74 Trough AXA (IU/ml) 1.15± ± Peak AXA (IU/ml) 1.98± ± Data given as mean ± SD or n (%). AXA, anti-factor Xa activity. and 0.59±0.38 IU/ml, respectively; P=0.146; Figure 3A). Peak AXA was significantly higher in the 5-mg b.i.d. group than in the 2.5-mg b.i.d. group (2.05±0.73 IU/ml vs. 1.51±0.65 IU/ml, respectively; P<0.001) and the under-dosed 2.5 mg b.i.d. group (2.05±0.73 IU/ml vs. 1.23±0.46 IU/ml, respectively; P<0.01; Figure 3B). The relationship between steady-state trough and peak AXA and PT in the overall patients is shown in Figure 4. A significant and moderate positive correlation was observed between AXA and PT both at trough and at peak (r=0.60, P<0.001 and r=0.69, P<0.0001, respectively). The relationship between steady-state trough and peak AXA and aptt in the overall patients is shown in Figure 5. A significant but weak positive correlation was observed between AXA and aptt both at trough and at peak (r=0.28, P<0.01 and r=0.37, P<0.001, respectively). The results of the multiple regression analysis to evaluate the factors affecting trough and peak AXA are shown in Table 2. High dose, age, and serum creatinine were significantly related to both trough and peak AXA, but female sex and body weight were not related to either trough or peak AXA. Outcome Assessment The average follow-up period was 286±152 days, and no thromboembolism was observed. Steady-state trough and peak AXA were previously examined in 7 patients with bleeding events (Table 3). In these 7 patients, major bleeding events were observed in 2 patients, and both patients had upper gastrointestinal tract bleeding. Non-major bleeding events, including nasal bleeding, gingival bleeding, and hemosputum, were observed in 5 patients. In these 7 patients with bleeding events, trough AXA was not significantly different from that in patients without bleeding events (1.15±0.54 IU/ml vs. 0.91±0.60 IU/ml, respectively; P=0.24). Similarly, peak AXA was not significantly different between patients with and without bleeding events (1.98±0.63 IU/ml vs. 1.81±0.75 IU/ml, respectively; P=0.49). Furthermore, age, sex, body weight, dose, mean creatinine clearance, mean CHADS2 score, and concurrent antiplatelet drugs were not significantly different between patients with and without bleeding events. Discussion To the best of our knowledge, this study provides the first report of the distribution of AXA in Japanese NVAF patients on apixaban in daily clinical practice. Chromogenic AXA assay is reported to be the most appropriate method to measure the pharmacodynamics of apixaban because of the strong correla- tion with plasma apixaban concentration. 9,11 13,15,16 In this study, the relationship between AXA and PT or aptt was also evaluated, but no strong positive correlations were observed. Another advantage of chromogenic AXA assay is that it is less sensitive than PT to sample collection conditions and variations in the amount of clotting factor among patients. 12 Although chromogenic AXA assay is not yet widely available, ready-to-use liquid reagents are readily accepted, because they reduce setup time and simplify analysis. 9,11 In the present study, AXA at baseline was 0.01±0.02 IU/ml, and the absence of detectable AXA activity likely excludes the presence of physiologically important apixaban activity. AXA increased significantly from trough to peak both after first intake and in the steady state. Trough AXA at steady-state was significantly higher than both baseline and first trough AXA, probably due to the b.i.d. administration, and it achieved steady state approximately 3 days after the first dose with mild accumulation. 17,18 Frost et al reported steady-state mean trough and peak AXA in healthy subjects who received apixaban 2.5 mg twice daily (0.24 IU/ml and 1.12 IU/ml, respectively). 15 The AXA levels in that study seem to be considerably lower than the present levels, but the healthy subjects in the Frost et al study were very young, in excellent physical condition, and very different from Japanese NVAF patients. Thus, the distribution of AXA in the present study is valuable as an index for monitoring precise coagulation function on apixaban therapy in real clinical practice. In addition, the recommended dose of 5 mg b.i.d. may be reduced to 2.5 mg b.i.d. by the attending physician depending on the individual bleeding risk in real daily practice, while 2.5 mg b.i.d. is recommended strictly for patients with at least 2 of the following conditions: age 80 years, body weight 60 kg, or serum creatinine 1.5 mg/dl. It should be noted that, in the present study, however, the under-dosed 2.5-mg b.i.d. group had significantly lower peak AXA than the 5-mg b.i.d. group, even though these patients had characteristics very close to the criteria for dose reduction; thus, dose reduction against the recommendation may result in insufficient efficacy of apixaban. The 2.5-mg b.i.d. group, however, also had significantly lower peak AXA than the 5-mg b.i.d. group. Therefore, peak AXA seems to be more sensitive than trough AXA for reflecting the difference in pharmacodynamics on apixaban therapy. The relatively lower pharmacodynamics of apixaban at peak in the 2.5-mg b.i.d. group may relate to the safety profile in elderly or frail patients on apixaban therapy. 1,19 Furthermore, the present results showed that individual factors included in the dose reduction criteria have different effects on AXA. In particular, body weight was not related to either trough or peak AXA in the present study. Upreti et al

7 2590 OSANAI H et al. reported that, compared with the reference weight group (65 85 kg), AXA at 3 h after dose with 10 mg apixaban in healthy subjects was approximately 30% lower and higher in the high- ( 120 kg) and low- ( 50 kg) body weight groups, respectively. 16 They concluded that the modest change in pharmacodynamics of apixaban is unlikely to require dose adjustment for apixaban based on body weight alone. In the present study, the average body weight of the patients overall was 56.8 kg (range, kg), with small deviation and without extremely high body weight, reflecting the characteristics of Japanese NVAF patients in daily clinical practice. 3,7,20 Therefore, it is possible that body weight had limited effect on both trough and peak AXA in the present study. In contrast, given that age and serum creatinine were significantly related to both trough and peak AXA, measurement of AXA may be important to avoid excessive effects if the patient has large deviation in these factors, such as a very high age or serum creatinine. The present study had several limitations. This study was performed at a single institution with a limited number of subjects, and the observation period was relatively short. In this study, both trough and peak AXA had a tendency to be high in patients with bleeding events compared with those without events, but there was no significant difference. This may be due to the small number of bleeding events. Therefore, further research is required to ensure that monitoring of AXA can improve the efficacy and safety outcomes for patients on apixaban therapy. The peak time was defined as 3 h after drug treatment according to the previous study, 18 but the actual peak concentration may differ among individual patients. Furthermore, the different chromogenic AXA assays available on the market may have linear or exponential relationships with plasma apixaban concentration depending on the reagent, and there is interassay variability. 11 The HemosIL Liquid Heparin Kit was reported to have an exponential correlation (correlation coefficient=0.99 and coefficient of variation=0.9%), so that there may be decreased sensitivity for higher plasma concentration of apixaban. 11 Conclusions The distribution of AXA in patients with NVAF and taking apixaban in daily clinical practice was investigated, both in the acute phase and at steady state. Trough AXA was significantly higher than the baseline level. Peak AXA was significantly lower in patients taking 2.5 mg b.i.d. with or without underdose than in patients taking 5 mg b.i.d., although there were no significant differences in trough AXA between the 3 groups. Other than the dose, age and serum creatinine were significantly related to both trough and peak AXA on multiple regression analysis. In patients taking apixaban in daily clinical practice, measurement of AXA clearly showed the pharmacodynamic profile of this drug. Disclosures H.O. and M.A. have received lecture fees from Bristol-Myers Squibb, Pfizer, Bayer Yakuhin, and Boehringer Ingelheim. References 1. Granger CB, Alexander JH, McMurray JV, Lopes RD, Hylek EM, Hanna M, et al. Apixaban versus warfarin in patients with atrial fibrillation. N Engl J Med 2011; 365: Connolly SJ, Eikelboom J, Joyner C, Diener HC, Hart R, Golitsyn S, et al. Apixaban in patients with atrial fibrillation. N Engl J Med 2011; 364: Ogawa S, Shinohara Y, Kanmuri K. Safety and efficacy of the oral direct factor Xa inhibitor apixaban in Japanese patients with nonvalvular atrial fibrillation: The ARISTOTLE-J study. Circ J 2011; 75: JCS Joint Working Group. Guidelines for pharmacotherapy of atrial fibrillation (2013): Digest version. Circ J 2014; 78: Connolly SJ, Ezekowitz MD, Yusuf S, Eikelboom J, Oldgren J, Parekh A, et al. Dabigatran versus warfarin in patients with atrial fibrillation. N Engl J Med 2009; 361: Patel MR, Mahaffey KW, Garg J, Pan G, Singer DE, Hacke W, et al. Rivaroxaban versus warfarin in nonvalvular atrial fibrillation. N Engl J Med 2011; 365: Hori M, Matsumoto M, Tanahashi N, Momomura S, Uchiyama S, Goto S, et al; J-ROCKET AF Study Investigators. Rivaroxaban vs. warfarin in Japanese patients with non-valvular atrial fibrillation in relation to age. Circ J 2014; 786: Giugliano RP, Ruff CT, Braunwald E, Murphy SA, Wiviott SD, Halperin JL, et al. Edoxaban versus warfarin in patients with atrial fibrillation. N Engl J Med 2013; 369: Cuker A, Deborah M, Siegal DM, Crowther MA, Garcia DA. Laboratory measurement of the anticoagulant activity of the non-vitamin K oral anticoagulants. 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