Effects of lepirudin, argatroban and melagatran and additional influence of phenprocoumon on ecarin clotting time

Thrombosis Research 111 (2003) 89 – 94

Regular Article

Effects of lepirudin, argatroban and melagatran and additional influence of phenprocoumon on ecarin clotting time Tivadar Fenyvesi a,*, Ingrid Jo¨rg a, Christel Weiss b, Job Harenberg a b

a Fourth Department of Medicine, University Hospital Mannheim, Theodor Kutzer Ufer 1-3, 68167 Mannheim, Germany Institute for Biometrics and Medical Statistics, University Hospital Mannheim, Theodor Kutzer Ufer 1-3, 68167 Mannheim, Germany

Received 26 May 2003; received in revised form 6 August 2003; accepted 11 August 2003

Abstract Introduction: Direct thrombin inhibitors (DTI) prolong the ecarin clotting time (ECT). Oral anticoagulants (OA) decrease prothrombin levels and thus interact with actions of DTIs on the ECT method during concomitant therapy. Materials and methods: Actions of lepirudin, argatroban and melagatran on ECT were investigated in normal plasma (NP) and in plasma of patients (n = 23 each) on stable therapy with phenprocoumon (OACP). Individual line characteristics were tested statistically. Results: Control ECT in OACP was prolonged compared to NP (50.1 F 0.9 vs. 45.7 F 0.8 s; p < 0.001). Lepirudin prolonged the ECT linearly. Argatroban and melagatran delivered biphasic dose – response curves. OA showed additive effects on the ECT of lepirudin but not of argatroban and melagatran. Both in NP and OACP, the first and second slopes of melagatran were steeper compared to argatroban (primary analysis; p < 0.001). When using the same drug, slopes in OACP were steeper than in NP (secondary analysis; p < 0.001). At similar molar concentrations, the crossing points of both slopes were significantly higher with melagatran (323.1 F 11.0 s in NP and 333.2 F 8.2 s in OACP) than with argatroban (219.6 F 14.7 and 248.4 F 15.2 s) corresponding to ratios of 7.1 F 0.2 and 6.7 F 0.2 (melagatran) vs. 4.8 F 0.3 and 4.9 F 03 with argatroban ( p < 0.0001). Discussion: The patterns of interactions between vitamin K antagonists and DTI effects are different for bivalent (increase of slope without affecting linearity) and monovalent inhibitors (slight increase or alteration of nonlinear slopes), but there are also differences between the two monovalent inhibitors on thrombin inhibition as determined by ECT. D 2003 Elsevier Ltd. All rights reserved. Keywords: Direct thrombin inhibitors; Ecarin clotting time; Oral anticoagulants; Enhancing effects

1. Introduction Recently, a snake venom based testing method, the ecarin clotting time (ECT), was refined [1] to overcome the limitations of traditional monitoring measures such as the activated partial thromboplastin time (aPTT) methods. A toxin of Echis carinatus cleaves prothrombin to meizothrombin and other active intermediates. Heparins and direct thrombin inhibitors are mostly monitored by the aPTT [2,3]. Limitations of aPTT methods include nonlinear dose – effect relationships with plateau effect, variability among different testing instruments, reagents and different lots of the same reagent [4]. The ECT has a linear dose – response relationship towards the direct thrombin inhibitor lepirudin [1,5] and is there* Corresponding author. Tel.: +49-621-383-3378. E-mail address: [email protected] (T. Fenyvesi). 0049-3848/$ - see front matter D 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2003.08.013

fore more accurate in monitoring of this direct thrombin inhibitor. ECT is also more sensitive towards new DTIs like argatroban and melagatran than the aPTT [3,6]. ECT is insensitive against heparins, however, because heparins require antithrombin which cannot react with meizothrombin or other intermediates like meizothrombin(desF1) with thrombin activity of the prothrombin – thrombin conversion [7]. Hirudin is contained in the saliva of the medical leech Hirudo medicinalis. It is a tadpole-like protein molecule occurring in two variants with 65 or 64 amino acids (molecular mass: 6.9 kDa) [8]. It is a bivalent inhibitor of the active catalytic site and the anion binding exosite (also called fibrinogen recognition site) of thrombin. Lepirudin is a recombinant hirudin (r-hirudin). The direct thrombin inhibitors lepirudin [9,10] and argatroban [11] (an arginin derivative, hydrated mole mass 0.526 kDa, monovalent active site inhibitor) are established to maintain effective anticoagulation in patients with heparin-induced thrombo-

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cytopenia without and with thrombosis (HIT type II). Melagatran (mole mass 0.478 kDa), a synthetic small molecular active site inhibitor applied in form of an orally available prodrug, ximelagatran, is currently under investigation for prophylaxis and treatment of venous thromboembolism in clinical trials [12 – 15]. Direct thrombin inhibitors and heparins increase the anticoagulant effects of oral vitamin K antagonists in various clotting time measurement techniques [16]. These interactions are of considerable extent with prothrombin time [17 – 19] and weaker within aPTT [20] or ECT methods [21,22]. There are marked differences regarding the extent of increasing effects between direct thrombin inhibitors and vitamin K antagonists within the ECT method in literature [19,20]. Clinical relevance of such additive effects arises during concomitant treatment periods. Such periods appear, for instance, when oral anticoagulants are discontinued to introduce direct thrombin inhibitors for the duration of diagnostic or therapeutic interventions. In the present study, we describe effects of three direct thrombin inhibitors (lepirudin, argatroban and melagatran) on the ECT in normal plasma (NP) and in plasma of patients on steady-state oral anticoagulation with phenprocoumon (orally anticoagulated plasma, OACP). We hypothesised that monovalent and bivalent inhibitors may interact differently with decarboxylated precursors of thrombin in OACP.

From the two methods currently described and available [1,5], the method with the higher detection sensitivity was chosen. This method is carried out according to Ref. [1]. 2.1. Statistical analyses All data are given as mean values F standard deviations of means (S.E.M.). For all parameters analyzed, Duncan– Scheffe test was performed using SAS software and level of significance was set at p < 0.001. 2.2. Approach to line characteristics The linear concentration – effect relationships of lepirudin were characterised by slope and intersection. The values obtained for NP and OACP were tested with the tests described above. For both values, level of significance was set at p < 0.001. The nonlinear curves of argatroban and melagatran were considered to consist of two parts: a linear acceding part and a plateau phase. They were fitted separately to linear equations delivering the characteristic parameter slope. To find out the y-values assigned to the crossing points of the two slopes, equations of both sections of each individual curve were transformed, equated and dissolved for y. The corresponding y-value was considered as a characteristic value attaching the two curve phases to each other (Fig. 1). The differences of the y-values were analyzed between argatroban and melagatran (primary anal-

2. Materials and methods Blood samples ( f 10 ml) of 23 healthy volunteers (normal plasma, NP) and of 23 patients (INR: 2.63 F 0.13; mean F S.E.M.; range: 1.4 – 3.3) on steady-state treatment (orally anticoagulated plasma, OACP) with the vitamin K antagonist phenprocoumon (Hoffmann La Roche, Basel, Switzerland) were collected by clean cubital vein punction into plastic vials with sodium citrate (3.8%; plasma/citrate: 9/1, v/v). All patients were outpatients and were within the therapeutic range. The time in therapeutic range was comparable. None of the patients was near to an acute thrombotic event. None of the patients received any concomitant treatment potentially interfering with phenprocoumon. The centrifuged plasma samples (1800
g, 10 min) were either tested immediately or shock frozen in liquid nitrogen and stored at  80 jC for analyses within 4 weeks. Plasma samples were spiked with concentrations ranging from 300 to 3000 ng/ml of r-hirudin (LepirudinR, Aventis, Frankfurt/ Main, Germany; molecular mass 6.9 kDa) and argatroban (by courtesy of Mitsubishi Chemical, Tokyo, Japan; molecular mass 0.526 kDa), and 30– 1000 ng/ml of melagatran (kindly provided by Astra Zeneca, Moelndal, Sweden; molecular mass 0.43 kDa). All ECT measurements were carried out in a KC 10a microk device from Amelung. (Lemgo, Germany) [23]. The Ecarin reagentR (lot No 8303/116-08) was kindly provided by Pentapharm (Basel, Switzerland).

Fig. 1. Exemplification of the approach to individual line characteristics of nonlinear concentration – response relationships. Individual relationships with argatroban.

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ysis) and between NP and OACP (secondary analysis). Primary and secondary analyses were also carried out for the linear phase and plateau phase slopes of argatroban and melagatran. Quality control for all curve characterising equations was R2 (mean>0.950).

3. Results 3.1. ECT expressed in seconds All immediate acting thrombin inhibitors affected the ECT in a concentration-dependent manner in both materials (normal and OA plasma). The dose –response relationship with lepirudin was linear. Argatroban and melagatran had nonlinear concentration – effect relationships. The steeper initial part showed a transition to a flatter relationship from about 1000 ng/ml with argatroban and 300 ng/ml with melagatran. Normal ECT range in our study was 45.7 F 0.8 s in NP. In OACP, this value was prolonged to 50.1 F 0.9 s ( p < 0.001). Lepirudin had a linear dose –response relationship with NP and OACP (Fig. 2A). A concentration of 500 ng/ml prolonged ECT to 102.9 F 3.4 s in NP and to 123.2 F 3.0 s in OACP ( p < 0.001). A higher therapeutic concentration (2000 ng/ml) of lepirudin delivered ECT values of 271.7 F 8.0 s in NP and 341.4 F 10.3 s in OACP ( p < 0.0001). Argatroban (500 ng/ml) lead to ECT values of 163.6 F 9.7 s in NP and 171.1 F 11.2 s in OACP ( p < 0.001; Fig. 3A); 2000 ng/ml prolonged the ECT to 317.8 F 18.5 and 345.6 F 19.3 s in NP and OACP, respectively ( p < 0.001). Melagatran at 100 ng/ml delivered clotting times of 179.2 F 4.0 s in NP and 190.0 F 4.0 s in OACP ( p < 0.0001; Fig. 4A); 300 ng/ml prolonged ECT to 345.7 F 9.1 s in NP and 361.4 F 7.8 s in OACP ( p < 0.0001).

Fig. 2. Concentration – ECT relationships of lepirudin expressed in seconds (A) and as ECT ratio (B) in normal plasma (NP, continuous line, n = 23) and plasma samples of patients on oral anticoagulation with phenprocoumon (OACP, discontinuous line, n = 23). Data is given as mean F S.E.M.

3.3. Individual normalised ECT ratios 3.2. Fitting of concentration– coagulation time relationships Significant deviations of the slopes and intersections of dose –response lines of lepirudin were found between NP and OACP. Also, the linear (slope 1) and plateau phase slopes (slope 2) showed significant deviations between argatroban (slope 1: 0.24 F 0.02 s/ng ml in NP and 0.26 F 0.02 s/ng ml in OACP) and melagatran (slope 1: 1.32 F 0.04 s/ng ml in NP and 1.38 s/ng ml in OACP) in the same group (primary analysis, p < 0.0001; for slope 2, see Table 1). The same finding occurred between both groups using the same drug (secondary analysis, p < 0.001; for details, see Table 1) Within the same group (NP or OACP), the y-values belonging to the crossing points of the slopes of both curve phases were 219.6 F 14.7 s (NP) and 248.4 F 15.2 s (OACP) with argatroban and 323.1 F 11.0 s (NP) and 333.2 F 8.2 s (OACP) in the case of melagatran (primary analysis, p < 0.0001). The secondary analysis between NP and OACP using the same drug resulted in significant deviations, too ( p < 0.001; for details, see Table 1).

Clotting times were transformed into individual normalised ratios based on the individual control value of each volunteer or patient, respectively. Control ratio is 1 in all cases. Concentration – ECT ratio lines of lepirudin were divergent with NP and OACP (Fig. 2B). A concentration of 500 ng/ml increased the ECT ratio by 2.3 F 0.1-fold in NP and 2.5 F 0.1-fold in OACP. A higher therapeutic concentration (2000 ng/ml) of lepirudin delivered ECT ratios of 6.0 F 0.2 in NP and 6.9 F 0.2 in OACP ( p < 0.001). In the cases of both argatroban and melagatran, the concentration – ECT ratio curves of NP and OACP plasmas were almost identical, without any tendency towards a divergence (Figs. 3B and 4B). Argatroban (500 ng/ml) provided ECT ratios of 3.6 F 0.2 in NP and 3.4 F 0.2 in OACP samples (n.s., Fig. 3B). At 2000 ng/ml, it increased the ECT ratio to 7.0 F 0.4 and 6.9 F 0.4 in NP and OACP, respectively (n.s.). Melagatran (100 ng/ml) increased the ratios by about four times (3.9 F 0.1 in NP and 3.8 F 0.1 in OACP, n.s.;

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Fig. 3. Concentration – ECT relationships of argatroban expressed in seconds (A) and as ECT ratio (B) in normal plasma (NP, continuous line, n = 23) and plasma samples of patients on oral anticoagulation with phenprocoumon (OACP, discontinuous line, n = 23). Data is given as mean F S.E.M.

Fig. 4B). A high therapeutic concentration of 300 ng/ml delivered ECT ratios of 7.6 F 0.2 in NP and 7.3 F 0.2 in OACP (n.s.). 3.4. Fitting of concentration – coagulation time ratio relationships Also in the case of the ratios, significant deviations of the slopes and intersections of dose – response lines of lepirudin were found between NP and OACP. The linear and plateau phase slopes showed significant deviations between argatroban (slope 1: 0.0053 F 0.0005 and 0.0051 F 0.0004 ratio/ ng ml in NP and OACP) and melagatran (slope 1: 0.029 F 0.001 and 0.028 F 0.001 ratio/ng ml in NP and OACP) in the same group (primary analysis, p < 0.0001; for slope 2, see Table 2). The same was found between both groups using the same drug (secondary analysis, p < 0.001; for details, see Table 2). Within the same group (NP or OACP), the y-values belonging to the crossing points of the slopes of both curve phases were elevated with a very high significance in the case of melagatran (7.1 F 0.24 in NP and

Fig. 4. Concentration – ECT relationships of melagatran expressed in seconds (A) and as ECT ratio (B) in normal plasma (NP, continuous line, n = 23) and plasma samples of patients on oral anticoagulation with phenprocoumon (OACP, discontinuous line, n = 23). Data is given as mean F S.E.M.

6.7 F 0.18) compared to argatroban (4.8 F 0.33 in NP and 4.9 F 0.32 in OACP) in OACP; primary analysis, p < 0.0001). The secondary analysis using the same drug Table 1 Synopsis of important statistical parameters of individual concentration – ECT [s] curve characteristic parameters (slopes [s/ng ml], intersections, crossing points of slopes hy-valuei) with lepirudin, argatroban and melagatran (n.a.: not applicable) Parameter

Slope part 1

Slope part 2

y-value of transition

Group

NP

OACP

NP

OACP

NP

Lepirudin Mean S.E.M.

0.11 0.0031

0.15 0.005

n.a. n.a.

n.a. n.a.

Argatroban Mean 0.24 S.E.M. 0.02

0.26 0.022

0.08 0.005

0.08 0.006

219.6 14.7

248.4 15.2

Melagatran Mean 1.31 S.E.M. 0.04

1.38 0.03

0.35 0.01

0.41 0.01

323.1 11.0

333.2 8.2

OACP

n.a. n.a.

T. Fenyvesi et al. / Thrombosis Research 111 (2003) 89–94 Table 2 Synopsis of important statistical parameters of individual concentration – ECT [ratio] curve characteristic parameters (slopes [ratio/ng ml], intersections, crossing points of slopes hy-valuei) with lepirudin, argatroban and melagatran (n.a.: not applicable) Parameter

Slope part 1

Slope part 2

y-value of transition

Group

NP

OACP

NP

OACP

NP

Lepirudin Mean S.E.M.

0.0025 0.0001

0.003 0.0001

n.a. n.a.

n.a. n.a.

n.a. n.a.

Argatroban Mean 0.0053 S.E.M. 0.0005

0.0051 0.0004

0.0018 0.0001

0.0016 0.0001

4.8 0.33

4.9 0.32

Melagatran Mean 0.029 S.E.M. 0.001

0.028 0.001

0.0078 0.0003

0.0082 0.0003

7.1 0.24

6.7 0.18

OACP

showed nonsignificant deviations between NP and OACP; for details, see Table 2.

4. Discussion Our study demonstrated different patterns of interactions between vitamin K antagonists and DTIs not only between bivalent and monovalent inhibitors, but also between the two monovalent inhibitors. The slow-acting bivalent inhibitor lepirudin exerted linear concentration – effect relationships, according to those described in literature [1,5,19,20]. The monovalent compounds argatroban and melagatran, however, showed nonlinear dose – effect relationships, possibly due to their different mechanism and kinetics [24] of action. These differences might be due to the differing binding modes of the compounds. Lepirudin is a bivalent ligand for both the catalytic active site and the anion binding exosite of thrombin [25,26], while argatroban and melagatran are monovalent inhibitors of the catalytic active site alone [27]. In case of monovalent active site inhibitors like argatroban and melagatran, the interaction between enzyme (thrombin) and inhibitor may come to a saturation state, because here, above a certain threshold concentration, the coagulation curve is changing. Increasing concentrations of the inhibitor lead to much less prolongation of clotting times, compared to the linear acceding phase. Lepirudin, a recombinant hirudin, not only inhibits the active catalytic site, but also binds to the fibrinogen-binding site ( = anion binding site or exosite 1). The saturation of the active catalytic site would not hinder further effects of lepirudin on this binding site, possibly explaining the linear concentration– effect relationship. A suboptimal gamma-carboxylation of factor II during vitamin K antagonism could increase the accessibility of this binding site to lepirudin [28]. Feedback mechanisms could explain the shape of the monovalent relationships. This could include a positive

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feedback of meizothrombin (the enzyme primarily generated within the ECT method) on F XI which is responsible for generation of thrombin (enzyme generated secondarily) within the clot, as well as feedback to F VII, F IX and F X [29]. At higher concentrations of argatroban or melagatran, the inhibitor possibly reacts with the thrombin generated by feedback mechanism. Deficiencies of active factors influenced by stable oral anticoagulation could influence feedback mechanisms in a different manner for each combination of direct thrombin inhibitor and binding sites. The precise mechanistic backgrounds remain to be investigated, however. Differing data are reported in literature about the extent of enhancement of direct thrombin inhibitor effects by oral anticoagulants [19,20]. In the case of lepirudin, with increasing concentrations, the results of the present work indicate enhancements by phenprocoumon effects which might be not negligible in clinical practice. There should be an effort to consider these interactions during overlapping applications, e.g. when monitoring patients who are switched from vitamin K antagonists to lepirudin or vice versa. Such therapeutic switches occur when outpatients receiving oral anticoagulants are hospitalised for surgical or invasive diagnostic procedures or the treatment of venous thromboembolism is switched from a direct thrombin inhibitor to a vitamin K antagonist. The aPTT is prolonged by oral anticoagulation due to a decrease of factors IX, X and II [30,31]. Within the test system of ECT, only the decrease in the level of F II affects coagulation times during OA therapy. There is so far neither a standardisation of the various aPTT reagents nor an adaptation of the reesult interpretation on OA effects, probably due to the varying effects of the reagents. This may not account for ECT favorising this method for determination of the effects of DTIs during concomitant oral anticoagulation. According to the results presented herein, appropriate clinical studies to validate therapeutic ranges for ECT ratio using appropriate control samples may be required. One attempt is currently being made in an international collaborative study [32].

Acknowledgements The authors would like to thank Mrs. Christina Giese, Mrs. Antje Hagedorn and Mrs. Inge Tra¨ger for excellent laboratory work and patient care. This study was supported by a grant of the D. Hopp Stiftung and the Faculty of Clinical Medicine Mannheim, University of Heidelberg.

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