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Effects of Lepirudin, and Melagatran and Additional

Influence of on Ecarin Clotting Time

Tivadar Fenyvesi* M.D., Ingrid Jörg* M.D., Christel Weiss + Ph.D., Job Harenberg* M.D.

Key words: direct inhibitors, ecarin clotti ng time, oral , enhancing effects

*IV. Department of Medicine +Institute for Biometrics and Medical Statistics University Hospital Mannheim

Theodor Kutzer Ufer 1 - 3 68167 Mannheim Germany

*Corresponding author Phone: +49 -621 -383 -3378 E-ma il: tivadar.fenyvesi @med1.ma.uni -heidelberg.de

Version 06.08.03 for Thromb Res

Review Copy

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Abstract

Introduction: Direct thrombin inhibitors (DTI) prolong the

ecarin clotting time (ECT). Oral anticoagulants (OA) decrease

prothrombin levels and thus interacts 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±0.9 vs. 45.7±0.8 sec; 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±11.0 s in NP and 333.2±8.2 s in OACP) than withReview argatroban (219.6±14.7 Copyand 248.4±15.2 s) corresponding to ratios of 7.1±0.2 and 6.7±0.2 (melagatran)

versus 4.8±0.3 and 4.9±03 with argatroban (p<0.0001).

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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 non-linear

slopes), but there are also differences between the two

monovalent inhibitors on thrombin inhibition as determined by

ECT.

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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 non-linear

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 therefore 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 , however, because heparins

require which can’t 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 medicinalisReview. It is a tadpole-like proteine Copy 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

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recognition site) of thrombin. Lepirudin is a recombinant

(r-hirudin). The direct thrombin inhibitors lepirudin 9

10 and argatroban 11 (an arginin derivative, hydrated mole mass

0.526 kD, monovalent active site inhibitor) are established to

maintain effective anticoagulation in patients with -

induced 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, , is currently under

investigation for prophylaxis and treatment of venous

thromboembolism in clinical trials 12 13 14 15. Direct thrombin

inhibitors and heparins increase the effects of

oral vitamin K antagonists in various clotting time

measurement techniques 16. These interactions are of

considerable extent with prothrombin time 17 18 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 theReview present study, we describe Copyeffects of three direct thrombin inhibitors (lepirudin, argatroban and melagatran) on

the ECT in normal plasma (NP) and in plasma of patients on

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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.

2. Materials and Methods

Blood samples (~10 ml) of 23 healthy volunteers (normal

plasma, NP) and of 23 patients (INR: 2.63 ± 0.13; mean ±

s.e.m.; range: 1.4 - 3.3 ) on steady-state treatment (orally

anticoagulated plasma, OACP) with the

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 (1800g, 10 min) were either

tested immediately or shock frozen in liquid nitrogen and

stored at –80°C for analyses within 4 weeks. Plasma samples

were spiked with concentrations ranging from 300 ng/ml to 3000 ng/ml of Reviewr-hirudin (Lepirudin, Aventis,Copy Frankfurt/Main, Germany; molecular mass 6.9 kDa) and argatroban (by courtesy

of Mitsubishi Chemical Corp., Tokyo, Japan; molecular mass

0.526 kDa), and 30 ng/ml to 1000 ng/ml of melagatran (kindly

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provided by Astra Zeneca, Moelndal, Sweden; molecular mass

~0.478 kDa). All ECT measurements were carried out in a KC 10a

micro device from Amelung Comp. (Lemgo, Germany)23. The Ecarin

reagent® (lot No 8303/116-08) was kindly provided by Pentapharm

Ltd. (Basel, Switzerland). 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 1.

Statistical analyses

All data are given as mean values ± 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.

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. TheReview non -linear curves of argatroban Copy 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

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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 analysis) and between NP and OACP (secondary analysis). Primary and secondary analysis was also carried out for the linear phase and plateau phase slopes of argatroban and melagatran. Quality control for all curve characterising equ ations was R 2 (mean > 0.950).

3. Results

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 non-linear

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 ± 0.8 sec in NP. In OACP, this

value was prolonged to 50.1 ± 0.9 sec (p < 0.001). Lepirudin Reviewhad a linear dose-response relationshipCopy with NP and OACP (Fig. 2 A). A concentration of 500 ng/ml prolonged ECT to

102.9 ± 3.4 sec in NP and to 123.2 ± 3.0 sec in OACP (p <

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0.001). A higher therapeutic concentration (2000 ng/ml) of

lepirudin delivered ECT values of 271.7 ± 8.0 sec in NP and

341.4 ± 10.3 sec in OACP (p < 0.0001).

500 ng/ml argatroban lead to ECT values of 163.6 ± 9.7 sec in

NP and 171.1 ± 11.2 sec in OACP (p < 0.001; Fig. 3 A). 2000

ng/ml prolonged the ECT to 317.8 ± 18.5 sec and 345.6 ± 19.3 in

NP and OACP, respectively (p < 0.001).

Melagatran at 100 ng/ml delivered clotting times of 179.2 ± 4.0

sec in NP and 190.0 ± 4.0 sec in OACP (p < 0.0001; Fig. 4 A).

300 ng/ml prolonged ECT to 345.7 ± 9.1 sec in NP and 361.4 ±

7.8 sec in OACP (p < 0.0001).

Fitting of concentration- 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±0.02 s/ng•ml in NP and 0.26±0.02 s/ng•ml in

OACP) and melagatran (slope 1: 1.32±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 bothReview groups using the same drug Copy (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

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slopes of both curve phases were 219.6±14.7 s (NP) and

248.4±15.2 s (OACP) with argatroban and 323.1±11.0 s (NP) and

333.2±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).

Individual normalised ECT ratios

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. 2 B). A concentration of 500 ng/ml increased

the ECT ratio by 2.3 ± 0.1-fold in NP and 2.5 ± 0.1-fold in

OACP. A higher therapeutic concentration (2000 ng/ml) of

lepirudin delivered ECT ratios of 6.0 ± 0.2 in NP and 6.9 ± 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

(Fig. 3 B, 4 B). 500 ng/ml argatroban provided ECT ratios of 3.6 ± 0.2 Reviewin NP and 3.4 ± 0.2 in OACP Copy samples (n. s. Fig. 3 B). At 2000 ng/ml it increased the ECT ratio to 7.0 ± 0.4 and

6.9 ± 0.4 in NP and OACP, respectively (n. s.).

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Melagatran (100 ng/ml) increased the ratios by about 4 times

(3.9 ± 0.1 in NP and 3.8 ± 0.1 in OACP; n. s.; Fig. 4 B). A

high therapeutic concentration of 300 ng/ml delivered ECT

ratios of 7.6 ± 0.2 in NP and 7.3 ± 0.2 in OACP (n. s.).

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±0.0005 and 0.0051±0.0004 ratio/ng•ml in NP and OACP)

and melagatran (slope 1: 0.029±0.001 and 0.028±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±0.24 in

NP and 6.7±0.18) compared to argatroban (4.8±0.33 in NP and

4.9±0.32 in OACP) in OACP; primary analysis, p < 0.0001). The secondary Reviewanalysis using the same drug Copy resulted showed non- significant deviations between NP and OACP, for details see

table 2.

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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 non-linear dose-effect relationships, possibly due to

their different mechanism and kinetics24 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 accedingReview phase. Lepirudin, a recombinantCopy hirudin, not only inhibits the active catalytic site, but also binds to the

fibrinogen-binding site (= anion binding site or exosite 1).

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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

sub-optimal 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 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 feedbacks to F VII, F IX and F X29. 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,Review the results of the Copypresent work indicate enhancements by phenprocoumon effects which might be not

negligible in clinical practice. There should be an effort to

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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.

AcknowledgementsReview Copy

The authors would like to thank to Mrs. Christina Giese, Mrs.

Antje Hagedorn and Mrs. Inge Träger for excellent laboratory

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work and patient care. This study was supported by a grant of

the Faculty of Clinical Medicine Mannheim, University of

Heidelberg.

Legends to figures and tables

Figure 1: Exemplification of the approach to individual line

characteristics of non-linear concentration response

relationships. Individual relationships with argatroban.

Figure 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 ± s.e.m.

Figure 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 ± s.e.m.

Figure 4: Concentration-ECT relationships of melagatran expressed Reviewin seconds (A) and as ECT ratio Copy (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 ± s.e.m.

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Table 1: Synopsis of important statistical parameters of

individual concentration-ECT [sec] curve characteristic

parameters (slopes [s/ng•ml], intersections, crossing points

of slopes ) with lepirudin, argatroban and melagatran

(n. a.: not applicable).

Table 2: Synopsis of important statistical parameters of

individual concentration-ECT [ratio] curve characteristic

parameters (slopes [ratio/ng•ml], intersections, crossing

points of slopes ) with lepirudin, argatroban and

melagatran (n. a.: not applicable).

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Table 1:

lepirudin Parameter Slope part 1 Slope part 2 y-value of transition Group NP OACP NP OACP NP OACP Mean 0.11 0.15 n. a. n. a. n. a. s.e.m. 0.0031 0.005 n. a. n. a. n. a.

argatroban Parameter Slope part 1 Slope part 2 y-value of transition Group NP OACP NP OACP NP OACP Mean 0.24 0.26 0.08 0.08 219.6 248.4 s.e.m. 0.02 0.022 0.005 0.006 14.7 15.2

melagatran Parameter Slope part 1 Slope part 2 y-value of transition Group NP OACP NP OACP NP OACP Mean 1.31 1.38 0.35 0.41 323.1 333.2 s.e.m. 0.04 0.03 0.01 0.01 11.0 8.2

Table 2:

lepirudin Parameter Slope part 1 Slope part 2 y-value of transition Group NP OACP NP OACP NP OACP Mean 0.0025 0.003 n. a. n. a. n. a. s.e.m. 0.0001 0.0001 n. a. n. a. n. a.

argatroban Parameter Slope part 1 Slope part 2 y-value of transition Group NP OACP NP OACP NP OACP Mean 0.0053 0.0051 0.0018 0.0016 4.8 4.9 s.e.m. 0.0005 0.0004 0.0001 0.0001 0.33 0.32

melagatran Parameter Slope part 1 Slope part 2 y-value of transition Group NP OACP NP OACP NP OACP Mean 0.029 0.028 0.0078 0.0082 7.1 6.7 s.e.m. 0.001 0.001 0.0003 0.0003 0.24 0.18

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