THROMBOSIS 0049-3848/92
RESEARCH 66; 271-275,1992 $5.00 + .OO Printed in the USA.
Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
BRIFF
COMMUNICATION
PHARMACOKINETIC INVESTIGATIONS OF THE u-HUMAN THROMBIN-HIRUDIN COMPLEX IN RHESUS MONKEYS
H. GrWch, M. Hropot, G. Berscheid, P. Crause, V. Malerczyk, G. Apidopoulos, G. Haun and B. Husak Hoechst AG, Frankfurt am Main, FRG (Received
23.7.1991;
accepted
in revised form 24.2.19920
by Editor F. Markwardt)
INTRODUCTION On the excretion of hirudin after parenterai administration has been reported manifold (1,2,3,4). The pharmacokinetics of hirudin in man and animals seem to be very similar. There are only a few reports on the formation of the thrombin-hirudin complex (THC). After parenteral administration of hirudin THC is formed in small amounts (56). Markwardt et al.,(l) reported on the formation of the THC by a ratio of l:l, having a molecular weight of 43 kDa. Bichler et al.,(6) determined THC by means of an ELISA. Grotsch et al.,(lO) demonstrated a possible way of elimination by proteolytic digestion in liver and kidney. The aim of this study was to investigate the pharmacokinetics and pharmacodynamics of the THC in rhesus monkeys in comparison to hirudin. To complete the interpretation of the ll-tC data, the rhesus monkeys were treated with hirudin one week in advance. Analysis of the thrombin time and the partial thromboplastin time was included. In addition to the chromogenic thrombin substrate assay (8,9), two ELISA assays for hirudin and the THC (5) were performed.
MATERIAI AND METHODS THC used for the experiment was prepared from a-human thrombin (kindly supplied by Dr. Riomisch, Behringwerke, Marburg, FRG) and rDNA hirudin (HBW 023 Hoechst AG, Frankfurt/M, FRG). Hirudin was added to thrombin in excess of a five-fold amount for a quantitative reaction. The solution was cleared by centriigation at 6OOOg_. THC was separated from unbound hirudin by gel permeation chromatography on Sephacryl S 200. Fractions containing THC in the absence of free hirudin were combined, dialysed against water and concentrated with a DIAFLO YMlO membrane. The ELl$A used for the determination of THC was provided from purified anti a-human thrombin (antibody 1) and HRP labelled sheep anti-hirudin (antibody 2). The assay procedure was in accordance to Bichler et al.,(6). The ELISA for hirudin was provided from purified Tripalmitoyl-S-cysteinylserylglycin-hirudin (monoclonal mouse, antibody 1) and Tripalmitoyl-S-cysteinylserylglycin-hitudin (polyclonal sheep, antibody 2) and anti-sheep IGG HRP labelled. ____________________~~~_____~~~~~~~~~~~_~~~~~~~~~~~~--~~~~~~~~~~ Key words: Hirudin; thrombin-hirudin complex; rhesus monkeys; chromogenic thrombin substrate assay; ELISA for hirudin and THC; clotting times.
271
THROMBIN-HIRUDIN
272
KINETICS
Vol. 66, Nos. 213
The assay procedure was in accordance to Spinner et al.,(7). The experiments were performed in 3 rhesus monkeys with 5.5, 7.8 and 11.4 kg body weight. Before the hirudin and THC were administered, the animals received 20 ml tap water per kg body weight by stomach tube. During the 24 hour observation period food and water were withdrawn. Each of the animals was treated in the first week with 0.1 mg hirudin per kg b.wt. followed in the second week by 0.4 mg THC per kg b.wt. (corresponding to 0.087mg hirudin/kg), intravenously. Blood specimens were taken at 0, 5, 15, 30, 60, 120, 180, 240, 360, 420, 480, 560 minutes and 24 hours after administration. Citrated plasma samples were collected for PlT, TT and analysis for hirudin and THC.
The pharmacokinetic parameters from hirudin and the THC are listed in Table 1. The half life of the THC is markedly prolonged as compared to hirudin. Total clearance is reduced by factor 5 whereas the renal clearance has been diminished below lml per minute. The urinary excretion of hirudin amounts between 36 and 70% of the administered dose within a 24 hour period. In contrast, the urinary excretion of hirudin, administered as THC was only between 15 and 18% of the administered dose of MC. The elimination of the hirudin and THC followed a two-compartment body model. The effects of hirudin and THC on TT and PTT are shown in Table 3 and Figure 2. The mean plasma concentrations of hirudin and THC are shown in Figure 1. After intravenous administration of hirudin maximum effects of TT and PTT were achieved at 5 minutes and between 5 and 60 minutes, respectively. The THC is stable in aqueous solution and does not show any prolonged clotting time in the concentration range yielded in the plasma samples. However, the THC causes a prolonged effect on PTT, reaching the maximum between 60 and 180 minutes. ll was influenced only short-lived between 5 and 10 minutes. The explanation can be seen under the consideration of degradation of both compounds as tested in tissue homogenates (10). As shown in Figure 2A hirudin liberated from THC is disposed to prolong the PTT. Though, the complex is inactive, it shows an increase in PTT beginning 5 minutes after the administration and lasting to about 300 minutes. The maximum of the clotting time is at about 180 minutes, whereas the effect of hirudin is already over at that time.
TABLE 1 Pharmacokinetics of hirudin and THC
CLtot(ml/min) CLren(ml/min) MT-vss(h)
Vol. 66, Nos. 213
THROMEIN-HIRUDIN
KINETICS
TABLE 2 Urinary excretion of hirudin and THC in rhesus monkeys (n = 3) THC values d in terms of hirudin. animal 1 2 3 Hirudin (ug, 0- 8h) 293.0 320.0 370.0 Hirudin (% of dose) 25.7 41 .o 67.3 Hirudin tug, 8- 24h) 117.0 18.0 16.0 Hirudin (% of dose) 10.3 2.3 2.9 Hirudin (% 0- 24h) 36.0 43.7 70.2 THC (ug, 0- 8h) 59.0 53.0 50.0 10.4 8.0 13.9 THC (% of dose) 49.4 23.5 15.2 THC tug, 8- 24h) 4.6 4.2 THC (% of dose) 6.7 14.7 15.0 18.1 THC (% 0- 24h)
P-l-t-all
TABLE 3 T (set) for hirudil n and THC in rhesu nonkeys (mean :! SD, n = 3) hirudin sampling THC time(h) P-IT 0.000 32.1 f 1.7 31.1 f 1.0 63.8 + 2.3 38.2 f 1.8 0.083 0.250 54.0 f 4.5 40.7 f 5.4 0.500 52.5 f 7.0 40.0 f 3.3 45.8 f 3.7 1.000 41 .o + 3.9 35.2 * 3.0 48.9 * 6.6 2.000 50.9 f. 7.7 3.000 34.8 f 2.7 47.5 f 4.6 4.000 32.9 + 4.1 48.1 f 7.4 33.5 f 2.3 5.000 33.3 f 3.4 43.9 ?r 2.5 6.000 33.1 f 3.1 39.7 f 1.1 7.000 38.7 f 3.4 8.000 32.4 f 2.8 31.2 f 2.9 24.000 28.7 f 3.5 l-r 24.5 + 0.5 25.7 ?r0.9 0.000 48.7 f 6.4 0.083 >300 38.2 + 5.2 0.250 >300 29.8 + 1.O >300 0.500 27.8 & 1.I 96.7 *20.3 1.000 27.9 f 1.2 38.9 + 4.3 2.000 27.1 f 0.7 29.8 f 0.6 3.000 26.9 + 1.6 28.0 + 1.O 4.000 26.7 f 1.O 27.0 f 2.3 5.000 26.8 + 1.I 26.2 f 2.9 6.009 25.2 f 2.4 25.7 + 2.9 7.000 25.7 f 2.1 25.3 + 3.2 8.000 26.3 + 2.1 25.2 + 1.3 24.000
273
THROMBIN-HIRUDIN
274
500
-
Hirudln (cl% Imarl
-
HlrudInozhr.aMr~
+
HhUdiB@L-l%)
+
mudln
@Ll2A)
TEC
(CM.marl
TEC
(ELBA)
600 400
Vol. 66, Nos. 2/3
KINETICS
-+-
TBC
(chr. anaT)
-*-
-a-
TEC
(ELBA)
-8
200 0
0
2
4
6
10 11 I4 lb
8
l8
10
0
1? U
1
4
6
8
lhl
10 II lh)
14 lb
l8
20 11 !U
600 500 -
Himdin (chrs fnIaT)
400
+
HimdIn @LISA)
200
+
THC
(chc q)
-+
TIE
(ELBA)
100 100 0
0
2
4
6
Fig.1. (l-3) Hirudin and THC plasma concentrations in rhesus monkeys. Determination by ELISA and ChrOItlOgdC thrombin substrate assay (chr. assay). THC in terms of hirudin.
2IOl21416l8202224 lhl
2B 60 -
li Himdin
+TlK
n n n n
’
-
IO-
oLa B 0
2
’
4
6
20-
n mn m a
210111416l2r)2224 (hl
-
01, 0
’
2
’
4
HImdIn
n
b
’
’
-+THC
m
’
’
2lOl214l6l4201124 lb1
n c n n
Fig.2. PlT(A) and TT(B) after intravenous administration of hirudin and THC in rhesus monkeys (means, n=3).
Vol. 66, Nos. 213
THROMBIN-HIRUDIN
KINETICS
275
Pharmacokinetics and pharmacodynamics of a-human thrombin-hirudin complex and hirudin were examined in rhesus monkeys. The data presented for hirudin are in accordance with the data as described for rhesus monkeys (11). According to (41, pharmacokinetics and pharmacodynamics in man and animals are very similar. For hirudin, a half life of about one hour was determined, whereas after THC a prolonged half life was found of about 2.5 hours. The prolongation of blood clotting may be explained by the disintegration and therefore degradation of the THC, thus hirudin becomes liberated. THC was formed in a small amount between 6 and 40 ng/ml after administration of the hiruUin. THC found after the hirudin administration measured by ELISA technique was in a close range between a five hundredth and a thousandth of the total plasma concentration of hirudin. THC was tolerated well in the rhesus monkeys, no side effects were seen during the observation time.
1. Markwardt, F. Hirudin and Derivates as Anticoagulant Agents. Thumb Hemost66, 141- 152, 1991. 2. Markwardt, F., Fink, G., Kaiser, B., KlGcking, HP., Nowak, G., Richter, M., Sturzebecher, Pharmacological survey of recombinant hirudin. Pharmazie43, 202- 207, 1988.
J.
3. Markwardt, F., Nowak, G., Stutzebecher, Uta., Walsmann, P. Studies on the pharmacokinetics hirudin. BiomedB&him Acta 46, 237- 244, 1987. 4. Meyer, BH., Luus, HG., MDller, FO., Badenhorst, PN., Rothig, HJ. The pharmacology hirudin, a new anticoagulant. S Ati Med J 78, 268- 270, 1990.
of
of recombinant
5. Berscheid, G., Grotsch, H., Neubauer, H., Punter, J., Reindl, J., Seipp, P. Determination of rDNA hirudin and a-human thrombin-hirudin complex in plasma samples: enzyme linked immunosorbent assays for hirudin and complex vs. chromogenic thrombin substrate assay. Thromb Res submitted 1992. 6. Bichler, J., Siebeck, M., Maschler, R., Pelzer, H., Fritz, H. Determination of thrombin-hirudin complex in plasma with an enzyme linked immunosorbent assay. Blood coagulation and Fibrinolysis 2, 129 133, 1991. 7. Spinner, S., Scheffauer, F., Maschler, R., Stoffler, G. Hirudin catching ELISA for quantitating the anticoagulant in biological fluids. Thromb Res 51, 617- 625, 1988. 8. Griessbach, Uta., Sturzebecher, J., Markwardt, F. Assay of hirudin in plasma using a chromogenic thrombin substrate. Thromb Res 37, 347- 350, 1985. 9. Grotsch, H., Damm, D., Ben Youssef, R., Haertel, D. Comparison of two different methods for the determination of r DNA- Hirudin in plasma samples: HPLC vs a chromogenic thrombin substrate assay. Thromb Res 64,273- 277, 1991. 10. Grotsch, H., Hropot, M. Degradation of rDNA hirudin and a-human thrombin-hirudin and kidney homogenates from the Wistar rat. Thromb Res 64, 763- 767, 1991.
complex in liver
11. Hropot, M., Apidoupoulus, G., Crause, P., Grotsch, H., Habermann, P., Husak, B., Klaus, E., Malerczyk, V., Tripier, D. Pharmakodynamik und Pharmakokinetik von rekombinantem Hirudin HIBW 023 nach einmaliger intravenoser oder subkutaner Applikation an Rhesus-Affen. Haemosfaseologie 11, 129131,199l.
RESEARCH 66; 271-275,1992 $5.00 + .OO Printed in the USA.
Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
BRIFF
COMMUNICATION
PHARMACOKINETIC INVESTIGATIONS OF THE u-HUMAN THROMBIN-HIRUDIN COMPLEX IN RHESUS MONKEYS
H. GrWch, M. Hropot, G. Berscheid, P. Crause, V. Malerczyk, G. Apidopoulos, G. Haun and B. Husak Hoechst AG, Frankfurt am Main, FRG (Received
23.7.1991;
accepted
in revised form 24.2.19920
by Editor F. Markwardt)
INTRODUCTION On the excretion of hirudin after parenterai administration has been reported manifold (1,2,3,4). The pharmacokinetics of hirudin in man and animals seem to be very similar. There are only a few reports on the formation of the thrombin-hirudin complex (THC). After parenteral administration of hirudin THC is formed in small amounts (56). Markwardt et al.,(l) reported on the formation of the THC by a ratio of l:l, having a molecular weight of 43 kDa. Bichler et al.,(6) determined THC by means of an ELISA. Grotsch et al.,(lO) demonstrated a possible way of elimination by proteolytic digestion in liver and kidney. The aim of this study was to investigate the pharmacokinetics and pharmacodynamics of the THC in rhesus monkeys in comparison to hirudin. To complete the interpretation of the ll-tC data, the rhesus monkeys were treated with hirudin one week in advance. Analysis of the thrombin time and the partial thromboplastin time was included. In addition to the chromogenic thrombin substrate assay (8,9), two ELISA assays for hirudin and the THC (5) were performed.
MATERIAI AND METHODS THC used for the experiment was prepared from a-human thrombin (kindly supplied by Dr. Riomisch, Behringwerke, Marburg, FRG) and rDNA hirudin (HBW 023 Hoechst AG, Frankfurt/M, FRG). Hirudin was added to thrombin in excess of a five-fold amount for a quantitative reaction. The solution was cleared by centriigation at 6OOOg_. THC was separated from unbound hirudin by gel permeation chromatography on Sephacryl S 200. Fractions containing THC in the absence of free hirudin were combined, dialysed against water and concentrated with a DIAFLO YMlO membrane. The ELl$A used for the determination of THC was provided from purified anti a-human thrombin (antibody 1) and HRP labelled sheep anti-hirudin (antibody 2). The assay procedure was in accordance to Bichler et al.,(6). The ELISA for hirudin was provided from purified Tripalmitoyl-S-cysteinylserylglycin-hirudin (monoclonal mouse, antibody 1) and Tripalmitoyl-S-cysteinylserylglycin-hitudin (polyclonal sheep, antibody 2) and anti-sheep IGG HRP labelled. ____________________~~~_____~~~~~~~~~~~_~~~~~~~~~~~~--~~~~~~~~~~ Key words: Hirudin; thrombin-hirudin complex; rhesus monkeys; chromogenic thrombin substrate assay; ELISA for hirudin and THC; clotting times.
271
THROMBIN-HIRUDIN
272
KINETICS
Vol. 66, Nos. 213
The assay procedure was in accordance to Spinner et al.,(7). The experiments were performed in 3 rhesus monkeys with 5.5, 7.8 and 11.4 kg body weight. Before the hirudin and THC were administered, the animals received 20 ml tap water per kg body weight by stomach tube. During the 24 hour observation period food and water were withdrawn. Each of the animals was treated in the first week with 0.1 mg hirudin per kg b.wt. followed in the second week by 0.4 mg THC per kg b.wt. (corresponding to 0.087mg hirudin/kg), intravenously. Blood specimens were taken at 0, 5, 15, 30, 60, 120, 180, 240, 360, 420, 480, 560 minutes and 24 hours after administration. Citrated plasma samples were collected for PlT, TT and analysis for hirudin and THC.
The pharmacokinetic parameters from hirudin and the THC are listed in Table 1. The half life of the THC is markedly prolonged as compared to hirudin. Total clearance is reduced by factor 5 whereas the renal clearance has been diminished below lml per minute. The urinary excretion of hirudin amounts between 36 and 70% of the administered dose within a 24 hour period. In contrast, the urinary excretion of hirudin, administered as THC was only between 15 and 18% of the administered dose of MC. The elimination of the hirudin and THC followed a two-compartment body model. The effects of hirudin and THC on TT and PTT are shown in Table 3 and Figure 2. The mean plasma concentrations of hirudin and THC are shown in Figure 1. After intravenous administration of hirudin maximum effects of TT and PTT were achieved at 5 minutes and between 5 and 60 minutes, respectively. The THC is stable in aqueous solution and does not show any prolonged clotting time in the concentration range yielded in the plasma samples. However, the THC causes a prolonged effect on PTT, reaching the maximum between 60 and 180 minutes. ll was influenced only short-lived between 5 and 10 minutes. The explanation can be seen under the consideration of degradation of both compounds as tested in tissue homogenates (10). As shown in Figure 2A hirudin liberated from THC is disposed to prolong the PTT. Though, the complex is inactive, it shows an increase in PTT beginning 5 minutes after the administration and lasting to about 300 minutes. The maximum of the clotting time is at about 180 minutes, whereas the effect of hirudin is already over at that time.
TABLE 1 Pharmacokinetics of hirudin and THC
CLtot(ml/min) CLren(ml/min) MT-vss(h)
Vol. 66, Nos. 213
THROMEIN-HIRUDIN
KINETICS
TABLE 2 Urinary excretion of hirudin and THC in rhesus monkeys (n = 3) THC values d in terms of hirudin. animal 1 2 3 Hirudin (ug, 0- 8h) 293.0 320.0 370.0 Hirudin (% of dose) 25.7 41 .o 67.3 Hirudin tug, 8- 24h) 117.0 18.0 16.0 Hirudin (% of dose) 10.3 2.3 2.9 Hirudin (% 0- 24h) 36.0 43.7 70.2 THC (ug, 0- 8h) 59.0 53.0 50.0 10.4 8.0 13.9 THC (% of dose) 49.4 23.5 15.2 THC tug, 8- 24h) 4.6 4.2 THC (% of dose) 6.7 14.7 15.0 18.1 THC (% 0- 24h)
P-l-t-all
TABLE 3 T (set) for hirudil n and THC in rhesu nonkeys (mean :! SD, n = 3) hirudin sampling THC time(h) P-IT 0.000 32.1 f 1.7 31.1 f 1.0 63.8 + 2.3 38.2 f 1.8 0.083 0.250 54.0 f 4.5 40.7 f 5.4 0.500 52.5 f 7.0 40.0 f 3.3 45.8 f 3.7 1.000 41 .o + 3.9 35.2 * 3.0 48.9 * 6.6 2.000 50.9 f. 7.7 3.000 34.8 f 2.7 47.5 f 4.6 4.000 32.9 + 4.1 48.1 f 7.4 33.5 f 2.3 5.000 33.3 f 3.4 43.9 ?r 2.5 6.000 33.1 f 3.1 39.7 f 1.1 7.000 38.7 f 3.4 8.000 32.4 f 2.8 31.2 f 2.9 24.000 28.7 f 3.5 l-r 24.5 + 0.5 25.7 ?r0.9 0.000 48.7 f 6.4 0.083 >300 38.2 + 5.2 0.250 >300 29.8 + 1.O >300 0.500 27.8 & 1.I 96.7 *20.3 1.000 27.9 f 1.2 38.9 + 4.3 2.000 27.1 f 0.7 29.8 f 0.6 3.000 26.9 + 1.6 28.0 + 1.O 4.000 26.7 f 1.O 27.0 f 2.3 5.000 26.8 + 1.I 26.2 f 2.9 6.009 25.2 f 2.4 25.7 + 2.9 7.000 25.7 f 2.1 25.3 + 3.2 8.000 26.3 + 2.1 25.2 + 1.3 24.000
273
THROMBIN-HIRUDIN
274
500
-
Hirudln (cl% Imarl
-
HlrudInozhr.aMr~
+
HhUdiB@L-l%)
+
mudln
@Ll2A)
TEC
(CM.marl
TEC
(ELBA)
600 400
Vol. 66, Nos. 2/3
KINETICS
-+-
TBC
(chr. anaT)
-*-
-a-
TEC
(ELBA)
-8
200 0
0
2
4
6
10 11 I4 lb
8
l8
10
0
1? U
1
4
6
8
lhl
10 II lh)
14 lb
l8
20 11 !U
600 500 -
Himdin (chrs fnIaT)
400
+
HimdIn @LISA)
200
+
THC
(chc q)
-+
TIE
(ELBA)
100 100 0
0
2
4
6
Fig.1. (l-3) Hirudin and THC plasma concentrations in rhesus monkeys. Determination by ELISA and ChrOItlOgdC thrombin substrate assay (chr. assay). THC in terms of hirudin.
2IOl21416l8202224 lhl
2B 60 -
li Himdin
+TlK
n n n n
’
-
IO-
oLa B 0
2
’
4
6
20-
n mn m a
210111416l2r)2224 (hl
-
01, 0
’
2
’
4
HImdIn
n
b
’
’
-+THC
m
’
’
2lOl214l6l4201124 lb1
n c n n
Fig.2. PlT(A) and TT(B) after intravenous administration of hirudin and THC in rhesus monkeys (means, n=3).
Vol. 66, Nos. 213
THROMBIN-HIRUDIN
KINETICS
275
Pharmacokinetics and pharmacodynamics of a-human thrombin-hirudin complex and hirudin were examined in rhesus monkeys. The data presented for hirudin are in accordance with the data as described for rhesus monkeys (11). According to (41, pharmacokinetics and pharmacodynamics in man and animals are very similar. For hirudin, a half life of about one hour was determined, whereas after THC a prolonged half life was found of about 2.5 hours. The prolongation of blood clotting may be explained by the disintegration and therefore degradation of the THC, thus hirudin becomes liberated. THC was formed in a small amount between 6 and 40 ng/ml after administration of the hiruUin. THC found after the hirudin administration measured by ELISA technique was in a close range between a five hundredth and a thousandth of the total plasma concentration of hirudin. THC was tolerated well in the rhesus monkeys, no side effects were seen during the observation time.
1. Markwardt, F. Hirudin and Derivates as Anticoagulant Agents. Thumb Hemost66, 141- 152, 1991. 2. Markwardt, F., Fink, G., Kaiser, B., KlGcking, HP., Nowak, G., Richter, M., Sturzebecher, Pharmacological survey of recombinant hirudin. Pharmazie43, 202- 207, 1988.
J.
3. Markwardt, F., Nowak, G., Stutzebecher, Uta., Walsmann, P. Studies on the pharmacokinetics hirudin. BiomedB&him Acta 46, 237- 244, 1987. 4. Meyer, BH., Luus, HG., MDller, FO., Badenhorst, PN., Rothig, HJ. The pharmacology hirudin, a new anticoagulant. S Ati Med J 78, 268- 270, 1990.
of
of recombinant
5. Berscheid, G., Grotsch, H., Neubauer, H., Punter, J., Reindl, J., Seipp, P. Determination of rDNA hirudin and a-human thrombin-hirudin complex in plasma samples: enzyme linked immunosorbent assays for hirudin and complex vs. chromogenic thrombin substrate assay. Thromb Res submitted 1992. 6. Bichler, J., Siebeck, M., Maschler, R., Pelzer, H., Fritz, H. Determination of thrombin-hirudin complex in plasma with an enzyme linked immunosorbent assay. Blood coagulation and Fibrinolysis 2, 129 133, 1991. 7. Spinner, S., Scheffauer, F., Maschler, R., Stoffler, G. Hirudin catching ELISA for quantitating the anticoagulant in biological fluids. Thromb Res 51, 617- 625, 1988. 8. Griessbach, Uta., Sturzebecher, J., Markwardt, F. Assay of hirudin in plasma using a chromogenic thrombin substrate. Thromb Res 37, 347- 350, 1985. 9. Grotsch, H., Damm, D., Ben Youssef, R., Haertel, D. Comparison of two different methods for the determination of r DNA- Hirudin in plasma samples: HPLC vs a chromogenic thrombin substrate assay. Thromb Res 64,273- 277, 1991. 10. Grotsch, H., Hropot, M. Degradation of rDNA hirudin and a-human thrombin-hirudin and kidney homogenates from the Wistar rat. Thromb Res 64, 763- 767, 1991.
complex in liver
11. Hropot, M., Apidoupoulus, G., Crause, P., Grotsch, H., Habermann, P., Husak, B., Klaus, E., Malerczyk, V., Tripier, D. Pharmakodynamik und Pharmakokinetik von rekombinantem Hirudin HIBW 023 nach einmaliger intravenoser oder subkutaner Applikation an Rhesus-Affen. Haemosfaseologie 11, 129131,199l.
