SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 17, NO. 2, 1991
Pharmacokinetics of Hirudin
The outstanding anticoagulant properties of both naturally occurring hirudin and its recombinant variants have already been described by several investigators during the past decade. 1-4 Before hirudin can be introduced into clinical practice, the fate of this specific thrombin inhibitor has to be clarified in vivo. Studies in experimental animals and in man, directed toward the determination of pharmacokinetic parameters, such as blood levels, distribution, metabolic changes, and excretion, are one of the prerequisites for starting clinical trials with this anticoagulant agent. This review will give an account of the pharmacokinetic properties of hirudin so far known. The determination of pharmacokinetic parameters required specific and sensitive methods by which hirudin could be quantified in biologic fluids. Hirudin assays were either based on its inhibitory effect on thrombin, determined by clotting tests or by chromogenic substrate methods (for details see Stürzebecher in this issue of Seminars), or based on immunologic methods5 or labeling tests with 125 I. 6 The pharmacokinetic parameters were calculated from the quantity of hirudin found in plasma and urine. Hirudin levels in plasma decreased in a biexponential manner and could be described by the equation
It served to calculate the fractional efflux (k12), reflux (k21) and elimination (k13) rate constants. The half-lives (t1/2) were derived from the elimination constants a and β and the area under the plasma concentration time curve (AUC), which was estimated by means of the trapezoidal rule. Total clearance from plasma (Cltot) was calculated by dividing the intravenous dose (D) by the AUC value.
From the Institute of Pharmacology and Toxicology, Medical Academy Erfurt, Erfurt, Germany. Reprint requests: Dr. Nowak, Nordhäuser Strasse 74, D-5010 Erfurt, Germany.
The quotient D/Cpo represents the apparent volume of distribution (Vc), and the volume in steady state (Vdss) was calculated according to the equation Vdss = (k21 + k12)/k21 X Vc. After extravascular administration of hirudin, the peak plasma concentration (Cmax) and the time after dosing when C max occurred (tmax) were mea sured.
NATIVE HIRUDIN Rats The first pharmacokinetic data of hirudin were obtained from experimental studies in rats. 7,8 After an intravenous bolus injection of 50 µg/kg, the elimination half-life amounted to approximately 1 hour.7 The fol lowing pharmacokinetic parameters of additional special investigations with 125I-labeled hirudin are listed in Ta ble 1.9 The elimination half-life was 65 minutes. From the microparameters (k12, k13, k10) it was concluded that there is a rapid transfer between the vascular and the extravascular compartments. The subcutaneous injection of 1 mg hirudin/kg led to a maximum hirudin plasma concentration (Cmax) of 0.84 µg/ml, 1 hour (tmax) after administration. Compared with the intravenous application, the elimination half-life (126 minutes) was markedly prolonged. The total urinary excretion of active hirudin amounted to 2 to 4% of the dose administered,8 whereas measurement of the radio activity revealed a recovery of 15% of radiolabeled hirudin in urine.9
Dogs To complete the pharmacokinetic data of hirudin and, simultaneously, to exclude possible species differ ences, corresponding studies were performed in Beagle dogs. After an intravenous bolus injection of hirudin (0.5 mg/kg), the blood levels decreased rapidly; after 60
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SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 17, NO. 2, 1991
TABLE 1. Pharmacokinetic Parameters After a Single Intravenous Injection of 1 mg Hirudin/kg in Rats A (µg/ml)
1.87
B (µg/ml)
0.79
α (h - 1 )
3.36
β (h - 1 )
0.64
t½α (h)
0.21
t½β ( h )
1.08
C1 tot (1/h)
0.17
k12 (h - 1 )
0.59
k21 (h - 1 )
0.65
-1
k10 (h )
1.48
Vc(l)
0.116
Vdss (1)
0.222
AUCo-5 (µg/ml X h)
1.74
C1 (ml X min - 1 )
2.09
minutes, they were below 10% of the initial concentra tion. The distribution phase lasted 60 minutes and was followed by the elimination phase. Table 2 gives the corresponding pharmacokinetic data. Comparison with values obtained with 125I-labeled hirudin revealed no marked differences, except for the total clearance data that were significantly lower in case of 125I-hirudin. Within the first hour after application, more than 50% and after 5 hours about 70% (85% in case of
TABLE 2. Pharmacokinetics of Hirudin in Dogs After an Intravenous Bolus Injection of 0.5 mg/kg* Detection Method Chromogenic Substrate Method
125
I-Hirudin
A (µg/ml)
3.5
3.11
B (µg/ml)
1.0
0.33
α (h - 1 )
4.12
3.33
β (h - 1 )
0.72
0.73
t½α (h)
0.17
0.21
t½β (h)
0.95
0.94
k12 (h )
1.37
0.60
k21 (h-1)
1.46
0.98
k10 (h )
1.99
2.48
AUC (µg/ml X h) Vc(l)
1.43 2.2
1.38 2.83
Vdss (1)
4.4
4.55
-1
-1
C1 tot (ml/min)
178
117
*Comparison with two different studies with different detection methods9'10
125
I-hirudin) of the administered dose were excreted in the urine in active form. In continuous infusion experi ments, different doses of hirudin were studied during different times. Infusion of 0.5 mg hirudin/kg x h over 1 hour, or 1.0 mg/kg x h over 2 hours resulted in a steady-state blood level of 1.0 or 3.5 fig/ml plasma about 15 to 30 minutes after the beginning of the infusion. When hirudin was administered slowly (0.1 mg/kg x h over 5 hours), a blood level of 0.35 µg/ml plasma was maintained over a prolonged period of time. 10 After infusion, the hirudin levels dropped sharply and, after 60 to 120 minutes, they decreased slowly with a half-life corresponding to the elimination phase following intravenous bolus injection. The amount of hirudin excreted in the urine corresponded to that found after bolus injection. The urinary recovery of native hirudin with this route of administration was about 70%. On subcutaneous injection of a single dose of 0.5 mg hirudin/kg, the blood levels increased rapidly and reached a maximum (C max = 0.3 µg/ml plasma) after about 120 minutes (t max ). Thereafter, the hirudin activity in blood decreased slowly. After 8 hours, an antithrombin activity was measured that corresponded to a hirudin concentration in plasma of 0.15 µg/ml. The half-life was approximately 182 minutes. From the values AUC intravenous/AUC subcutaneous, an absorption quotient of 0.6 was calculated for 6 hours; for 12 hours it was 0.9. About 7% of the hirudin administered was excreted per hour: after 8 hours, 50% of the administered dose was found in urine in active form. Enteral application of hirudin in dogs was per formed by means of a duodenal tube. By the detection method used, no hirudin activity was found in blood samples taken every 2 hours throughout the experiment (12 hours). However, small amounts of hirudin were found in the urine. In total, only 0.5% of the adminis tered dose was excreted via the kidneys within 24 hours.9
Human Volunteers Important information on the pharmacokinetic be havior of native hirudin in vivo was obtained in pilot studies in man. Healthy volunteers participated in these studies to complement the pharmacokinetic understand ing of native hirudin.11 The hirudin levels in plasma after the intravenous injection of 1000 ATU hirudin/kg were investigated in three volunteers. After the initial disap pearance, which was interpreted as a distribution phase (60 minutes), first-order elimination kinetics followed. The half-life of the elimination phase (t1/2β) was 0.84 ± 0.19 hours. The volume of distribution (Vdss) at steady-state, calculated from the course of the hirudin level in plasma, amounted to 12.9 ± 2.8 1. The area
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147
under the plasma concentration time curve (AUC) was 5.69 ± 1.72 ATU/ml x h. The total systemic clearance (Cltot) was 230 ± 98 ml/min. After subcutaneous injection of the same dose (1000 ATU/kg) in three volunteers, the hirudin levels in blood increased within 60 minutes and reached plateau values in plasma of 0.5 ATU/ml. This fell below the detection limit after 5 hours. The elimination half-life after subcu taneous application was similar to that after intravenous injection (t1/2β = 0.64 h). The subcutaneous absorption rate constant (kA) was calculated to be 1.1 h - 1 , indicat ing a rapid absorption of the drug. The subcutaneous bioavailability was calculated to be 36%. The cumulative urinary excretion of hirudin showed that 30 to 40% of the administered amount of hirudin was excreted within 24 hours in the urine in active form. The largest percentage was excreted within the first 2 hours after the intravenous injection and after 4 hours following subcutaneous injection. Similar kinetic parameters have been determined by further pharmacokinetic studies in human volunteers with native hirudin, where the elimination half-life after subcutaneous injection of hirudin (1000 ATU/kg) amounted to 91 minutes, thus being nearly twice as long as in the case of intravenous injection (50.4 and 64.7 minutes, respectively).12
RECOMBINANT HIRUDIN Rats, Rabbits, Dogs The pharmacokinetic characteristics of recombinant (rh) hirudin was intensively studied in various animal
species.13-17 Table 3 shows the pharmacokinetic param eters obtained in some animal species after a single intravenous bolus injection of r-hirudin. The elimination half-lives (t1/2β) were almost identical at the respective dosages (0.5 to 2 mg/kg) in the different animal species studied. Similar to native hirudin, r-hirudin was relatively rapidly distributed into the extravascular compartment. Compared with other animal species, a particularly high blood level was found in rabbits (higher AUC values). Compared with native hirudin, longer elimination phase half-lives were found. The total clearance of r-hirudin was correspondingly low in rabbits. The total clearance in rats and dogs was found to be approximately within the same order of magnitude, related to the available blood volume. As with native hirudin, a relatively small amount of active r-hirudin was found in the urine of rats, 16 whereas in dogs almost the total quantity of administered r-hirudin was eliminated through the kidneys in active form; more than 70% within the first hour after application, about 95% after 5 hours. A similarly high urinary elimination was found after the infusion of r-hirudin at the same dose (0.5 mg/kg) over 60 minutes. After subcutaneous admin istration of 0.5 mg/kg r-hirudin, the blood levels in creased rapidly within the first 30 minutes. The time (tmax) was 90 minutes with a C max of 0.3 µg/ml. Thereafter, the hirudin blood levels decreased slowly, with an apparent elimination half-life of 5.75 hours. A plasma level of 0.15 µg/ml was found after 8 hours. During this time, about 80% of the administered dose had been excreted via the kidneys.
TABLE 3. Pharmacokinetic Data of Recombinant Hirudin in Different Animal Species after a Single Intravenous Bolus Injection Rat (mg/kg) 1.0
2.0
Rabbit (mg/kg) 1.0
Dog (mg/kg) 0.5
A (µg/ml)
2.45
10.46
12.55
2.5
B (µg/ml)
0.82
1.50
1.43
0.30
α (h - 1 )
4.75
8.25
6.52
2.77
β (h -1 )
0.65
0.59
0.60
0.57
t½α (h)
0.15
0.08
0.11
0.25
t½β
1.07
1.18
1.16
1.22
k12 (h - 1 )
1.93
4.15
2.67
0.57
k21 (h - 1 )
1.68
1.55
1.21
0.81
k10 (h )
1.8
3.14
3.24
1.96
AUC (µg/ml X h)
1.64
3.81
4.31
1.54
Vc(l/kg)
0.31
0.17
0.07
0.18
Vdss(l/kg)
0.66
0.63
0.23
C1 tot (ml/min)
3.05
2.09
12.38
(h)
-1
0.28 183
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PHARMACOKINETICS OF HIRUDIN—NOWAK
SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 17, NO. 2, 1991
The administration of r-hirudin to nephrectomized dogs was followed by a higher hirudin blood level than found in animals with normal renal functions. These experiments confirmed that after 1 hour, 75 to 80% and after 120 minutes about 85% of the applied dose were distributed into the extravascular compartment. Then the blood level remained nearly constant, which speaks in favor of the exclusive renal elimination of r-hirudin.13
Primates Similar pharmacokinetic data of r-hirudin and com parable elimination half-lives were also found in primates.17 All pharmacokinetic studies with hirudin confirmed that it is distributed in the extracellular space of the organism. Hence, besides the rapid renal elimina tion, primarily distribution phenomena are responsible for the decrease in blood levels after an intravenous administration.
Special Studies In special studies the placental transfer of r-hirudin was examined in pregnant rabbits on the 27th day of gestation.16 r-Hirudin (0.5 mg/kg) was administered into the marginal ear vein 20, 15, and 2 hours before the dissection. The concentration of r-hirudin found in maternal blood plasma was about 60 times higher than in the fetal circulation, which was confirmed by heart puncture. The r-hirudin content in amniotic fluid amounted to 0.015%. Therefore there is only a very slight placental transfer of r-hirudin to the fetal organism. After intratracheal instillation of r-hirudin, the ab sorption amounted to about 30%. The plasma level may be described by the equation C P = 0.2 x e-0.21 x t-0.21 x e-1.2 x t. Accordingly, the elimination half-life of about 200 minutes was still longer than that after subcutaneous injection. After rectal administration, no marked absorption of r-hirudin could be observed. To influence the pharmacokinetic behavior, a mod ified hirudin bound to a high molecular weight vehicle was used. 18,19 Dextran, which has a molecular weight of 70,000 d, was solely distributed in blood plasma. At a dose of dextran-r-hirudin of 1000 ATU/kg, a signifi cantly higher plasma level was obtained than with r-hirudin alone, whereas unbound r-hirudin administered at the same dose fell below the detection limit after 1 hour. The elimination half-life of dextran-hirudin amounted to about 7 hours. The distribution volume of 9.7% of body mass proved to be only somewhat higher than the blood volume.19 Studies with dextran-bound r-hirudin in rats and rabbits have shown that dextran 40-hirudin had an elimination half-life of about 6.5 hours. With dextran 70-hirudin that time was prolonged
to 7.5 hours.19 This confirmed that dextran-bound r-hirudin disposes of the same inhibitory activity as unbound r-hirudin. Thus, similar covalently bonded preparations of hirudin may be of interest for clinical use.
Human Volunteers The pharmacokinetics of r-hirudin were studied in healthy volunteers after single intravenous, subcutane ous, and intramuscular doses of 0.1 mg/kg.20 The pharmacokinetic data obtained after an intravenous bolus injection are summarized in Table 4. The intravenous route of administration of r-hirudin in man revealed distribution and excretion characteristics similar to those of native hirudin. After a subcutaneous injection of 0.1 mg/kg r-hirudin the tmax amounted to 120 minutes, while C max was 0.15 µg/ml. Afterward, the hirudin blood levels decreased slowly. During this phase, an apparent elim ination half-life of approximately 180 minutes was found. Nearly similar adsorption and elimination values have been recorded after the intramuscular administra tion of 0.1 mg/kg. All three routes of administration were followed by the excretion of r-hirudin in the urine in active form (35 to 40% within 24 hours). A delayed excretion of r-hirudin into the urine could be demon strated after subcutaneous or intramuscular administra tion because of the delayed adsorption into the blood. Another study on the kinetics of r-hirudin in man showed that after subcutaneous administration of 0.1 to 0.4 mg/kg r-hirudin, the maximum plasma levels were 0.45 µg/ml at a mean tmax of 120 minutes.21 In these studies, an elimination half-life of 105 and an absorption
TABLE 4. Pharmacokinetics of Recombinant Hirudin in Human Volunteers After a Single Intravenous Dose of 0.1 mg/kg A (µg/ml)
1.0
B (µg/ml)
0.25
α (h - 1 )
4.62
-1
β (h )
0.75
t½α (h)
0.15
t½β (h)
1.91
k12 (h - 1 )
1.93
k21 (h-1)
1.68
-1
k10 (h )
1.8
AUC (µg/ml X h)
0.54
Vc(l/kg)
4.4
V dss (1/kg) C1 tot (ml/min)
8.9 168
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half-life of 55 minutes were determined. The apparent distribution volume Vdss of 31.9 1 was markedly increased, compared with findings described previously.21 The Cltot value of 208 ml/min corresponded relatively well with the data determined by us before.20 The quantity of active hirudin excreted in the urine amounted to 35% of the dose administered. This corroborates previous findings.21
CONCLUSION Pharmacokinetic studies with hirudin confirmed its distribution into the extracellular space of the body. After intravenous administration, the elimination half-life was about 1 hour; an extravascular route of administration prolonged this time to 2 to 3 hours. Hirudin was exclusively eliminated via the kidneys and it was partly recovered in unchanged form in the urine. Marked species differences were found in the renal excretion. Only a slight percentage of active hirudin was excreted by rats, whereas nearly complete excretion of active hirudin was found in dogs. After the administration of hirudin to man (0.1 mg/kg) only about one third of the dose was excreted in the urine. The enteral resorption of hirudin remained low and the placental transfer was small in experimental animals.
REFERENCES 1. Markwardt F: Die antagonistische Wirkung des Hirudins gegen Thrombin in vivo. Naturwisswnschaften 43:111, 1956. 2. Markwardt F: Development of hirudin as an antithrombotic agent. Semin Thromb Hemost 15:269-282, 1989. 3. Walenga JM, R Pifarre, DA Hoppensteadt, J Fareed: Development of recombinant hirudin as a therapeutic anticoagulant and antithrombotic agent: Some objective considerations. Semin Thromb Hemost 15:316-333, 1989. 4. Talbot M: Biology of recombinant hirudin (CGP 39393): A new prospect in the treatment of thrombosis. Semin Thromb Hemost 15:293-301, 1989. 5. Spinner S, G Stöffler, E Fink: Quantitative enzyme-linked immunosorbent assay (ELISA) for hirudin. J Immunol Methods 87:79— 93, 1986.
149
6. Richter M, P Walsmann, U Cyranka, F Markwardt: 125I-Markierung von Hirudin. Pharmazie 41:510, 1986. 7. Markwardt F, G Nowak, J Hoffmann: The influence of drugs on disseminated intravascular coagulation (DIC). Effects of naturally occurring and synthetic thrombin inhibitors. Thromb Res 11:275283, 1977. 8. Markwardt F, J Hauptmann, G Nowak, C Klessen, P Walsmann: Pharmacological studies on the antithrombotic action of hirudin in experimental animals. Thromb Haemost 47:226-229, 1982. 9. Richter M, U Cyranka, G Nowak, P Walsmann: Pharmacokinetics of 125-I hirudin in rats and dogs. Folia Haematol (Leipz) 115:6469, 1988. 10. Markwardt F, G Nowak, U Stürzebecher, P Walsmann: Studies on the pharmacokinetics of hirudin. Biomed Biochim Acta 46:237244, 1987. 11. Markwardt F, G Nowak, J Stürzebecher, U Grieβpbach, P Walsmann, P Vogel: Pharmacokinetics and anticoagulant effect of hirudin in man. Thromb Haemost 52:160-163, 1984. 12. Bichler J, B Fichtl, M Siebeck, H Fritz: Pharmacokinetics and pharmacodynamics of hirudin in man after single subcutaneous and intravenous bolus administration. Arzneimittelforsch 38:704-710, 1988. 13. Nowak G, F Markwardt, E Fink: Pharmacokinetic studies with recombinant hirudin in dogs. Folia Haematol (Leipz) 115:70-74, 1988. 14. Kaiser B, A Simon, F Markwardt: Antithrombotic effects of recombinant hirudin in experimental angioplasty and intravascular thrombolysis. Thromb Haemost 63:44-47, 1990. 15. Markwardt F, B Kaiser, G Nowak: Studies on antithrombotic effects of recombinant hirudin. Thromb Res 54:377-388, 1989. 16. Markwardt F, G Fink, B Kaiser, H.-P Klöcking, G Nowak, M Richter, J Stürzebecher: Pharmacological survey of recombinant hirudin. Pharmazie 43:202-207, 1988. 17. Markwardt F, XQ Huán, JM Walenga, M Muench, D Hoppensteadt: Pharmacokinetics of recombinant hirudin in primates. (Abst). Fed Proc 1:484, 1988. 18. Richter M, P Walsmann, F Markwardt: Plasma level of dextranr-hirudin. Pharmazie 44:73-74, 1989. 19. Markwardt F, M Richter, P Walsmann, G Riesener, M Paintz: Preparation of dextran-bound recombinant hirudin and its pharmacokinetic behaviour. Biochim, Biophys Acta 49:1103-1108, 1990. 20. Markwardt F, G Nowak, J Stürzebecher, G Vogel: Clinicopharmacological studies with recombinant hirudin. Thromb Res 52:393-400, 1988. 21. Bichler J, M Siebeck, M Spannagl, P Close, H Fritz, W Schramm: Pharmakokinetische und gerinnungshemmende Eigenschaften von rekombinantem Hirudin (CGP 39 393) nach zweimaliger Gabe an gesunden Probanden. In: 6. Kongreβ der Gesellschaft für Thrombose- und Hämostaseforschung. Kiel, 21.-24.2.1990. Hauptvorträge, Ausgewählte freie Vorträge. Schattauer, Stuttgart, 1990, p 190.
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PHARMACOKINETICS OF HIRUDIN—NOWAK
Pharmacokinetics of Hirudin
The outstanding anticoagulant properties of both naturally occurring hirudin and its recombinant variants have already been described by several investigators during the past decade. 1-4 Before hirudin can be introduced into clinical practice, the fate of this specific thrombin inhibitor has to be clarified in vivo. Studies in experimental animals and in man, directed toward the determination of pharmacokinetic parameters, such as blood levels, distribution, metabolic changes, and excretion, are one of the prerequisites for starting clinical trials with this anticoagulant agent. This review will give an account of the pharmacokinetic properties of hirudin so far known. The determination of pharmacokinetic parameters required specific and sensitive methods by which hirudin could be quantified in biologic fluids. Hirudin assays were either based on its inhibitory effect on thrombin, determined by clotting tests or by chromogenic substrate methods (for details see Stürzebecher in this issue of Seminars), or based on immunologic methods5 or labeling tests with 125 I. 6 The pharmacokinetic parameters were calculated from the quantity of hirudin found in plasma and urine. Hirudin levels in plasma decreased in a biexponential manner and could be described by the equation
It served to calculate the fractional efflux (k12), reflux (k21) and elimination (k13) rate constants. The half-lives (t1/2) were derived from the elimination constants a and β and the area under the plasma concentration time curve (AUC), which was estimated by means of the trapezoidal rule. Total clearance from plasma (Cltot) was calculated by dividing the intravenous dose (D) by the AUC value.
From the Institute of Pharmacology and Toxicology, Medical Academy Erfurt, Erfurt, Germany. Reprint requests: Dr. Nowak, Nordhäuser Strasse 74, D-5010 Erfurt, Germany.
The quotient D/Cpo represents the apparent volume of distribution (Vc), and the volume in steady state (Vdss) was calculated according to the equation Vdss = (k21 + k12)/k21 X Vc. After extravascular administration of hirudin, the peak plasma concentration (Cmax) and the time after dosing when C max occurred (tmax) were mea sured.
NATIVE HIRUDIN Rats The first pharmacokinetic data of hirudin were obtained from experimental studies in rats. 7,8 After an intravenous bolus injection of 50 µg/kg, the elimination half-life amounted to approximately 1 hour.7 The fol lowing pharmacokinetic parameters of additional special investigations with 125I-labeled hirudin are listed in Ta ble 1.9 The elimination half-life was 65 minutes. From the microparameters (k12, k13, k10) it was concluded that there is a rapid transfer between the vascular and the extravascular compartments. The subcutaneous injection of 1 mg hirudin/kg led to a maximum hirudin plasma concentration (Cmax) of 0.84 µg/ml, 1 hour (tmax) after administration. Compared with the intravenous application, the elimination half-life (126 minutes) was markedly prolonged. The total urinary excretion of active hirudin amounted to 2 to 4% of the dose administered,8 whereas measurement of the radio activity revealed a recovery of 15% of radiolabeled hirudin in urine.9
Dogs To complete the pharmacokinetic data of hirudin and, simultaneously, to exclude possible species differ ences, corresponding studies were performed in Beagle dogs. After an intravenous bolus injection of hirudin (0.5 mg/kg), the blood levels decreased rapidly; after 60
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145
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SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 17, NO. 2, 1991
TABLE 1. Pharmacokinetic Parameters After a Single Intravenous Injection of 1 mg Hirudin/kg in Rats A (µg/ml)
1.87
B (µg/ml)
0.79
α (h - 1 )
3.36
β (h - 1 )
0.64
t½α (h)
0.21
t½β ( h )
1.08
C1 tot (1/h)
0.17
k12 (h - 1 )
0.59
k21 (h - 1 )
0.65
-1
k10 (h )
1.48
Vc(l)
0.116
Vdss (1)
0.222
AUCo-5 (µg/ml X h)
1.74
C1 (ml X min - 1 )
2.09
minutes, they were below 10% of the initial concentra tion. The distribution phase lasted 60 minutes and was followed by the elimination phase. Table 2 gives the corresponding pharmacokinetic data. Comparison with values obtained with 125I-labeled hirudin revealed no marked differences, except for the total clearance data that were significantly lower in case of 125I-hirudin. Within the first hour after application, more than 50% and after 5 hours about 70% (85% in case of
TABLE 2. Pharmacokinetics of Hirudin in Dogs After an Intravenous Bolus Injection of 0.5 mg/kg* Detection Method Chromogenic Substrate Method
125
I-Hirudin
A (µg/ml)
3.5
3.11
B (µg/ml)
1.0
0.33
α (h - 1 )
4.12
3.33
β (h - 1 )
0.72
0.73
t½α (h)
0.17
0.21
t½β (h)
0.95
0.94
k12 (h )
1.37
0.60
k21 (h-1)
1.46
0.98
k10 (h )
1.99
2.48
AUC (µg/ml X h) Vc(l)
1.43 2.2
1.38 2.83
Vdss (1)
4.4
4.55
-1
-1
C1 tot (ml/min)
178
117
*Comparison with two different studies with different detection methods9'10
125
I-hirudin) of the administered dose were excreted in the urine in active form. In continuous infusion experi ments, different doses of hirudin were studied during different times. Infusion of 0.5 mg hirudin/kg x h over 1 hour, or 1.0 mg/kg x h over 2 hours resulted in a steady-state blood level of 1.0 or 3.5 fig/ml plasma about 15 to 30 minutes after the beginning of the infusion. When hirudin was administered slowly (0.1 mg/kg x h over 5 hours), a blood level of 0.35 µg/ml plasma was maintained over a prolonged period of time. 10 After infusion, the hirudin levels dropped sharply and, after 60 to 120 minutes, they decreased slowly with a half-life corresponding to the elimination phase following intravenous bolus injection. The amount of hirudin excreted in the urine corresponded to that found after bolus injection. The urinary recovery of native hirudin with this route of administration was about 70%. On subcutaneous injection of a single dose of 0.5 mg hirudin/kg, the blood levels increased rapidly and reached a maximum (C max = 0.3 µg/ml plasma) after about 120 minutes (t max ). Thereafter, the hirudin activity in blood decreased slowly. After 8 hours, an antithrombin activity was measured that corresponded to a hirudin concentration in plasma of 0.15 µg/ml. The half-life was approximately 182 minutes. From the values AUC intravenous/AUC subcutaneous, an absorption quotient of 0.6 was calculated for 6 hours; for 12 hours it was 0.9. About 7% of the hirudin administered was excreted per hour: after 8 hours, 50% of the administered dose was found in urine in active form. Enteral application of hirudin in dogs was per formed by means of a duodenal tube. By the detection method used, no hirudin activity was found in blood samples taken every 2 hours throughout the experiment (12 hours). However, small amounts of hirudin were found in the urine. In total, only 0.5% of the adminis tered dose was excreted via the kidneys within 24 hours.9
Human Volunteers Important information on the pharmacokinetic be havior of native hirudin in vivo was obtained in pilot studies in man. Healthy volunteers participated in these studies to complement the pharmacokinetic understand ing of native hirudin.11 The hirudin levels in plasma after the intravenous injection of 1000 ATU hirudin/kg were investigated in three volunteers. After the initial disap pearance, which was interpreted as a distribution phase (60 minutes), first-order elimination kinetics followed. The half-life of the elimination phase (t1/2β) was 0.84 ± 0.19 hours. The volume of distribution (Vdss) at steady-state, calculated from the course of the hirudin level in plasma, amounted to 12.9 ± 2.8 1. The area
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under the plasma concentration time curve (AUC) was 5.69 ± 1.72 ATU/ml x h. The total systemic clearance (Cltot) was 230 ± 98 ml/min. After subcutaneous injection of the same dose (1000 ATU/kg) in three volunteers, the hirudin levels in blood increased within 60 minutes and reached plateau values in plasma of 0.5 ATU/ml. This fell below the detection limit after 5 hours. The elimination half-life after subcu taneous application was similar to that after intravenous injection (t1/2β = 0.64 h). The subcutaneous absorption rate constant (kA) was calculated to be 1.1 h - 1 , indicat ing a rapid absorption of the drug. The subcutaneous bioavailability was calculated to be 36%. The cumulative urinary excretion of hirudin showed that 30 to 40% of the administered amount of hirudin was excreted within 24 hours in the urine in active form. The largest percentage was excreted within the first 2 hours after the intravenous injection and after 4 hours following subcutaneous injection. Similar kinetic parameters have been determined by further pharmacokinetic studies in human volunteers with native hirudin, where the elimination half-life after subcutaneous injection of hirudin (1000 ATU/kg) amounted to 91 minutes, thus being nearly twice as long as in the case of intravenous injection (50.4 and 64.7 minutes, respectively).12
RECOMBINANT HIRUDIN Rats, Rabbits, Dogs The pharmacokinetic characteristics of recombinant (rh) hirudin was intensively studied in various animal
species.13-17 Table 3 shows the pharmacokinetic param eters obtained in some animal species after a single intravenous bolus injection of r-hirudin. The elimination half-lives (t1/2β) were almost identical at the respective dosages (0.5 to 2 mg/kg) in the different animal species studied. Similar to native hirudin, r-hirudin was relatively rapidly distributed into the extravascular compartment. Compared with other animal species, a particularly high blood level was found in rabbits (higher AUC values). Compared with native hirudin, longer elimination phase half-lives were found. The total clearance of r-hirudin was correspondingly low in rabbits. The total clearance in rats and dogs was found to be approximately within the same order of magnitude, related to the available blood volume. As with native hirudin, a relatively small amount of active r-hirudin was found in the urine of rats, 16 whereas in dogs almost the total quantity of administered r-hirudin was eliminated through the kidneys in active form; more than 70% within the first hour after application, about 95% after 5 hours. A similarly high urinary elimination was found after the infusion of r-hirudin at the same dose (0.5 mg/kg) over 60 minutes. After subcutaneous admin istration of 0.5 mg/kg r-hirudin, the blood levels in creased rapidly within the first 30 minutes. The time (tmax) was 90 minutes with a C max of 0.3 µg/ml. Thereafter, the hirudin blood levels decreased slowly, with an apparent elimination half-life of 5.75 hours. A plasma level of 0.15 µg/ml was found after 8 hours. During this time, about 80% of the administered dose had been excreted via the kidneys.
TABLE 3. Pharmacokinetic Data of Recombinant Hirudin in Different Animal Species after a Single Intravenous Bolus Injection Rat (mg/kg) 1.0
2.0
Rabbit (mg/kg) 1.0
Dog (mg/kg) 0.5
A (µg/ml)
2.45
10.46
12.55
2.5
B (µg/ml)
0.82
1.50
1.43
0.30
α (h - 1 )
4.75
8.25
6.52
2.77
β (h -1 )
0.65
0.59
0.60
0.57
t½α (h)
0.15
0.08
0.11
0.25
t½β
1.07
1.18
1.16
1.22
k12 (h - 1 )
1.93
4.15
2.67
0.57
k21 (h - 1 )
1.68
1.55
1.21
0.81
k10 (h )
1.8
3.14
3.24
1.96
AUC (µg/ml X h)
1.64
3.81
4.31
1.54
Vc(l/kg)
0.31
0.17
0.07
0.18
Vdss(l/kg)
0.66
0.63
0.23
C1 tot (ml/min)
3.05
2.09
12.38
(h)
-1
0.28 183
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SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 17, NO. 2, 1991
The administration of r-hirudin to nephrectomized dogs was followed by a higher hirudin blood level than found in animals with normal renal functions. These experiments confirmed that after 1 hour, 75 to 80% and after 120 minutes about 85% of the applied dose were distributed into the extravascular compartment. Then the blood level remained nearly constant, which speaks in favor of the exclusive renal elimination of r-hirudin.13
Primates Similar pharmacokinetic data of r-hirudin and com parable elimination half-lives were also found in primates.17 All pharmacokinetic studies with hirudin confirmed that it is distributed in the extracellular space of the organism. Hence, besides the rapid renal elimina tion, primarily distribution phenomena are responsible for the decrease in blood levels after an intravenous administration.
Special Studies In special studies the placental transfer of r-hirudin was examined in pregnant rabbits on the 27th day of gestation.16 r-Hirudin (0.5 mg/kg) was administered into the marginal ear vein 20, 15, and 2 hours before the dissection. The concentration of r-hirudin found in maternal blood plasma was about 60 times higher than in the fetal circulation, which was confirmed by heart puncture. The r-hirudin content in amniotic fluid amounted to 0.015%. Therefore there is only a very slight placental transfer of r-hirudin to the fetal organism. After intratracheal instillation of r-hirudin, the ab sorption amounted to about 30%. The plasma level may be described by the equation C P = 0.2 x e-0.21 x t-0.21 x e-1.2 x t. Accordingly, the elimination half-life of about 200 minutes was still longer than that after subcutaneous injection. After rectal administration, no marked absorption of r-hirudin could be observed. To influence the pharmacokinetic behavior, a mod ified hirudin bound to a high molecular weight vehicle was used. 18,19 Dextran, which has a molecular weight of 70,000 d, was solely distributed in blood plasma. At a dose of dextran-r-hirudin of 1000 ATU/kg, a signifi cantly higher plasma level was obtained than with r-hirudin alone, whereas unbound r-hirudin administered at the same dose fell below the detection limit after 1 hour. The elimination half-life of dextran-hirudin amounted to about 7 hours. The distribution volume of 9.7% of body mass proved to be only somewhat higher than the blood volume.19 Studies with dextran-bound r-hirudin in rats and rabbits have shown that dextran 40-hirudin had an elimination half-life of about 6.5 hours. With dextran 70-hirudin that time was prolonged
to 7.5 hours.19 This confirmed that dextran-bound r-hirudin disposes of the same inhibitory activity as unbound r-hirudin. Thus, similar covalently bonded preparations of hirudin may be of interest for clinical use.
Human Volunteers The pharmacokinetics of r-hirudin were studied in healthy volunteers after single intravenous, subcutane ous, and intramuscular doses of 0.1 mg/kg.20 The pharmacokinetic data obtained after an intravenous bolus injection are summarized in Table 4. The intravenous route of administration of r-hirudin in man revealed distribution and excretion characteristics similar to those of native hirudin. After a subcutaneous injection of 0.1 mg/kg r-hirudin the tmax amounted to 120 minutes, while C max was 0.15 µg/ml. Afterward, the hirudin blood levels decreased slowly. During this phase, an apparent elim ination half-life of approximately 180 minutes was found. Nearly similar adsorption and elimination values have been recorded after the intramuscular administra tion of 0.1 mg/kg. All three routes of administration were followed by the excretion of r-hirudin in the urine in active form (35 to 40% within 24 hours). A delayed excretion of r-hirudin into the urine could be demon strated after subcutaneous or intramuscular administra tion because of the delayed adsorption into the blood. Another study on the kinetics of r-hirudin in man showed that after subcutaneous administration of 0.1 to 0.4 mg/kg r-hirudin, the maximum plasma levels were 0.45 µg/ml at a mean tmax of 120 minutes.21 In these studies, an elimination half-life of 105 and an absorption
TABLE 4. Pharmacokinetics of Recombinant Hirudin in Human Volunteers After a Single Intravenous Dose of 0.1 mg/kg A (µg/ml)
1.0
B (µg/ml)
0.25
α (h - 1 )
4.62
-1
β (h )
0.75
t½α (h)
0.15
t½β (h)
1.91
k12 (h - 1 )
1.93
k21 (h-1)
1.68
-1
k10 (h )
1.8
AUC (µg/ml X h)
0.54
Vc(l/kg)
4.4
V dss (1/kg) C1 tot (ml/min)
8.9 168
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half-life of 55 minutes were determined. The apparent distribution volume Vdss of 31.9 1 was markedly increased, compared with findings described previously.21 The Cltot value of 208 ml/min corresponded relatively well with the data determined by us before.20 The quantity of active hirudin excreted in the urine amounted to 35% of the dose administered. This corroborates previous findings.21
CONCLUSION Pharmacokinetic studies with hirudin confirmed its distribution into the extracellular space of the body. After intravenous administration, the elimination half-life was about 1 hour; an extravascular route of administration prolonged this time to 2 to 3 hours. Hirudin was exclusively eliminated via the kidneys and it was partly recovered in unchanged form in the urine. Marked species differences were found in the renal excretion. Only a slight percentage of active hirudin was excreted by rats, whereas nearly complete excretion of active hirudin was found in dogs. After the administration of hirudin to man (0.1 mg/kg) only about one third of the dose was excreted in the urine. The enteral resorption of hirudin remained low and the placental transfer was small in experimental animals.
REFERENCES 1. Markwardt F: Die antagonistische Wirkung des Hirudins gegen Thrombin in vivo. Naturwisswnschaften 43:111, 1956. 2. Markwardt F: Development of hirudin as an antithrombotic agent. Semin Thromb Hemost 15:269-282, 1989. 3. Walenga JM, R Pifarre, DA Hoppensteadt, J Fareed: Development of recombinant hirudin as a therapeutic anticoagulant and antithrombotic agent: Some objective considerations. Semin Thromb Hemost 15:316-333, 1989. 4. Talbot M: Biology of recombinant hirudin (CGP 39393): A new prospect in the treatment of thrombosis. Semin Thromb Hemost 15:293-301, 1989. 5. Spinner S, G Stöffler, E Fink: Quantitative enzyme-linked immunosorbent assay (ELISA) for hirudin. J Immunol Methods 87:79— 93, 1986.
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6. Richter M, P Walsmann, U Cyranka, F Markwardt: 125I-Markierung von Hirudin. Pharmazie 41:510, 1986. 7. Markwardt F, G Nowak, J Hoffmann: The influence of drugs on disseminated intravascular coagulation (DIC). Effects of naturally occurring and synthetic thrombin inhibitors. Thromb Res 11:275283, 1977. 8. Markwardt F, J Hauptmann, G Nowak, C Klessen, P Walsmann: Pharmacological studies on the antithrombotic action of hirudin in experimental animals. Thromb Haemost 47:226-229, 1982. 9. Richter M, U Cyranka, G Nowak, P Walsmann: Pharmacokinetics of 125-I hirudin in rats and dogs. Folia Haematol (Leipz) 115:6469, 1988. 10. Markwardt F, G Nowak, U Stürzebecher, P Walsmann: Studies on the pharmacokinetics of hirudin. Biomed Biochim Acta 46:237244, 1987. 11. Markwardt F, G Nowak, J Stürzebecher, U Grieβpbach, P Walsmann, P Vogel: Pharmacokinetics and anticoagulant effect of hirudin in man. Thromb Haemost 52:160-163, 1984. 12. Bichler J, B Fichtl, M Siebeck, H Fritz: Pharmacokinetics and pharmacodynamics of hirudin in man after single subcutaneous and intravenous bolus administration. Arzneimittelforsch 38:704-710, 1988. 13. Nowak G, F Markwardt, E Fink: Pharmacokinetic studies with recombinant hirudin in dogs. Folia Haematol (Leipz) 115:70-74, 1988. 14. Kaiser B, A Simon, F Markwardt: Antithrombotic effects of recombinant hirudin in experimental angioplasty and intravascular thrombolysis. Thromb Haemost 63:44-47, 1990. 15. Markwardt F, B Kaiser, G Nowak: Studies on antithrombotic effects of recombinant hirudin. Thromb Res 54:377-388, 1989. 16. Markwardt F, G Fink, B Kaiser, H.-P Klöcking, G Nowak, M Richter, J Stürzebecher: Pharmacological survey of recombinant hirudin. Pharmazie 43:202-207, 1988. 17. Markwardt F, XQ Huán, JM Walenga, M Muench, D Hoppensteadt: Pharmacokinetics of recombinant hirudin in primates. (Abst). Fed Proc 1:484, 1988. 18. Richter M, P Walsmann, F Markwardt: Plasma level of dextranr-hirudin. Pharmazie 44:73-74, 1989. 19. Markwardt F, M Richter, P Walsmann, G Riesener, M Paintz: Preparation of dextran-bound recombinant hirudin and its pharmacokinetic behaviour. Biochim, Biophys Acta 49:1103-1108, 1990. 20. Markwardt F, G Nowak, J Stürzebecher, G Vogel: Clinicopharmacological studies with recombinant hirudin. Thromb Res 52:393-400, 1988. 21. Bichler J, M Siebeck, M Spannagl, P Close, H Fritz, W Schramm: Pharmakokinetische und gerinnungshemmende Eigenschaften von rekombinantem Hirudin (CGP 39 393) nach zweimaliger Gabe an gesunden Probanden. In: 6. Kongreβ der Gesellschaft für Thrombose- und Hämostaseforschung. Kiel, 21.-24.2.1990. Hauptvorträge, Ausgewählte freie Vorträge. Schattauer, Stuttgart, 1990, p 190.
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