In vitro study of r-hirudin permeability through membranes of different haemodialysers

Elke Bucha, Reiner Kreml and Goetz Nowak

Max-Planck-Gesellschaft, Research Unit Pharmacological Haemostaseology, Friedrich-Schiller-University, Jena, Germany

Correspondence and offprint requests to: E. Bucha, MPG-AG `Pharmakologische Hömostaseologie', POB, D-07740 Jena, Germany.



   Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. After introducing the specific thrombin inhibitor recombinant hirudin (r-hirudin) into clinical practice in cases of heparin-induced thrombocytopenia (HIT, type II) the possibility of its use as an anticoagulant during haemodialysis treatment in HIT II patients is being discussed more frequently. On the one hand, the efficient, safe and routine use of r-hirudin during haemodialyses, including the maintenance of a therapeutic blood level, presupposes that no r-hirudin will leave the circulation by passing through the dialyser membrane. On the other hand, it is important to have dialysers whose permeability to r-hirudin allows its efficient removal from the human body because, to date, no antidote is commercially available in cases of dangerously high blood concentrations of r-hirudin.

Methods. An in vitro circulation model was used to study the r-hirudin permeability of some low- and high-flux dialysers. As r-hirudin-containing vehicles, both albumin-containing saline solution and bovine blood were circulated in the blood space of the system for 2 h. Transmembrane r-hirudin passage was tested by measuring r-hirudin concentration both in the blood and dialysate space fluids using the ecarin clotting time (ECT).

Results. Low-flux dialysers with membranes made from polysulfone or regenerated cellulose proved to be almost impermeable to r-hirudin. In contrast, other low-flux membranes were partly permeable to r-hirudin (e.g. Hemophan) or even almost completely permeable (e.g. cellulose acetate). All high-flux dialysers tested were permeable to r-hirudin.

Conclusions. Only low-flux dialysers with polysulfone or regenerated cellulose membranes proved to be suitable for r-hirudin use in routine haemodialysis therapy. Other low-flux, and all high-flux, capillaries are permeable to r-hirudin and offer the possibility of lowering toxic r-hirudin concentrations after overdosing.

Keywords: haemodialysis; membrane permeability; r-hirudin



   Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recombinant hirudin (r-hirudin), a tight binding, specific thrombin inhibitor became available in 1997 for clinical use in cases of heparin-induced thrombocytopenia (HIT, type II/HITT). r-Hirudin is a single chain miniprotein composed of 65 amino acids (molecular mass: ~7 kDa). It inhibits thrombin directly, without the need for any cofactors and without the existence of endogenous antagonists [1]. Previously, r-hirudin had shown its anticoagulant efficacy in cases of different cardiological indications, including deep venous thrombosis, as well as its applicability during haemodialysis [210].

Owing to the missing cross-reactivity between r-hirudin and heparins or heparinoids it has, to date, been the only alternative for the immediate treatment of patients with HIT/HITT [11].

Clinical evidence of HIT/HITT has also been described in patients undergoing regular haemodialysis therapy and requiring an efficient anticoagulation for this treatment [12,13].

To use r-hirudin as an anticoagulant during haemodialysis in HIT II patients some aspects have to be taken into consideration. Owing to the possibility of elimination of r-hirudin only via the kidneys, its retention time may be prolonged considerably depending on the extent of renal dysfunction [1416]. Therefore, the dose has to be adjusted to the individual plasma concentration of r-hirudin. This is now possible using the ecarin clotting time (ECT) [17]. Clinical studies have shown therapeutic concentrations of r-hirudin during haemodialysis ranging between 400 and 1000 ng/ml in blood and between 600 and 1500 ng/ml in plasma [9,10].

For the efficient, safe and routine use of r-hirudin in haemodialysis it is essential that no r-hirudin leaves the circulation through the dialyser membrane, in order to not disturb the correlation between residual renal clearance function, r-hirudin dosage and r-hirudin blood concentration. This was first described for polysulfone low-flux dialysers used in experimental haemodialyses in dogs [18]. However, the early phase of the first long-term r-hirudin treatment of a haemodialysis patient with HIT demonstrated that a low-flux dialyser partly permeable to r-hirudin makes r-hirudin anticoagulated haemodialyses more difficult even with an increased dosage [10].

However, dialysers that allow r-hirudin to pass through their membranes are also of interest. Because no antidote for r-hirudin has been commercially available to date, this would be the only possibility of providing efficient detoxication in a case of excessively increased blood levels associated with bleeding events.

Based on these two important facts connected with the clinical use of r-hirudin, the permeability of different commercially available low- and high-flux dialysers was studied in an in vitro circulation system using albumin-containing saline solution or bovine blood as r-hirudin-containing vehicles.



   Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The following materials were used in the study:

Dialyser: A-2008C, Fresenius AG, Bad Homburg, Germany

Bloodline tubing system: FV 153 PAED.S/FA 126 PAED.S, Fresenius AG (shortened version)

Dialysers: See Table 1Go(all dialysers used in the study were kindly provided by the manufacturers)


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Table 1. Dialysers tested with regard to their r-hirudin permeability
 
Dialysate: SK-F 203 acid bicarbonate hemodialysis concentrate (mixed with water 1:27.6) and BC-F 8.4 alkaline bicarbonate hemodialysis concentrate (1:34), Fresenius AG

r-Hirudin: HBW 023, Behring Werke AG, Marburg, Germany (specific activity: 13647 ATU/mg)

r-Hirudin-containing vehicles: Saline (0.9%) containing 1% bovine serum albumin (NaCl–BSA solution) and r-hirudin anticoagulated bovine blood

Blood donor: Variegated mountain cattle, conventional stable-keeping (Federal Institute of Veterinary Medicine, Jena, Germany).

Determination of r-hirudin concentration using ecarin clotting time
Blood samples were centrifuged at 1000 g for 15 min and 0.2 ml of the gained plasma was then mixed with 0.08 ml Tris buffer (pH 7.5) and pre-incubated for 2 min at 37°C. Following addition of 0.02 ml ecarin start reagent (10 EU ecarin dissolved in 1 ml 0.9% NaCl solution containing 50 mmol/l CaCl2), the ecarin clotting time (ECT) was measured using a CL-4 coagulometer (Behnk Elektronik, Norderstedt, Germany). The concentration of r-hirudin was determined using a standard curve. To determine the r-hirudin concentration in NaCl–BSA solution and in the dialysate, the samples were added to the reaction mixture as part of the buffer addition, using a standard pooled plasma.

Test design
After the dialyser had been prepared according to clinical guidelines, the filled dialysate space was closed to allow only the substance, and not the fluid, to permeate through the membrane. Afterwards both albumin-containing saline solution and bovine blood (300 ml) were circulated as r-hirudin-containing vehicles in the blood space of the system for 2 h at a flow rate of 200 ml/min. The blood had freshly been drawn via V. jugularis from the donor cattle.

In all experiments 9–15 mg r-hirudin (30–50 µg/ml) were used which were comparable with r-hirudin doses in clinical use in haemodialysis and would guarantee effective anticoagulation of the blood during the experiments even after a possible loss of r-hirudin through the membranes.

To determine the time course of the r-hirudin concentration, samples were taken immediately before and throughout the experiments. At the end of the experiments the r-hirudin concentration was also measured in the dialysate fluid. Therefore, the relationship between the concentration in the dialysate and blood space at the end of circulation could be used as a measure of r-hirudin permeation through the membranes; the quotient of the values was termed the `permeability factor' (PF). In addition, a quantitative r-hirudin balance was calculated by comparing the absolute r-hirudin content in the used initial solution with the amount of r-hirudin found in the blood and dialysate fluid at the end of the experiments. Each of the dialysers was tested twice making statistical calculations unnecessary.



   Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The dialysers studied were classified according to their permeability to r-hirudin, represented as the quotient of the dialysate and blood space r-hirudin concentrations measured at the end of the experiments (Table 2Go). Only low-flux dialysers with membranes made from polysulfone or regenerated cellulose (including Cuprophan) proved to be almost impermeable to r-hirudin. Almost no r-hirudin could be determined in the dialysate fluid (PF: 0–0.03). In contrast, other low-flux dialysers, such as Hemophan, were partly permeable (PF: 0.59/0.76) or, as measured for cellulose acetate, almost completely permeable (0.88/0.82). In all the low-flux dialysers tested no correlation was found to exist between r-hirudin permeability and ultrafiltration coefficient.


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Table 2. Diffusion of r-hirudin through different haemodialysers and loss of r-hirudin during 2-h circulation in an in vitro circulation system (without dialysate flow)
 
All the high-flux dialysers tested allowed r-hirudin to pass through their membranes.

Comparing the circulation tests using NaCl–BSA solution and bovine blood as r-hirudin-containing vehicles no qualitative differences were revealed.

Any loss of r-hirudin in the dialysers studied was ascertained using the absolute amounts of r-hirudin determined before and at the end of the 2-h circulation (Table 2Go). The smallest losses were observed in dialysers with cellulose acetate-N and AM-Bio 65 membranes, whereas the most distinct differences were found in experiments using cellulose acetate, Hemophan and cellulose diacetate dialysers. In circulating r-hirudinized blood, dialysers with polyamide, Gambrane and AN-69HF membranes also showed r-hirudin losses >3.0 mg/m2.



   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effective anticoagulation is an essential prerequisite of haemodialysis therapy in patients with renal insufficiency. Therefore, a diagnosis of HIT II requires the immediate exchange of heparin for another sufficient anticoagulant, which must not show any cross-reaction to the antibodies. Up until now, the only alternative to heparin in such cases has been r-hirudin, which is now available for clinical use. Its application in patients to be haemodialysed requires consideration of its pharmacokinetic behaviour, in particular, the markedly prolonged half-life if the exclusive, renal elimination pathway is impaired. In earlier studies, a strong correlation between residual renal clearance function, r-hirudin elimination and effective r-hirudin dosage in patients with impaired renal function was observed [9,17]. This correlation is disturbed when some r-hirudin leaves the circulation via the dialyser membrane during haemodialysis treatments in an unknown, membrane-dependent manner. This was observed in a HIT patient during three r-hirudin anticoagulated haemodialyses using Hemophan low-flux dialysers [10]. All other previously performed r-hirudin anticoagulated haemodialyses in dogs or humans used only low-flux dialysers of polysulfone or regenerated cellulose membranes [69,18]. Comparison of the time courses for r-hirudin concentration in the blood showed a markedly stronger decrease when using Hemophan low-flux dialysers than when using polysulfone or regenerated cellulose membrane low-flux dialysers.

Actually, the in vitro circulation experiments demonstrate that only the latter membrane types are impermeable to r-hirudin. Membranes of all the other low-flux dialysers tested were permeable to r-hirudin to different degrees. Therefore, even in a case of the application of r-hirudin as an infusion no uniform dose schedule for all types of dialyser is possible. Knowledge of the ultrafiltration coefficient of the dialysers does not allow any statement about their permeability to r-hirudin to be made because no correlation was found.

The membranes of all the high-flux dialysers studied were permeable to r-hirudin. In experimental animal haemodialyses performed in beagles, it had already been demonstrated that by using high-flux dialysers consisting of polysulfone or polyacrylonitrile membranes r-hirudin could be removed from the body [18]. Haemofiltration using polysulfone membranes also proved successful in the rapid and effective elimination of r-hirudin from the circulation [19]. Following r-hirudin administration >80% of the total r-hirudin content of the body is located in the extravasal space. Therefore, this r-hirudin also has to be taken into account when lowering the r-hirudin concentration of the blood using high-flux dialysers, because there is a concentration equilibrium between the extra-and intravasal spaces. In cases of subtherapeutic blood concentrations of r-hirudin its reduction by high-flux haemodialysis is minimal, caused by the very low concentration difference between blood and dialysate. Therefore, it will be almost compensated for by the movement of r-hirudin from the extravasal fluid into the blood. This may explain the lack of an efficient r-hirudin reduction in patients with plasma concentrations ranging around or below 200 ng/ml as described by Vanholder et al. [16].

In the in vitro study described, a loss of r-hirudin was found when comparing the amounts determined before and after the experiments. This can be explained mainly by an unspecific adsorption of r-hirudin to the membrane surface. Interpreting the r-hirudin balance of the test using bovine blood, one should take into account that plasma proteins may have influenced the adsorptive processes unspecifically (r-hirudin does not undergo a plasma protein binding), or that part of the r-hirudin may have been consumed by preventing coagulation-activating processes at the surface of the dialyser membranes. The described in vitro investigations concerning the permeability to r-hirudin of some commercially available dialysers, revealed that r-hirudin shows a special permeability behaviour caused by its molecular structure (nearly 30% of the amino acids have acid side chains). To use r-hirudin as an anticoagulant during haemodialyses in patients suffering from HIT II only low-flux dialysers with membranes made from polysulfone or regenerated cellulose are of high advantage concerning r-hirudin dosage, because the different r-hirudin permeability of the other dialysers has not been taken into account. High-flux dialysers seem to be suitable as an antidote to excessive r-hirudin blood concentrations after overdosage.



   References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 31.12.98
Accepted in revised form: 7. 7.99