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The in vitro anticoagulant effects of Danaparoid, Fondaparinux, and Lepirudin in children compared to adults
Thrombosis Research (2008) 122, 709–714
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REGULAR ARTICLE
The in vitro anticoagulant effects of Danaparoid, Fondaparinux, and Lepirudin in children compared to adults Vera Ignjatovic a,b,c,⁎, Robyn Summerhayes a,c , Yan Yan Yip b , Paul Monagle a,b a b c
Department of Clinical Haematology, Royal Children's Hospital, Melbourne, Australia Department of Pathology, University of Melbourne, Australia Murdoch Children's Research Institute, Melbourne, Australia
Received 25 June 2007; received in revised form 8 November 2007; accepted 4 February 2008 Available online 1 April 2008
KEYWORDS Children; Danaparoid; Fondaparinux; Lepirudin
Abstract Introduction: Major physiological differences in the coagulation system of children compared to that of adults are well documented. We have previously investigated the age-related differences in response to Unfractionated Heparin (UFH). However, the impact of developmental haemostasis on more recent anticoagulant drugs is unknown. A number of these drugs are approved for use in specific indications in adults and none are approved for use in children. This study aimed to determine whether age-related differences in effect and impact on monitoring tests exist in vitro for danaparoid, fondaparinux and lepirudin. Materials and Methods: Plasma samples were obtained from healthy children and pooled into age-specific pools, in order to obtain sufficient quantity of plasma required for the analysis of the three drugs. Each age-specific pool was spiked with different concentrations of danaparoid, fondaparinux and lepirudin and response was measured using standard techniques. All experiments were repeated using three separate plasma pools. The effect of each drug in children's plasma was compared to the effect in the respective adult plasma pool. Results: Age-related differences in effect on thrombin potential and monitoring tests were observed only with the drug lepirudin. Specifically, APTT for children up to 5 years of age was increased compared to adults; all children had lower ECT results
Abbreviations: APTT, Activated Partial Thromboplastin Time; ECT, Ecarin Clotting Time; ETP, Endogenous Thrombin Potential; UFH, Unfractionated Heparin. ⁎ Corresponding author. Department of Clinical Haematology, Royal Children's Hospital, Parkville, Victoria, 3010, Australia. Tel.: +61 3 8344 3750; fax: +61 3 8344 4004. E-mail address: [email protected] (V. Ignjatovic). 0049-3848/$ - see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2008.02.001
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V. Ignjatovic et al. compared to adults; children up to 10 years of age had increased inhibition of ETP as compared to adults. Conclusions: This study confirms age-related differences in response to anticoagulants with predominant anti-IIa effect and highlights the need for further research into this area. © 2008 Elsevier Ltd. All rights reserved.
Introduction Thromboembolic disease remains a major health problem in our society. Historically, therapies for thromboembolic disease, including heparin and warfarin, are commonly complicated by bleeding. Since bleeding and clotting are at opposite ends of the physiological spectrum, the search for an ideal anticoagulant that inhibits clotting, while not increasing clinical bleeding is ongoing. The general increase in the understanding of the coagulation system has led to a rapid increase in the development of new anticoagulants. Many of these new drugs have specific target proteins within the coagulation system and a number of these drugs, such as danaparoid, fondaparinux and lepirudin are approved for use in specific indications in adults. However, these anticoagulants are not approved for use in children. Danaparoid is a low molecular weight heparinoid which acts as a Factor Xa (FXa) and Factor IIa (FIIa) inhibitor in a ratio of 28:1 [1]. Lepirudin is a recombinant, desulfated form of hirudin; a direct thrombin inhibitor that binds to both fibrin-bound, as well as fluid-phase thrombin [2]. Fondaparinux is synthetic analogue of the pentasaccharide sequence required for the binding of Heparin to Antithrombin (AT) and as such is an AT-dependent specific FXa inhibitor. Danaparoid and lepirudin have been used for the treatment of Heparin Induced Thrombocytopenia (HIT) in children [3–8]. However, there are no studies comparing the age-related effect of these drugs. Hirudin has been studied in cord plasma, with outcomes suggesting that dosing strategies for neonates should not be derived from studies performed in adults [9]. This is due to the fact that higher doses of the drug were required to achieve the same level of anticoagulation as compared to adults. To date there is no published data on fondaparinux in the paediatric setting. Paralleling the development of new anticoagulants is the expansion in the understanding of Developmental Haemostasis. This concept was established by Andrew et al. and confirms fundamental differences in the haemostatic system of neonates and children compared to adults [10–12]. Differences in weight-adjusted dosing for Unfractionated
Heparin (UFH), warfarin and Low Molecular Weight Heparin (LMWH), have been well documented. More importantly, we have recently shown, first in vitro and then ex vivo, the impact of Developmental Haemostasis on age-related effect and monitoring of UFH therapy in children [13,14]. This study was designed to determine whether age-related differences in effect and impact on monitoring tests exist in vitro for danaparoid, lepirudin and fondaparinux, as an initial step prior to studying these drugs in vivo. Materials and Methods Plasma samples were obtained from healthy children and adults, without previous thromboembolic events and not receiving any form of anticoagulant therapy. Paediatric samples were collected from healthy children about to undergo minor day surgery, while the adults were healthy volunteers. Informed consent was obtained from the parents of children and from the adult participants themselves. This study was approved by the Royal Children's Hospital Ethics in Human Research Committee (EHRC #24124B). Blood samples were collected in tubes containing 0.105 mol/L (i.e., 3.2%) trisodium citrate anticoagulant in a ratio of 9 volumes of whole blood to 1 volume anticoagulant. Citrated samples were centrifuged at 3000 rpm for 10 minutes at 10°C (Megafuge 1.0R, Heraeus) and platelet-poor plasma was frozen at -70°C for batchtesting. Plasma samples were pooled into the following age-related pools: b1 years old, 1-5 years old, 6-10 years old, 11-16 years old and Adults (N18 years old). Each age-specific plasma pool consisted of a minimum of fifteen individual donors and was not significantly different between the different pools. Three separate groups of donors were used to create three separate plasma pools for each age-group. Each age-specific plasma pool was divided into sub-pools and spiked with different concentrations of: danaparoid (0.3, 0.5, 0.7, 1.0 and 1.5 anti-FXa U/ml plasma), lepirudin (0.375, 0.75, 1.5, 2.25 and 3.0 µg/ml plasma) and fondaparinux (0.5, 1.0, 1.5, 2.0 and 4.0 µg/ml plasma). Unspiked, age-specific plasma pools were used as controls. Danaparoid (Orgaran), lepirudin (Refludan) and fondaparinux (Arixtra) were obtained from Organon, Australia; Pharmion, Australia and Sanofi-Synthelabo, Australia, respectively. The Activated Partial Thromboplastin Time (APTT) was measured using a commercially available STA® PTT-A reagent. The Ecarin Clotting Time (ECT) was determined using a commercially available Ecarin reagent, in combination with the STA® analyser (Diagnostica STAGO, France). The maximum measurement times for the APTT and ECT assays was modified allowing for measurement of results up to 600 sec. This is the upper limit of time measurement possible on the STA® analyser.
Age-related in vitro effect of novel anticoagulants
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Colorimetric assay for Lepirudin was performed using the combination of the STA® analyser (Diagnostica STAGO, France) and the Chromogenic Hirudin Test (Chromogenix, Helena Laboratories, Australia). Anti-Xa assays were performed using: COAMATIC® and COATEST® chromogenic anti-Xa test kits (Chromogenix, Helena Laboratories, Australia), and analysed using the fully automated STA® coagulation analyser (Diagnostica STAGO, France). These two anti-Xa assays measure plasma concentration of anticoagulants; however there are major differences between these methods relating to the presence/absence of exogenous AT, as well as presence/absence of additional Dextran Sulphate (DS). The COAMATIC® assay does not contain exogenous AT, while it does use additional DS. On the other hand, the COATEST® assay uses exogenous AT, while it does not use any additional DS. Endogenous Thrombin Potential (ETP) was quantitated using a non-automated microplate method, based on a modification of a previously described technique.12 Plasma samples were defibrinated by incubation with Reptilase for 30 minutes at 37 oC. The sample was then incubated for a further 10 minutes on ice, with any clot formed being wound out using a wire loop. 50 µl of defibrinated plasma was then mixed with 25 µl of APTT reagent (Diagnostica Stago) at 37 °C for 3 minutes before 25 µl of 0.04 M CaCl2 was added. At 15, 30, 45, 60, 75, 90, 120 and 180 seconds following addition of CaCl2, 10 µl of this reaction mixture was taken out and mixed with 190 µl of 0.005 M Na2 EDTA on ice, to stop thrombin generation. 10 µl of each time-sample EDTA solution was then taken out and mixed with 10 µl of 0.00016 M chromogenic substrate S-2238 (Chromogenix, Italy) in buffer. Following 10 minute incubation, 40 µl of 50% (v/v) acetic acid was added to each sample to stop any further coagulation from taking place. The amount of thrombin generated was quantitated by measuring absorbance of the reaction mixture at 405 nm, and comparing it to the absorbance produced by reaction of S-2238 with known amount of thrombin. Standard curve was prepared using dilutions of human alpha thrombin (Enzyme Research Laboratories, Lot HT 2344PAL). Since thrombin-bound to α2macroglobulin retains activity against small substrates 13, formation of thrombin-α2macroglobulin complexes was quantitated by a previously described method (thrombin generations carried out as above, except that thrombin not bound to α2macroglobulin was neutralised with antithrombin and heparin prior to addition of S-2238). This was subtracted from the total thrombin activity to determine the free thrombin activity.12 Amidolytic activities were plotted against time to produce thrombin generation curves for all plasma pools. The ability of plasma to generate thrombin is expressed as TP, corresponding to the area under the thrombin generation curve, calculated using the trapezoidal rule.
Statistical analysis The results are expressed as the mean and standard deviation (SD) of the three different experimental runs performed. Differences between each age-group were compared to that observed in adults, using one-way analysis of variance, with subsequent twosided, unpaired t tests performed if significance was observed. A result of p b 0.05 was considered statistically significant. Statistical software package STATA, Release 8 0 (Stata Corporation, College station, TX) was used for data processing and analysis.
Results The results of this study are summarised in Figs. 1–3, which represent danaparoid, lepirudin and fondaparinux, respectively.
Figure 1 Age-related response to danaparoid. Results shown are APTT, Coamatic Anti-Xa, Coatest Anti-Xa and ETP in panels A, B, C and D, respectively. For each age, points on the curve represent the mean value for the three plasma pools.
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V. Ignjatovic et al. Danaparoid APTT results in all samples spiked with danaparoid showed a linear increase in relation to increasing danaparoid concentration. (Fig. 1A) In addition, the APTTresults for adult plasma were significantly lower compared to children's plasma, for the same concentration of the drug, across all concentrations tested (p range b 0.0001 to 0.0405). The anti-Xa response, as measured by both the Coamatic and Coatest assays (Fig. 1B and C, respectively) to danaparoid also increased in a dosedependent manner. In this case, age-related differences were not observed. Fig. 1D shows that the ETP decreased significantly with increasing concentration of danaparoid, for each individual age-group. However, there were no agerelated differences in the ETP response to this drug.
Figure 2 Age-related response to lepirudin. Results shown are APTT, ECT, ETP and Chromogenic assay for lepirudin in panels A, B, C and D, respectively. For each age, points on the curve represent the mean value for the three plasma pools.
Figure 3 Age-related response to fondaparinux. Results shown are Coamatic Anti-Xa, Coatest Anti-Xa and ETP in panels A, B and C, respectively. For each age, points on the curve represent the mean value for the three plasma pools.
Age-related in vitro effect of novel anticoagulants Lepirudin Fig. 2A shows the linear increase in APTT in response to lepirudin. This response was found to vary significantly with age. Specifically, the APTT for the b 1 year and 1-5 year old age-groups was found to be increased compared to adults (p range 0.0007 to 0.0424). The ECT increased with increasing concentrations of lepirudin (Fig. 2B). In addition, there were age-related differences in response, with children having significantly lower ECT results compared to adults (p range b 0.0001 to 0.0490). Chromogenic assay for lepirudin showed good correlation between the spiked and measured concentration of the drug (Fig. 2C). The results did not vary for the different plasma pools tested, at various concentrations of lepirudin. The ETP response to lepirudin is shown in Fig. 2D. Increasing concentrations of lepirudin resulted in a decrease in ETP across all age-groups. In addition, for the b 1, 1-5 and 6-10 year olds, this inhibition was significantly increased compared to the age-specific baseline, for the same lepirudin concentration as compared to older children and adults, across all concentrations tested. (p range 0.001 to 0.0470)
Fondaparinux Fig. 3A and B show the anti-Xa response to fondaparinux, respectively. Overall, there were no age-related differences in the anti-Xa response to this drug. Fig. 3C summarizes the ETP response to fondaparinux. For all age-groups tested, the inhibition of thrombin correlated with increasing concentrations of fondaparinux up to 2 µg/ml. However, there was no additional ETP decrease with higher concentrations of fondaparinux. In addition, there were no age-related differences in terms of the ETP response to this drug.
Discussion The coagulation system changes significantly with age, a phenomenon known as Developmental Haemostasis. This is the first study to consider age-related differences in effect and impact on routine monitoring tests for a number of novel anticoagulants. Our results confirm that in vitro, the effect of danaparoid and fondaparinux, two drugs with predominant anti-Xa activity, on total thrombin potential appear to be minimally affected by Developmental Haemostasis, despite some differences in results of non- specific monitoring tests. On the other hand, lepirudin, which has pure anti-IIa activity showed significant age-related differences in effect on thrombin potential and monitoring tests. We have previously shown in vitro and in vivo that the pharmacodynamics of UFH changes with age and that this is reflected in the results of UFH monitoring tests [13,14]. The results of the current study are consistent with the age-related differences in response to UFH, which has a combination of anti-Xa and anti-IIa activity, [13,14] and suggest that the major source
713 of age-related differences relates to interactions between anticoagulants and thrombin (factor IIa) rather than factor Xa. This hypothesis will need to be confirmed with further in vivo studies. An age-related effect of danaparoid was only observed with the APTT, with both the anti-Xa and ETP comparable between adults and children, for the same dose of the drug. The APTT may be sensitive to small amounts of anti-IIa activity that are not reflected in the measurement of the global thrombin potential (ETP). The fact that the measured anti-Xa reflected the “spiked” anti-Xa in each age-group suggests that in these plasma samples from healthy children there is no age-related difference in binding of danaparoid to other plasma proteins. However, ex vivo studies in sick children are required to confirm this before clinical dosing recommendations could be confirmed. Lepirudin is usually monitored quantitatively using the APTT and functionally using the Activated Clotting Time (ACT) or ECT [15]. For both of these tests, we observed age-related differences in response, for the same dose of the drug. Further, the impact on ETP was significantly increased in younger children, for the same dose of lepirudin. This suggests that the response to lepirudin in children is different to adults and that the safety and effectiveness data from adults cannot be extrapolated to children. A previous study reported that increased doses of hirudin were required to achieve the same level of anticoagulation in cord plasma compared to adult plasma, the opposite to our findings (18). However, cord plasma is known to be a different system to plasma from children. In terms of fondaparinux, the results of this study indicate that in vitro there are no age-related differences in response to this drug. Similarly as for danaparoid, this is probably related to the fact that this drug functions by inhibiting FXa, and that the critical age-related factor appears to be Thrombin (IIa). In three separate experiments, using three entirely separate plasma pools for each age-group, we observed consistent and reproducible age-related differences in response to lepirudin compared to adults. These findings need to be confirmed using ex vivo samples, as in vitro spiking of plasma with anticoagulants allows for a limited interpretation of the results. Specifically, in vitro testing does not account for possible plasma binding and endothelial interactions, such as release of TFPI. In conclusion, our study confirms the age-related differences in response to lepirudin, an anticoagulant with predominant anti-IIa effect and reemphasizes the need for further research into this area. Specifically, our results emphasize the need
714 for ex vivo studies to be performed. In the future separate pharmacokinetic and efficacy studies should also be performed in the clinical setting in children, especially when drugs with similar mode of action are considered for clinical use.
Acknowledgments The authors acknowledge assistance of the members of Anaesthetic, Surgical and Dermatology departments of the Royal Children's Hospital (RCH), Melbourne, for facilitation of sample collection. The support of the RCH Core Laboratory staff in handling specimens is also acknowledged Dr Ignjatovic is supported by the Sanofi-Aventis Clinical Research Fellowship.
References [1] Subramaniam B, Park KW. Direct Thrombin Inhibitors. Int Anesthesiol Clin 2005;43(2):39—53. [2] Gurm HS, Bhatt DL. Thrombin, an ideal target for pharmacological inhibition: A review of direct thrombin inhibitors. Am Heart J 2005;149:S43—53. [3] Bidlingmaier C, Magnani H, Girisch M, Kurnik K. Safety and efficacy of Danaparoid (Orgaran) for use in children. Acta Haematol 2006;115(3-4): 237—47. [4] Martchenke J, Boshkov L. Heparin-induced thrombocytopenia in neonates. J Neon Nurs 2005;24(5):33—7. [5] Ranze O, Ranze P, Magnani HN, Greinacher A. Heparin-induced thrombocytopenia in paediatric patients: a review of the literature and a new case treated with danaparoid sodium. Eur J Pediatr 1999;158(Suppl 3): S130—3.
V. Ignjatovic et al. [6] Risch L, Fischer JE, Herklotz R, Huber A. Heparin-induced thrombocytopenia in paediatrics: clinical characteristics, therapy and outcomes. Intensive Care Med 2004;30(8): 1615—24. [7] Saxon BR, Black MD, Edgell D, Noel D, Leaker MT. Pediatric heparin-induced thrombocytopenia: management with Danaparoid (Oragaran). Ann Thorac Surg 1999;68 (3):1076—8. [8] Zohrer' B, Zenz W, Rettenbacher A, Covi P, Kurnik K, Kroll H. Danaparoid sodium (Orgaran) in four children with heparininduced thrombocytopenia type II. Acta Paedagog 2001;90 (7):765—71. [9] Baier K, Cvirn G, Fritsch P, Kostenberger M, Gallistl S, Leschnik B, et al. Higher concentrations of Heparin and Hirudin are required to inhibit thrombin generation in Tissue Factor-Activated cord plasma than in adult plasma. Paediatr Respir 2005;57(5):685—9. [10] Andrew M, Paes B, Milner R, Jonston M, Mitchell L, Tollefsen D, et al. Development of the human coagulation system in the full-term infant. Blood 1987;70(1):165—72. [11] Andrew M, Paes B, Milner R, Jonston M, Mitchell L, Tollefsen D, et al. Development of the Human coagulation system in the healthy premature infant. Blood 1988;72(5): 1651—7. [12] Andrew M, Vegh P, Johnston M, Bowker J, Ofosu F, Mitchell L, et al. Maturation of the hemostatic system during childhood. Blood 1992;80(8):1998—2005. [13] Ignjatovic V, Furmedge J, Newall F, Chan A, Berry L, Fong C, et al. Age-related differences in Heparin response. Thromb Res 2006;118(6):741—5. [14] Ignjatovic V, Summerhayes R, Gan A, Than J, Chan A, Cochrane A, et al. Monitoring Unfractionated Heparin (UFH) therapy: which Anti Factor Xa assay is appropriate? Thromb Res 2006;120:347—51. [15] Fritsma GA. Direct Thrombin Inhibitors. Clin Lab Sci 2004;17(2): 118—23.
www.elsevier.com/locate/thromres
REGULAR ARTICLE
The in vitro anticoagulant effects of Danaparoid, Fondaparinux, and Lepirudin in children compared to adults Vera Ignjatovic a,b,c,⁎, Robyn Summerhayes a,c , Yan Yan Yip b , Paul Monagle a,b a b c
Department of Clinical Haematology, Royal Children's Hospital, Melbourne, Australia Department of Pathology, University of Melbourne, Australia Murdoch Children's Research Institute, Melbourne, Australia
Received 25 June 2007; received in revised form 8 November 2007; accepted 4 February 2008 Available online 1 April 2008
KEYWORDS Children; Danaparoid; Fondaparinux; Lepirudin
Abstract Introduction: Major physiological differences in the coagulation system of children compared to that of adults are well documented. We have previously investigated the age-related differences in response to Unfractionated Heparin (UFH). However, the impact of developmental haemostasis on more recent anticoagulant drugs is unknown. A number of these drugs are approved for use in specific indications in adults and none are approved for use in children. This study aimed to determine whether age-related differences in effect and impact on monitoring tests exist in vitro for danaparoid, fondaparinux and lepirudin. Materials and Methods: Plasma samples were obtained from healthy children and pooled into age-specific pools, in order to obtain sufficient quantity of plasma required for the analysis of the three drugs. Each age-specific pool was spiked with different concentrations of danaparoid, fondaparinux and lepirudin and response was measured using standard techniques. All experiments were repeated using three separate plasma pools. The effect of each drug in children's plasma was compared to the effect in the respective adult plasma pool. Results: Age-related differences in effect on thrombin potential and monitoring tests were observed only with the drug lepirudin. Specifically, APTT for children up to 5 years of age was increased compared to adults; all children had lower ECT results
Abbreviations: APTT, Activated Partial Thromboplastin Time; ECT, Ecarin Clotting Time; ETP, Endogenous Thrombin Potential; UFH, Unfractionated Heparin. ⁎ Corresponding author. Department of Clinical Haematology, Royal Children's Hospital, Parkville, Victoria, 3010, Australia. Tel.: +61 3 8344 3750; fax: +61 3 8344 4004. E-mail address: [email protected] (V. Ignjatovic). 0049-3848/$ - see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2008.02.001
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V. Ignjatovic et al. compared to adults; children up to 10 years of age had increased inhibition of ETP as compared to adults. Conclusions: This study confirms age-related differences in response to anticoagulants with predominant anti-IIa effect and highlights the need for further research into this area. © 2008 Elsevier Ltd. All rights reserved.
Introduction Thromboembolic disease remains a major health problem in our society. Historically, therapies for thromboembolic disease, including heparin and warfarin, are commonly complicated by bleeding. Since bleeding and clotting are at opposite ends of the physiological spectrum, the search for an ideal anticoagulant that inhibits clotting, while not increasing clinical bleeding is ongoing. The general increase in the understanding of the coagulation system has led to a rapid increase in the development of new anticoagulants. Many of these new drugs have specific target proteins within the coagulation system and a number of these drugs, such as danaparoid, fondaparinux and lepirudin are approved for use in specific indications in adults. However, these anticoagulants are not approved for use in children. Danaparoid is a low molecular weight heparinoid which acts as a Factor Xa (FXa) and Factor IIa (FIIa) inhibitor in a ratio of 28:1 [1]. Lepirudin is a recombinant, desulfated form of hirudin; a direct thrombin inhibitor that binds to both fibrin-bound, as well as fluid-phase thrombin [2]. Fondaparinux is synthetic analogue of the pentasaccharide sequence required for the binding of Heparin to Antithrombin (AT) and as such is an AT-dependent specific FXa inhibitor. Danaparoid and lepirudin have been used for the treatment of Heparin Induced Thrombocytopenia (HIT) in children [3–8]. However, there are no studies comparing the age-related effect of these drugs. Hirudin has been studied in cord plasma, with outcomes suggesting that dosing strategies for neonates should not be derived from studies performed in adults [9]. This is due to the fact that higher doses of the drug were required to achieve the same level of anticoagulation as compared to adults. To date there is no published data on fondaparinux in the paediatric setting. Paralleling the development of new anticoagulants is the expansion in the understanding of Developmental Haemostasis. This concept was established by Andrew et al. and confirms fundamental differences in the haemostatic system of neonates and children compared to adults [10–12]. Differences in weight-adjusted dosing for Unfractionated
Heparin (UFH), warfarin and Low Molecular Weight Heparin (LMWH), have been well documented. More importantly, we have recently shown, first in vitro and then ex vivo, the impact of Developmental Haemostasis on age-related effect and monitoring of UFH therapy in children [13,14]. This study was designed to determine whether age-related differences in effect and impact on monitoring tests exist in vitro for danaparoid, lepirudin and fondaparinux, as an initial step prior to studying these drugs in vivo. Materials and Methods Plasma samples were obtained from healthy children and adults, without previous thromboembolic events and not receiving any form of anticoagulant therapy. Paediatric samples were collected from healthy children about to undergo minor day surgery, while the adults were healthy volunteers. Informed consent was obtained from the parents of children and from the adult participants themselves. This study was approved by the Royal Children's Hospital Ethics in Human Research Committee (EHRC #24124B). Blood samples were collected in tubes containing 0.105 mol/L (i.e., 3.2%) trisodium citrate anticoagulant in a ratio of 9 volumes of whole blood to 1 volume anticoagulant. Citrated samples were centrifuged at 3000 rpm for 10 minutes at 10°C (Megafuge 1.0R, Heraeus) and platelet-poor plasma was frozen at -70°C for batchtesting. Plasma samples were pooled into the following age-related pools: b1 years old, 1-5 years old, 6-10 years old, 11-16 years old and Adults (N18 years old). Each age-specific plasma pool consisted of a minimum of fifteen individual donors and was not significantly different between the different pools. Three separate groups of donors were used to create three separate plasma pools for each age-group. Each age-specific plasma pool was divided into sub-pools and spiked with different concentrations of: danaparoid (0.3, 0.5, 0.7, 1.0 and 1.5 anti-FXa U/ml plasma), lepirudin (0.375, 0.75, 1.5, 2.25 and 3.0 µg/ml plasma) and fondaparinux (0.5, 1.0, 1.5, 2.0 and 4.0 µg/ml plasma). Unspiked, age-specific plasma pools were used as controls. Danaparoid (Orgaran), lepirudin (Refludan) and fondaparinux (Arixtra) were obtained from Organon, Australia; Pharmion, Australia and Sanofi-Synthelabo, Australia, respectively. The Activated Partial Thromboplastin Time (APTT) was measured using a commercially available STA® PTT-A reagent. The Ecarin Clotting Time (ECT) was determined using a commercially available Ecarin reagent, in combination with the STA® analyser (Diagnostica STAGO, France). The maximum measurement times for the APTT and ECT assays was modified allowing for measurement of results up to 600 sec. This is the upper limit of time measurement possible on the STA® analyser.
Age-related in vitro effect of novel anticoagulants
711
Colorimetric assay for Lepirudin was performed using the combination of the STA® analyser (Diagnostica STAGO, France) and the Chromogenic Hirudin Test (Chromogenix, Helena Laboratories, Australia). Anti-Xa assays were performed using: COAMATIC® and COATEST® chromogenic anti-Xa test kits (Chromogenix, Helena Laboratories, Australia), and analysed using the fully automated STA® coagulation analyser (Diagnostica STAGO, France). These two anti-Xa assays measure plasma concentration of anticoagulants; however there are major differences between these methods relating to the presence/absence of exogenous AT, as well as presence/absence of additional Dextran Sulphate (DS). The COAMATIC® assay does not contain exogenous AT, while it does use additional DS. On the other hand, the COATEST® assay uses exogenous AT, while it does not use any additional DS. Endogenous Thrombin Potential (ETP) was quantitated using a non-automated microplate method, based on a modification of a previously described technique.12 Plasma samples were defibrinated by incubation with Reptilase for 30 minutes at 37 oC. The sample was then incubated for a further 10 minutes on ice, with any clot formed being wound out using a wire loop. 50 µl of defibrinated plasma was then mixed with 25 µl of APTT reagent (Diagnostica Stago) at 37 °C for 3 minutes before 25 µl of 0.04 M CaCl2 was added. At 15, 30, 45, 60, 75, 90, 120 and 180 seconds following addition of CaCl2, 10 µl of this reaction mixture was taken out and mixed with 190 µl of 0.005 M Na2 EDTA on ice, to stop thrombin generation. 10 µl of each time-sample EDTA solution was then taken out and mixed with 10 µl of 0.00016 M chromogenic substrate S-2238 (Chromogenix, Italy) in buffer. Following 10 minute incubation, 40 µl of 50% (v/v) acetic acid was added to each sample to stop any further coagulation from taking place. The amount of thrombin generated was quantitated by measuring absorbance of the reaction mixture at 405 nm, and comparing it to the absorbance produced by reaction of S-2238 with known amount of thrombin. Standard curve was prepared using dilutions of human alpha thrombin (Enzyme Research Laboratories, Lot HT 2344PAL). Since thrombin-bound to α2macroglobulin retains activity against small substrates 13, formation of thrombin-α2macroglobulin complexes was quantitated by a previously described method (thrombin generations carried out as above, except that thrombin not bound to α2macroglobulin was neutralised with antithrombin and heparin prior to addition of S-2238). This was subtracted from the total thrombin activity to determine the free thrombin activity.12 Amidolytic activities were plotted against time to produce thrombin generation curves for all plasma pools. The ability of plasma to generate thrombin is expressed as TP, corresponding to the area under the thrombin generation curve, calculated using the trapezoidal rule.
Statistical analysis The results are expressed as the mean and standard deviation (SD) of the three different experimental runs performed. Differences between each age-group were compared to that observed in adults, using one-way analysis of variance, with subsequent twosided, unpaired t tests performed if significance was observed. A result of p b 0.05 was considered statistically significant. Statistical software package STATA, Release 8 0 (Stata Corporation, College station, TX) was used for data processing and analysis.
Results The results of this study are summarised in Figs. 1–3, which represent danaparoid, lepirudin and fondaparinux, respectively.
Figure 1 Age-related response to danaparoid. Results shown are APTT, Coamatic Anti-Xa, Coatest Anti-Xa and ETP in panels A, B, C and D, respectively. For each age, points on the curve represent the mean value for the three plasma pools.
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V. Ignjatovic et al. Danaparoid APTT results in all samples spiked with danaparoid showed a linear increase in relation to increasing danaparoid concentration. (Fig. 1A) In addition, the APTTresults for adult plasma were significantly lower compared to children's plasma, for the same concentration of the drug, across all concentrations tested (p range b 0.0001 to 0.0405). The anti-Xa response, as measured by both the Coamatic and Coatest assays (Fig. 1B and C, respectively) to danaparoid also increased in a dosedependent manner. In this case, age-related differences were not observed. Fig. 1D shows that the ETP decreased significantly with increasing concentration of danaparoid, for each individual age-group. However, there were no agerelated differences in the ETP response to this drug.
Figure 2 Age-related response to lepirudin. Results shown are APTT, ECT, ETP and Chromogenic assay for lepirudin in panels A, B, C and D, respectively. For each age, points on the curve represent the mean value for the three plasma pools.
Figure 3 Age-related response to fondaparinux. Results shown are Coamatic Anti-Xa, Coatest Anti-Xa and ETP in panels A, B and C, respectively. For each age, points on the curve represent the mean value for the three plasma pools.
Age-related in vitro effect of novel anticoagulants Lepirudin Fig. 2A shows the linear increase in APTT in response to lepirudin. This response was found to vary significantly with age. Specifically, the APTT for the b 1 year and 1-5 year old age-groups was found to be increased compared to adults (p range 0.0007 to 0.0424). The ECT increased with increasing concentrations of lepirudin (Fig. 2B). In addition, there were age-related differences in response, with children having significantly lower ECT results compared to adults (p range b 0.0001 to 0.0490). Chromogenic assay for lepirudin showed good correlation between the spiked and measured concentration of the drug (Fig. 2C). The results did not vary for the different plasma pools tested, at various concentrations of lepirudin. The ETP response to lepirudin is shown in Fig. 2D. Increasing concentrations of lepirudin resulted in a decrease in ETP across all age-groups. In addition, for the b 1, 1-5 and 6-10 year olds, this inhibition was significantly increased compared to the age-specific baseline, for the same lepirudin concentration as compared to older children and adults, across all concentrations tested. (p range 0.001 to 0.0470)
Fondaparinux Fig. 3A and B show the anti-Xa response to fondaparinux, respectively. Overall, there were no age-related differences in the anti-Xa response to this drug. Fig. 3C summarizes the ETP response to fondaparinux. For all age-groups tested, the inhibition of thrombin correlated with increasing concentrations of fondaparinux up to 2 µg/ml. However, there was no additional ETP decrease with higher concentrations of fondaparinux. In addition, there were no age-related differences in terms of the ETP response to this drug.
Discussion The coagulation system changes significantly with age, a phenomenon known as Developmental Haemostasis. This is the first study to consider age-related differences in effect and impact on routine monitoring tests for a number of novel anticoagulants. Our results confirm that in vitro, the effect of danaparoid and fondaparinux, two drugs with predominant anti-Xa activity, on total thrombin potential appear to be minimally affected by Developmental Haemostasis, despite some differences in results of non- specific monitoring tests. On the other hand, lepirudin, which has pure anti-IIa activity showed significant age-related differences in effect on thrombin potential and monitoring tests. We have previously shown in vitro and in vivo that the pharmacodynamics of UFH changes with age and that this is reflected in the results of UFH monitoring tests [13,14]. The results of the current study are consistent with the age-related differences in response to UFH, which has a combination of anti-Xa and anti-IIa activity, [13,14] and suggest that the major source
713 of age-related differences relates to interactions between anticoagulants and thrombin (factor IIa) rather than factor Xa. This hypothesis will need to be confirmed with further in vivo studies. An age-related effect of danaparoid was only observed with the APTT, with both the anti-Xa and ETP comparable between adults and children, for the same dose of the drug. The APTT may be sensitive to small amounts of anti-IIa activity that are not reflected in the measurement of the global thrombin potential (ETP). The fact that the measured anti-Xa reflected the “spiked” anti-Xa in each age-group suggests that in these plasma samples from healthy children there is no age-related difference in binding of danaparoid to other plasma proteins. However, ex vivo studies in sick children are required to confirm this before clinical dosing recommendations could be confirmed. Lepirudin is usually monitored quantitatively using the APTT and functionally using the Activated Clotting Time (ACT) or ECT [15]. For both of these tests, we observed age-related differences in response, for the same dose of the drug. Further, the impact on ETP was significantly increased in younger children, for the same dose of lepirudin. This suggests that the response to lepirudin in children is different to adults and that the safety and effectiveness data from adults cannot be extrapolated to children. A previous study reported that increased doses of hirudin were required to achieve the same level of anticoagulation in cord plasma compared to adult plasma, the opposite to our findings (18). However, cord plasma is known to be a different system to plasma from children. In terms of fondaparinux, the results of this study indicate that in vitro there are no age-related differences in response to this drug. Similarly as for danaparoid, this is probably related to the fact that this drug functions by inhibiting FXa, and that the critical age-related factor appears to be Thrombin (IIa). In three separate experiments, using three entirely separate plasma pools for each age-group, we observed consistent and reproducible age-related differences in response to lepirudin compared to adults. These findings need to be confirmed using ex vivo samples, as in vitro spiking of plasma with anticoagulants allows for a limited interpretation of the results. Specifically, in vitro testing does not account for possible plasma binding and endothelial interactions, such as release of TFPI. In conclusion, our study confirms the age-related differences in response to lepirudin, an anticoagulant with predominant anti-IIa effect and reemphasizes the need for further research into this area. Specifically, our results emphasize the need
714 for ex vivo studies to be performed. In the future separate pharmacokinetic and efficacy studies should also be performed in the clinical setting in children, especially when drugs with similar mode of action are considered for clinical use.
Acknowledgments The authors acknowledge assistance of the members of Anaesthetic, Surgical and Dermatology departments of the Royal Children's Hospital (RCH), Melbourne, for facilitation of sample collection. The support of the RCH Core Laboratory staff in handling specimens is also acknowledged Dr Ignjatovic is supported by the Sanofi-Aventis Clinical Research Fellowship.
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