Original article 303
Investigation of the antiangiogenic behaviors of rivaroxaban and low molecular weight heparins Celal Yavuz, Ahmet Caliskan, Oguz Karahan, Suleyman Yazici, Orkut Guclu, Sinan Demirtas and Binali Mavitas Antithrombotic agents play important roles in the prophylactic and therapeutic management of many cardiovascular disorders. Therefore, many researchers have focused on developing new strategies for anticoagulation. New oral anticoagulants and factor Xa inhibitors are products of such research. Although they are identified as advantageous, there are limited data available about their multisystemic interactions. Thus, the antiangiogenic behaviors of oral factor Xa inhibitors and low molecular weight heparins (LMWHs) were investigated in this study. The chick chorioallantoic membrane (CAM) model was designed to investigate the antiangiogenic potential of new oral factor Xa inhibitors (rivaroxaban) and LMWHs (enoxaparin sodium and tinzaparin sodium). Four different molar concentrations (10S4, 10S5, 10S6, and 10S7 mmol/l) were studied for each drug. Each concentration was studied on 20 fertilized eggs. Vessel structures were evaluated under a stereoscopic microscope, and vessel formation was scaled according to previous literature. Both enoxaparin and tinzaparin sodium have increased antiangiogenic efficacy on CAM in a dosedependent manner. However, this increased efficacy did not
reach significant levels (average score < 0.5). On the contrary, while rivaroxaban showed dose-dependent antiangiogenic properties similar to enoxaparin and tinzaparin, a significant average antiangiogenic score (0.7) was detected at 10S4 mmol/l concentrations. New oral anticoagulants seem to be more favorable. However, their safety for the cardiovascular system needs to be clarified through microsystem studies on, for example, angiogenesis. Blood Coagul Fibrinolysis 25:303–308 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Introduction
factor Xa inhibitors have been advanced and are now licensed for thromboprophylaxis in some clinical conditions. Hence, recent studies have claimed that LMWHs are safer and have a superior antithrombotic effect [8,9].
Angiogenesis protects the tissues against hypoxia in ischemic conditions, and the cellular mechanisms can be continued with neo-angiogenesis and neo-vasculogenesis. The investigations were focused on ways to get therapeutic benefits in low-perfusion situations with neo-angiogenesis [1]. Antithrombotic strategies play an important role in the management of various cardiovascular disorders. Most of the previous studies claimed that adverse clinical outcomes can be reduced with antithrombotic drugs [2,3]. However, the safety and effectiveness of these drugs have been discussed in recent reports [3]. In particular, bleeding disorders are reportedly the most common adverse effect of antithrombotic agents [4]. Important anticoagulant agents with antithrombotic validity are low molecular weight heparins (LMWHs) [5]. Initially, unfractionated heparin (UFH) was widely used for thromboprophylaxis in cardiovascular disorders. However, antithrombotic agents more powerful than the standard UFH were produced later: enoxaparin sodium produced with benzylation was followed by alkaline depolymerization and tinzaparin sodium produced with enzymatic depolarization [6,7]. Afterwards, anticoagulation strategies with LMWH became a favorable treatment modality for thromboprophylaxis [7]. However, oral 0957-5235 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
Blood Coagulation and Fibrinolysis 2014, 25:303–308 Keywords: antiangiogenesis, factor Xa inhibitors, low molecular weight heparin, rivaroxaban Department of Cardiovascular Surgery, Medical School of Dicle University, Diyarbakir, Merkez, Turkey Correspondence Oguz Karahan, MD, Medical School of Dicle University, Department of Cardiovascular Surgery, 21280, Diyarbakir, Turkey Tel: +0090 412 248 80 01 1108; fax: +0090 412 48 84 40; e-mail: [email protected] Received 13 July 2013 Revised 26 September 2013 Accepted 28 September 2013
Angiogenesis is an important determinant for cardiovascular disorders [10]. Moreover, the inhibition of angiogenesis is not desirable during the treatment of cardiovascular disorders [10]. Khurana et al. [10] reported that inhibition of angiogenesis is likely to be a viable therapeutic strategy for cardiovascular disease. The chick chorioallantoic membrane (CAM) model is the standardized method for investigating the antiangiogenic potential of agents, especially drugs [11]. Despite these studies, there are not sufficient data to clarify the angiogenic behaviors of new oral anticoagulants and LMWHs. Therefore, the antiangiogenic potential of rivaroxaban, enoxaparin sodium, and tinzaparin sodium were investigated in this study.
Material and methods Study design
This study was designed as an in-vivo chick embryo chorioallantoic membrane model study. The study DOI:10.1097/MBC.0000000000000019
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304 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 4
protocol was approved in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals prepared by the Local Animal Ethics Committee. Preparation of drug pellets
Pellets containing agar only were utilized to confirm the antiangiogenic potential of agar as negative control group. Enoxaparin sodium (Oksapar 6000 anti-Xa IU/0.6 ml; Koc¸ak Pharmaceutical Inc., Istanbul, Turkey), tinzaparin sodium (Innohep 10000 IU/2 ml, Leo Pharmamaceutical, Ballerup, Denmark), and rivaroxaban (Xarelto, Bayer Schering Pharma, Berlin, Germany) were studied. All drugs were in their commercially available form as soluble infusions. The agarose (Merck, Darmstadt, Germany) was added to distilled water to obtain a 1.5% [w/v (weight-for-volume)] solution. The agarose solution was autoclaved at 1218C under one bar standard atmospheric pressure to provide dissolution and sterilization for 15 min. Next, the solution was aliquoted into sterile containers and allowed to cool down to 378C. The drugs were added at this stage. The agar and drug preparations step were standardized, according to the previously defined studies by Dogan et al. [7] and Katrancioglu et al. [12]. Appropriate volumes of solutions were used to achieve four different concentrations of the drugs (104, 105, 106, and 107 mmol/l).
Study protocol
The albumen (5 ml) was removed from the eggshell on the fifth day of incubation via a syringe (Fig. 1a), and a shell piece approximately 2–3 cm in diameter was removed from the contrary side of the eggs for confirmation of the normal development of the chick embryo (Fig. 1b). After this step, malformed or dead embryos were excluded. The apertures occurring on the eggshells after removal were sealed with gelatin. Afterwards, the eggs were placed into the incubators for an additional 72 h to have the CAM reach 2 cm in diameter. The gelatin seals were removed on the eighth day for drug pellet placement in each egg (Fig. 1c); thereafter, the eggs were incubated for an additional 24 h to evaluate the antiangiogenic effects of the drug pellets. Eggs with inflammation and embryo toxicity caused by the pellets were excluded. After election, 20 eggs were used for each group. After the exclusion, the remaining eggs were scored on a stereoscopic microscope following the method of Bu¨rgermeister et al. [13]. Angiogenesis scoring
Supplying and storing of fertilized egg
The antiangiogenic effects of the drugs in the CAM were evaluated under a stereoscopic microscope and assessed according to the scoring system used in previous reports [7,12,14]. This system is based on the evaluation of the capillaries’ density changes around the drug pellets and extension area classification. The scoring system is as follows.
Ross 308 strain fertilized hen eggs obtained from Yemsel Poultry Company (Kayseri, Turkey) were used in the experiment. The eggs were placed horizontally in temperature-controlled (37.58C) and humidity-controlled (80 5%) incubators (Prodi, Egg Incubator – CT120 SH; Ankara, Turkey) for 4 days before the experiment was initiated.
(1) A score of 0 (Fig. 2a) indicates the absence of any demonstrable antiangiogenic effect (normal development of embryo and normal capillary surrounding). (2) A score of 0.5 indicates a weak change [no significant capillary lack but an area (not larger than the pellet area) with reduced capillary density].
Fig. 1
(a)
(b)
(c)
(a) Removal of the albumen (b). Normal development of the chick embryo (c). The placement of the drug pellet.
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Antiangiogenic potential of factor Xa inhibitors Yavuz et al. 305
(3) A score of 1 (Fig. 2b) reflects a mild-to-moderate change [minimally, a capillary-free area or an area (less than double the size of the pellet) with a significantly decreased capillary density]. (4) A score of 2 means a strong antiangiogenic effect (a marked capillary-free area around the drug pellet, which is equal to or more than double the size of the pellet itself). The average score formula is as follows: Number of eggs ðScore 2Þ 2 þ Number of eggs ðScore 1Þ 1 Total eggs number ðScore 0; 1; 2Þ
According to this classification system, an average score less than 0.5 indicates that there is not an antiangiogenic effect; an average score ¼ 0.5–1 means a weak antiangiogenic effect; an average score more than 1 indicates a strong antiangiogenic effect. Statistical analysis
A statistical analysis was performed using the Kruskal– Wallis analysis of variance test and the Mann–Whitney U test to compare the antiangiogenic scores for each drug. A P value was considered statistically significant at P < 0.05.
Results As a negative control, pellets containing 10 ml agar (without drugs) were utilized to confirm the antiangiogenic potential of agar; an antiangiogenic effect (Fig. 2a) was not observed (average score ¼ 0.05). Each drug pellet was evaluated separately, and results obtained with different solutions were compared. Different concentrations (104, 105, 106, and 107 mmol/l) of each drug were compared. Despite the slightly increased potential, there was no statistical significant difference between 104, 105, 106, and 107 mmol/l concentrations of enoxaparin sodium (Fig. 3) and tinzaparin sodium (Fig. 4). Nevertheless, rivaroxaban inhibits angiogenesis significantly at a 104 mmol/l concentration (average score ¼ 0.7). The dose-dependent antiangiogenic behaviors of rivaroxaban are demonstrated in Fig. 5.
The antiangiogenic score of rivaroxoban was higher than that of enoxaparin sodium and tinzaparin sodium in all concentrations (Fig. 6). In fact, statistical difference (P < 0.05) was observed between drug average score’s in each concentration. The average score of all drugs was detected as 0 (no antiangiogenic effect) at 107 mmol/l concentrations. As expected, the maximum average antiangiogenic scores were obtained at 104 mmol/l concentrations for all drugs. The average score’s of each drug for all concentrations were schematized with a scatter graph in Fig. 6.
Discussion Thrombotic events are closely associated with cardiovascular disorders. Therefore, new treatment strategies are currently being developed for eventually replace UFH for the anticoagulant management of cardiovascular diseases [15]. Fareed et al. [15] Despite the newly developed anticoagulant agents are clearly defined, it is not possible that these agents can alternative for heparin in all kind of cardivascular disorders. Nevertheless, superiority and effectiveness studies are important to clarify the longterm results of new drugs [8,9]. UFH, the standard until now, is gradually being replaced by LMWHs after recent trials showing that LMHWs were as effective and rapidacting as UFH [15,16]. Moreover, LWMHs can be given with invasive methods either intravenously or subcutaneously as similar as UFH [15]. Oral anticoagulants were favored throughout the past decades, especially for outpatient thromboprophylaxis [15,16]. One new generation oral anticoagulant is rivaroxaban, which binds with a part of factor Xa. After blocking factor Xa, factor II (prothrombin) cannot be catalyzed, and thrombin is not presented [17]. Additionally, rivoroxaban, which can be monitored with anti-factor Xa assays similar to those of LMWHs, does not inhibit or induce major cytochrome P450 enzymes [17]. The role of new vessel formation within flow-disrupted organ systems has become one of the outstanding puzzles
Fig. 2
(a)
(b)
Formation of the capillaries after pellet application (x8) (a). Normal development of the chick embryo with normal angiogenesis after the application of the drug-free pellet (b). Moderate antiangiogenic efficacy with a rivaroxaban pellet with 105mmol/l concentration (score: 1).
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306 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 4
Fig. 3 2.0 P = 1.000
Antiangiogenic score
Antiangiogenic score
2.0
1.5
1.0
0.5
0
1.0
0.5
0
10–7
µmol/l
P = 0.001*
n = 20
P = 0.853
10–6 µmol/l
n = 20
10–4 µmol/l
n = 20
2.0
Antiangiogenic score
2.0
Antiangiogenic score
1.5
1.5
1.0
0.5
0
1.5
1.0
0.5
0
10 µmol/l –5
n = 20
Antiangiogenic effects of enoxaparin sodium in different concentrations.
in the biology of cardiovascular disease [10]. Despite all the advances, neo-angiogenesis is not suggested during the treatment of cardiovascular disorders [10]. Therefore, problems regarding angiogenic behaviors have been investigated as both medical and surgical strategies for
the management of many cardiovascular diseases [14,18]. Similarly, the antiangiogenic activities of cardiovascular drugs have been investigated in previous studies [7,19], as has the angiogenesis regulation of cardiovascular medications [19,20]. LMWH’s antiangiogenic potential was
Fig. 4 2.0 P = 1.000
Antiangiogenic score
Antiangiogenic score
2.0
1.5
1.0
0.5
1.0
0.5
0
0
10–7 µmol/l 2.0
n = 20
P = 0.006*
P = 0.321
1.5
1.0
0.5
0
10–6 µmol/l
n = 20
10–4 µmol/l
n = 20
2.0
Antiangiogenic score
Antiangiogenic score
1.5
1.5
1.0
0.5
0
10 µmol/l –5
n = 20
Antiangiogenic effects of tinzaparin sodium in different concentrations.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Antiangiogenic potential of factor Xa inhibitors Yavuz et al. 307
Fig. 5 2.0 P = 0.599
Antiangiogenic score
Antiangiogenic score
2.0
1.5
1.0
0.5
0
1.0
0.5
0
10–7
µmol/l
n = 20
P = 0.001*
P = 0.078
10–6 µmol/l
n = 20
10–4 µmol/l
n = 20
2.0
Antiangiogenic score
2.0
Antiangiogenic score
1.5
1.5
1.0
0.5
0
1.5
1.0
0.5
0
10 µmol/l –5
n = 20
Antiangiogenic effects of rivaroxaban in different concentrations.
investigated and compared in previous CAM model trials [7]. Notably, Dogan et al. [7] reported that antiangiogenic scores of tinzaparin sodium were significantly elevated in a dose-dependent manner and that enoxaparin sodium has marked antiangiogenic behavior at 100 IU doses (highest dose). However, Katrancioglu et al. [14] detected no significant difference between the antiangiogenic scores of enoxaparin sodium and tinzaparin sodium. Nevertheless, they added that tinzaparin sodium shows a significant dose-dependent antiangiogenic effect [14]. The 107 mmol/l concentrations of LMWHs were not Fig. 6 Rivaroxaban Enoxaparin Tinzaparin
2.0
1.5
1.0
P = 0.041 P = 0.054 AS:0.7 P = 0.036 P = 0.438
P = 0.022
0.5
P = 0.044
AS:0.45
*AS:0.3
AS:0.3
AS:0.1 AS:0.05 –6
–5
The antiangiogenic behavior of new generation oral antifactor Xa inhibitors is still unclear. Important reports indicate that thrombin regulates immune responses, endothelial functions, and angiogenic factors [21,22]. These drugs affect the thrombin pathways, so the antiangiogenic effects of these drugs should be investigated. The antiangiogenic effects of rivaroxaban are significantly related to drug concentration (antiangiogenic effects of 104 vs. 104mmol/l, P ¼ 0.001). In particular, a marked average antiangiogenic score was obtained at 104 mmol/l concentration (average score ¼ 0.7). In summary, antiocoagulant drugs are important in cardiovascular disorder management, and so the possible antiangiogenic potential of anticoagulant drugs should never be neglected. Their superiority and safety can be well understood through clarification of their antiangiogenic potential. Although they seem to be well tolerated for therapeutic use, these findings should be supported with further clinical studies.
AS:0.45
AS:0.35
AS:0.15
0
studied in those reports. Moreover, antiangiogenic behavior was not observed at 107 mmol/l concentrations (average score ¼ 0).
–4
10 µmol/l concentrations 10 µmol/l concentrations 10 µmol/l concentrations
The comparison of average antiangiogenic scores of drugs in each concentration. AS, average score.
Acknowledgements All named authors had full access to the study data, approved the mention of their names in the article, and shared responsibility for the final decision to submit for publication. Study concept and design: C.Y., A.C., and O.K. Analysis and interpretation of the data: S.Y. Drafting of the article: O.K. and O.G. Technical and material
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308 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 4
support: S.D. Moderation and coordination of the study: B.M. Funding: The authors received no financial support for the research and/or authorship of this article.
9
10 11
Conflicts of interest
The authors declare no conflicts of interest with respect to the authorship and/or publication of this article.
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13
References 1
2
3
4 5 6
7
8
Tateishi-Yuyama E, Matsubara H, Murohara T, Ikeda U, Shintani S, Masaki H, et al. Therapeutic Angiogenesis using Cell Transplantation (TACT) Study Investigators. Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial. Lancet 2002; 360:427–435. Brieger D. Antithrombotic strategies to reduce adverse clinical outcomes in patients with acute coronary syndrome. Am J Cardiol 2012; 110:1200– 1206. Fosbol EL, Wang TY, Li S, Piccini JP, Lopes RD, Shah B, et al. Safety and effectiveness of antithrombotic strategies in older adult patients with atrial fibrillation and non-ST elevation myocardial infarction. Am Heart J 2012; 163:720–728. Weinberger J. Adverse effects and drug interactions of antithrombotic agents used in prevention of ischaemic stroke. Drugs 2005; 65:461–471. Weitz JI. Low-molecular-weight heparins. N Engl J Med 1997; 337:688– 699. Sandercock PA, Counsell C, Tseng MC. Low-molecular-weight heparins or heparinoids versus standard unfractionated heparin for acute ischaemic stroke. Cochrane Database Syst Rev 2008;CD000119. Dogan OT, Polat ZA, Karahan O, Epozturk K, Altun A, Akkurt I, et al. Antiangiogenic activities of bemiparin sodium, enoxaparin sodium, nadroparin calcium and tinzaparin sodium. Thromb Res 2011; 128:e29– e32. Ghosh K, Ghosh K. Advances in haematological pharmacotherapy in 21st century. Indian J Hematol Blood Transfus 2010; 26:30–40.
14
15
16 17
18
19 20 21
22
Fox BD, Kahn SR, Langleben D, Eisenberg MJ, Shimony A. Efficacy and safety of novel oral anticoagulants for treatment of acute venous thromboembolism: direct and adjusted indirect meta-analysis of randomised controlled trials. BMJ 2012; 345:e7498. Khurana R, Simons M, Martin JF, Zachary IC. Role of angiogenesis in cardiovascular disease: a critical appraisal. Circulation 2005; 112:1813–1824. Karahan O, Yavuz C, Demirtas S, Caliskan A, Atahan E. The investigation of the antiangiogenic potential of amiodarone HCl in the chick embryo chorioallantoic membrane model. Biomedical Research 2013; 24:131–134. Katrancioglu N, Karahan O, Kilic AT, Altun A, Katrancioglu O, Polat ZA. The antiangiogenic effects of levosimendan in a CAM assay. Microvasc Res 2012; 83:263–266. Bu¨rgermeister J, Paper DH, Vogl H, Linhardt RJ, Franz G. LaPSvS1, a (1>3)-beta-galactan sulfate and its effect on angiogenesis in vivo and in vitro. Carbohydr Res 2002; 337:1459–1466. Katrancioglu N, Karahan O, Kilic AT, Altun A, Katrancioglu O, Polat ZA. Comparison of the antiangiogenic effects of heparin sodium, enoxaparin sodium, and tinzaparin sodium by using chorioallantoic membrane assay. Blood Coagul Fibrinolysis 2012; 23:218–221. Fareed J, Hoppensteadt DA, Bick RL. Management of thrombotic and cardiovascular disorders in the new millennium. Clin Appl Thromb Hemost 2003; 9:101–108. Russell TM. Warfarin and beyond: an update on oral anticoagulation therapy. US Pharm 2011; 36:26–43. Firriolo FJ, Hupp WS. Beyond warfarin: the new generation of oral anticoagulants and their implications for the management of dental patients. Oral Surg Oral Med Oral Pathol Oral Radiol 2012; 113:431–441. Yener A, Bardakcı Y, Iriz E, Ozogul C, Yener N, Bayram H, et al. Intraoperative changes in the pericardium: a study via electron microscope. Turkish J Thorac Cardiovasc Surg 2011; 19:576–585. Presta M, Leali D, Stabile H, Ronca R, Camozzi M, Coco L, et al. Heparin derivatives as angiogenesis inhibitors. Curr Pharm Des 2003; 9:553–566. Miura S, Saku K. Regulation of angiogenesis and angiogenic factors by cardiovascular medications. Curr Pharm Des 2007; 13:2113–2117. Zhou Q, Bea F, Preusch M, Wang H, Isermann B, Shahzad K, et al. Evaluation of plaque stability of advanced atherosclerotic lesions in apo Edeficient mice after treatment with the oral factor Xa inhibitor rivaroxaban. Mediators Inflamm 2011; 2011: 432080. Maragoudakis ME, Tsopanoglou NE, Andriopoulou P. Mechanism of thrombin-induced angiogenesis. Biochem Soc Trans 2002; 30:173–177.
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Investigation of the antiangiogenic behaviors of rivaroxaban and low molecular weight heparins Celal Yavuz, Ahmet Caliskan, Oguz Karahan, Suleyman Yazici, Orkut Guclu, Sinan Demirtas and Binali Mavitas Antithrombotic agents play important roles in the prophylactic and therapeutic management of many cardiovascular disorders. Therefore, many researchers have focused on developing new strategies for anticoagulation. New oral anticoagulants and factor Xa inhibitors are products of such research. Although they are identified as advantageous, there are limited data available about their multisystemic interactions. Thus, the antiangiogenic behaviors of oral factor Xa inhibitors and low molecular weight heparins (LMWHs) were investigated in this study. The chick chorioallantoic membrane (CAM) model was designed to investigate the antiangiogenic potential of new oral factor Xa inhibitors (rivaroxaban) and LMWHs (enoxaparin sodium and tinzaparin sodium). Four different molar concentrations (10S4, 10S5, 10S6, and 10S7 mmol/l) were studied for each drug. Each concentration was studied on 20 fertilized eggs. Vessel structures were evaluated under a stereoscopic microscope, and vessel formation was scaled according to previous literature. Both enoxaparin and tinzaparin sodium have increased antiangiogenic efficacy on CAM in a dosedependent manner. However, this increased efficacy did not
reach significant levels (average score < 0.5). On the contrary, while rivaroxaban showed dose-dependent antiangiogenic properties similar to enoxaparin and tinzaparin, a significant average antiangiogenic score (0.7) was detected at 10S4 mmol/l concentrations. New oral anticoagulants seem to be more favorable. However, their safety for the cardiovascular system needs to be clarified through microsystem studies on, for example, angiogenesis. Blood Coagul Fibrinolysis 25:303–308 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Introduction
factor Xa inhibitors have been advanced and are now licensed for thromboprophylaxis in some clinical conditions. Hence, recent studies have claimed that LMWHs are safer and have a superior antithrombotic effect [8,9].
Angiogenesis protects the tissues against hypoxia in ischemic conditions, and the cellular mechanisms can be continued with neo-angiogenesis and neo-vasculogenesis. The investigations were focused on ways to get therapeutic benefits in low-perfusion situations with neo-angiogenesis [1]. Antithrombotic strategies play an important role in the management of various cardiovascular disorders. Most of the previous studies claimed that adverse clinical outcomes can be reduced with antithrombotic drugs [2,3]. However, the safety and effectiveness of these drugs have been discussed in recent reports [3]. In particular, bleeding disorders are reportedly the most common adverse effect of antithrombotic agents [4]. Important anticoagulant agents with antithrombotic validity are low molecular weight heparins (LMWHs) [5]. Initially, unfractionated heparin (UFH) was widely used for thromboprophylaxis in cardiovascular disorders. However, antithrombotic agents more powerful than the standard UFH were produced later: enoxaparin sodium produced with benzylation was followed by alkaline depolymerization and tinzaparin sodium produced with enzymatic depolarization [6,7]. Afterwards, anticoagulation strategies with LMWH became a favorable treatment modality for thromboprophylaxis [7]. However, oral 0957-5235 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
Blood Coagulation and Fibrinolysis 2014, 25:303–308 Keywords: antiangiogenesis, factor Xa inhibitors, low molecular weight heparin, rivaroxaban Department of Cardiovascular Surgery, Medical School of Dicle University, Diyarbakir, Merkez, Turkey Correspondence Oguz Karahan, MD, Medical School of Dicle University, Department of Cardiovascular Surgery, 21280, Diyarbakir, Turkey Tel: +0090 412 248 80 01 1108; fax: +0090 412 48 84 40; e-mail: [email protected] Received 13 July 2013 Revised 26 September 2013 Accepted 28 September 2013
Angiogenesis is an important determinant for cardiovascular disorders [10]. Moreover, the inhibition of angiogenesis is not desirable during the treatment of cardiovascular disorders [10]. Khurana et al. [10] reported that inhibition of angiogenesis is likely to be a viable therapeutic strategy for cardiovascular disease. The chick chorioallantoic membrane (CAM) model is the standardized method for investigating the antiangiogenic potential of agents, especially drugs [11]. Despite these studies, there are not sufficient data to clarify the angiogenic behaviors of new oral anticoagulants and LMWHs. Therefore, the antiangiogenic potential of rivaroxaban, enoxaparin sodium, and tinzaparin sodium were investigated in this study.
Material and methods Study design
This study was designed as an in-vivo chick embryo chorioallantoic membrane model study. The study DOI:10.1097/MBC.0000000000000019
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
304 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 4
protocol was approved in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals prepared by the Local Animal Ethics Committee. Preparation of drug pellets
Pellets containing agar only were utilized to confirm the antiangiogenic potential of agar as negative control group. Enoxaparin sodium (Oksapar 6000 anti-Xa IU/0.6 ml; Koc¸ak Pharmaceutical Inc., Istanbul, Turkey), tinzaparin sodium (Innohep 10000 IU/2 ml, Leo Pharmamaceutical, Ballerup, Denmark), and rivaroxaban (Xarelto, Bayer Schering Pharma, Berlin, Germany) were studied. All drugs were in their commercially available form as soluble infusions. The agarose (Merck, Darmstadt, Germany) was added to distilled water to obtain a 1.5% [w/v (weight-for-volume)] solution. The agarose solution was autoclaved at 1218C under one bar standard atmospheric pressure to provide dissolution and sterilization for 15 min. Next, the solution was aliquoted into sterile containers and allowed to cool down to 378C. The drugs were added at this stage. The agar and drug preparations step were standardized, according to the previously defined studies by Dogan et al. [7] and Katrancioglu et al. [12]. Appropriate volumes of solutions were used to achieve four different concentrations of the drugs (104, 105, 106, and 107 mmol/l).
Study protocol
The albumen (5 ml) was removed from the eggshell on the fifth day of incubation via a syringe (Fig. 1a), and a shell piece approximately 2–3 cm in diameter was removed from the contrary side of the eggs for confirmation of the normal development of the chick embryo (Fig. 1b). After this step, malformed or dead embryos were excluded. The apertures occurring on the eggshells after removal were sealed with gelatin. Afterwards, the eggs were placed into the incubators for an additional 72 h to have the CAM reach 2 cm in diameter. The gelatin seals were removed on the eighth day for drug pellet placement in each egg (Fig. 1c); thereafter, the eggs were incubated for an additional 24 h to evaluate the antiangiogenic effects of the drug pellets. Eggs with inflammation and embryo toxicity caused by the pellets were excluded. After election, 20 eggs were used for each group. After the exclusion, the remaining eggs were scored on a stereoscopic microscope following the method of Bu¨rgermeister et al. [13]. Angiogenesis scoring
Supplying and storing of fertilized egg
The antiangiogenic effects of the drugs in the CAM were evaluated under a stereoscopic microscope and assessed according to the scoring system used in previous reports [7,12,14]. This system is based on the evaluation of the capillaries’ density changes around the drug pellets and extension area classification. The scoring system is as follows.
Ross 308 strain fertilized hen eggs obtained from Yemsel Poultry Company (Kayseri, Turkey) were used in the experiment. The eggs were placed horizontally in temperature-controlled (37.58C) and humidity-controlled (80 5%) incubators (Prodi, Egg Incubator – CT120 SH; Ankara, Turkey) for 4 days before the experiment was initiated.
(1) A score of 0 (Fig. 2a) indicates the absence of any demonstrable antiangiogenic effect (normal development of embryo and normal capillary surrounding). (2) A score of 0.5 indicates a weak change [no significant capillary lack but an area (not larger than the pellet area) with reduced capillary density].
Fig. 1
(a)
(b)
(c)
(a) Removal of the albumen (b). Normal development of the chick embryo (c). The placement of the drug pellet.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Antiangiogenic potential of factor Xa inhibitors Yavuz et al. 305
(3) A score of 1 (Fig. 2b) reflects a mild-to-moderate change [minimally, a capillary-free area or an area (less than double the size of the pellet) with a significantly decreased capillary density]. (4) A score of 2 means a strong antiangiogenic effect (a marked capillary-free area around the drug pellet, which is equal to or more than double the size of the pellet itself). The average score formula is as follows: Number of eggs ðScore 2Þ 2 þ Number of eggs ðScore 1Þ 1 Total eggs number ðScore 0; 1; 2Þ
According to this classification system, an average score less than 0.5 indicates that there is not an antiangiogenic effect; an average score ¼ 0.5–1 means a weak antiangiogenic effect; an average score more than 1 indicates a strong antiangiogenic effect. Statistical analysis
A statistical analysis was performed using the Kruskal– Wallis analysis of variance test and the Mann–Whitney U test to compare the antiangiogenic scores for each drug. A P value was considered statistically significant at P < 0.05.
Results As a negative control, pellets containing 10 ml agar (without drugs) were utilized to confirm the antiangiogenic potential of agar; an antiangiogenic effect (Fig. 2a) was not observed (average score ¼ 0.05). Each drug pellet was evaluated separately, and results obtained with different solutions were compared. Different concentrations (104, 105, 106, and 107 mmol/l) of each drug were compared. Despite the slightly increased potential, there was no statistical significant difference between 104, 105, 106, and 107 mmol/l concentrations of enoxaparin sodium (Fig. 3) and tinzaparin sodium (Fig. 4). Nevertheless, rivaroxaban inhibits angiogenesis significantly at a 104 mmol/l concentration (average score ¼ 0.7). The dose-dependent antiangiogenic behaviors of rivaroxaban are demonstrated in Fig. 5.
The antiangiogenic score of rivaroxoban was higher than that of enoxaparin sodium and tinzaparin sodium in all concentrations (Fig. 6). In fact, statistical difference (P < 0.05) was observed between drug average score’s in each concentration. The average score of all drugs was detected as 0 (no antiangiogenic effect) at 107 mmol/l concentrations. As expected, the maximum average antiangiogenic scores were obtained at 104 mmol/l concentrations for all drugs. The average score’s of each drug for all concentrations were schematized with a scatter graph in Fig. 6.
Discussion Thrombotic events are closely associated with cardiovascular disorders. Therefore, new treatment strategies are currently being developed for eventually replace UFH for the anticoagulant management of cardiovascular diseases [15]. Fareed et al. [15] Despite the newly developed anticoagulant agents are clearly defined, it is not possible that these agents can alternative for heparin in all kind of cardivascular disorders. Nevertheless, superiority and effectiveness studies are important to clarify the longterm results of new drugs [8,9]. UFH, the standard until now, is gradually being replaced by LMWHs after recent trials showing that LMHWs were as effective and rapidacting as UFH [15,16]. Moreover, LWMHs can be given with invasive methods either intravenously or subcutaneously as similar as UFH [15]. Oral anticoagulants were favored throughout the past decades, especially for outpatient thromboprophylaxis [15,16]. One new generation oral anticoagulant is rivaroxaban, which binds with a part of factor Xa. After blocking factor Xa, factor II (prothrombin) cannot be catalyzed, and thrombin is not presented [17]. Additionally, rivoroxaban, which can be monitored with anti-factor Xa assays similar to those of LMWHs, does not inhibit or induce major cytochrome P450 enzymes [17]. The role of new vessel formation within flow-disrupted organ systems has become one of the outstanding puzzles
Fig. 2
(a)
(b)
Formation of the capillaries after pellet application (x8) (a). Normal development of the chick embryo with normal angiogenesis after the application of the drug-free pellet (b). Moderate antiangiogenic efficacy with a rivaroxaban pellet with 105mmol/l concentration (score: 1).
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306 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 4
Fig. 3 2.0 P = 1.000
Antiangiogenic score
Antiangiogenic score
2.0
1.5
1.0
0.5
0
1.0
0.5
0
10–7
µmol/l
P = 0.001*
n = 20
P = 0.853
10–6 µmol/l
n = 20
10–4 µmol/l
n = 20
2.0
Antiangiogenic score
2.0
Antiangiogenic score
1.5
1.5
1.0
0.5
0
1.5
1.0
0.5
0
10 µmol/l –5
n = 20
Antiangiogenic effects of enoxaparin sodium in different concentrations.
in the biology of cardiovascular disease [10]. Despite all the advances, neo-angiogenesis is not suggested during the treatment of cardiovascular disorders [10]. Therefore, problems regarding angiogenic behaviors have been investigated as both medical and surgical strategies for
the management of many cardiovascular diseases [14,18]. Similarly, the antiangiogenic activities of cardiovascular drugs have been investigated in previous studies [7,19], as has the angiogenesis regulation of cardiovascular medications [19,20]. LMWH’s antiangiogenic potential was
Fig. 4 2.0 P = 1.000
Antiangiogenic score
Antiangiogenic score
2.0
1.5
1.0
0.5
1.0
0.5
0
0
10–7 µmol/l 2.0
n = 20
P = 0.006*
P = 0.321
1.5
1.0
0.5
0
10–6 µmol/l
n = 20
10–4 µmol/l
n = 20
2.0
Antiangiogenic score
Antiangiogenic score
1.5
1.5
1.0
0.5
0
10 µmol/l –5
n = 20
Antiangiogenic effects of tinzaparin sodium in different concentrations.
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Antiangiogenic potential of factor Xa inhibitors Yavuz et al. 307
Fig. 5 2.0 P = 0.599
Antiangiogenic score
Antiangiogenic score
2.0
1.5
1.0
0.5
0
1.0
0.5
0
10–7
µmol/l
n = 20
P = 0.001*
P = 0.078
10–6 µmol/l
n = 20
10–4 µmol/l
n = 20
2.0
Antiangiogenic score
2.0
Antiangiogenic score
1.5
1.5
1.0
0.5
0
1.5
1.0
0.5
0
10 µmol/l –5
n = 20
Antiangiogenic effects of rivaroxaban in different concentrations.
investigated and compared in previous CAM model trials [7]. Notably, Dogan et al. [7] reported that antiangiogenic scores of tinzaparin sodium were significantly elevated in a dose-dependent manner and that enoxaparin sodium has marked antiangiogenic behavior at 100 IU doses (highest dose). However, Katrancioglu et al. [14] detected no significant difference between the antiangiogenic scores of enoxaparin sodium and tinzaparin sodium. Nevertheless, they added that tinzaparin sodium shows a significant dose-dependent antiangiogenic effect [14]. The 107 mmol/l concentrations of LMWHs were not Fig. 6 Rivaroxaban Enoxaparin Tinzaparin
2.0
1.5
1.0
P = 0.041 P = 0.054 AS:0.7 P = 0.036 P = 0.438
P = 0.022
0.5
P = 0.044
AS:0.45
*AS:0.3
AS:0.3
AS:0.1 AS:0.05 –6
–5
The antiangiogenic behavior of new generation oral antifactor Xa inhibitors is still unclear. Important reports indicate that thrombin regulates immune responses, endothelial functions, and angiogenic factors [21,22]. These drugs affect the thrombin pathways, so the antiangiogenic effects of these drugs should be investigated. The antiangiogenic effects of rivaroxaban are significantly related to drug concentration (antiangiogenic effects of 104 vs. 104mmol/l, P ¼ 0.001). In particular, a marked average antiangiogenic score was obtained at 104 mmol/l concentration (average score ¼ 0.7). In summary, antiocoagulant drugs are important in cardiovascular disorder management, and so the possible antiangiogenic potential of anticoagulant drugs should never be neglected. Their superiority and safety can be well understood through clarification of their antiangiogenic potential. Although they seem to be well tolerated for therapeutic use, these findings should be supported with further clinical studies.
AS:0.45
AS:0.35
AS:0.15
0
studied in those reports. Moreover, antiangiogenic behavior was not observed at 107 mmol/l concentrations (average score ¼ 0).
–4
10 µmol/l concentrations 10 µmol/l concentrations 10 µmol/l concentrations
The comparison of average antiangiogenic scores of drugs in each concentration. AS, average score.
Acknowledgements All named authors had full access to the study data, approved the mention of their names in the article, and shared responsibility for the final decision to submit for publication. Study concept and design: C.Y., A.C., and O.K. Analysis and interpretation of the data: S.Y. Drafting of the article: O.K. and O.G. Technical and material
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308 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 4
support: S.D. Moderation and coordination of the study: B.M. Funding: The authors received no financial support for the research and/or authorship of this article.
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Conflicts of interest
The authors declare no conflicts of interest with respect to the authorship and/or publication of this article.
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References 1
2
3
4 5 6
7
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Tateishi-Yuyama E, Matsubara H, Murohara T, Ikeda U, Shintani S, Masaki H, et al. Therapeutic Angiogenesis using Cell Transplantation (TACT) Study Investigators. Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial. Lancet 2002; 360:427–435. Brieger D. Antithrombotic strategies to reduce adverse clinical outcomes in patients with acute coronary syndrome. Am J Cardiol 2012; 110:1200– 1206. Fosbol EL, Wang TY, Li S, Piccini JP, Lopes RD, Shah B, et al. Safety and effectiveness of antithrombotic strategies in older adult patients with atrial fibrillation and non-ST elevation myocardial infarction. Am Heart J 2012; 163:720–728. Weinberger J. Adverse effects and drug interactions of antithrombotic agents used in prevention of ischaemic stroke. Drugs 2005; 65:461–471. Weitz JI. Low-molecular-weight heparins. N Engl J Med 1997; 337:688– 699. Sandercock PA, Counsell C, Tseng MC. Low-molecular-weight heparins or heparinoids versus standard unfractionated heparin for acute ischaemic stroke. Cochrane Database Syst Rev 2008;CD000119. Dogan OT, Polat ZA, Karahan O, Epozturk K, Altun A, Akkurt I, et al. Antiangiogenic activities of bemiparin sodium, enoxaparin sodium, nadroparin calcium and tinzaparin sodium. Thromb Res 2011; 128:e29– e32. Ghosh K, Ghosh K. Advances in haematological pharmacotherapy in 21st century. Indian J Hematol Blood Transfus 2010; 26:30–40.
14
15
16 17
18
19 20 21
22
Fox BD, Kahn SR, Langleben D, Eisenberg MJ, Shimony A. Efficacy and safety of novel oral anticoagulants for treatment of acute venous thromboembolism: direct and adjusted indirect meta-analysis of randomised controlled trials. BMJ 2012; 345:e7498. Khurana R, Simons M, Martin JF, Zachary IC. Role of angiogenesis in cardiovascular disease: a critical appraisal. Circulation 2005; 112:1813–1824. Karahan O, Yavuz C, Demirtas S, Caliskan A, Atahan E. The investigation of the antiangiogenic potential of amiodarone HCl in the chick embryo chorioallantoic membrane model. Biomedical Research 2013; 24:131–134. Katrancioglu N, Karahan O, Kilic AT, Altun A, Katrancioglu O, Polat ZA. The antiangiogenic effects of levosimendan in a CAM assay. Microvasc Res 2012; 83:263–266. Bu¨rgermeister J, Paper DH, Vogl H, Linhardt RJ, Franz G. LaPSvS1, a (1>3)-beta-galactan sulfate and its effect on angiogenesis in vivo and in vitro. Carbohydr Res 2002; 337:1459–1466. Katrancioglu N, Karahan O, Kilic AT, Altun A, Katrancioglu O, Polat ZA. Comparison of the antiangiogenic effects of heparin sodium, enoxaparin sodium, and tinzaparin sodium by using chorioallantoic membrane assay. Blood Coagul Fibrinolysis 2012; 23:218–221. Fareed J, Hoppensteadt DA, Bick RL. Management of thrombotic and cardiovascular disorders in the new millennium. Clin Appl Thromb Hemost 2003; 9:101–108. Russell TM. Warfarin and beyond: an update on oral anticoagulation therapy. US Pharm 2011; 36:26–43. Firriolo FJ, Hupp WS. Beyond warfarin: the new generation of oral anticoagulants and their implications for the management of dental patients. Oral Surg Oral Med Oral Pathol Oral Radiol 2012; 113:431–441. Yener A, Bardakcı Y, Iriz E, Ozogul C, Yener N, Bayram H, et al. Intraoperative changes in the pericardium: a study via electron microscope. Turkish J Thorac Cardiovasc Surg 2011; 19:576–585. Presta M, Leali D, Stabile H, Ronca R, Camozzi M, Coco L, et al. Heparin derivatives as angiogenesis inhibitors. Curr Pharm Des 2003; 9:553–566. Miura S, Saku K. Regulation of angiogenesis and angiogenic factors by cardiovascular medications. Curr Pharm Des 2007; 13:2113–2117. Zhou Q, Bea F, Preusch M, Wang H, Isermann B, Shahzad K, et al. Evaluation of plaque stability of advanced atherosclerotic lesions in apo Edeficient mice after treatment with the oral factor Xa inhibitor rivaroxaban. Mediators Inflamm 2011; 2011: 432080. Maragoudakis ME, Tsopanoglou NE, Andriopoulou P. Mechanism of thrombin-induced angiogenesis. Biochem Soc Trans 2002; 30:173–177.
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