ORIGINAL ARTICLE
JOURNAL OF OCULAR PHARMACOLOGY AND THERAPEUTICS Volume 00, Number 00, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/jop.2013.0234
Serum D-Dimer Levels to Evaluate the Risk for Arterial Thromboembolism After Intravitreal Injection of Bevacizumab and Ranibizumab Donghyun Jee,1,2 Masahiro Zako,3 and Tae Yoon La1,2
Abstract
Purpose: There are concerns about arterial thromboembolic event after intravitreal injection of bevacizumab or ranibizumab. Motivated by the fact that D-dimer was a sensitive biomarker for thromboembolism, we evaluated serum D-dimer levels in patients with age-related macular degeneration (AMD) after intravitreal injection of bevacizumab and ranibizumab. Methods: In this prospective, nonrandomized, uncontrolled study, 122 patients (122 eyes) with AMD were enrolled. Sixty-two eyes received intravitreal injections of bevacizumab and 60 eyes received intravitreal injections of ranibizumab monthly for 3 months. Serum D-dimer levels were measured in patients before intravitreal injection and 1 day, 1 week, 1 month, and 3 months thereafter. Results: Serum D-dimer levels were not significantly altered following injection of either bevacizumab or ranibizumab. Subgroup analysis for patients at risk for thromboembolic events revealed that serum D-dimer levels showed no significant change after injection of ranibizumab. However, D-dimer levels significantly increased at 1 day (P = 0.041) and 1 week (P = 0.022) after injection of bevacizumab. Conclusions: Serum D-dimer levels were not changed after injection with either bevacizumab or ranibizumab. In subgroup analysis, bevacizumab injection in patients at risk of thromboembolism increased serum D-dimer levels.
which blocks physiologic VEGF.7 This blockage of physiologic VEGF increases the risk of a thromboembolic event or cardiovascular accident.7,8 Because VEGF not only stimulates endothelial cell proliferation, but also maintains vascular integrity, VEGF inhibition could cause endothelial dysfunction leading to thrombosis.9 A recent meta-analysis of prospective clinical trials using anti-VEGF antibodies indicated an association between intravitreal injection of anti-VEGF antibodies and the subsequent incidence of cardiovascular accidents.10 Another study reported that the rate of arterial thromboembolic events was marginally higher in treatment arms receiving higher doses of anti-VEGF antibodies, although this trend was not significant.11 Thromboembolism is a major cause of death in the United States and a leading cause of morbidity, with an annual incidence of about 1 case per 1,000 individuals.12,13 Although a thromboembolic event can be fatal, few studies have investigated thromboembolism after intravitreal antiVEGF therapy. One possible reason is that thromboembolic
Introduction
I
nhibitors of vascular endothelial growth factor (VEGF) are widely used to treat patients with wet, agerelated macular degeneration (AMD).1,2 Neutralization of all isoforms of VEGF-A significantly improves the ability to minimize vision loss, and restore sight in patients with AMD.3,4 Commonly used antibody-derived drugs for treating AMD are bevacizumab (Avastin; Hoffmann La Roche, Basel, Switzerland) and ranibizumab (Lucentis; Genetech, Inc., San Francisco, CA). Bevacizumab is a full-length antibody that was developed as a potential therapeutic agent for use in oncology.5 Ranibizumab is an antigen-binding fragment (Fab) of an antibody, and was developed as part of an anti-VEGF program for AMD treatment.6 Although anti-VEGF antibodies provide remarkable clinical benefits for AMD patients, concerns remain about the potential systemic off-target effects of anti-VEGF inhibition. Intravitreally injected drugs enter the systemic circulation, 1 2 3
Department of Ophthalmology and Visual Science, St. Vincent Hospital, Suwon, Korea. College of Medicine, Catholic University of Korea, Seoul, Korea. Department of Ophthalmology, Aichi Medical University, Aichi, Japan.
1
2
events after intravitreal injection of anti-VEGF occur at extremely low rates. To identify low rates of events to evaluate safety, highly sensitive methods are needed. D-dimer is the most reliable and sensitive biomarker for thromboembolic events.14 It is regarded as a valuable biomarker for diagnosing and managing a vast array of thrombosis-related clinical conditions.15 D-dimer is a specific degradation product of fibrin clots. This small protein fragment, which is present in blood clots degraded by fibrinolysis, is known as ‘‘D-dimer’’ because it contains 2 crosslinked D-fragments of fibrinogen. D-dimer levels are elevated when the coagulation system is activated, such as in cases of thrombosis. Measurement of D-dimer levels is a gold standard for diagnosing thrombosis and identifying increased risk of thrombotic events.15–19 Elevated serum Ddimer levels indicate a high risk of thrombosis.20 Many long-term prospective studies have demonstrated an association between D-dimer and thromboembolism or coronary heart disease.21 In this study, we evaluated serum D-dimer levels after intravitreal injection of bevacizumab or ranibizumab in patients with AMD.
Methods This prospective, nonrandomized study examined patients who received 3 monthly intravitreal injections of bevacizumab or ranibizumab for wet AMD between March and November 2010. The decision about which drug to use was made by the physician (D. Jee), taking into account the patient’s wish, economic status, and insurance status. We excluded patients with ocular neovascularization or rubeosis iridis, or who had undergone intraocular surgery within the last 6 months. Patient with underlying thromboembolic disease was defined as those with present or past history for stroke, coronary heart disease, and venous thromboembolic disease. The study design followed the tenets of the Declaration of Helsinki for biomedical research and was approved by the institutional review board of the Catholic University of Korea in Seoul, Korea. Informed consent was obtained from the patients. To evaluate the change of Ddimer levels after injection, we assume the large effect size (d = 0.8) as a clinically important difference. Using 2-sided significance level 0.05 and power 80%, we obtained *60 eyes per each group. All patients received an intravitreal injection monthly for 3 months. We collected blood samples from patients to examine serum D-dimer levels before the initial injection and 1 day, 1 week, 1 month, and 3 months after initial injection (Fig. 1). D-dimer levels were measured by a medical technologist who is masked to the clinical status of patients or injected drugs in a clinical laboratory using VIDAS D-dimer ELISA method (VIDAS, BioMerieux, craponne, France).17 All patients were instilled with proparacaine (Alcaine; Alcon, Fort Worth, TX) and had skin draping that contained 10% povidone. After inserting a speculum, a 5% povidone solution was applied to the conjunctival sac. After sterile drape placement, the conjunctiva was displaced and bevacizumab (1.25 mg in 0.05 mL) or ranibizumab (0.05 mg in 0.05 mL) was injected perpendicular to the sclera at the inferotemporal position of the pars plana (3.5 mm posterior to the surgical limbus) by using a 30-gauge needle attached to a 1-mL tuberculin syringe. Using an operating microscope,
JEE, ZAKO, AND LA
FIG. 1. Timing of intravitreal injection of anti–vascular endothelial growth factor and blood sampling to examine serum D-dimer levels. Intravitreal injection was performed monthly for 3 months, and blood sampling was conducted before 1st injection, and 1 day, 1 week, 1 month, and 3 months after initial injection. the location of the needle tip was identified as being in the center of the vitreous cavity. All patients underwent clinical examinations, including best-corrected visual acuity measurement, slit-lamp biomicroscopy, intraocular pressure measurement, and fundus examination. We assessed differences between the serum D-dimer levels before and after intravitreal injection within each group by repeated measured analysis of variance (ANOVA) by using commercial software (SPSS version 17.0; SPSS, Inc., Chicago, IL). We also evaluated differences in D-dimer levels between the groups receiving bevacizumab and ranibizumab by repeated measured ANOVA. P < 0.05 was considered statistically significant.
Results We enrolled 122 eyes from 122 patients who received intravitreal injections of bevacizumab or ranibizumab from March to November 2010. All eyes were divided into 2 groups: bevacizumab (62 eyes) and ranibizumab (60 eyes). The bevacizumab and ranibizumab groups were well-balanced overall for age, gender, and underlying arterial thromboembolic disease (Table 1). In both groups, D-dimer levels showed no significant change before and after injection at any point (Fig. 2). No significant difference was seen between the bevacizumab and ranibizumab groups (Table 2).
Table 1. Clinical Characteristics of Subjects in the Study Bevacizumab Ranibizumab (n = 62) (n = 60) Age (years) 68.33 – 6.27 Gender (male/female) 38/24 Diabetes 11 Hypertension 28 Smoking 18 Underlying 12 (19.3%) thromboembolic diseases Stroke 4 Coronary heart 10 disease 1 Venous thromboembolic disease
P
69.36 – 8.99 40/20 9 33 21 11 (18.3%)
0.463 0.5364 0.682 0.277 0.479 0.785
6 8
0.475 0.663
2
0.549
D-DIMER AFTER ANTI-VEGF INJECTION
3
FIG. 2. D-dimer levels after intravitreal injection of bevacizumab (B) or ranibizumab ( ). No significant change was detected at any time after intravitreal injection in either bevacizumab or ranibizumab groups. Repeated measured analysis of variance (ANOVA).
Subgroup analysis for patients with risk of thromboembolic diseases was performed. D-dimer levels showed no significant change after ranibizumab injection, but D-dimer levels at 1 day and 1 week after bevacizumab injection were significantly higher than that before injection (P = 0.041 and P = 0.022, respectively; Fig. 3). D-dimer levels were significantly higher at 1 day (P = 0.038) and 1 week (P = 0.017, Table 3) after bevacizumab injection than after ranibizumab injection. During the follow-up period, none of the patients developed any severe local adverse events, such as endophthalmitis, uveitis, or retinal tears, or systemic side effects, such as cerebrovascular attacks or myocardial infarction.
Discussion Our study demonstrated that D-dimer levels were unchanged after intravitreal injections of either bevacizumab or ranibizumab. In patients with thromboembolic risk factors, intravitreal injections of bevacizumab resulted in transient increases in D-dimer levels. Although the exact mechanism of D-dimer elevation by bevacizumab remains to be elucidated, we have several hypotheses. One possible reason is the entry of different amounts of anti-VEGF agents into systemic circulation. Intravitreally administered bevacizumab might be transferred across the blood-retinal barrier into the systemic vascular system, while ranibizumab might remain localized in the injected eye. In a recent study in which the pharmacokinetics of intravitreally administered ranibizumab and bevacizumab were compared in rabbits, ranibizumab was not detected in serum, while
small amounts of bevacizumab were detected.22,23 In another study, it was reported that intravitreal bevacizumab appeared in plasma and in the fellow eye via systemic circulation.24 The pharmacokinetic difference in these 2 anti-VEGF agents could be due to differences in molecular structure. Bevacizumab is a full-length antibody, while ranibizumab is a Fab fragment. Ranibizumab does not possess the fragment crystallizable (Fc) region, which is important in transferring intravitreally injected full-length immunoglobulin G (IgG) across the blood-retinal barrier into the systemic blood system.25 In a study using Fc-receptor-knockout mice, IgG without Fc receptor did not penetrate the blood-retinal barrier. Thus, bevacizumab may enter the systemic circulation more easily. Systemic bevacizumab might disrupt physiological VEGF function, which could lead to increased D-dimer levels. A recent report demonstrated significantly reduced VEGF plasma levels after intravitreal bevacizumab injection; these were not seen after intravitreal ranibizumab injection.26 Another possible reason is that the half-life of ranibizumab is shorter than of that of bevacizumab when exposed to systemic circulation. Antigen-binding fragments, such as ranibizumab, tend to have a shorter half-life than full-length antibodies, such as bevacizumab. The systemic half-life of a Fab is a few hours, whereas the half-life of full-length IgG is *3 weeks.27 The longer half-life of bevacizumab might be involved in the transient increase in serum D-dimer levels in this study.
Table 3. D-Dimer (mg/L) Levels in Patients with Underlying Arterial Thromboembolic Disease After Bevacizumab or Ranibizumab Injection
Table 2. D-Dimer (mg/L) Levels in Patients After Bevacizumab or Ranibizumab Injection
Preoperative Postoperative 1 day 1 week 1 month 3 months
FIG. 3. D-dimer levels after intravitreal injection of bevacizumab (B) or ranibizumab ( ) in patients with underlying arterial thromboembolic disease. D-dimer levels were not significantly changed after ranibizumab injection, but Ddimer levels at 1 day and 1 week after bevacizumab injection significantly increased compared with before injection. *P < 0.05, repeated measured ANOVA.
Bevacizumab (n = 62)
Ranibizumab (n = 60)
P
182 – 206 215 – 325 231 – 280 210 – 167 192 – 227
206 – 127 192 – 194 211 – 233 185 – 241 208 – 316
0.345 0.106 0.672 0.089 0.715
Continuous variables are expressed as n or mean – standard deviation. Repeated measured analysis of variance (ANOVA).
Preoperative Postoperative 1 day 1 week 1 month 3 months
Bevacizumab (n = 12)
Ranibizumab (n = 11)
P
215 – 242 356 – 225 412 – 268 327 – 274 281 – 266
221 – 264 238 – 165 217 – 192 263 – 271 241 – 304
0.654 0.038a 0.017a 0.147 0.526
Continuous variables are expressed as n or mean – standard deviation. a P < 005, repeated measured ANOVA.
4
The exact mechanism of how anti-VEGF agents induce thromboembolism is unclear. One possible mechanism is that suppression of physiological VEGF causes damage to vascular endothelial cells, especially to the balanced coagulative and fibrinolytic systems. This develops into thromboembolism, resulting in elevated D-dimer levels. Our hypothesis is supported by the finding that D-dimer levels are positively associated with VEGF levels.28,29 AMD is associated with strokes.30,31 The Atherosclerosis Risk in Communities Study showed an almost 2-fold risk of stroke in AMD patients over 10 years (hazard ratio, 1.87; 95% confidence interval [CI], 1.21–2.88).32 Hu et al. reported that neovascular AMD is associated with a higher risk of stroke over 5 years (hazard ratio, 2.42; 95% CI, 1.47– 3.98).30 Thus, the suppression of physiological VEGF by intravitreal injection of anti-VEGF for AMD treatment could induce thromboembolic events, because patients with AMD is more susceptible to stroke. D-dimer levels were not elevated at 3 months after intravitreal injection in this study. Our study had a schedule of 3 monthly injections as a loading dose. Cumulative doses from 3 monthly injections might have an adverse effect on D-dimer levels. However, we found no cumulative effect from repeated injections. We presume that D-dimer levels might be normalized after repeated and regular inhibition of VEGF by a complex feedback mechanism. In a recent study, pro re nata (PRN; as needed) anti-VEGF therapy was associated with more thromboembolic events than a monthly regimen.33 This implies that abrupt inhibition of physiologic VEGF is associated with higher risk of thromboembolism than repeated inhibition at regular intervals. The risk of thromboembolism after intravitreal injection of anti-VEGF has been described in previous reports.7,34,35 Comparison of AMD Treatments Trials (CATT) study—a randomized, controlled clinical trial—reported that thromboembolic events were more frequent in a bevacizumab group than in a ranibizumab group.33 Our results correspond with these previous results. The D-dimer assay has a high negative predictive value. It is highly sensitive but not specific to thromboembolic disease.36 D-dimer levels can also be elevated in malignancy, pregnancy, recent trauma or surgery, bleeding, and sepsis, because all these diseases have a common underlying process of thromboembolism.16 Therefore, D-dimer elevation by bevacizumab in patients with underlying thromboembolic disease does not indicate an elevated postoperative thromboembolism risk. However, intravitreally injected bevacizumab and ranibizumab seem to be safe from thromboembolic complications considering the high negative predictive value of the D-dimer test. D-dimer assays can be used to exclude a diagnosis of thromboembolic events after intravitreal injection of anti-VEGF. One limitation of our study is that actual thromboembolic events were not evaluated. However, a number of previous studies demonstrated that D-dimer levels are associated with thromboembolism risk.14–18,20 We are continuing to follow the patients in this study to evaluate thromboembolic events. Another limitation is the study design that means a nonrandomized design, no clinical trial, and a small number of cases. To our knowledge, this is the first study that measured Ddimer levels after intravitreal injection of anti-VEGF agents in patients with AMD. Intravitreal injection of bevacizumab
JEE, ZAKO, AND LA
and ranibizumab did not increase D-dimer levels overall, whereas D-dimer levels were transiently increased in patients with thromboembolic risk factors after intravitreal injection of bevacizumab. Intravitreal injection of ranibizumab is safe with respect to the risk of thromboembolic complications. The results from this study contribute to the understanding of systemic safety concerns after intravitreal injection of anti-VEGF agents for the treatment of AMD.
Author Disclosure Statement None of the authors have any financial or proprietary interest in any material or method mentioned.
References 1. Rosenfeld, P.J., Brown, D.M., Heier, J.S., Boyer, D.S., Kaiser, P.K., Chung, C.Y., and Kim, R.Y. Ranibizumab for neovascular age-related macular degeneration. N. Engl. J. Med. 355:1419–1431, 2006. 2. Schmucker, C., Ehlken, C., Hansen, L.L., Antes, G., Agostini, H.T., and Lelgemann, M. Intravitreal bevacizumab (Avastin) vs. ranibizumab (Lucentis) for the treatment of age-related macular degeneration: a systematic review. Curr. Opin. Ophthalmol. 21:218–226, 2010. 3. Brown, D.M., Kaiser, P.K., Michels, M., Soubrane, G., Heier, J.S., Kim, R.Y., Sy, J.P., and Schneider, S. Ranibizumab versus verteporfin for neovascular age-related macular degeneration. N. Engl. J. Med. 355:1432–1444, 2006. 4. Schouten, J.S., La Heij, E.C., Webers, C.A., Lundqvist, I.J., and Hendrikse, F. A systematic review on the effect of bevacizumab in exudative age-related macular degeneration. Graefes Arch. Clin. Exp. Ophthalmol. 247:1–11, 2009. 5. Ferrara, N., Hillan, K.J., Gerber, H.P., and Novotny, W. Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat. Rev. Drug Discov. 3:391– 400, 2004. 6. Ferrara, N., Damico, L., Shams, N., Lowman, H., and Kim, R. Development of ranibizumab, an anti-vascular endothelial growth factor antigen binding fragment, as therapy for neovascular age-related macular degeneration. Retina. 26:859–870, 2006. 7. Csaky, K., and Do, D.V. Safety implications of vascular endothelial growth factor blockade for subjects receiving intravitreal anti-vascular endothelial growth factor therapies. Am. J. Ophthalmol. 148:647–656, 2009. 8. Schmidt-Erfurth, U. Clinical safety of ranibizumab in agerelated macular degeneration. Expert. Opin. Drug Saf. 9: 149–165, 2010. 9. Kilickap, S., Abali, H., and Celik, I. Bevacizumab, bleeding, thrombosis, and warfarin. J. Clin. Oncol. 21:3542, 2003; author reply 3543. 10. Ueta, T., Yanagi, Y., Tamaki, Y., and Yamaguchi, T. Cerebrovascular accidents in ranibizumab. Ophthalmology. 116:362, 2009. 11. Alexander, S.L., Linde-Zwirble, W.T., Werther, W., Depperschmidt, E.E., Wilson, L.J., Palanki, R., Saroj, N., Butterworth, S.L., and Ianchulev, T. Annual rates of arterial thromboembolic events in medicare neovascular age-related macular degeneration patients. Ophthalmology. 114:2174– 2178, 2007. 12. Mannucci, P. Venous thrombosis: the history of knowledge. Pathophysiol. Haemost. Thromb. 32:209–212, 2002. 13. Thorneycroft, I.H., and Goldzieher, J.W. Venous thromboembolism: a review. J. Reprod. Med. 48(11 Suppl):911– 920, 2003.
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14. Lippi, G., Cervellin, G., Franchini, M., and Favaloro, E.J. Biochemical markers for the diagnosis of venous thromboembolism: the past, present and future. J. Thromb. Thrombolysis. 30:459–471, 2010. 15. Hargett, C.W., and Tapson, V.F. Clinical probability and D-dimer testing: how should we use them in clinical practice? Semin. Respir. Crit. Care Med. 29:15–24, 2008. 16. Tripodi, A. D-dimer testing in laboratory practice. Clin. Chem. 57:1256–1262, 2011. 17. Cosmi, B., and Palareti, G. Update on the predictive value of D-dimer in patients with idiopathic venous thromboembolism. Thromb. Res. 125(Suppl 2):S62–S65, 2010. 18. Verhovsek, M., Douketis, J.D., Yi, Q., Shrivastava, S., Tait, R.C., Baglin, T., Poli, D., and Lim, W. Systematic review: D-dimer to predict recurrent disease after stopping anticoagulant therapy for unprovoked venous thromboembolism. Ann. Intern. Med. 149:481–490, W494, 2008. 19. Brill-Edwards, P., and Lee, A. D-dimer testing in the diagnosis of acute venous thromboembolism. Thromb. Haemost. 82:688–694, 1999. 20. Wada, H., Kobayashi, T., Abe, Y., Hatada, T., Yamada, N., Sudo, A., Uchida, A., and Nobori, T. Elevated levels of soluble fibrin or D-dimer indicate high risk of thrombosis. J. Thromb. Haemost. 4:1253–1258, 2006. 21. Lowe, G.D. Can haematological tests predict cardiovascular risk? The 2005 Kettle Lecture. Br. J. Haematol. 133: 232–250, 2006. 22. Bakri, S.J., Snyder, M.R., Reid, J.M., Pulido, J.S., Ezzat, M.K., and Singh, R.J. Pharmacokinetics of intravitreal ranibizumab (Lucentis). Ophthalmology. 114:2179–2182, 2007. 23. Bakri, S.J., Snyder, M.R., Reid, J.M., Pulido, J.S., and Singh, R.J. Pharmacokinetics of intravitreal bevacizumab (Avastin). Ophthalmology. 114:855–859, 2007. 24. Nomoto, H., Shiraga, F., Kuno, N., Kimura, E., Fujii, S., Shinomiya, K., Nugent, A.K., Hirooka, K., and Baba, T. Pharmacokinetics of bevacizumab after topical, subconjunctival, and intravitreal administration in rabbits. Invest. Ophthalmol. Vis. Sci. 50:4807–4813, 2009. 25. Kim, H., Robinson, S.B., and Csaky, K.G. FcRn receptormediated pharmacokinetics of therapeutic IgG in the eye. Mol. Vis. 15:2803–2812, 2009. 26. Carneiro, A.M., Costa, R., Falcao, M.S., Barthelmes, D., Mendonca, L.S., Fonseca, S.L., Goncalves, R., Goncalves, C., Falcao-Reis, F.M., and Soares, R. Vascular endothelial growth factor plasma levels before and after treatment of neovascular age-related macular degeneration with bevacizumab or ranibizumab. Acta Ophthalmol. 90:e25–e30, 2011. 27. Meyer, C.H., and Holz, F.G. Preclinical aspects of antiVEGF agents for the treatment of wet AMD: ranibizumab and bevacizumab. Eye (Lond). 25:661–672, 2011.
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28. Matsuyama, W., Hashiguchi, T., Mizoguchi, A., Iwami, F., Kawabata, M., Arimura, K., and Osame, M. Serum levels of vascular endothelial growth factor dependent on the stage progression of lung cancer. Chest. 118:948–951, 2000. 29. Tseng, C.S., Lo, H.W., Teng, H.C., Lo, W.C., and Ker, C.G. Elevated levels of plasma VEGF in patients with dengue hemorrhagic fever. FEMS Immunol. Med. Microbiol. 43:99–102, 2005. 30. Hu, C.C., Ho, J.D., and Lin, H.C. Neovascular age-related macular degeneration and the risk of stroke: a 5-year population-based follow-up study. Stroke. 41:613–617, 2010. 31. Sun, C., Klein, R., and Wong, T.Y. Age-related macular degeneration and risk of coronary heart disease and stroke: the Cardiovascular Health Study. Ophthalmology. 116:1913–1919, 2009. 32. Wong, T.Y., Klein, R., Sun, C., Mitchell, P., Couper, D.J., Lai, H., Hubbard, L.D., and Sharrett, A.R. Age-related macular degeneration and risk for stroke. Ann. Intern. Med. 145:98–106, 2006. 33. Martin, D.F., Maguire, M.G., Ying, G.S., Grunwald, J.E., Fine, S.L., and Jaffe, G.J. Ranibizumab and bevacizumab for neovascular age-related macular degeneration. N. Engl. J. Med. 364:1897–1908, 2011. 34. Tolentino, M. Systemic and ocular safety of intravitreal anti-VEGF therapies for ocular neovascular disease. Surv. Ophthalmol. 56:95–113, 2011. 35. Schmucker, C., Ehlken, C., Agostini, H.T., Antes, G., Ruecker, G., Lelgemann, M., and Loke, Y.K. A safety review and meta-analyses of bevacizumab and ranibizumab: off-label versus goldstandard. PloS One. 7:e42701, 2012. 36. Wells, P.S., Anderson, D.R., Rodger, M., Forgie, M., Kearon, C., Dreyer, J., Kovacs, G., Mitchell, M., Lewandowski, B., and Kovacs, M.J. Evaluation of D-dimer in the diagnosis of suspected deep-vein thrombosis. N. Engl. J. Med. 349:1227–1235, 2003.
Received: February 10, 2014 Accepted: August 10, 2014
Address correspondence to: Prof. Tae Yoon La College of Medicine Catholic University of Korea Seoul 137-701 Republic of Korea E-mail: [email protected]
JOURNAL OF OCULAR PHARMACOLOGY AND THERAPEUTICS Volume 00, Number 00, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/jop.2013.0234
Serum D-Dimer Levels to Evaluate the Risk for Arterial Thromboembolism After Intravitreal Injection of Bevacizumab and Ranibizumab Donghyun Jee,1,2 Masahiro Zako,3 and Tae Yoon La1,2
Abstract
Purpose: There are concerns about arterial thromboembolic event after intravitreal injection of bevacizumab or ranibizumab. Motivated by the fact that D-dimer was a sensitive biomarker for thromboembolism, we evaluated serum D-dimer levels in patients with age-related macular degeneration (AMD) after intravitreal injection of bevacizumab and ranibizumab. Methods: In this prospective, nonrandomized, uncontrolled study, 122 patients (122 eyes) with AMD were enrolled. Sixty-two eyes received intravitreal injections of bevacizumab and 60 eyes received intravitreal injections of ranibizumab monthly for 3 months. Serum D-dimer levels were measured in patients before intravitreal injection and 1 day, 1 week, 1 month, and 3 months thereafter. Results: Serum D-dimer levels were not significantly altered following injection of either bevacizumab or ranibizumab. Subgroup analysis for patients at risk for thromboembolic events revealed that serum D-dimer levels showed no significant change after injection of ranibizumab. However, D-dimer levels significantly increased at 1 day (P = 0.041) and 1 week (P = 0.022) after injection of bevacizumab. Conclusions: Serum D-dimer levels were not changed after injection with either bevacizumab or ranibizumab. In subgroup analysis, bevacizumab injection in patients at risk of thromboembolism increased serum D-dimer levels.
which blocks physiologic VEGF.7 This blockage of physiologic VEGF increases the risk of a thromboembolic event or cardiovascular accident.7,8 Because VEGF not only stimulates endothelial cell proliferation, but also maintains vascular integrity, VEGF inhibition could cause endothelial dysfunction leading to thrombosis.9 A recent meta-analysis of prospective clinical trials using anti-VEGF antibodies indicated an association between intravitreal injection of anti-VEGF antibodies and the subsequent incidence of cardiovascular accidents.10 Another study reported that the rate of arterial thromboembolic events was marginally higher in treatment arms receiving higher doses of anti-VEGF antibodies, although this trend was not significant.11 Thromboembolism is a major cause of death in the United States and a leading cause of morbidity, with an annual incidence of about 1 case per 1,000 individuals.12,13 Although a thromboembolic event can be fatal, few studies have investigated thromboembolism after intravitreal antiVEGF therapy. One possible reason is that thromboembolic
Introduction
I
nhibitors of vascular endothelial growth factor (VEGF) are widely used to treat patients with wet, agerelated macular degeneration (AMD).1,2 Neutralization of all isoforms of VEGF-A significantly improves the ability to minimize vision loss, and restore sight in patients with AMD.3,4 Commonly used antibody-derived drugs for treating AMD are bevacizumab (Avastin; Hoffmann La Roche, Basel, Switzerland) and ranibizumab (Lucentis; Genetech, Inc., San Francisco, CA). Bevacizumab is a full-length antibody that was developed as a potential therapeutic agent for use in oncology.5 Ranibizumab is an antigen-binding fragment (Fab) of an antibody, and was developed as part of an anti-VEGF program for AMD treatment.6 Although anti-VEGF antibodies provide remarkable clinical benefits for AMD patients, concerns remain about the potential systemic off-target effects of anti-VEGF inhibition. Intravitreally injected drugs enter the systemic circulation, 1 2 3
Department of Ophthalmology and Visual Science, St. Vincent Hospital, Suwon, Korea. College of Medicine, Catholic University of Korea, Seoul, Korea. Department of Ophthalmology, Aichi Medical University, Aichi, Japan.
1
2
events after intravitreal injection of anti-VEGF occur at extremely low rates. To identify low rates of events to evaluate safety, highly sensitive methods are needed. D-dimer is the most reliable and sensitive biomarker for thromboembolic events.14 It is regarded as a valuable biomarker for diagnosing and managing a vast array of thrombosis-related clinical conditions.15 D-dimer is a specific degradation product of fibrin clots. This small protein fragment, which is present in blood clots degraded by fibrinolysis, is known as ‘‘D-dimer’’ because it contains 2 crosslinked D-fragments of fibrinogen. D-dimer levels are elevated when the coagulation system is activated, such as in cases of thrombosis. Measurement of D-dimer levels is a gold standard for diagnosing thrombosis and identifying increased risk of thrombotic events.15–19 Elevated serum Ddimer levels indicate a high risk of thrombosis.20 Many long-term prospective studies have demonstrated an association between D-dimer and thromboembolism or coronary heart disease.21 In this study, we evaluated serum D-dimer levels after intravitreal injection of bevacizumab or ranibizumab in patients with AMD.
Methods This prospective, nonrandomized study examined patients who received 3 monthly intravitreal injections of bevacizumab or ranibizumab for wet AMD between March and November 2010. The decision about which drug to use was made by the physician (D. Jee), taking into account the patient’s wish, economic status, and insurance status. We excluded patients with ocular neovascularization or rubeosis iridis, or who had undergone intraocular surgery within the last 6 months. Patient with underlying thromboembolic disease was defined as those with present or past history for stroke, coronary heart disease, and venous thromboembolic disease. The study design followed the tenets of the Declaration of Helsinki for biomedical research and was approved by the institutional review board of the Catholic University of Korea in Seoul, Korea. Informed consent was obtained from the patients. To evaluate the change of Ddimer levels after injection, we assume the large effect size (d = 0.8) as a clinically important difference. Using 2-sided significance level 0.05 and power 80%, we obtained *60 eyes per each group. All patients received an intravitreal injection monthly for 3 months. We collected blood samples from patients to examine serum D-dimer levels before the initial injection and 1 day, 1 week, 1 month, and 3 months after initial injection (Fig. 1). D-dimer levels were measured by a medical technologist who is masked to the clinical status of patients or injected drugs in a clinical laboratory using VIDAS D-dimer ELISA method (VIDAS, BioMerieux, craponne, France).17 All patients were instilled with proparacaine (Alcaine; Alcon, Fort Worth, TX) and had skin draping that contained 10% povidone. After inserting a speculum, a 5% povidone solution was applied to the conjunctival sac. After sterile drape placement, the conjunctiva was displaced and bevacizumab (1.25 mg in 0.05 mL) or ranibizumab (0.05 mg in 0.05 mL) was injected perpendicular to the sclera at the inferotemporal position of the pars plana (3.5 mm posterior to the surgical limbus) by using a 30-gauge needle attached to a 1-mL tuberculin syringe. Using an operating microscope,
JEE, ZAKO, AND LA
FIG. 1. Timing of intravitreal injection of anti–vascular endothelial growth factor and blood sampling to examine serum D-dimer levels. Intravitreal injection was performed monthly for 3 months, and blood sampling was conducted before 1st injection, and 1 day, 1 week, 1 month, and 3 months after initial injection. the location of the needle tip was identified as being in the center of the vitreous cavity. All patients underwent clinical examinations, including best-corrected visual acuity measurement, slit-lamp biomicroscopy, intraocular pressure measurement, and fundus examination. We assessed differences between the serum D-dimer levels before and after intravitreal injection within each group by repeated measured analysis of variance (ANOVA) by using commercial software (SPSS version 17.0; SPSS, Inc., Chicago, IL). We also evaluated differences in D-dimer levels between the groups receiving bevacizumab and ranibizumab by repeated measured ANOVA. P < 0.05 was considered statistically significant.
Results We enrolled 122 eyes from 122 patients who received intravitreal injections of bevacizumab or ranibizumab from March to November 2010. All eyes were divided into 2 groups: bevacizumab (62 eyes) and ranibizumab (60 eyes). The bevacizumab and ranibizumab groups were well-balanced overall for age, gender, and underlying arterial thromboembolic disease (Table 1). In both groups, D-dimer levels showed no significant change before and after injection at any point (Fig. 2). No significant difference was seen between the bevacizumab and ranibizumab groups (Table 2).
Table 1. Clinical Characteristics of Subjects in the Study Bevacizumab Ranibizumab (n = 62) (n = 60) Age (years) 68.33 – 6.27 Gender (male/female) 38/24 Diabetes 11 Hypertension 28 Smoking 18 Underlying 12 (19.3%) thromboembolic diseases Stroke 4 Coronary heart 10 disease 1 Venous thromboembolic disease
P
69.36 – 8.99 40/20 9 33 21 11 (18.3%)
0.463 0.5364 0.682 0.277 0.479 0.785
6 8
0.475 0.663
2
0.549
D-DIMER AFTER ANTI-VEGF INJECTION
3
FIG. 2. D-dimer levels after intravitreal injection of bevacizumab (B) or ranibizumab ( ). No significant change was detected at any time after intravitreal injection in either bevacizumab or ranibizumab groups. Repeated measured analysis of variance (ANOVA).
Subgroup analysis for patients with risk of thromboembolic diseases was performed. D-dimer levels showed no significant change after ranibizumab injection, but D-dimer levels at 1 day and 1 week after bevacizumab injection were significantly higher than that before injection (P = 0.041 and P = 0.022, respectively; Fig. 3). D-dimer levels were significantly higher at 1 day (P = 0.038) and 1 week (P = 0.017, Table 3) after bevacizumab injection than after ranibizumab injection. During the follow-up period, none of the patients developed any severe local adverse events, such as endophthalmitis, uveitis, or retinal tears, or systemic side effects, such as cerebrovascular attacks or myocardial infarction.
Discussion Our study demonstrated that D-dimer levels were unchanged after intravitreal injections of either bevacizumab or ranibizumab. In patients with thromboembolic risk factors, intravitreal injections of bevacizumab resulted in transient increases in D-dimer levels. Although the exact mechanism of D-dimer elevation by bevacizumab remains to be elucidated, we have several hypotheses. One possible reason is the entry of different amounts of anti-VEGF agents into systemic circulation. Intravitreally administered bevacizumab might be transferred across the blood-retinal barrier into the systemic vascular system, while ranibizumab might remain localized in the injected eye. In a recent study in which the pharmacokinetics of intravitreally administered ranibizumab and bevacizumab were compared in rabbits, ranibizumab was not detected in serum, while
small amounts of bevacizumab were detected.22,23 In another study, it was reported that intravitreal bevacizumab appeared in plasma and in the fellow eye via systemic circulation.24 The pharmacokinetic difference in these 2 anti-VEGF agents could be due to differences in molecular structure. Bevacizumab is a full-length antibody, while ranibizumab is a Fab fragment. Ranibizumab does not possess the fragment crystallizable (Fc) region, which is important in transferring intravitreally injected full-length immunoglobulin G (IgG) across the blood-retinal barrier into the systemic blood system.25 In a study using Fc-receptor-knockout mice, IgG without Fc receptor did not penetrate the blood-retinal barrier. Thus, bevacizumab may enter the systemic circulation more easily. Systemic bevacizumab might disrupt physiological VEGF function, which could lead to increased D-dimer levels. A recent report demonstrated significantly reduced VEGF plasma levels after intravitreal bevacizumab injection; these were not seen after intravitreal ranibizumab injection.26 Another possible reason is that the half-life of ranibizumab is shorter than of that of bevacizumab when exposed to systemic circulation. Antigen-binding fragments, such as ranibizumab, tend to have a shorter half-life than full-length antibodies, such as bevacizumab. The systemic half-life of a Fab is a few hours, whereas the half-life of full-length IgG is *3 weeks.27 The longer half-life of bevacizumab might be involved in the transient increase in serum D-dimer levels in this study.
Table 3. D-Dimer (mg/L) Levels in Patients with Underlying Arterial Thromboembolic Disease After Bevacizumab or Ranibizumab Injection
Table 2. D-Dimer (mg/L) Levels in Patients After Bevacizumab or Ranibizumab Injection
Preoperative Postoperative 1 day 1 week 1 month 3 months
FIG. 3. D-dimer levels after intravitreal injection of bevacizumab (B) or ranibizumab ( ) in patients with underlying arterial thromboembolic disease. D-dimer levels were not significantly changed after ranibizumab injection, but Ddimer levels at 1 day and 1 week after bevacizumab injection significantly increased compared with before injection. *P < 0.05, repeated measured ANOVA.
Bevacizumab (n = 62)
Ranibizumab (n = 60)
P
182 – 206 215 – 325 231 – 280 210 – 167 192 – 227
206 – 127 192 – 194 211 – 233 185 – 241 208 – 316
0.345 0.106 0.672 0.089 0.715
Continuous variables are expressed as n or mean – standard deviation. Repeated measured analysis of variance (ANOVA).
Preoperative Postoperative 1 day 1 week 1 month 3 months
Bevacizumab (n = 12)
Ranibizumab (n = 11)
P
215 – 242 356 – 225 412 – 268 327 – 274 281 – 266
221 – 264 238 – 165 217 – 192 263 – 271 241 – 304
0.654 0.038a 0.017a 0.147 0.526
Continuous variables are expressed as n or mean – standard deviation. a P < 005, repeated measured ANOVA.
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The exact mechanism of how anti-VEGF agents induce thromboembolism is unclear. One possible mechanism is that suppression of physiological VEGF causes damage to vascular endothelial cells, especially to the balanced coagulative and fibrinolytic systems. This develops into thromboembolism, resulting in elevated D-dimer levels. Our hypothesis is supported by the finding that D-dimer levels are positively associated with VEGF levels.28,29 AMD is associated with strokes.30,31 The Atherosclerosis Risk in Communities Study showed an almost 2-fold risk of stroke in AMD patients over 10 years (hazard ratio, 1.87; 95% confidence interval [CI], 1.21–2.88).32 Hu et al. reported that neovascular AMD is associated with a higher risk of stroke over 5 years (hazard ratio, 2.42; 95% CI, 1.47– 3.98).30 Thus, the suppression of physiological VEGF by intravitreal injection of anti-VEGF for AMD treatment could induce thromboembolic events, because patients with AMD is more susceptible to stroke. D-dimer levels were not elevated at 3 months after intravitreal injection in this study. Our study had a schedule of 3 monthly injections as a loading dose. Cumulative doses from 3 monthly injections might have an adverse effect on D-dimer levels. However, we found no cumulative effect from repeated injections. We presume that D-dimer levels might be normalized after repeated and regular inhibition of VEGF by a complex feedback mechanism. In a recent study, pro re nata (PRN; as needed) anti-VEGF therapy was associated with more thromboembolic events than a monthly regimen.33 This implies that abrupt inhibition of physiologic VEGF is associated with higher risk of thromboembolism than repeated inhibition at regular intervals. The risk of thromboembolism after intravitreal injection of anti-VEGF has been described in previous reports.7,34,35 Comparison of AMD Treatments Trials (CATT) study—a randomized, controlled clinical trial—reported that thromboembolic events were more frequent in a bevacizumab group than in a ranibizumab group.33 Our results correspond with these previous results. The D-dimer assay has a high negative predictive value. It is highly sensitive but not specific to thromboembolic disease.36 D-dimer levels can also be elevated in malignancy, pregnancy, recent trauma or surgery, bleeding, and sepsis, because all these diseases have a common underlying process of thromboembolism.16 Therefore, D-dimer elevation by bevacizumab in patients with underlying thromboembolic disease does not indicate an elevated postoperative thromboembolism risk. However, intravitreally injected bevacizumab and ranibizumab seem to be safe from thromboembolic complications considering the high negative predictive value of the D-dimer test. D-dimer assays can be used to exclude a diagnosis of thromboembolic events after intravitreal injection of anti-VEGF. One limitation of our study is that actual thromboembolic events were not evaluated. However, a number of previous studies demonstrated that D-dimer levels are associated with thromboembolism risk.14–18,20 We are continuing to follow the patients in this study to evaluate thromboembolic events. Another limitation is the study design that means a nonrandomized design, no clinical trial, and a small number of cases. To our knowledge, this is the first study that measured Ddimer levels after intravitreal injection of anti-VEGF agents in patients with AMD. Intravitreal injection of bevacizumab
JEE, ZAKO, AND LA
and ranibizumab did not increase D-dimer levels overall, whereas D-dimer levels were transiently increased in patients with thromboembolic risk factors after intravitreal injection of bevacizumab. Intravitreal injection of ranibizumab is safe with respect to the risk of thromboembolic complications. The results from this study contribute to the understanding of systemic safety concerns after intravitreal injection of anti-VEGF agents for the treatment of AMD.
Author Disclosure Statement None of the authors have any financial or proprietary interest in any material or method mentioned.
References 1. Rosenfeld, P.J., Brown, D.M., Heier, J.S., Boyer, D.S., Kaiser, P.K., Chung, C.Y., and Kim, R.Y. Ranibizumab for neovascular age-related macular degeneration. N. Engl. J. Med. 355:1419–1431, 2006. 2. Schmucker, C., Ehlken, C., Hansen, L.L., Antes, G., Agostini, H.T., and Lelgemann, M. Intravitreal bevacizumab (Avastin) vs. ranibizumab (Lucentis) for the treatment of age-related macular degeneration: a systematic review. Curr. Opin. Ophthalmol. 21:218–226, 2010. 3. Brown, D.M., Kaiser, P.K., Michels, M., Soubrane, G., Heier, J.S., Kim, R.Y., Sy, J.P., and Schneider, S. Ranibizumab versus verteporfin for neovascular age-related macular degeneration. N. Engl. J. Med. 355:1432–1444, 2006. 4. Schouten, J.S., La Heij, E.C., Webers, C.A., Lundqvist, I.J., and Hendrikse, F. A systematic review on the effect of bevacizumab in exudative age-related macular degeneration. Graefes Arch. Clin. Exp. Ophthalmol. 247:1–11, 2009. 5. Ferrara, N., Hillan, K.J., Gerber, H.P., and Novotny, W. Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat. Rev. Drug Discov. 3:391– 400, 2004. 6. Ferrara, N., Damico, L., Shams, N., Lowman, H., and Kim, R. Development of ranibizumab, an anti-vascular endothelial growth factor antigen binding fragment, as therapy for neovascular age-related macular degeneration. Retina. 26:859–870, 2006. 7. Csaky, K., and Do, D.V. Safety implications of vascular endothelial growth factor blockade for subjects receiving intravitreal anti-vascular endothelial growth factor therapies. Am. J. Ophthalmol. 148:647–656, 2009. 8. Schmidt-Erfurth, U. Clinical safety of ranibizumab in agerelated macular degeneration. Expert. Opin. Drug Saf. 9: 149–165, 2010. 9. Kilickap, S., Abali, H., and Celik, I. Bevacizumab, bleeding, thrombosis, and warfarin. J. Clin. Oncol. 21:3542, 2003; author reply 3543. 10. Ueta, T., Yanagi, Y., Tamaki, Y., and Yamaguchi, T. Cerebrovascular accidents in ranibizumab. Ophthalmology. 116:362, 2009. 11. Alexander, S.L., Linde-Zwirble, W.T., Werther, W., Depperschmidt, E.E., Wilson, L.J., Palanki, R., Saroj, N., Butterworth, S.L., and Ianchulev, T. Annual rates of arterial thromboembolic events in medicare neovascular age-related macular degeneration patients. Ophthalmology. 114:2174– 2178, 2007. 12. Mannucci, P. Venous thrombosis: the history of knowledge. Pathophysiol. Haemost. Thromb. 32:209–212, 2002. 13. Thorneycroft, I.H., and Goldzieher, J.W. Venous thromboembolism: a review. J. Reprod. Med. 48(11 Suppl):911– 920, 2003.
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14. Lippi, G., Cervellin, G., Franchini, M., and Favaloro, E.J. Biochemical markers for the diagnosis of venous thromboembolism: the past, present and future. J. Thromb. Thrombolysis. 30:459–471, 2010. 15. Hargett, C.W., and Tapson, V.F. Clinical probability and D-dimer testing: how should we use them in clinical practice? Semin. Respir. Crit. Care Med. 29:15–24, 2008. 16. Tripodi, A. D-dimer testing in laboratory practice. Clin. Chem. 57:1256–1262, 2011. 17. Cosmi, B., and Palareti, G. Update on the predictive value of D-dimer in patients with idiopathic venous thromboembolism. Thromb. Res. 125(Suppl 2):S62–S65, 2010. 18. Verhovsek, M., Douketis, J.D., Yi, Q., Shrivastava, S., Tait, R.C., Baglin, T., Poli, D., and Lim, W. Systematic review: D-dimer to predict recurrent disease after stopping anticoagulant therapy for unprovoked venous thromboembolism. Ann. Intern. Med. 149:481–490, W494, 2008. 19. Brill-Edwards, P., and Lee, A. D-dimer testing in the diagnosis of acute venous thromboembolism. Thromb. Haemost. 82:688–694, 1999. 20. Wada, H., Kobayashi, T., Abe, Y., Hatada, T., Yamada, N., Sudo, A., Uchida, A., and Nobori, T. Elevated levels of soluble fibrin or D-dimer indicate high risk of thrombosis. J. Thromb. Haemost. 4:1253–1258, 2006. 21. Lowe, G.D. Can haematological tests predict cardiovascular risk? The 2005 Kettle Lecture. Br. J. Haematol. 133: 232–250, 2006. 22. Bakri, S.J., Snyder, M.R., Reid, J.M., Pulido, J.S., Ezzat, M.K., and Singh, R.J. Pharmacokinetics of intravitreal ranibizumab (Lucentis). Ophthalmology. 114:2179–2182, 2007. 23. Bakri, S.J., Snyder, M.R., Reid, J.M., Pulido, J.S., and Singh, R.J. Pharmacokinetics of intravitreal bevacizumab (Avastin). Ophthalmology. 114:855–859, 2007. 24. Nomoto, H., Shiraga, F., Kuno, N., Kimura, E., Fujii, S., Shinomiya, K., Nugent, A.K., Hirooka, K., and Baba, T. Pharmacokinetics of bevacizumab after topical, subconjunctival, and intravitreal administration in rabbits. Invest. Ophthalmol. Vis. Sci. 50:4807–4813, 2009. 25. Kim, H., Robinson, S.B., and Csaky, K.G. FcRn receptormediated pharmacokinetics of therapeutic IgG in the eye. Mol. Vis. 15:2803–2812, 2009. 26. Carneiro, A.M., Costa, R., Falcao, M.S., Barthelmes, D., Mendonca, L.S., Fonseca, S.L., Goncalves, R., Goncalves, C., Falcao-Reis, F.M., and Soares, R. Vascular endothelial growth factor plasma levels before and after treatment of neovascular age-related macular degeneration with bevacizumab or ranibizumab. Acta Ophthalmol. 90:e25–e30, 2011. 27. Meyer, C.H., and Holz, F.G. Preclinical aspects of antiVEGF agents for the treatment of wet AMD: ranibizumab and bevacizumab. Eye (Lond). 25:661–672, 2011.
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28. Matsuyama, W., Hashiguchi, T., Mizoguchi, A., Iwami, F., Kawabata, M., Arimura, K., and Osame, M. Serum levels of vascular endothelial growth factor dependent on the stage progression of lung cancer. Chest. 118:948–951, 2000. 29. Tseng, C.S., Lo, H.W., Teng, H.C., Lo, W.C., and Ker, C.G. Elevated levels of plasma VEGF in patients with dengue hemorrhagic fever. FEMS Immunol. Med. Microbiol. 43:99–102, 2005. 30. Hu, C.C., Ho, J.D., and Lin, H.C. Neovascular age-related macular degeneration and the risk of stroke: a 5-year population-based follow-up study. Stroke. 41:613–617, 2010. 31. Sun, C., Klein, R., and Wong, T.Y. Age-related macular degeneration and risk of coronary heart disease and stroke: the Cardiovascular Health Study. Ophthalmology. 116:1913–1919, 2009. 32. Wong, T.Y., Klein, R., Sun, C., Mitchell, P., Couper, D.J., Lai, H., Hubbard, L.D., and Sharrett, A.R. Age-related macular degeneration and risk for stroke. Ann. Intern. Med. 145:98–106, 2006. 33. Martin, D.F., Maguire, M.G., Ying, G.S., Grunwald, J.E., Fine, S.L., and Jaffe, G.J. Ranibizumab and bevacizumab for neovascular age-related macular degeneration. N. Engl. J. Med. 364:1897–1908, 2011. 34. Tolentino, M. Systemic and ocular safety of intravitreal anti-VEGF therapies for ocular neovascular disease. Surv. Ophthalmol. 56:95–113, 2011. 35. Schmucker, C., Ehlken, C., Agostini, H.T., Antes, G., Ruecker, G., Lelgemann, M., and Loke, Y.K. A safety review and meta-analyses of bevacizumab and ranibizumab: off-label versus goldstandard. PloS One. 7:e42701, 2012. 36. Wells, P.S., Anderson, D.R., Rodger, M., Forgie, M., Kearon, C., Dreyer, J., Kovacs, G., Mitchell, M., Lewandowski, B., and Kovacs, M.J. Evaluation of D-dimer in the diagnosis of suspected deep-vein thrombosis. N. Engl. J. Med. 349:1227–1235, 2003.
Received: February 10, 2014 Accepted: August 10, 2014
Address correspondence to: Prof. Tae Yoon La College of Medicine Catholic University of Korea Seoul 137-701 Republic of Korea E-mail: [email protected]
