EVALUATION OF INTRAVITREAL BEVACIZUMAB FOR ITS SYSTEMIC SIDE EFFECT THROMBOSIS AFTER CHRONIC ADMINISTRATION
Anila Naz, Rahila Najam, Bushra Riaz, Arsalan Ahmed
Department of Pharmacology, Faculty of Pharmacy, University of Karachi, Karachi-75270, Pakistan.Department of Pharmacology, Faculty of Pharmacy, Jinnah University for Women, Karachi, Pakistan.Layton Rehmatullah Benevolent Trust, Lahore, Pakistan.
Keywords: Bevacizumab, thrombosis, intravitreal.
Abstract

Bevacizumab targets Vascular endothelial growth factor-A (VEGF-A). Bevacizumab specifically binds to the VEGF-A protein, thereby inhibiting the process of angiogenesis.Thrombosis and hypertension are the major systemic side effects of bevacizumab.As thrombosis and hypertension are the major systemic side effects of bevacizumab whether this drug couldpredispose a patient to thrombosis or not after intravitreal administration, as it is absorbed even through intravitreal administration. We determined the safety of the drug.This study was conducted at Al Ibrahim eye hospital for 3months. The drug wasintravitrealy administered by Professor Dr.P.S.Mahar.For this 10 patients were administered three doses of intravitrealbevacizumab at monthlyinterval and followed for chronic effects of drug. Blood samples were taken to determine fibrinogen level, platelet count,prothrombin time (PT), activated partial thromboplastin time (APTT) andsodium level by kit method. Blood pressure was also monitored of all the patients before and after the drug administration.There has been significant decrease seen in fibrinogen level. Non-significant rise in the PT.Platelet counts decrease insignificantly. Slight increase is noted in sodium level.Slight increase is noted in diastolic blood pressure where as systolic blood pressure is insignificantly increased.Thus results of our study indicate that there may be bleeding tendency after bevacizumab so careful monitoring is required in patients receiving this drug, as well as monitoring of blood pressure is required.

Article Information

Identifiers and Pagination:
Year:2014
Volume:6
First Page:179
Last Page:186
Publisher Id:JAppPharm (2014 ). 6:3. 179-186
Article History:
Received:March 10, 2014
Accepted:March 24, 2014
Collection year:2014
First Published:April 1, 2014

INTRODUCTION:

Bevacizumab is a humanized monoclonal antibody that targets Vascular endothelial growth factor-A (VEGF-A), an isoform of Vascular endothelial growth factor(VEGF) that is responsible for stimulating abnormal blood vessel growth and blood vessel leakage in disease like diabetic retinopathy and retinal vein occlusion. Its role in the cause and progression of choroidal neovascularization in neovascular age-related macular degeneration has become increasingly important, hence by specifically blocking VEGF, there is reduction in pathological angiogenesis.

Four major steps of angiogenesis have been recognized; namely vasodilation and hyper permeability, vessel destabilization and matrix degradation, endothelial cell proliferation and migration, lumen formation and vessel stabilization. Angiogenesis is a complex biological process involving a delicate balance and interplay between a variety of molecular angiogenic and angiostatic factors. The VEGF-A is believed to be prime regulator of angiogenesis and takes part in all four stages of angiogenesis [1]. Intravitrealbevacizumab has been described in some recent articles for treatment of proliferative diabetic retinopathy complicated byvitreous hemorrhage [2], macular edema in central retinal vein occlusion, andneovascular age-related macular degeneration [3]. Invivo studies revealed that in human nonproliferative diabetic retinopathy there was increased expression of VEGF-A [4]. Upregulation of VEGFR-1,VEGFR-2 and VEGFR-3 was also observed [4,5].

The role of VEGF-A in the development of choroidal neovascularization has been established. Increased VEGF-A expression was noted in surgically excised choroidal neovascularization, retinal pigment epithelium and vitreous of age-related macular degeneration [6].

The permeability inducing actions of VEGF appear to induce the development of macular edema. VEGF may cause macular edema that accompanies diabetic retinopathy, retinal vein occlusion and uveitis. Human eyes with macular edema secondary to uveitis, retinal vein accession and diabetic retinopathy have shown increased retinal levels of VEGF [7]. When VEGF was blocked, blood-retinal barrier breakdown could be both prevented and reversed through VEGF inhibition.

Bevacizumb(Avastin) is full length, recombinant humanized Anti-VEGF monoclonal antibody that binds to all isoforms of VEGF-A. It binds directly to the VEGF ligand (which is expressed by both normal and tumor cells) to prevent its interaction with receptors on the surface of endothelial cells, there by inhibiting the biologic activity of VEGF as observed in invitro and invivo assay systems [8].

Angiogenesis is an essential process in tumor development [9,10]. The VEGF ligand is the predominant regulator of tumor angiogenesis [10]. Avastin directly targets the VEGF ligand to specifically inhibit angiogenesis [11]. Maintaining VEGF ligand inhibition may prevent tumor vessel regrowth over time [12,13].

It is the FDA’s approved Anti-VEGF agent in the treatment of patients with metastatic colorectal cancer [14], lung cancer, metastatic renal cell cancer FDA, 2009),glioblastomamultiforme[15]. It has shown promising result, through intravitreal route in the treatment of neovascular ocular diseases like choroidal neovascularization, macular edema and diabetic retinopathy[16,17].

As thrombosis      and hypertension are the major systemic side effects of bevacizumab whether this drug couldpredispose a patient to thrombosis or not after intravitreal administration, as it is absorbed even through intravitreal administration. We determined the safety of the drug.

Since no work has been carried out before on this issue in our local setup, this study would be important in decision making regarding the safety of intravitrealbevacizumab in choroidal and retinal neovascular disorders.

MATERIAL AND METHODS:

Inorder to evaluate the systemic side effects of intravitrealbevacizumab total 10 patients were selected. Patients were selected from Al Ibrahim eye hospital, meeting the inclusion and exclusion criteria receiving Bevacizumab (Avastin) injection. The purpose, procedure, risks and benefits of the study was explained to the patients. Informed consent was taken. In operation theater before injecting the inravitrealbevacizumab,eye ball was anesthetized with topical proparacaine drops sterilized with povidone iodine 5%.

Inclusion criteria:

Patients having neovascularization in retina and choroid of eye like diabetic retinopathy and age related macular degeneration diseases including both males and females having age above 40years. Hypertensive and non hypertensive,diabetic and non diabetic patients were included.

Exclusion criteria:

Patients having any hematological disorders like thrombosis. History of previous Bevacizumab injection.

Drug:

Patients received the drug Bevacizumab. This drug wasintravitrealy administered by Professor Dr.P.S.Mahar. The dose of intravitrealbecizumab administered to the patient is 1.25mg in 0.05ml with 1ml syringe.

Experimental protocol:

These 10 patients were administered three doses of intravitrealbevacizumab at monthlyinterval and followed for chronic effects of drug. Blood samples were taken before and after one week of drug administration to determine fibrinogen level, platelet count,prothrombin time (PT), activated partial thromboplastin time (APTT) andsodium level by kit method. Blood pressure was also monitored of all the patients before and after the drug administration.

Sample collection:

Blood samples were collected in three types of tubes:

BD vacutainer EDTA.K2 tubes for collection of pure blood to study hematological parameter like platelet count.

Bio vac 3.2% sodium citrate tubes for collection of plasma to study coagulation parameters like
PTand APTT and fibrinogen level. Prothrombintimeand activated partial prothrombin time was checked manually using standard reagent kits of DiagnosticaStago France.

B-ject Gel clot activator vaccum tube for collection of serum to study sodium level.

After the collection of samples 3 ml of blood n 3.2% sodium citrate tubes, plasma was separated out and 4 ml of blood in Gel tubes, Serum was separated out, by centrifuging the blood samples in 800 centrifuge machine (china) at 4000 RPM for 5 minutes. The separated plasma and serum was stored in 2-8 °°C and within3hours all the coagulation and electrolyte estimation were performed.

The estimation of platelet was directly done on automated MS4E Vet Hematology analyzer model# 3MSR0214 by MeletSchloesing Switzerland.

After separating the plasma the fibrinogen level was measured by humaclot duo (coagulation analyzer, model# 18650) (Human Germany) using standard reagent kits of Human Germany. Hemostat fibrinogen (manual and automated determination of plasma fibrinogen) was used[18].

Neoplastin CI Plus was used for the determination ofprothrombin time [19]. C.K.Prest was used for the determination of Kaolin-activated partial thromboplastin time [20,21].

After separating the serum, the sodium level was measured by Microlab 300 semi-automated clinical chemistry analyzer by Merck Netherlands.

RESULT

DISCUSSION

Scutz in the study reported that Bevacizumab treatment is associated with significant increase in the risk of arterial thrombosis[22]. In the present study, determination of fibrinogen level, platelet count,prothrombin time (PT), activated partial thromboplastin time (APTT) andsodium level was conducted by kit method. Blood pressure was also monitored of all the patients before and after the drug administration.

There has been significant decrease in the fibrinogen levels seen after chronic administration. Fibrinogen is a key protein in the coagulation pathway, interacting in multiple processes of platelet aggregation, clot formation and wound healing, and contributing to the final step of the coagulation cascade[23]. Low level of fibrinogen may lead to predisposition to bleeding. Thus if a patient receiving intravitrealbevacizumab have any history of thrombosis should receive chronic dosing of intravitrealbevacizumab.

There has been some rise in the PT but it is non-significant. Decreased level of fibrinogen and platelet count may be causing this rise of PT. The results of our study indicate that there is slight risk of bleeding after using intravitrealbevacizumab in neovascularization of choroid and retina. APTT is non-significantly decreased after the injection in chronic stages.

Platelet counts decrease after the injection. Platelets are meant for maintaining hemostasis. They help in wound healing and prevent bleeding.One physiological function of platelets may be to act as scavangers of circulating VEGF to restrict angiogenic activity to sites of wound healing. This may also be occurring in the pathological situation of neovascularization [24].

Slight increase is observed in sodium levels after the injection. Slight increase is noted in diastolic blood pressure. Insignificant increase is seen in systolic blood pressure.This increase in blood pressure may be associated with elevated level of sodium. Raiser et al, study supports our results, reporting that there is a risk of dysregualtion of blood pressure or persistence of hypertension in hypertensive patients after intravitrealbevacizumab injections [25].

Mouradet al.,(2008) reported that bevacizumab treatment resulted in endothelial dysfunction and capillary rarefaction; both changes are closely associated and could be responsible for the rise in blood pressure observed in most patients [26].

The results of our study indicate that there may be bleeding tendency after bevacizumab because fibrinogen levels and platelet counts have decreased after chronic treatment, so careful monitoring is required in patients receiving this drug, as well as monitoring of blood pressure is required. In our study intravitreal administration of bevacizumabproduced a slight increase in systolic and diastolic blood pressure. This work could be further extended to reassess the results with an increased sample size and also determine the effects in complicated patients having secondary problems.

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17.    Rosenfeld PJ, Moshfeghi AA, Puliafito CA, Optical coherence tomography findings after an intravitreal injection of bevacizumab (avastin) for neovascular age-related macular degeneration. Ophthalmic Surg Lasers Imaging 2005 Jul-Aug;36(4):331-5.

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Prof. Dr. Cornelia M. Keck (Philipps-Universität Marburg)
Marburg, Germany

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Welcome to the research group of Prof. Dr. Cornelia M. Keck in Marburg. Cornelia M. Keck is a pharmacist and obtained her PhD in 2006 from the Freie Universität (FU) in Berlin. In 2009 she was appointed as Adjunct Professor for Pharmaceutical and Nutritional Nanotechnology at the University Putra Malaysia (UPM) and in 2011 she obtained her Venia legendi (Habilitation) at the Freie Universität Berlin and was appointed as a Professor for Pharmacology and Pharmaceutics at the University of Applied Sciences Kaiserslautern. Since 2016 she is Professor of Pharmaceutics and Biopharmaceutics at the Philipps-Universität Marburg. Her field of research is the development and characterization of innovative nanocarriers for improved delivery of poorly soluble actives for healthcare and cosmetics. Prof. Keck is executive board member of the German Association of Nanotechnology (Deutscher Verband Nanotechnologie), Vize-chairman of the unit „Dermocosmetics“ at the German Society of Dermopharmacy, active member in many pharmaceutical societies and member of the BfR Committee for Cosmetics at the Federal Institute for Risk Assessment (BfR).

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