OUTLINE OF PROPOSED RESEARCH TOPIC

· PROPOSED TOPIC FOR RESEARCH :

A study on the effect of intravitreal anti-vascular endothelial growth factor on central

corneal thickness & intra ocular pressure in patients attending a tertiary care centre in
eastern India.

. OBJECTIVES OF PROPOSED RESEARCH :

GENERAL OBJECTIVES : 1. To assess any toxic effect of anti-VEGF after intravitreal
injection.
2. To compare IOP & CCT variations in a population attending a
tertiary care centre in Eastern India
3. To estimate the prevalence of problems requiring anti-VEGF
Injections.
SPECIFIC OBJECTIVES : 1 Effect of anti-VEGF on central corneal thickness (CCT).
2. Effect of anti-VEGF on intra-ocular pressure (IOP).
3. Correlation between changes in IOP & CCT if any.
· RESEARCH HYPOTHESIS : The study is aimed to observe whether there is any correlation

existing between intravitreal injection of anti-VEGF and post

injection changes in IOP & CCT in patients treated in NRS

Medical college & Hospital for age related macular degenara-

tion (ARMD),retinal venous obstruction(RVO), diabetic

retinopathy, macular oedema or central serous chorioretino-

pathy (CRCS).

· ALTERNATE HYPOTHESIS :There may be direct relationship between intravitreal anti-

VEGF and change in CCT & IOP .

· NULL HYPOTHESIS : There may not be any correlation between intravitreal anti
VEGF injection & and change in CCT & IOP.
 BACKGROUND OF RESEARCH :

The development of a vascular supply is a fundamental requirement for organ development and
differentiation in multicellular organisms. Angiogenesis is also implicated in the pathogenesis of
a variety of disorders. These include: proliferative retinopathies, age-related macular degenera-
tion, In the case of proliferative retinopathies and age-related macular degeneration, the new
blood vessels are directly responsible for many of the destructive events. Several factors have
previously been identified as potential positive regulators of angiogenesis viz; aFGF, bFGF,
EGF, TNF-alfa, TGF-J3, PGE2, monobutyrin, HGF, TNF-ALFAPD-ECGF, angiogenin and in-
terleukin-8 & VEGF. There is potential clinical applications of VEGF and VEGF antagonists .
The pivotal role played by VEGF in physiological angiogenesis is emphasised by the recent find-
ing that heterozygous mutations inactivating the VEGF gene result in embryonic lethality (Fer-
rara et aI., 2004) [1]

Also, VEGF administration has a therapeutic effect in animal models of coronary or limb is-
chemia (Simon et al)(Ferrara, 1993)[2][3]. Furthermore, recent studies point to VEGF as a crucial
mediator of neovascularisation associated with tumours and proliferative retinopathies (Aiello et
aI., 1994)[4].VEGF is a potent mitogen (ED50 2 -1 0 pM) for vascular endothelial cells but it is
devoid of consistent and appreciable mitogenic activity for other cell types(Ferrara and Henzel,
1989)[5];( Plouet et aI 1989)[6];. VEGF is also able to induce a marked angiogenic response in a
variety of in vivo models including the chick chorioallentoic membrane(Leung et al.,1989[7];),
the rabbit cornea (Phillips et al., 1995) [8], the primate iris (Tolentino et al., in press) [9], the rabbit
bone (Connolly et al.,1989a)[10], etc .. VEGF has been independently purified and clones as a vas-
cular permeability factor (VPF) based on its ability to induce vascular leakage in the guinea pig
skin (Connolly et aI.[11], 1993; Keck et aI., 1989)[12].

Vascular Endothelial Growth Factor has been identified as an endothelial cell-specific mitogen
and angiogenic stimulator in vivo. Multiple biologically active forms of VEGF- A are generated
by both alternative mRNA splicing and post-translational modification (proteolytic cleavage), and
two of these forms (VEGF165 and VEGF121) have been detected in choroidal neovascular le-
sions. Anti-VEGFs (namely Ranibizumab, Bevacizumab, Pegabtinib) are stable small RNA-like
molecules that bind to the 165-kDa isoform of human VEGF and inactivate it, thereby reducing
the retinal and choroidal angiogenesis and halting the increase in vascular permeability.

The CCT and IOP has been compared in many studies with patients age , sex ,IOP & CCT and its
significance determined. The central corneal thickness was seen to vary with various epidemio-
logical parameters in a normal Indian population . The mean was found to be 537.62 microns (+/-
21.71 ).Males have been found to have significantly thicker cornea ( 540.3 +/-22.7 ) than female
( 524.6 +/-17.3 )and the average CCT decreased significantly with age. The 16– 30 age group has
the highest mean CCT of 543.2 and the 60-75 age group has the lowest mean CCT of 515.6 ,the
difference of which was significant (P< 0.05 ). It was also detected that in normal subject who had
thicker corneas, IOP where found to be higher with 0.4 mm of Hg increase in IOP for every 10
micron increase in CCT. The implications of these deferences are tremendous in the increasing
era of refractive surgeries as well as in the diagnosis of glaucoma .The variations in the CCT based
on various epidemiological parameters have been scarcely studied over an Indian population
which could provide valuable information regarding the natural progression of physiological
changes of cornea as well as give clue to the causes of changes in pathological processes . Data
taken from 10th International Conference on CLINICAL & EXPERIMENTAL OPHTHALMOLOGY
[13]
,November 21-23 ,2016 Dubai UAE

The anterior corneal surface is the major refractive element of the human eye, contributing about
80% of the dioptric power of the whole eye in the non-accommodated state. The structure of the
human cornea has a non-uniform curvature with variable thickness throughout. It is thinner at the
centre and thickens towards the edges. The viscous fluid in the anterior chamber of the eye exerts
pressure on the cornea that inflates it and gives its shape.

Corneal thickness is an important indicator of corneal health, and its alteration may be indicative
of different pathologies . Its measurement is the essential factor to assign to the corneal
status,

endothelial pump mechanism and in wide range of disorders; as ectatic dystrophies, contact lenses
related complication, dry eye, and diabetes mellitus. In addition, it is an increasingly important
procedure in the evaluation of patients with ocular hypertension or glaucoma . The curvature of
the internal and external surfaces significantly affects its refractive power but the influence of
the refractive error on corneal thickness has not yet been clearly established. Corneal thickness
can influence the IOP measurement. Thin cornea can underestimate IOP and cornea with higher
thickness can over estimate IOP. Technologically advanced instruments using pachymetry can
give adjusted IOP for a certain corneal thickness.Central Corneal Thickness (CCT ) can affect the
measurement of applanation tonometry. It also affects the decision to perform some keratorefrac-
tive surgical procedures .Various methods have been used in the past to measure the central
corneal thickness. They include Optical Pachymetry ,Ultrasound Pachymetry , Optical Coherence
Tomography (OCT ), Laser Inferometry and Ultrasound Biomicroscopy .Ultrasound pachymetry
is considered to be the standard of measuring CCT.

Pachymetry- derived from Greek words Pachos = thick+ metry = to measure. It is an instru-
ment to measure corneal thickness. Corneal thickness is an important indicator of health status of
cornea especially the corneal endothelial pump function. Thickness of cornea was first reported in
older text book on physiological optics(Helmholtz & Gulstrand ). CCT reading of 0.7 mm or more
is indicative of endothelial decompensation.

The PITX2/PITX2 mutation seen in Axenfield –Rieger malformation results in reduced corneal
thickness. Techniques of pachymetric measurement include Spot measurement & wide area map-
ing. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Wet
AMD induces growth of abnormal blood vessels that obscure vision. Anti VEGF are widely used
to treat AMD, primarily based on the positive results of the Minimally Classic/Occult Trial of the
Anti-VEGF Antibody Ranibizumab in theTreatment of Neovascular AMD (MARINA)[15]and Anti-
VEGF Antibody for the Treatment of Predominantly Classic Choroidal Neovascularizationin
AMD (ANCHOR)[14] studies, and it was approved by the US Food and Drug Administration for
this purpose in 2006. Ranibizumab is a fragment of a recombinant, humanised monoclonal anti-
body derived from the parent antibody Bevacizumab. It binds to vascular endothelial growth factor
(VEGF) and inhibits VEGF signalling. Moreover, after intravitreal injection of Ranibizumab into
rabbit eyes, the antibody was detected in the aqueous humour and was found to reach a peak con-
centration of 17.9 mg/mL3 days after drug administration. Furthermore, it was previously shown
that VEGF and its receptor are present in the corneal endothelium; therefore, it is reasonable to as -
sume that anti VEGF may influence VEGF function in the corneal endothelium. Intracameral in-
jections of Bevacizumab for treatment of neovascular glaucoma can cause corneal endothelial cell
loss as determined by specular microscopy, although the endothelial cell loss is less as compared
to that occurring after cataract surgeries or glaucoma surgeries. Although the pharmacokinetic
profile of Ranibizumab after intravitreal injection in human eyes has not been determined defini -
tively, Ranibizumab has been detected in the aqueous humour after intravitreal standard injections
in animal models. Moreover, VEGF and its receptors were expressed in the corneal endothelium.
Therefore, Ranibizumab in the aqueous humour after intravitreal injections may affect the function
of VEGF in the corneal endothelium.

There has been a paucity of studies to elucidate the possible endothelial toxicity of anti-VEGF
molecules in the Indian population, and it is this void that we have attempted to fill in this study.
Clinical efficacy of intravitreal anti-VEGF drugs has been widely demonstrated in several angio-
genesis-driven eye diseases including diabetic macular edema and the neovascular form of age-re-
lated macular degeneration. Pegaptanib, Ranibizumab, and Aflibercept have been approved for use
in the eye, whereas Bevacizumab is widely used by ophthalmologists to treat patients in an
off label use fashion. These drugs are active in the nanomolar to picomolar range. Intravitreal
anti-VEGF agents are the result of innovative biotechnology processes aimed at creating high-
affinity targeted drugs. Different structural features of these molecules represent the fundamental
basis for the comprehension of their clinical pharmacology. Pegaptanib was the first aptamer ap-
proved for use in humans. It is a 40-kDa RNA polyethylene glycol linked molecule having a
VEGF-binding sequence of 27 nucleotides plus an additional 3′-3′-terminal deoxythymidine.
Ranibizumab is a 48-kDa recombinant humanised immunoglobulin G1κ isotype monoclonal anti-
[20]
body fragment (Fab) devoid of the Fc portion Aflibercept is a 115-kDa fusion protein obtained
combining the Fc portion of a full monoclonal antibody and the two highest affinity domains of
[19]
VEGF receptor type-1 (R1) and VEGFR2 . Bevacizumab is a fully humanised IgG1 of 148 kDa
[18]
administered by intravenous route in cancer patients . This drug is also widely used via the in-
travitreal route by ophthalmologists to treat patients “off-label” since there is no Federal Drug Ad-
ministration (FDA) approval for it to be used as the treatment of wet AMD or diabetic macular
edema (DME). Intravitreal anti-VEGF drugs are the mainstay of important angiogenesis-driven eye
diseases. Efficacy of anti-VEGF therapy has been proven by several independent phase III clinical
trials. For instance, the CRUISE[16] and BRAVO[17]studies demonstrated the efficacy of monthly
ranibizumab for macular edema after central RVO Specifically, patients with macular edema due
to RVO experienced clinically and statistically significant improvements in best-corrected visual
acuity (BCVA).
METHODOLOGY:

STUDY DESIGN : It is a clinico-observational prospective study

STUDY SETTING AND TIMELINES: One Year & six months .(one year data collection and
six months

data analysis)

PLACE OF STUDY : The study will be conducted in NRS Medical College & Hospital

in the department of Ophthalmology

PERIOD OF STUDY: February 2020 to August 2021

STUDY POPULATION : Patients attending Eye OPD who fulfil the inclusion criteria.

SAMPLE SIZE : 300

STUDY DESIGN Single centred ,hospital based clinico-observational study.

CONTROL The normal other eye of the patient.

INCLUSION CRITERIA :- 1. New patients who are diagnosed to have age related macular

degeneration (ARMD), retinal venous obstruction(RVO),

diabetic retinopathy, macular oedema or central serous

chorio-retinopathy (CRCS) etc requiring anti VEGF

injection

2 .Patients who will need single intravitreal anti-VEGF

injection in a 30 days span .

EXCLUSION CRITERIA :- Patients with the following conditions will be excluded from the

study.

(1) Pre existing glaucoma .

(2) H/O previous glaucoma shunt surgery .

(3) Aphakic & vitrectomised patients.

(4) Corneal scaring .

(5) Pre existing corneal disease like Fuchs endothelial dystrophy,

 (6) Contact lens users

(7) Ocular trauma that disrupts the structural integrity of cornea

(8) Previous recipient of cornea

(9) Previous recipient of anti VEGF,

(9) uncooperative patients who may affect the data collection

and analysis

STUDY METHOD :- Patients who express their consent who are within the age group

of 40-80 years including male & female willing to come for

regular follow up, are included in the study if they demonstrate

focal or diffuse macular oedema on Optical Coherence Tomogra-

phy (OCT) due to any of the causes in the inclusion criteria

requiring anti VEGF intravitreal injection patients are to be

excluded as per exclusion criteria. The study will

be conducted prospectively over a time period 18

months period . Adequate number of eyes( 300)

requiring intravitreal anti –VEGF drugs for macular

oedema, age related macular degeneration or retinal

venous occlusion are to be selected (as per selection criteria) for

evaluation and follow up. Sample size is calculated in order to

achieve a power of study 80% at P > 0.05 amongst the number

of patients attending retina clinic of our tertiary care Hospital

( NRS Medical college & Hospital ) keeping in

mind the etiolologies in question .Institutional

Review Board (IRB ) / Ethics Committee approval

needs to be cleared before the commencement of the

study.

Detailed medical history& ocular history will be taken on

first visit & following examination performed.

1. Snellens best corrected visual acquity ( BCVA )

2. Intraocular pressure ( IOP ) with non contact tonometry

(NCT) TOPCON tonometer CT-1P.

3. Slit lamp examination of anterior segment

4. Fundal examination

5. Central corneal thickness (CCT) using TOPCON Comput-

erised Tonometer CT-1P.

At second visit ,injection antiVEGF will be administered through pars

Plana into the vitreous cavity with a 30 G needle at a distance

of 3.5 mm from the limbus in peudophakics & 4 mm in phakics.

Post operatively E/D moxifloxacin 0.5% QDS ,E/D Nepafenac 0.09%

TDS for 7 days & Tab Acetazolamide 250 mg will be given if

needed 3 more visits on 3rd ,15th and 1month will be scheduled post

operatively whence CCT & IOP will be measured .

PLAN FOR DATA ANALYSIS: Properly planned collected data will be analysed in appropriate

statistical way after the data collection phase is completed.

REVIEW OF LITERATURES

Although the pharmacokinetic profile of Ranibizumab after intravitreal injection in human eyes
has not been determined definitively, Ranibizumab has been detected in the aqueous hu-
[21][22]
mor after intravitreal standard injections in animal models . In a rabbit study, Ranibizumab was
present in the aqueous humour of eyes after a 0.5-mg intravitreal injection of Ranibizumab but was
not detected in the serum or the fellow untreated eyes. Concentrations exceeding 0.1 mg/mL of
Ranibizumab were maintained in the ocular compartments for 29 days.Previously, many studies have
shown that VEGF and its receptors are expressed in cornea. Gan et al in 2004[23] found out that VEGF
is expressed in rabbit eyes in corneal epithelium, and endothelium but not in corneal stroma. They
also observed that VEGFR-2 was present in the corneal epithelial and endothelial cells but absent in
keratinocytes. Phillip et al[24] also found out that VEGF and VEGFR are present in corneal epithelium
and endothelium but not on keratinocytes, and that their expression was increased in patients with in-
flamed and injured cornea, thereby indicating that VEGF may be involved in neovascularisation of
injured and inflamed human corneas. Yoeruek et al[29]. in 2007 did an in vivo study and reported that
all human corneal cell lines were immunopositive for VEGF, VEGFR-1 and VEGFR-2. It is therefore
reasonable to consider that Ranibizumab may have cytotoxic effects on corneal endothelium.J. Benitz
herreros et al. in 2010[25] conducted the morphometric analysis of corneal endothelium after 0.5 mg
intravitreal ranibizumab injection in AMD patients using corneal specular microscopy on American

population. In their study, there was no significant difference in the corneal endothelial cell density
and central corneal thickness at 6 months. Consuelo Perez-Rico et al[26] conducted a prospective ob-
servational case series study in 2010 on effect of intravitreal Ranibizumab on corneal endothelium in
patients of age-related macular degeneration, which showed that there was no significant effect on
endothelial cell count, coefficient of variation, % of hexagonal cells and central corneal thickness af-
ter a follow-up of 6 months. Chun-Chi Chiang et al. in 2008[27] and Tseng JJ et al. in 2012 [28]
con-
ducted a six monthly follow-up study to determine the effect of 2.5 mg intravitreal Bevacizumab on
 central corneal thickness. This study showed that there was no significant difference in central
corneal thickness before and after injection.

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